Molecules for regulating the immune response

Abstract

This disclosure provides, in particular, molecules comprising a binding domain that specifically binds to a target antibody and at least one modified Fc domain. This disclosure also provides methods and compositions that enable selective depletion and / or neutralization of pathogenic antibodies. This disclosure provides molecules for selectively depleting and / or neutralizing target antibodies. In some embodiments, the molecules described herein include a first polypeptide comprising a first Fc domain and a binding domain that specifically binds to a target antibody, and a second polypeptide comprising a second Fc domain.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 517,101 and U.S. Provisional Application No. 63 / 517,104, filed on August 1, 2023. The entire contents of the aforementioned applications are incorporated herein by reference. [Background technology]

[0002] Uncontrolled immune responses to pathogens or autoantigens are associated with many diseases, including autoimmune diseases, chronic inflammatory disorders, and allergies. Autoimmune diseases develop when the body's immune system attacks its own healthy cells. Autoimmune diseases include various types such as Graves' disease, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, eclampsia, multiple sclerosis, and vasculitis. Autoantibodies are produced by pathogenic plasma cells that target autoantigens. Autoantibodies are considered markers of diseases associated with uncontrolled immune responses (e.g., autoimmune diseases), and methods to target and deplete these antibodies in patients have been investigated. However, therapeutic approaches to autoimmune diseases and other diseases associated with uncontrolled immune responses often do not selectively deplete pathogenic autoantibodies, but rather lead to the depletion of antibodies that provide an appropriate immune response to invading pathogens. [Overview of the project]

[0003] This disclosure provides molecules for selectively depleting and / or neutralizing target antibodies. The molecules described herein include, in some embodiments, a first polypeptide comprising a first Fc domain and a binding domain specifically binding to a target antibody, and a second polypeptide comprising a second Fc domain. In some embodiments, the first and second Fc domains form a homodimer or heterodimer of the first and second polypeptides. In some embodiments, the second polypeptide further comprises a binding domain specifically binding to a target antibody, and the molecule is a homodimer. In some embodiments, the second polypeptide further comprises a binding domain specifically binding to a target antibody, and the molecule is a heterodimer. In some embodiments, the second polypeptide does not contain a binding domain specifically binding to a target antibody, and the molecule is a heterodimer.

[0004] In some embodiments, the first and / or second Fc domains contain one or more mutant amino acid residues, resulting in increased binding affinity to internalized receptors (e.g., FcγRIIB) compared to the corresponding wild-type Fc domains.

[0005] In some embodiments, when one or two molecules bind to a target antibody, an immune complex is formed. In some embodiments, the immune complex formed by one molecule and a target antibody as described herein has improved binding kinetics with FcγRIIB compared to an immune complex containing the target antibody bound to one corresponding molecule having a wild-type Fc domain. In some embodiments, the immune complex formed by two molecules and a target antibody as described herein has improved binding kinetics with FcγRIIB compared to an immune complex containing the target antibody bound to two corresponding molecules having wild-type Fc domains. Such improved binding kinetics increase the clearance of the immune complex.

[0006] In one embodiment, the present disclosure provides a molecule comprising a first polypeptide comprising a first Fc domain and a binding domain that specifically binds to a target antibody; and a second polypeptide comprising a second Fc domain, wherein the first Fc domain and the second Fc domain form a homodimer or heterodimer of the first polypeptide and the second polypeptide, and the first and / or second Fc domains comprise one or more mutant amino acid residues having increased binding affinity to FcγRIIB compared to the corresponding wild-type Fc domain, and when the two molecules bind to a target antibody, an immune complex is formed having improved binding kinetics with FcγRIIB compared to an immune complex comprising the target antibody bound to two corresponding molecules having wild-type Fc domains.

[0007] In some embodiments, the second polypeptide further comprises a binding domain that specifically binds to a target antibody, and the molecule is a homodimer. In some embodiments, the second polypeptide further comprises a binding domain that specifically binds to a target antibody, and the molecule is a heterodimer. In some embodiments, the second polypeptide does not contain a binding domain that specifically binds to a target antibody, and the molecule is a heterodimer.

[0008] In some embodiments, when two molecules bind to the target antibody, an immune complex is formed that exhibits improved binding dynamics with FcγRIIB compared to an immune complex containing the target antibody bound to only one molecule.

[0009] In some embodiments, the improved binding kinetics include an increase in the association rate, a decrease in the dissociation rate, and / or a change in the equilibrium dissociation constant. In some embodiments, the improved binding kinetics result in an increase in the avidity, stability, strength, frequency, and / or duration of binding between the immune complex and FcγRIIB.

[0010] In some embodiments, the target antibody is a pathogenic antibody. In some embodiments, the target antibody is an autoantibody. In some embodiments, the target antibody is a secreted antibody. In some embodiments, the target antibody is a membrane-bound antibody or an autoreactive B cell receptor.

[0011] In some embodiments, the first and / or the second Fc domain comprising one or more mutated amino acid residues has no increased binding affinity for FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn as compared to the corresponding wild-type Fc domain. In some embodiments, the first and / or the second Fc domain comprising one or more mutated amino acid residues has a decreased binding affinity for FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn as compared to the corresponding wild-type Fc domain. In some embodiments, the first and / or the second Fc domain comprising one or more mutated amino acid residues has substantially no binding affinity for FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn as compared to the corresponding wild-type Fc domain.

[0012] In some embodiments, the improved binding kinetics include a binding affinity of the immune complex for FcγRIIB that is at least 10% higher. In some embodiments, the at least 10% higher binding affinity includes a binding affinity that is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more. In some embodiments, the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.001 μM. In some embodiments, the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.001 μM. In some embodiments, the molecule binds to FcγRIIB with an affinity in the range of about 0.1 μM to 0.01 μM. In some embodiments, the binding affinity includes the binding affinity for a cell line that overexpresses FcγRIIB (e.g., a CHO cell line) when measured by flow cytometry.

[0013] In some embodiments, the molecule does not bind to complement (C1q).

[0014] In some embodiments, the molecule preferentially binds to immune cells that express FcγRIIB rather than immune cells that express FcγRIIA. In some embodiments, the molecule has substantially no binding affinity for cells that do not express FcγRIIB. In some embodiments, the immune cells that express FcγRIIB include B cells, monocytes, and / or basophils. In some embodiments, the immune cells that do not express FcγRIIB include T cells, NK cells, neutrophils, and / or eosinophils. <​​​​​​​​​In some embodiments, the molecule inhibits B cells by crosslinking FcγRIIB with the B cell receptor. In some embodiments, the molecule crosslinks FcγRIIB with the B cell receptor. In some embodiments, an immune complex of one or two molecules with an anti-TSHR autoantibody crosslinks FcγRIIB with the B cell receptor.

[0018] In some embodiments, the first Fc domain and the second Fc domain each include an immunoglobulin constant region comprising a CH2 domain and a CH3 domain.

[0019] In some embodiments, the first and / or second Fc domains are, according to the EU numbering scheme, the following amino acid mutations: E233V, L234D, L235F, G236R, G237D, S239L, S267D, H268P, S298G, T299A, A327L, L328A, A330H, E333I, R292Q, E2 Includes one or more of the following: 33P, P238D, H268D, P271G, A330R, L234Y, T250V, V264I, T307P, Q311R, A330K, P343R, M428L, N434A, Y436T, Q438R, S440E, G236N, S267E, L235R, D270E, E233D, and G237D.

[0020] In some embodiments, the first and / or second Fc domain is a set of amino acid mutations according to the EU numbering scheme: (i) E233V, L234D, L235F, G236R, G237D, S239L, S267D, H268P, S298G, T299A, A327L, L328A, A330H, and E333I; (ii) E233V, L234D, L235F, G236 R, G237D, S239L, S267D, R292Q, H268P, S298G, T299A, A327L, L328A, A330H, and E333I; (iii) E233V, L234D, L 235F, G236R, G237D, S239L, H268P, R292Q, S298G, T299A, A327L, L328A, A330H, and E333I; (iv) E233P, G237D, P238D, H268D, P271G, and A330R; (v) L234Y, P238D, T250V, V264I, T307P, Q311R, A330K, P343R, M428L, N434A, Y436T, Q438R, and S440E; (vi) L234D, G236N, and S267E; (vii) L235R; (viii) G236N and S267E; (ix) P238D and D2 70E; (x)P238D and P271G; (xi)P238D, D270E and P271G; (xii)G237D, P238D, P271G and A330R; (xiii)G237D, P238D, D270E, P271G and A330R; (xiv)E233D, G237D, P238D, H268D, P271G and A330R; and (xv)P238D, including one or more of the above.

[0021] In some embodiments, the first and / or second Fc domains include the mutant amino acid residue P238D, according to the EU numbering scheme.

[0022] In some embodiments, the first and / or second Fc domain does not contain the following mutant amino acid residues: S267E and L328F, according to the EU numbering scheme.

[0023] In some embodiments, the first Fc domain includes a sequence selected from sequence numbers 103, 105, 107, 109, 111-113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139-149, 163-164, 374-376, 378, or a fragment or variant thereof (for example, a sequence selected from sequence numbers 107, 109, 113, 115, 119, 131, 139, 140, 142, 148, 374, or 378). In some embodiments, the second Fc domain includes a sequence selected from sequence numbers 104, 106, 108, 110, 111, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139-149, 163-164, 374-375, 377, 379, or a fragment or variant thereof (for example, a sequence selected from sequence numbers 108, 110, 114, 116, 120, 132, 139, 140, 142, 148, 374, or 379).

[0024] In some embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 107 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 108; or (ii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 108 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 107.

[0025] In some embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 109 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 110; or (ii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 109.

[0026] In some embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 113 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 114; or (ii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 114 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 113.

[0027] In some embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 119 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 120; or (ii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 120 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 119.

[0028] In some embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 131 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 132; or (ii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 132 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 131.

[0029] In some embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 378 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 379; or (ii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 379 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 378.

[0030] In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 374, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 374.

[0031] In some embodiments, the binding domain includes an antigen, or a fragment or variant thereof, to which the target antibody binds. In some embodiments, the antigen is an autoantigen, and the target antibody is an autoantibody. In some embodiments, the first polypeptide includes a binding domain containing a first antigen domain, and the second polypeptide further includes a binding domain containing a second antigen domain. In some embodiments, the first antigen domain and the second antigen domain are the same. In some embodiments, the first antigen domain and the second antigen domain are different. In some embodiments, the first antigen domain includes two or more antigen domains, and / or the second antigen domain includes two or more antigen domains. In some embodiments, the first and / or second antigen domains include two or more (e.g., 2, 3, 4, 5, 6, or 7) epitopes of the same antigen. In some embodiments, the first and / or second antigen domain includes two or more (e.g., 2, 3, 4, 5, 6, or 7) epitopes of different antigens.

[0032] In some embodiments, the binding domain includes an antibody variable domain, such as Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, sdAb, scFv, SMIP, diabody, triabody, tetrabody, minibody, nanobody, maxibody, tandab, DVD, BiTe, TandAb, VHH, peptide sequence, or mimotope, or any combination thereof. In some embodiments, the binding domain binds to the Fc domain of the target antibody (e.g., CH2 or CH3). In some embodiments, the antibody variable domain includes Fab. In some embodiments, the second polypeptide of the molecule further includes a second binding domain containing an antibody variable domain. In some embodiments, the second binding domain is the same as the binding domain of the first polypeptide. In some embodiments, the second binding domain binds to a different target antibody than the binding domain of the first polypeptide.

[0033] In some embodiments, the first and second Fc domains form a heterodimer as a result of a knob-in-hole (KIH) mutation. In some embodiments, the KIH mutation includes Y349T and T394F according to the EU numbering scheme. In some embodiments, the first Fc domain includes the Y349T mutation, and the second Fc domain includes the T394F mutation. In some embodiments, the first Fc domain includes the T394F mutation, and the second Fc domain includes the Y349T mutation. In some embodiments, the KIH mutation includes T366W, S354C, T366S, L368A, Y407V, and Y349C according to the EU numbering scheme. In some embodiments, the first Fc domain includes the T366W and S354C mutations according to the EU numbering scheme, and the second Fc domain includes the T366S, L368A, Y407V, and Y349C mutations. In some embodiments, the first Fc domain includes the T366S, L368A, Y407V, and Y349C mutations according to the EU numbering scheme, and the second Fc domain includes the T366W and S354C mutations.

[0034] In some embodiments, the first and / or second Fc domains contain an IgG1 isotype. In some embodiments, the first and / or second Fc domains contain a human IgG1 isotype.

[0035] In some embodiments, the first and / or second Fc domains include one or more mutant amino acid residues that extend the half-life. In some embodiments, the first and / or second Fc domains include one of the following mutant amino acid residues: M252Y, S254T, and T256E, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domains include a combination of the following mutant amino acid residues: M252Y, S254T, and T256E, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domains include one or more of the following mutant amino acid residues: M428L and N434S, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domains include a combination of the following mutant amino acid residues: M428L and N434S, according to the EU numbering scheme.

[0036] In some embodiments, the binding domain is covalently bonded to the first Fc domain. In some embodiments, the C-terminus of the binding domain is covalently bonded to the N-terminus of the first Fc domain. In some embodiments, the N-terminus of the binding domain is covalently bonded to the C-terminus of the first Fc domain. In some embodiments, the binding domain is covalently bonded to the first Fc domain via a linker.

[0037] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 150 (GGGGS), SEQ ID NO: 151 (GGGGSGGGGS), SEQ ID NO: 152 (GGGGSGGGGSGGGGS), SEQ ID NO: 153 (VDGGGGSGGGGSGGGGSG), SEQ ID NO: 154 (GGGGSGGGGSGGGGSGGGGS), SEQ ID NO: 155 (GGGGSGGGGSGGGGSGGGGSSGGGGS), SEQ ID NO: 156 (GSGGS), SEQ ID NO: 157 (GGSG), SEQ ID NO: 158 (GGSGG), SEQ ID NO: 159 (GSGSG), SEQ ID NO: 160 (GSGGG), SEQ ID NO: 161 (GGGSG), or SEQ ID NO: 162 (GSSSG).

[0038] In another aspect, the Disclosure provides nucleic acids comprising a nucleotide sequence encoding a molecule of the Disclosure.

[0039] In another aspect, the Disclosure provides a host cell comprising a nucleic acid containing a nucleotide sequence encoding a molecule of the Disclosure.

[0040] In another embodiment, the Disclosure provides a vector comprising a nucleic acid containing a nucleotide sequence encoding a molecule of the Disclosure. In some embodiments, the vector includes a viral vector. In some embodiments, the viral vector includes a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, or an adenovirus vector.

[0041] In another aspect, the present disclosure provides an immune complex comprising (i) a target antibody; and (ii) two molecules, each comprising a first polypeptide comprising a first Fc domain and a binding domain that binds to the target antibody; and a second polypeptide comprising a second Fc domain, wherein the first Fc domain and the second Fc domain form a homodimer or heterodimer of the first polypeptide and the second polypeptide, and the first and / or second Fc domains comprise one or more mutant amino acid residues having increased binding affinity to FcγRIIB compared to the corresponding wild-type Tc domain, wherein the immune complex has improved binding kinetics with FcγRIIB compared to an immune complex comprising the target antibody bound to two corresponding molecules having wild-type Fc domains.

[0042] In some embodiments, the immune complex exhibits improved binding kinetics with FcγRIIB compared to an immune complex containing only the target antibody and one molecule. In some embodiments, the immune complex exhibits improved binding kinetics with FcγRIIB compared to an immune complex containing only the target antibody and one molecule. In some embodiments, the immune complex exhibits improved binding kinetics with FcγRIIB compared to the anti-TSHR autoantibody alone. In some embodiments, the binding domain of each of the two molecules is bound to the target antibody. In some embodiments, the improved binding kinetics include an increased association rate, a decreased dissociation rate, and / or a change in the equilibrium dissociation constant. In some embodiments, the improved binding kinetics result in increased avidity, stability, strength, frequency, and / or duration of binding between the immune complex and FcγRIIB.

[0043] In some embodiments, the target antibody is a pathogenic antibody. In some embodiments, the target antibody is an autoantibody. In some embodiments, the target antibody is a secreted antibody. In some embodiments, the target antibody is a membrane-bound antibody or an autoreactive B cell receptor.

[0044] In some embodiments, the first and / or second Fc domains containing one or more mutant amino acid residues do not show increased binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn compared to the corresponding wild-type Fc domain. In some embodiments, the first and / or second Fc domains containing one or more mutant amino acid residues show decreased binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn compared to the corresponding wild-type Fc domain. In some embodiments, the first and / or second Fc domains, which include one or more mutant amino acid residues, have negligible or no binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn compared to the corresponding wild-type Fc domains.

[0045] In some embodiments, the improved binding kinetics include at least 10% higher binding affinity of the immune complex to FcγRIIB. In some embodiments, the at least 10% higher binding affinity includes at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more binding affinity. In some embodiments, the molecule binds to FcγRIIB with affinity in the range of about 1 μM to 0.001 μM. In some embodiments, the molecule binds to FcγRIIB with affinity in the range of about 1 μM to 0.001 μM. In some embodiments, the molecule binds to FcγRIIB with affinity in the range of about 0.1 μM to 0.01 μM. In some embodiments, the binding affinity includes binding affinity to a cell line overexpressing FcγRIIB (e.g., CHO cell line) as measured by flow cytometry.

[0046] In some embodiments, the immune complex preferentially binds to immune cells expressing FcγRIIB rather than to immune cells expressing FcγRIIA. In some embodiments, the immune complex crosslinks FcγRIIB with B cell receptors on B cells.

[0047] In another embodiment, the Disclosure provides a pharmaceutical composition comprising a molecule of the Disclosure or a nucleic acid encoding such molecule, and a pharmaceutically acceptable carrier.

[0048] In another aspect, the Disclosure provides a method for generating the molecule of the Disclosure, comprising expressing a nucleic acid encoding the molecule in a host cell and recovering the molecule.

[0049] In another embodiment, the Disclosure provides a method for reducing the antibody titer of a circulating target antibody in a subject diagnosed with an autoimmune disease, comprising administering the pharmaceutical composition of the Disclosure to the subject, wherein the target antibody is a circulating pathogenic antibody. In some embodiments, the antibody titer is reduced within less than one hour (e.g., less than 30 minutes) after administration of the pharmaceutical composition. In some embodiments, the antibody titer in or from the subject after administration is reduced compared to before administration.

[0050] In another embodiment, the Disclosure provides a method for treating a subject suffering from or susceptible to an autoimmune disease, the method comprising administering to the subject a pharmaceutical composition comprising the molecule of the Disclosure or a nucleic acid encoding the molecule. In some embodiments, the autoimmune disease is associated with the target antibody, which is a pathogenic autoantibody targeted by the binding domain of the molecule. In some embodiments, the titer of the circulating pathogenic autoantibody decreases within less than one hour (e.g., less than 30 minutes) after administration of the pharmaceutical composition to the subject. In some embodiments, the titer of the antibody in or from the subject after administration decreases compared to before administration. In some embodiments, the antibody titer in or from the subject is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the antibody titer before administration. In some embodiments, the reduction in antibody titer persists for a long period of time. In some embodiments, the duration includes at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, or longer. In some embodiments, the circulating pathogenic autoantibody is removed from the subject within 30 minutes of administering the pharmaceutical composition to the subject. In some embodiments, the molecule in the pharmaceutical composition neutralizes circulating pathogenic autoantibodies in the subject. In some embodiments, at least two molecules in the pharmaceutical composition form an immune complex with the circulating pathogenic autoantibodies when the binding domain of the molecules binds to the circulating pathogenic autoantibodies. In some embodiments, the circulating pathogenic autoantibodies are removed from the subject by FcγRIIB-mediated intracellular uptake of the immune complex by B cells expressing FcγRIIB.In some embodiments, the circulating pathogenic autoantibodies are removed from the subject by FcγRIIB-mediated intracellular uptake of the immune complex by hepatic sinusoidal endothelial cells expressing FcγRIIB. In some embodiments, when the pharmaceutical composition is administered to the subject, it reduces the pathogenic autoantibody stimulating activity in the subject's serum. In some embodiments, the pharmaceutical composition is administered to the subject intravenously, intramuscularly, or subcutaneously. In some embodiments, the subject is a human.

[0051] In another aspect, the present disclosure relates to a composition for reducing the titer of a target antibody in the serum of a subject requiring a reduction in the titer of the target antibody in the serum, the composition comprising a plurality of molecules, each molecule comprising (a) a first polypeptide comprising a first Fc domain and a binding domain that specifically binds to the target antibody; and (b) a second polypeptide comprising a second Fc domain, wherein the first Fc domain and the second Fc domain form a homodimer or heterodimer of the first polypeptide and the second polypeptide, and the first and / or second Fc domains are one or more The present invention provides a composition comprising the above-mentioned mutant amino acid residues, which have increased binding affinity to FcγRIIB compared to the corresponding wild-type Fc domain, wherein upon administration of the plurality of molecules, the molecules bind to the target antibody to form an immune complex comprising two molecules bound to the target antibody, and the immune complex binds to FcγRIIB expressed on the surface of hepatic sinusoidal endothelial cells (LSEC) with higher avidity compared to an immune complex comprising two corresponding molecules having wild-type Fc domains, undergoes endocytosis, thereby reducing the titer of the target antibody in the serum of the subject. [Brief explanation of the drawing]

[0052] [Figure 1] This document shows schematic diagrams of exemplary molecules and exemplary mechanisms described herein for the selective depletion of target antibodies (e.g., autoantibodies).

[0053] [Figure 2]This specification shows exemplary molecular forms using single-domain antigens (e.g., TSHR autoantigen domains) described herein.

[0054] [Figure 3] This specification shows exemplary molecular forms using the multi-domain antigens (e.g., PLA2R autoantigen domains) described herein. A list of exemplary PLA2R autoantigen domains available for use in the molecules described herein is also provided.

[0055] [Figure 4] The following are exemplary molecular forms described herein.

[0056] [Figure 5] The following are exemplary molecular forms described herein.

[0057] [Figure 6] The following are exemplary molecular forms described herein.

[0058] [Figure 7] The following are exemplary molecular forms described herein.

[0059] [Figure 8A] This illustrates an exemplary mechanism by which anti-TSHR autoantibodies stimulate cAMP production in cells.

[0060] [Figure 8B] The results of the cAMP assay after the addition of gradually increasing concentrations of agonists (M22, K1-18, and TSH) are shown.

[0061] [Figure 9] The results of cAMP assays after administration of an exemplary molecule (variant D3) alone, or with an anti-TSHR autoantibody (M22) or TSH are shown.

[0062] [Figure 10]The results of cAMP assays are shown for the exemplary molecule (variant D3) alone or after administration with an anti-TSHR autoantibody (M22 or K1-18).

[0063] [Figure 11] The results of cAMP assays using serum samples from healthy donors or patients containing anti-TSHR autoantibodies with and without the exemplary molecule (variant D3) are shown.

[0064] [Figure 12] The results of the cAMP assay are shown, in which an exemplary molecule (variant D3) reduced TSHR activity (measured by downstream cAMP activity) in individual patient serum samples (A) and pooled patient serum samples (B).

[0065] [Figure 13] Some exemplary mechanisms of action of the molecules described herein, which include mutations in the Fc domain that increase affinity for FcγRIIB, are shown below, including (A) neutralization of autoantibodies, (B) elimination of autoantibodies by targeting FcγRIIB isoform 2 on hepatic sinusoidal endothelial cells, (C) targeting pathogenic B cells that produce targeted autoantibodies (e.g., anti-TSHR autoantibodies) by targeting FcγRIIB isoform 1 to the B cell receptor (BCR), resulting in apoptosis and inhibition of B cells, and (D) prevention of binding to FcγRIIB on T cells and T cell activation.

[0066] [Figure 14] The results of binding assays are shown, in which exemplary molecules are captured on an SPR sensor chip and the binding activity of trastuzumab control (A), variant B3 (B), variant D3 (C), and variant E3 (D) to the activating receptor FcγRIIA167H is measured using FcγR as the analyte.

[0067] [Figure 15]The results of binding assays are shown, in which exemplary molecules are captured on an SPR sensor chip and the binding activity of trastuzumab control (A), variant B3 (B), variant D3 (C), and variant E3 (D) to the activating receptor FcγRIIA167R is measured using FcγR as the analyte.

[0068] [Figure 16] The results of binding assays are shown, in which exemplary molecules are captured on an SPR sensor chip and the binding activity of trastuzumab control (A), variant B3 (B), variant D3 (C), and variant E3 (D) to the inhibitory receptor FcγRIIB is measured using FcγR as the analyte.

[0069] [Figure 17] The results of a binding assay are shown, in which His-tagged FcγR is captured on an SPR sensor chip, and the binding activity of trastuzumab control (A), variant B3 (B), variant D3 (C), and variant E3 (D) to the activating receptor FcγRIIA167H is measured using exemplary molecules as analytes.

[0070] [Figure 18] The results of a binding assay are shown, in which His-tagged FcγR is captured on an SPR sensor chip, and the binding activity of trastuzumab control (A), variant B3 (B), variant D3 (C), and variant E3 (D) to the activating receptor FcγRIIA167R is measured using exemplary molecules as analytes.

[0071] [Figure 19] The results of a binding assay are shown, in which His-tagged FcγR is captured on an SPR sensor chip, and the binding activity of trastuzumab control (A), variant B3 (B), variant D3 (C), and variant E3 (D) to the inhibitory receptor FcγRIIB is measured using exemplary molecules as analytes.

[0072] [Figure 20]The following are ELISA results showing the binding of exemplary molecules to C1q.

[0073] [Figure 21] This report presents the results of a binding assay measuring the binding of anti-TSHR autoantibodies (M22) to FcγRIIB-expressing CHO cells when M22 is pre-complexed with exemplary molecules so that most M22 binds to two molecules (when added in a 4:1 molar ratio of molecules to M22), compared to when M22 primarily binds to one molecule (when added in a 1:1 molar ratio of molecules to M22). Results are also shown when the anti-FcγRIIB blocking antibody 2B6 is included in the 4:1 experiment.

[0074] [Figure 22] This relates to FcγRIIB protein expression among various immune cell types. Figure A is a modified version based on Kerntke, et al., (2020) Frontiers in immunology 11:489401, incorporated herein by reference, and shows that B cells highly express FcγRIIB. Figure B shows that the immune complex of M22 and exemplary molecules bound most strongly to B cells and unclassified cells. Unclassified cells represent cells that could not be classified as monocytes, B cells, NK cells, or T cells because they are negative for CD16, CD19, CD56, and CD3. Such cells may be non-classical monocytes or basophils.

[0075] [Figure 23] This study demonstrates that M22, pre-complexed with exemplary molecules, binds to FcγRIIB-expressing cells: B cells (A) and monocytes (B). Results are also shown when the anti-FcγRIIB blocking antibody 2B6 is included in the experiment.

[0076] [Figure 24] This shows that M22, pre-complexed with exemplary molecules, binds to NK cells (A) and unclassified cells (B). Results are also shown when the anti-FcγRIIB blocking antibody 2B6 is included in the experiment.

[0077] [Figure 25] This shows that variant D3 does not strongly bind to CHO-FcγRIIB+ cells. In contrast, variant B3 binds to CHO-FcγRIIB+ cells at a low concentration of 1 nM. Binding of variant D3 to CHO-FcγRIIB+ cells was evident only at 1 μM. Binding of variants B3 and D3 is completely blocked by the anti-FcγRIIB blocking antibody 2B6.

[0078] [Figure 26] This shows an example of how molecules bind to B cells.

[0079] [Figure 27] The diagram shows exemplary molecular binding to both classical cells (CD14+) (A) and unclassified cells (B). Unclassified cells represent cells that could not be classified as monocytes, B cells, NK cells, or T cells because they are negative for CD16, CD19, CD56, and CD3. Such cells may be non-classical monocytes or basophils.

[0080] [Figure 28] This study demonstrates the binding activity of trastuzumab (positive control) to the inhibitory receptor FcγRIIB when trastuzumab is captured on an SPR sensor chip and FcγR is used as the analyte.

[0081] [Figure 29] Exemplary molecules were captured on an SPR sensor chip, and the binding activity of variants X1(A), X2(B), X3(C), and X5(D) to the inhibitory receptor FcγRIIB is shown when FcγR is used as the analyte.

[0082] [Figure 30] Exemplary molecules were captured on an SPR sensor chip, and the binding activity of variants X6(A) and X7(B) to the inhibitory receptor FcγRIIB was demonstrated using FcγR as the analyte.

[0083] [Figure 31] This study shows the binding activity of trastuzumab (positive control) to the activating receptor FcγRIIA167R when trastuzumab is captured on an SPR sensor chip and FcγR is used as the analyte.

[0084] [Figure 32] Exemplary molecules were captured on an SPR sensor chip, and the binding activity of variants X1(A), X2(B), X3(C), and X5(D) to the activating receptor FcγRIIA167R was demonstrated using FcγR as the analyte.

[0085] [Figure 33] Exemplary molecules were captured on an SPR sensor chip, and the binding activity of variants X6(A) and X7(B) to the activating receptor FcγRIIA167R was demonstrated using FcγR as the analyte.

[0086] [Figure 34] This study shows the binding activity of trastuzumab (positive control) to the activating receptor FcγRIIA167H when trastuzumab is captured on an SPR sensor chip and FcγR is used as the analyte.

[0087] [Figure 35] Exemplary molecules were captured on an SPR sensor chip, and the binding activity of variants X1(A), X2(B), X3(C), and X5(D) to the activating receptor FcγRIIA167H when FcγR was used as the analyte is shown.

[0088] [Figure 36] Exemplary molecules were captured on an SPR sensor chip, and the binding activity of variants X6(A) and X7(B) to the activating receptor FcγRIIA167H was demonstrated using FcγR as the analyte.

