IG-like fusion protein for treating Graves' disease

Abstract

A composition is provided comprising a first polypeptide comprising a first fragment and a dimerization domain of the N-terminal extracellular domain of TSHR or an analog or derivative thereof, and a second polypeptide comprising a second fragment and a dimerization domain of the N-terminal extracellular domain of TSHR or an analog or derivative thereof. A polypeptide comprising a fragment of the N-terminal extracellular domain of TSHR comprising at least one mutation that increases solubility, decreases aggregation, or both is also provided. A pharmaceutical composition comprising a composition, a polypeptide, a nucleic acid system and molecules encoding the composition and the polypeptide of the present invention, as well as a method of treatment using the composition or polypeptide and a method for determining suitability for treatment, and a method for producing the composition or protein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 464,673, filed on May 8, 2023, and U.S. Provisional Patent Application No. 63 / 609,409, filed on December 13, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (CNPY - P - 006 - PCT.xml, size: 112,853 bytes, creation date: May 6, 2024) is hereby incorporated by reference in its entirety.

[0003] The present invention is in the field of fusion protein production and the treatment of Graves' disease (GD).

Background Art

[0004] Graves' disease is an autoimmune disease that mainly affects the thyroid gland and presents as hyperthyroidism, diffuse goiter, thyroid eye disease (Graves' orbitopathy) (GO), and in some cases, a dermopathy called pretibial or localized myxedema (PTM). Graves' disease is the most common cause of hyperthyroidism (60 - ~80% of all cases), occurs at all ages, is most common in people aged 20 - 50 years, and is more common in women. In addition, there are strong genetic background components associated with Graves' disease, as well as environmental factors including pregnancy (mainly postpartum), iodine excess, infection, emotional stress, and smoking.

[0005] Autoantibodies, mainly thyroid - stimulating immunoglobulin (TSI), also known as thyroid - stimulating hormone receptor antibody (TRAb), bind to the thyroid - stimulating hormone receptor (TSHR) on the thyroid cell membrane, stimulate the action of thyroid - stimulating hormone (TSH), result in both thyroid hormone synthesis and thyroid growth, and ultimately cause hyperthyroidism.

[0006] Symptoms of hyperthyroidism may include insomnia, hand tremors, hyperactivity, alopecia, excessive sweating, heat intolerance, and weight loss despite increased appetite. Further signs, most commonly, include diffusely enlarged, non-tender thyroid gland, delayed eyelid movement, excessive lacrimation (due to GO), cardiac arrhythmias, and hypertension. Patients with thyroid toxicity may experience behavioral and personality changes such as psychosis, agitation, and depression. In mild hyperthyroidism, patients may experience less obvious signs, such as anxiety, restlessness, irritability, and emotional instability.

[0007] Currently, there is no available cure for Graves' disease, and therefore, current treatment is directed towards targeting the symptoms presented. Initially, symptomatic patients with cardiac lesions, especially those with a heart rate exceeding 90 beats / min, those with a history of cardiovascular disease, and elderly patients should be initiated with beta-adrenergic blockers such as atenolol. There are three main treatment methods to reduce thyroid hormone synthesis: oral antithyroid drugs (ATDs) / thionamide, radioactive iodine (RAI), and thyroidectomy. The latter two approaches ultimately lead to hypothyroidism in patients, resulting in lifelong thyroid hormone replacement.

[0008] The primary goal of ATD treatment is to achieve normalization of thyroid hormone production and induce remission of the disease, and in some cases, to prepare the patient for radioactive iodine ablation or surgery. ATD therapies such as methimazole (MMI) or its derivative called carbimazole, and propylthiouracil (PTU) block thyroid hormone synthesis and are associated with several rare side effects such as agranulocytosis, hepatotoxicity, and pancreatitis. Because ATD controls symptoms but does not cure the disease, relapses are common, and the remission rate is about 50-60%.

[0009] Because the success of each treatment option varies, patients are often subjected to two or more approaches if the first treatment attempt does not prove to be completely successful. The risk of relapse or subsequent hypothyroidism is considerable, and the general effectiveness of the treatments available for Graves' disease is lower than desired. As a result, there is an urgent need for alternative and safe therapies that provide sufficient and long-lasting effects. Specifically, there is a great unmet need for novel therapies that target the TSH autoantibodies that cause Graves' disease. In addition, there is a great need for drugs that can directly target the autoreactive B cells / plasma cells that are the source of these autoantibodies, and thus potentially offer a cure for the condition.

[0010] Currently, there are no treatments that target the causes of GD, such as autoreactive antibodies or antibody-producing B cells. Improved treatment approaches that target the mechanistic causes of GD are greatly needed. [Overview of the project]

[0011] The present invention provides polypeptides comprising fragments of the extracellular domain of a thyroid-stimulating hormone receptor (TSHR) containing at least one mutation that increases solubility, reduces aggregation, or both. The present invention further provides compositions comprising fragments of a first human receptor target of a Graves' disease (GD) autoantibody and fragments of a second human receptor target of a GD autoantibody. Polypeptides and compositions further comprising effector moieties that are not unmodified Fc domains are also provided. Methods for treating GD by administering the pharmaceutical compositions of the present invention are also provided, as are nucleic acid molecules and systems encoding the polypeptides and compositions of the present invention, methods for producing those polypeptides and compositions, and methods for determining suitability for treatment by the methods of the present invention.

[0012] According to a first embodiment, a polypeptide is provided comprising a fragment of the N-terminal extracellular domain of a thyroid-stimulating hormone receptor (TSHR) and at least one mutation that increases the solubility of the fragment, decreases the aggregation of the fragment, or both.

[0013] In another embodiment, a polypeptide of the present invention including an effector portion is provided. In some embodiments, the effector portion is an effector portion that is not an unmodified Fc domain.

[0014] According to some embodiments, the polypeptide comprises a deletion of the C-peptide region of the N-terminal extracellular domain of TSHR.

[0015] According to some embodiments, the N-terminal extracellular domain of TSHR contains the amino acid sequence provided in SEQ ID NO: 1.

[0016] According to some embodiments, the mutation is the deletion of amino acids 297-346 in SEQ ID NO: 1.

[0017] According to some embodiments, the polypeptide includes or consists of SEQ ID NO: 3.

[0018] According to some embodiments, the mutation is the deletion of amino acids 297-393 in SEQ ID NO: 1.

[0019] According to some embodiments, the polypeptide comprises or consists of Sequence ID No. 83.

[0020] According to some embodiments, the mutation is the deletion of amino acids 261-393 in SEQ ID NO: 1.

[0021] According to some embodiments, the polypeptide comprises or consists of Sequence ID No. 84.

[0022] According to some embodiments, the mutation is a deletion of amino acids 242-393 in SEQ ID NO: 1.

[0023] According to some embodiments, the polypeptide comprises or consists of SEQ ID NO: 85.

[0024] According to some embodiments, the polypeptide further comprises at least one mutation in the ligand binding domain of TSHR, and the mutation reduces the binding of TSHR in the N-terminal extracellular domain to TSH.

[0025] According to some embodiments, the mutation is a mutation of K163, E231 or both in SEQ ID NO: 1.

[0026] According to some embodiments, K163 is mutated to alanine, E231 is mutated to lysine, or both.

[0027] According to some embodiments, the polypeptide comprises or consists of a sequence selected from SEQ ID NOs: 86-88.

[0028] According to some embodiments, the polypeptide further comprises an effector portion.

[0029] According to some embodiments, the effector portion comprises the Fc domain of the human antibody heavy chain.

[0030] According to some embodiments, the polypeptide comprises or consists of a sequence selected from SEQ ID NOs: 6, 9-13, and 71-82.

[0031] According to some embodiments, the effector portion is not the Fc domain.

[0032] According to some embodiments, the effector portion comprises an Fc domain comprising at least one mutation that increases antibody-dependent cell-mediated cytotoxicity (ADCC).

[0033] According to some embodiments, the effector portion can induce cell death by binding to the fragment.

[0034] According to some embodiments, the effector portion is selected from an Fc domain containing at least one mutation that increases ADCC, amatoxin / amanitin, anthracycline, anthramycin-based dimer, calicheamycin, camptothecin or its analogues, duocalmycin, triptolide, and tubulin inhibitors.

[0035] According to some embodiments, the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0036] According to some embodiments, the effector portion contains tesirin. According to some embodiments, the effector portion is tesirin.

[0037] According to some embodiments, the effector portion includes an Fc domain containing SEQ ID NO: 63 or SEQ ID NO: 65, the Fc domain containing multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 63 or SEQ ID NO: 65.

[0038] According to some embodiments, the effector portion is conjugated into a polypeptide by a linker.

[0039] According to another embodiment, a. A first polypeptide comprising a fragment of the N-terminal extracellular domain of a thyroid-stimulating hormone receptor (TSHR) or an analog or derivative thereof, and a first dimerization domain, b. A second polypeptide comprising a fragment of the N-terminal extracellular domain of TSHR or an analog or derivative thereof and a second dimerization domain, A composition is provided which includes a first and second dimerization domain configured to dimerize with respect to each other.

[0040] According to some embodiments, dimerization involves forming a covalent bond between a first dimerization domain and a second dimerization domain.

[0041] According to some embodiments, the protein complex includes an immunoglobulin scaffold.

[0042] According to some embodiments, a. The first dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the second dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains dimerize by disulfide bonds, or b. The first and second dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, and neither the first nor the second dimerization domain contains a CH1 domain or a CL domain.

[0043] According to some embodiments, fragments and dimerization domains of the first, second, or both polypeptide chains are separated by a linker.

[0044] According to some embodiments, the first polypeptide chain, the second polypeptide chain, or both further comprise the Fc region of a human antibody heavy chain.

[0045] According to some embodiments, the first dimerization domain and the second dimerization domain each comprise the Fc region of a human antibody heavy chain.

[0046] According to some embodiments, the Fc region can induce cytotoxicity against cells that bind to the protein complex.

[0047] According to some embodiments, the first polypeptide chain comprises a first CH3 domain of the immunoglobulin heavy chain, a first CH2 domain of the immunoglobulin heavy chain, or both, and the second polypeptide chain comprises a second CH3 domain of the immunoglobulin heavy chain, a second CH2 domain of the immunoglobulin heavy chain, or both.

[0048] According to some embodiments, the first CH3 domain comprises at least a first mutation, and the second CH3 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

[0049] According to some embodiments, the first CH2 domain comprises at least a first mutation, and the second CH2 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

[0050] According to some embodiments, the first mutation is selected from the mutations provided in Table 1, and the second mutation is a mutation provided in Table 1 that corresponds to the first mutation.

[0051] According to some embodiments, the first mutation is the T366W mutation within the CH3 domain, and the second mutation is a combination of the T366S mutation, the L368A mutation, and the Y407V mutation.

[0052] According to some embodiments, the Fc region of the first, second, or both polypeptide chains is separated from the fragment or dimerization domain by a linker.

[0053] According to some embodiments, the Fc region includes at least one mutation that increases ADCC or CDC.

[0054] According to some embodiments, the Fc region is an Fc region containing SEQ ID NO: 63 or SEQ ID NO: 65, and includes a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E and G16A / S47E / H48F / S104T / I112E within SEQ ID NO: 63 or SEQ ID NO: 65.

[0055] According to some embodiments, the dimerization domain of the first, second, or both polypeptide chains is C-terminal to the fragment and N-terminal to the Fc region.

[0056] According to some embodiments, the composition lacks an antibody-variable domain.

[0057] According to some embodiments, the first polypeptide chain and the second polypeptide chain each contain or consist of the polypeptide of the present invention.

[0058] According to some embodiments, the composition further comprises a third polypeptide comprising a fragment of TSHR or an analog or derivative thereof and a third dimerizing domain, the first polypeptide further comprising a fourth dimerizing domain, and the third and fourth dimerizing domains can be dimerized with respect to each other.

[0059] According to some embodiments, a. The third dimerization domain includes the first hinge domain of the immunoglobulin heavy chain, the fourth dimerization domain includes the second hinge domain of the heavy chain, and the first and second dimerization domains dimerize by disulfide bonds, or b. The third and fourth dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, with neither the first nor the third polypeptide containing a CH1 domain or a CL domain.

[0060] According to some embodiments, the composition further comprises a fourth polypeptide comprising a fragment of TSHR or an analog or derivative thereof and a fifth dimerization domain, the second polypeptide further comprising a sixth dimerization domain, and the fifth and sixth dimerization domains can be dimerized with respect to each other.

[0061] According to some embodiments, a. The fifth dimerization domain includes the first hinge domain of the immunoglobulin heavy chain, the sixth dimerization domain includes the second hinge domain of the heavy chain, and the first and second dimerization domains dimerize by disulfide bonds, or b. The fifth and sixth dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, with neither the first nor the third polypeptide containing a CH1 domain or a CL domain.

[0062] According to some embodiments, the first polypeptide and the second polypeptide are either both CH1 domain-free, or both CL domain-free.

[0063] According to some embodiments, the third and fourth dimerization domains or the fifth and sixth dimerization domains include mutations that enable dimerization of the third and fourth dimerization domains as well as dimerization of the fifth and sixth dimerization domains, and that inhibit dimerization of the third dimerization domain to the fifth or sixth dimerization domain and dimerization of the sixth dimerization domain to the third or fourth dimerization domain.

[0064] According to some embodiments, the fragment is a polypeptide of the present invention.

[0065] According to some embodiments, the first polypeptide chain and the second polypeptide chain comprise different TSHR fragments.

[0066] According to some embodiments, TSHR lacks its endogenous signal peptide and contains a heterologous signal peptide.

[0067] According to some embodiments, at least one of the fragments includes a mutation that increases the solubility of the composition, decreases the aggregation of the composition, or both.

[0068] According to some embodiments, at least one of the fragments comprises a mutation in the ligand-binding domain of TSHR, the mutation reducing the binding of the N-terminal extracellular domain of TSHR to TSH.

[0069] According to some embodiments, the analog or its derivative contains at least 85% identity with TSHR.

[0070] According to some embodiments, the fragment comprises at least 20 consecutive amino acid TSHRs.

[0071] According to some embodiments, the fragment comprises at least one B cell receptor (BCR)-specific epitope target of the autoantibody.

[0072] According to some embodiments, the composition comprises a first or second polypeptide containing a sequence selected from SEQ ID NOs: 6, 9-13 and 71-82.

[0073] According to some embodiments, the hinge domain, CH2 domain, or CH3 domain contains at least one mutation that increases or decreases antibody-dependent cell-mediated cytotoxicity (ADCC).

[0074] According to some embodiments, at least one mutation that reduces ADCC is a. Hinge domain mutations including L19A and L20A mutations in Sequence ID No. 22, and b. Mutations in the CH2 domain, including the N59A mutation in Sequence ID No. 36. Selected from.

[0075] According to some embodiments, the composition further comprises at least one effector portion capable of inducing cell death of cells bound to the composition.

[0076] According to some embodiments, the effects portion is not in the Fc domain.

[0077] According to some embodiments, the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0078] According to some embodiments, the effector portion contains tesirin. According to some embodiments, the effector portion is tesirin.

[0079] In another embodiment, a pharmaceutical composition is provided comprising a polypeptide or composition of the present invention and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0080] In another embodiment, a method is provided for treating Graves' disease (GD) in a subject requiring treatment for GD, comprising administering a composition comprising a fragment of the N-terminal extracellular domain of TSHR or an analog or derivative thereof to the subject, thereby treating GD.

[0081] According to some embodiments, TSHR may lack a signal peptide and contain or consist of SEQ ID NO: 1, or TSHR may contain a signal peptide and contain or consist of SEQ ID NO: 2, or both.

[0082] According to some embodiments, the composition comprises the polypeptide of the present invention.

[0083] According to some embodiments, the composition is the composition of the present invention.

[0084] According to some embodiments, the composition is the pharmaceutical composition of the present invention.

[0085] According to some embodiments, the method further includes reducing the level of circulating antibodies against TSHR in a subject.

[0086] According to some embodiments, the treatment includes reducing the concentration of circulating autoantibodies against TSHR.

[0087] According to some embodiments, the composition comprises an Fc region, and treatment involves killing B cells that produce anti-TSHR autoantibodies.

[0088] According to some embodiments, the B cells are autoreactive B cells that produce autoantibodies against fragments of the composition.

[0089] In another embodiment, nucleic acid molecules encoding the polypeptide of the present invention are provided.

[0090] In another embodiment, a nucleic acid system is provided, comprising nucleic acid molecules, wherein a first nucleic acid molecule encodes a first polypeptide of the composition of the present invention, and a second nucleic acid molecule encodes a second polypeptide of the composition of the present invention.

[0091] According to some embodiments, the nucleic acid system further comprises a third nucleic acid molecule encoding a third polypeptide of the composition of the present invention, a fourth nucleic acid molecule encoding a fourth polypeptide of the composition of the present invention, or both.

[0092] In another aspect, a method is provided for producing a polypeptide or composition of the present invention, comprising expressing a nucleic acid molecule or nucleic acid system of the present invention in a cell, wherein the nucleic acid system is configured to produce a polypeptide encoded in the cell, thereby producing a polypeptide or composition of the present invention.

[0093] In another embodiment, a method for producing a protein, To obtain a first fragment of the extracellular domain of TSHR or an analog or derivative thereof, and a second fragment of the extracellular domain of TSHR or an analog or derivative thereof, The first fragment is linked to the first dimerization domain to produce the first polypeptide chain, the second fragment is linked to the second dimerization domain to produce the second polypeptide chain, the first and second dimerization domains are capable of dimerizing with each other, and the first polypeptide and the second polypeptide are brought into contact under conditions sufficient to induce dimerization, or The method involves culturing host cells containing one or more vectors comprising nucleic acid sequences encoding at least two polypeptide chains, wherein the two polypeptide chains are i. Obtain a first fragment of the extracellular domain of TSHR or an analog or derivative thereof, and a second fragment of the extracellular domain of TSHR or an analog or derivative thereof, and ii. To produce a first polypeptide chain by linking a first fragment to a first dimerization domain, and to produce a second polypeptide chain by linking a second fragment to a second dimerization domain, wherein the first and second dimerization domains are capable of dimerizing with each other. A method is provided for producing a protein, which involves culturing, and thereby producing a protein.

[0094] According to some embodiments, the protein complex is the protein complex of the present invention.

[0095] According to some embodiments, this method a. Linking a third dimerization domain to the first dimerization domain or first fragment within the first polypeptide chain to obtain a third fragment of the extracellular domain of TSHR or its analogue or derivative; linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain; the third dimerization domain and the fourth dimerization domain being able to dimerize with each other; and contacting the first, second, and third polypeptides under conditions sufficient to induce dimerization, or b. In host cells, i. To obtain a third fragment of TSHR or an analog or derivative thereof, and ii. Linking the third fragment to the fourth dimerization domain to produce a third polypeptide chain. The further comprising expressing a nucleic acid sequence encoding a third polypeptide chain produced by, The first polypeptide chain further comprises a third dimerization domain, and the third and fourth dimerization domains can dimerize with each other.

[0096] According to some embodiments, this method a. Linking the sixth dimerization domain to the second dimerization domain or second fragment within the second polypeptide chain to obtain a fourth fragment of the extracellular domain of TSHR or its analogue or derivative, linking the fourth fragment to the fifth dimerization domain to produce a fourth polypeptide chain, wherein the fifth dimerization domain and the sixth dimerization domain can dimerize with each other, and the first, second, third and fourth polypeptides are brought into contact under conditions sufficient to induce dimerization, or b. In host cells, i. Obtain a fourth fragment of the extracellular domain of TSHR or an analog or derivative thereof, and ii. Linking the fourth fragment to the fifth dimerization domain to produce the fourth polypeptide chain. The further comprising expressing a nucleic acid sequence encoding a fourth polypeptide chain produced by, The second polypeptide chain further comprises a sixth dimerization domain, and the fifth and sixth dimerization domains can dimerize with each other.