[0089] [Figure 37] The exemplary in vivo activity of the exemplary molecules described herein is shown. A shows a schematic diagram of the administration of the exemplary molecule in wild-type BALB / c mice (M22 antibody was administered one day prior to administration of the exemplary molecule). B-C show the serum concentrations (ng / mL) of the M22 antibody measured over time when different exemplary molecules were administered at t=0.

[0090] [Figure 38] The exemplary in vivo activity of the exemplary molecules described herein is shown. A shows a schematic diagram of the administration of the exemplary molecule in wild-type BALB / c mice (M22 antibody was administered one day prior to administration of the exemplary molecule). B-C show the serum concentrations (ng / mL) of the exemplary molecules measured over time when different exemplary molecules were administered at t=0.

[0091] [Figure 39] The results of a pK experiment (A) measuring the molecular concentration (ng / mL) in serum over time, with each point representing the median value for 5 mice (wild-type BALB / c mice) and the bar indicating the mean half-life of the molecule represented by SEM (B).

[0092] [Figure 40] The exemplary in vivo activity of the exemplary molecules described herein is shown. A shows a schematic diagram of the administration of the exemplary molecule in B-hFcRn mice (mice containing the human FcRn gene) (M22 antibody was administered one day prior to administration of the exemplary molecule). B-C show the serum concentrations (ng / mL) of the M22 antibody measured over time when different exemplary molecules were administered at t=0.

[0093] [Figure 41]The exemplary in vivo activity of the exemplary molecules described herein is shown. A shows a schematic diagram of the administration of the exemplary molecule in B-hFcRn mice (mice containing the human FcRn gene) (M22 antibody was administered one day prior to administration of the exemplary molecule). B-C show the serum concentrations (ng / mL) of the exemplary molecule ("ASP") measured over time when different exemplary molecules were administered at t=0.

[0094] [Figure 42] The results of a pK experiment (A) measuring the concentration (ng / mL) of a molecule ("ASP") in serum over time, with each point representing the median value for 5 mice (B-hFcRn) and the bar indicating the mean half-life of the molecule represented by SEM (B).

[0095] [Figure 43] The exemplary in vivo activity of the exemplary molecules described herein is shown. A shows a schematic diagram of the administration of the exemplary molecule in huFcγR-huFcRn mice (mice containing human FcRn and FcγR genes) (M22 antibody was administered one day prior to administration of the exemplary molecule). B-C show the serum concentrations (ng / mL) of the M22 antibody measured over time when different exemplary molecules were administered at t=0.

[0096] [Figure 44] The exemplary in vivo activity of the exemplary molecules described herein is shown. A shows a schematic diagram of the administration of the exemplary molecules in huFcγR-huFcRn mice (mice containing human FcRn and FcγR genes) (M22 antibody was administered one day prior to administration of the exemplary molecule). B-C show the serum concentrations (ng / mL) of the exemplary molecules measured over time when different exemplary molecules ("ASP") were administered at t=0.

[0097] [Figure 45] The results of a pK experiment (A) measuring the concentration (ng / mL) of a molecule ("ASP") in serum over time, with each point representing the median value for five mice (huFcγR-huFcRn mice) and the bar indicating the mean half-life of the molecule represented by SEM (B).

[0098] [Figure 46] This section presents exemplary results from experiments testing the formation of an immune complex between an exemplary molecule (variant D3) and the M22 antibody. The M22 antibody and the exemplary molecule were mixed in various ratios, and complex formation was evaluated by HPLC-SEC.

[0099] [Figure 47] The following are exemplary results from an experiment testing the formation of immune complexes between exemplary molecules and the M22 antibody. Patient serum samples were incubated with a fluorescently labeled molecule, and it was shown that the molecule formed complexes as 2:1 and 1:1 (molecule:autoantibody) complexes (represented by characteristic peaks).

[0100] [Figure 48] The results of an ELISA assay measuring inflammatory cytokines to evaluate the immune response to a representative molecule are shown. A shows the level of IL-6 secreted into the supernatant of human PBMCs cultured with the representative molecule and M22 antibody. B shows the level of MCP-1 secreted into the supernatant of human PBMCs cultured with the representative molecule and M22 antibody.

[0101] [Figure 49] The results of experiments measuring the activation of monocytes (A) and NK cells (B) after culturing with exemplary molecules and the M22 antibody are shown.

[0102] [Figure 50] The results of an experiment measuring the activation of TH-P immune cells cultured with an exemplary molecule-M22 immune complex (molecule:M22 ratio was 4:1) are shown.

[0103] [Figure 51] The results of experiments using AC-SINS (affinity-captured self-interacting nanoparticle spectroscopy) to identify the self-association tendencies of exemplary molecules are shown.

[0104] [Figure 52] The results of experiments using DSC to measure the thermal stability of exemplary molecular variants D3(A) and E3(B) are shown.

[0105] [Figure 53] The results of Western blot analysis measuring the phosphorylation of FcγRIIB in B cells pre-complexed with M22 (4:1) or incubated with exemplary molecules as free drugs in the presence of activated anti-IgG / IgM F(ab)2 or anti-IgM F(ab)2 are shown.

[0106] [Figure 54] The results of binding assays are shown, in which molecules are captured on an SPR sensor chip and the binding activity of trastuzumab control, variant G1, variant G2, variant G3, variant G6, variant G7, and variant G8 to the activating receptor FcγRIIA167R is measured using FcγR as the analyte.

[0107] [Figure 55] The results of binding assays are shown, in which molecules are captured on an SPR sensor chip and the binding activity of variants G9, G10, G11, G12, G13, G14, and G4 to the activating receptor FcγRIIA167R is measured using FcγR as the analyte.

[0108] [Figure 56] The results of binding assays are shown, in which molecules are captured on an SPR sensor chip and the binding activity of trastuzumab control, variant G1, variant G2, variant G3, variant G6, variant G7, and variant G8 to the activating receptor FcγRIIA167H is measured using FcγR as the analyte.

[0109] [Figure 57]The results of binding assays are shown, in which molecules are captured on an SPR sensor chip and the binding activity of variants G9, G10, G11, G12, G13, G14, and G4 to the activating receptor FcγRIIA167H is measured using FcγR as the analyte.

[0110] [Figure 58] The results of binding assays are shown, in which molecules are captured on an SPR sensor chip and the binding activity of trastuzumab control, variant G1, variant G2, variant G3, variant G6, variant G7, and variant G8 to the inhibitory receptor FcγRIIB is measured using FcγR as the analyte.

[0111] [Figure 59] The results of binding assays measuring the binding activity of variants G9, G10, G11, G12, G13, G14, and G4 to the inhibitory receptor FcγRIIB, using FcγR as the analyte, are shown.

[0112] [Figure 60] This bar graph shows the mean fluorescence intensity (MFI) of an Alexa Fluor647-labeled M22 autoantibody detected by flow cytometry at increasing concentrations of free molecules bound to ectopically expressed FcγRIIB in genetically modified CHO-K1 cell lines (CHO-FcγRIIB). FcγRIIB-dependent binding of exemplary molecules was evaluated using pretreatment of CHO-FcγRIIB cells with 10 μg / mL anti-FcγRIIB blocking antibody clone 2B6. MFI values ​​were calculated from single living cells. Each condition was evaluated individually.

[0113] [Figure 61]This image shows a flow cytometry half-offset histogram of the fluorescence signal of Alexa Fluor647-labeled M22 autoantibody, indicating the detection of binding of free molecules to ectopically expressed FcγRIIB in genetically modified CHO-K1 cell lines (CHO-FcγRIIB) at increasing concentrations. FcγRIIB-dependent binding of exemplary molecules was evaluated using pretreatment of CHO-FcγRIIB cells with 10 μg / mL anti-FcγRIIB blocking antibody clone 2B6. Signals were calculated from single living cells. Each condition was evaluated individually.

[0114] [Figure 62] This bar graph shows the mean fluorescence intensity (MFI) of an Alexa Fluor647-labeled M22 autoantibody detected by flow cytometry at increasing concentrations of free molecules bound to ectopically expressed FcγRIIA167R in a genetically modified CHO-K1 cell line (CHO-FcγRIIA167R). FcγRIIA-dependent binding of the molecule was evaluated using pretreatment of CHO-FcγRIIA167R cells with 10 μg / mL anti-FcγRIIA blocking antibody clone IV.3. MFI values ​​were calculated from single living cells. Each condition was evaluated individually. Data represent biological replication and mean ± sd for n=2.

[0115] [Figure 63] This image shows a flow cytometry half-offset histogram of the fluorescence signal of Alexa Fluor647-labeled M22 autoantibody, indicating the detection of binding of the free molecule to ectopically expressed FcγRIIA167R in the genetically modified CHO-K1 cell line (CHO-FcγRIIA167R) at increasing concentrations. FcγRIIA-dependent binding of the molecule was evaluated using pretreatment of CHO-FcγRIIA167R cells with 10 μg / mL anti-FcγRIIA blocking antibody clone IV.3. Signals were calculated from single living cells. Representative data from one of two independent experiments. [Modes for carrying out the invention]

[0116] definition To make this disclosure easier to understand, certain terms are first defined below. Further definitions of the terms below, and any additional terms, are provided throughout this specification. Publications and other reference materials referenced herein to provide background and additional details relating to their implementation are incorporated herein by reference.

[0117] In this specification, the articles "a" and "an" are used to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0118] Administration: As used herein, generally refers to the administration of a composition to a subject or system. Those skilled in the art will recognize various routes that may be used for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be intraocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by intrabronchial instillation), buccal mucosa, percutaneous (e.g., one or more of the following, or including, topical, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, subarachnoid, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosa, nasal, oral, rectal, subcutaneous, sublingual, topical, trachea (e.g., by intratracheal instillation), transvaginal, vitreous, etc. In some specific embodiments, administration may be parenteral (e.g., by intravenous injection). In some embodiments, administration may include intermittent administration (e.g., multiple doses spaced apart) and / or cyclical administration (e.g., individual doses spaced apart by a common period). In some embodiments, administration may include continuous administration over at least a selected period (e.g., perfusion).

[0119] Affinity: As is known in the art, "affinity" is a measure of the firmness with which two or more binding partners (e.g., an antibody and a target antigen) associate with each other. Those skilled in the art will be familiar with the various assays available for assessing affinity, and also with appropriate controls for such assays. In some embodiments, affinity is assessed by quantitative assays. In some embodiments, affinity is assessed over multiple concentrations (e.g., one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitors (e.g., relevant, e.g., those that may be present in a physiological context). In some embodiments, affinity is assessed in comparison to a reference (e.g., a "positive control" reference with a known affinity above a certain threshold, or a "negative control" reference with a known affinity below a certain threshold). In some embodiments, affinity may be assessed in comparison to a contemporaneous reference. In some embodiments, affinity may be assessed in comparison to a past reference. Typically, when affinity is assessed in comparison to a reference, it is assessed under equivalent conditions.

[0120] Approximately or about: When used herein and applied to one or more values ​​of interest, means a value similar to the given reference value. In some embodiments, the terms “approximately” or “about” mean a range of values ​​that are within 20% (more than or less than) of the given reference value in either direction, unless otherwise specified or evident from the context (except where such a number exceeds 100% of the possible reference value).

[0121] Antibody: As used herein, refers to a polypeptide containing a canonical immunoglobulin sequence element sufficient to result in specific binding to a particular target antigen. As known in the art, an intact antibody, such as one produced naturally, is a tetrameric drug composed of two identical heavy-chain polypeptides and two identical light-chain polypeptides that associate with each other to form a structure commonly referred to as a "Y-shaped" structure. Each heavy chain contains at least four domains: an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH1, CH2, and carboxy-terminal CH3 (located at the base of the trunk portion of the Y). A short region known as the "switch" connects the heavy-chain variable and constant domains. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region conjugate the two heavy-chain polypeptides together in the intact antibody. Each light chain consists of two domains. In other words, an amino-terminal variable (VL) domain is followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch." Intact antibody tetramers consist of two heavy-light chain dimers, in which the heavy and light chains are linked to each other by one disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other, thereby linking the dimers together to form a tetramer. Naturally produced antibodies are also typically glycosylated at the CH2 domain. Each domain in natural antibodies has a structure characterized by an "immunoglobulin fold" formed by two beta sheets (e.g., 3, 4, or 5-strand sheets) bundled together in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops or "CDRs" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4) known as "complementarity-determining regions." When the natural antibody folds, the FR region forms a β-sheet that confers a structural framework to the domain, and the CDR loop regions of both the heavy and light chains come together in three-dimensional space to form a single hypervariable antigen-binding site located at the tip of a Y-shaped structure.The Fc region of naturally occurring antibodies is located at the base of the Y structure and binds to complement system elements, including, for example, effector cells that mediate cytotoxicity, and also to receptors on effector cells. The affinity and / or other binding properties of the Fc region to Fc receptors can be regulated through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to this disclosure include a glycosylated Fc domain, which includes an Fc domain that has been modified or manipulated in terms of glycosylation. In some embodiments, any polypeptide, or a polypeptide complex containing a sufficient immunoglobulin domain sequence as found in naturally occurring antibodies, may be referred to and / or used as an “antibody,” regardless of whether such polypeptides are produced naturally (e.g., by organisms that react to antigens) or by recombinant operations, chemosynthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence characteristic of mouse antibodies, rabbit antibodies, primate antibodies, or human antibodies. In some embodiments, the antibody sequence elements are human, humanized, primated, chimeric, and the like, as are known in the art. Furthermore, as used herein, the term “antibody” may refer to any construct or format known or developed in the art for utilizing the structural and functional features of an antibody in an alternative presentation, in a suitable embodiment (unless otherwise stated or evident from the context). For example, in some embodiments, the antibodies utilized in accordance with this disclosure may be intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies; or forms used herein in a broad sense, selected from, but not limited to, antibody fragments (preferably those fragments exhibiting desired antigen-binding activity) that encompass a variety of antibody structures.For example, the antibodies described herein may be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as any other immunological binding moieties known in the art, including, for example, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, sdAb, scFv, SMIP, diabody, triabody, tetrabody, minibody, nanobody, maxibody, tandab, DVD, BiTe, TandAb, or any combination thereof. The subunit structures and three-dimensional configurations of different classes of antibodies are known in the art. In some embodiments, antibodies may lack covalent modifications (e.g., glycan binding) that they would have if naturally produced. In some embodiments, antibodies may include covalent modifications, e.g., glycan binding, cargo moieties (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0122] Antigen-binding domain: The “antigen-binding domain” refers to the portion of an antibody that binds to the antigen to which an intact antibody binds. Antigen-binding domains of antibodies include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to the antigen to form a complex. Exemplary antigen-binding domains include, but are not limited to, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, sdAb, scFv, SMIP, diabody, triabody, tetrabody, minibody, nanobody, maxibody, tandab, DVD, BiTe, TandAb, or any combination thereof. In some embodiments, the antigen-binding domain of the antibody described herein is scFv. In some embodiments, the antigen-binding domain of the antibody described herein is only the VHH domain. As with complete antibody molecules, the antigen-binding domain may be monospecific or multispecific (e.g., bispecific). The multispecific antigen-binding domain of an antibody may include at least two different variable domains, each of which can specifically bind to a different antigen or to a different epitope of the same antigen.

[0123] Antibody heavy chain: As used herein, this refers to the larger of the two types of polypeptide chains present in naturally produced intact antibodies.

[0124] Antibody light chain: As used herein, this refers to the smaller of the two types of polypeptide chains present in naturally produced intact antibodies.

[0125] Synthetic antibody: As used herein, this refers to an antibody produced using recombinant DNA technology. This term should also be interpreted to mean an antibody produced by the synthesis of an antibody-encoding DNA molecule, and an antibody on which the DNA molecule expresses an antibody protein, or an amino acid sequence that identifies an antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0126] Antigen: As used herein, the term “antigen” refers to a molecule (e.g., a peptide, polypeptide, or polysaccharide) that elicits a specific immune response. Antigen-specific immune responses, also known as adaptive immune responses, are mediated by lymphocytes (e.g., T cells, B cells, NK cells) that express antigen receptors (e.g., T cell receptors, B cell receptors). In some embodiments, the antigen is a T cell antigen and elicits a cellular immune response. In some embodiments, the antigen is a B cell antigen and elicits a humoral (i.e., antibody) response. In some embodiments, the antigen is both a T cell antigen and a B cell antigen. As used herein, the term “antigen” encompasses both full-length polypeptides and parts or immunogenic fragments of polypeptides, as well as peptide epitopes within polypeptides (e.g., peptide epitopes conjugated by major histocompatibility complex (MHC) molecules (e.g., MHC class I, or MHC class II)). In some embodiments, the antigen is an autoantigen. In some embodiments, the antigen is tissue-specific or non-specific and is identified, for example, from cells or tissues that are targets of an autoimmune response, or from healthy cells or tissues.

[0127] Autoantigen: As used herein, “autoantigen” refers to an antigen that triggers an autoimmune response. An autoantigen is an endogenous (self) antigen that is recognized by the immune system as non-self, i.e., an exogenous pathogen. An autoantigen may be a protein, an immunogenic fragment of a protein, or a complex of proteins that is recognized by the immune system of a person suffering from or susceptible to an autoimmune disease.

[0128] Autoimmune disease: As used herein, “autoimmune disease” refers to an immune response to an autoantigen (or self-antigen).

[0129] Associated: The term “associated” is used herein when two events or entities are related to each other if the existence, level, degree, type and / or form of one is related to that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microorganism, etc.) is considered associated with a particular disease, disorder, or condition if its existence, level and / or form correlates (e.g., across a relevant population) with the onset, susceptibility, severity and stage of a disease, disorder, or condition.

[0130] Binding domain: As used herein, refers to a portion or element that specifically binds to a target portion or element. Typically, the interaction between the binding domain and its target is non-covalent. In some embodiments, the binding domain may be, or include, a portion or element of any chemical classification, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, and small molecules. In some embodiments, the binding domain may be, or include, a polypeptide (or a complex thereof). In some embodiments, the binding domain may be, or include, a target-binding portion of an antibody drug, a cytokine, a ligand (e.g., a receptor ligand), a receptor, a toxin, etc. In some embodiments, the binding domain may be, or include, an aptamer. In some embodiments, the binding domain may be, or include, a peptide nucleic acid (PNA). In some embodiments, the binding domain may be an antigen (e.g., an autoantigen). In some embodiments, the binding domain binds to an antibody (i.e., a “target antibody”).

[0131] The effective dose for therapeutic or prophylactic use will depend, for example, on the stage and severity of the disease or disorder being treated, the subject's age, weight, and overall health, as well as the prescribing physician's judgment. The dose will also depend on the chosen drug, method of administration, timing and frequency of administration, the presence, nature, and extent of any adverse side effects that may be associated with the administration of a particular drug, and the desired physiological effect. It will be understood by those skilled in the art that various diseases or disorders may require long-term treatment involving multiple doses, each or in a variety of processes, using the molecules of the present invention.

[0132] To code: As used herein, “to code” refers to the inherent properties and resulting biological properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and which acts as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is usually a nucleotide sequence listed in a sequence listing, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, can be said to code for a protein, or other products of that gene or cDNA.

[0133] Epitope: As used herein, refers to a portion that is specifically recognized by an immunoglobulin (e.g., antibody) binding component. In some embodiments, an epitope consists of multiple chemical atoms or chemical groups on an antigen. In some embodiments, such chemical atoms or chemical groups are surface-exposed when the antigen adopts a relevant three-dimensional structure. In some embodiments, such chemical atoms or chemical groups are physically close to each other in space when the antigen adopts such a three-dimensional structure. In some embodiments, at least some of such chemical atoms or chemical groups are physically separated from each other when the antigen adopts a different three-dimensional structure (e.g., linearized or denatured).

[0134] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from a nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence includes one or more of the following: (1) generation of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0135] Fragment: As used herein, the terms “fragment” or “part” refer to a structure that includes a distinct part of the whole but lacks one or more parts found in the whole structure. In some embodiments, a fragment consists of such a distinct part. In some embodiments, a fragment consists of or includes characteristic structural elements or parts found in the whole. In some embodiments, the nucleotide fragment contains or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., nucleic acids) found throughout the nucleotide. In some embodiments, the nucleotide fragment contains or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more monomeric units (e.g., nucleic acids) found throughout the nucleotide. In some embodiments, the polypeptide or protein fragment contains or comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., amino acids) found throughout the polypeptide or protein.In some embodiments, a polypeptide or protein fragment contains or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more monomeric units (e.g., amino acids) found in the polypeptide or protein as a whole. In some embodiments, the whole substance or entity may be referred to as the “parent” of the fragment.

[0136] Identity: As used herein, the term “identity” refers to the overall relationship between macromolecules, e.g., between nucleic acid molecules (e.g., between DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, macromolecules are considered “substantially identical” to one another if their sequences are identical by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. For example, the calculation of the identity percentage of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for the purpose of best comparison (for example, gaps can be introduced in one or both of the first and second sequences for best alignment, and non-identical sequences can be ignored for the purpose of comparison). In some embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. For example, the percentage of identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the comparison of nucleic acid sequences by the ALIGN program uses a PAM120 weighted residue table, a gap length penalty 12, and a gap penalty 4.Alternatively, the percentage of identity between two nucleotide sequences can also be determined using the GAP program in the GCG software package and the NWSgapdna.CMP matrix.

[0137] Human antibodies: As used herein, these are intended to include antibodies having variable and constant regions generated (or assembled) from human immunoglobulin sequences. In some embodiments, an antibody (or antibody component) may be considered "human" even if, for example, one or more CDRs and a particular CDR3 contain residues or elements whose amino acid sequences are not encoded by human germline immunoglobulin sequences (e.g., including sequence diversity, which may have been introduced (inherently), for example, by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).

[0138] Immune cells: As used herein, this refers to cells involved in an immune response, e.g., promoting an immune response. Examples of immune cells include, but are not limited to, T lymphocytes, natural killer (NK) cells, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, plasma cells, or B lymphocytes. Sources of immune cells (e.g., T lymphocytes) can be obtained from the subject.

[0139] Immune mediators: As used herein, the term “immune mediator” refers to any molecule that influences cells and processes involved in the immune response. Examples of immune mediators include cytokines, chemokines, soluble proteins, enzymes, and cell surface markers.

[0140] Immune response: As used herein, refers to a cellular and / or systemic response to an antigen that occurs when immune cells identify an antigen molecule as foreign, induce antibody formation, and / or activate themselves or other immune cells to eliminate the antigen.

[0141] Immunoglobulin or Ig: As used herein, this refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody found in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response to most targets. It is the most efficient immunoglobulin in agglutination, complement binding, and other antibody responses and is important for protection against bacteria and viruses. IgD is an immunoglobulin whose antibody function is unknown, but it may function as an antigen receptor. IgE is an immunoglobulin that mediates immediate-type hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0142] Improved, Increased, or Reduced: As used herein, the terms “improved,” “increased,” or “reduced,” or grammatically equivalent terms, indicate a value compared to an equivalent reference measure. For example, in some embodiments, an evaluation value achieved by the drug of interest may be “improved” compared to one obtained by an equivalent reference drug. Alternatively or additionally, in some embodiments, an evaluation value achieved in the subject or system of interest may be “improved” compared to a value obtained in the same subject or system under different conditions (e.g., before or after an event such as administration of the drug of interest) or in a different equivalent subject (e.g., in the presence of one or more indicators of the particular disease, disorder, or condition of interest, or in an equivalent subject or system different from the subject or system of interest that has been previously exposed to the condition or drug, etc.). In some embodiments, the comparative term refers to a statistically significant difference (e.g., a rate and / or magnitude large enough to achieve a statistical significance). A person skilled in the art will know, or can easily determine, the degree and / or rate of difference necessary or sufficient to achieve such a statistically significant difference in a given context.

[0143] Isolated: As used herein, this refers to something that has been modified or removed from its natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide that has been partially or completely separated from its naturally occurring coexisting substances is “isolated.” Isolated nucleic acids or polypeptides may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0144] K D :As used herein, this refers to the dissociation constant of the binder (e.g., the antibody or its binding component) from a complex with its partner (e.g., the epitope to which the antibody or its binding component binds).

[0145] Koff: As used herein, Koff refers to the dissociation rate constant of the dissociation of a binder (e.g., an antibody or its binding component) from a complex with its partner (e.g., an epitope to which the antibody or its binding component binds).

[0146] Kon: As used herein, Kon refers to the binding rate constant of the association of a binder (e.g., an antibody or its binding component) from a complex with its partner (e.g., an epitope to which the antibody or its binding component binds).

[0147] To modulate: As used herein, the term “to modulate” means to mediate a detectable increase or decrease in the level of response and / or a change in the nature of the response in an object compared to the level and / or nature of the response in the object in the absence of treatment and / or compared to the level and / or nature of the response in an otherwise identical but untreated object. The term encompasses, in an object, preferably a human, disrupting and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response.

[0148] Nucleic acid: As used herein, refers to a polymer of at least three nucleotides. In some embodiments, nucleic acid comprises DNA. In some embodiments, nucleic acid comprises RNA. In some embodiments, nucleic acid is single-stranded. In some embodiments, nucleic acid is double-stranded. In some embodiments, nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite links and / or one or more peptide bonds, such as in "peptide nucleic acid". In some embodiments, nucleic acid comprises one or more, or all, native residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, nucleic acid comprises one or more, or all, non-native residues. In some embodiments, the non-natural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the non-natural residues include one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those of the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence containing one or more introns.In some embodiments, nucleic acids can be prepared by isolation from natural sources, enzymatic synthesis (e.g., polymerization based on a complementary template, such as in vivo or in vitro replication in recombinant cells or systems, or by chemical synthesis). In some embodiments, the nucleic acid has a residue length of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000.

[0149] Operatively linked: As used herein, this means a functional link between, for example, a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, if a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are adjacent to each other in the same reading frame and, if necessary, are used to link two protein-coding regions.

[0150] Pharmaceutical composition: As used herein, refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent of interest exists in a unit dose suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, such as: oral administration, e.g., liquid drugs (aqueous solutions or non-aqueous solutions or suspensions), tablets, e.g., those targeting oral absorption, sublingual absorption, and systemic absorption, pills, powders, granules, pastes for application to the tongue; parenteral administration, e.g., parenteral administration by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions or sustained-release formulations; topical application, e.g., creams, ointments, or sustained-release patches, or sprays applied to the skin, lungs, or mouth; vaginal or rectal use, e.g., pessaries, creams, or foams; sublingual use; ocular use; transdermal use; or transnasal, transpulmonary, and other mucosal surface use.

[0151] Polynucleotide: As used herein, refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides and can be hydrolyzed to monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed to nucleosides. Polynucleotides as used herein include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes, using conventional cloning techniques and PCR, and synthetic means.

[0152] Protein: as used herein, refers to polypeptides (i.e., a series of at least two amino acids linked to one another by peptide bonds). Therefore, proteins and polypeptides as used herein are interchangeable. Proteins may contain non-amino acid portions (e.g., glycoproteins, proteoglycans, etc.) and / or may be processed or modified. Those skilled in the art will understand that “protein” may be a complete polypeptide (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those skilled in the art will understand that a protein may contain two or more polypeptides linked, for example, by one or more disulfide bonds, or associated by other covalent or noncovalent means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, proteins may contain native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than approximately 100 amino acids, less than approximately 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, their biologically active portions, and / or characteristic portions thereof.

[0153] Specific Binding: As used herein, the term “specifically binding” in relation to an antigen-binding domain, for example, an antigen-binding domain found in an antibody, refers to an antigen-binding domain that recognizes a specific antigen but substantially does not recognize or bind to other molecules in the sample. For example, an antigen-binding domain that specifically binds to an antigen of one species may also bind to one or more antigens of other species. However, such cross-reactivity itself does not change the classification of the antigen-binding domain as specific. In another example, an antigen-binding domain that specifically binds to an antigen may also bind to different alleles of the antigen. However, such cross-reactivity itself does not change the classification of the antigen-binding domain as specific. In some cases, the terms “specific binding” or “specifically binding” may be used in relation to the interaction between an antigen-binding domain and a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope). For example, an antigen-binding domain recognizes and binds to a specific protein structure rather than the protein in general. If the antigen-binding domain is specific to epitope "A", then in reactions involving labeled "A" and the antigen-binding domain, the presence of a molecule containing epitope A (or free unlabeled A) reduces the amount of labeled A bound to the antigen-binding domain.

[0154] Subject: As used herein, subject means an organism, e.g., a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, gerbil, cat, or dog). In some embodiments, a human subject is an adult, adolescent, or child subject. In some embodiments, a subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., an autoimmune disease. In some embodiments, a subject is susceptible to a disease, disorder, or condition, and in some embodiments, a susceptible subject is predisposed to developing a disease, disorder, or condition and / or exhibits increased risk (compared to the mean risk observed in a reference subject or reference population). In some embodiments, a subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any specific symptoms (e.g., clinical symptoms of a disease) or features of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, the subject is an individual to be diagnosed and / or treated.

[0155] Target: When used herein, this refers to cells, tissues, organs, or sites in the body that are the target of the methods, systems, and / or compositions provided, for example, cells, tissues, organs, or sites in the body that require treatment or are preferentially bound by the molecules described herein.

[0156] To treat: As used herein, the terms “to treat,” “to treat,” or “to treat” mean partial or complete reduction, improvement, delay, inhibition, prevention, mitigation, and / or reduction of incidence and / or severity of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects that do not exhibit any signs or characteristics of a disease, disorder, and / or condition (e.g., prophylactically). In some embodiments, treatment may be administered to subjects exhibiting only early or mild signs or characteristics of a disease, disorder, and / or condition, for example, to reduce the risk of developing a condition associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects exhibiting established, severe, and / or late signs of a disease, disorder, or condition. As used herein, “therapeutic agent” is any agent used to treat a subject.

[0157] Vector: As used herein, the term “vector” refers to a composition of substances comprising isolated nucleic acids that can be used to deliver isolated nucleic acids into the interior of cells. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes plasmids or viruses that autonomously replicate. The term should also be interpreted to include non-plasmid and non-viral components that facilitate the delivery of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0158] Throughout this disclosure, various aspects may be presented in range form. It should be understood that descriptions in range form are merely for convenience and conciseness and should not be interpreted as immutable limitations on ranges. Therefore, a range description should be considered to specifically disclose not only the individual numbers within that range, but also all possible subranges. For example, a range description such as 1 to 6 should be considered to have not only the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6, but also specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6. This applies regardless of the width of the range.

[0159] Detailed explanation Immune system and pathogenic antibodies The methods described herein are useful for treating or improving autoimmune diseases or diseases associated with an overactive / uncontrolled immune system (e.g., type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and / or vasculitis).

[0160] In some embodiments, the molecules described herein may be used to treat diseases associated with unregulated immune responses involving an immune system overwhelmed by a characteristic population of target antibodies (i.e., pathogenic antibodies) (e.g., autoimmune diseases). The molecules described herein provide a mechanism for depleting specific populations of target antibodies associated with various diseases.

[0161] An unregulated immune response to pathogens or autoantigens is associated with many diseases, including autoimmune diseases, chronic inflammatory disorders, and allergies. Autoimmune diseases develop when the body's immune system attacks its own healthy cells. Examples of autoimmune diseases include Graves' disease, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, eclampsia, multiple sclerosis, and vasculitis. Pathogenic antibodies (e.g., autoantibodies) are produced by pathogenic plasma cells and target autoantigens and healthy cells.

[0162] In general, immune responses can be usefully defined in terms of their integrated functional end effects. Dhabar et al., Immunol Res. 58(2-3):193 (2014) propose that immune responses can be classified as immunoprotective, immunopathological, and immunomodulatory. While these categories provide useful building blocks for organizing the idea, the overall in vivo immune response may consist of several types of responses with different amounts of dominance from each category.

[0163] Immune defense responses are defined as responses that promote efficient wound healing, eliminate infections and cancer, and mediate vaccine-induced immune memory. These responses are associated with cytokines and mediators such as IFN-γ, IL-12, IL-2, granzyme B, and CD107. Immunopathological responses are defined as responses to autoimmune diseases such as multiple sclerosis, arthritis, and lupus, or to harmless antigens (asthma, allergies), and responses involving chronic, non-dissipating inflammation. These responses may also be associated with molecules involved in immune defense responses, but may also include immune mediators such as TNF-α, IL-10, IL-13, IL-17, IL-4, IgE, and histamine. Immunomodulatory responses are defined as those involving factors that regulate (primarily downregulate) the function of immune cells and other immune cells. Recent research suggests that there are arms of the immune system that function to inhibit immune responses, for example, regulatory CD4, among others. + CD25 + FoxP3 + T cells, IL-10, and TGF-beta have been shown to have immunomodulatory / inhibitory functions.

[0164] The target antibodies described herein induce, for example, an immunopathological response to autologous or autoantigens or other antigens associated with chronic, non-dissipating inflammation. This disclosure provides molecules that, when administered to a subject, selectively deplete these target antibodies, thereby treating the underlying disease.

[0165] autoimmune disease In some embodiments, diseases associated with an uncontrolled immune response are autoimmune diseases.

[0166] Autoimmune diseases develop when the body's immune system attacks its own healthy cells. Examples of autoimmune diseases include Graves' disease, membranous nephropathy, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, eclampsia, multiple sclerosis, and vasculitis. The development of autoimmune diseases involves two common elements: (i) loss of tolerance to autoantigens, and (ii) immune-mediated damage to healthy cells. The National Institutes of Health (NIH) estimates that as many as 23.5 million Americans suffer from autoimmune diseases (NIH The Autoimmune Diseases Coordinating Committee_2005).

[0167] An exemplary autoimmune disease is Graves' disease. Graves' disease is an autoimmune thyroid disorder caused by antibodies that stimulate thyrotropin or thyroid-stimulating hormone (TSH) receptors (TSHR) localized in thyroid follicular cells or thyroid cells. These antibodies can bind to TSH receptors in retroorbital tissue, causing Graves' ophthalmopathy or thyroid eye disease (see Burch and Cooper, JAMA 314(23):2544 (2015)). Therefore, stimulating thyrotropin receptor antibodies are a major cause of Graves' disease and GO, and are an important indicator in diagnosis and prediction of clinical severity.

[0168] The thyrotropin receptor, or thyroid-stimulating hormone receptor (TSHR), is the primary autoantigen causing Graves' hyperthyroidism and related eye diseases (Graves' ophthalmopathy or thyroid eye disease). TSHR peptides are recognized by the immune system upon ingestion and presented on antigen-presenting cells (APCs) via MHC class II. Helper T cells recognize the TSHR autoantigen by binding to its fragments on APCs. Activation of helper T cells leads to binding to B cells, where the inflammatory cytokines interleukin II and gamma interferon cause the B cells to mature into TSHR antibody-secreting plasma cells. The synthesized TSHR antibodies bind to TSHR expressed by thyroid cells and orbital target cells (fibroblasts, preadipocytes). This activates pathways such as the Gαs adenylyl cyclase (AC) pathway, stimulating protein kinase A and inducing gene activation by the cAMP response element-binding (CREB) protein. Additional pathways, including the Gαq protein kinase C (PKC) pathway, are also activated, leading to the activation of protein kinase B (Akt) and the induction of mammalian target of rapamycin (mTOR). This induction of gene expression can lead to differentiation into preadipocytes and the synthesis of glycosaminoglycans in the orbital space, potentially causing edema and subsequent fibrosis, which are the clinical phenotypes of thyroid eye disease (see George et al., Front Endocrinol, 11:629925 (2021); and Hansen et al., International Journal of Molecular Sciences 24.7:6835 (2023)).

[0169] Graves' disease can be diagnosed by clinical features, high levels of thyroxine (T4) and triiodothyronine (T3), and undetectable levels of TSH. Levels of TSHR antibodies are also an important indicator (see Burch and Cooper, JAMA 314(23):2544 (2015)). Standard treatment for hyperthyroidism due to Graves' disease includes antithyroid drugs to normalize thyroid hormone production, thyroid atrophy using RAIs, or surgical removal of the thyroid (see Burch and Cooper, JAMA 314(23):2544 (2015)). However, these therapies do not target the stimulant TSHR antibodies associated with the disease.

[0170] Other treatments recently developed for Graves' disease and other autoimmune diseases involving pathogenic plasma cells that produce autoantibodies include antibodies targeting B cell / plasma cell markers, such as anti-CD20 antibodies (e.g., rituximab), anti-CD19 antibodies, anti-CD38 antibodies (e.g., daratumumab), FcRn inhibitors, and plasma exchange. However, such strategies target all B cells or plasma cells, not just the cells that produce pathogenic autoantibodies. In the case of CD38, targeting such cells also depletes other CD38+ cells, including monocytes, T cells, and NK cells. FcRn inhibitors result in pan-IgG depletion and, in many cases, incomplete depletion of autoantibodies.

[0171] Another exemplary autoimmune disease is membranous nephropathy (MN). Membranous nephropathy (MN) is an autoimmune disorder caused by antibodies against the M-type phospholipase A2 receptor (PLA2R). MN presents as a slowly progressing kidney disease. Patients with MN may exhibit symptoms of nephrotic syndrome, including edema and proteinuria.

[0172] The binding of PLA2R antibodies to the glomerular basement membrane (GBM) leads to the formation of immune complexes in the GBM. Deposition of IgG and complement system components in the GBM due to PLA2R antibody binding has been shown to contribute to GBM thickening and damage to the glomerular filtration barrier (see Gu et al., Biomolecules 11(4):513(2021)).

[0173] Treatment for membranous nephropathy may include non-immunosuppressive therapy for nephrotic symptoms. For example, treatment for edema may include a sodium-restricted diet and loop diuretics. Statin therapy is recommended for patients with persistent proteinuria and hypercholesterolemia. Early immunosuppressive treatment for membranous nephropathy included the use of prednisolone and other immunosuppressants, such as cyclophosphamide. However, such treatments have been found to have adverse effects, including anemia, leukopenia, infections, and infertility (see Ronco et al., Nat Rev Dis Primers 7:69 (2021)).

[0174] This disclosure recognizes that essential immunity can be preserved and further selectively introduced to enhance the potency of antibodies targeting exogenous pathogens, such as viral antigens. The molecules described herein include further selectivity to target target antibodies associated with autoimmune diseases and diseases associated with an overactive or unregulated immune system. In some embodiments, this strategy includes molecules containing a binding domain that binds to a target antibody, and the target antibody, upon binding to one or two molecules, forms an immune complex that is selectively targeted and destroyed by the immune system.

[0175] Exemplary molecules This disclosure provides molecules for selectively depleting and / or neutralizing target antibodies. The molecules described herein include, in some embodiments, a first polypeptide comprising a first Fc domain and a binding domain specifically binding to a target antibody, and a second polypeptide comprising a second Fc domain. In some embodiments, the first and second Fc domains form a homodimer or heterodimer of the first and second polypeptides. In some embodiments, the second polypeptide further comprises a binding domain specifically binding to a target antibody, and the molecule is a homodimer. In some embodiments, the second polypeptide further comprises a binding domain specifically binding to a target antibody, and the molecule is a heterodimer. In some embodiments, the second polypeptide does not contain a binding domain specifically binding to a target antibody, and the molecule is a heterodimer.

[0176] In some embodiments, the first and / or second Fc domains contain one or more mutant amino acid residues, resulting in increased binding affinity to internalized receptors (e.g., FcγRIIB) compared to the corresponding wild-type Fc domains.

[0177] In some embodiments, when one or two molecules bind to a target antibody, an immune complex is formed. In some embodiments, the immune complex formed by one molecule and a target antibody as described herein has improved binding kinetics with FcγRIIB compared to an immune complex containing the target antibody bound to one corresponding molecule having a wild-type Fc domain. In some embodiments, the immune complex formed by two molecules and a target antibody as described herein has improved binding kinetics with FcγRIIB compared to an immune complex containing the target antibody bound to two corresponding molecules having wild-type Fc domains. Such improved binding kinetics increase the clearance of the immune complex.

[0178] Joint domain This disclosure provides molecules comprising a binding domain that specifically binds to a target antibody (e.g., an autoantibody, e.g., an autoantibody secreted or expressed on B cells). The binding domain may include any domain that binds to a target antibody associated with autoimmune diseases and / or diseases characterized by an unregulated or hyperactive immune response. In some embodiments, the binding domain comprises an antigen (e.g., an autoantigen), or a fragment or variant thereof. In some embodiments, the binding domain comprises a binding domain that targets any portion or region or epitope on the target antibody (e.g., an autoantibody). In some embodiments, the binding domain comprises an antibody-variable domain, e.g., Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, sdAb, scFv, SMIP, diabody, triabody, tetrabody, minibody, nanobody, maxibody, tandab, DVD, BiTe, TandAb, VHH, peptide sequence, or mimotope or any combination thereof.

[0179] In some embodiments, the binding domains described herein (e.g., antigen domains) prevent the target antibody from binding to its alloantigen.

[0180] Antigen domain In some embodiments, the binding domain comprises an antigen domain, or a fragment or variant thereof. Such an antigen domain is targeted by specific antibodies (e.g., autoantibodies) associated with various diseases (e.g., autoimmune diseases and / or diseases characterized by uncontrolled or hyperactive immune responses).

[0181] In some embodiments, the antigen domain includes two or more antigen domains of the same antigen (e.g., 2, 3, 4, 5, 6, or 7 or more antigen domains). In some embodiments, the antigen domain includes two or more antigen domains of one or more different antigens (e.g., 2, 3, 4, 5, 6, or 7 or more antigen domains).

[0182] In some embodiments, the molecules described herein include a first polypeptide having a binding domain containing an antigen domain and a second polypeptide not containing a binding domain (e.g., an antigen domain), i.e., a monovalent molecule.

[0183] In some embodiments, the molecules described herein include a first polypeptide having a binding domain containing a first antigen domain and a second polypeptide having a binding domain containing a second antigen domain, i.e., a divalent molecule. In some embodiments, the first antigen domain and the second antigen domain are the same. In some embodiments, the first antigen domain and the second antigen domain are different, i.e., a bispecific molecule. In some embodiments, the first antigen domain and the second antigen domain are different fragments or domains of the same antigen (e.g., including different epitopes of the same antigen).

[0184] Many diseases are known to be associated with specific target antibodies. For example, in some embodiments, the antigen domain includes a TSHR autoantigen domain, or a fragment or variant thereof. Such an antigen domain is targeted by anti-TSHR autoantibodies known to be associated with autoimmune diseases such as Graves' disease and thyroid eye disease. As another example, in some embodiments, the antigen domain includes one or more PLA2R autoantigen domains, or fragments or variants thereof. Such an antigen domain is targeted by anti-PLA2R autoantibodies known to cause autoimmune diseases such as membranous nephropathy.

[0185] i.TSHR In some embodiments, the TSHR autoantigen domain (or a fragment or variant thereof) may be used in a molecule to target anti-TSHR autoantibodies known to cause autoimmune diseases such as Graves' disease and thyroid eye disease.

[0186] TSHR belongs to a family of leucine-rich repeat-containing class A G protein-coupled receptors, including follicle-stimulating hormone. The first 21 amino acids (shown in SEQ ID NO: 9) form a signal peptide, which is ultimately cleaved. The remaining amino acid sequence consists of an N-terminal leucine-rich repeat domain (LRD, amino acids 22-281), a hinge or cleavage domain (CD, amino acids 282-409), and a transmembrane domain (TMD, amino acids 410-764). TSHR-stimulating autoantibodies involved in Graves' disease and hyperthyroidism bind to the LRD (see Miller-Gallacher et al., Journal of Molecular Endocrinology 62(3):117(2019), which is incorporated in its entirety herein by reference).

[0187] In some embodiments, the autoantigen domain includes the thyroid-stimulating hormone receptor (TSHR), or a fragment or variant thereof. In some embodiments, the TSHR autoantigen domain includes a fragment or variant of SEQ ID NO: 9. In some embodiments, the TSHR autoantigen domain includes the TSHR leucine-rich repeat domain corresponding to amino acids 22-260 of SEQ ID NO: 9, i.e., "TSHR260" as shown in SEQ ID NO: 1. Such domains are known to interact with certain stimulant antibodies, including the autoantibody known as M22 (see Miller-Gallacher et al., Journal of Molecular Endocrinology 62(3):117-128 (2019)). Such domains are also known to interact with antagonist antibodies such as K1-70 (see Miller-Gallacher et al., Journal of Molecular Endocrinology 62(3):117 (2019)). In some embodiments, the TSHR autoantigen domain includes a fragment of TSHR corresponding to amino acids 22-289 of SEQ ID NO: 9, i.e., "TSHR289" as shown in SEQ ID NO: 5. In some embodiments, the autoantigen domain includes an amino acid sequence, or a fragment thereof, that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5. In some embodiments, the autoantigen domain includes the amino acid sequence, or a fragment thereof, of SEQ ID NO: 1 or SEQ ID NO: 5.

[0188] In some embodiments, the TSHR autoantigen domain contains one or more mutations that increase stability while retaining binding to irritating anti-TSHR autoantibodies (e.g., M22) or TSH-blocking anti-TSHR autoantibodies (e.g., K1-70). Specific mutations introduced into TSHR260 have been shown to produce TSHR260 variants that are approximately 900 times more heat-stable than wild-type TSHR and TSHR260 (see Miller-Gallacher et al., Journal of Molecular Endocrinology 62(3):117-128 (2019)).

[0189] In some embodiments, the autoantigen domain comprises a human TSHR variant containing one or more of the following mutations to the amino acid sequence of SEQ ID NO: R112P, D143P, V169R, I253R, H63S, or any combination thereof. In some embodiments, the autoantigen domain comprises a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: R112P and D143P (see, for example, SEQ ID NOs: 2 and 6). In some embodiments, the autoantigen domain comprises a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: R112P, D143P, V169R, and I253R (see, for example, SEQ ID NOs: 3 and 7). In some embodiments, the autoantigen domain comprises a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: R112P, D143P, and H63S (see, for example, SEQ ID NOs: 4 and 8).

[0190] In some embodiments, the antigen domain comprises a human TSHR variant containing one or more of the following mutations to the amino acid sequence of SEQ ID NO: S94, G194P, K218P, V87P, G137P, G188P, or any combination thereof. In some embodiments, the autoantigen domain comprises a human TSHR variant containing the mutation S94P to the amino acid sequence of SEQ ID NO: SEQ ID NO: SEQ ID NO: 307 (see, for example, SEQ ID NO: 307). In some embodiments, the autoantigen domain comprises a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: S94P and G194P (see, for example, SEQ ID NO: 308). In some embodiments, the autoantigen domain comprises a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: SEQ ID NO: S94P, G194P, and K218P (see, for example, SEQ ID NO: 309). In some embodiments, the autoantigen domain includes a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1, 2, 3, 4, 5 (see, e.g., SEQ ID NO: 310). In some embodiments, the autoantigen domain includes a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5 (see, e.g., SEQ ID NO: 311). In some embodiments, the autoantigen domain includes a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5 (see, e.g., SEQ ID NO: 312). In some embodiments, the autoantigen domain includes a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5 (see, e.g., SEQ ID NO: 313). In some embodiments, the autoantigen domain includes a human TSHR variant containing the mutation G194P to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5 (see, for example, SEQ ID NO: 314).In some embodiments, the autoantigen domain includes a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5: G194P and K218P (see, for example, SEQ ID NO: 315). In some embodiments, the autoantigen domain includes a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5: V87P, S94P, G137P, G194P, and K218P (see, for example, SEQ ID NO: 316). In some embodiments, the autoantigen domain includes a human TSHR variant containing the following mutations to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5: V87P, S94P, G137P, G188P, G194P, and K218P (see, for example, SEQ ID NO: 317).

[0191] In some embodiments, the autoantigen domain comprises a human TSHR variant containing a sequence that is at least 90% identical to one of sequence numbers 1-8 and 307-317 (shown in Table 1 below). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0192] ii. PLA2R In some embodiments, the antigen domain comprises one or more PLA2R autoantigen domains (or fragments or variants thereof). In some embodiments, one or more PLA2R autoantigen domains may be used in the molecule to target anti-PLA2R autoantibodies known to cause autoimmune diseases such as membranous nephropathy.

[0193] In some embodiments, the PLA2R autoantigen domain comprises two or more fragments of the full-length PLA2R protein sequence. The PLA2R protein includes a cysteine-rich (CysR) domain, a fibronectin type II (FnII) domain, eight consecutive C-type lectin domains (CTLDs), and an intracellular C-terminal tail. PLA2R belongs to the mannose receptor family, which also includes Endo180, DEC-205, and FcRY, and is a subgroup of the C-type lectin superfamily. Anti-PLA2R autoantibodies (e.g., those involved in MN) have been shown to primarily bind to the epitope region of PLA2R located within the 28-amino acid peptide of the CysR domain shown in Sequence ID No. 15 (see Fresquet et al., Proceedings of the National Academy of Sciences 119(29):e2202209119(2022)). In some embodiments, the PLA2R autoantigen domain comprises a fragment of PLA2R that includes all or some of the specific domains of the PLA2R protein, including but not limited to a cysteine-rich (CysR) domain, a fibronectin type II (FnII) domain, one or more eight consecutive type C lectin domains (CTLDs), and / or an intracellular C-terminal tail.

[0194] In some embodiments, the antigen domain includes a sequence, fragment, or variant thereof having at least 90% identity with SEQ ID NO: 15. In some embodiments, the autoantigen domain includes the amino acid sequence, fragment, or variant thereof according to SEQ ID NO: 15. In some embodiments, the autoantigen domain includes at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21, or at least 22, or at least 23 or more consecutive amino acid residues of SEQ ID NO: 15. In some embodiments, the autoantibody domain includes one or more fragments within SEQ ID NO: 15.

[0195] In some embodiments, the antigen domain includes SEQ ID NO: 15 (a fragment within the CysR region), or a fragment or variant thereof, and one or more additional fragments within the PLA2R protein (e.g., one or more fragments containing amino acid sequences within one or more CTLD domains).

[0196] In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 15) corresponding to amino acid positions 38-65 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 16) corresponding to amino acid positions 38-165 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 380) corresponding to amino acid positions 38-164 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 17) corresponding to amino acid positions 38-169 and 1107-1246 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes three fragments of PLA2R (SEQ ID NO: 18) corresponding to amino acid positions 38-169, 223-367, and 1107-1246 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes three fragments of PLA2R (SEQ ID NO: 19) corresponding to amino acid positions 38-367 and 1107-1246 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 20) corresponding to amino acid positions 1107-1379 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 381) corresponding to amino acid positions 1107-1380 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 382) corresponding to amino acid positions 1117-1380 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 21) corresponding to amino acid positions 38-170 and 223-368 of the full-length PLA2R protein sequence according to SEQ ID NO: 14, separated by a GGGGS linker (SEQ ID NO: 150).In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 22) corresponding to amino acid positions 38-368 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 383) corresponding to amino acid positions 38-360 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 23) corresponding to amino acid positions 38-367 and 1107-1379 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 24) corresponding to amino acid positions 21-164 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes three fragments of PLA2R (SEQ ID NO: 25) corresponding to amino acid positions 38-169, 223-367, and 1107-1379 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 26) corresponding to amino acid positions 30-164 of the full-length PLA2R protein sequence according to SEQ ID NO: 14.

[0197] In some embodiments, the PLA2R autoantigen domain includes mutations that increase stability while retaining binding to irritating anti-PLA2R autoantibodies.

[0198] In some embodiments, the autoantigen domain comprises a human PLA2R variant containing one or more of the following mutations: K74V, S150V, or any combination thereof. In some embodiments, the autoantigen domain comprises a human PLA2R variant containing the K74V mutation relative to the amino acid sequence of SEQ ID NO: 26 (see, for example, SEQ ID NO: 27). In some embodiments, the autoantigen domain comprises a human PLA2R variant containing the S150V mutation relative to the amino acid sequence of SEQ ID NO: 26 (see, for example, SEQ ID NO: 28).

[0199] In some embodiments, the autoantibody-binding domain comprises a human PLA2R autoantigen domain variant or fragment thereof, which includes one or more mutations in any one amino acid sequence of SEQ ID NOs. 14-26 or 380-383.

[0200] In some embodiments, the autoantigen domain comprises a human PLA2R variant containing a sequence that is at least 90% identical to one of sequence numbers 14-28 or 380-383 (shown in Table 2 below). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0201] Antibody variable domain In some embodiments, the binding domain includes an antibody variable domain (e.g., Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, sdAb, scFv, SMIP, diabody, triabody, tetrabody, minibody, nanobody, maxibody, tandab, DVD, BiTe, TandAb, VHH, peptide sequence, or mimotope or any combination thereof).

[0202] In some embodiments, the antibody-variable domain includes Fab. In some embodiments, the molecule described herein includes a first polypeptide containing a first binding domain and a second polypeptide containing a second binding domain, wherein the first and / or second binding domains contain an antibody-variable domain (e.g., Fab). In some embodiments, the first and second binding domains bind to different target antibodies. In some embodiments, the first and second binding domains bind to the same target antibody. In some embodiments, the first and second binding domains bind to different epitopes of the same target antibody.

[0203] In some embodiments, the antibody variable domain targets the Fc domain of the target antibody (e.g., CH2, CH3 domain, etc.).

[0204] In some embodiments, the antibody-variable domain binds to target antibodies associated with autoimmune diseases and / or diseases characterized by uncontrolled or hyperactive immune responses. In some embodiments, the antibody-variable domain targets, for example, the Fc domain of an IgE antibody that is characteristic of allergies.

[0205] FC Domain In some embodiments, the molecule described herein comprises a first polypeptide containing a binding domain linked to a first Fc domain and a second polypeptide containing a second Fc domain. In some embodiments, the Fc domain described herein contains one or more mutations that modify the binding affinity to a specific Fc receptor (e.g., FcγRIIB, FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn).

[0206] In some embodiments, the first Fc domain and the second Fc domain are the same (e.g., in a homodimer molecule). In some embodiments, the first Fc domain and the second Fc domain are different (e.g., in a heterodimer molecule).

[0207] In some embodiments, the Fc domain contains one or more mutant amino acid residues and has reduced binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn compared to the corresponding wild-type Fc domain. In some embodiments, the Fc domain contains one or more mutant amino acid residues and has substantially no binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn compared to the corresponding wild-type Fc domain.

[0208] In some embodiments, the first and / or second Fc domains of the molecule include modifications (e.g., one or more mutations) that enhance binding to internally localized receptors. In some embodiments, the first and / or second Fc domains of the molecule include modifications (e.g., one or more mutations) that reduce binding to specific Fc receptors. In some embodiments, the first and / or second Fc domains of the molecule include modifications (e.g., one or more mutations) that enhance other features of the molecules described herein (e.g., extension of half-life, heterodimerization, etc.).

[0209] The Fc domain contained in the molecule may contain one of the five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In some embodiments, the conventional antibody contains an IgG antibody. In some embodiments, the Fc domain described herein contains a specific isotype selected from the group of IgG isotypes: IgG1, IgG2, IgG3, and IgG4. In some embodiments, the molecule contains a first and / or second Fc domain which is the IgG1 isotype. In some embodiments, the molecule contains a first and / or second Fc domain which is the human IgG1 isotype. Furthermore, in some embodiments, the Fc domain may contain any specific heavy chain constant domains corresponding to different classes of immunoglobulins, including α, δ, ε, γ, and μ, respectively. In some embodiments, the conventional antibody is an intact IgG1 antibody or other antibody class or isotype as described herein (see, for example, Hudson et al., Nat. Med. 9:129 (2003); Pluckthun, The Pharmacology of Monoclonal Antibodies 113:269 (1994); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444 (1993); WO1993 / 01161; and U.S. Patents No. 5,571,894, No. 5,869,046, No. 6,248,516, and No. 5,587,458, respectively, which are incorporated herein by reference).

[0210] The Fc domain of antibodies contained in the molecules described herein binds to complement system elements, including, for example, effector cells that mediate cytotoxicity, and also to receptors on effector cells. As is known in the art, the affinity and / or other binding attributes of the Fc domain to Fc receptors can be modulated through glycosylation or other modifications. In some embodiments, the molecules described herein include glycosylated Fc domains, which include modified or manipulated glycosylation Fc domains. In some embodiments, the molecules are naturally produced (e.g., generated by organisms that react to antigens) or generated by genetic engineering, chemosynthesis, or other artificial systems or methodologies.

[0211] In some embodiments, one or more modifications made to the Fc domain increase the clearance of the immune complex formed by one or more molecules described herein bound to a target antibody. In some embodiments, one or more modifications made to the Fc domain may also induce selective targeting and / or clearance of the immune complex formed by one or more molecules described herein bound to a target antibody. For example, in some embodiments, when two molecules bind to a target antibody, an immune complex is formed with improved binding kinetics to one or more Fc receptors (e.g., FcγRIIB) compared to an immune complex containing a target antibody bound to two corresponding molecules having wild-type Fc domains. Binding kinetics may be characterized, for example, by an increased association rate, a decreased dissociation rate, and / or a change in the equilibrium dissociation constant. In some embodiments, the Fc domain preferentially binds to immune cells expressing FcγRIIB rather than immune cells expressing FcγRIIA. In some embodiments, the Fc domain has substantially no binding affinity to cells that do not express FcγRIIB (e.g., T cells, NK cells, neutrophils, and / or eosinophils). In some embodiments, the cells expressing FcγRIIB are B cells, monocytes, and / or basophils.

[0212] In some embodiments, improved binding kinetics include an immune complex binding affinity of at least 10% higher to one or more Fc receptors (e.g., FcγRIIB). In some embodiments, improved binding kinetics include a binding affinity of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more.

[0213] In some embodiments, the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.001 μM. In some embodiments, the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.01 μM. In some embodiments, the molecule binds to FcγRIIB with an affinity in the range of about 0.1 μM to 0.01 μM.

[0214] In some embodiments, the Fc domain described herein includes one or more modifications such that the molecule described herein does not activate immune cells (for example, does not activate immune cells to secrete inflammatory cytokines, such as IL-6).

[0215] Exemplary Fc domain sequences for use in accordance with the present disclosure are shown in Table 3 below. It will be understood that any of these Fc domain sequences can be used for the first or second polypeptide of the molecules of the present disclosure. It will also be understood that any exemplary Fc domain sequence having a knob mutation (identified by reference to "knob") can be used with an exemplary Fc domain sequence having a hole mutation (identified by reference to "hole") in preparing a heterodimeric molecule. In some embodiments, the Fc domain sequences shown in Table 3 can be used in pairs, for example, but not limited to, based on the reference numbers found in Table 3, when preparing a heterodimeric molecule (e.g., human IgG1 Fc 1.1 knob can be used with human IgG1 Fc 1.1 hole, human IgG1 Fc 1.2 knob can be used with human IgG1 Fc 1.2 hole, etc.). References to Fc domain sequences useful for "antigen arm for antigen depletion" (i.e., in a polypeptide also including an autoantibody binding domain) or "free arm for antigen depletion" (i.e., in a polypeptide not including an autoantibody binding domain) in Table 3 are intended to be exemplary and non-limiting, i.e., an Fc domain sequence identified in Table 3 as being useful for "antigen arm for antigen depletion" may, in some embodiments, also be used for "free arm for antigen depletion", and it will also be understood that an Fc domain sequence described in Table 3 as being useful for "free arm for antigen depletion" may, in some embodiments, also be used for "antigen arm for antigen depletion". [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-7

[0216] In some embodiments, the first Fc domain comprises a sequence selected from SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 376, and SEQ ID NO: 378, and the second Fc domain comprises a sequence selected from SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 377, and SEQ ID NO: 379.

[0217] In some embodiments, the first Fc domain includes sequences selected from SEQ ID NOs: 103, 105, 113, 115, 117, 121, 123, 125, 376, and 378, and the second Fc domain includes sequences selected from SEQ ID NOs: 104, 106, 114, 116, 118, 122, 124, 126, 377, and 379.