[0097] According to some embodiments, the method further includes inducing at least one mutation in the extracellular domain of a protein, or shortening the protein and removing a portion thereof.

[0098] According to some embodiments, the method further includes measuring the aggregation of mutant or truncated proteins and selecting mutant or truncated proteins with reduced aggregation.

[0099] In another embodiment, a method for producing a protein, a. Obtaining a fragment of the extracellular domain of TSHR or its analogue or derivative, b. Producing a mutant fragment by generating at least one mutation in the fragment, c. Measuring the solubility, aggregation, or both of the mutant fragments. d. Select at least one mutant fragment that increases solubility, decreases aggregation, or both, compared to the obtained fragment. A method is provided that includes and thereby produces a protein.

[0100] According to some embodiments, the method further comprises ligating an effector portion to at least one polypeptide chain or mutant fragment, the effector portion being able to kill cells bound to at least one polypeptide.

[0101] According to some embodiments, the effects portion is not in the Fc domain.

[0102] According to some embodiments, the effector portion includes the Fc domain of the antibody heavy chain.

[0103] According to some embodiments, the effector portion comprises a molecule selected from an Fc domain containing at least one mutation that increases ADCC or CDC, alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0104] According to some embodiments, the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0105] According to some embodiments, the effector portion is an Fc domain containing at least one mutation that increases ADCC or CDC.

[0106] According to some embodiments, the effector portion is an Fc domain containing SEQ ID NO: 63 or SEQ ID NO: 65, and the Fc domain contains multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 63 or SEQ ID NO: 65.

[0107] In another embodiment, a protein produced by the method of the present invention is provided.

[0108] In another aspect, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising: receiving a sample from the object; contacting the sample with the protein or composition of the present invention; and determining the binding of autoantibodies in the sample to the protein or composition, wherein the binding of autoantibodies to the protein or composition indicates that the object is suitable to be treated by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0109] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given merely as examples, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0110] [Figure 1A] This figure shows one possible embodiment of the quadruple-chain therapeutic agent of the present invention, and is a diagram showing a general embodiment of the molecule for treating GD. [Figure 1B] This figure shows one possible embodiment of the quadruple-chain therapeutic agent of the present invention, wherein each of the four chains comprises a different protein fragment. [Figure 1C] This figure shows one possible embodiment of the quadruple-chain therapeutic agent of the present invention, in which all four protein fragments are identical. [Figure 1D] This figure shows one possible embodiment of the quadruple-chain therapeutic agent of the present invention, wherein two heavy chains and two light chains are identical. [Figure 1E] This figure shows one possible embodiment of the quadruple-chain therapeutic agent of the present invention, wherein two heavy chains are different and two light chains are identical. [Figure 1F]This figure shows one possible embodiment of the quadruple-chain therapeutic agent of the present invention, in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments.

[0111] [Figure 2A-1] A diagram of possible embodiments of the double-chain therapeutic agent of the present invention, showing a general embodiment of a molecule having two heavy chains for treating GD. [Figure 2A-2] Same as above. [Figure 2B-1] This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, in which at least one of the CH1, CH2, or CH3 domains is excluded. [Figure 2B-2] Same as above. [Figure 2B-3] Same as above. [Figure 2B-4] Same as above. [Figure 2B-5] Same as above. [Figure 2B-6] Same as above. [Figure 2C] This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, wherein the two protein fragments are identical. [Figure 2D] This figure shows possible embodiments of the double-stranded therapeutic agent of the present invention, where two protein fragments represent different embodiments. [Figure 2E] This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, in which the two protein fragments are different and the molecule does not contain a CH1 domain. [Figure 2F] This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, in which the two protein fragments are different and the molecule does not contain a CH1 domain or a hinge domain. [Figure 2G] This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, in which a general embodiment is shown in which two tandem fragments are contained in each heavy chain and linked via a linker. [Figure 2H]This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, which has a tandem fragment configuration in which all subunits are the same. [Figure 2I] This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, which is a tandem fragment configuration in which all subunits are identical without the CH1 domain. [Figure 2J] This figure shows a possible embodiment of the double-chain therapeutic agent of the present invention, which is a tandem fragment configuration in which each heavy chain contains two identical different fragments. [Figure 2K] This figure shows a possible embodiment of the double-chain therapeutic agent of the present invention, in which each heavy chain contains two identical different fragments without a CH1 domain, representing a tandem fragment configuration. [Figure 2L] This figure shows a possible embodiment of the double-chain therapeutic agent of the present invention, in which the two heavy chains have a tandem fragment configuration containing different fragments that are not the same. [Figure 2M] This figure shows a possible embodiment of the double-stranded therapeutic agent of the present invention, which comprises a tandem fragment configuration in which two heavy chains contain different fragments that are not identical and lack a CH1 domain. [Figure 2N] A diagram illustrating possible embodiments of the double-chain therapeutic agent of the present invention, showing a general embodiment of a molecule having one heavy chain and one light chain.

[0112] [Figure 3A] A diagram of one possible embodiment of the triple-chain therapeutic agent of the present invention, showing a general embodiment of a molecule having two heavy chains and one light chain. [Figure 3B] This figure shows one possible embodiment of the triple-stranded therapeutic agent of the present invention, wherein the three protein fragments are identical. [Figure 3C] This figure shows one possible embodiment of the triple-stranded therapeutic agent of the present invention, in which each protein fragment represents a different embodiment. [Figure 3D] This figure shows one possible embodiment of the triple-stranded therapeutic agent of the present invention, in which two of the protein fragments are the same and the third is different.

[0113] [Figure 4] This figure shows an embodiment of the quadruple-stranded therapeutic agent for treating GD of the present invention, similar to that shown in Figure 1, but comprising four different immunoglobulin scaffolds in which each of the four chains promotes the formation of different protein fragments and quadruple-stranded molecules.

[0114] [Figure 5A] This figure shows a general embodiment of the quadruple-chain therapeutic agent of the present invention, which is a general embodiment of four chains in which two chains contain two dimerization domains and two chains contain a single dimerization domain. [Figure 5B] This figure shows a general embodiment of the quadruple-chain therapeutic agent of the present invention, which has an optional linker for separating various domains and fragments.

[0115] [Figure 6A] This figure shows an embodiment of a single-chain therapeutic agent of the present invention, comprising a single-chain molecule containing a fragment from TSHR. [Figure 6B] This figure shows a single-chain therapeutic agent of the present invention, and is a diagram of a fragment embodiment including shortening of TSHR. [Figure 6C] This figure shows an embodiment of a single-chain molecule containing three different fragments, representing a single-chain therapeutic agent of the present invention. [Figure 6D] This figure shows an embodiment of a single-chain molecule containing four different fragments, representing a single-chain therapeutic agent of the present invention. [Figure 6E] This figure shows an embodiment of a single-chain molecule containing a fragment and a heavy chain constant region, representing a single-chain therapeutic agent of the present invention. [Figure 6F] This figure shows an embodiment of a single-chain molecule containing a fragment and a CH3-CH2 fragment of the heavy chain constant region, representing a single-chain therapeutic agent of the present invention. [Figure 6G-1] This figure shows a single-chain therapeutic agent of the present invention, and illustrates single-chain molecules 6A and 6C-6F having amino acid (AA) linkers that separate various domains. [Figure 6G-2] Same as above.

[0116] [Figure 7A] This is a photograph of an SDS-PAGE gel showing the molecule under reducing conditions, and it shows CRD-238, not a fusion with IgG. [Figure 7B] This is a photograph of an SDS-PAGE gel showing molecules under reducing conditions, specifically CRD-240, which is a fusion of IgG1 with CH1-CH2-CH3. [Figure 7C] This is a photograph of an SDS-PAGE gel showing molecules under reducing conditions, specifically a fusion of IgG1-derived molecules with a hinge-CH2-CH3 group that does not contain CH1.

[0117] [Figure 8A] This is a scatter plot of the percentage of autoantibody depletion from serum samples derived from GD patients due to the addition of CRD-239. [Figure 8B] This is a scatter plot of the relative binding affinities of CRD-239 and CRD-240 to autoantibodies in nine serum samples from GD patients.

[0118] [Figure 9] This bar graph summarizes the increase in MFI relative to background for CRD-239 that binds to 11 different hybridomas.

[0119] [Figure 10] This is a bar chart showing the internalization ratios of CRD-239 and the anti-AChR-Fc molecule (CRD-509) for hybridomas expressing BCR against the TSHR extracellular domain or AChR.

[0120] [Figure 11] This is a bar chart showing the depletion percentage of anti-TSHR autoantibodies from the serum of GD patients contacted with various molecules of the present invention.

[0121] [Figure 12A] This bar graph summarizes the increase in MFI relative to background for the binding of various molecules of the present invention to two hybridomas. [Figure 12B] This is a bar chart of the percentage of anti-TSHR B cell hybridoma cytotoxicity after contact with either CRD-527, CRD-527-Tecillin, or an unrelated molecule conjugated to Tecillin (CRD-509). [Modes for carrying out the invention]

[0122] The present invention provides compositions comprising, in some embodiments, a fragment of a first human receptor target or an analog or derivative of a Graves' disease (GD) autoantibody and a fragment of a second human protein receptor target or an analog or derivative of a GD autoantibody. Also provided are protein complexes comprising at least two polypeptide chains, the first chain comprising a fragment of the first human protein target or an analog or derivative of a GD autoantibody and a first dimerization domain, and the second chain comprising a fragment of the second human protein target or an analog or derivative of a GD autoantibody and a second dimerization domain that can dimerize with the first dimerization domain. Polypeptides comprising fragments of the extracellular domain of a thyroid-stimulating hormone receptor (TSHR) comprising at least one mutation that increases solubility, reduces aggregation, or both are also provided. Polypeptides, compositions, and protein complexes of the present invention further comprising effector moieties that are not unmodified Fc domains are also provided. The present invention further relates to a pharmaceutical composition comprising a composition and / or protein complex, a nucleic acid encoding the polypeptide of the composition and / or protein complex, a treatment method using the composition and / or protein complex and a method for determining suitability for treatment, and a method for producing the composition and / or protein complex.

[0123] In a first embodiment, a composition is provided comprising a fragment of a first protein target of a GD autoantibody or an analog or derivative thereof.

[0124] In another embodiment, a composition is provided comprising a fragment of a first protein target of a GD autoantibody and a fragment of a second protein target of a GD autoantibody or an analog or derivative thereof.

[0125] In another embodiment, a protein is provided comprising a fragment of a first protein target of a GD autoantibody or an analog or derivative thereof.

[0126] In another embodiment, a protein is provided comprising a fragment of a first protein target of a GD autoantibody or an analog or derivative thereof, and a fragment of a second protein target of a GD autoantibody or an analog or derivative thereof.

[0127] In another embodiment, a protein complex comprising at least two polypeptide chains is provided, the first polypeptide chain comprising a fragment of a first protein target of a GD autoantibody or an analog or derivative thereof and a first dimerization domain, and the second polypeptide chain comprising a fragment of a second protein target of a GD autoantibody or an analog or derivative thereof and a second dimerization domain.

[0128] In some embodiments, the composition comprises a protein complex comprising at least two polypeptide chains, the first polypeptide chain comprising a fragment of a first protein target of the GD autoantibody or an analog or derivative thereof and a first dimerization domain, and the second polypeptide chain comprising a fragment of a second protein target of the GD autoantibody or an analog or derivative thereof and a second dimerization domain. In some embodiments, the composition comprises the protein complex of the present invention. In some embodiments, the composition comprises the protein of the present invention. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is a fusion protein.

[0129] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to polymers of amino acid residues. In other embodiments, the terms “peptide,” “polypeptide,” and “protein” as used herein encompass natural peptides, peptide mimetics (typically including non-peptide bonds or other synthetic modifications), as well as peptide analogs, peptoids, and semipeptoids, or any combination thereof. In other embodiments, the peptides, polypeptides, and proteins described have modifications that make them more stable in the body or more permeable to cells. In one embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, peptides are not cyclic peptides. In some embodiments, fragments are not cyclic peptides. In some embodiments, extracellular domains are not cyclic peptides.

[0130] In some embodiments, the protein complex is an immunoglobulin (Ig)-like complex. In some embodiments, the protein complex includes an Ig-like scaffold. In some embodiments, the protein complex includes an Ig-like backbone. In some embodiments, the protein complex is an IgFc fusion complex. In some embodiments, the composition lacks an antibody-variable domain. In some embodiments, the protein complex lacks an antibody-variable domain. In some embodiments, the composition lacks a variable domain. In some embodiments, the protein complex lacks a variable domain. In some embodiments, the first chain lacks a variable domain. In some embodiments, the second chain lacks a variable domain. In some embodiments, the protein complex is a multi-chain complex. In some embodiments, the composition is a therapeutic composition. In some embodiments, the protein complex is a therapeutic complex. In some embodiments, the composition is for use in therapeutic methods. In some embodiments, the protein complex is for use in therapeutic methods. In some embodiments, the composition is for use in the manufacture of pharmaceuticals. In some embodiments, the protein complex is for use in the manufacture of pharmaceuticals. In some embodiments, the composition is for use in treating GD. In some embodiments, the protein complex is for use in treating GD. In some embodiments, the protein complex is for use in diagnosing GD. In some embodiments, the protein complex is for use in determining appropriate treatment for GD. In some embodiments, the protein complex is for use in characterizing the serological response to GD. In some embodiments, the protein complex is for use in determining the autoantibody titer of GD.

[0131] As used herein, the term “polypeptide chain” refers to a polymer of amino acids linked by peptide bonds from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). In some embodiments, the polypeptide chain is a recombinant polypeptide. In some embodiments, the polypeptide chain contains at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the polypeptide chain contains at least 290 amino acids. In some embodiments, the polypeptide chain contains at least 300 amino acids. In some embodiments, the polypeptide chain contains up to 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 amino acids. Each possibility represents a distinct embodiment of the present invention.

[0132] As used herein, the term “recombinant polypeptide” refers to a protein encoded by recombinant DNA and therefore not found in nature. In some embodiments, the protein complex is not found in nature. In some embodiments, the polypeptide chain is not found in nature. In some embodiments, the recombinant polypeptide is a synthetic polypeptide. The term “recombinant DNA” refers to a DNA molecule formed by laboratory methods. Generally, this recombinant DNA is in the form of a vector, plasmid, or virus used to express recombinant proteins in cells. The production of recombinant proteins by cell expression is well known in the art, and the polypeptides of the present invention may be produced using any method of recombinant protein expression. Cell-free expression systems for recombinant protein production may also be used.

[0133] As used herein, the term “expression” refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. Therefore, the expression of a nucleic acid molecule may refer to the transcription of a nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or the translation of RNA into a precursor or mature protein (polypeptide). In some embodiments, the nucleic acid molecule of the present invention is expressed in a cell to produce the polypeptide of the present invention. In some embodiments, the nucleic acid complex of the present invention is expressed in a cell to produce the protein complex of the present invention. In some embodiments, RNA is a vector.

[0134] The expression of DNA sequences or RNA within cells is well known to those skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct modification of the cell's genome. In some embodiments, the DNA sequence resides in an expression vector, such as a plasmid or viral vector. In some embodiments, a Kozak sequence is inserted upstream of the transcription start codon. In some embodiments, the Kozak sequence enhances the amount of protein being expressed.

[0135] In some embodiments, the protein complex comprises at least two polypeptide chains. In some embodiments, the protein complex comprises at least three polypeptide chains. In some embodiments, the protein complex comprises at least four polypeptide chains. In some embodiments, the protein complex comprises or consists of two polypeptide chains. In some embodiments, the protein complex comprises or consists of three polypeptide chains. In some embodiments, the protein complex comprises or consists of four chains. In some embodiments, the polypeptide chains are the same. In some embodiments, the polypeptide chains are different. In some embodiments, at least two of the polypeptide chains are the same. In some embodiments, at least two of the polypeptide chains are different.

[0136] protein In some embodiments, the protein is a mammalian protein. In some embodiments, the mammal is a human. In some embodiments, the protein is a transmembrane protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a subunit within a receptor. In some embodiments, the protein is a cell surface protein. In some embodiments, the cell surface protein is an endogenous membrane protein. In some embodiments, the cell surface protein is a plasma membrane-embedded protein. In some embodiments, the cell surface protein is a membrane-fixed protein. In some embodiments, the protein is a GD-related protein. In some embodiments, the protein is a synthetic protein. In some embodiments, the protein is a naturally occurring protein. In some embodiments, the protein is a target of a GD autoantibody. In some embodiments, the protein is a thyroid-stimulating hormone receptor (TSHR).

[0137] As used herein, the term “receptor” refers to a protein expressed on the surface of a cell that can bind to a ligand. In some embodiments, the receptor is a protein that can transmit a signal to the cytoplasm of a cell. In some embodiments, the receptor includes a ligand-binding domain. In some embodiments, the receptor includes a transmembrane domain. In some embodiments, the receptor includes an intracellular domain. In some embodiments, the ligand is thyroid-stimulating hormone (TSH).

[0138] In some embodiments, the fragment includes the extracellular domain (ECD) of the protein. In some embodiments, the extracellular domain is the N-terminal ECD. In some embodiments, the fragment includes a fragment of the extracellular domain of the protein. In some embodiments, the fragment consists of the extracellular domain of that fragment. In some embodiments, the fragment consists of the extracellular domain of the protein. In some embodiments, the fragment consists of a fragment of the extracellular domain of the protein. In some embodiments, the fragment includes the transmembrane domain of the protein. In some embodiments, the fragment lacks the transmembrane domain of the protein. In some embodiments, the fragment lacks the intracellular domain of the protein. In some embodiments, the chain lacks the transmembrane domain. In some embodiments, the chain lacks the intracellular domain. In some embodiments, the fragment includes a sequence derived from a homologous human protein. In some embodiments, the fragment includes a sequence derived from a homologous non-human protein. In some embodiments, the fragment includes a mutation in the human protein.

[0139] In some embodiments, the fragment contains at least five amino acids of the protein. In some embodiments, the fragment contains at least ten amino acids of the protein. In some embodiments, the fragment contains at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 amino acids. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragment contains at least 290 amino acids. In some embodiments, the fragment contains at least 300 amino acids. In some embodiments, the amino acids of the protein are the sequential amino acids of the protein. In some embodiments, the fragment constitutes less than 100% of the protein. In some embodiments, the fragment constitutes less than 100% of the extracellular domain of the protein. In some embodiments, the fragment contains less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50% of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragment contains less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50% of the extracellular domain of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragment contains 5-500, 5-250, 5-100, 5-50, 10-500, 10-250, 10-100, 10-50, 20-500, 20-250, 20-200, 20-50, 25-500, 25-250, 25-100, 25-50, 50-500, 50-250, 50-100, 100-500, or 100-250 amino acids. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragment contains 290-350 amino acids. In some embodiments, the fragment contains 290-310 amino acids.In some embodiments, the fragment contains 5 to 50 amino acids. In some embodiments, the fragment contains up to 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 225, 230, 240, 250, 260, 270, 275, 280, 290, 300, 3 It contains 10, 320, 325, 330, 340, 350, 360, 370, 375, 380, 390, 400, 410, 420, 425, 430, 440, 450, 460, 470, 475, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acids. Each possibility represents a distinct embodiment of the present invention.

[0140] In some embodiments, the chain comprises at least one fragment. In some embodiments, the chain comprises at least two fragments. In some embodiments, the fragments are separated by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker includes increased solubility compared to the protein region excluded from the chain. In some embodiments, the protein region is replaced by a non-protein protein region. In some embodiments, the replacement region includes increased solubility compared to the replaced protein region. In some embodiments, the replacement region includes increased protein stability compared to the replaced protein region.