[0218] In some embodiments, the first Fc domain includes sequences selected from SEQ ID NOs: 107, 109, 119, 127, 129, 131, 133, 135, and 137, and the second Fc domain includes sequences selected from SEQ ID NOs: 108, 110, 120, 128, 130, 132, 134, 136, and 138.

[0219] In some embodiments, the first Fc domain includes the sequence of sequence number 103, and the second Fc domain includes the sequence of sequence number 104.

[0220] In some embodiments, the first Fc domain includes the sequence of sequence number 105, and the second Fc domain includes the sequence of sequence number 106.

[0221] In some embodiments, the first Fc domain includes the sequence of sequence number 107, and the second Fc domain includes the sequence of sequence number 108.

[0222] In some embodiments, the first Fc domain includes the sequence of sequence number 109, and the second Fc domain includes the sequence of sequence number 110.

[0223] In some embodiments, the first Fc domain includes the sequence of sequence number 113, and the second Fc domain includes the sequence of sequence number 114.

[0224] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 115, and the second Fc domain comprises the sequence of SEQ ID NO: 116.

[0225] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 117, and the second Fc domain comprises the sequence of SEQ ID NO: 118.

[0226] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 119, and the second Fc domain comprises the sequence of SEQ ID NO: 120.

[0227] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 121, and the second Fc domain comprises the sequence of SEQ ID NO: 122.

[0228] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 123, and the second Fc domain comprises the sequence of SEQ ID NO: 124.

[0229] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 125, and the second Fc domain comprises the sequence of SEQ ID NO: 126.

[0230] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 127, and the second Fc domain comprises the sequence of SEQ ID NO: 128.

[0231] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 129, and the second Fc domain comprises the sequence of SEQ ID NO: 130.

[0232] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 131, and the second Fc domain comprises the sequence of SEQ ID NO: 132.

[0233] [[ID=�6]] In some embodiments, the first Fc domain comprises the sequence of SEQ ID NO: 133, and the second Fc domain comprises the sequence of SEQ ID NO: 134.

[0234] In some embodiments, the first Fc domain includes the sequence of sequence number 135, and the second Fc domain includes the sequence of sequence number 136.

[0235] In some embodiments, the first Fc domain includes the sequence of sequence number 137, and the second Fc domain includes the sequence of sequence number 138.

[0236] In some embodiments, the first Fc domain includes the sequence of sequence number 376, and the second Fc domain includes the sequence of sequence number 377.

[0237] In some embodiments, the first Fc domain includes the sequence of sequence number 378, and the second Fc domain includes the sequence of sequence number 379.

[0238] In some embodiments, the first Fc domain includes the sequence of sequence number 111, and the second Fc domain includes the sequence of sequence number 111.

[0239] In some embodiments, the first Fc domain includes the sequence of sequence number 139, and the second Fc domain includes the sequence of sequence number 139.

[0240] In some embodiments, the first Fc domain includes the sequence of sequence number 140, and the second Fc domain includes the sequence of sequence number 140.

[0241] In some embodiments, the first Fc domain includes the sequence of sequence number 141, and the second Fc domain includes the sequence of sequence number 141.

[0242] In some embodiments, the first Fc domain includes the sequence of sequence number 142, and the second Fc domain includes the sequence of sequence number 142.

[0243] In some embodiments, the first Fc domain includes the sequence of sequence number 143, and the second Fc domain includes the sequence of sequence number 143.

[0244] In some embodiments, the first Fc domain contains the sequence of sequence number 144, and the second Fc domain contains the sequence of sequence number 144.

[0245] In some embodiments, the first Fc domain contains the sequence of sequence number 145, and the second Fc domain contains the sequence of sequence number 145.

[0246] In some embodiments, the first Fc domain contains the sequence of sequence number 146, and the second Fc domain contains the sequence of sequence number 146.

[0247] In some embodiments, the first Fc domain includes the sequence of sequence number 147, and the second Fc domain includes the sequence of sequence number 147.

[0248] In some embodiments, the first Fc domain contains the sequence of sequence number 148, and the second Fc domain contains the sequence of sequence number 148.

[0249] In some embodiments, the first Fc domain includes the sequence of sequence number 149, and the second Fc domain includes the sequence of sequence number 149.

[0250] In some embodiments, the first Fc domain includes the sequence of sequence number 163, and the second Fc domain includes the sequence of sequence number 163.

[0251] In some embodiments, the first Fc domain contains the sequence of sequence number 164, and the second Fc domain contains the sequence of sequence number 164.

[0252] In some embodiments, the first Fc domain contains the sequence of sequence number 374, and the second Fc domain contains the sequence of sequence number 374.

[0253] In some embodiments, the first Fc domain includes the sequence of sequence number 375, and the second Fc domain includes the sequence of sequence number 375.

[0254] Hinge arrangement In some embodiments, the Fc domain includes a hinge sequence. In some embodiments, the Fc domain includes the amino acid sequence of SEQ ID NO: 299 (DKTHTCPPCP). In some embodiments, the Fc domain includes the amino acid sequence of SEQ ID NO: 300 (EPKSSDKTHTCPPCP). In some embodiments, the Fc domain includes the amino acid sequence of SEQ ID NO: 301 (ERKCCVECPPCP). In some embodiments, the Fc domain includes the amino acid sequence of SEQ ID NO: 302 (ELKTRPLGDTTHTCPPCP). In some embodiments, the Fc domain includes the amino acid sequence of SEQ ID NO: 303 (ELKTRPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3). In some embodiments, the Fc domain includes the amino acid sequence of SEQ ID NO: 304 (ESKYGPPCPPCP).

[0255] In this regard, it should be understood that any of the exemplary Fc domain sequences shown in Table 3 may be modified by replacing the hinge sequence of SEQ ID NO: 299 (DKTHTCPPCP) or SEQ ID NO: 300 (EPKSSDKTHTCPPCP) with the hinge sequences of SEQ ID NO: 301 (ERKCCVECPPCP), SEQ ID NO: 302 (ELKTRPLGDTTHTCPPCP), SEQ ID NO: 303 (ELKTRPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3), SEQ ID NO: 304 (ESKYGPPCPPCP), or any other suitable hinge sequence, including variants of the SEQ ID NOs: 299-304 hinge sequences containing one, two, three, four, five or more mutations.

[0256] Mutations to increase binding to the internal localization receptor In some embodiments, additional mutations are introduced into the Fc domain of the molecules described herein to target cell surface receptors (i.e., endocytosis receptors) that bind to ligands, cause them to move internally, and target them to lysosomes. By modifying the Fc domain to increase binding to endocytosis receptors, the molecules described herein and autoantibodies bound to them are targeted for internal movement and lysosomal degradation.

[0257] In some embodiments, the molecule comprises a first and / or second Fc domain containing one or more mutant amino acid residues that modify binding to an internal localization receptor on a cell, allowing the internal localization receptor to deliver its cargo to the cell's lysosomes for degradation. In some embodiments, the modification of binding to the internal localization receptor includes increased binding to the internal localization receptor. While we do not wish to be bound by any theory, when a molecule bound to an autoantibody binds to an internal localization receptor on a cell, the internal localization receptor allows the molecule to move internally, and the autoantibody is delivered to the cell's lysosomes for degradation.

[0258] Examples of internally distributed receptors include, but are not limited to, FcγRIIB, FcRn, ASGPR, BCMA, CD38, SLAMF7, GPCR5D, or CD138.

[0259] In some embodiments, the first and / or second Fc domains contain one or more mutant amino acid residues that increase binding to human FcγR, particularly FcγRIIB. In some embodiments, such mutations include at least one of the following mutant amino acid residues: S267E and L328F, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domains contain a combination of the following mutant amino acid residues: S267E and L328F, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domains contain the mutant amino acid residue P238D, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domains contain at least one of the following mutant amino acid residues: L234A, L235A, and P238D, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domains contain the following mutant amino acid residues: L234A, L235A, and P238D, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domain includes at least one of the following mutant amino acid residues: L234A, L235A, P238D, and P329G, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domain includes at least one of the following mutant amino acid residues: L234A, L235A, P238D, and P329G, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domain includes at least one of the following mutant amino acid residues: L234A, L235A, and P238D, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domain includes the following mutant amino acid residues: L234A, L235A, and P238D, according to the EU numbering scheme. In some embodiments, the first and / or second Fc domain includes at least one of the following mutant amino acid residues: N297A and P238D, according to the EU numbering scheme.

[0260] In some embodiments, one or more Fc mutations are introduced to increase binding to the neonatal receptor (FcRn). In some embodiments, the Fc domain is the IgG1 Fc domain. Human IgG1 spontaneously binds to FcRn at acidic pH, which allows it to bind to FcRn, translocate into the cell, recirculate back to the cell surface, and avoid degradation by lysosomes. In some embodiments, the Fc mutations include mutations that increase binding to FcRn in a neutral pH environment (e.g., the extracellular environment). While we do not wish to be bound by any theory, the molecules described herein include such mutations to increase the binding of the Fc domain to FcRn on the cell surface in a neutral pH environment to increase receptor-mediated internal translocation into cells and the transport of (molecule-bound) autoantibodies to lysosomes.

[0261] In some embodiments, the first and / or second Fc domains include one or more mutant amino acid residues that increase binding to FcRn at neutral or near-neutral pH (e.g., pH between approximately 6.8 and 7.5). In some embodiments, the first and / or second Fc domains include a human IgG1 isotype and remain bound to FcRn even when entering an environment with an acidic pH and / or a low calcium concentration (e.g., cellular endosomes). In some embodiments, the first and / or second Fc domains include at least one of the following mutant amino acid residues according to the EU numbering scheme: M252Y, S254T, T256E, H433K, and N434F. In some embodiments, such mutations include combinations (i.e., "MST-HN") containing the following mutations according to the EU numbering scheme: M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the first and / or second Fc domain comprises the following combination of mutant amino acid residues according to the EU numbering scheme: M252Y, S254T, T256E, H433K, and N434F (i.e., "MST-HN").

[0262] In some embodiments, the first and / or second Fc domains include at least one mutant amino acid sequence that reduces binding to one or more Fc-gamma receptors (FcγRs). Such modifications may also interfere with immune crosslinking (i.e., between the molecule, autoantibodies, and FcγRs) that leads to an inflammatory response. Such mutations may focus on the molecule's primary mechanism of action, namely, targeted internal translocation and subsequent degradation of autoantibodies. In some embodiments, the first and / or second Fc domains include at least one of the following mutant amino acid residues: G236R and L328R, according to the EU numbering scheme.

[0263] In some embodiments, the molecules described herein may include any combination of the above Fc mutations that modify binding to an internal localization receptor or an Fc receptor. In some embodiments, the molecules described herein include an Fc domain comprising the "MST-HN" modification described herein in combination with the "RR" mutation described herein. In some embodiments, the molecules described herein include an Fc domain comprising the "MST-HN" modification described herein in combination with the "P238D" mutation described herein. In some embodiments, the molecules described herein include an Fc domain comprising the "MST-HN" modification described herein in combination with the "RR" mutation and the "P238D" mutation described herein.

[0264] i. Exemplary FcγRIIB mutation In some embodiments, the Fc domain contains one or more amino acid mutations that increase affinity for FcγRIIB. In some embodiments, FcγRIIB is human FcγRIIB. In some embodiments, FcγRIIB is mouse FcγRIIB.

[0265] In some embodiments, the Fc domain is utilized in molecules described herein that include one or more mutations that improve the binding kinetics of an immune complex containing one or more molecules bound to a target antibody to FcγRIIB. In some embodiments, the improved binding kinetics include a binding affinity of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more. In some embodiments, the improved binding kinetics include an increase in the avidity, stability, strength, frequency, and / or duration of binding between the immune complex and FcγRIIB. In some embodiments, the improved binding kinetics include an increase in the association rate, a decrease in the dissociation rate, and / or a change in the equilibrium dissociation constant.

[0266] In some embodiments, the molecules described herein having first and second Fc domains include one or more mutations in the first and / or second Fc domains to increase binding to FcγRIIB, and when the two molecules bind to a target antibody, an immune complex is formed with improved binding kinetics to FcγRIIB compared to an immune complex containing a target antibody bound to two corresponding molecules having wild-type Fc domains. While we do not wish to be bound by any theory, the molecules described herein may have one or more mutations that increase binding affinity to FcγRIIB, although the binding affinity of the molecules alone to FcγRIIB is moderate. In some embodiments, the molecules bind to FcγRIIB with affinity in the range of about 1 μM to 0.001 μM. In some embodiments, the molecules bind to FcγRIIB with affinity in the range of about 1 μM to 0.01 μM. In some embodiments, the molecules bind to FcγRIIB with affinity in the range of about 0.1 μM to 0.01 μM. In some embodiments, when such mutations are introduced into the Fc domain of the molecules described herein, they confer an avidity-mediated binding effect to FcγRIIB when two or more molecules are present in the immune complex with the target antibody. In some embodiments, the molecules described herein exhibit increased binding to FcγRIIB when the immune complex contains two molecules bound to the target antibody compared to an immune complex with only one molecule bound to the target antibody. While we do not wish to be bound by any theory, such avidity-mediated effects allow for selective binding and depletion of the immune complex, as well as weaker binding (and therefore depletion) of the molecule if it is not part of the immune complex. These features allow the molecules described herein to circulate longer in the target bloodstream before being eliminated by internal translocation and degradation via FcγRIIB.

[0267] Furthermore, this disclosure provides Fc domain mutations that achieve binding affinity to FcγRIIB, conferring avidity-mediated effects that utilize the advantages and additional selectivity described herein. Exemplary Fc domain mutations that can be used to achieve these binding dynamics with FcγRIIB include, for example, in some embodiments, the following mutations according to the EU numbering scheme: E233V, L234D, L235F, G236R, G237D, S239L, S267D, H268P, S298G, T299A, A327L, L328A, A330H, E3 Includes one or more of the following: 33I, R292Q, E233P, P238D, H268D, P271G, A330R, L234Y, T250V, V264I, T307P, Q311R, A330K, P343R, M428L, N434A, Y436T, Q438R, S440E, G236N, S267E, L235R, D270E, E233D, and G237D.

[0268] In some embodiments, the Fc domain mutation includes one or more of the following mutations: E233V, L234D, L235F, G236R, G237D, S239L, S267D, H268P, S298G, T299A, A327L, L328A, A330H, or E333I. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: E233V, L234D, L235F, G236R, G237D, S239L, S267D, H268P, S298G, T299A, A327L, L328A, A330H, and E333I (see, for example, SEQ ID NOs: 113, 114, and 139).

[0269] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: E233V, L234D, L235F, G236R, G237D, S239L, S267D, R292Q, H268P, S298G, T299A, A327L, L328A, A330H, or E333I. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: E233V, L234D, L235F, G236R, G237D, S239L, S267D, R292Q, H268P, S298G, T299A, A327L, L328A, A330H, and E333I (see, for example, SEQ ID NOs: 115, 116, and 140).

[0270] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: E233V, L234D, L235F, G236R, G237D, S239L, H268P, R292Q, S298G, T299A, A327L, L328A, A330H, or E333I. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: E233V, L234D, L235F, G236R, G237D, S239L, H268P, R292Q, S298G, T299A, A327L, L328A, A330H, and E333I (see, for example, SEQ ID NOs: 117, 118, and 141).

[0271] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: L234Y, P238D, T250V, V264I, T307P, Q311R, A330K, P343R, M428L, N434A, Y436T, Q438R, or S440E. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: L234Y, P238D, T250V, V264I, T307P, Q311R, A330K, P343R, M428L, N434A, Y436T, Q438R, and S440E (see, for example, SEQ ID NOs. 119, 120, and 142).

[0272] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: L234D, G236N, or S267E. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: L234D, G236N, and S267E (see, for example, SEQ ID NOs. 121, 122, and 143).

[0273] In some embodiments, the Fc domain mutation includes L235R according to the EU numbering scheme (see, for example, SEQ ID NOs: 123, 124, and 144).

[0274] In some embodiments, the Fc domain mutation includes one or both of the following mutations G236N and S267E, according to the EU numbering scheme. In some embodiments, the Fc domain includes the following set of mutations: G236N and S267E, according to the EU numbering scheme (see, for example, SEQ ID NOs. 125, 126, and 145).

[0275] In some embodiments, the Fc domain mutation includes one or both of the following mutations P238D and D270E, according to the EU numbering scheme. In some embodiments, the Fc domain includes the following set of mutations: P238D and D270E, according to the EU numbering scheme (see, for example, SEQ ID NOs. 127, 128, and 146).

[0276] In some embodiments, the Fc domain mutation includes one or both of the following mutations P238D and P271G, according to the EU numbering scheme. In some embodiments, the Fc domain includes the following set of mutations: P238D and P271G, according to the EU numbering scheme (see, for example, Sequence IDs 129, 130, and 147).

[0277] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: P238D, D270E, or P271G. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: P238D, D270E, and P271G (see, for example, Sequence IDs 131, 132, and 148).

[0278] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: G237D, P238D, P271G, or A330R. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: G237D, P238D, P271G, and A330R (see, for example, Sequence IDs 133, 134, and 149).

[0279] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: G237D, P238D, D270E, P271G, or A330R. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: G237D, P238D, D270E, P271G, and A330R (see, for example, Sequence IDs 135, 136, and 163).

[0280] In some embodiments, the Fc domain mutation includes one or more of the following mutations according to the EU numbering scheme: E233D, G237D, P238D, H268D, P271G, or A330R. In some embodiments, the Fc domain includes the following set of mutations according to the EU numbering scheme: E233D, G237D, P238D, H268D, P271G, and A330R (see, for example, Sequence IDs 137, 138, and 164).

[0281] In some embodiments, the Fc domain mutation includes P238D according to the EU numbering scheme (see, for example, SEQ ID NOs. 107 and 108).

[0282] In some embodiments, Fc domains having mutations that increase binding affinity to FcγRIIB also exhibit reduced or undetectable binding to specific activated Fc receptors. In some embodiments, the activated Fc receptor comprises one or more of FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn. Such binding properties lead to additional beneficial properties of the molecules described herein, including a lower risk of toxicity, for example, due to a lower risk of activating the innate immune response (via the activated Fc receptor) depending on the molecule introduced.

[0283] In some embodiments, the Fc domain described herein includes one or more modifications such that the molecule described herein does not activate immune cells (for example, does not activate immune cells to secrete inflammatory cytokines, such as IL-6).

[0284] Mutation for heterodimerization In some embodiments, an Fc mutation is introduced to facilitate heterodimerization of two polypeptides, where each polypeptide contains an Fc domain, and the first and second Fc domains heterodimerize to form a complete molecule.

[0285] In particular, generating heterodimerized Fc domains of two different polypeptides from a single composition presents challenges because random pairing of different polypeptides can result in undesirable species. The presence of erroneous pairing byproducts significantly reduces the yield, requiring advanced purification procedures to isolate the desired antibody drug in such situations. Generally, the same problem of erroneous pairing byproducts persists even when recombinant expression techniques are used. One approach to address the erroneous pairing byproduct problem is known as the "knob-into-hole technique" (KIH), which aims to force pairing of two different polypeptides containing Fc domains by introducing mutations into the CH3 region of the Fc domain to alter the contact interface. In one CH3 region, a large amino acid is replaced with an amino acid having a short side chain to create a "hole," while an amino acid with a large side chain is introduced into the other CH3 region to create a "knob." For example, it has been observed that co-expression of two heavy chains of an antibody having such modifications with two light chains leads to a higher yield of heterodimer formation compared to homodimer formation (see Ridgway et al., Protein Eng. 9:617 (1996) and WO1996 / 027011, incorporated herein by reference). In some embodiments, the molecules described herein utilize the KIH technology described, for example, in WO1998 / 050431, which is incorporated entirely herein by reference.

[0286] As described herein, the molecule comprises a first Fc domain and a second Fc domain. In some embodiments, the first Fc domain and / or the second Fc domain comprises a CH2 region variant and / or a CH3 region variant, each of which independently comprises at least one different amino acid substitution such that heterodimerization of the first and second Fc domains of the molecule of the present invention is promoted more than homodimerization, and such variants independently generate a heterodimer domain pair.

[0287] As described herein, the first and / or second Fc domains of the molecules described herein may include certain mutations utilizing the KIH technique, including but not limited to CH3 modifications. In some embodiments, the molecule includes first and second Fc domains that form a heterodimer using a knob-in-hole (KIH) modification. In some embodiments, the KIH mutations include Y349T and T394F according to the EU numbering scheme. In some embodiments, the first Fc domain includes the Y349T mutation and the second Fc domain includes the T394F mutation. In some embodiments, the first Fc domain includes the T394F mutation and the second Fc domain includes the Y349T mutation. In some embodiments, the KIH mutations include T366W, S354C, T366S, L368A, Y407V, and Y349C according to the EU numbering scheme. In some embodiments, the first Fc domain includes the T366W and S354C mutations, and the second Fc domain includes the T366S, L368A, Y407V, and Y349C mutations. In some embodiments, the first Fc domain includes the T366S, L368A, Y407V, and Y349C mutations, and the second Fc domain includes the T366W and S354C mutations.

[0288] Those skilled in the art will understand that other known KIH mutations or other Fc modifications are known in the art to promote heterodimerization, and that charge-to-charge swap designs (e.g., the "DD-KK" mutation pair) and isotype-strand swap designs (e.g., "SEED Fc") (see Ha et al., Frontiers in Immunology 7:394 (2016), which is incorporated herein by reference) may be used in the molecules described herein.

[0289] Mutations for half-life extension In some embodiments, the first and / or second Fc domain of the molecule contains one or more mutant amino acid residues that extend the half-life. In some embodiments, the first and / or second Fc domain contains one of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: M252Y, S254T, and T256E ("MST" or "YTE"). In some embodiments, the first and / or second Fc domain contains a combination of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: M252Y, S254T, and T256E. In some embodiments, the first and / or second Fc domain contains one of the following mutant amino acid residues, according to the EU numbering scheme: M428L and N434S ("L / S"). In some embodiments, the first and / or second Fc domain contains a combination of the following mutant amino acid residues, according to the EU numbering scheme: M428L and N434S.

[0290] In some embodiments, the first and / or second Fc domain includes one of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: T250Q and M428L ("QL"). In some embodiments, the first and / or second Fc domain includes one of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: H433K and N434F ("KF"). In some embodiments, the first and / or second Fc domain includes one of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: T307A, E380A and N434A ("AAA"). In some embodiments, the first and / or second Fc domain includes the following mutant amino acid residue that extends the half-life, according to the EU numbering scheme: V308P. In some embodiments, the first and / or second Fc domain includes one of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: M252Y, V308P, and N434Y ("YPY"). In some embodiments, the first and / or second Fc domain includes one of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: H285D, T307Q, and A378V ("DQV"). In some embodiments, the first and / or second Fc domain includes one of the following mutant amino acid residues that extend the half-life, according to the EU numbering scheme: L309D, Q311H, and N434S ("DHS"). Exemplary Fc mutations are described, for example, in Liu et al., Antibodies 9(4):64 (2020), which is incorporated herein by reference in its entirety. Linker

[0291] The molecules described herein include an Fc domain linked to a binding domain. In some embodiments, the binding domain (e.g., an antigen domain) is directly linked to the Fc domain. In some embodiments, the binding domain (e.g., an antigen domain) is linked to the Fc domain via a linker. Various linkers are expected to be used in the molecules described herein. The linkers may be between the binding domain and the Fc domain, but they may also be between one domain of the molecule, for example, linking one or more antigen domains within the binding domain.

[0292] In some embodiments, the linker includes a flexible linker to provide flexibility to the molecule (e.g., between the binding domain and the Fc domain). In some embodiments, the flexible linker includes at least one flexible amino acid (e.g., Gly).

[0293] An example of a flexible linker is glycine polymer (G). n , glycine-serine polymer (e.g., (GS) n (GSGGS: Sequence ID 156) n , and (GGGS: Sequence ID 151) n Examples include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art (wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Because glycine and glycine-serine polymers are relatively unstructured, they may be able to act as neutral chains between their constituents. Glycine has even greater access to the phi-psy space than alanine and is less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11:173-142 (1992)). In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 150 (GGGGS), SEQ ID NO: 151 (GGGGSGGGGS), SEQ ID NO: 152 (GGGGSGGGGSGGGGS), or SEQ ID NO: 153 (VDGGGGSGGGGSGGGGSG).

[0294] Further examples of flexible linkers include, but are not limited to, sequence numbers 157 (GGSG), 158 (GGSGG), 159 (GSGSG), 160 (GSGGG), 161 (GGGSG), and 162 (GSSSG).

[0295] Further exemplary linkers include: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 154) and GGGGSGGGGSGGGGSGGGGSSGGGGS (SEQ ID NO: 155).

[0296] Those skilled in the art will recognize that the molecular designs described herein may include a linker that is entirely or partially flexible, so that the linker may include not only a flexible linker but also one or more parts that impart a less flexible structure to provide the desired molecular structure.

[0297] Suitable linkers can be easily selected and may have various lengths, such as 1 amino acid (e.g., Gly) to 20 or more amino acids, 2 amino acids to 15 amino acids, or 3 amino acids to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids).

[0298] In some embodiments, the linker may be an amino acid-free synthetic linker, such as a polyethylene (PEG) linker, or a chemical conjugation, such as another known synthetic linker commonly used in antibody-drug conjugates, or may include such a linker. In this context, it should also be understood that the molecules described herein also include molecules in which the components of a first or second polypeptide are linked by a chemical conjugation, such as "click" chemistry or other chemistry, using optionally intervening amino acids or synthetic linkers.

[0299] In some embodiments, the molecules described herein are fusion proteins in which the first and second polypeptides can be encoded by a single nucleic acid sequence. In some embodiments, the molecules described herein are chemically conjugated molecules comprising components conjugated using synthetic chemistry.

[0300] Exemplary configuration Various configurations of the molecules described herein are anticipated. Such configurations include various elements of the molecules described herein, comprising a first polypeptide containing a binding domain (e.g., an antigen domain) linked to a first Fc domain, and a second polypeptide containing a second Fc domain. Exemplary binding domains, Fc domains, and linkers are described. Such components can be assembled in different configurations to produce the molecules described herein.

[0301] Various combinations of specific binding domains, Fc domains, and linkers are incorporated herein. An exemplary configuration is shown in Figure 2, in which the molecule comprises a first polypeptide containing an antigen domain linked to a first Fc domain and a second polypeptide containing a second Fc domain. The antigen domain shown in Figure 2 may be a variant of the antigen domain described herein or, for example, a TSHR autoantigen domain. Another exemplary configuration is shown in Figure 3, in which the molecule comprises a first polypeptide containing an antigen domain linked to a first Fc domain and a second polypeptide containing a second Fc domain. The antigen domain shown in Figure 3 may be a variant of the antigen domain described herein or, for example, one or more PLA2R autoantigen domains. Figure 3 is intended to show binding domains containing one or more exemplary PLA2R autoantigen domains, e.g., CysR, CysR-CTLD1, CysR-CTLD1-CTLD7, CysR-FnII-CTLD1-CTLD7-CTLD8, CysR-CTLD1-CTLD7-CTLD8, or bispecific CysR-FnII-CTLD1×CTLD7-CTLD8. In some embodiments, the molecule may be as shown in Figure 2 or Figure 3, i.e., it does not contain a second antigen domain linked to a second Fc domain, so that the molecule is monovalent (e.g., has one binding domain). In some embodiments, the molecule shown in Figure 2 or Figure 3 may contain a second antigen domain linked to a second Fc domain, so that the molecule is divalent (e.g., has two binding domains). In some such embodiments, the molecule may be a divalent molecule, and the first and second antigen domains are the same. In some such embodiments, the molecule may be a divalent molecule, and the first antigenic domain and the second antigenic domain may be different. The differences may be different domains of the same antigen, different epitopes on the same antigen, and / or different antigens.

[0302] In some embodiments, the molecules described herein have a configuration, for example, as shown in Figure 4. In such a configuration, the C-terminus of the antigen domain (A) is linked to the N-terminus of the first Fc domain (Fc1) via an optional linker (L) (first polypeptide), which forms a heterodimer with the second Fc domain (Fc2) of the second polypeptide.

[0303] In some embodiments, the molecules described herein have a configuration, for example, as shown in Figure 5. In such a configuration, the C-terminus of an antigen domain (A) is linked to the N-terminus of a first Fc domain (Fc1) via an optional linker (L) (first polypeptide), and this molecule comprises a second binding domain containing an antibody variable domain, the C-terminus of the antibody variable domain (e.g., containing HC and LC)Fab being linked to the N-terminus of a second Fc domain (Fc2) (second polypeptide).

[0304] In some embodiments, the molecules described herein have a configuration, for example, as shown in Figure 6. In such a configuration, the C-terminus of a first Fc domain (Fc1) is linked to the N-terminus of an antigen domain (A) via an optional linker (L) (first polypeptide), which forms a heterodimer with a second Fc domain (Fc2) of a second polypeptide.

[0305] In some embodiments, the molecules described herein have a configuration, for example, as shown in Figure 7A. In such a configuration, the molecule comprises two antigen domains, where the C-terminus of a first Fc domain (Fc1) is linked to the N-terminus of a first antigen domain (A') via an optional linker (L') (first polypeptide), and the C-terminus of a second Fc domain (Fc2) is linked to the N-terminus of a second antigen domain (A') via an optional linker (L') (second polypeptide). In some embodiments, the two antigen domains are the same. In some embodiments, the two antigen domains are different.

[0306] In some embodiments, the molecules described herein have a configuration, for example, as shown in Figure 7B. In such a configuration, the molecule comprises two antigen domains, where the C-terminus of a first antigen domain (A) is linked to the N-terminus of a first Fc domain (Fc1) via an optional linker (L) (first polypeptide), and the C-terminus of a second antigen domain (A) is linked to the N-terminus of a second Fc domain (Fc2) via an optional linker (L) (second polypeptide). In some embodiments, the two antigen domains are the same. In some embodiments, the two antigen domains are different.