[0141] In some embodiments, a protein is the target of the antibody. As used herein, the term “antibody” includes all classes of IgA, IgD, IgE, IgG, and IgM, and all their subclasses. In some embodiments, the antibody is a circulating antibody. In some embodiments, the antibody is a naturally occurring antibody. In some embodiments, the antibody is an autoantibody.

[0142] As used herein, the term “autoantibody” refers to an antibody produced by the subject’s own immune system against at least one of the subject’s own proteins. In some embodiments, the autoantibody is an autoreactive antibody. In some embodiments, the autoantibody targets an autoantigen. An autoantigen is also known as an autoantigen. In some embodiments, the autoantibody is associated with GD. In some embodiments, the autoantibody characterizes GD. In some embodiments, the autoantibody is an autoantibody of GD. In some embodiments, the autoantibody is produced by autoreactive B cells. In some embodiments, the protein is the antigen of the antibody. In some embodiments, the fragment contains the antigen of the antibody. In some embodiments, the fragment contains at least one antigen of the antibody. In some embodiments, the fragment contains at least two antigens of the antibody. In some embodiments, the fragment contains at least one, two, three, four, five, six, seven, eight, nine, or ten antigens of the antibody. Each possibility represents a distinct embodiment of the invention. In some embodiments, the antigen of the antibody is an autoantigen. In some embodiments, the antigen is an epitope. In some embodiments, the antigen contains at least one epitope. In some embodiments, the epitope comprises at least five amino acids. In some embodiments, the epitope comprises five to six amino acids. In some embodiments, the epitope comprises five to ten amino acids. In some embodiments, the epitope is a simple epitope. In some embodiments, a simple epitope is a linear epitope. In some embodiments, the epitope is a complex epitope. In some embodiments, a complex epitope is a 3D epitope. In some embodiments, a complex epitope is a discontinuous epitope. In some embodiments, a discontinuous epitope comprises at least two discontinuous portions of amino acids that bind to form an epitope. In some embodiments, a linker sequence is located between the two portions of the epitope.

[0143] As used herein, the term “analog” includes any peptide having a substantially identical amino acid sequence to that of a protein, but in which one or more residues are conservedly substituted with functionally similar residues. In some embodiments, the analog exhibits similar functionality to the original protein. Examples of conservative substitutions include substitution of a nonpolar (hydrophobic) residue, e.g., isoleucine, valine, leucine, or methionine; substitution of a polar (hydrophilic) residue, e.g., between arginine and lysine, glutamine and asparagine, or glycine and serine; substitution of a basic residue, e.g., between lysine, arginine, or histidine; or substitution of an acidic residue, e.g., between aspartic acid or glutamic acid. Each possibility represents a distinct embodiment of the invention. In some embodiments, the substitution lies outside the antigenic region of the protein. In some embodiments, the substitution lies outside the epitope of the antibody. In some embodiments, the analog is still a target of the antibody. In some embodiments, the analog retains the binding of an autoantibody. The analog may have deletions or mutations that result in an amino acid sequence different from the canonical amino acid sequence of the protein. Furthermore, the analog may be analogous to a protein fragment, in which case the fragment must contain at least 50 consecutive amino acids of the protein or at least one epitope of the antibody. In some embodiments, the analog is an analog to the canonical sequence of the protein.

[0144] In some embodiments, the analog to the protein includes an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the analog to the protein includes an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the analog to the protein includes an amino acid sequence having at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the analog can still bind to GD autoantibodies. In some embodiments, the analog can still capture GD autoantibodies. In some embodiments, the analog can still treat GD. In some embodiments, the analog includes at least one substitution. In some embodiments, the analogues include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. Each possibility represents a distinct embodiment of the invention. In some embodiments, the substitutions are variations of the canonical sequence.

[0145] As used herein, the term “derivative” refers to any polypeptide based on a protein that still retains antibody binding. A derivative may not be merely a fragment of a protein, but may have no replaced or removed amino acids (analogs), and rather may have additional modifications made to the protein, such as post-translational modifications. Furthermore, a derivative may be a derivative of a protein fragment, in which case the fragment must contain at least 50 consecutive amino acids of the protein or at least one epitope of the antibody. In some embodiments, the derivative is a derivative of the canonical sequence of a protein.

[0146] In some embodiments, the protein derivative comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the protein derivative comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the protein derivative comprises an amino acid sequence having at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the derivative can still bind to a GD autoantibody. In some embodiments, the derivative can still capture a GD autoantibody. In some embodiments, the derivative can still treat GD. In some embodiments, the derivative is a mutant protein or fragment.

[0147] The canonical amino acid sequences of known proteins are well known in the art. They can be found in various databases, including UniProt, NCBI, and UCSC Genome Browser. Any sequence accepted as a canonical sequence may be used. As a non-limiting example, human TSHR is encoded by the TSHR gene, its canonical nucleic acid sequence can be found in Entrez gene 7253, its canonical protein-coding mRNA sequence can be found in NM_000369, NM_001018036, and NM_001142626, and its canonical amino acid sequence can be found in NP_000360, NP_001018046, and NP001136098, as well as UniProt number P16473. In some embodiments, the canonical sequence is identical to a sequence present in at least 50, 60, 70, 75, 80, 90, 95, 97, or 99 percent of the population. Each possibility represents a distinct embodiment of the invention. In some embodiments, the canonical sequence is identical to the most common sequence present in the population. In some embodiments, the population is a disease population. In some embodiments, the population is a population with an autoimmune disease.

[0148] In some embodiments, the canonical amino acid sequence of the N-terminal extracellular domain of TSHR is GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYL The extracellular domain comprises or consists of NKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMG (SEQ ID NO: 1). In some embodiments, the extracellular domain lacks a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the TSHR signal peptide comprises or consists of MRPADLLQLVLLLDLPRDLG (SEQ ID NO: 15).

[0149] In some embodiments, the canonical amino acid sequence of the N-terminal extracellular domain of TSHR is MRPADLLQLVLLLDLPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAF NGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMG (SEQ ID NO: 2) is included or consists of. In some embodiments, the extracellular domain lacks a signal peptide. In some embodiments, the extracellular domain further includes a signal peptide. In some embodiments, the TSHR signal peptide includes or consists of MRARPRPRPLWATVLALGALAGVGVG (SEQ ID NO: 16). In some embodiments, the TSHR signal peptide is used.

[0150] In some embodiments, the signal peptide is a heterologous signal peptide. In some embodiments, the signal peptide is a signal peptide of an antibody chain. In some embodiments, the signal peptide is of the antibody heavy chain. In some embodiments, the signal peptide is of the antibody light chain. In some embodiments, the signal peptide is of the kappa light chain. In some embodiments, the signal peptide is of the lambda light chain. In some embodiments, the heavy chain signal peptide contains MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 17). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 17. In some embodiments, the light chain signal peptide contains MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 18). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 18. In some embodiments, the signal peptide contains or consists of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 19). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 19. In some embodiments, the signal peptide contains or consists of MGWSCIILFLVATATGVHS (SEQ ID NO: 20). In some embodiments, the light chain signal peptide is SEQ ID NO: 20.

[0151] In some embodiments, the protein is a derivative of the N-terminal extracellular domain of TSHR. In some embodiments, the protein is a derivative of SEQ ID NO: 1. In some embodiments, the derivative is a variant of SEQ ID NO: 1. In some embodiments, the derivative includes a deletion of the C-peptide region of the N-terminal extracellular domain of TSHR. In some embodiments, the deletion of the C-peptide region increases the solubility of the extracellular domain of TSHR. In some embodiments, the deletion of the C-peptide region increases the expression of the extracellular domain of TSHR. In some embodiments, the C-peptide region includes amino acids 297-346 of SEQ ID NO: 1. In some embodiments, the C-peptide region includes amino acids 317-366 of SEQ ID NO: 2. In some embodiments, the C-peptide region consists of amino acids 297-346 of SEQ ID NO: 1. In some embodiments, the C-peptide region consists of amino acids 317-366 of SEQ ID NO: 2. In some embodiments, the C-peptide region includes the amino acid sequence ALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGF (SEQ ID NO: 57). In some embodiments, the C-peptide region consists of SEQ ID NO: 57. In some embodiments, peptides lacking the C-peptide region include GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVNGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMG (SEQ ID NO: 3). In some embodiments, the peptide of the present invention includes SEQ ID NO: 3. In some embodiments, the peptide lacking the C-peptide region is represented by SEQ ID NO: 3.In some embodiments, the peptide of the present invention is comprised of SEQ ID NO: 3.

[0152] In some embodiments, the derivative is a shortened version of SEQ ID NO: 1. In some embodiments, the derivative is a shortened version of the extracellular domain of TSHR. In some embodiments, the mutation is a shortened version. In some embodiments, the shortened version is a C-terminal shortened version. In some embodiments, the derivative is the N-terminal fragment of SEQ ID NO: 1. In some embodiments, the shortened version is a deletion of the C-terminal fragment of SEQ ID NO: 1. In some embodiments, the derivative is a shortened version or a derivative of a shortened version.

[0153] In some embodiments, the mutation is the deletion of amino acids 297–393 in SEQ ID NO: 1. In some embodiments, the shortening is a C-terminal shortening starting from amino acid 297 to the C-terminus of SEQ ID NO: 1. It will be understood that all numbers given for SEQ ID NO: 1 are equivalent to the same positions in SEQ ID NO: 2. The positions in SEQ ID NO: 2 can be achieved by adding 20 bases to the positions in SEQ ID NO: 1 (by adding a 20-mer signal peptide in SEQ ID NO: 2). In some embodiments, the N-terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVN (SEQ ID NO: 83) or a variant thereof. In some embodiments, the N-terminal fragment comprises SEQ ID NO: 83 or a variant thereof. In some embodiments, the polypeptide comprises SEQ ID NO: 83. In some embodiments, the polypeptide comprises SEQ ID NO: 83.

[0154] In some embodiments, the mutation is the deletion of amino acids 261-393 of SEQ ID NO: 1. In some embodiments, the shortening is a C-terminal shortening starting from amino acid 261 of SEQ ID NO: 1 to the C-terminus. In some embodiments, the N-terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYP (SEQ ID NO: 84) or a variant thereof. In some embodiments, the N-terminal fragment consists of SEQ ID NO: 84 or a variant thereof. In some embodiments, the fragment contains or consists of amino acids 1-296 of SEQ ID NO: 1. In some embodiments, the polypeptide contains SEQ ID NO: 84. In some embodiments, the polypeptide consists of SEQ ID NO: 84.

[0155] In some embodiments, the mutation is the deletion of amino acids 261-393 of SEQ ID NO: 1. In some embodiments, the shortening is a C-terminal shortening starting from amino acid 261 of SEQ ID NO: 1 to the C-terminus. In some embodiments, the N-terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYP (SEQ ID NO: 84) or a variant thereof. In some embodiments, the N-terminal fragment consists of SEQ ID NO: 84 or a variant thereof. In some embodiments, the fragment contains or consists of amino acids 1-260 of SEQ ID NO: 1. In some embodiments, the polypeptide contains SEQ ID NO: 84. In some embodiments, the polypeptide consists of SEQ ID NO: 84.

[0156] In some embodiments, the mutation is the deletion of amino acids 242-393 of SEQ ID NO: 1. In some embodiments, the shortening is a C-terminal shortening starting from amino acid 242 of SEQ ID NO: 1 to the C-terminus. In some embodiments, the N-terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLK (SEQ ID NO: 85) or a variant thereof. In some embodiments, the N-terminal fragment consists of SEQ ID NO: 85 or a variant thereof. In some embodiments, the fragment contains or consists of amino acids 1-241 of SEQ ID NO: 1. In some embodiments, the polypeptide includes SEQ ID NO: 85. In some embodiments, the polypeptide consists of SEQ ID NO: 85.

[0157] In some embodiments, the first protein and the second protein are the same protein. In some embodiments, the first and second proteins originate from the same protein, but the fragments are different fragments. In some embodiments, the fragments are different fragments. In some embodiments, the fragments contain or consist of different sequences. In some embodiments, the first and second proteins are different proteins. In some embodiments, the first fragment includes GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVN (SEQ ID NO: 58). In some embodiments, the first fragment consists of SEQ ID NO: 58. In some embodiments, the second fragment includes GQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMG (SEQ ID NO: 59). In some embodiments, the second fragment consists of sequence number 59. In some embodiments, the first and second fragments are located on a polypeptide chain.

[0158] In some embodiments, the protein or fragment contains a mutation. In some embodiments, the extracellular domain variant contains a mutation. In some embodiments, the mutation reduces aggregation. In some embodiments, the mutation reduces molecular cleavage. In some embodiments, the protein or fragment contains a mutation that reduces aggregation. In some embodiments, aggregation is protein aggregation. In some embodiments, aggregation is fragment aggregation. In some embodiments, aggregation is extracellular domain aggregation. In some embodiments, aggregation is complex aggregation. In some embodiments, aggregation is polypeptide aggregation. In some embodiments, reducing aggregation includes increasing solubility. In some embodiments, reducing aggregation includes increasing stability. In some embodiments, the protein or fragment contains a mutation that increases solubility. In some embodiments, the protein or fragment contains a mutation that increases the stability of the protein or fragment. In some embodiments, the protein is a polypeptide. In some embodiments, the mutation is an insertion. In some embodiments, the protein is a surface protein and contains a mutation that increases solubility. In some embodiments, the fragment is the extracellular domain of a surface protein and contains an insertion that increases solubility. In some embodiments, the fragment is the extracellular domain of a surface protein and contains a deletion that increases solubility.

[0159] In some embodiments, the mutation is selected from the cysteine ​​263 mutation (C263), cysteine ​​264 mutation (C264), cysteine ​​281 mutation (C281), lysine 293 mutation (K293), glycine 347 mutation (G347), glutamine 348 mutation (Q348), glutamic acid 349 mutation (E349), cysteine ​​370 mutation (C370), cysteine ​​378 mutation (C378), and cysteine ​​mutation 388 mutation (C388) in SEQ ID NO: 1. In some embodiments, at least one mutation is selected from the C283 mutation, C284 mutation, C301 mutation, K313 mutation, G367 mutation, Q368 mutation, E369 mutation, C390 mutation, C398 mutation, and C408 mutation in SEQ ID NO: 2. It will be understood that C263 in SEQ ID NO: 1 and C283 in SEQ ID NO: 2 are the same residues as in SEQ ID NO: 20, which contains the N-terminal 20-amino acid signal peptide. The same applies to the correspondence of all other residues between SEQ ID NO: 1 and SEQ ID NO: 2. For simplicity, all further references to amino acids are given with respect to SEQ ID NO: 1, but it will be understood that the corresponding amino acids in SEQ ID NO: 2 are also intended. In some embodiments, the mutation is a mutation at C263. In some embodiments, the mutation is a mutation at C264. In some embodiments, the mutation is a mutation at C281. In some embodiments, the mutation is a mutation at K293. In some embodiments, the mutation is a mutation at G347. In some embodiments, the mutation is a mutation at Q348. In some embodiments, the mutation is a mutation at E349. In some embodiments, the mutation is a mutation at C370. In some embodiments, the mutation is a mutation at C378. In some embodiments, the mutation is a mutation at C388. In some embodiments, C263 is mutated to valine (C263V). In some embodiments, C264 is mutated to valine (C264V). In some embodiments, C281 is mutated to valine (C281V). In some embodiments, K293 is mutated to alanine (K293A). In some embodiments, G347 is mutated to asparagine (G347N).In some embodiments, Q348 is mutated to glutamate (Q348E). In some embodiments, E349 is mutated to threonine (E349T). In some embodiments, C370 is mutated to valine (C370V). In some embodiments, C378 is mutated to valine (C378V). In some embodiments, C388 is mutated to valine (C388V).

[0160] In some embodiments, the mutation is multiple mutations. In some embodiments, the multiple mutations are at least 2, 3, 4, 5, 6, 7, 8, or 9 mutations. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the multiple mutations are selected from the mutations of C263, C264, C281, K293, G347, Q348, E349, C370, C378, and C388 in SEQ ID NO: 1. In some embodiments, the multiple includes the mutations of C263 and C264. In some embodiments, the multiple includes the mutations of G347, Q348, and E349. In some embodiments, the multiple includes the mutations of C263, C264, C281, G347, Q348, E349, C370, C378, and C388 in SEQ ID NO: 1.

[0161] In some embodiments, the fragment includes an extracellular functional domain. In some embodiments, the functional domain is a ligand-binding domain. In some embodiments, the ligand is TSH. In some embodiments, the fragment further includes at least one mutation in the ligand-binding domain. In some embodiments, variants of the fragment include at least one mutation in the ligand-binding domain. In some embodiments, at least one mutation reduces binding to TSH. In some embodiments, the reduction is neutralized. In some embodiments, the reduction includes a reduction of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% in binding. Each possibility represents a distinct embodiment of the invention. In some embodiments, the reduction is at least a 50% reduction. In some embodiments, the reduction is at least a 90% reduction. In some embodiments, the binding is the binding of TSHR to TSH. In some embodiments, binding is the binding of the N-terminal extracellular domain of TSHR to TSH. In some embodiments, binding is the binding of a peptide to TSH. In some embodiments, binding is the binding of a fragment to TSH. In some embodiments, mutations that increase solubility also decrease ligand binding. In some embodiments, deletion of the C peptide region decreases ligand binding. In some embodiments, deletion of amino acids 297-346 of SEQ ID NO: 1 decreases ligand binding. In some embodiments, SEQ ID NO: 3 includes a decrease in ligand binding.

[0162] In some embodiments, at least one mutation is selected from the lysine 163 (K163) and glutamate 231 (E231) mutations in SEQ ID NO: 1. In some embodiments, at least one mutation is selected from the K183 and E251 mutations in SEQ ID NO: 2. It will be understood that K163 in SEQ ID NO: 1 and K183 in SEQ ID NO: 2 are the same residue, and in exactly the same way, E231 in SEQ ID NO: 1 and E251 in SEQ ID NO: 2 are the same residue. K183 and E231 are known in the art to be important for ligand binding and are evolutionarily conserved residues. Mutations in either of these residues, particularly mutations that neutralize or alter the charge, reduce binding to TSH. In some embodiments, the mutation is the K163 mutation in SEQ ID NO: 1. In some embodiments, the mutation is the E231 mutation in SEQ ID NO: 1. In some embodiments, K163 is mutated to a non-positively charged amino acid. In some embodiments, K163 is mutated to an uncharged amino acid. In some embodiments, K163 is mutated to a negatively charged amino acid. In some embodiments, K163 is mutated to alanine. In some embodiments, at least one mutation is K163A in SEQ ID NO: 1. In some embodiments, E231 is mutated to a non-negatively charged amino acid. In some embodiments, E231 is mutated to a non-charged amino acid. In some embodiments, E231 is mutated to a positively charged amino acid. In some embodiments, E231 is mutated to alanine. In some embodiments, E231 is mutated to lysine or arginine. In some embodiments, E231 is mutated to lysine. In some embodiments, at least one mutation is E231A in SEQ ID NO: 1. In some embodiments, at least one mutation is E231K in SEQ ID NO: 1.

[0163] In some embodiments, the variant of the fragment includes sequence number 86. In some embodiments, the variant of the fragment consists of sequence number 86. In some embodiments, sequence number 86 includes the K163A mutation. In some embodiments, the variant of the fragment includes sequence number 87. In some embodiments, the variant of the fragment consists of sequence number 87. In some embodiments, sequence number 87 includes the K163R mutation. In some embodiments, the variant of the fragment includes sequence number 88. In some embodiments, the variant of the fragment consists of sequence number 88. In some embodiments, sequence number 88 includes the E231K mutation. In some embodiments, the fragment includes or consists of sequences selected from sequence numbers 86-88. In some embodiments, the polypeptide includes or consists of sequence number 86. In some embodiments, the polypeptide includes or consists of sequence number 87. In some embodiments, the polypeptide includes or consists of sequence number 88.

[0164] Since the protein complex of the present invention is intended to bind to antibodies and B cells, it will be understood by those skilled in the art that it is advantageous that it does not bind to itself or to other copies of the therapeutic molecule. Therefore, mutations and shortenings that reduce aggregation but do not interfere with autoantibody binding are advantageous. Similarly, if the fragment contains a complete ligand-binding domain, it may be advantageous to disable ligand binding. The endogenous ligand may be present in circulation, and ligand binding may capture it and prevent it from reaching the intended target receptor. In some embodiments, the fragment includes a shortening of the extracellular domain. In some embodiments, the fragment consists of a shortening of the extracellular domain. In some embodiments, the shortening lacks at least one extracellular functional domain. In some embodiments, the shortening lacks at least two extracellular functional domains.

[0165] In some embodiments, the derivative is a derivative of the shortened sequence. In some embodiments, the derivative contains at least 85% identity with the shortened sequence and does not further contain a stretch of homologous / identical amino acids to the sequence from TSHR. Thus, it will be understood that a sequence having sequence identity to the shortened sequence is not the unshortened sequence. In some embodiments, the shortened sequence contains at least one mutation. In some embodiments, the derivative contains at least 85% identity with SEQ ID NO: 1. In some embodiments, the derivative contains at least 85% identity with SEQ ID NO: 2. In some embodiments, the derivative contains at least 85% identity with SEQ ID NO: 3.

[0166] Dimerization domain In some embodiments, the dimerizing domains can dimerize with each other. In some embodiments, the first dimerizing domain can dimerize with the second dimerizing domain. In some embodiments, the first and second dimerizing domains can dimerize with each other. In some embodiments, dimerization is possible because it is configured to dimerize. In some embodiments, dimerization occurs under physiological conditions. In some embodiments, dimerization occurs in a body fluid. In some embodiments, the body fluid is blood. In some embodiments, the body fluid is plasma. In some embodiments, the body fluid is serum. In some embodiments, dimerization occurs within a subject. In some embodiments, dimerization occurs in vivo. In some embodiments, dimerization occurs in vitro.

[0167] As used herein, the term “dimerizing domain” refers to an amino acid sequence that, upon contact with another amino acid sequence (another dimerizing domain), binds to it and forms a dimer. Dimerizing domains are well known in the art, as many protein sequences are known to bind to one another. In some embodiments, dimerization involves the formation of a covalent bond between dimerizing domains. In some embodiments, dimerization involves an electrostatic bond. In some embodiments, dimerization does not involve an electrostatic bond. In some embodiments, dimerization is reversible. In some embodiments, dimerization is irreversible. In some embodiments, dimerization involves a bond formed between dimerizing domains. In some embodiments, the bond is a chemical bond. In some embodiments, the bond is a disulfide bond. In some embodiments, the bond is a peptide bond. Examples of dimerizing domains include the hinge domain of an antibody heavy chain, the CH1 / CL domain of an antibody heavy / light chain, and the ECD domain of TCR alpha / beta. Furthermore, the upper hinge domain can be manipulated by cysteine ​​substitution / mutation to serine to prevent dimerization. In some embodiments, the dimerizing domain contains or consists of the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 21).

[0168] In some embodiments, the dimerization domain includes or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, the Ig hinge domain is a heavy chain hinge domain. In some embodiments, Ig is human Ig. In some embodiments, the immunoglobulin is selected from IgA, IgD, IgE, IgG, and IgM. In some embodiments, the immunoglobulin is IgG. In some embodiments, IgG is IgG1. In some embodiments, IgG is IgG2. In some embodiments, IgG is IgG3. In some embodiments, IgG is selected from IgG1 and IgG3. In some embodiments, IgG is IgG4. In some embodiments, both the first and second dimerization domains are Ig hinge domains. In some embodiments, the first and second dimerization domains are identical. In some embodiments, the first and second dimerization domains are at least 95% identical. In some embodiments, the first and second dimerization domains are at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, or 100% identical. Each possibility represents a distinct embodiment of the present invention.

[0169] In some embodiments, the hinge domain includes the amino acid sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 22). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the IgG1 hinge includes or consists of SEQ ID NO: 22. In some embodiments, the hinge domain includes the amino acid sequence EPKCCVECPPCPAPPAAAP (SEQ ID NO: 23). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgG2 hinge includes or consists of SEQ ID NO: 23. In some embodiments, the hinge domain includes the amino acid sequence ESKYGPPCPPCPAPEFLGGP (SEQ ID NO: 24). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the IgG4 hinge includes or consists of SEQ ID NO: 24. In some embodiments, the hinge domain contains the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGP (SEQ ID NO: 25). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IgG3 hinge contains or consists of SEQ ID NO: 25. In some embodiments, the hinge domain contains the CPXCP (SEQ ID NO: 26) motif. In some embodiments, X in SEQ ID NO: 26 is selected from P and R. In some embodiments, SEQ ID NO: 26 is CPPCP (SEQ ID NO: 27). In some embodiments, SEQ ID NO: 26 is CPRCP (SEQ ID NO: 28). In some embodiments, the hinge domain contains EPKSCDKTHTCPPCP (SEQ ID NO: 29). Thus, it will be understood that the hinge region can be considered to terminate after the CPXCP motif.

[0170] In some embodiments, the dimerization domain includes or consists of an Ig CH1 domain. In some embodiments, the dimerization domain includes or consists of an Ig heavy chain CH1 domain. In some embodiments, the dimerization domain includes or consists of an Ig light chain. In some embodiments, the dimerization domain includes or consists of a light chain CL domain. In some embodiments, the CL domain is a CL kappa domain. In some embodiments, the CL domain is a CL lambda domain. It is well known in the art that the CH1 domain of the Ig heavy chain dimerizes with the light chain CL domain. In some embodiments, the first dimerization domain includes or consists of a CH1 domain, and the second dimerization domain includes or consists of a CL domain. In some embodiments, both the first and second dimerization domains include a hinge domain. In some embodiments, neither the first nor the second dimerization domains contain a CH1 domain. In some embodiments, neither the first nor the second dimerization domain contains a CL domain. In some embodiments, neither the first nor the second polypeptide chain contains a CH1 domain. In some embodiments, neither the first nor the second polypeptide chain contains a CL domain.

[0171] In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 30). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 30. In some embodiments, SEQ ID NO: 30 is the IgG1 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 31). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 31. In some embodiments, SEQ ID NO: 31 is the IgG2 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 32). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 32. In some embodiments, SEQ ID NO: 32 is the IgG3 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 33), and in some embodiments, the Ig CH1 domain consists of SEQ ID NO: 33. In some embodiments, SEQ ID NO: 33 is the IgG4 CH1 domain.

[0172] In some embodiments, the Ig CL kappa domain comprises the amino acid sequence AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSGDTKSFNRGEC (SEQ ID NO: 34). In some embodiments, the Ig CL kappa domain consists of SEQ ID NO: 34. In some embodiments, the Ig CL lambda domain comprises the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSGDKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 35). In some embodiments, the Ig CL lambda domain consists of SEQ ID NO: 35.

[0173] In some embodiments, the dimerization domain is an Fc domain. In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is an antibody heavy chain Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is an IgG1 heavy chain Fc domain. In some embodiments, the Fc domain includes a constant region of the antibody heavy chain. In some embodiments, the Fc domain includes CH1, hinge, CH2, and CH3 domains. In some embodiments, the Fc domain includes hinge CH2 and CH3 domains. In some embodiments, the Fc domain includes CH2 and CH3 domains.

[0174] Effects pedal section In some embodiments, the composition includes an effector moiety. In some embodiments, the first polypeptide chain includes an effector moiety. In some embodiments, the second polypeptide chain includes an effector moiety. In some embodiments, both the first and second polypeptide chains include an effector moiety. As used herein, the term “moiety” refers to a part of a molecule that may include either an entire functional group or a portion of a functional group as a substructure. The term “moiety” may also refer to a part of a molecule that exhibits a particular set of chemical and / or pharmacological properties similar to the corresponding molecule. As used herein, the term “effector moiety” refers to a molecule or fragment of a molecule that performs a cytotoxic effect. In some embodiments, the effector moiety is an effector molecule.

[0175] In some embodiments, the effector portion can induce a cytotoxic effect. In some embodiments, the effector portion is configured to induce a cytotoxic effect. In some embodiments, the effector portion can induce death. In some embodiments, the effector portion is configured to induce death. In some embodiments, death is cell death. In some embodiments, death is apoptosis. In some embodiments, death is necrosis. In some embodiments, death is cell-mediated death. In some embodiments, death is phagocytosis. In some embodiments, the cytotoxic effect is toward target cells. In some embodiments, death is present in the target cells. In some embodiments, the cytotoxic effect occurs upon binding. In some embodiments, death occurs upon binding. In some embodiments, the cytotoxic effect is toward target cells bound to the composition. In some embodiments, death is the death of target cells bound to the composition. In some embodiments, the cytotoxic effect is toward cells bound by a protein complex. In some embodiments, the cytotoxic effect is toward cells bound to a protein complex. In some embodiments, death is the death of cells bound to a protein complex. In some embodiments, death is the death of cells bound to a protein complex. In some embodiments, the cytotoxic effect is a direct effect. In some embodiments, the cytotoxic effect is an indirect effect. In some embodiments, binding of the composition is binding to a fragment. In some embodiments, binding to a protein complex is binding to a fragment. In some embodiments, the fragment is at least one of the fragments. In some embodiments, the fragment is one of the fragments. In some embodiments, the fragment is both fragments.

[0176] In some embodiments, the effector portion is a cytotoxic portion. In some embodiments, the effector portion is a toxin. In some embodiments, the effector portion is a poison. In some embodiments, the effector portion is a chemotherapeutic agent. In some embodiments, the effector portion is an anticancer agent. In some embodiments, the effector portion is an engager. In some embodiments, the engager binds to cytotoxic cells. In some embodiments, binding to cytotoxic cells means recruiting cytotoxic cells. In some embodiments, binding means being bound.

[0177] In some embodiments, the effector portion recruits a cytotoxic agent. In some embodiments, the cytotoxic agent is a cytotoxic cell. In some embodiments, the cytotoxic cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is a CD8-positive T cell. In some embodiments, the effector portion induces antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector portion induces complement-dependent cell-mediated cytotoxicity (CDC).

[0178] In some embodiments, the effector portion binds to a receptor on the cell surface of a cytotoxic cell. Examples of receptors include, but are not limited to, CD3, CD8, CD56, CD14, and CD16. In some embodiments, the receptor is a marker of a cytotoxic cell. In some embodiments, the receptor is specific to a cytotoxic cell. In some embodiments, the receptor is CD3. In some embodiments, the effector portion is an activator that binds to CD3. In some embodiments, the engager is an activator that binds to CD3. In some embodiments, CD3 is human CD3. In some embodiments, the activator that binds to CD3 is an anti-CD3 antibody or its antigen-binding fragment. In some embodiments, the receptor is CD16. In some embodiments, the effector portion is an activator that binds to CD16. In some embodiments, the engager is an activator that binds to CD16. In some embodiments, CD16 is human CD16. In some embodiments, the activator that binds to CD16 is an anti-CD16 antibody or its antigen-binding fragment. In some embodiments, the antibody of the antigen-binding fragment is a single-chain antibody. In some embodiments, the antibody of the antigen-binding fragment is a single-domain antibody. In some embodiments, the antibody of the antigen-binding fragment is a single-strand variable fragment (scFv). Anti-CD3 agents are well known in the art, and any such conjugate may be used. For example, an anti-human CD3 scFv known as OKT3 may be used as the agent. In some embodiments, the cytotoxic moiety is selected from alpha-amanitin, a radioactive moiety, and an anti-CD3 conjugate.Other examples of human anti-CD3 antibodies include muromonab (trade name Orthoclone OKT3), a mouse monoclonal anti-human CD3 antibody (DrugBank accession number DB00075); teplizumab, a humanized version of the mouse OKT3 anti-CD3 monoclonal antibody (DrugBank accession number DB06606); UCHT1, a mouse monoclonal anti-human CD3 antibody; UCHT1 mutant 9, a humanized version of the UCHT1 clone; and bispecific CD19-CD3 blinatumomab (DrugBank accession number DB09052). Examples of human anti-CD16 include AFM13, a bispecific tetravalent native cell engager (ICE®) that targets CD30 on tumor cells and CD16A on NK cells and macrophages, and GTB-3550 (CD16 / IL-15 / CD33), a triplicate killer cell engager.

[0179] In some embodiments, the composition includes an Fc region. In some embodiments, the effector portion is an Fc domain. The terms Fc region and Fc domain are used interchangeably herein. In some embodiments, the effector domain includes an Fc domain. In some embodiments, the Fc domain is both a dimerization domain and an effector portion. In some embodiments, the effector portion is not an Fc region. In some embodiments, the effector portion does not include an Fc region. In some embodiments, not an Fc region is not an unmodified Fc region. In some embodiments, the composition includes an effector portion that is not an Fc region. In some embodiments, the composition includes an effector portion other than an Fc region. In some embodiments, the composition lacks an Fc region. In some embodiments, the composition includes an effector portion that is a dimerization domain and an effector portion that does not include an Fc domain. In some embodiments, the protein includes an effector portion that is not an Fc region. In some embodiments, the protein includes an effector portion other than an Fc region. In some embodiments, the protein lacks an Fc region. In some embodiments, the protein is a dimerized domain containing an Fc region further conjugated to the effector portion. In some embodiments, the engager is an Fc region. In some embodiments, the engager is not an Fc region. In some embodiments, the composition contains an effector portion that is superior to Fc in killing cells. In some embodiments, superior killing means superior killing B cells. In some embodiments, Fc is unmodified Fc. In some embodiments, Fc is non-mutant Fc. In some embodiments, Fc is naturally occurring Fc. In some embodiments, Fc is not naturally occurring Fc. In some embodiments, Fc is human Fc. In some embodiments, superior Fc is Fc containing at least one mutation that increases ADCC. In some embodiments, the Fc region is an Fc domain. In some embodiments, the Fc region is an Fc fragment.In some embodiments, the first polypeptide chain includes an Fc region. In some embodiments, the second polypeptide chain includes an Fc region. In some embodiments, both the first and second polypeptide chains include an Fc region. In some embodiments, the Fc region is the Fc region of an antibody heavy chain. In some embodiments, the antibody heavy chain is a human antibody heavy chain. In some embodiments, the heavy chain is an IgG heavy chain. In some embodiments, IgG is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, IgG is selected from IgG1 and IgG3. In some embodiments, IgG is IgG1. In some embodiments, IgG is IgG2. In some embodiments, IgG is IgG3. In some embodiments, IgG is IgG4.

[0180] In some embodiments, the Fc region can induce a cytotoxic effect. In some embodiments, the Fc domain includes DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 63). In some embodiments, the Fc domain contains EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 64). It will be understood that SEQ ID NO: 64 contains five additional N-terminal amino acids compared to SEQ ID NO: 63. Thus, the numbering herein is given with respect to SEQ ID NO: 63, but the numbering for SEQ ID NO: 64 can be found by adding 5. In some embodiments, the Fc region can induce a cytotoxic effect. In some embodiments, the Fc domain includes DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 65).In some embodiments, the Fc domain contains EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66). It will be understood that SEQ ID NO: 66 contains five additional N-terminal amino acids compared to SEQ ID NO: 65. Thus, the numbering herein is given with respect to SEQ ID NO: 65 (or the equivalent SEQ ID NO: 63), but the numbering for SEQ ID NO: 66 can be found by adding 5. SEQ ID NO: 63 and SEQ ID NO: 65 differ by two amino acids. These two sequences are interchangeable, and it will be understood that if a mutation is given to sequence number 63, it will also apply to sequence number 65, and vice versa. Similarly, sequence numbers 64 and 66 also differ by only two amino acids, and these two sequences are interchangeable.

[0181] In some embodiments, the Fc domain consists of SEQ ID NO: 63. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 63. In some embodiments, the Fc domain includes or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97 or 99% homology with SEQ ID NO: 63. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 64. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 64. In some embodiments, the Fc domain includes or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97 or 99% homology with SEQ ID NO: 64. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 65. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 65. In some embodiments, the Fc domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology with SEQ ID NO: 65. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain comprises SEQ ID NO: 66. In some embodiments, the Fc domain of IgG1 comprises or consists of SEQ ID NO: 66. In some embodiments, the Fc domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology with SEQ ID NO: 66. Each possibility represents a distinct embodiment of the present invention.

[0182] In some embodiments, the Fc region can induce a cytotoxic effect. In some embodiments, the Fc region is configured to induce a cytotoxic effect. In some embodiments, the cytotoxic effect is directed towards target cells. In some embodiments, the cytotoxic effect occurs upon binding. In some embodiments, the cytotoxic effect is directed towards cells bound by a protein complex. In some embodiments, the cytotoxic effect is directed towards cells that bind to a protein complex. In some embodiments, the cytotoxic effect is mediated by the binding of immune cells to the Fc region. In some embodiments, the cytotoxic effect is mediated by the activation of immune cells by the Fc region. In some embodiments, the cytotoxic effect is mediated by the recruitment of immune cells by the Fc region. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are macrophages. In some embodiments, the T cells are cytotoxic T cells. In some embodiments, the T cells are CD8-positive T cells. In some embodiments, the Fc region induces antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the Fc region induces complement-dependent cell injury (CDC).

[0183] In some embodiments, the Fc region includes an Ig CH2 domain. In some embodiments, the Fc region includes an Ig heavy chain CH2 domain. In some embodiments, the Fc region includes an Ig CH3 domain. In some embodiments, the Fc region includes an Ig heavy chain CH3 domain. In some embodiments, the Fc region includes or consists of both an Ig CH2 domain and an Ig CH3 domain. In some embodiments, the Fc region includes or consists of both an Ig heavy chain CH2 domain and an Ig heavy chain CH3 domain. In some embodiments, the first chain includes a first portion of the Fc region, and the second chain includes a second portion of the Fc region. In some embodiments, the first portion includes a CH2 domain, a CH3 domain, or both. In some embodiments, the second portion includes a CH2 domain, a CH3 domain, or both. In some embodiments, the interface between the first portion of the Fc region and the second portion of the Fc region produces a functional Fc region. In some embodiments, the interface includes contact. In some embodiments, the interface includes adjacent positioning. In some embodiments, the interface involves the formation of the protein complex of the present invention. In some embodiments, the interface involves the dimerization of the first and second dimerization domains. In some embodiments, the CH2 domain is an Ig CH2 domain. In some embodiments, the CH2 domain is a heavy chain CH2 domain. In some embodiments, the CH3 domain is an Ig CH3 domain. In some embodiments, the CH3 domain is a heavy chain CH3 domain.

[0184] In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 36). In some embodiments, the CH2 domain consists of SEQ ID NO: 36. In some embodiments, SEQ ID NO: 36 is the IgG1 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO: 37). In some embodiments, the CH2 domain consists of SEQ ID NO: 37. In some embodiments, SEQ ID NO: 37 is the IgG2 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 38). In some embodiments, the CH2 domain consists of SEQ ID NO: 38. In some embodiments, SEQ ID NO: 38 is the IgG4 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO: 39). In some embodiments, the CH2 domain consists of SEQ ID NO: 39. In some embodiments, SEQ ID NO: 39 is the IgG3 CH2 domain.