[0307] Here, Figures 4–7 label the antigen (e.g., any antigen described herein) as "A" or "A'", and this disclosure also includes any binding domain described herein.

[0308] Furthermore, or alternatively, in some embodiments, the molecule may include a first binding domain containing a first antibody-variable domain and a second binding domain containing a second antibody-variable domain, wherein the first and second antibody-variable domains can bind to the same or different target antigens.

[0309] In some embodiments, the molecule comprises a first polypeptide containing an amino acid sequence that is at least 90% identical to (a) any one of the sequences of SEQ ID NOs: 103, 105, 107, 109, 111-113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139-149, 163-164, 374-376, or 378 (for example, a sequence selected from SEQ ID NOs: 107, 109, 113, 115, 119, 131, 139, 140, 142, 148, 374, or 378). (b) a second polypeptide comprising an amino acid sequence that is at least 90% identical to any one of the following: (d) a sequence of sequence numbers 104, 106, 108, 110, 111, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139-149, 163-164, 374-375, 377, or 379 (for example, a sequence selected from sequence numbers 108, 110, 114, 116, 120, 132, 139, 140, 142, 148, 374, or 379).

[0310] In some embodiments, the molecule is a first polypeptide containing one amino acid sequence from among (a) SEQ ID NOs: 103, 105, 107, 109, 111-113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139-149, 163-164, 374-376, or 378 (for example, a sequence selected from SEQ ID NOs: 107, 109, 113, 115, 119, 131, 139, 140, 142, 148, 374, or 378). (b) a second polypeptide comprising any one amino acid sequence of sequence numbers 104, 106, 108, 110, 111, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139-149, 163-164, 374-375, 377, or 379 (for example, a sequence selected from sequence numbers 108, 110, 114, 116, 120, 132, 139, 140, 142, 148, 374, or 379).

[0311] In some embodiments, the molecule contains an amino acid sequence that is at least 90% identical to (a) any one of the sequences 104, 106, 108, 110, 111, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139-149, 163-164, 374-375, 377, or 379 (for example, a sequence selected from sequences 108, 110, 114, 116, 120, 132, 139, 140, 142, 148, 374, or 379). (b) comprising a first polypeptide and a second polypeptide having an amino acid sequence that is at least 90% identical to any one of sequence numbers 103, 105, 107, 109, 111-113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139-149, 163-164, 374-376, or 378 (for example, a sequence selected from sequence numbers 107, 109, 113, 115, 119, 131, 139, 140, 142, 148, 374, or 378).

[0312] In some embodiments, the molecule contains (a) one amino acid sequence of SEQ ID NOs: 104, 106, 108, 110, 111, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139-149, 163-164, 374-375, 377, or 379 (for example, a sequence selected from SEQ ID NOs: 108, 110, 114, 116, 120, 132, 139, 140, 142, 148, 374, or 379). (b) a first polypeptide and a second polypeptide comprising any one amino acid sequence of sequence numbers 103, 105, 107, 109, 111-113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139-149, 163-164, 374-376, or 378 (for example, a sequence selected from sequence numbers 107, 109, 113, 115, 119, 131, 139, 140, 142, 148, 374, or 378).

[0313] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 103 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 104; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 104 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 103.

[0314] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 105 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 106; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 106 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 105.

[0315] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 107 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 108; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 108 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 107.

[0316] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 109 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 110; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 109.

[0317] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 113 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 114; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 114 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 113.

[0318] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 115 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 116; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 116 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 115.

[0319] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 117 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 118; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 118 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 117.

[0320] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 119 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 120; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 120 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 119.

[0321] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 121 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 122; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 122 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 121.

[0322] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 123 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 124; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 124 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 123.

[0323] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 125 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 126; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 126 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 125.

[0324] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 127 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 128; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 128 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 127.

[0325] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 129 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 130; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 130 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 129.

[0326] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 131 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 132; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 132 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 131.

[0327] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 133 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 134; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 134 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 133.

[0328] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 135 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 136; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 136 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 135.

[0329] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 138; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 138 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 137.

[0330] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 376 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 377; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 377 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 376.

[0331] In some embodiments, the molecule comprises a first and a second polypeptide, (i) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 378 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 379; or (ii) the first polypeptide comprising the amino acid sequence of SEQ ID NO: 379 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 378.

[0332] In some embodiments, the molecule comprises a first and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 374, and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 374.

[0333] Characteristics of an example molecule The molecules described herein may be identified, evaluated, and / or characterized for one or more of their physical / chemical properties and / or biological activities. Those skilled in the art will recognize a variety of approaches, including specific assays that can be used for such identification, evaluation, and / or characterization. The binding domains of the molecules described herein can be selected according to a variety of criteria, including, but not limited to, binding affinity or response potency (e.g., neutralization / removal of a target antibody).

[0334] The binding domains described herein can be selected, in particular, based on their binding properties to their targets. The binding properties of the antigen-binding domains described herein can be measured by methods known in the art, for example, one of the following: BIACORE analysis, enzyme-linked immunosorbent assay (ELISA), X-ray crystallography, sequence analysis, and scanning mutagenesis. The binding interaction between the antibody and the target antigen can be analyzed using surface plasmon resonance (SPR). SPR or biomolecular interaction analysis (BIA) detects biospecific interactions in real time without labeling the interacting substances. When the mass at the binding surface of the BIA chip changes (indicating a binding event), the refractive index of light near the surface changes. This change in refractive index generates a detectable signal and is measured as an indicator of real-time reactions between biomolecules. Methods using SPR are described, for example, in U.S. Patent No. 5,641,640, Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345 (1991); Szabo et al., Curr. Opin. Struct. Biol. 5:699-705 (1995), and online resources provided by BIAcore (Cytiva, USA). In addition, KinExA (binding equilibrium exclusion) assays available from Sapidyne Instruments (Boise, Idaho) and / or Octet BLI (biolayer interference) from Sartorius (Goettingen, Germany) may also be used.

[0335] Using information from SPR or similar BIA methods, the equilibrium dissociation constant (K D ) for the binding of the antigen-binding domain to the target antigen, as well as accurate and quantitative measurements of kinetic parameters including K on and K off can be provided. Such data can be used to compare different molecules. Information from SPR can also be used to develop structure-activity relationships (SAR). Specific binding parameters, such as mutant amino acids at a given position correlated with high affinity, can be identified.

[0336] In some embodiments, the binding domains described herein exhibit high affinity with respect to binding to the target antibody. In various embodiments, the K D of the binding domains described herein for the target antibody is about 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , or 10 -15 M or less or any range therebetween. In certain cases, the K D of the binding domains described herein for the target antibody is between 0.001 and 1 nM, for example, 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM or any range therebetween.

[0337] In some embodiments, the binding domain (e.g., antigen domain) binds to the target with high binding affinity. In various embodiments, the K D of the antigen domains described herein is about 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 、10-10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , or 10 -15 The binding domain is less than M or any range between them. In some embodiments, the binding domain is an antigen domain that binds to the target antibody with a binding affinity equal to or higher than the affinity of the native antigen domain to the target antibody, or includes such an antigen domain. In certain cases, the binding affinity of the antigen domain to the target antibody is K D nM is between 0.001 and 1 nM, for example, 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM, or any range in between.

[0338] In some embodiments, the molecule is characterized by its ability to selectively reduce or deplete circulating target antibodies in a sample or patient.

[0339] In some embodiments, the level of target antibodies (e.g., antibodies associated with autoimmune diseases and / or diseases characterized by an overactive or unregulated immune system) in or from a biological sample of a subject after administration of the molecule described herein decreases compared to the level before administration. In some embodiments, the level of target antibodies decreases by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the level before administration of the molecule. In some embodiments, the level of target antibodies persists for a long period of time. In some embodiments, the duration includes at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, or longer.

[0340] In some embodiments, the molecules described herein have increased affinity for FcγRIIB. In some embodiments, FcγRIIB is human FcγRIIB. In some embodiments, FcγRIIB is mouse FcγRIIB. In some embodiments, the increased affinity for FcγRIIB is provided by mutating one or both of the Fc domains of the molecule, as described herein.

[0341] In some embodiments, the molecules described herein include one or more mutations in one of the Fc domains that improve the binding kinetics of an immune complex comprising one or more molecules and a target antibody to FcγRIIB. In some embodiments, the improved binding kinetics include a binding affinity of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more. In some embodiments, the improved binding kinetics include an increase in the avidity, stability, strength, frequency, and / or duration of binding between the immune complex and FcγRIIB. In some embodiments, the improved binding kinetics include an increase in the association rate, a decrease in the dissociation rate, and / or a change in the equilibrium dissociation constant.

[0342] In some embodiments, the molecules described herein having first and second Fc domains include one or more mutations in the first and / or second Fc domains to increase binding to FcγRIIB, and when the two molecules bind to a target antibody, an immune complex is formed that has improved binding kinetics with FcγRIIB compared to an immune complex containing a target antibody bound to two corresponding molecules having wild-type Fc domains.

[0343] In some embodiments, the molecules described herein, when not present in an immune complex, have a moderate binding affinity to FcγRIIB (for example, the Fc domain of the molecule has a slightly increased binding affinity to FcγRIIB compared to a molecule containing a wild-type Fc domain). In some embodiments, the molecules bind to FcγRIIB with affinities ranging from about 1 μM to 0.001 μM. In some embodiments, the molecules bind to FcγRIIB with affinities ranging from about 1 μM to 0.01 μM. In some embodiments, the molecules bind to FcγRIIB with affinities ranging from about 0.1 μM to 0.01 μM. In some embodiments, the molecules described herein exhibit avidity-mediated binding to FcγRIIB when a portion of an immune complex containing two molecules is bound to the target antibody. In some embodiments, the molecules described herein exhibit increased binding to FcγRIIB when present as an immune complex having two molecules bound to the target antibody compared to the same immune complex having only one molecule bound to the target antibody. In some embodiments, the molecules described herein exhibit increased binding to FcγRIIB when they exist as an immune complex having two molecules bound to a target antibody, compared to the target antibody alone. While we do not wish to be bound by any theory, such avidity-mediated effects allow for the selective binding and depletion of the immune complex (i.e., the two molecules described herein and one target antibody), as well as weaker binding of the molecule if it is not part of the immune complex. These features allow the molecules described herein to circulate longer in the target bloodstream before being eliminated by internal translocation and degradation via FcγRIIB.

[0344] Furthermore, this disclosure provides molecules comprising an Fc domain that exhibits increased binding affinity to FcγRIIB and reduced or undetectable binding to specific activated Fc receptors. In some embodiments, the activated Fc receptor comprises one or more of FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn. In some embodiments, the Fc domain of the molecules described herein comprises one or more mutant amino acid residues that exhibit reduced binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn compared to the corresponding wild-type Fc domain. In some embodiments, the Fc domain of the molecules described herein contains one or more mutant amino acid residues and has substantially no binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIA176F, FcγRIIIA176V, FcγRIIIB, and / or FcRn compared to the corresponding wild-type Fc domain. Such binding properties are beneficial in the molecules described herein and may reduce the risk of toxicity because they have a lower risk of activating the innate immune response (via activated Fc receptors) depending on the molecule introduced into the target body.

[0345] In some embodiments, the molecules described herein preferentially bind to immune cells expressing FcγRIIB rather than to immune cells expressing FcγRIIA. In some embodiments, the molecules described herein have substantially no binding affinity to cells that do not express FcγRIIB (e.g., T cells, NK cells, neutrophils, and / or eosinophils). Immune cells known to express FcγRIIB include B cells, monocytes, and / or basophils.

[0346] In some embodiments, the molecules described herein prevent the target antibody from binding to its alloantigen. In some embodiments, the molecules described herein neutralize the target antibody. In some embodiments, one or more molecules described herein form an immune complex with the target antibody, and the immune complex is internalized and degraded by immune cells expressing FcγRIIB. In some embodiments, the immune complex comprises one or more molecules (e.g., two or more molecules) bound to the target antibody and is removed from circulation, e.g., destroyed, by one of the mechanisms predicted in Figure 13. FcγRIIB is an internalized receptor that binds to its target, causes the complex to internalize, and transports the target to lysosomes for degradation. While we do not wish to be bound by any theory, molecules described herein with increased FcγRIIB binding can deplete the target antibody and / or antigen-specific B cells producing the target antibody by various mechanisms, including those shown in Figures 13A–D. Figure 13B shows a potential mechanism of action, including antibody elimination by targeting FcγRIIB isoform 2 on hepatic sinusoidal endothelial cells (LSEC). In this exemplary mechanism of action, the molecule's binding domain (e.g., antigen domain) binds to the target antibody, and the Fc domain binds to FcγRIIB isoform 2 on hepatic sinusoidal endothelial cells. The target antibody is translocated into the hepatic sinusoidal endothelial cells and targeted to lysosomes for degradation. In another exemplary mechanism shown in Figure 13C, the molecule described herein may also target pathogenic B cells producing the target antibody by targeting FcγRIIB isoform 1 on B cells containing an antigen-specific B cell receptor (BCR) (e.g., the target antibody expressed on the surface of B cells), thereby resulting in B cell apoptosis and inhibition. In another exemplary mechanism shown in Figure 13D, the molecule described herein can target FcγRIIB on T cells to prevent T cell activation.

[0347] In some embodiments, the Fc domain described herein includes one or more modifications such that the molecule described herein does not activate immune cells (for example, does not activate immune cells to secrete inflammatory cytokines, such as IL-6).

[0348] In some embodiments, the molecules described herein do not bind to specific components of the complement system (e.g., C1q). In some embodiments, the molecules described herein do not bind to C1q. In some embodiments, the molecules described herein do not activate the complement system.

[0349] Method for generating exemplary molecules This disclosure features a method comprising generating the molecules described herein.

[0350] The molecules described herein may be produced using recombinant methods and compositions (see, for example, U.S. Patent No. 4,816,567). In some embodiments, isolated nucleic acids encoding the molecules described herein may be provided. Such nucleic acids may encode an amino acid sequence comprising a first and / or second polypeptide. In further embodiments, one or more vectors comprising such nucleic acids may be provided. A vector can be a nucleic acid molecule capable of propagating another nucleic acid it is linked to. The term may include vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can induce the expression of functionally linked nucleic acids.

[0351] In further embodiments, host cells containing such nucleic acids may be provided. The host cells may be cells into which exogenous nucleic acids have been introduced, and may include offspring of such cells. The host cells may include primary transformed cells and offspring derived therefrom, regardless of passage number, and may include “transformers” and “transformed cells.” The offspring may contain mutations, although their nucleic acid content may not be exactly the same as that of the parent cells. Mutant offspring having the same function or biological activity as those screened or selected in the originally transformed cells are included herein. In one such embodiment, the host cells may include a vector containing a nucleic acid encoding an amino acid sequence comprising a first polypeptide and a second polypeptide of the molecule (e.g., transformed therein). In some embodiments, the first vector contains a nucleic acid encoding an amino acid sequence comprising a first polypeptide of the molecule, and the second vector contains a nucleic acid encoding an amino acid sequence comprising a second polypeptide of the molecule. In some embodiments, the host cells may be eukaryotic cells, e.g., Chinese hamster ovary (CHO) cells, lymphoid cells (e.g., Y0, NS0, Sp20 cells), or human fetal kidney (HEK293) cells. In some embodiments, methods for generating the molecules and / or antigen-binding domains described herein may be provided, which may include culturing host cells containing nucleic acids encoding the molecules and / or antigen-binding domains provided above under conditions suitable for the expression of the molecules and / or antigen-binding domains, and optionally recovering the molecules from the host cells or host cell culture medium.

[0352] With regard to the recombinant production of molecules and / or antigen-binding domains, for example, isolated nucleic acids encoding molecules and / or antigen-binding domains may be inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures.

[0353] Suitable host cells for cloning or expressing antibody-coding vectors include prokaryotic or eukaryotic cells as described herein. For example, the molecule and / or antigen-binding domain may be produced in bacteria if glycosylation and Fc effector function are not required (see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology 248:245-254 (2003)). After expression, the molecule and / or antigen-binding domain may be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0354] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts may also be suitable hosts for cloning or expressing molecular and / or antigen-binding domain coding vectors (see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006)). Suitable host cells for the expression of glycosylated antibodies may also be derived from multicellular organisms, including invertebrates and vertebrates. Examples of invertebrates include plant cells and insect cells (see, e.g., U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429). Examples of vertebrate cells include mammalian cell lines, monkey kidney CV1 cell line (COS-7) transformed with SV40; human fetal kidney cells (e.g., 293 or 293T cells as described in Graham et al., J. Gen Virol. 36:59-74 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT060562); TR1 cells; MRC 5 cells; FS4 cells; Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NS0, and Sp2 / 0 (e.g., Yazaki and Wu, Methods in Molecular Biology). (See 248:255-268 (2003)) can be cited.

[0355] The molecules described herein can be purified by any technique. For example, without being bound by theory, the molecules described herein can be recovered and purified from recombinant cultures by well known methods including, but not limited to, protein A purification, protein G purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography. High-performance liquid chromatography ("HPLC") can also be used for purification. For example, see Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), each of which is incorporated herein by reference in its entirety, e.g., Chapters 1, 4, 6, 8, 9, and 10.

[0356] As discussed herein, some or all of the molecular components may also be optionally linked via amino acids or synthetic linkers using "click" chemistry or other chemistry. Such molecules may be prepared by recombinant means, and then chemically modified for conjugation. For example, suitable methods used for preparing antibody-drug conjugates are well known in the art.

[0357] The purified molecules and antigen-binding domains thus contained can be characterized, for example, by ELISA, ELISPOT, flow cytometry, immunocytology, BIACORE analysis, Octet BLI analysis, KINEXA binding equilibrium exclusion assay, SDS-PAGE, and Western blotting, or by HPLC analysis, as well as several other functional assays disclosed herein. The contents of all references cited throughout this application (including references, issued patents, published patent applications, and concurrently pending patent applications) are expressly incorporated herein by reference.

[0358] Purpose This disclosure provides a technology for the selective depletion of targeted antibodies associated with autoimmune diseases and / or diseases related to an overactive or unregulated immune system.

[0359] In some embodiments, the molecule can be used in combination with the second therapy by being administered before, simultaneously with, or after the administration of the second therapy.

[0360] In some embodiments, the molecules of the present disclosure are used to treat subjects suffering from autoimmune diseases who would benefit from the selective neutralization and / or depletion of autoantibodies. Such molecules are constructed to include binding domains that bind to internally distributed receptors and autoantibodies. The molecules may also include modifications to enhance binding to internally distributed receptors (e.g., FcγRIIB).

[0361] For use in therapeutic methods, the molecules of this disclosure are formulated, administered, and given in a manner consistent with the preferred medical practice. Factors to be considered in this context include the specific disease or disorder being treated, the specific subject being treated, the clinical condition of the individual subject, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to the healthcare professional.

[0362] In some embodiments, the present disclosure provides a method for treating a disease. In some embodiments, the method comprises administering a therapeutically effective amount of the molecule described herein to a subject having such a disease. In some embodiments, a composition comprising the molecule described herein in a pharmaceutically acceptable form is administered to the subject. In some embodiments, the disease to be treated is an autoimmune disease. In some embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the subject. The “subject” may be a mammal, including a human.

[0363] Any such method may optionally include administering an effective amount of at least one composition or pharmaceutical composition containing at least one molecule described herein to a subject requiring such adjustment, treatment, diagnosis, and / or therapy (e.g., a subject suffering from an autoimmune disease and / or a disease related to an overactive or uncontrolled immune system).

[0364] In some embodiments, the methods provided include therapeutic methods comprising administering an effective amount of a composition to be delivered to a target, such that the molecule binds to a target antibody and an internally distributed receptor (e.g., FcγRIIB) on the cell, so that the complex is internally distributed and targeted to lysosomes. In some embodiments, the effective amount of the composition comprises an effective amount of the molecule described herein, and when the two molecules bind to the target antibody, an immune complex is formed that has improved binding kinetics with FcγRIIB compared to an immune complex containing the target antibody bound to two corresponding molecules having wild-type Fc domains. Such activity mediates the elimination of the immune complex containing the target antibody when bound to the molecule. Furthermore, molecules targeting an internally distributed receptor, e.g., FcγRIIB, can inhibit B cells on which the target antibody is expressed (e.g., as described in Chu et al., Mol Immunol 45:3926-3933 (2008), which is incorporated herein in whole by reference).

[0365] The therapeutic methods described herein may optionally further include concurrent or combination therapies for treating such diseases, further comprising administering the composition comprising the molecules described herein and administering at least one additional therapeutic agent before, simultaneously with, and / or after.

[0366] This disclosure also provides a method for treating subjects suffering from or susceptible to autoimmune diseases and / or diseases associated with an overactive or uncontrolled immune system, for example, by administering to the subject a pharmaceutical composition comprising the molecules described herein, or nucleic acid molecules encoding such molecules. In some embodiments, such treatment reduces or improves one or more signs or symptoms of autoimmune diseases and / or diseases associated with an overactive or uncontrolled immune system.

[0367] For example, in patients with Graves' disease, treatment with a pharmaceutical composition comprising the molecules described herein can reduce or improve one or more signs or symptoms of Graves' disease, such as ophthalmos, ptosis, diplopia, and irritation of the periorbital tissues and conjunctiva (in Graves' ophthalmopathy), hyperthyroidism, palpitations, tremors, heat intolerance, weight loss, and anxiety.

[0368] In some embodiments, treatment with the molecules described herein reduces the level of the target antibody in a subject or biological sample compared to the level before administration. In some embodiments, the level of the target antibody is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the level before administration. In some embodiments, treatment with the molecules described herein reduces the target antibody in a subject for a duration of, for example, at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, or longer.

[0369] Pharmaceutical composition In some embodiments, the molecules described herein may be formulated as pharmaceutical compositions and administered to a subject (for example, to treat autoimmune diseases). In various embodiments, the molecules described herein may be incorporated into pharmaceutical compositions. Such pharmaceutical compositions may be useful, for example, for the prevention and / or treatment of diseases, such as autoimmune diseases. Pharmaceutical compositions may be formulated by methods known to those skilled in the art (for example, those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985)).

[0370] In some embodiments, the pharmaceutical composition comprises a nucleic acid molecule containing a nucleotide sequence encoding the molecule described herein and a pharmaceutically acceptable carrier. In some embodiments, the molecule is expressed in a host cell containing a nucleic acid molecule containing a nucleotide sequence encoding the molecule described herein. In some embodiments, the molecule described herein is encoded by a vector (e.g., a viral vector such as a retroviral vector, lentiviral vector, adeno-associated virus (AAV) vector, or adenovirus vector).

[0371] In some embodiments, the pharmaceutical composition comprises a first molecule described herein or a nucleic acid molecule encoding said molecule, further comprising a nucleic acid molecule encoding a therapeutic agent or a second therapeutic agent that selectively depletes a target antibody (e.g., an autoantibody), and a pharmaceutically acceptable carrier.

[0372] In some embodiments, the pharmaceutical composition may be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic agent, and absorption retarder. The compositions of this disclosure may also include a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt.

[0373] In some embodiments, compositions containing the molecules described herein, for example, sterile formulations for injection, can be formulated according to conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, an isotonic solution containing physiological saline or glucose and other additives such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection in combination with an optionally suitable solubilizer, such as an alcohol such as ethanol and / or a polyalcohol such as propylene glycol or polyethylene glycol and / or a nonionic surfactant such as polysorbate 80 or HCO-50.

[0374] As disclosed herein, pharmaceutical compositions may be any form known in the art. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., solutions for injection and infusion), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.

[0375] The selection or use of a particular form may depend, in part, on the intended mode of administration and therapeutic use. For example, compositions containing a composition intended for systemic or local delivery may be in the form of an injectable or injectable solution. Thus, compositions may be formulated for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration refers to modes of administration other than enteral and local administration, which are usually by injection, and include, but are not limited to, injections and infusions into the vein, nasal cavity, eye, lung, muscle, artery, subarachnoid space, joint, orbit, heart, skin, lung, peritoneal, trachea, subcutaneous, subepidermal, joint, subcapsular, subarachnoid, intrathecal, epidural, brain, skull, carotid artery, and sternal regions.

[0376] The route of administration may be parenteral, for example, by injection, nasal administration, pulmonary administration, or transdermal administration. Administration may be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0377] In some embodiments, the pharmaceutical compositions of this disclosure may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile solutions for injection can be prepared by incorporating the compositions described herein in the required amounts in a suitable solvent, along with one or a combination of the components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the compositions described herein into a sterile vehicle, the sterile vehicle comprising a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for preparing sterile solutions for injection, the preparation method includes vacuum drying and freeze-drying, thereby obtaining the compositions described herein and any desired additional components (see below) from a pre-sterile filtered solution. Appropriate fluidity of the solution can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Sustained absorption of the injectable composition can be brought about by including absorption-delaying reagents, such as monostearate and gelatin, in the composition.

[0378] In some embodiments, the compositions described herein can be therapeutically delivered to a target by topical administration. As used herein, “topical administration” or “topical delivery” refers to delivery that does not rely on the transport of the composition or drug to its intended target tissue or site via the vascular system. For example, a composition may be delivered by injection or implantation of the composition or drug, or by injection or implantation of a device containing the composition or drug. In some embodiments, after topical administration near the target tissue or site, the composition or drug, or one or more of its components, may be diffused to the intended target tissue or site other than the administration site.

[0379] In some embodiments, the composition may be formulated with a carrier that protects the compound from rapid release, such as a controlled-release formulation, which includes an embedding and a microencapsulation delivery system. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid, may be used. Many methods for preparing such formulations are known to those skilled in the art. See, for example, JR Robinson (1978) “Sustained and Controlled Release Drug Delivery Systems,” Marcel Dekker, Inc., New York.

[0380] In some embodiments, the administration of the molecules described herein is achieved by administering the nucleic acid encoding the molecules to a subject. In some embodiments, the nucleic acid is RNA (e.g., mRNA). In some embodiments, the RNA encoding the molecules described herein is associated with a delivery agent, i.e., a substance or element that is non-covalently or covalently bonded to the molecule, or administered co-administered with the molecule, and performs one or more functions that increase the stability and / or potency of the bioactive agent beyond what would be achieved if the bioactive agent were delivered (e.g., administered to a subject) in the absence of the delivery agent. For example, the delivery agent may prevent the degradation of RNA (e.g., in the blood), facilitate the entry of RNA into cells or a cell compartment of interest (e.g., the cytoplasm), and / or enhance binding to specific cells containing the molecular target to be regulated. Those skilled in the art are aware of the numerous delivery agents that can be used to deliver inhibitory RNA, e.g., mRNA. See Kanasty et al., Nat Mater. 12(11):967-77 (2013). In some embodiments, for example, to administer RNA systemically, the RNA may be associated with a delivery agent such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. While not wishing to be bound by any theory, it is thought that positively charged cationic delivery systems facilitate the binding of negatively charged RNA and further enhance interactions with negatively charged cell membranes, thereby enabling efficient uptake of RNA by cells. Lipids (e.g., cationic or neutral lipids), dendrimers, or polymers may be bound to the inhibitory RNA or form vesicles or micelles that encapsulate the inhibitory RNA. Methods for generating and administering complexes containing cationic agents and RNA are known in the art. In some embodiments, it is envisioned that one of the delivery agents described in particular in U.S. Publication No. 2016 / 0298124 may be used. In some embodiments, the RNA encoding the molecules described herein is administered in association with lipids or lipid-containing particles.In some embodiments, RNA is administered in association with a cationic polymer (which may be a polypeptide or non-polypeptide polymer), lipids, peptides, PEG, cyclodextrin, or a combination thereof, which may be in the form of nanoparticles or microparticles. The lipids or peptides may be cationic. The nanoparticles may have a covalently or non-covalently bonded targeting moiety and / or a cell-permeable or membrane-active moiety. Nanoparticles such as lipid nanoparticles are described in Tatiparti et al., Nanomaterials 7:77 (2017).

[0381] The nucleic acids encoding the molecules described herein can be incorporated into gene constructs used as part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce molecules within cells. Expression constructs of such components can be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering component genes to cells in vivo. Approaches include insertion of target genes into viral vectors, including recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1 (HSV-1), or plasmids of recombinant bacteria or eukaryotes. Viral vectors can directly transfect cells. Plasmid DNA can be delivered using, for example, cationic liposomes (lipofectin) or derivatization, polylysine conjugates, gramicidin S, artificial viral envelopes, or other such intracellular carriers, as well as direct injection or CaPO4 precipitation of gene constructs (see, e.g., WO2004 / 060407).Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are known to those skilled in the art (e.g., Eglitis et al., Science 230:1395-1398 (1985); Danos and Mulligan Proc.Natl.Acad.Sci.USA 85:6460-6464 (1988); Wilson et al., Proc.Natl.Acad.Sci.USA 85:3014-3018 (1988); Armentano et al., Proc.Natl.Acad.Sci.USA 87:6141-6145 (1990); Huber et al., Proc.Natl.Acad.Sci.USA 88:8039-8043 (1991); Ferry et al., Proc.Natl.Acad.Sci.USA 88:8377-8381(1991);Chowdhury et al.Science 254:1802-1805(1991);van Beusechem et al.,Proc.Natl.Acad.Sci.USA 89:7640-7644(1992);Kay et al.,Human Gene Therapy 3:641-647 (1992);Dai et al.,Proc.Natl.Acad.Sci.USA 89:10892-10895(1992);Hwu et al.,J Immunol See 150:4104-4115 (1993); U.S. Patents 4,868,116 and 4,980,286; and PCT Publications WO1989 / 07136, WO1989 / 02468, WO1989 / 05345, and WO1992 / 07573). Another viral gene delivery system utilizes adenovirus-derived vectors (see, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992)). Suitable adenovirus vectors derived from adenovirus strain Ad5 dl324 or other adenovirus strains (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art.Another viral vector system useful for delivering target genes is adeno-associated virus (AAV). See, for example, Flotte et al., Am J Respir Cell Mol Biol 7:349-356 (1992); Samulski et al., J Virol 63:3822-3828 (1989); and McLaughlin et al., J Virol 62:1963-1973 (1989).