[0185] In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40). In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41). In some embodiments, the CH3 domain consists of SEQ ID NO: 40. In some embodiments, the CH3 domain consists of SEQ ID NO: 41. In some embodiments, SEQ ID NO: 40 is the IgG1 CH3 domain. In some embodiments, SEQ ID NO: 41 is the IgG1 CH3 domain. In some embodiments, the sequence of SEQ ID NO: 40 is a sequence found primarily in European and American humans. In some embodiments, SEQ ID NO: 41 is a sequence primarily found in Asian humans. In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42). In some embodiments, the CH3 domain consists of SEQ ID NO: 42. In some embodiments, SEQ ID NO: 42 is the IgG2 CH3 domain. In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 43). In some embodiments, the CH3 domain consists of SEQ ID NO: 43. In some embodiments, SEQ ID NO: 43 is the IgG4 CH3 domain.In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 44). In some embodiments, the CH3 domain consists of SEQ ID NO: 44. In some embodiments, SEQ ID NO: 44 is the IgG3 CH3 domain.

[0186] In some embodiments, Fc contains a mutation. In some embodiments, the CH3 domain contains a mutation. In some embodiments, the first CH3 domain contains a first mutation. In some embodiments, the second CH3 domain contains a second mutation. In some embodiments, the CH2 domain contains a mutation. In some embodiments, the first CH2 domain contains a first mutation. In some embodiments, the second CH2 domain contains a second mutation. In some embodiments, both the CH2 and CH3 domains contain mutations. In some embodiments, the first CH2 domain and the first CH3 domain each contain a first mutation. In some embodiments, the second CH2 domain and the second CH3 domain each contain a second mutation. In some embodiments, the mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the second mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the mutation enables heterodimerization. In some embodiments, the mutation enables heterodimerization of the first and second chains. In some embodiments, enabling means promoting. In some embodiments, enabling means enhancing.

[0187] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutation or modification may be used to construct the polypeptide of the present invention. In some embodiments, an IgG-derived region is replaced with an IgA-derived region. In some embodiments, a TCR-derived region is inserted into a first CH3 domain, and a TCRb-derived region is inserted into a second CH3 domain. In some embodiments, the mutation is an insertion of a TCR-derived region. In some embodiments, the TCR is selected from TCR and TCRb. In some embodiments, the mutation is an insertion of a different Ig-derived region. Examples of these mutations can be found in Table 1. In some embodiments, the mutation is selected from the mutations in Table 1. In some embodiments, the first mutation is selected from the group of mutations provided in the row and second column of Table 1, and the second mutation is the group of mutations provided in the same row of Table 1 in the third column. The mutations in Table 1 are provided with IgG1 Kabat numbering unless otherwise specified, and the corresponding mutations can be produced in other IGs, specifically in other IgGs. In some embodiments, the first mutation is T366Y and the second mutation is Y407T. In some embodiments, the first mutations are S354C and T366W and the second mutations are Y349C, T366S, L368A and Y407V. In some embodiments, the first mutations are S364H and F405A and the second mutations are Y349T and T392F. In some embodiments, the first mutations are T350V, L351Y, F405A and Y407V and the second mutations are T350V, T366L, K392L and T394W. In some embodiments, the first mutations are K392D and K409D, and the second mutations are E356K and D399K. In some embodiments, the first mutations are D221E, P228E and L368E, and the second mutations are D221R, P228R and K409R. In some embodiments, the first mutations are K360E and K409W, and the second mutations are Q347R, D399V and F405T.In some embodiments, the first mutations are K360E, K409W, and Y349C, and the second mutations are Q347R, D399V, F405T, and S354C. In some embodiments, the first mutation is F405L, and the second mutation is K409R. In some embodiments, the first mutations are K360D, D399M, and Y407A, and the second mutations are E345R, Q347R, T366V, and K409V. In some embodiments, the first mutations are Y349S, K370Y, T366M, and K409V, and the second mutations are E356G, E357D, S364Q, and Y407A. In some embodiments, the first mutation is T366K, and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, as well as Y349D and R355D. In some embodiments, the first mutations are T366K and C351K, and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, as well as Y349D and R355D. In some embodiments, the first mutations are L351D and L368E, and the second mutations are L351K and T366K. In some embodiments, the first mutations are L368D and K370S, and the second mutations are E357Q and S364K. In some embodiments, the first mutation is T366W, and the second mutations are T366S, L368A, and Y407V. In some embodiments, Ig is IgG2, and the first mutations are C223E, P228E, and L368E, and the second mutations are C223R, E225R, P228R, and K409R. In some embodiments, the first mutation is S354C or T366W, and the second mutations are Y349C, T366S, L368A, or Y407V. In some embodiments, the first mutation is S364H or F405A, and the second mutation is Y349T or T392F.In some embodiments, the first mutation is T350V, L351Y, F405A, or Y407V, and the second mutation is T350V, T366L, K392L, or T394W. In some embodiments, the first mutation is K392D or K409D, and the second mutation is E356K or D399K. In some embodiments, the first mutation is D221E, P228E, or L368E, and the second mutation is D221R, P228R, or K409R. In some embodiments, the first mutation is K360E or K409W, and the second mutation is Q347R, D399V, or F405T. In some embodiments, the first mutation is K360E, K409W, or Y349C, and the second mutation is Q347R, D399V, F405T, or S354C. In some embodiments, the first mutation is K360D, D399M, or Y407A, and the second mutation is E345R, Q347R, T366V, or K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, or K409V, and the second mutation is E356G, E357D, S364Q, or Y407A. In some embodiments, the first mutation is L351D or L368E, and the second mutation is L351K or T366K. In some embodiments, the first mutation is L368D or K370S, and the second mutation is E357Q or S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A, or Y407V. In some embodiments, Ig is IgG2, and the first mutation is C223E, P228E, or L368E, and the second mutation is C223R, E225R, P228R, or K409R. In some embodiments, the CH3 domain contains or consists of GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45).In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 46). In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47). In some embodiments, the CH3 domain contains or consists of GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 48). In some embodiments, the first strand contains SEQ ID NO: 60, and the second strand contains SEQ ID NO: 61. In some embodiments, SEQ ID NOs: 60 and 61 heterodimerize, reducing homodimerization.

[0188] [Table 1]

[0189] In some embodiments, the Fc domain includes at least one mutation that increases effector function. In some embodiments, the Fc domain includes at least one mutation that increases CDC, ADCC, or both. In some embodiments, the Fc domain includes at least one mutation that increases CDC. In some embodiments, the Fc domain includes at least one mutation that increases ADCC. In some embodiments, the Fc domain includes at least one mutation that increases antibody effector function. In some embodiments, the Fc domain includes at least one mutation that increases antibody stability. In some embodiments, stability is half-life. In some embodiments, half-life is circulating half-life. In some embodiments, half-life is blood half-life. In some embodiments, blood is serum.

[0190] In some embodiments, the mutation reduces effector function. In some embodiments, the effector function includes ADCC, CDC, or both. In some embodiments, the reduced effector function includes reduced cytotoxicity. In some embodiments, reduction is abolition. In some embodiments, Fc is derived from IgG1 or IgG3, and the mutation reduces effector function. In some embodiments, Fc is derived from IgG1 and includes at least one mutation that reduces effector function. Mutations that reduce effector function are well known in the art, and any such mutation can be used. An example of such a mutation can be found in Saunders, 2019, "Conceptual approaches to modulating antibody effector functions and circulation half-life," Front Immunol., Jun 7;10:1296, which is incorporated herein by reference in its entirety.

[0191] It is known to those skilled in the art that IgG2 and IgG4 have significantly reduced effector function and are generally not cytotoxic in nature. Furthermore, mutations in IgG4, such as S228P and L235E, are known to further reduce effector function. Furthermore, mutations that reduce the cytotoxicity / effector function of IgG1 and IgG3 are well known in the art. In some embodiments, IgG contains at least one mutation. In some embodiments, the mutation is multiple mutations. In some embodiments, the mutation reduces cytotoxicity. In some embodiments, the mutation increases stability. In some embodiments, the mutation reduces aggregation. In some embodiments, the Fc domain contains at least one mutation that reduces antibody effector function. In some embodiments, the Fc domain contains at least one mutation that reduces ADCC. In some embodiments, the at least one mutation that reduces ADCC is an LALA mutation. As used herein, an LALA mutation refers to a mutation of two consecutive leucine residues to an alanine residue. In some embodiments, the LALA mutation is located within the hinge domain. In some embodiments, the hinge domain is the hinge domain of IgG1. In some embodiments, the LALA mutation is the mutation to A19 and A20 of L19 and L20 of SEQ ID NO: 22. In some embodiments, the LALA mutation hinge includes the L19A and L20A mutations of SEQ ID NO: 22. In some embodiments, the Fc domain includes a hinge domain containing EPKSCDKTHTCPPCPAPEAA (SEQ ID NO: 49). In some embodiments, the Fc domain containing the LALA mutation includes SEQ ID NO: 49. In some embodiments, the Fc domain includes a hinge domain consisting of SEQ ID NO: 49. In some embodiments, the LALA mutation hinge domain consists of SEQ ID NO: 49. In some embodiments, the LALA mutation is the L234A and L235A mutations of Fc. In some embodiments, at least one mutation that reduces ADCC is the N297A mutation. In some embodiments, the N297A mutation is located within the CH2 domain.In some embodiments, the N297A mutation is a mutation of asparagine 59 to alanine in SEQ ID NO: 36. In some embodiments, the N297A mutation CH2 domain includes the N59A mutation in SEQ ID NO: 36. In some embodiments, the Fc domain includes a CH2 domain containing SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 50). In some embodiments, the Fc domain includes a CH2 domain consisting of SEQ ID NO: 50. In some embodiments, the N297A mutation CH2 domain consists of SEQ ID NO: 50.

[0192] In some embodiments, the mutations that reduce cytotoxicity include LALA mutations. In some embodiments, the mutations that reduce cytotoxicity include PG-LALA mutations. In some embodiments, the mutation is a mutation of proline 329 to glycine in the IgG1 human heavy chain (P329G). In some embodiments, the P to G mutation is a mutation of P109 to G in SEQ ID NO: 63. In some embodiments, the mutation is a mutation of leucine 234 to alanine in the IgG1 human heavy chain (L234A). In some embodiments, the L to A mutation is a mutation of L14 to A in SEQ ID NO: 63. In some embodiments, the mutation is a mutation of leucine 235 to alanine in the IgG1 human heavy chain (L235A). In some embodiments, the L to A mutation is a mutation of L15 to A in SEQ ID NO: 63. In some embodiments, the mutations include P109G, L14A, and L15A in SEQ ID NO: 63. In some embodiments, the multiple mutations include L14A and L15A of SEQ ID NO: 63. In some embodiments, the multiple mutations include P329G, L234A and L235A of the IgG1 human heavy chain. In some embodiments, the multiple mutations include L234A and L235A of the IgG1 human heavy chain. It will be understood by those skilled in the art that parallel mutations can also be performed on the IgG3 heavy chain or the heavy chain of non-human IgG1. In some embodiments, the multiple mutations that reduce cytotoxicity include YTE mutations. In some embodiments, the mutation is a mutation of methionine 252 to tyrosine (M252Y) of the IgG1 human heavy chain. In some embodiments, the M to Y mutation is a mutation of M32 to Y of SEQ ID NO: 63. In some embodiments, the mutation is a mutation of serine 254 to threonine (S254T) of the IgG1 human heavy chain. In some embodiments, the S to T mutation is a mutation of S34 to T of SEQ ID NO: 63. In some embodiments, the mutation is a mutation of threonine 256 to glutamate in the IgG1 human heavy chain (T256E). In some embodiments, the T-to-E mutation is a mutation of T36 to E in SEQ ID NO: 63. In some embodiments, multiple mutations include M32Y, S34T, and T36E in SEQ ID NO: 63.In some embodiments, the multiple mutations include M252Y, S254T, and T256E of the IgG1 human heavy chain. In some embodiments, the mutation is the mutation (N297) at asparagine 297 of the IgG1 human heavy chain. In some embodiments, asparagine is mutated to alanine (N297A). In some embodiments, asparagine is mutated to glutamine (N297Q). In some embodiments, asparagine is N77 of SEQ ID NO: 63 (N77A or N77Q).

[0193] In some embodiments, the mutation increases the half-life of a molecule, peptide, polypeptide, or protein complex. In some embodiments, the mutation that increases the half-life is the mutation that increases binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutation that increases binding to FcRn is selected from the mutations provided in Table 4. In some embodiments, the mutation is a mutation of asparagine 434 to histidine (N434H). In some embodiments, the N434H mutant Fc domain contains the N214H mutation of SEQ ID NO: 63 or 65. In some embodiments, the mutation is a mutation of valine 308 to proline (V308P). In some embodiments, the H435A mutant Fc domain contains the H215A mutation of SEQ ID NO: 63 or 65. In some embodiments, the mutation attenuates binding to FcRN. In some embodiments, the mutation that attenuates binding is a mutation of histidine 435 to alanine (H435A). In some embodiments, the H435A mutant Fc domain contains the H215A mutation of SEQ ID NO: 63 or 65. In some embodiments, the mutations that increase binding to FcRn are multiple mutations. In some embodiments, the multiple mutations include, or consist of, a mutation of methionine 252 to tyrosine (M252Y), a mutation of serine 234 to threonine, and a mutation of threonine 256 to glutamate (T256E) (also named YTE). In some embodiments, the M252Y / S254T / T256E mutant Fc domain contains the M32Y, S34T, and T35E mutations of SEQ ID NO: 63 or 65. In some embodiments, the multiple mutations include, or consist of, a mutation of methionine 428 to leucine (M428L) and a mutation of asparagine 434 to serine (N434S) (also named LS). In some embodiments, the M428L / N434S mutant Fc domain includes the M208L and N214S mutations of SEQ ID NO: 63 or 65. In some embodiments, multiple mutations include or consist of M428L and alanine mutation (N434A) (also named LA) of asparagine 434.In some embodiments, the M428L / N434A mutant Fc domain includes the M208L and N214A mutations of SEQ ID NO: 63 or 65. In some embodiments, a plurality of mutations include, or consist of, a mutation of threonine 250 to glutamine (T250Q) and a mutation of methionine 428 to leucine (M428L) (also named QL). In some embodiments, the T250Q / M428L mutant Fc domain includes the T30Q and M208L mutations of SEQ ID NO: 63 or 65. In some embodiments, a plurality of mutations include, or consist of, a mutation of histidine 433 to lysine (H433K) and a mutation of asparagine 434 to phenylalanine (N434F). In some embodiments, the H433K / N434F mutant Fc domain includes the H213K and N214F mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality includes or consists of M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the M252Y / S254T / T256E / H433K / N434F mutant Fc domain includes the M32Y, S34T, T35E, H213K, and N214F mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality includes or consists of the mutation of threonine 307 to alanine (T307A), the mutation of glutamate 380 to alanine (E380A), and the mutation of asparagine 434 to alanine (N434A). In some embodiments, the T307A / E380A / N434A mutant Fc domain includes the T87A, E160A, and N214A mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality includes or consists of a mutation of methionine 252 to tyrosine (M252Y), a mutation of valine 308 to protein (V308P), and a mutation of asparagine 343 to tyrosine (N343Y). In some embodiments, the M252Y / V308P / N343Y mutant Fc domain includes the M32Y, V88P, and N123Y mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality includes or consists of M252Y, a mutation of valine 308 to proline (V308P), and a mutation of asparagine 434 to tyrosine (N434Y).In some embodiments, the M252Y / V308P / N434Y mutant Fc domain includes the M32Y, V88P, and N214Y mutations of SEQ ID NO: 63 or 65. In some embodiments, a plurality of mutations include, or consist of, a mutation of histidine 258 to aspartic acid (H258D), a mutation of threonine 307 to glutamine (T307Q), and a mutation of alanine 378 to valine (A378V). In some embodiments, the H258D / T307Q / A378V mutant Fc domain includes the H38D, T87Q, and A158V mutations of SEQ ID NO: 63 or 65. In some embodiments, a plurality of mutations include, or consist of, a mutation of leucine 309 to aspartic acid (L309D), a mutation of glutamine 311 to histidine (Q311H), and a mutation of asparagine 434 to serine (N434S). In some embodiments, the L309D / Q311H / N434S mutant Fc domain includes the L89D, Q91H, and N214A mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality of attenuating binding includes or consists of the mutation of isoleucine 253 to alanine (I253A), H435A, and histidine 436 to alanine (H436A). In some embodiments, the I253A / H435A / H436A mutant Fc domain includes the I33A, H215A, and H216A mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality of attenuating binding includes or consists of I253A, the mutation of histidine 310 to alanine (H310A), and H435A. In some embodiments, the I253A / H310A / H435A mutant Fc domain includes the I33A, H90A, and H215A mutants of SEQ ID NO: 63 or 65.

[0194] [Table 2]

[0195] In some embodiments, the mutation is one that reduces binding to the Fc receptor. In some embodiments, the Fc receptor is FcγR. In some embodiments, FcγR is FcγRI. In some embodiments, the mutation is one that reduces binding to C1q. In some embodiments, the mutation that reduces binding to the Fc receptor reduces ADCC. In some embodiments, the mutation is a mutation at N297. Since N-glycan binds to N297, the mutation in this case disables glycosylation of this residue. In some embodiments, the mutation at N297 is a mutation to alanine (N297A). In some embodiments, the mutation at N297 is a mutation to glutamine (N297Q). In some embodiments, the mutation at N297 is a mutation to glycine (N297G). In some embodiments, the N297A mutant CH2 domain contains the N59A mutation of SEQ ID NO: 36. In some embodiments, the N297A mutant Fc domain contains the N77A mutation of SEQ ID NO: 63 or 65. In some embodiments, the N297Q mutant CH2 domain includes the N59Q mutation of SEQ ID NO: 36. In some embodiments, the N297Q mutant Fc domain includes the N77Q mutation of SEQ ID NO: 63 or 65. In some embodiments, the N297G mutant CH2 domain includes the N59G mutation of SEQ ID NO: 36. In some embodiments, the N297G mutant Fc domain includes the N77G mutation of SEQ ID NO: 63 or 65. In some embodiments, the mutations are multiple mutations that reduce binding to the Fc receptor. In some embodiments, the multiple mutations include, or consist of, mutations from glycine 236 to arginine (G236R) and mutations from leucine 328 to arginine (L328R). In some embodiments, the G236R / L328R mutant Fc includes a hinge domain containing the G21R mutation of SEQ ID NO: 22 and a CH2 domain containing the L90R mutation of SEQ ID NO: 36. In some embodiments, the G236R / L328R mutant Fc domain includes the G16R and L108R mutations of SEQ ID NO: 63 or 65. In some embodiments, multiple mutations include, or consist of, a mutation from serine 298 to glycine (S298G) and a mutation from threonine 299 to alanine (T299A).In some embodiments, the S298G / T299A mutant CH2 domain includes the S60G and T61A mutations of SEQ ID NO: 36. In some embodiments, the S298G / T299A mutant Fc domain includes the S78G and T79A mutations of SEQ ID NO: 63 or 65. In some embodiments, multiple mutations include or consist of leucine 234 to phenylalanine (L234F), leucine 235 to glutamic acid (L235E), and aspartic acid 265 to arginine (D265A). In some embodiments, the L234F / L235E / D265A mutant Fc includes a hinge domain containing the L19F and L20E mutations of SEQ ID NO: 22 and a CH2 domain containing the D27A mutation of SEQ ID NO: 36. In some embodiments, the L234F / L235E / D265A mutant Fc domain includes the L14F, L15E, and D45A mutations of SEQ ID NO: 63 or 65. In some embodiments, multiple mutations include, or consist of, a mutation from leucine 234 to alanine (L234A), a mutation from leucine 235 to alanine (L235A), and a mutation from proline 329 to glycine (P329G). In some embodiments, the L234A / L235A / P329G mutant Fc domain includes a hinge domain containing the L19A and L20A mutations of SEQ ID NO: 22, and a CH2 domain containing the P91G mutation of SEQ ID NO: 36. In some embodiments, the L234A / L235A / P329G mutant Fc domain includes the L14A, L15A, and P109G mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality includes or consists of L234F, L235E, and proline 331 to serine mutation (P331S). In some embodiments, the L234F / L235E / P331S mutant Fc includes a hinge domain containing the L19F and L20E mutations of SEQ ID NO: 22 and a CH2 domain containing the P93S mutation of SEQ ID NO: 36. In some embodiments, the L234F / L235E / P331S mutant Fc domain includes the L14F, L15E, and P111S mutations of SEQ ID NO: 63 or 65.In some embodiments, the multiple mutations include, or consist of, a mutation from leucine 235 to alanine (L235A), a mutation from glycine 237 to alanine (G237A), and a mutation from glutamic acid 318 to alanine (E318A). In some embodiments, the L235A / G237A / E318A mutation Fc includes a hinge domain containing the L20A and G22A mutations of SEQ ID NO: 22 and a CH2 domain containing the E80A mutation of SEQ ID NO: 36. In some embodiments, the L235A / G237A / E318A mutation Fc domain includes the L15A, G17A, and E98A mutations of SEQ ID NO: 63 or 65.

[0196] In some embodiments, Fc is modified to reduce its binding to the Fc receptor. In some embodiments, the modification is the removal of glycosylation. In some embodiments, Fc glycosylation is removed enzymatically. In some embodiments, enzymatic deglycosylation is carried out using a deglycosylase. In some embodiments, enzymatic deglycosylation is carried out using a cleaving enzyme that cleaves the sugar. Examples of enzymes for deglycosylation include, but are not limited to, peptide-N-glycosidase F (PNGase) and endoglycosidase H (EndoH). Kits for deglycosylation are also commercially available.

[0197] In some embodiments, the mutation is a mutation that increases binding to the Fc receptor. In some embodiments, the Fc receptor is selected from FcγRI, FcγRIIA, FcγRIIIA, and FcγRIIIB. In some embodiments, the Fc receptor is FcγRI. In some embodiments, the mutation is a mutation of serine 267 to glutamate (S267E). In some embodiments, the S267E mutant CH2 domain contains the S29E mutation of SEQ ID NO: 36. In some embodiments, the S267E mutant Fc domain contains the S47E mutation of SEQ ID NO: 63 or 65. In some embodiments, the mutation is a mutation of proline 238 to aspartate (P238D). In some embodiments, the P238D mutant hinge domain contains the P23D mutation of SEQ ID NO: 22. In some embodiments, the P238D mutant Fc domain contains the P18D mutation of SEQ ID NO: 63 or 65. In some embodiments, the mutation is multiple mutations that increase binding to the Fc receptor. In some embodiments, the multiple variants include or consist of S267E and a mutation of leucine 328 to phenylalanine (L328F) (also known as SELF). In some embodiments, the S267E / L328F mutant CH2 domain includes the S29E and L90F mutations of SEQ ID NO: 36. In some embodiments, the S267E / L328F mutant Fc domain includes the S47E and L108F mutations of SEQ ID NO: 63 or 65. In some embodiments, the multiple variants include or consist of S267E, as well as a mutation of histidine 268 to phenylalanine (H268F) and a mutation of serine 324 to threonine (S324T) (also known as EFT). In some embodiments, the S267E / H268F / S324T mutant CH2 domain includes the S29E, H30F, and S86T mutations of SEQ ID NO: 36. In some embodiments, the S267E / H268F / S324T mutant Fc domain includes the S47E, H48F, and S104T mutations of sequence number 63 or 65.In some embodiments, the plurality includes or consists of mutations of glycine 237 to aspartic acid (G237D), P238D, proline 271 to glycine (P271G), and alanine 330 to arginine (A330R) (also named V9). In some embodiments, the G237D / P238D / P271G / A330R mutant polypeptide includes a mutant hinge domain containing the G22D and P23D mutations of SEQ ID NO: 22, and a mutant CH2 domain containing the P33G and A92R mutations of SEQ ID NO: 36. In some embodiments, the G237D / P238D / P271G / A330R mutant Fc domain includes the G17D, P18D, P51G, and A110R mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality includes or consists of G237D, P238D, a mutation of histidine 268 to aspartic acid (H268D), P271G, and A330R (also named V11). In some embodiments, the G237D / P238D / H268D / P271G / A330R mutant polypeptide includes a mutant hinge domain containing the G22D and P23D mutations of SEQ ID NO: 22, and a mutant CH2 domain containing the H30D, P33G, and A92R mutations of SEQ ID NO: 36. In some embodiments, the G237D / P238D / H268D / P271G / A330R mutant Fc domain includes the G17D, P18D, H48D, P51G, and A110R mutations of SEQ ID NO: 63 or 65. In some embodiments, the plurality includes or consists of mutations of glutamic acid 233 to aspartic acid (E233D), G237D, P238D, H268D, P271G, and A330R (also named V12). In some embodiments, the E233D / G237D / P238D / H268D / P271G / A330R mutant polypeptide includes a mutant hinge domain containing the E18D, G22D, and P23D mutations of SEQ ID NO: 22, and a mutant CH2 domain containing the H30D, P33G, and A92R mutations of SEQ ID NO: 36.In some embodiments, the E233D / G237D / P238D / H268D / P271G / A330R mutant Fc domain includes the E13D, G17D, P18D, H48D, P51G, and A110R mutants of SEQ ID NO: 63 or 65.

[0198] The S267E mutation was found to enhance affinity for inhibitory FcγRIIB and activated FcγRIIa. The SELF mutation in hIgG1 resulted in a substantially 430-fold increase in binding to FcγRIIB compared to human WT IgG1, with minimal modification to binding to FcγRI and FcγRIIA-H131. The EFT mutation was found to increase FcγRIIB binding by 18-fold compared to human WT IgG1. EFT also increased CDC, ADCC, and antibody-dependent phagocytic (ADCP) activity through enhanced binding to C1q and activator FcG receptors. In some embodiments, the mutations that increase ADCC are multiple mutations in EFT. P238D demonstrated enhanced binding to FcγRIIB with an affinity increase of approximately 4.3-fold compared to WT human IgG1. P238D also significantly reduced binding to all other activated Fcg receptors. V9 significantly enhanced antibody affinity to hFcγRIIB by approximately 32-fold compared to WT IgG1. V9 was also found to decrease affinity to the hFcγRIIAR131 allele by approximately 3-fold compared to WT IgG1. V11 significantly enhanced antibody affinity to hFcγRIIB by approximately 96-fold compared to human WT IgG1, while decreasing affinity to hFcγRIIA R131 by approximately 3-fold. V12 demonstrated a significant enhancement of binding to FcγRIIB by 217-fold compared to human WT IgG1. The V12 mutation also showed no detectable binding to the FcγRIIIA allotype and reduced FcγRI binding (0.061-fold change compared to wt IgG1) and FcγRIIA-H131 (0.068-fold change compared to wt IgG1). It should be noted that V12 slightly improves binding to FcγRIIA-R131, with a twofold increase in binding compared to WT hIgG1.

[0199] Mutations that result in the functions listed above are well known in the art, and any such mutations can be used. Examples of such mutations can be found at least in KOSaunders, 2019, "Conceptual approaches to modulating antibody effector functions and circulation half-life", Front, Immunol., June 7, 2019; 10:1296, which is incorporated herein by reference in its entirety. Table 1 of Saunders provides Fc modifications that enhance antibody effector function. Table 2 of Saunders provides Fc modifications that improve antibody circulation half-life. Table 3 of Saunders provides Fc modifications that inhibit antibody effector function. It will be understood by those skilled in the art that parallel mutations can also be performed in IgG3 heavy chains or non-human IgG1 heavy chains. It will be understood that the numbers given herein refer to full-length IgG including the variable domain. The numbers may be shifted to correspond to the positions of these amino acids only in the Fc portion of IgG.

[0200] In some embodiments, the mutation increases effector function. In some embodiments, the mutation increases ADCC. In some embodiments, the mutation is not one that increases CDC. In some embodiments, the mutation increases ADCC but not CDC. It will be understood by those skilled in the art that unmodified Fc is not sufficiently cytotoxic to overcome the booster effect produced by the molecules of the present invention, but Fc containing a mutation that increases ADCC is sufficiently cytotoxic to overcome the booster effect. In some embodiments, the effector function includes ADCC. In some embodiments, the effector function includes ADCC but does not include CDC. In some embodiments, the increased effector function includes increased cytotoxicity. In some embodiments, Fc is derived from IgG1 or IgG3, and the mutation increases effector function. In some embodiments, Fc is derived from IgG1 and includes at least one mutation that increases effector function. Mutations that increase effector function are well known in the art, and any such mutation can be used. Examples of such mutations can be found in Liu, 2020, "Fc-engineering for modulated effector functions-improving antibodies for cancer treatment," Antibodies (Basel), December 2020;9(4):64, which is incorporated herein by reference in its entirety.

[0201] In some embodiments, the mutations that increase ADCC are multiple mutations that increase ADCC. In some embodiments, the multiple mutations include a mutation of leucine 235 to valine (L235V), a mutation of phenylalanine 243 to leucine (F243L), a mutation of arginine 292 to proline (R292P), a mutation of tyrosine 300 to leucine (Y300L), and a mutation of proline 296 to leucine (P396L) within human IgG1. In some embodiments, the multiple mutations include a mutation of leucine 15 to valine (L15V), a mutation of phenylalanine 23 to leucine (F23L), a mutation of arginine 72 to proline (R72P), a mutation of tyrosine 80 to leucine (Y80L), and a mutation of proline 176 to leucine (P176L) within Sequence ID No. 63. In some embodiments, the multiple mutations include a mutation of serine 239 to aspartic acid (S239D) and a mutation of isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include a mutation of serine 19 to aspartic acid (S19D) and a mutation of isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 63. In some embodiments, the S239D / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include a mutation of serine 239 to aspartic acid (S239D), a mutation of alanine 330 to leucine (A330L), and a mutation of isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include a mutation of serine 19 to aspartic acid (S19D), alanine 110 to leucine (A110L), and isoleucine 112 to glutamic acid (I112E) in SEQ ID NO: 63. In some embodiments, the S239D / A330L / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include a mutation of glycine 236 to alanine (G236A), alanine 330 to leucine (A330L), and isoleucine 332 to glutamic acid (I332E) in human IgG1.In some embodiments, the multiple mutations include a mutation of glycine 16 to alanine (G16A), a mutation of alanine 110 to leucine (A110L), and a mutation of isoleucine 112 to glutamate (I112E) in SEQ ID NO: 63. In some embodiments, the multiple mutations include a mutation of serine 298 to alanine (S298A), a mutation of glutamate 333 to alanine (E333A), and a mutation of lysine 334 to alanine (K334A) in human IgG1. In some embodiments, the multiple mutations include a mutation of serine 78 to alanine (S78A), a mutation of glutamate 113 to alanine (E113A), and a mutation of lysine 114 to alanine (K114A) in SEQ ID NO: 63. In some embodiments, the multiple mutations include a mutation of proline 247 to isoleucine (P247I) and a mutation of alanine 339 to glutamine (A339Q) within human IgG1. In some embodiments, the multiple mutations include a mutation of proline 27 to isoleucine (P27I) and a mutation of alanine 119 to glutamine (A119Q) within SEQ ID NO: 63. In some embodiments, the multiple mutations include a mutation of glycine 236 to alanine (G236A), serine 239 to aspartic acid (S239D), and isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include a mutation of glycine 16 to alanine (G16A), serine 19 to aspartic acid (S19D), and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 63. In some embodiments, the G236A / S239D / I332E mutation also increases ADCP.In some embodiments, the mutations include mutations in the first heavy chain of human IgG1, such as a mutation of lysine 234 to tyrosine (L234Y), a mutation of lysine 235 to glutamine (L235Q), a mutation of glycine 236 to tryptophan (G236W), a mutation of serine 239 to methionine (S239M), a mutation of histidine 268 to aspartic acid (H268D), a mutation of aspartic acid 270 to glutamic acid (D270E), and a mutation of serine 298 to alanine (S298A), as well as mutations in the second heavy chain of IgG1, such as a mutation of aspartic acid 270 to glutamic acid (D270E), a mutation of lysine 326 to aspartic acid (K26D), a mutation of alanine 330 to methionine (A330M), and a mutation of lysine 334 to glutamic acid (K334E). In some embodiments, multiple mutations include mutations in the first chain of SEQ ID NO: 63, specifically the mutation of lysine 14 to tyrosine (L14Y), lysine 15 to glutamine (L15Q), glycine 16 to tryptophan (G16W), serine 19 to methionine (S19M), histidine 48 to aspartic acid (H48D), aspartic acid 50 to glutamic acid (D50E), and serine 78 to alanine (S78A), as well as mutations in the second chain of SEQ ID NO: 63, specifically the mutation of aspartic acid 50 to glutamic acid (D50E), lysine 326 to aspartic acid (K106D), alanine 110 to methionine (A110M), and lysine 114 to glutamic acid (K114E). It will be understood that all of the mutations listed above given for SEQ ID NO: 63 also apply to SEQ ID NO: 65. In fact, they also apply to sequence numbers 64 and 66, but all numbering given above in this specification must be incremented by 5 for these sequences.

[0202] In some embodiments, the ADCC-enhanced Fc domain contains the L15V / F23L / R72P / Y80L / P176L mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 63 and 65. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 67). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs. 67. In some embodiments, the Fc containing the L15V / F23L / R72P / Y80L / P176L mutation is sequence number 67. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to sequence number 67 and contains the L15V / F23L / R72P / Y80L / P176L mutation.

[0203] In some embodiments, the ADCC-enhanced Fc domain contains the S19D / A110L / I112E mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 63 and 65. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 68). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs. 68. In some embodiments, the Fc containing the S19D / A110L / I112E mutation is sequence number 68. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97, or 99% identical to sequence number 68 and contains the S19D / A110L / I112E mutation.

[0204] In some embodiments, the ADCC-enhanced Fc domain contains the G16A / A110L / I112E mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 63 and 65. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 70). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs. 70. In some embodiments, the Fc containing the G16A / A110L / I112E mutation is SEQ ID NO: 70. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 70 and contains the G16A / A110L / I112E mutation.

[0205] In some embodiments, the mutation increases CDC. In some embodiments, multiple mutations increase CDC. In some embodiments, multiple mutations include a mutation in human IgG1 of glycine 236 to alanine (G236A), serine 267 to glutamate (S267E), histidine 268 to phenylamine (H268F), serine 324 to threonine (S324T), and isoleucine 332 to glutamate (I332E). In some embodiments, multiple mutations include a mutation in SEQ ID NO: 63 of glycine 16 to alanine (G16A), serine 47 to glutamate (S47E), histidine 48 to phenylamine (H48F), serine 104 to threonine (S104T), and isoleucine 112 to glutamate (I112E). It will be understood that all the mutations listed above given for sequence number 63 also apply to sequence number 65. In fact, they also apply to sequence numbers 64 and 66, but all the numbering given above in this specification must be increased by 5 for these sequences.

[0206] In some embodiments, the CDC-enhanced Fc domain contains the G16A / S47E / H48F / S104T / I112E mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 63 and 65. In some embodiments, the CDC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVTNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 69). In some embodiments, the CDC-enhanced Fc domain consists of SEQ ID NOs. 69. In some embodiments, the Fc containing the G16A / S47E / H48F / S104T / I112E mutation is SEQ ID NO: 69. In some embodiments, the Fc domain with increased CDC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 69 and contains the G16A / S47E / H48F / S104T / I112E mutation.

[0207] In some embodiments, the effector domain is selected from SEQ ID NOs: 67-70. In some embodiments, the effector domain includes any one of SEQ ID NOs: 67-70. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 67-70. In some embodiments, the effector domain is selected from SEQ ID NOs: 67, 68, and 70. In some embodiments, the effector domain includes any one of SEQ ID NOs: 67, 68, and 10. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 67, 68, and 70. In some embodiments, the effector domain has at least 75, 80, 85, 90, 92, 95, 97, or 99% identity with any one of SEQ ID NOs: 67, 68, and 70 and retains increased ADCC compared to the control Fc domain. In some embodiments, the control Fc domain is an unmodified Fc domain. In some embodiments, the unmodified Fc is a naturally occurring Fc. In some embodiments, the unmodified Fc is a naturally occurring human Fc.

[0208] In some embodiments, Fc is modified to increase ADCC. In some embodiments, the modification is the removal of fucosylation. In some embodiments, Fc fucosylation is removed enzymatically. In some embodiments, Fc is defucosylated. In some embodiments, the method includes performing defucosylation of the molecule. In some embodiments, the molecule of the present invention is produced in a cell line engineered to produce a defucosylated molecule.

[0209] In some embodiments, the mutation increases CDC. In some embodiments, multiple mutations increase CDC. In some embodiments, multiple mutations include a mutation in human IgG1 of glycine 236 to alanine (G236A), serine 267 to glutamate (S267E), histidine 268 to phenylamine (H268F), serine 324 to threonine (S324T), and isoleucine 332 to glutamate (I332E). In some embodiments, multiple mutations include a mutation in SEQ ID NO: 63 of glycine 16 to alanine (G16A), serine 47 to glutamate (S47E), histidine 48 to phenylamine (H48F), serine 104 to threonine (S104T), and isoleucine 112 to glutamate (I112E). In some embodiments, the multiple mutations include a mutation of lysine 326 to tryptophan (K326W) and a mutation of glutamate 333 to serine (E333S) within human IgG1. In some embodiments, the multiple mutations include a mutation of lysine 106 to tryptophan (K106W) and a mutation of glutamate 113 to serine (E113S) within SEQ ID NO: 63. In some embodiments, the multiple mutations include a mutation of glutamate 345 to arginine (E345R), glutamate 430 to glycine (E430G), and serine 440 to tyrosine (S440Y) within human IgG1. In some embodiments, the multiple mutations include a mutation of glutamate 125 to arginine (E125R), glutamate 210 to glycine (E210G), and serine 220 to tyrosine (S220Y) within SEQ ID NO: 63. It will be understood that all the mutations listed above given for sequence number 63 also apply to sequence number 65. In fact, they also apply to sequence numbers 64 and 66, but all the numbering given above in this specification must be increased by 5 for these sequences.

[0210] In some embodiments, the effector portion is a drug. In some embodiments, the protein is a TSHR-ECD drug conjugate. In some embodiments, the protein is a TSHR-Fc drug conjugate. In some embodiments, the complex is a TSHR-ECD drug conjugate. In some embodiments, the complex is a TSHR-Fc drug conjugate. In some embodiments, the effector portion is cytotoxic. In some embodiments, the effector portion is radioactive. In some embodiments, the effector portion is a radioactive portion. In some embodiments, the effector portion is radiolabeled. In some embodiments, the effector portion is a chemotherapeutic agent. In some embodiments, the effector portion is not a chemotherapeutic agent. In some embodiments, the effector portion is toxic to non-replicating cells. In some embodiments, the toxicity is lethal. In some embodiments, the effector portion is sufficient to kill cells. Drug conjugation, particularly drug conjugation to antibody backbones, is well known in the art, and any conjugation method may be used.