[0382] A drug solution may contain a therapeutically effective amount of the composition described herein. Such an effective amount can be readily determined by those skilled in the art, partly based on the effect of the administered composition, or, if two or more drugs are used, the combined effect of the composition and one or more additional drugs. The therapeutically effective amount of the composition described herein may also vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the composition (and one or more additional drugs) to elicit a desired response in the individual, e.g., improvement of at least one state parameter of an autoimmune disease, e.g., improvement of at least one symptom. For example, the therapeutically effective amount of the composition described herein can suppress (reduce the severity of, or eliminate the occurrence of) any one of the symptoms of a particular disorder and / or a particular disorder known in the art or described herein, and / or prevent it. The therapeutically effective amount is also the amount in which the therapeutically beneficial effect outweighs the toxic or adverse effect of the composition.

[0383] Appropriate human doses of any of the compositions described herein can be further evaluated, for example, in a Phase I dose-escalation study. See, for example, van Gurp et al., Am J Transplantation 8(8):1711-1718 (2008); Hanouska et al., Clin Cancer Res 13(2, part 1):523-531 (2007); and Hetherington et al., Antimicrobial Agents and Chemotherapy 50(10):3499-3500 (2006).

[0384] The toxicity and therapeutic efficacy of the composition can be determined by known pharmaceutical procedures in cell culture or experimental animals (e.g., animal models of any of the cancers described herein). These procedures include, for example, LD 50 (Lethal dose for 50% of the population) and ED 50 It can be used to determine the effective therapeutic dose (for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, or ratio LD50. 50 / ED 50 It is expressed as follows. Compositions described herein that exhibit a high therapeutic index are preferred. Compositions exhibiting toxic side effects may be used, but care must be taken to mitigate side effects by designing a delivery system that targets such compounds to the site of the affected tissue and by minimizing any damage that may occur to normal cells.

[0385] Those skilled in the art will understand that data obtained from cell culture assays and animal experiments can be used when formulating a range of dosages for human use. Appropriate dosages of the compositions described herein are generally ED with little or no toxicity. 50 The blood concentration of the composition containing [the substance] is within the specified range. The dose may vary within this range depending on the dosage form used and the route of administration utilized. The therapeutically effective dose of the compositions described herein can first be estimated from a cell culture assay. The IC determined in cell culture 50 Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the antibody concentration that achieves median inhibition of symptoms. Such information can be used to more accurately determine an effective dose in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography. In some embodiments, if local administration (e.g., local administration to the eye or joint) is desired, cell culture or animal modeling can be used to determine the dose required to achieve a therapeutically effective concentration at the local site.

[0386] All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference. Furthermore, materials, methods, and examples are illustrative and not intended to limit. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Similar or equivalent methods and materials may be used, but preferred methods and materials are described herein.

[0387] This disclosure is further illustrated by the following embodiments. The embodiments are provided for illustrative purposes only and should not be construed as limiting the scope or content of this disclosure in any way. [Examples]

[0388] Example 1: Generation of exemplary molecules targeting anti-TSHR autoantibodies This example demonstrates the generation and testing of exemplary molecules described herein that target and selectively deplete circulating autoantibodies. In this example, the targeted autoantibodies are anti-TSHR autoantibodies associated with various diseases such as Graves' disease (GD) and thyroid eye disease.

[0389] Generation of an exemplary molecule Autoantibody binding domain The exemplary molecules in this example were designed to include an autoantigen domain as an autoantibody-binding domain. Specifically, human TSHR was selected as the autoantigen. Fragments and mutations of the human wild-type TSHR sequence were tested for their ability to selectively target and bind to anti-TSHR autoantibodies.

[0390] Two fragments of wild-type TSHR were used as starting points (as shown in SEQ ID NOs: 1 and 5). The fragment labeled "260 WT" or "TSHR260" (SEQ ID NO: 1) is a fragment of the complete TSHR sequence (SEQ ID NO: 9) corresponding to amino acids 22-260 of SEQ ID NO: 9. The fragment labeled "289 WT" or "TSHR289" (SEQ ID NO: 5) is a fragment of the complete TSHR sequence (SEQ ID NO: 9) corresponding to amino acids 22-289 of SEQ ID NO: 9.

[0391] To optimize stability and expression, the TSHR fragment was further mutated. The mutations tested included the "2P" mutation, containing the following mutant amino acid residues: R112P and D143P, relative to the wild-type TSHR sequence; the "2P2R" mutation, containing the following mutant amino acid residues: R112P, D143P, V169R, and I253R; the "2P1S" mutation, containing the following mutant amino acid residues: R112P, D143P, and H63S; and the "2P2R aglycosylation" mutation, which includes an N-to-Q mutation (containing the sequence motif NXS or NXT, where X can be any amino acid other than P) at all N-linked glycosylation sites to remove the "2P2R" mutation and glycosylation. The sequences of THSR260 wild-type and variants 2P, 2P2R, and 2P1S are shown in Table 4. [Table 4]

[0392] Additional TSHR260 variants were generated by introducing the following mutant amino acid residues: "SP" mutation containing S94P; "SP GP" mutation containing S94P and G194P; "SP GP KP" mutation containing S94P, G194P, and K218P; "VP SP KP" mutation containing V87P, S94P, and K218P; "VP SP GP" mutation containing V87P, S94P, and G194P; "GP KP" mutation containing G194P and K218P; "VP SP GP KP" mutation containing V87P, S94P, G137P, and K218P; and "SP GP GP KP" mutation containing S94P, G137P, G188P, and K218P.

[0393] FC Domain The exemplary molecules in this embodiment were designed to include an Fc domain with specific mutations and modifications.

[0394] In this example, we selected the human IgG1 Fc domain contained in the molecules generated and tested. Human IgG1 spontaneously binds to FcRn at acidic pH, which allows it to bind to FcRn, migrate into cells, recirculate back to the cell surface, and avoid degradation by lysosomes.

[0395] In some molecules, Fc mutations have been introduced to increase the binding of the Fc domain to human FcγRIIB. Such mutations include S267E and L328F and / or P238D, according to EU numbering. These mutations enable binding to FcγRIIB at neutral pH. While we do not wish to be bound by any theory, once bound, the molecule / autoantibody complex may be translocated into the cell and targeted to lysosomes for degradation. Such a mechanism would destroy the autoantibody while simultaneously retaining the free molecule in circulation.

[0396] In some molecules, Fc mutations have been introduced to increase the binding of the Fc domain to the human neonatal receptor (FcRn). In particular, mutations have been introduced to increase the binding of the Fc domain to FcRn in a neutral pH environment (e.g., the extracellular environment). Such mutations include combinations (i.e., "MST-HN") containing the following mutations according to EU numbering: M252Y, S254T, T256E, H433K, N434F. While we do not wish to be bound by any theory, the molecules described herein may include such mutations to increase the binding of the Fc domain to FcRn on the cell surface in a neutral pH environment in order to increase receptor-mediated internal translocation into cells and the transport of (molecule-bound) autoantibodies to lysosomes.

[0397] Furthermore, mutations were introduced to eliminate binding affinity to the human Fc-gamma receptor (FcγR) along with the MST-HN mutation. Such mutations include the following: G236R and L328R ("RR"), introduced to reduce binding to one or more Fc-gamma receptors. This modification interferes with immune crosslinking (i.e., crosslinking between molecules, autoantibodies, and FcγR) that leads to inflammatory responses. Such mutations may focus on the primary mechanism of action of the molecule against targeted internal translocation and subsequent degradation of autoantibodies.

[0398] In this embodiment, Fc mutations are also introduced into specific molecules to facilitate heterodimerization of two polypeptides, so that each polypeptide contains an Fc domain, and the first and second Fc domains heterodimerize to produce a complete molecule. Such mutations are known as knob-in-hole (KIH) modifications. Specific mutations used in this embodiment include the T366W and S354C mutations (first Fc domain), as well as the T366S, L368A, Y407V, and Y349C mutations (second Fc domain). Those skilled in the art will understand that this disclosure encompasses molecules in which the T366W and S354C mutations are contained in the second Fc domain (instead of the first Fc domain), and the T366S, L368A, Y407V, and Y349C mutations are contained in the first Fc domain (instead of the second Fc domain). Those skilled in the art will also understand that other known KIH mutations or other Fc modifications for promoting heterodimerization are known in the art and may be used in the molecules described in this embodiment and present disclosure.

[0399] In some molecules, Fc mutations were also introduced to extend the molecular half-life. Specific mutations used in this embodiment include the following: M428L and N434S ("LS") and M252Y, S254T and T256E ("MST" or "YTE"). Those skilled in the art will understand that other known mutations or modifications for extending half-life are known in the art and may be used in the molecules described in this embodiment and in this disclosure.

[0400] Table 5 below shows exemplary Fc domain sequences used in the molecules prepared and tested in this example. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11]

[0401] Additional antigen-binding domainsIn one exemplary molecule in this example, an additional antigen-binding domain was included in a second polypeptide covalently bonded to a second Fc domain. In this example, a second antigen-binding domain specific to the asialoglycoprotein receptor (ASGPR) was used. ASGPR is a membrane protein located on mammalian hepatocytes that targets a target glycoprotein and removes it from circulation. While we do not wish to be bound by any theory, such an antigen-binding domain was introduced to provide a secondary mechanism that allows the molecule to target autoantibodies for internal translocation and degradation in lysosomes. A molecule bound to a target autoantibody can target ASGPR on hepatocytes, and binding to ASGPR causes internal translocation of the complex (i.e., removal of the target autoantibody). The antigen-binding domain for ASGPR used in this example includes anti-ASGPR Fab(4F3), which contains the heavy chain sequence shown in SEQ ID NO: 209 and the light chain sequence shown in SEQ ID NO: 210 (see Table 6 below). [Table 6]

[0402] Using the first and second polypeptides having the above modifications, exemplary molecules listed in Table 7 below were generated based on the exemplary molecular forms shown in Figure 4 (variants A1-A4, B1-B4, D1-D4, and E1-E4) and Figure 5 (variant C1 containing anti-ASPGR Fab in the second polypeptide). These sequences were encoded by expression plasmids (e.g., pTT5, pcDNA), which were then transfected into suitable host cells (e.g., CHO, HEK293) and expressed using standard transfection techniques. After expression for 5-14 days, or when cell viability decreased, the cells were harvested. The conditioned medium was then purified using standard chromatography techniques such as protein A affinity, ion exchange chromatography, and size exclusion chromatography to produce molecules with a purity of over 95% (assessed by HPLC). The molecules were then buffer-exchanged with appropriate formulation buffers and stored at 4°C or -80°C before use. [Table 7-1] [Table 7-2]

[0403] The sequences of a subset of exemplary molecules generated in this example are shown in Table 8 below (with exemplary signal peptides). Table 8 also includes the sequences of several additional exemplary molecules (variants G1-G14) tested in Example 10. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12] [Table 8-13] [Table 8-14] [Table 8-15] [Table 8-16] [Table 8-17] [Table 8-18] [Table 8-19] [Table 8-20] [Table 8-21] [Table 8-22] [Table 8-23] [Table 8-24]

[0404] Example 2: Generation of exemplary molecules targeting anti-PLA2R autoantibodies This example demonstrates the generation and testing of exemplary molecules described herein that target and selectively deplete circulating autoantibodies. In this example, the targeted autoantibodies are anti-PLA2R autoantibodies associated with various diseases such as membranous nephropathy (MN).

[0405] Generation of an exemplary molecule Autoantibody binding domainThe exemplary molecules in this example were designed to include an autoantigen domain as an autoantibody-binding domain. Specifically, human PLA2R was selected as the autoantigen. Various fragments and mutations of the human wild-type PLA2R sequence were tested for their ability to selectively target and bind to anti-PLA2R autoantibodies.

[0406] A fragment of wild-type PLA2R was used as a starting point (as shown in SEQ ID NOs. 15-19; see Table 2). In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 15) corresponding to amino acid positions 38-65 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes a fragment of PLA2R (SEQ ID NO: 16) corresponding to amino acid positions 38-165 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 17) corresponding to amino acid positions 38-169 and 1107-1246 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes three fragments of PLA2R (SEQ ID NO: 18) corresponding to amino acid positions 38-169, 223-367, and 1107-1246 of the full-length PLA2R protein sequence according to SEQ ID NO: 14. In some embodiments, the PLA2R autoantigen domain includes two fragments of PLA2R (SEQ ID NO: 19) corresponding to amino acid positions 38-367 and 1107-1246 of the full-length PLA2R protein sequence according to SEQ ID NO: 14.

[0407] Table 2 shows additional exemplary PLA2R sequences for use according to this embodiment (see SEQ ID NOs. 20-28 and 380-380).

[0408] FC Domain The exemplary molecules in this embodiment were designed to include an Fc domain with specific mutations and modifications.

[0409] In this example, we selected the human IgG1 Fc domain contained in the molecules generated and tested. Human IgG1 spontaneously binds to FcRn at acidic pH, which allows it to bind to FcRn, migrate into cells, recirculate back to the cell surface, and avoid degradation by lysosomes.

[0410] In some molecules, Fc mutations have been introduced to increase the binding of the Fc domain to human FcγRIIB. Such mutations include S267E and L328F and / or P238D, according to EU numbering. These mutations enable binding to FcγRIIB at neutral pH. While we do not wish to be bound by any theory, once bound, the molecule / autoantibody complex may be translocated into the cell and targeted to lysosomes for degradation. Such a mechanism would destroy the autoantibody while simultaneously retaining the free molecule in circulation.

[0411] In some molecules, Fc mutations have been introduced to increase the binding of the Fc domain to the human neonatal receptor (FcRn). In particular, mutations have been introduced to increase the binding of the Fc domain to FcRn in a neutral pH environment (e.g., the extracellular environment). Such mutations include combinations (i.e., "MST-HN") containing the following mutations according to EU numbering: M252Y, S254T, T256E, H433K, N434F. While we do not wish to be bound by any theory, the molecules described herein may include such mutations to increase the binding of the Fc domain to FcRn on the cell surface in a neutral pH environment in order to increase receptor-mediated internal translocation into cells and the transport of (molecule-bound) autoantibodies to lysosomes.

[0412] Furthermore, mutations were introduced to eliminate binding affinity to the human Fc-gamma receptor (FcγR) along with the MST-HN mutation. Such mutations include the following: G236R and L328R ("RR"), introduced to reduce binding to one or more Fc-gamma receptors. This modification interferes with immune crosslinking (i.e., crosslinking between molecules, autoantibodies, and FcγR) that leads to inflammatory responses. Such mutations may focus on the primary mechanism of action of the molecule against targeted internal translocation and subsequent degradation of autoantibodies.

[0413] In this embodiment, Fc mutations are also introduced into specific molecules to facilitate heterodimerization of two polypeptides, so that each polypeptide contains an Fc domain, and the first and second Fc domains heterodimerize to produce a complete molecule. Such mutations are known as knob-in-hole (KIH) modifications. Specific mutations used in this embodiment include the T366W and S354C mutations (first Fc domain), as well as the T366S, L368A, Y407V, and Y349C mutations (second Fc domain). Those skilled in the art will understand that this disclosure encompasses molecules in which the T366W and S354C mutations are contained in the second Fc domain (instead of the first Fc domain), and the T366S, L368A, Y407V, and Y349C mutations are contained in the first Fc domain (instead of the second Fc domain). Those skilled in the art will also understand that other known KIH mutations or other Fc modifications for promoting heterodimerization are known in the art and may be used in the molecules described in this embodiment and present disclosure.

[0414] In some molecules, Fc mutations were also introduced to extend the molecular half-life. Specific mutations used in this embodiment include the following: M252Y, S254T, and T256E ("MST" or "YTE"). Those skilled in the art will understand that other known mutations or modifications for extending half-life (e.g., M428L and N434S ("LS") mutations) are known in the art and may be used in the molecules described in this embodiment and the present disclosure.

[0415] Exemplary Fc domain sequences for use according to this embodiment are shown in Table 3 above.

[0416] Using the first and second polypeptides having the above modifications, exemplary molecules listed in Table 9A below, based on the exemplary molecular form shown in Figure 4, and exemplary molecules listed in Table 9B below, based on the exemplary molecular form shown in Figure 7B, were generated. These sequences were encoded by an expression plasmid (e.g., pTT5, pcDNA), which was then transfected into suitable host cells (e.g., CHO, HEK293) and expressed using standard transfection techniques. After expression for 5–14 days, or when cell viability decreased, the cells were harvested. The conditioned medium was then purified using standard chromatography techniques such as protein A affinity, ion exchange chromatography, and size exclusion chromatography to produce molecules with a purity of over 95% (assessed by HPLC). The molecules were then buffer-exchanged with a suitable formulation buffer and stored at 4°C or -80°C before use. [Table 9A-1] [Table 9A-2] [Table 9B]

[0417] The sequences of the exemplary molecules generated in this example are shown in Table 9C below (with exemplary signal peptides). [Table 9C-1] [Table 9C-2] [Table 9C-3] [Table 9C-4] Table 9C-5 Table 9C-6 Table 9C-7 Table 9C-8 Table 9C-9

Table 9C-10

Table 9C-11

Table 9C-12

Table 9C-14

Table 9C-19

[0418] Example 3: In vitro neutralization of anti-TSHR autoantibody This example demonstrates the functionality of the exemplary molecule (variant D3) described in Example 1. Specifically, the exemplary molecule was tested for its ability to target and neutralize patient-derived monoclonal anti-TSHR autoantibodies via its anti-TSHR autoantibody domain. The molecule's effect on TSHR activity was also tested.

[0419] Determination of EC80 of monoclonal autoantibodies M22, K1-18, or the native ligand TSH in CHO-TSHR cells. To determine the potency of stimulating anti-TSHR autoantibodies or the natural ligand thyroid-stimulating hormone (TSH), Chinese hamster ovary (CHO) cells (Eurofins DiscoverX) overexpressing wild-type TSHR were co-incubated with multiple concentrations of monoclonal autoantibodies M22 or K1-18, or TSH, followed by a blank negative control, on an 11-point dilution curve (diluted in PBS). CHO-TSHR cells were isolated using TrypLE® (Thermo Fisher) 16–20 hours prior to the assay. Cells were then seeded at 20,000 cells per well in 100 μl of 96-well flat-bottom opaque white-walled plates (Corning). After overnight incubation at 37°C and 5% CO2, serial dilutions of monoclonal autoantibodies or TSH were prepared in 96-well round-bottom plates in PBS at pH 7.4 at room temperature. The assay plates containing the cells were then removed from the incubator, and the culture medium was aspirated from all wells using a multichannel pipette. Next, serially diluted monoclonal autoantibodies or TSH were added to assay plates containing CHO-TSHR cells and incubated at 37°C for 30 minutes. Then, the luminescence signal detecting cAMP produced by CHO-TSHR cells was measured according to the manufacturer's protocol for Biologics' HitHunter® cAMP Assay. Briefly, the cells were lysed by adding 60 μl of “cAMP detection solution” containing lysis buffer. The plates were then incubated at room temperature for 1 hour (protected from light), after which “cAMP solution A” was added, and the mixture was incubated at room temperature for 3 hours, protected from light. Finally, the plates were read using a standard luminescence plate reader. A schematic diagram of the anti-TSHR autoantibody-TSHR-cAMP pathway is shown in Figure 8A. Figure 8B shows cAMP activity with increasing concentrations of agonists (M22, K1-18, or TSH).

[0420] Neutralization of CHO-TSHR cells by monoclonal autoantibodies M22, K1-18, or molecules of the native ligand TSH. To determine the efficacy of molecular variant D3 in depleting the thyroid-stimulating hormone (TSH) stimulating activity of monoclonal autoantibodies M22, K1-18, or their native ligands, cAMP levels produced by CHO cells overexpressing wild-type TSHR were measured by ELISA. CHO-TSHR cells were isolated using TrypLE® (Thermo Fisher) 16–20 hours prior to the assay. The cells were then seeded at 20,000 cells per well in 100 μl of 96-well flat-bottom opaque white-walled plates (Corning). After incubation overnight at 37°C in 5% CO2, the molecule (variant D3) was titrated using an 11-point 3-fold serial dilution curve. Each dilution was then mixed with a single concentration of M22, K1-18, or TSH, pre-determined as the EC80 for this assay. These mixtures were incubated at room temperature for 10 minutes before being incubated with CHO-TSHR cells. Next, the assay plate containing the cells was removed from the incubator, and the culture medium was aspirated from all wells using a multichannel pipette. Then, 30 µl of PBS was added to each well, and 15 µl of the 3-fold dilution mix was transferred to each well to the final 1x concentration. The cells were then incubated with the immune complex at 37°C for 30 minutes. Next, the luminescence signal to detect cAMP produced by CHO-TSHR cells was measured according to the manufacturer's protocol for Biologics' HitHunter® cAMP Assay. Briefly, the cells were lysed by adding 60 µl of “cAMP detection solution” containing lysis buffer. The plate was then incubated at room temperature for 1 hour (protected from light), and “cAMP solution A” was added. The mixture was then incubated at room temperature for 3 hours, protected from light. Finally, the plate was read with a standard luminescence plate reader.

[0421] The results of the cAMP assay are shown in Figures 9 and 10. Figure 9 shows that the exemplary molecular variant D3 blocks TSHR-stimulated M22 autoantibody-induced cAMP signaling without affecting TSH signaling. Figure 10 shows that the exemplary molecular variant D3 also blocks TSHR-stimulated K1-18 autoantibody-induced cAMP signaling.

[0422] Neutralization of polyclonal patient serum variant D3 in CHO-TSHR cells To determine the efficacy of exemplary molecular variant D3 in depleting the activity of polyclonal anti-TSHR antibodies in patient serum, variant D3 was diluted in human serum in a 1:10 ratio to a final concentration of 200 nM. These mixtures were incubated at room temperature for 10 minutes and then co-incubated with Chinese hamster ovary (CHO) cells (Eurofins DiscoverX) overexpressing wild-type TSHR. 16–20 hours prior to the assay, the CHO-TSHR cells were isolated using TrypLE® (Thermo Fisher). The cells were then seeded at 40,000 cells per well in 100 μl of 96-well flat-bottom opaque white-walled plates (Corning). After incubation overnight at 37°C in 5% CO2, a variant D3 dilution was prepared using patient serum containing polyclonal anti-TSHR antibodies. The assay plate containing the cells was then removed from the incubator, and the culture medium was aspirated from all wells using a multichannel pipette. Next, the cells were incubated with the molecular / patient serum mixture at 37°C for 30 minutes. Then, the absorbance signal to quantify cAMP produced by CHO-TSHR cells was measured according to the manufacturer's protocol for the Cyclic AMP XP Assay Kit (Cell Signaling Technologies).

[0423] In short, after co-incubating with molecular / patient serum, the cells were washed in PBS and then lysed with 1× lysis buffer. Next, 50 μl of the cell lysate was incubated with 50 μl of HRP-binding target solution in a prepared antibody-coated plate at room temperature for 3 hours. Then, the plate was washed four times with 200 μl / well of 1× wash buffer, and 100 μl of TMB substrate was added. After incubation with the TMB substrate at room temperature for 5–30 minutes, 100 μl / well of stop solution was added, and the plate was read using a plate reader at both 450 nm and 570 nm.

[0424] The results of the cAMP assay are shown in Figure 11. The data indicate that molecular variant D3 neutralized polyclonal anti-TSHR antibodies in each patient's serum by reducing cAMP activity in each serum sample.

[0425] Neutralization of polyclonal patient serum with exemplary molecules in CHO-TSHR cells (Quidel Turbo TSI) To determine the efficacy of exemplary molecular variant D3 against the depletion of activity in patient serum containing polyclonal anti-TSHR antibody, the molecule was diluted in human serum in a 1:10 ratio to a final concentration of 200 nM. These mixtures were incubated at room temperature for 10 minutes and then co-incubated with Chinese hamster ovary (CHO) cells (Thyretain Turbo TSI, Quidel Ortho) overexpressing MC4 mutant TSHR. First, the cAMP reagent, standard panel, and controls were thawed at 37°C for 7–10 minutes and equilibrated to room temperature. The standards and samples were mixed by gentle pipetting, and 5 μl of each standard or sample was added to the bottom corner of each well in a white-walled 96-well plate (Corning). The Turbo TSI cells were thawed in a 37°C water bath for 2 minutes, and the entire contents were transferred to one bottle of 5 mL of cAMP reagent and mixed by inversion. Next, 50 μl of the cAMP reagent and Turbo TSI cell mixture was transferred to each well of a white-walled 96-well plate containing 5 μl of standard or sample. Patient serum mixtures, with or without cells and molecules, were incubated at room temperature for 60 minutes. The plates were then read using a standard plate reader or luminometer.

[0426] Figure 12A shows that exemplary molecular variant D3 reduced the TSHR-stimulating activity of patient serum autoantibodies in 19 / 19 patients (including reductions to background levels in 95% of donor serum (18 / 19 donors)) and pooled samples (Figure 12B).

[0427] Autoantibody-molecular complex recirculation assay Cells expressing FcRn are seeded in a 96-well plate, incubated for 1 hour, then serum-starved and divided into two groups (pH 6.0 and pH 7.0). Various dilutions of the molecule containing the above-mentioned modification to enhance FcRn binding are added along with various dilutions of anti-TSHR autoantibodies (e.g., M22, K1-70, K-18), and the cells are co-incubated for 4 hours. The cells are then washed in HBSS, the pH is adjusted to neutral (pH 7.4), and incubated at 37°C for 4 hours or overnight. The cell supernatant is collected and analyzed by anti-human IgG ELISA to quantify the amount of anti-TSHR autoantibodies recycled into the supernatant.

[0428] The anti-TSHR monoclonal autoantibody M22 is labeled with a fluorescent tag (i.e., PE). Next, a molecule containing the above-mentioned modification to enhance FcγRIIB binding is co-incubated with the labeled M22 in either a 1:1 or 4:1 ratio (molecule:autoantibody) for 15 minutes at room temperature to form a complex. Cells expressing FcγRIIB are seeded in a 96-well plate and stained with Live / Dead Violet Fixable Dye. The pre-complexed molecule and autoantibody are then incubated with the cells at varying concentrations at either 4°C (to evaluate binding) or 37°C (to evaluate uptake). After incubation, the cells are washed with PBS (binding) or acidic medium to remove the surface-bound complex (uptake). Cell fluorescence is measured by flow cytometry.

[0429] SEC-based evaluation of immune complexes in vitro To assess the size of the immune complex formed when anti-TSHR autoantibodies bind to a molecule, the molecule is incubated with serum from Graves' disease / thyroid eye disease patients containing anti-TSHR autoantibodies, or with anti-TSHR monoclonal autoantibodies (M22, K1-70, K1-18, CS-17) as a positive control. The size of the immune complex formed between the molecule and the autoantibodies present in the patient's serum is estimated using an SEC-based method similar to that described in Boysen et al., Journal of Immunology Research 2:1-9, 2016, which is incorporated herein by reference.

[0430] Example 4: Testing and characterization of exemplary molecules targeting anti-TSHR autoantibodies Identification of Fc mutations that confer specific binding to FcγRIIB. The exemplary molecules described above were tested for their ability to selectively bind to FcγRIIB via their Fc domains, as well as for whether or not they had reduced or absent binding affinity to other Fc receptors, FcγRIIA167H and FcγRIIA167R.

[0431] FcγRIIB isoform 2 is an endocytosis receptor that binds to its target, causes the complex to move internally, and transports the target to lysosomes for degradation. While we do not wish to be bound by any theory, the molecules described herein with increased FcγRIIB binding can deplete antigen-specific B cells producing anti-TSHR autoantibodies and / or anti-TSHR autoantibodies through various mechanisms, including those shown in Figures 13A–D. Figure 13B illustrates a potential mechanism of action, including the elimination of autoantibodies by targeting FcγRIIB isoform 2 on hepatic sinusoidal endothelial cells (LSECs). In this exemplary mechanism, the autoantigen domain (TSHR or a fragment or variant thereof) binds to the autoantibody (anti-TSHR autoantibody), and the Fc domain binds to FcγRIIB isoform 2 on hepatic sinusoidal endothelial cells. The autoantibody is then moved into the hepatic sinusoidal endothelial cells and targeted to lysosomes for degradation. In another exemplary mechanism shown in Figure 13C, the molecules described herein may target pathogenic B cells that produce a targeted autoantibody (e.g., anti-TSHR autoantibody) by targeting FcγRIIB isoform 1 to the B cell receptor (BCR), thereby resulting in apoptosis and inhibition of B cells. In another exemplary mechanism shown in Figure 13D, the molecules described herein: an autoantibody immune complex can target FcγRIIB on dendritic cells to reduce / inhibit antigen presentation to T cells.

[0432] Binding assays using SPR were performed to determine the binding affinity of exemplary molecules. His-tagged FcγRIIA167H, FcγRIIA167R, or FcγRIIB were captured on a CM5 SPR tip pre-conjugated with an anti-His capture antibody via standard amine coupling. Subsequently, molecules of increasing concentration were injected into the captured FcγR, and single dissociation was performed using single-cycle kinetics. The data were then analyzed using steady-state analysis suitable for low-affinity interactions. In particular, plots of the equilibrium response against molecular concentration were generated. The KD value corresponds to the concentration that yields 50% of the maximum response. The molecules shown include control IgG1 (trastuzumab antibody), variant B3, variant D3, and variant E3 against various Fc receptors: FcγRIIA167H, FcγRIIA167R, and FcγRIIB. Molecular variant B3 was designed to have improved binding to FcγRIIB (inhibitory Fcγ receptor) by introducing the mutation S267E / L328F. Molecular variant D3 contains the mutation P238D in its Fc domain to increase binding to FcγRIIB. Molecular variant E3 contains the P238D mutation and the LS mutation in its Fc domain to extend half-life. Trastuzumab and exemplary molecules with a wild-type Fc domain (WT IgG1 Fc) were used as positive controls.

[0433] The results of the binding assays are shown in Tables 10 and 11, and Figures 14-19. Specifically, Figures 14-16 show the binding activity of trastuzumab (positive control) (A), variant B3 (B), variant D3 (C), and variant E3 (D) to the activating receptor FcγRIIA167H (Figure 14), the activating receptor FcγRIIA167R (Figure 15), and the inhibitory receptor FcγRIIB (Figure 16) when exemplary molecules were captured on an SPR sensor chip and FcγR was used as the analyte. Table 11 shows the K for each assay. D Show the value.

[0434] Figures 17-19 and Table 10 show the binding activity of trastuzumab (positive control) (A), variant B3 (B), variant D3 (C), and variant E3 (D) to FcγRIIA167H (Figure 17), FcγRIIA167R (Figure 18), and FcγRIIB (Figure 19) when His-tagged FcγR was captured on an SPR sensor chip and exemplary molecules were used as analytes. Table 10 shows the untreated K for each assay. D Show the value.

[0435] The results show that exemplary molecular variant D3 (P238D mutation) showed increased binding to FcγRIIB and no or significantly reduced binding to FcγRIIA167R and FcγRIIA167H compared to the control (wild-type IgG1 Fc). In contrast, exemplary molecular variant B3 (SE / LF mutation) showed increased binding to both FcγRIIA167R and FcγRIIB.

[0436] While we do not wish to be bound by any theory, molecular variant D3, which has selective affinity for inhibitory FcγRIIB, presents certain advantages, such as enabling avidity-induced FcγRIIB-mediated intracellular uptake and degradation. Furthermore, this molecule has a relatively small size and little to no affinity for activating the Fcγ receptor, which partially reduces its toxicity risk. [Table 10] [Table 11]

[0437] Complementary symbiosis Exemplary molecular variants B3 and D3 were tested for their complement-binding ability. The antibody rituximab was used as a positive control. IgG4 was used as a negative control. Additional molecules were generated using the TSHR260 2P2R autoantigen and the IgG1 Fc domain containing an Fc mutation (LALAPG) that removes effector activity ("Fc null" molecules).

[0438] Various dilutions of one of the exemplary molecules or controls were tested for binding to C1q by ELISA. Here, serial dilutions of the molecule or control (at 0.01 μg / mL, 0.1 μg / mL, 1.0 μg / mL, 10.0 μg / mL, 100.0 μg / mL, and 1000 μg / mL) were coated onto plates overnight at 4°C. After blocking and subsequent washing steps, 5 ug / mL of human C1q was added to the ELISA plate at room temperature for 1 hour. The plate was washed, and a 1:200 dilution of sheep polyclonal anti-C1q HRP antibody was added and incubated for a further 1 hour. The ELISA plate was developed by adding TMB. The color development was then stopped by adding 3N HCl. The absorbance of the plate was read at 450 nm using the prepared plate.

[0439] The assay results show that variant D3 and the Fc null molecule do not bind to C1q. This is in contrast to WT IgG1 Fc and variant B3 (Figure 20).

[0440] Exemplary molecular testing of avidity-mediated effects In this experiment, CHO cells expressing FcγRIIB (CHO-FcγRIIB+) were incubated with and without the 2B6 antibody, an anti-FcγRIIB antibody that blocks the binding of FcγRIIB to the antibody's Fc domain, along with exemplary molecules and M22 (anti-TSHR autoantibody) in either the same molar ratio or a 4-molar excess (i.e., a 1:1 molecule:M22 molar ratio, or a 4:1 molecule:M22 molar ratio). The exemplary molecules tested included (i) a molecule containing the TSHR260 2P2R autoantigen and an Fc domain containing the LALAPG mutation ("Fc null"), and therefore used as a negative control; (ii) a molecule containing the TSHR260 2P2R autoantigen and the wild-type Fc domain WT IgG1 Fc, and used as a positive control; (iii) variant B3 containing a mutation (S267E / L328F) that enhances binding to FcγRIIB; (iv) variant D3 containing a mutation that eliminates binding affinity to the Fcγ-activated receptor FcγRIIA and moderately enhances binding to FcγRIIB; (v) variant E3 being the same as variant D3 but also containing an LS mutation that extends the half-life; and (vi) variant F3 containing IgG1 having only the TSHR260 2P2R autoantigen and the LS mutation.

[0441] These results demonstrate that variant D3 binds to FcγRIIB-expressing CHO cells with increased avidity (Figure 21). Specifically, variant D3 doubles the binding of M22 to FcγRIIB when M22 is pre-complexed so that most M22 binds to two molecules ("4:1") compared to when M22 is primarily bound to one molecule ("1:1"). WT IgG1 Fc showed only a 1.5-fold increase, and variant B3 (SE / LF Fc) showed no increase in binding. Variant E3 showed no increase in avidity compared to WT IgG1. Furthermore, the binding of exemplary molecules is completely blocked by the blocking anti-FcγRIIB antibody (clone 2B6). Thus, variant D3 exhibits an unparalleled potent avidity-mediated binding effect to FcγRIIB.

[0442] Binding specificity for FcγRIIB-expressing cells The ability of exemplary molecules to selectively bind to cells expressing FcγRIIB was also tested. Various immune cell types are known to express FcγRIIB at different levels. FcγRIIB is known to be mainly expressed in B cells, minimally expressed in non-classical monocytes, and either not expressed or expressed at low levels in T cells, NK cells, and classical monocytes (Figure 22B; modified from Kerntke, et al., Frontiers in immunology. 11:489401, 2020, incorporated herein by reference). Each cell type (monocytes, B cells, NK cells, and T cells) was incubated with exemplary molecular variants D3 and M22 (anti-TSHR autoantibodies) in a 4 molar excess of the molecule (i.e., 4:1 molecule:M22). Cells were characterized to have specific markers for monocytes, B cells, NK cells, and T cells, namely CD16, CD19, CD56, and CD3, respectively. Unclassified cells represent cells that could not be classified as monocytes, B cells, NK cells, or T cells because they are negative for CD16, CD19, CD56, and CD3. Such cells may be non-classical monocytes or basophils.

[0443] Figure 22A shows that the M22 immune complex bound to variant D3 binds only to primary cells expressing FcγRIIB (i.e., B cells and unclassified cells that may be non-classical monocytes or basophils).

[0444] Further binding studies were performed to compare the selectivity of exemplary molecular variants D3 and B3, and other exemplary molecules, including WT IgG1 Fc (containing TSHR260 2P2R), to Fc-expressing cells compared with an Fc null variant (as a negative control). Binding was evaluated in B cells, T cells, monocytes, non-classical monocytes, and NK cells. Each cell type was incubated with the exemplary molecules and M22 (anti-TSHR autoantibody) in a 4 molar excess of the molecule (i.e., 4:1 molecule:M22).

[0445] Figure 23 (exemplary molecular binding to B cells (A) and monocytes (B)) and Figure 24 (NK cells (A) and unclassified cells (CD3, CD19, CD14, CD56, and CD16 negative, potentially basophils) (B)) show that variants D3 and E3 exhibit cell binding specific to cells expressing FcγRIIB. Furthermore, cell binding is blocked by the anti-FcγRIIB antibody 2B6 at molar ratios of variant D3:M22 or variant E3:M22 (4:1).

[0446] Binding of free molecules to FcγRIIB In this experiment, CHO cells expressing FcγRIIB (CHO-FcγRIIB+) were incubated with and without 2B6 antibody (an anti-FcγRIIB antibody that blocks the binding of FcγRIIB to the antibody's Fc domain) at different concentrations of exemplary molecules. The exemplary molecules tested included variant B3, which contains a mutation that enhances binding to FcγRIIB (S267E / L328F; positive control), and variant D3, which contains a mutation that eliminates binding affinity to the Fcγ-activating receptor FcγRIIA and moderately enhances binding to FcγRIIB. Binding of the exemplary molecules to CHO-FcγRIIB+ cells was evaluated using anti-TSHR antibody (CS-17).

[0447] Figure 25 shows that variant D3, as a free molecule, does not strongly bind to CHO-FcγRIIB+ cells. Variant B3 binds to CHO-FcγRIIB+ cells at a low concentration of 1 nM. Binding of variant D3 to CHO-FcγRIIB+ cells was evident only at >1 μM. Binding of variant D3 is completely blocked by the anti-FcγRIIB blocking antibody 2B6.

[0448] FcγRIIB+ Binding of Free Molecules to Primary Cells Exemplary molecules were also tested for their ability to bind to human primary cells expressing FcγRIIB. Exemplary molecules included (i) a molecule containing the TSHR260 2P2R autoantigen and an Fc domain containing the LALAPG mutation ("Fc null"), and therefore used as a negative control; (ii) variant B3 containing a mutation (S267E / L328F) that enhances binding to FcγRIIB; and (iii) variant D3 containing a mutation that eliminates binding affinity to FcγRIIA and moderately enhances binding to FcγRIIB.

[0449] After incubating the molecules with human PBMCs, binding was evaluated by quantifying the binding of the anti-TSHR antibody CS-17 to cells bound to the exemplary molecules.

[0450] Figure 26 shows exemplary molecular binding to B cells. Similar to Figure 25, variant D3 shows detectable binding to cells only at 1 μM, while variant B3 at 1 nM binds to B cells.

[0451] Figure 27 shows exemplary molecular binding to classical monocytes (CD14+) and unclassified cells likely to be basophils (negative for CD3, CD19, CD14, CD56, and CD16). Similar to Figure 26 in B cells, the 1 μM variant D3 binds weakly to monocytes, while the 10 nM variant B3 binds strongly to monocytes.

[0452] Example 5: Testing and characterization of exemplary molecules targeting anti-PLA2R autoantibodies Testing of Fc mutations that confer specific binding to FcγRIIB In Example 2, the exemplary molecules described above were tested to determine whether mutations that enhance affinity for FcγRIIB are available, regardless of the antigen fragment (or other autoantibody targeting domain) contained in the molecule, and whether avidity-mediated or selective binding to FcγRIIB can be conferred.

[0453] Binding assays using SPR were performed to determine the binding affinity of exemplary molecules. Molecules were captured on a CM5 SPR chip. Subsequently, incremental concentrations of FcγR analytes (FcγRIIA167H, FcγRIIA167R, or FcγRIIB) were injected into the captured molecules, and single dissociation was performed using single-cycle kinetics. The data were then analyzed using steady-state analysis suitable for low-affinity interactions. In particular, plots of the equilibrium response against molecular concentration were generated. The KD value is equivalent to the concentration that yields 50% of the maximum response. The molecules shown include control IgG1 (trastuzumab antibody), variant X1, variant X2, variant X3, variant X5, variant X6, and variant X7 against various Fc receptors: FcγRIIA167H, FcγRIIA167R, and FcγRIIB. All molecules contain the P238D mutation in their Fc domain to increase binding to FcγRIIB. Trastuzumab was used as a positive control.

[0454] The results of the binding assay are shown in Tables 12-15 and Figures 28-36 below. [Table 12] [Table 13] [Table 14] [Table 15]

[0455] Specifically, Figures 28-30 and Table 12 show the binding activity of trastuzumab (positive control) (Figure 28), variant X1 (Figure 29A), variant X2 (Figure 29B), variant X3 (Figure 29C), variant X5 (Figure 29D), and variant X6 (Figure 30A) and variant X7 (Figure 30B) to the inhibitory receptor FcγRIIB when molecules were captured on an SPR sensor chip and FcγRIIB was used as the analyte. Table 12 shows the K for each assay. DShow the value.

[0456] Specifically, Figures 31-33 and Table 13 show the binding activity of trastuzumab (positive control) (Figure 31), variant X1 (Figure 32A), variant X2 (Figure 32B), variant X3 (Figure 32C), variant X5 (Figure 32D), and variant X6 (Figure 33A) and variant X7 (Figure 33B) to the activating receptor FcγRIIA167R when molecules were captured on an SPR sensor chip and FcγRIIA167R was used as the analyte. Table 13 shows the K for each assay. D Show the value.

[0457] Specifically, Figures 34-36 and Table 14 show the binding activity of trastuzumab (positive control) (Figure 34), variant X1 (Figure 35A), variant X2 (Figure 35B), variant X3 (Figure 35C), variant X5 (Figure 35D), and variant X6 (Figure 36A) and variant X7 (Figure 36B) to the activating receptor FcγRIIA167H when molecules were captured on an SPR sensor chip and FcγRIIA167H was used as the analyte. Table 14 shows the K for each assay. D Show the value.

[0458] Table 15 shows an overview of the relative binding affinity for each FcγR receptor.

[0459] The binding assay results indicate that molecules with the P238D mutation (i.e., all variants tested) showed similar selectivity for FcγRIIB compared to FcγRIIA(167R and 167H). Specifically, all molecules showed enhanced binding to FcγRIIB and a significant decrease in binding to FcγRIIA167R, and did not bind to FcγRIIA167H.

[0460] While we do not wish to be bound by any theory, this embodiment demonstrates that the P238D mutation can confer selective affinity to inhibitory FcγRIIBs of the molecules described herein, accompanied by various autoantigens, suggesting that the effect is antigen-independent. Furthermore, as shown in previous embodiments, introducing the P238D mutation confers a "moderate" binding affinity, thereby creating an avidity-mediated effect in the molecules described herein, enabling avidity-induced FcγRIIB-mediated intracellular uptake and degradation. Moreover, this embodiment demonstrates that such avidity-mediated effects can be utilized in molecules with different antigens, thereby targeting different autoantibodies, and that the risk of intracellular toxicity is low, partly due to the relatively small size of the molecules and their little to no affinity for activated Fcγ receptors.

[0461] Example 6: In vivo testing of exemplary molecules targeting anti-TSHR autoantibodies In this example, the in vivo activity of the exemplary molecule described in Example 1 is investigated. To evaluate the clearance activity of the exemplary molecule against anti-TSHR monoclonal autoantibodies, a mouse model is injected with an anti-TSHR monoclonal autoantibody (e.g., M22), followed by injection of the molecule or a control, and the clearance of the anti-TSHR monoclonal autoantibody is evaluated over time.

[0462] An in vitro preliminary study using ELISA to evaluate the detection of anti-TSHR monoclonal autoantibodies and molecules. To test the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for detecting molecules in mouse serum, the ELISA for detecting anti-TSHR monoclonal autoantibody (M22) or the molecule must first be performed in vitro using a known concentration.

[0463] A typical ELISA protocol is as follows: Coat the plate with capture antibody overnight at 4°C. Block the plate with blocking buffer (PBS containing 5% BSA volume / volume) at room temperature for 2 hours. Wash the plate with washing buffer (PBS + 0.05% Tween® 20 or similar), add the sample, and incubate at room temperature for 1 hour. Wash the plate, add the detection antibody solution, and incubate at room temperature for 1 hour. Wash the plate, protect it from light, and incubate with TMB substrate at room temperature for 15 minutes. Stop the reaction with 2M H2SO4 and read the result with a microplate reader set to 450nm-570nm.

[0464] In vitro preliminary study to evaluate labeling and detection of anti-TSHR monoclonal autoantibodies To detect the anti-TSHR monoclonal autoantibody M22, various concentrations of M22 were titrated in buffer, buffer containing 1% mouse serum, or buffer containing 10% mouse serum, and detected using ELISA. The capture antibody used to conjugate to the anti-TSHR monoclonal autoantibody M22 was goat anti-human Fc, and the detection antibody was HRP-labeled goat anti-human kappa or lambda light chain antibody, depending on the light chain of the detection antibody.

[0465] As described above, in addition to using the ELISA method to detect anti-TSHR monoclonal autoantibodies, anti-TSHR monoclonal autoantibodies may also be labeled with biotin for detection. In this in vitro preliminary test, anti-TSHR monoclonal autoantibodies are labeled with biotin in PBS according to the manufacturer's protocol. Then, the detection sensitivity is evaluated by titrating the labeled anti-TSHR monoclonal autoantibodies at various concentrations in buffer, buffer containing 1% mouse serum, or buffer containing 10% mouse serum. The capture antibody used to conjugate anti-TSHR monoclonal autoantibody M22 was goat anti-human Fc, and the detection antibody was streptavidin poly-HRP.

[0466] In vitro preliminary tests to evaluate the detection of exemplary molecules For the detection of exemplary molecules, exemplary molecules at various concentrations were titrated in buffer, buffer containing 1% mouse serum, or buffer containing 10% mouse serum, and detected using ELISA. The capture antibody used to bind to the exemplary molecules was goat anti-human Fc, and the detection antibodies were mouse anti-human TSHR CS-17, followed by HRP-labeled anti-mouse IgG.

[0467] Preliminary study to evaluate molecular pK in vivo To evaluate the clearance of the exemplary molecule in wild-type mice, the exemplary molecule was injected at a concentration of 1 in 20 mM histidine, 150 mM NaCl, pH 6.0, and serum was collected 2, 6, 12, 24, 48, 72 hours, or 7 days after injection. The exemplary molecule in the mouse serum was detected using the method described above, with serum from mice not injected with the exemplary molecule used as a negative control, and serum from those mice with the exemplary molecule ex vivo added used as a positive control.

[0468] A preliminary study to evaluate the clearance of anti-TSHR monoclonal autoantibodies in vivo. To evaluate the clearance of the anti-TSHR monoclonal autoantibody M22 in wild-type mice, mice were injected with 1.5 ug, 15 ug, or 150 ug of M22 in 20 mM histidine, 150 mM NaCl, and pH 6.0, respectively. Serum was collected 12, 24, 48, 72 hours, or 7 days after injection. M22 in the mouse serum was detected using the method described above. Serum from mice not injected with the anti-TSHR monoclonal autoantibody was used as a negative control, and the serum was used as a positive control by adding the anti-TSHR monoclonal autoantibody ex vivo to the same serum.

[0469] Evaluation of the clearance of anti-TSHR monoclonal autoantibodies by exemplary molecules in vivo. Based on the clearance information of the anti-TSHR monoclonal autoantibody M22 and the exemplary molecule evaluated in this study, mice are injected with 1 concentration of anti-TSHR monoclonal autoantibody in 20 mM histidine, 150 mM NaCl, pH 6.0. Twenty-four hours after injection, mice are injected with a 4-fold molar excess of the molecule or a vehicle as a control. The clearance of the anti-TSHR monoclonal autoantibody and the exemplary molecule is evaluated using the method described above.

[0470] Example 7: In vivo testing of exemplary molecules targeting anti-TSHR autoantibodies This example investigates the in vivo activity of exemplary molecular variants B3 (TSHR260 2P2R containing Fc with the S267E / L328F mutation), variant D3 (TSHR260 2P2R containing Fc with the P238D mutation), variant E3 (TSHR260 2P2R containing Fc with the P238D and LS mutations), and Fc null molecule (TSHR260 2P2R containing Fc with the LALAPG mutation).

[0471] Molecular and M22 clearance in wild-type mice In this experiment, wild-type BALBC mice were injected with M22 (TSHR autoantibody), and 24 hours later, the mice were injected with the molecule. Serum was collected from the mice, and the concentrations of the molecule and M22 were analyzed at -10 minutes, 10 minutes, 30 minutes, 3 hours, 12 hours, 48 ​​hours, and 7 days after molecular administration (see drug administration schematics in Figures 37A and 38A).

[0472] result Figures 37B-C show the levels of M22 at different time points. Figures 38B-C show the levels of the molecule at different time points. These results show that, in contrast to the Fc null molecule, M22 is undetectable 30 minutes after administration of variant D3 with a 4 molar excess (molar ratio of molecule:M22 of 4:1) (Figures 37B-C). With variant D3 at a 1:4 molar ratio, some M22 remains after all molecule:M22 complexes have disappeared. This amount is consistent with the proportion of unbound M22 remaining in circulation.

[0473] Under the 4:1 condition, the molecule survived for up to 7 days, indicating a long half-life of the molecule in serum (Figures 38B-C). This is equivalent to the pK experiment in which the molecule was administered in the absence of M22, as shown in Figure 39. Furthermore, variants B3 and D3 were not detected under the 1:4 molar ratio condition (Figures 38B-C).

[0474] pK experiments performed in the absence of M22 showed that variant B3 has a shorter half-life compared to variant D3, which is likely due to its high affinity for FcγRIIA and FcγRIIB (Figure 39A-B). The Fc null molecule has a reduced volume of distribution and decreased clearance due to the lack of binding to FcγR. Variant D3 has a half-life of approximately 9 days, which is similar to most human antibodies in wild-type mice. The dots represent the median of 5 mice, and the bars represent SEM (Figure 39B).

[0475] Molecular and M22 clearance in B-hFcRn mice In this experiment, B-hFcRn mice were injected with M22 (TSHR autoantibody), and 24 hours later, the mice were injected with a molecule. Serum was collected from the mice, and the concentrations of the molecule and M22 were analyzed at -10 minutes, 10 minutes, 30 minutes, 3 hours, 12 hours, 48 ​​hours, and 7 days after molecular administration.

[0476] result The results are shown in Figures 40B-C. These results indicate that, in contrast to the Fc null molecule, M22 is undetectable 10 minutes after administration of variant D3 or variant E3 in a 4 molar excess (a molar ratio of 4:1 molecules to M22).

[0477] Furthermore, variants D3 and E3 remain after all molecules:M22 complexes have disappeared, but all molecules have long half-lives as shown in Figures 41B-C. Variant E3 (with LS mutation) showed an extended half-life compared to variant D3 (without LS mutation).

[0478] The results of pK experiments in the absence of M22 are shown in Figures 42A-B. In humanized FcRn mice (B-hFcRn), variant E3 (with LS mutation) showed an extended half-life compared to variant D3 (without LS mutation). The Fc null molecule showed a decrease in distribution volume and reduced clearance due to the lack of binding to FcγR. The half-life may be slightly underestimated due to the limited time points after the distribution phase. The dots represent the median of 5 mice, and the bars represent SEM (Figure 42B).

[0479] Molecular and M22 clearance in hFcγR / hFcRn mice In this experiment, hFcγR / hFcRn mice were injected with M22 (TSHR autoantibody), and 24 hours later, the mice were injected with a molecule. Serum was collected from the mice, and the concentrations of the molecule and M22 were analyzed at -10 minutes, 10 minutes, 30 minutes, 3 hours, 12 hours, 48 ​​hours, and 7 days after molecular administration.

[0480] result Figures 43B-C show the levels of M22 at different time points. Figures 44B-C show the levels of the molecule at different time points. These results indicate that M22 is undetectable 12 hours after administration of variant D3 in a 4-molar excess (molar ratio of molecule:M22) in contrast to the Fc null molecule (Figures 43A-C). Administration of variant E3 in a 4-molar excess significantly slows down M22 clearance compared to variant D3, with some M22 surviving beyond the point observed without molecular administration (vehicle).

[0481] The results of pK experiments in the absence of M22 are shown in Figures 45A-B. When administered intravenously to hFcγR / hFcRn mice, variant E3 (with LS mutation) showed an extended half-life of approximately 18 days compared to variant D3 (without LS mutation), which had a half-life of approximately 14 days. When administered via sc, both variants E3 and D3 showed high levels of bioavailability, reaching serum concentrations similar to those of intravenously administered molecules 48 hours after injection, and subsequently following similar depletion rates.

[0482] Example 8: Additional tests to examine the activity of exemplary molecules This example examines other embodiments of the exemplary molecules described herein.

[0483] Immune complex formation: IC evaluation In this experiment, patient-derived recombinant autoantibodies M22, K1-70, and K1-18 were mixed with variant D3 (and / or variant B3) in various ratios and evaluated for complex formation by HPLC-SEC. All combinations tested between monoclonal autoantibodies and exemplary molecules showed up to two peaks representing 2:1 molecule:autoantibody and 1:1 molecule:autoantibody complexes (data for variant D3 and variant D3:M22 complexes are shown in Figure 46).

[0484] In another experiment, a preliminary study using patient serum samples incubated with the fluorescently labeled variant D3 molecule showed high background due to the endogenous fluorescence of the serum. Nevertheless, similar 2:1 and 1:1 complexes, as well as potentially larger complexes, were observed (Figure 47).

[0485] Measurement of inflammatory cytokine activation To evaluate the activity and immune response to exemplary molecules, human PBMCs were cultured overnight with the molecule:M22 complex in 160 nM molecule:40 nM M22, and levels of IL-6 and MCP-1 in the supernatant were measured by ELISA. The assay results showed that the molecule:M22 complex did not induce secretion of the inflammatory cytokine IL-6, while positive controls (goat IgG immune complex, anti-IgM / G Fab2, and anti-CD3 / CD28 antibody) induced cytokine secretion of IL-6 or MCP-1 (Figures 48A-B, respectively).

[0486] Measurement of monocyte and NK cell activationIn another experiment, human PBMCs were cultured overnight with a 160 nM molecule:40 nM M22 molecule:M22 complex, and cell type activation was measured by flow cytometry. The results of this experiment showed that treatment with variant D3:autoantibody complex did not result in the activation of monocytes or NK cells (Figures 49A and 49B, respectively). Variant B3:autoantibody complex slightly, but in some cases statistically significant, increased activation.

[0487] Overall, the M22 immune complex with variant D3 molecules in a 4:1 ratio (double-capped M22, or one exemplary molecule bound to each Fab of the M22 antibody) did not result in activation in PBMCs when measured by IL-6 or MCP-1 secretion (Figure 48), nor did it result in immune cell activation in the M22 immune complex when measured by activation markers for monocytes, NK cells, B cells, and T cells (Figure 49).

[0488] Measurement of THP-1 immune cell activation In this experiment, THP-1 cells (monocyte cell line) were cultured with a 4:1 ratio of the molecule:M22 immune complex (double-capped M22). Phosphorylation of Syk, downstream of the activated Fc receptor, was measured by flow cytometry. Exemplary molecules, variant B3, variant D3, Fc null molecule, and IgG1 WT Fc were tested. The results are shown in Figures 50A-B. The results indicate that variant D3 does not induce immune cell activation in its immune complex with M22. Specifically, the 4:1 ratio variant D3:M22 immune complex (double-capped M22) does not induce activation of THP-1 cells. THP-1 cells cultured with variant D3:M22 complex exhibited background levels of Syk phosphorylation, while THP-1 cells cultured with variant B3 showed elevated pSyk levels comparable to those activated by large immune complexes ("large ICs") formed by human IgG:goat anti-human IgG complexes (positive control). These results suggest that exemplary molecular variant D3, when conjugated with autoantibodies, may be well-tolerated and not induce significant immune responses.

[0489] Feasibility assessment of exemplary molecules Development potential refers to the likelihood that a biologic candidate has the potential to become a manufacturable, safe, and effective drug. Therefore, assays were performed to evaluate the development potential of exemplary molecules. TSHR variants D3 and E3 were tested using the following assays: AC-SINS to evaluate self-association (Sule et al., Mol.Pharm. 10(4):1322-1331, 2013), BVP-ELISA to evaluate multispecificity (Hotzel et al., MAbs 4(6):753-760, 2012), and DSC to evaluate thermal stability.

[0490] Measurement of self-referential tendencies using AC-SINS AC-SINS (Affinity Capture Self-Interacting Nanoparticle Spectroscopy) can identify antibodies and biologics that exhibit a firm low self-association tendency even at low concentrations (5-50 ug / mL). Briefly, variants D3 and E3 (50 ug / mL in PBS, pH 7.5) were captured on gold nanoparticles, and the plasmon wavelength of the TSHR variant-gold composite was measured at 25°C. This plasmon wavelength redshifts as the distance between particles decreases due to attractive self-interactions. The measured plasmon wavelength shift of variants D3 and E3 showed a 1 nm shift (see Table 16 and Figure 51 below), which indicates a "well-behaving" molecule that shows no tendency toward self-association (unlike the 16 nm plasmon wavelength shift for IgG controls, which are known to self-associate and aggregate). [Table 16]

[0491] The normalization factor is calculated as 1 / (blank sample OD450~OD620), and the baculovirus score is calculated as (250ug / mL sample OD450~OD620) × normalization factor.

[0492] DSC for measuring thermal stability:To evaluate the thermal stability of the TSHR variants, MicroCal® VP-Capillary Differential Scanning Calorimetry (DSC) was used. Briefly, variants D3 and E3 were tested at 1 mg / mL in 20 mM histidine, 150 mM NaCl, pH 6.0. The temperature range started at 10°C and ended at 95°C, with a scan rate of 1.5°C / min. The results show Tm initiation between 48.4–49.3°C, Tm1 between 58.2–59°C, and Tm2 between 77.6–81.9°C (Figures 52A–B).

[0493] Example 9: Inhibition of B cells This example demonstrates the phosphorylation of FcγRIIB in B cells in the presence of an exemplary molecule (variant D3) pre-complexed with M22. More specifically, this example demonstrates the effective crosslinking of the variant D3 / M22 complex with the B cell receptor (BCR) in the presence of a multispecific anti-IgG / IgM F(ab)2 activating reagent. Figure 53 shows the phosphorylation of FcγRIIB when variant D3 is pre-complexed with M22, but not when variant D3 is used as a free drug. While we do not wish to be bound by any theory, it is thought that the phosphorylation of FcγRIIB in B cells requires the simultaneous occurrence of (i) binding of the Fc domain of variant D3 to FcγRIIB, binding of the TSHR antigen domain of variant D3 to the M22 antibody, and (iii) binding of the Fc domain of the M22 antibody to activated anti-IgG / IgM F(ab)2, which itself subsequently binds to IgM or IgG BCR found in purified naive B cells. No phosphorylation was detected when any of the crosslinks were removed. This result also indicates that anti-IgM F(ab)2 alone did not recruit the variant D3 / M22 complex. No phosphorylation was detected when using exemplary molecules with the Fc null mutation (see results with “Fc null” molecules including the LALAPG mutation). Variant B3, containing the high-affinity SE / LF Fc mutation, resulted in FcγRIIB phosphorylation even without complexation with M22.