[0211] In some embodiments, the effector moiety is amatoxin. In some embodiments, the effector moiety is amanitin. Amatoxins are a group of toxic compounds found in poisonous mushrooms. They consist of eight amino acid residues arranged in a macrocyclic motif and inhibit RNA polymerase. Amatoxins are also known as amanitin. In some embodiments, amanitin is selected from alpha-amanitin, beta-amanitin, gamma-amanitin, epsilon-amanitin, amanulin, amanulinic acid, amaninamide, amanin, and proamanulin. In some embodiments, amanitin is alpha-amanitin. In some embodiments, the effector moiety is alpha-amanitin.

[0212] In some embodiments, the chemotherapeutic agent is an anthracycline. In some embodiments, the effector portion is an anthracycline. Anthracyclines are a class of drugs extracted from Streptomyces bacteria that intercalate into DNA and cause cytotoxicity primarily by inhibiting topoisomerase. Examples of anthracyclines include, but are not limited to, doxorubicin, daunorubicin, epirubicin, nemorubicin, PNU-159682, radirubicin, and idarubicin. In some embodiments, the anthracycline is PNU-159682.

[0213] In some embodiments, the chemotherapeutic agent is an anthramycin dimer. In some embodiments, the anthramycin dimer is pyrrolobenzodiazepine (PBD). In some embodiments, the chemotherapeutic agent is PBD. In some embodiments, the anthramycin dimer is indolinobenzodiazepine dimer (IGN). In some embodiments, the chemotherapeutic agent is pyridinobenzodiazepine (PDD). In some embodiments, the anthramycin dimer is PDD. In some embodiments, the effector moiety is PBD. In some embodiments, the effector moiety is PDD. PBD and PDD are families of DNA minor glove binders that inhibit DNA and RNA synthesis. In some embodiments, PBD is a PBD dimer. Examples of PBD and PDD include, but are not limited to, anthramycin, SJG-136, NS694501, and FGX2-62. In some embodiments, PBD is anthramycin. In some embodiments, the effector moiety is anthramycin. In some embodiments, anthramycin is anthramycin-methyl ether (AME). In some embodiments, anthramycin is an anthramycin dimer. In some embodiments, PBD is tesirin (SG3249). In some embodiments, tesirin is SG3199. In some embodiments, the chemotherapeutic agent is SG3249. In some embodiments, the chemotherapeutic agent is SG3199. In some embodiments, the effector portion contains tesirin. In some embodiments, the effector portion consists of tesirin.

[0214] In some embodiments, the chemotherapeutic agent is calicheamicin. In some embodiments, the effector portion is calicheamicin. Calicheamicin is a class of antibiotics derived from the bacterium Micromonospora echinospora that bind to the DNA minor groove and cause strand breaks. Examples of calicheamicin include, but are not limited to, calicheamicin gamma 1, esperamycin, and ozogamicin.

[0215] In some embodiments, the chemotherapeutic agent is camptothecin or an analogue. In some embodiments, the effector portion is camptothecin or an analogue. In some embodiments, the effector portion is camptothecin. Examples of camptothecin analogues include, but are not limited to, exatecan, SN-38, and deluxtecan (Dxd). In some embodiments, the camptothecin analogue is Dxd. In some embodiments, the chemotherapeutic agent is Dxd. In some embodiments, the effector portion is Dxd.

[0216] In some embodiments, the chemotherapeutic agent is duocalmycin. In some embodiments, the effector portion is duocalmycin. Duocalmycin is a small molecule isolated from Streptomyces bacteria that binds to the DNA minor groove and alkylates adenine bases. Examples of duocalmycin include, but are not limited to, duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, duocalmycin TM, duocalmycin MA, and CC-1065.

[0217] In some embodiments, the chemotherapeutic agent is a triptolide. In some embodiments, the effector portion is a triptolide.

[0218] In some embodiments, the effector portion is a tubulin inhibitor. In some embodiments, the effector portion is a meitansinoid. In some embodiments, the meitansinoid is a thiol-containing meitansinoid. Meitansinoids or meitansin are known to be tubulin inhibitors that inhibit microtubule assembly by binding tubulin to the ryzoxin binding site. In some embodiments, the meitansinoid is meltansine (DM-1). In some embodiments, meltansine is emtansine. In some embodiments, the tubulin inhibitor is auristatin. In some embodiments, auristatin is selected from monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). In some embodiments, the tubulin inhibitor is tubulicine. In some embodiments, tubulicine is tubulicine A. In some embodiments, auristatin is MMAE. In some embodiments, auristatin is MMAF. In some embodiments, the effector portion is MMAE. In some embodiments, the effector portion is MMAF.

[0219] In some embodiments, the effector portion is a combination of parts. In some embodiments, the effector portion is a plurality of effector portions. In some embodiments, the effector portion is a combination of cytotoxic parts. In some embodiments, the effector portion includes at least two cytotoxic parts selected from the group consisting of amatoxin, anthracycline, pyrrolobenzodiazepine, calicheamycin, camptothecin, duochamycin, triptolide, and tubulin inhibitors. In some embodiments, the effector portion includes at least two cytotoxic parts selected from the group consisting of amatoxin, anthracycline, pyrrolobenzodiazepine, calicheamycin, camptothecin, duochamycin, triptolide, and maytansinoids.

[0220] Third and fourth chains In some embodiments, the protein complex further comprises a third polypeptide chain. In some embodiments, the third polypeptide chain comprises a third fragment of the protein target of the GD autoantibody. In some embodiments, the third fragment is different from the first fragment. In some embodiments, the third fragment is different from the second fragment. In some embodiments, the third fragment is the same as the first fragment. In some embodiments, the first fragment is the same as the second fragment. In some embodiments, the third fragment is the same as the first and second fragments. In some embodiments, the same means the same sequence. In some embodiments, different means different sequences.

[0221] In some embodiments, the third polypeptide further comprises a third dimerization domain. In some embodiments, the first polypeptide further comprises a fourth dimerization domain. In some embodiments, the third and fourth dimerization domains can dimerize with each other. In some embodiments, the third and fourth dimerization domains are configured to dimerize with each other. In some embodiments, the third dimerization domain is not configured to dimerize into the first dimerization domain. In some embodiments, the third dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize into the first dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, the domains configured to dimerize can dimerize. In some embodiments, the third and fourth dimerization domains are different from the first and second dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, and the third and fourth dimerization domains are CH1 / CL domains. In some embodiments, the first and second dimerization domains are CH1 / CL domains, and the third and fourth dimerization domains are hinge domains.

[0222] In some embodiments, the protein complex further comprises a fourth polypeptide chain. In some embodiments, the fourth polypeptide chain comprises a fourth fragment of the protein target of the GD autoantibody. In some embodiments, the fourth fragment is different from the first fragment. In some embodiments, the fourth fragment is different from the second fragment. In some embodiments, the fourth fragment is different from the third fragment. In some embodiments, the fourth fragment is the same as the first fragment. In some embodiments, the fourth fragment is the same as the second fragment. In some embodiments, the fourth fragment is the same as the third fragment. In some embodiments, the fourth fragment is the same as the first, second, and third fragments. In some embodiments, the first, second, and third fragments are all the same. In some embodiments, the first, second, third, and fourth fragments are all different. In some embodiments, the same means the same sequence. In some embodiments, different means different sequences. In some embodiments, different means different proteins. In some embodiments, different means the same protein but contains different sequences. In some embodiments, the different components are the same protein, but different regions of the protein. In some embodiments, at least two of the first, second, third, and fourth proteins are part of a single protein complex. In some embodiments, the protein complex is a mammalian complex. In some embodiments, the protein complex is a human complex.

[0223] In some embodiments, the fourth polypeptide further comprises a fifth dimerization domain. In some embodiments, the second polypeptide further comprises a sixth dimerization domain. In some embodiments, the fifth and sixth dimerization domains can dimerize with each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerize with each other. In some embodiments, the fifth dimerization domain is not configured to dimerize into the first dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize into the third dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize into the fourth dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the first dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the third dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the fourth dimerization domain. In some embodiments, the fifth and sixth dimerization domains are different from the first and second dimerization domains. In some embodiments, the fifth and sixth dimerization domains are different from the third and fourth dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, the third and fourth dimerization domains are CH1 / CL domains, and the fifth and sixth dimerization domains are CH1 / CL domains. In some embodiments, the first and second dimerization domains are CH1 / CL domains, the third and fourth dimerization domains are hinge domains, and the fifth and sixth dimerization domains are hinge domains. In some embodiments, neither the first polypeptide nor the second polypeptide contains a CH1 domain.In some embodiments, both the first and second polypeptides contain a CH1 domain. Both the first and second polypeptides contain a CL domain. In some embodiments, neither the first nor the second polypeptide contains a CL domain. In some embodiments, the first polypeptide contains a CH1 domain and the second polypeptide contains a CL domain. In some embodiments, the third polypeptide contains a CL domain and the fourth polypeptide contains a CH1 domain. In some embodiments, the first polypeptide contains a CL domain and the second polypeptide contains a CH1 domain. In some embodiments, the third polypeptide contains a CH1 domain and the fourth polypeptide contains a CL domain.

[0224] In some embodiments, the third and fourth dimerization domains include mutations that enable dimerization of the third and fourth dimerization domains and inhibit dimerization of the third dimerization domain to the fifth, sixth, or both dimerization domains. In some embodiments, the third and fourth dimerization domains include mutations that enable dimerization of the third and fourth dimerization domains and inhibit dimerization of the fourth dimerization domain to the fifth, sixth, or both dimerization domains. In some embodiments, the fifth and sixth dimerization domains include mutations that enable dimerization of the fifth and sixth dimerization domains and inhibit dimerization of the fifth dimerization domain to the third, fourth, or both dimerization domains. In some embodiments, the fifth and sixth dimerization domains include mutations that enable dimerization of the fifth and sixth dimerization domains and inhibit dimerization of the sixth dimerization domain to the third, sixth, or both dimerization domains.

[0225] Alternate configuration In some embodiments, the composition comprises a polypeptide chain comprising a fragment of a first protein target or an analog or derivative of the GD autoantibody and a fragment of a second protein target or an analog or derivative of the GD autoantibody. In some embodiments, the polypeptide chain is a single polypeptide chain. In some embodiments, the single chain comprises a fragment of the first protein and a fragment of the second protein. In some embodiments, the polypeptide chain further comprises a fragment of a third protein target or an analog or derivative of the GD autoantibody. In some embodiments, the polypeptide chain further comprises a fragment of a fourth protein target or an analog or derivative of the GD autoantibody. In some embodiments, the polypeptide chain further comprises an Fc region. In some embodiments, the polypeptide chain further comprises an effector portion.

[0226] In some embodiments, a fragment of the first protein target or its analogue or derivative of the GD autoantibody is separated by a linker from a fragment of the second protein target or its analogue or derivative of the GD autoantibody. In some embodiments, a fragment of the third protein target or its analogue or derivative of the GD autoantibody is separated by a linker from a fragment of the first or second protein target or its analogue or derivative of the GD autoantibody. In some embodiments, a fragment of the fourth protein target or its analogue or derivative of the GD autoantibody is separated by a linker from a fragment of the first, second or third protein target or its analogue or derivative of the GD autoantibody. In some embodiments, the fragment is separated by a linker from the Fc region. In some embodiments, the effector portion is separated and joined by a linker. In some embodiments, the effector portion is separated by the fragment by a linker.

[0227] In some embodiments, the fragment and dimerization domain are separated by a linker. In some embodiments, the dimerization domain and the Fc region are separated by a linker. In some embodiments, the fragment and the Fc region are separated by a linker. In some embodiments, the effector portion is linked by a linker. In some embodiments, the effector portion and the fragment are separated by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a bond. In some embodiments, the bond is a peptide bond. In some embodiments, the bond is an amino acid bond. In some embodiments, the linker is a flexible linker. The linker is well known in the art, and any linker may be used.

[0228] In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is a polyethylene glycol (PEG) linker. In some embodiments, the PEG linker is a Gly3-PEG-azide linker. In some embodiments, the linker is a dibenzocyclooctin group (DBCO) linker. In some embodiments, the DBCO linker is a DBCO-C6 linker. In some embodiments, the DBCO linker is a DBCO-Gly5-EDA linker. In some embodiments, the linker is a dimethylethylenediamine (DMEDA) linker. In some embodiments, the linker is an N-dimethylethylenediamine (DMAE) linker. In some embodiments, the linker is a glutathione linker. In some embodiments, the linker is a CLICK linker. In some embodiments, the CLICK linker is a CLICK-DBCO linker. In some embodiments, the CLICK linker is a CLICK-azide linker. In some embodiments, the linker is a disulfide linker. In some embodiments, the linker is a thiol linker. In some embodiments, the linker is an azido linker. In some embodiments, the linker is a maleimide (Mal) linker. In some embodiments, the Mal linker is a maleimide caproyl linker. In some embodiments, the Mal linker is a Mal-C6 linker. In some embodiments, the Mal linker is a Mal-Gly5-EDA linker. In some embodiments, the linker is a lysine linker. In some embodiments, the linker is an asparagine linker. In some embodiments, the linker is an acid-unstable linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is protease-cleavable. In some embodiments, the cleavable linker is a glutathione-cleavable linker. In some embodiments, the linker is an incleavable linker. Other examples of linkers include, for example, SPDB linkers, SMCC linkers, MCC linkers, and butanoic acid linkers.In some embodiments, the linker is a p-aminobenzyl (PAB) linker. In some embodiments, the linker is a p-aminocarbamate (PABC) linker. In some embodiments, the linker is a maleimidocaproyl (mc) linker. In some embodiments, the linker contains mc. In some embodiments, the linker is a Val-Cit-PAB linker. In some embodiments, the linker is a Val-Cit-PABC linker. In some embodiments, the linker is a Val-Cit-PAB-MMAE linker. In some embodiments, the linker is an mc-VC-PABC-MMAE linker. In some embodiments, the linker is an mc-MMAF linker. In some embodiments, the linker is a monomethyl auristatin E (MMAE) linker. Examples of peptide linkers include, but are not limited to, the Val-Cit-PAB linker, the Phe-Lys(Trt)-PAB linker, and the Ala-Ala-Asn-PAB linker. In some embodiments, the linker is a mixture of linkers. In some embodiments, the linker is a DBCO-PEG linker. In some embodiments, the linker is a PBCO-PEG-DMEDA linker. In some embodiments, the linker is a DBCO-PEG-VC-PAB-DMEDA linker. In some embodiments, VC in the linker is replaced with EVC. In some embodiments, VC in the linker is replaced with EVA. In some embodiments, the fragments and dimerization domains are linked by non-cleavable linkers. In some embodiments, the fragments and dimerization domains are linked by cleavable linkers. In some embodiments, the effector portions are linked by cleavable linkers. In some embodiments, the effector portions are linked by non-cleavable linkers.

[0229] In some embodiments, conjugation is linked. In some embodiments, conjugation is via bonding. In some embodiments, the conjugate is directly conjugated. In some embodiments, the conjugate is conjugated via a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the effector portion is conjugated by a linker.

[0230] In some embodiments, conjugation is the conjugation of an amino acid linker, partially, or both, and involves the extension of the amino acid sequence of the drug chain of the present invention. The nucleic acid molecule encoding the drug of the present invention can be modified to include a linker, partially, or both of the coding sequence, so that a complete conjugate is produced at translation. In some embodiments, the conjugate is a fusion protein. Methods for partially linking and conjugating are well known in the art, and any such method may be used. In some embodiments, the method is a combination of at least two methods. In particular, methods for linking and conjugating to an IgG scaffold are also well known. Methods for linking / conjugating include, but are not limited to, natural cysteine ​​reduction (including natural hinge reduction, also referred herein to as natural cysteine ​​conjugation), manipulated cysteine ​​reduction, disulfide crosslinking, lysine conjugation, and enzymatic conjugation. Examples of enzymatic conjugations include, but are not limited to, click chemistry, saltase-assisted SMAC technology, transglutaminase addition of amine azides, and glycan remodeling.

[0231] For example, natural cysteine ​​conjugation can be performed as follows: The CRD protein was reduced using TCEP and incubated at 37°C for 90 minutes. Then, DMA and linker payload were added, followed by incubation at room temperature for 2 hours. Finally, the conjugated material was purified by size exclusion chromatography.

[0232] In some embodiments, the conjugation is site-specific. In some embodiments, the conjugation is not random. In some embodiments, the conjugation or linkage is directed to the IgG backbone. In some embodiments, the conjugation or linkage is not directed to the TSHR fragment. In some embodiments, the conjugation or linkage does not interfere with antibody binding to the TSHR fragment. In some embodiments, the antibody is an autoantibody. In some embodiments, the conjugation or linkage is directed to the dimerization domain. In some embodiments, the conjugation or linkage is directed to the hinge region. In some embodiments, the conjugation or linkage is directed to the CH2 region. In some embodiments, the conjugation or linkage is directed to the CH3 region. In some embodiments, the conjugation or linkage is directed to the CH1 region. In some embodiments, the conjugation or linkage is directed to the CL region. In some embodiments, the linkage or conjugation is directed to a native amino acid residue. In some embodiments, the linkage or conjugation is directed to an engineered amino acid residue. In some embodiments, the residue is cysteine. Examples of manipulated cysteine ​​include, but are not limited to, A231C, S239C, N325C, L328C, D265C, and S442C in the IgG heavy chain. In some embodiments, the residue is lysine. In some embodiments, the residue is asparagine. In some embodiments, glycan remodeling is used to link to asparagine. In some embodiments, asparagine is N297 in the IgG heavy chain. In some embodiments, the residue is glutamine. In some embodiments, N297 is converted, manipulated, or mutated to glutamine (N297Q). In some embodiments, glutamine is Q295 in the IgG heavy chain. Examples of manipulated glutamine include, but are not limited to, Q297.Unless otherwise specified, the references provide Kabat numbering for IgG1, and the corresponding mutations can be produced in other IGs, specifically in other IgGs. In some embodiments, ligation or conjugation is to the C-terminus or N-terminus of the drug chain of the present invention. In some embodiments, ligation or conjugation is to the C-terminus. In some embodiments, ligation or conjugation is to the N-terminus. In some embodiments, the terminus is the terminus of the heavy chain. In some embodiments, the terminus is the terminus of the light chain. In some embodiments, conjugation or ligation is to multiple sites.

[0233] In some embodiments, the linker is long enough to inhibit steric hindrance between different parts of the chain. In some embodiments, the linker is long enough to inhibit steric hindrance between different parts of the conjugate. In some embodiments, the linker is long enough to allow binding to the antibody fragment without steric hindrance from another part of the chain. In some embodiments, the linker is long enough to allow binding to the antibody fragment without steric hindrance from another part of the conjugate. In some embodiments, the linker is long enough to allow binding to the cell fragment without steric hindrance from another part of the chain. In some embodiments, the linker is long enough to allow binding to the cell fragment without steric hindrance from another part of the conjugate. In some embodiments, the linker is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid lengths. Each possibility represents a distinct embodiment of the invention. In some embodiments, the linker is at least 1 amino acid length. In some embodiments, the linker is at least 5 amino acid length. In some embodiments, the linker is at least 10 amino acid length. In some embodiments, the linker is at least 15 amino acids long. In some embodiments, the linker is up to 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids long. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the linker is up to 10 amino acids long. In some embodiments, the linker is up to 20 amino acids long. In some embodiments, the linker is up to 50 amino acids long. In some embodiments, the linker is up to 100 amino acids long.

[0234] In some embodiments, the linker includes GGGGS (sequence number 7). In some embodiments, the linker consists of sequence number 7. In some embodiments, the linker includes (GGGGS)n, where n is an integer. In some embodiments, the linker consists of (GGGGS)n, where n is an integer. In some embodiments, the linker includes GGGS (sequence number 4). In some embodiments, the linker consists of sequence number 4. In some embodiments, the linker includes (GGGS)n, where n is an integer. In some embodiments, the linker consists of (GGGS)n, where n is an integer. In some embodiments, the linker includes or consists of GGGGSGGGGSGGGGSGGGGSGGGGS (sequence number 52). In some embodiments, the linker includes GSAGSAAGSGEF (sequence number 51). In some embodiments, the linker includes or consists of (GGGS)nGS, where n is an integer. In some embodiments, n is selected from 1, 2, 3, 4, and 5. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, the linker includes sequence number 8. In some embodiments, the linker consists of sequence number 8.