[0494] method: Purified primary B cells were thawed and resuspended in cold FACS buffer (PBS + 2% FBS) at 1e6 / mL. Equal volumes of purified primary B cells were distributed into 1.5 mL Eppendorf tubes for separate tests, and the cells were maintained on ice. Test molecules, either pre-complexed with M22 (TSHR autoantibody) (pre-mixed in a 4:1 molar ratio) or as free drugs (molecules only), were added to the purified primary B cells at 10 μg / mL, and the cells were incubated at room temperature for 10 minutes. Primary B cells were activated by adding anti-IgM or anti-IgG / IgM Fab(2) at 50 μg / mL and incubated at room temperature for 5 minutes. The cells were rotated at 600 × g for 2 minutes, and then lysed in 15 μL of cold RIPA buffer containing phosphatase and protease inhibitors. The cells were incubated on ice for 30 minutes in RIPA buffer containing phosphatase and protease inhibitors. The lysates were clarified at 10,000 × g at 4°C for 10 minutes. 4 × NuPage LDS sample buffer (+1% beta-mercaptoethanol) was added to each lysate at a rate of 5 μL per 15 μL of protein-containing sample.

[0495] The proteins were boiled at 95°C for 5 minutes using a heat block. The proteins were loaded onto 1.5 mm 15-well 4-12% Trisbis gels and electrophoresed at 200 V for 45 minutes. The gels were transferred to a PVDF membrane at 20 V for 7 minutes using a Thermo iBlot2.

[0496] The membrane was blocked in 5% milk with shaking for 30 minutes at room temperature, cut into separate blots at the 75kD mark, and explored overnight at 4°C in 5% milk with either Raptor (CST, 1:1000) or pCD32b (Abcam, 1:1000) while rotating the stage. The following morning, the blots were shaken in 5% milk at room temperature for 1 hour and detected with anti-rabbit IgG-HRP secondary antibody (1:5,000, Southern Biotechnology). After washing the blots three times with TBST, they were detected using a Pierce chemiluminescence kit and read using iBlot.

[0497] Example 10: Testing and characterization of exemplary molecules Identification of Fc mutations that confer specific binding to FcγRIIB. The exemplary molecules described above were tested for their ability to selectively bind to FcγRIIB via their Fc domains, as well as for whether or not they had reduced or absent binding affinity to other Fc receptors, FcγRIIA167H and FcγRIIA167R.

[0498] Binding assays using SPR were performed to determine the binding affinity of exemplary molecules. Molecules were captured on a CM5 SPR chip. Subsequently, incremental concentrations of FcγR analytes (FcγRIIA167H, FcγRIIA167R, or FcγRIIB) were injected into the captured molecules, and a single dissociation was performed using single-cycle kinetics. The data were then analyzed using steady-state analysis suitable for low-affinity interactions. In particular, plots of the equilibrium response against molecular concentration were generated. The KD value corresponds to the concentration that yields 50% of the maximum response. The molecules shown include control IgG1 (trastuzumab antibody), variants G1, G2, G3, G4, G6, G7, G8, G9, G10, G11, G12, G13, and G14 against various Fc receptors: FcγRIIA167H, FcγRIIA167R, and FcγRIIB. Exemplary molecules containing trastuzumab and the wild-type Fc domain (WT IgG1 Fc) were used as positive controls.

[0499] The results of the binding assays are shown in Table 13 and Figures 54-59 below. Specifically, Figure 54 shows the binding activity of trastuzumab (positive control) (A), variant G1 (B), variant G2 (C), variant G3 (D), variant G6 (E), variant G7 (F), and variant G8 (G) to the activating receptor FcγRIIA167R when exemplary molecules were captured on an SPR sensor chip and FcγR was used as the analyte. Figure 55 shows the binding activity of variant G9 (A), variant G10 (B), variant G11 (C), variant G12 (D), variant G13 (E), variant G14 (F), and variant G4 (G) to the activating receptor FcγRIIA167R when exemplary molecules were captured on an SPR sensor chip and FcγR was used as the analyte.

[0500] Figure 56 shows the binding activity of trastuzumab (positive control) (A), variant G1 (B), variant G2 (C), variant G3 (D), variant G6 (E), variant G7 (F), and variant G8 (G) to the activating receptor FcγRIIA167H when exemplary molecules are captured on an SPR sensor chip and FcγR is used as the analyte. Figure 57 shows the binding activity of variant G9 (A), variant G10 (B), variant G11 (C), variant G12 (D), variant G13 (E), and variant G14 (G) to the activating receptor FcγRIIA167H when exemplary molecules are captured on an SPR sensor chip and FcγR is used as the analyte.

[0501] Figure 58 shows the binding activity of trastuzumab (positive control) (A), variant G1 (B), variant G2 (C), variant G3 (D), variant G6 (E), variant G7 (F), and variant G8 (G) to the inhibitory receptor FcγRIIB when exemplary molecules are captured on an SPR sensor chip and FcγR is used as the analyte. Figure 59 shows the binding activity of variant G9 (A), variant G10 (B), variant G11 (C), variant G12 (D), variant G13 (E), and variant G14 (G) to the inhibitory receptor FcγRIIB when exemplary molecules are captured on an SPR sensor chip and FcγR is used as the analyte.

[0502] Table 13 below shows the kinetic response parameters of exemplary molecules that bind to the activating receptors FcγRIIA167R and FcγRIIA167H, and the inhibitory receptor FcγRIIB, where KD is the dissociation constant, NB indicates non-binding, and UTD indicates that KD could not be determined. [Table 13-1] [Table 13-2]

[0503] Exemplary molecular testing of avidity-mediated effects In this experiment, CHO cells expressing FcγRIIB (CHO-FcγRIIB+) were incubated with and without the 2B6 antibody, an anti-FcγRIIB antibody that blocks the binding of FcγRIIB to the antibody's Fc domain, along with exemplary molecules and M22 (TSHR autoantibody). The exemplary molecules tested included variants D3, B3, G1, G2, G5, G10, G11, G12, G13, and G14.

[0504] Figures 60-61 show that the Fc mutations in variants G1, G2, G5, and G11 result in a similar lack of or very weak binding of free molecules to FcγRIIB, compared to variant D3, which weakly binds to FcγRIIB in its free form only at the highest concentration tested (1 μM). In contrast, the Fc mutations in variants G10, G12, G13, and G14 result in increased molecular concentration-dependent binding to FcγRIIB. Variant G14, in particular, shows a similar binding profile to FcγRIIB compared to variant B3, which strongly binds to FcγRIIB in its free form in a concentration-dependent manner. The assay signal decreases upon pretreatment with anti-FcγRIIB antibody clone 2B6, indicating that the binding of all molecules tested is FcγRIIB-dependent. The Fc mutation D270E reduces the binding affinity to FcγRIIB compared to variants G10 and G12, which are the same molecule without the Fc mutation, as observed in variants G11 and G13. These observations are consistent with surface plasmon resonance (SPR) results, which evaluate the binding of the same molecule to FcγRIIB.

[0505] Figures 62-63 show that, similar to variant D3, the Fc mutations in variants G1, G2, and G5 result in a lack of binding to FcγRIIA167R, while the Fc mutations in variants G10, G11, and G13 show binding only at the highest concentration tested (1 μM). In contrast, the Fc mutations in variants G12 and G14 result in stronger concentration-dependent binding, which in variant G14 is already detectable at the lowest concentration tested (10 nM). The binding profile of variant G14 to FcγRIIA167R is similar to that of variant B3, which binds strongly to FcγRIIA167R. The assay signal decreases upon pretreatment with anti-FcγRIIA antibody clone IV.3, indicating that the binding of all molecules tested is dependent on FcγRIIA167R. The Fc mutation D270E reduces the binding affinity to FcγRIIA167R in association with variant G13 compared to variant G12, which contains the same mutation but lacks D270E. These observations are consistent with surface plasmon resonance (SPR) results evaluating the binding of the same molecule to FcγRIIA167R.

[0506] Equal parts Those skilled in the art will recognize many equivalents to the specific embodiments described herein, or can confirm them by routine experimentation alone. The scope is not intended to be limited to the above description, but rather as set forth in the following claims.

Claims

1. A first polypeptide comprising a first Fc domain and a binding domain that specifically binds to a target antibody; and Second polypeptide containing a second Fc domain A molecule containing, The first Fc domain and the second Fc domain form homodimers or heterodimers of the first polypeptide and the second polypeptide, and the first and / or second Fc domains contain one or more mutant amino acid residues and have increased binding affinity to FcγRIIB compared to the corresponding wild-type Fc domain. The molecules, when bound to the target antibody, form an immune complex having improved binding dynamics with FcγRIIB compared to an immune complex containing the target antibody bound to two corresponding molecules having wild-type Fc domains.

2. The molecule according to claim 1, wherein the second polypeptide further comprises a binding domain that specifically binds to a target antibody, and the molecule is a homodimer.

3. The molecule according to claim 1, wherein the second polypeptide further comprises a binding domain that specifically binds to a target antibody, and the molecule is a heterodimer.

4. The molecule according to claim 1, wherein the second polypeptide does not contain a binding domain that specifically binds to a target antibody, and the molecule is a heterodimer.

5. The molecule according to claim 1, wherein when two molecules bind to the target antibody, an immune complex is formed that has improved binding dynamics with FcγRIIB compared to an immune complex containing the target antibody bound to only one molecule.

6. The molecule according to any one of claims 1 to 5, wherein the improved binding dynamics include an increase in the association rate, a decrease in the dissociation rate, and / or a change in the equilibrium dissociation constant.

7. The molecule according to any one of claims 1 to 6, wherein the improved binding kinetics result in an increase in the avidity, stability, strength, frequency, and / or duration of binding between the immune complex and FcγRIIB.

8. The molecule according to any one of claims 1 to 7, wherein the target antibody is a pathogenic antibody.

9. The molecule according to any one of claims 1 to 8, wherein the target antibody is an autoantibody.

10. The target antibody is a secreted antibody, the molecule according to any one of claims 1 to 9.

11. The molecule according to any one of claims 1 to 8, wherein the target antibody is a membrane-bound antibody or an autoreactive B cell receptor.

12. The molecule according to any one of claims 1 to 11, wherein the first and / or second Fc domains comprising one or more mutant amino acid residues do not show increased binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIIIA176F, FcγRIIIIIA176V, FcγRIIIIB, and / or FcRn compared to the corresponding wild-type Fc domain.

13. The molecule according to any one of claims 1 to 12, wherein the first and / or second Fc domains, which contain one or more mutant amino acid residues, have reduced binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIIIA176F, FcγRIIIIIA176V, FcγRIIIIB, and / or FcRn compared to the corresponding wild-type Fc domain.

14. The molecule according to any one of claims 1 to 13, wherein the first and / or second Fc domains comprising one or more mutant amino acid residues have substantially no binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIIIA176F, FcγRIIIIIA176V, FcγRIIIIB, and / or FcRn compared to the corresponding wild-type Fc domain.

15. The molecule according to any one of claims 1 to 14, wherein the improved binding kinetics include at least a 10% higher binding affinity of the immune complex to FcγRIIB.

16. The molecule according to claim 15, wherein the aforementioned at least 10% higher binding affinity includes a binding affinity of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more.

17. The molecule according to claim 15, wherein the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.001 μM.

18. The molecule according to claim 15, wherein the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.001 μM.

19. The molecule according to claim 15, wherein the molecule binds to FcγRIIB with an affinity in the range of about 0.1 μM to 0.01 μM.

20. The molecule according to claim 15 or 16, wherein the binding affinity includes the binding affinity to a cell line overexpressing FcγRIIB (e.g., CHO cell line) as measured by flow cytometry.

21. The molecule is the molecule according to any one of claims 1 to 20, wherein the molecule does not bind to the complement (C1q).

22. The molecule according to any one of claims 1 to 21, wherein the molecule preferentially binds to immune cells expressing FcγRIIB rather than to immune cells expressing FcγRIIA.

23. The molecule according to claim 22, wherein the molecule has substantially no binding affinity to cells that do not express FcγRIIB.

24. The molecule according to claim 23, wherein the immune cells expressing FcγRIIB include B cells, monocytes and / or basophils.

25. The molecule according to claim 23 or 24, wherein the immune cells that do not express FcγRIIB include T cells, NK cells, neutrophils, and / or eosinophils.

26. The molecule according to any one of claims 1 to 25, wherein the molecule prevents the target antibody from binding to its homogenous antigen.

27. The molecule according to any one of claims 1 to 26, wherein the molecule does not activate immune cells (for example, does not activate immune cells for secreting inflammatory cytokines, such as IL-6).

28. The molecule is the molecule according to any one of claims 1 to 27, which inhibits B cells by crosslinking FcγRIIB with the B cell receptor.

29. The molecule according to claim 28, wherein the molecule crosslinks FcγRIIB with the B cell receptor.

30. The molecule according to claim 28, wherein one or two molecules of an immune complex with an anti-TSHR autoantibody crosslinks FcγRIIB with the B cell receptor.

31. The molecule according to any one of claims 1 to 30, wherein the first Fc domain and the second Fc domain each include an immunoglobulin constant region comprising a CH2 domain and a CH3 domain.

32. The first and / or second Fc domains are, according to the EU numbering scheme, the following amino acid mutations: E233V, L234D, L235F, G236R, G237D, S239L, S267D, H268P, S298G, T299A, A327L, L328A, A330H, E333I, R292Q, E233P, P238D, H The molecule according to claim 31, comprising one or more of 268D, P271G, A330R, L234Y, T250V, V264I, T307P, Q311R, A330K, P343R, M428L, N434A, Y436T, Q438R, S440E, G236N, S267E, L235R, D270E, E233D, and G237D.

33. The first and / or second Fc domain, according to the EU numbering scheme, has the following set of amino acid mutations: (i) E233V, L234D, L235F, G236R, G237D, S239L, S267D, H268P, S298G, T299A, A327L, L328A, A330H, and E333I; (ii) E233V, L234D, L235F, G236R, G237D, S239L, S267D, R292Q, H268P, S298G, T299A, A327L, L328A, A330H, and E333I; (iii) E233V, L234D, L235F, G236R, G237D, S239L, H268P, R292Q, S298G, T299A, A327L, L328A, A330H, and E333I; (iv) E233P, G237D, P238D, H268D, P271G, and A330R; (v) L234Y, P238D, T250V, V264I, T307P, Q311R, A330K, P343R, M428L, N434A, Y436T, Q438R, and S440E; (vi) L234D, G236N, and S267E; (vii) L235R; (viiii) G236N and S267E; (ix) P238D and D270E; (x) P238D and P271G; (xi) P238D, D270E and P271G; (xi) G237D, P238D, P271G, and A330R; (xiiii) G237D, P238D, D270E, P271G, and A330R; (xiv) E233D, G237D, P238D, H268D, P271G, and A330R; and The molecule according to claim 32, comprising one or more of (xv)P238D.

34. The molecule according to any one of claims 1 to 33, wherein the first and / or second Fc domain comprises the mutant amino acid residue P238D according to the EU numbering scheme.

35. The molecule according to any one of claims 1 to 34, wherein the first and / or second Fc domain does not contain the following mutant amino acid residues: S267E and L328F, according to the EU numbering scheme.

36. The molecule according to any one of claims 1 to 35, wherein the first Fc domain comprises a sequence selected from SEQ ID NOs: 103, 105, 107, 109, 111-113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139-149, 163-164, 374-376, 378, or a fragment or variant thereof (for example, a sequence selected from SEQ ID NOs: 107, 109, 113, 115, 119, 131, 139, 140, 142, 148, 374, or 378).

37. The molecule according to any one of claims 1 to 36, wherein the second Fc domain comprises a sequence selected from SEQ ID NOs: 104, 106, 108, 110, 111, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139-149, 163-164, 374-375, 377, 379, or a fragment or variant thereof (for example, a sequence selected from SEQ ID NOs: 108, 110, 114, 116, 120, 132, 139, 140, 142, 148, 374, or 379).

38. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 107, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 108; or (ii) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 108, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

107. The molecule according to any one of claims 1 to 35.

39. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 109, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 110; or (ii) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 110, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

109. The molecule according to any one of claims 1 to 35.

40. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 113, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 114; or (ii) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 114, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

113. The molecule according to any one of claims 1 to 35.

41. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 119, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 120; or (ii) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 120, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

119. The molecule according to any one of claims 1 to 35.

42. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 131, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 132; or (ii) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 132, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

131. The molecule according to any one of claims 1 to 35.

43. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 378, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 379; or (ii) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 379, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

378. The molecule according to any one of claims 1 to 35.

44. The molecule according to any one of claims 1 to 35, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 374, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

374.

45. The molecule according to any one of claims 1 to 44, wherein the binding domain comprises an antigen, or a fragment or variant thereof, to which the target antibody binds.

46. The molecule according to claim 45, wherein the antigen is an autoantigen and the target antibody is an autoantibody.

47. The molecule according to any one of claims 1 to 3 and 5 to 46, wherein the first polypeptide comprises a binding domain containing a first antigen domain, and the second polypeptide further comprises a binding domain containing a second antigen domain.

48. The molecule according to claim 47, wherein the first antigen domain and the second antigen domain are the same.

49. The molecule according to claim 47, wherein the first antigen domain and the second antigen domain are different.

50. The molecule according to any one of claims 47 to 49, wherein the first antigen domain comprises two or more antigen domains, and / or the second antigen domain comprises two or more antigen domains.

51. The molecule according to claim 50, wherein the first and / or second antigen domain comprises two or more (e.g., two, three, four, five, six, or seven) epitopes of the same antigen.

52. The molecule according to claim 50, wherein the first and / or second antigen domain comprises two or more (e.g., two, three, four, five, six, or seven) epitopes of different antigens.

53. The molecule according to any one of claims 1 to 52, wherein the binding domain includes an antibody variable domain, such as Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, sdAb, scFv, SMIP, diabody, triabody, tetrabody, minibody, nanobody, maxibody, tandab, DVD, BiTe, TandAb, VHH, peptide sequence, or mimotope, or any combination thereof.

54. The molecule according to claim 53, wherein the binding domain binds to the Fc domain (e.g., CH2 or CH3) of the target antibody.

55. The molecule according to claim 53 or 54, wherein the antibody variable domain comprises Fab.

56. The molecule according to claim 55, wherein the second polypeptide of the molecule further comprises a second binding domain containing an antibody variable domain.

57. The molecule according to claim 56, wherein the second binding domain is the same as the binding domain of the first polypeptide.

58. The molecule according to claim 57, wherein the second binding domain binds to a different target antibody than the binding domain of the first polypeptide.

59. The molecule according to any one of claims 1 to 58, wherein the first and second Fc domains form a heterodimer as a result of a knob-in-hole (KIH) mutation.

60. The molecule according to claim 59, wherein the KIH mutations include Y349T and T394F according to the EU numbering scheme.

61. The molecule according to claim 60, wherein the first Fc domain contains the Y349T mutation, and the second Fc domain contains the T394F mutation.

62. The molecule according to claim 59, wherein the KIH mutations include T366W, S354C, T366S, L368A, Y407V, and Y349C according to the EU numbering scheme.

63. The molecule according to claim 62, wherein the first Fc domain comprises the T366W and S354C mutations according to the EU numbering scheme, and the second Fc domain comprises the T366S, L368A, Y407V, and Y349C mutations.

64. The molecule according to any one of claims 1 to 63, wherein the first and / or second Fc domain comprises an IgG1 isotype.

65. The molecule according to claim 64, wherein the first and / or second Fc domain comprises a human IgG1 isotype.

66. The molecule according to any one of claims 1 to 65, wherein the first and / or second Fc domain comprises one or more mutant amino acid residues that extend the half-life.

67. The molecule according to claim 66, wherein the first and / or second Fc domain comprises one of the following mutant amino acid residues according to the EU numbering scheme: M252Y, S254T, and T256E.

68. The molecule according to claim 66 or 67, wherein the first and / or second Fc domain comprises, in accordance with the EU numbering scheme, the following combination of mutant amino acid residues: M252Y, S254T, and T256E.

69. The molecule according to claim 66, wherein the first and / or second Fc domain comprises one of the following mutant amino acid residues: M428L and N434S, according to the EU numbering scheme.

70. The molecule according to claim 66 or 69, wherein the first and / or second Fc domain comprises the following combination of mutant amino acid residues: M428L and N434S, in accordance with the EU numbering scheme.

71. The molecule according to any one of claims 1 to 70, wherein the binding domain is covalently bonded to the first Fc domain.

72. The molecule according to claim 71, wherein the C-terminus of the binding domain is covalently bonded to the N-terminus of the first Fc domain.

73. The molecule according to claim 71, wherein the N-terminus of the binding domain is covalently bonded to the C-terminus of the first Fc domain.

74. The molecule according to any one of claims 1 to 73, wherein the binding domain is covalently bonded to the first Fc domain via a linker.

75. The molecule according to claim 74, wherein the linker comprises the amino acid sequence of SEQ ID NO: 150 (GGGGGS), SEQ ID NO: 151 (GGGGGSGGGGGS), SEQ ID NO: 152 (GGGGGSGGGGGGGGGGGS), SEQ ID NO: 153 (VDGGGGGSGGGGGGGGGGGGGS), SEQ ID NO: 154 (GGGGGSGGGGGGGGGGGSGGGGGS), SEQ ID NO: 155 (GGGGGSGGGGGGGGGGGGGGGGGSGGGGGS), SEQ ID NO: 156 (GSGGGS), SEQ ID NO: 157 (GGSG), SEQ ID NO: 158 (GGSG), SEQ ID NO: 159 (GSSGSG), SEQ ID NO: 160 (GSGGGG), SEQ ID NO: 161 (GGGGSG), or SEQ ID NO: 162 (GSSSSG).

76. A nucleic acid comprising a nucleotide sequence encoding the molecule according to any one of claims 1 to 75.

77. A host cell comprising the nucleic acid described in claim 76.

78. A vector comprising the nucleic acid described in claim 76.

79. (i) Target antibody; and (ii) Two molecules An immune complex comprising, each molecule, A first polypeptide comprising a first Fc domain and a binding domain that binds to the target antibody; and Second polypeptide containing a second Fc domain Includes, The first Fc domain and the second Fc domain form homodimers or heterodimers of the first polypeptide and the second polypeptide, and the first and / or second Fc domains contain one or more mutant amino acid residues and have increased binding affinity to FcγRIIB compared to the corresponding wild-type Tc domain. The immune complex exhibits improved binding dynamics with FcγRIIB compared to an immune complex comprising the target antibody bound to two corresponding molecules having wild-type Fc domains.

80. The immune complex according to claim 79, wherein the immune complex has improved binding dynamics with FcγRIIB compared to an immune complex comprising only the target antibody and one molecule.

81. The immune complex according to claim 79, wherein the immune complex has improved binding dynamics with FcγRIIB compared to an immune complex comprising only the target antibody and one molecule.

82. The immune complex according to claim 79, wherein the immune complex has improved binding dynamics with FcγRIIB compared to the anti-TSHR autoantibody alone.

83. The immune complex according to any one of claims 79 to 82, wherein the binding domain of each of the two molecules is bound to the target antibody.

84. The improved binding dynamics include an increase in the association rate, a decrease in the dissociation rate, and / or a change in the equilibrium dissociation constant, according to any one of claims 79 to 83.

85. The immune complex according to any one of claims 79 to 84, wherein the improved binding dynamics result in an increase in the avidity, stability, strength, frequency, and / or duration of binding between the immune complex and FcγRIIB.

86. The immune complex according to any one of claims 79 to 85, wherein the target antibody is a pathogenic antibody.

87. The immune complex according to any one of claims 79 to 86, wherein the target antibody is an autoantibody.

88. The immune complex according to any one of claims 79 to 87, wherein the target antibody is a secreted antibody.

89. The immune complex according to any one of claims 79 to 86, wherein the target antibody is a membrane-bound antibody or an autoreactive B cell receptor.

90. The immunocomplex according to any one of claims 79 to 89, wherein the first and / or second Fc domains comprising one or more mutant amino acid residues do not show increased binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIA176F, FcγRIIA176V, FcγRIIB, and / or FcRn compared to the corresponding wild-type Fc domain.

91. The immunocomplex according to any one of claims 79 to 90, wherein the first and / or second Fc domains, each containing one or more mutant amino acid residues, have reduced binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIA176F, FcγRIIA176V, FcγRIIB, and / or FcRn compared to the corresponding wild-type Fc domain.

92. The immunocomplex according to any one of claims 79 to 91, wherein the first and / or second Fc domains comprising one or more mutant amino acid residues have negligible or no binding affinity to FcγRI, FcγRIIA167H, FcγRIIA167R, FcγRIIIIIA176F, FcγRIIIIIA176V, FcγRIIIIB, and / or FcRn compared to the corresponding wild-type Fc domain.

93. The immune complex according to any one of claims 79 to 92, wherein the improved binding dynamics include at least a 10% higher binding affinity of the immune complex to FcγRIIB.

94. The immunocomplex according to claim 93, wherein the binding affinity of at least 10% higher includes a binding affinity of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more.

95. The immunocomplex according to claim 94, wherein the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.001 μM.

96. The immunocomplex according to claim 94, wherein the molecule binds to FcγRIIB with an affinity in the range of about 1 μM to 0.001 μM.

97. The immunocomplex according to claim 94, wherein the molecule binds to FcγRIIB with an affinity in the range of about 0.1 μM to 0.01 μM.

98. The immune complex according to claim 93 or 94, wherein the binding affinity includes the binding affinity to a cell line overexpressing FcγRIIB (e.g., a CHO cell line) as measured by flow cytometry.

99. The immune complex according to any one of claims 79 to 98, wherein the immune complex preferentially binds to immune cells expressing FcγRIIB rather than to immune cells expressing FcγRIIA.

100. The immune complex according to any one of claims 79 to 99, wherein the immune complex crosslinks FcγRIIB with the B cell receptor on a B cell.

101. The immunocomplex according to any one of claims 79 to 100, wherein the molecule is the molecule described in any one of claims 1 to 61.

102. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 75 or a nucleic acid encoding a molecule according to any one of claims 1 to 75, and a pharmaceutically acceptable carrier.

103. A method for generating a molecule, comprising expressing the nucleic acid described in claim 76 in a host cell and recovering the molecule.

104. A method for reducing the antibody titer of a circulating target antibody in a subject diagnosed with an autoimmune disease, The method comprising administering the pharmaceutical composition according to claim 102 to the subject, wherein the target antibody is a circulating pathogenic antibody.

105. The method according to claim 104, wherein the antibody titer decreases within less than one hour (for example, less than 30 minutes) after administration of the pharmaceutical composition.

106. The method according to claim 105, wherein the antibody titer in the subject or a biological sample from the subject after administration is lower than that before administration.

107. A method of treating a person who has an autoimmune disease or is susceptible to it, The method comprising administering to the subject a pharmaceutical composition containing a molecule according to any one of claims 1 to 75 or a nucleic acid encoding a molecule according to any one of claims 1 to 75.

108. The method according to claim 107, wherein the autoimmune disease is related to the target antibody, and the target antibody is a pathogenic autoantibody targeted by the binding domain of the molecule.

109. The method according to claim 108, wherein the antibody titer of the circulating pathogenic autoantibody decreases within less than one hour (for example, less than 30 minutes) after administering the pharmaceutical composition to the subject.

110. The method according to any one of claims 107 to 109, wherein the antibody titer in the subject or a biological sample from the subject after administration is lower than before administration.

111. The method according to claim 109 or 110, wherein the antibody titer in or from the subject is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the antibody titer before administration.

112. The method according to any one of claims 109 to 111, wherein the decrease in antibody titer is sustained over a long period of time.

113. The method according to claim 112, wherein the duration includes at least one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, ten weeks, twelve weeks, or more.

114. The method according to any one of claims 109 to 113, wherein the circulating pathogenic autoantibody is removed from the subject within 30 minutes after administering the pharmaceutical composition to the subject.

115. The method according to any one of claims 109 to 114, wherein the molecule in the pharmaceutical composition neutralizes the circulating pathogenic autoantibody in the target.

116. The method according to any one of claims 109 to 115, wherein at least two molecules in the pharmaceutical composition form an immune complex with the circulating pathogenic autoantibody when the binding domains of the molecules bind to the circulating pathogenic autoantibody.

117. The method according to claim 116, wherein the circulating pathogenic autoantibody is removed from the target by FcγRIIB-mediated intracellular uptake of the immune complex by B cells expressing FcγRIIB.

118. The method according to claim 116 or 117, wherein the circulating pathogenic autoantibodies are removed from the target by FcγRIIB-mediated intracellular uptake of the immune complex by hepatic sinusoidal endothelial cells expressing FcγRIIB.

119. The method according to any one of claims 104 to 118, wherein when the pharmaceutical composition is administered to the subject, it reduces the pathogenic autoantibody stimulating activity in the serum of the subject.

120. The method according to any one of claims 104 to 119, wherein the pharmaceutical composition is administered intravenously, intramuscularly, or subcutaneously to the subject.

121. The method according to any one of claims 104 to 120, wherein the subject is a human.

122. A composition for reducing the titer of a target antibody in the serum of a subject requiring a reduction in the titer of the target antibody in the serum, wherein the composition is The present invention comprises multiple molecules, each molecule comprising (a) a first polypeptide comprising a first Fc domain and a binding domain that specifically binds to a target antibody; and (b) a second polypeptide comprising a second Fc domain, wherein the first Fc domain and the second Fc domain form homodimers or heterodimers of the first polypeptide and the second polypeptide. The first and / or second Fc domains contain one or more mutant amino acid residues and have increased binding affinity to FcγRIIB compared to the corresponding wild-type Fc domains. The composition, wherein, upon administration of the plurality of molecules, the molecules bind to the target antibody to form an immune complex comprising two molecules bound to the target antibody, and the immune complex binds to FcγRIIB expressed on the surface of hepatic sinusoidal endothelial cells (LSEC) with higher avidity compared to an immune complex comprising two corresponding molecules having wild-type Fc domains, undergoes endocytosis, thereby reducing the titer of the target antibody in the serum of the subject.

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