[0235] In some embodiments, the linker is a rigid linker. In some embodiments, the linker includes EAAAK (sequence number 62). In some embodiments, the linker consists of sequence number 62. In some embodiments, the rigid linker includes or consists of sequence number 62. In some embodiments, the linker includes (EAAAK)n, where n is an integer. In some embodiments, the linker consists of (EAAAK)n, where n is an integer. In some embodiments, the linker includes sequence number 14. In some embodiments, the linker consists of sequence number 14. In some embodiments, the linker includes A(EAAAK)nA, where n is an integer. In some embodiments, the linker consists of A(EAAAK)nA, where n is an integer. In some embodiments, the linker includes AEAAAKEAAAKEAAAKEAAAKA (sequence number 15). In some embodiments, the linker consists of sequence number 15.

[0236] In some embodiments, the dimerization domain is the C-terminus of the fragment. In some embodiments, the fragment is the C-terminus of the dimerization domain. In some embodiments, the Fc region is the C-terminus of the fragment. In some embodiments, the fragment is the C-terminus of the Fc region. In some embodiments, the dimerization domain is the C-terminus of the Fc region. In some embodiments, the Fc region is the C-terminus of the dimerization domain. In some embodiments, the dimerization domain is the N-terminus of the fragment. In some embodiments, the fragment is the N-terminus of the dimerization domain. In some embodiments, the Fc region is the N-terminus of the fragment. In some embodiments, the fragment is the N-terminus of the Fc region. In some embodiments, the dimerization domain is the N-terminus of the Fc region. In some embodiments, the Fc region is the N-terminus of the dimerization domain.

[0237] In some embodiments, the epitope extends to at least two fragments. In some embodiments, the epitope extends to first and second fragments. In some embodiments, the epitope extends to first and third fragments. In some embodiments, the epitope extends to first and fourth fragments. In some embodiments, the epitope extends to second and third fragments. In some embodiments, the epitope extends to second and fourth fragments. In some embodiments, the epitope extends to third and fourth fragments. In some embodiments, the epitope extends to two proteins. In some embodiments, the epitope extends to two proteins in a protein complex. In some embodiments, the epitope extends to three fragments. In some embodiments, the epitope extends to three proteins. In some embodiments, the epitope extends to four fragments. In some embodiments, the epitope extends to four proteins. In some embodiments, the epitope is a complex epitope. In some embodiments, the epitope is a B cell receptor (BCR) specific epitope.

[0238] In some embodiments, all fragments are derived from TSHR. In some embodiments, the fragments derived from TSHR are mutated. In some embodiments, the complex includes a TSHR shortening. In some embodiments, the complex includes a TSHR mutation. In some embodiments, the mutation is a point mutation. In some embodiments, the mutation is a deletion.

[0239] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 53. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 53.

[0240] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 54. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 54.

[0241] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPGDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 55. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 55.

[0242] In some embodiments, the first polypeptide includes a fragment linked to SEQ ID NO: 60. In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 60. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 60. In some embodiments, the first polypeptide includes a fragment linked to SEQ ID NO: 61. In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 61. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 61.

[0243] In some embodiments, the first polypeptide includes a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 56.

[0244] In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 53. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 54. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 55. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 9. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 10. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 53. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 54. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 55. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 9. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 10.

[0245] In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence provided in SEQ ID NO: 6, 9, 10, 11, 12 or 13. Each sequence represents a separate embodiment of the present invention. In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence selected from SEQ ID NO: 6 and 9-13. In some embodiments, the polypeptide chain comprises or consists of the amino acid sequences provided in SEQ ID NO: 6 and 9-13, or an analog or derivative thereof having at least 85% identity. Each sequence represents a separate embodiment of the present invention. In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence selected from Table 2. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 6 and 9-13. Each sequence represents a separate embodiment of the present invention. In some embodiments, the complex comprises or consists of two polypeptide chains provided in SEQ ID NO: 6, 9-13, or an analog or derivative thereof having at least 85% identity. Each sequence represents a separate embodiment of the present invention. In some embodiments, the two chains are the same chain. In some embodiments, the two chains are different chains.

[0246] In some embodiments, the polypeptide chain comprises or consists of amino acid sequences provided in SEQ ID NOs: 6, 9, 10, 11, 12, 13, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, or 82. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the polypeptide chain comprises or consists of amino acid sequences selected from SEQ ID NOs: 6, 9-13, and 71-72. In some embodiments, the polypeptide chain comprises or consists of amino acid sequences provided in SEQ ID NOs: 6, 9-13, and 71-82, or analogues or derivatives thereof having at least 85% identity. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the polypeptide chain comprises or consists of amino acid sequences selected from Table 2. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NOs: 6, 9-13, and 71-82. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the complex comprises or consists of two polypeptide chains provided in Sequence IDs 6, 9-13, 71-82, or analogues or derivatives thereof having at least 85% identity. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the two chains are the same chain. In some embodiments, the two chains are different chains.

[0247] In some embodiments, the polypeptide includes sequence number 6. In some embodiments, the polypeptide consists of sequence number 6. In some embodiments, the first or second polypeptide includes or consists of sequence number 6. In some embodiments, the polypeptide includes sequence number 9. In some embodiments, the polypeptide consists of sequence number 9. In some embodiments, the first or second polypeptide includes or consists of sequence number 9. In some embodiments, the polypeptide includes sequence number 10. In some embodiments, the polypeptide consists of sequence number 10. In some embodiments, the first or second polypeptide includes or consists of sequence number 10. In some embodiments, the polypeptide includes sequence number 11. In some embodiments, the polypeptide consists of sequence number 11. In some embodiments, the first or second polypeptide includes or consists of sequence number 11. In some embodiments, the polypeptide includes sequence number 12. In some embodiments, the polypeptide consists of sequence number 12. In some embodiments, the first or second polypeptide includes or consists of sequence number 12. In some embodiments, the polypeptide includes sequence number 13. In some embodiments, the polypeptide consists of sequence number 13. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 13. In some embodiments, the polypeptide includes SEQ ID NO: 71. In some embodiments, the polypeptide consists of SEQ ID NO: 71. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 71. In some embodiments, the polypeptide includes SEQ ID NO: 72. In some embodiments, the polypeptide consists of SEQ ID NO: 72. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 72. In some embodiments, the polypeptide includes SEQ ID NO: 73. In some embodiments, the polypeptide consists of SEQ ID NO: 73. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 73.In some embodiments, the polypeptide includes SEQ ID NO: 74. In some embodiments, the polypeptide consists of SEQ ID NO: 74. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 74. In some embodiments, the polypeptide includes SEQ ID NO: 75. In some embodiments, the polypeptide consists of SEQ ID NO: 75. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 75. In some embodiments, the polypeptide includes SEQ ID NO: 76. In some embodiments, the polypeptide consists of SEQ ID NO: 76. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 76. In some embodiments, the polypeptide includes SEQ ID NO: 77. In some embodiments, the polypeptide consists of SEQ ID NO: 77. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 77. In some embodiments, the polypeptide includes SEQ ID NO: 78. In some embodiments, the polypeptide consists of SEQ ID NO: 78. In some embodiments, the polypeptide includes SEQ ID NO: 78. In some embodiments, the polypeptide includes SEQ ID NO: 78. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 78. In some embodiments, the polypeptide includes SEQ ID NO: 79. In some embodiments, the polypeptide consists of SEQ ID NO: 79. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 79. In some embodiments, the polypeptide includes SEQ ID NO: 80. In some embodiments, the polypeptide consists of SEQ ID NO: 80. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 80. In some embodiments, the polypeptide includes SEQ ID NO: 81. In some embodiments, the polypeptide consists of SEQ ID NO: 81. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 81. In some embodiments, the polypeptide includes SEQ ID NO: 82. In some embodiments, the polypeptide consists of SEQ ID NO: 82. In some embodiments, the first or second polypeptide includes or consists of SEQ ID NO: 82.In some embodiments, the polypeptide includes a sequence selected from SEQ ID NOs: 9-13. In some embodiments, the polypeptide consists of a sequence selected from SEQ ID NOs: 9-13. In some embodiments, the first or second polypeptide includes or consists of a sequence selected from SEQ ID NOs: 9-13. In some embodiments, the polypeptide includes a sequence selected from SEQ ID NOs: 71-82. In some embodiments, the polypeptide consists of a sequence selected from SEQ ID NOs: 71-82. In some embodiments, the first or second polypeptide includes or consists of a sequence selected from SEQ ID NOs: 9-13 and 71-82. In some embodiments, the polypeptide includes a sequence selected from SEQ ID NOs: 9-13 and 71-82. In some embodiments, the polypeptide consists of a sequence selected from SEQ ID NOs: 9-13 and 71-82. In some embodiments, the first or second polypeptide includes or consists of a sequence selected from SEQ ID NOs: 9-13 and 71-82.

[0248] In some embodiments, the polypeptide comprises or consists of CRD-238. In some embodiments, the complex comprises or consists of CRD-240. In some embodiments, the complex comprises or consists of CRD-285. In some embodiments, the complex comprises or consists of CRD-286. In some embodiments, the complex comprises or consists of CRD-287. In some embodiments, the complex comprises or consists of CRD-527. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 70% identity with the sequences provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 75% identity with the sequences provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 80% identity with the sequences provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 85% identity with the sequences provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 90% identity with the sequences provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 95% identity with the sequences provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 97% identity with the sequences provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 99% identity with the sequences provided herein.

[0249] Pharmaceutical composition In another aspect, a pharmaceutical composition comprising the protein or polypeptide of the present invention is provided.

[0250] In another aspect, a pharmaceutical composition comprising the protein complex of the present invention is provided.

[0251] In another embodiment, a pharmaceutical composition comprising the composition of the present invention is provided.

[0252] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or adjuvant. As used herein, the terms “carrier,” “adjuvant,” or “excipient” refer to any component of the pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline solution. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol polyols; esters, such as ethyl oleate and ethyl laurate, and agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline and Ringer's solution; ethyl alcohol and phosphate buffer; and other non-toxic, suitable substances used in pharmaceutical formulations. Some non-limiting examples of substances that can function as carriers in this specification include sugars, starches, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers, and other non-toxic, pharmaceutically acceptable substances used in other pharmaceutical formulations. Wetting and lubricating agents such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions intended herein.Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those skilled in the art, for example, as described in The Merck Index, 13th edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); CTFA (Cosmetic Industry Association) International Cosmetic Ingredient Dictionary and Handbook, 10th edition (2004); and the "Inactive Ingredient Guide" of the Center for Drug Evaluation and Research (CDER) of the U.S. Food and Drug Administration (FDA), all of which are incorporated herein by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful for the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextrose solution, Hanks' solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th edition, Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2005), each of which is incorporated herein by reference in whole. The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, sustained-release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum. Examples of liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, and lamellar layers.Liposomes for use with the peptides described herein are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is generally determined considering factors such as liposome size and stability in blood. Various methods for preparing liposomes are available, as outlined in, for example, Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York. See also U.S. Patents No. 4,235,871, No. 4,501,728, No. 4,837,028, and No. 5,019,369.

[0253] The carriers may constitute approximately 0.1% to approximately 99.99999% by weight of the pharmaceutical compositions presented herein in total.

[0254] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the protein complex of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the conjugate of the present invention. The term "therapeutic amount" refers to the amount of drug effective in treating a disease or disorder in a mammal. In some embodiments, the therapeutic amount is the amount effective in the required dose and duration to achieve the desired therapeutic or prophylactic outcome. The exact dosage form and regimen are determined by the physician depending on the patient's condition. In some embodiments, the effective amount is sufficient to treat at least one symptom of the disease. In some embodiments, the disease is GD. In some embodiments, the disease is PF. In some embodiments, the disease is GD. In some embodiments, GD is characterized by autoantibodies against the protein. In some embodiments, GD is characterized by autoantibodies against TSHR.

[0255] As used herein, the terms “treatment” or “to treat” a disease, disorder, or condition encompass the alleviation of at least one symptom, a reduction in its severity, or the inhibition of its progression. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For a treatment to be effective, a useful composition or method herein may only reduce the severity of the disease, disorder, or condition, reduce the severity of its associated symptoms, or improve the quality of life of the patient or subject. Treatments for GD are well known in the art and may include any acceptable means for evaluating improvement of GD symptoms. These may include rituximab, steroids, steroid-sparing immunosuppressants (such as azathioprine, mycophenolate, and cyclophosphamide), dapsone, and intravenous immunoglobulin (IVIG). Treatments may include improving quality of life, suppressing blister formation, reducing autoantibodies, and killing autoreactive B cells.

[0256] In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the pharmaceutical composition is formulated for administration to a human. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0257] As used herein, terms such as “administer” and “dosage” refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice. One embodiment of this subject provides intravenous administration of a therapeutically effective amount of the composition of this subject to a patient in need. Other preferred routes of administration may include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal. In some embodiments, administering is intravenous administration. In some embodiments, administering is topical administration. In some embodiments, administering is selected from oral, intravenous, intramuscular, intraperitoneal, intratumoral, topical, or subcutaneous administration. In some embodiments, administering is administration to the site of disease.

[0258] The dosage administered depends on the recipient's age, health condition, and weight, as well as the type of concomitant treatments, if any, the frequency of those treatments, and the nature of the desired effect.

[0259] Treatment method In another embodiment, a method for treating a subject requiring treatment with GD is provided, comprising administering the protein or polypeptide of the present invention to the subject, thereby treating the subject with GD.

[0260] In another embodiment, a method for treating a subject requiring treatment with GD is provided, comprising administering the protein complex of the present invention to the subject, thereby treating the subject with GD.

[0261] In another embodiment, a method for treating GD in a subject requiring GD treatment is provided, comprising administering the composition of the present invention to the subject, thereby treating the subject with GD.

[0262] In some embodiments, administration refers to administering the pharmaceutical composition of the present invention. In some embodiments, GD is characterized by an antibody against a protein. In some embodiments, the protein is the target of the GD antibody. It will be understood by those skilled in the art that the protein complex is designed using a fragment of the protein targeted by the GD antibody in the subject. In some embodiments, the antibody is an autoantibody. In some embodiments, the disease is GD, and the autoantibody is against TSHR.

[0263] In some embodiments, the treatment includes reducing the antibody concentration. In some embodiments, the treatment includes a lower number of antibodies. In some embodiments, the antibody concentration is the circulating antibody concentration. In some embodiments, the treatment includes depleting the antibodies. In some embodiments, the treatment includes killing B cells. In some embodiments, the B cells are autoreactive B cells. In some embodiments, killing B cells is specific B cell death. In some embodiments, the treatment includes killing antibody-producing B cells. In some embodiments, the treatment includes killing antibody-producing B cells but not substantially killing other B cells. In some embodiments, the treatment includes killing B cells that produce antibodies against a protein complex. In some embodiments, the treatment includes killing B cells that produce antibodies against a fragment. In some embodiments, the treatment includes killing B cells that produce antibodies against a fragment of a protein complex.

[0264] In some embodiments, reducing an antibody involves binding to an antibody. In some embodiments, reducing means removing at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% of an antibody. Each possibility represents a distinct embodiment of the invention. In some embodiments, reducing means removing at least 80% of an antibody. In some embodiments, the antibody is an autoantibody. In some embodiments, the antibody is an antibody in a subject. In some embodiments, the antibody is a circulating antibody. In some embodiments, the autoantibody is an autoantibody against a protein or fragment. In some embodiments, the autoantibody is a cytotoxic autoantibody. In some embodiments, the autoantibody includes an IgG1 autoantibody. In some embodiments, the autoantibody includes IgG3. In some embodiments, the autoantibody includes IgG1 and IgG3 autoantibodies. In some embodiments, the autoantibody includes IgG1, IgG2, and IgG3 autoantibodies. In some embodiments, the autoantibody includes IgG1, IgG3, and IgG4 autoantibodies. In some embodiments, the autoantibodies include IgG1, IgG2, IgG3, and IgG4 autoantibodies. In some embodiments, reduction means removing at least 25% of the antibodies. In some embodiments, reduction means removing at least 50% of the antibodies. In some embodiments, reduction means removing at least 70% of the antibodies. In some embodiments, reduction means removing at least 75% of the antibodies. In some embodiments, reduction means removing at least 80% of the antibodies. In some embodiments, the percentage of antibodies is the percentage of autoantibodies. In some embodiments, the percentage of antibodies is the percentage of antibodies against a protein or fragment. In some embodiments, the percentage of antibodies is the percentage of antibodies associated with the disease.

[0265] In some embodiments, the method further includes reducing the antibody in the subject. In some embodiments, the reduction is performed before administration. In some embodiments, the antibody reduction is the reduction of circulating antibodies. In some embodiments, the antibody is an autoantibody. In some embodiments, the antibody is against a protein. In some embodiments, the antibody is against a protein from which a fragment originates. In some embodiments, the antibody is against a protein from which at least one of the fragments originates. In some embodiments, the reduction is the reduction of antibodies against all proteins from which at least one of the fragments originates. In some embodiments, the antibody is against a protein complex. Methods for reducing antibodies are well known in the art and include, for example, plasma ferresis, intravenous Ig (IVIg), antibody filtering, and B-cell targeted therapy, any of which may be used. In some embodiments, the method includes plasma ferresis of the antibody before administration. In some embodiments, the method includes administering B-cell targeted therapy before administering the therapeutic agent of the present invention. In some embodiments, the B-cell targeted therapy is anti-B-cell therapy. In some embodiments, the B-cell targeted therapy is B-cell lethal therapy. In some embodiments, B-cell targeted therapy is panB-cell therapy. In some embodiments, B-cell targeted therapy is not targeted therapy. As used herein, “targeted B-cell therapy” is a therapy that targets only specific B-cell clones that produce specific antibodies. In some embodiments, anti-B-cell therapy is anti-B-cell antibody. B-cell targeted antibodies are known in the art, and non-limiting examples include anti-CD20 antibodies. Anti-CD20 therapeutic antibodies are well known in the art and include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tiuxetan, tocitumomab, and ubrituximab. In some embodiments, B-cell targeted therapy is rituximab.

[0266] nucleic acid In another aspect, a nucleic acid system comprising at least two nucleic acid molecules is provided. The first nucleic acid molecule encodes the first polypeptide chain of the protein complex of the present invention, and the second nucleic acid molecule encodes the second polypeptide chain of the protein complex of the present invention.

[0267] In another aspect, a nucleic acid system comprising at least two nucleic acid molecules is provided. The first nucleic acid molecule encodes a first polypeptide chain comprising a fragment of the first human protein target of the GD autoantibody or its analog or derivative and a first dimerization domain, and the second nucleic acid molecule encodes a second polypeptide chain comprising a fragment of the second human protein target of the GD autoantibody or its analog or derivative and a second dimerization domain.

[0268] In another aspect, a nucleic acid molecule encoding the protein of the present invention is provided.

[0269] In another aspect, a nucleic acid molecule encoding the polypeptide chain of the composition of the present invention is provided.

[0270] In another aspect, a nucleic acid molecule encoding the composition of the present invention is provided.

[0271] In another aspect, a nucleic acid molecule encoding a fragment of the first protein target of the GD autoantibody or its analog or derivative and a fragment of the second human protein target of the GD autoantibody or its analog or derivative is provided.

[0272] In some embodiments, the nucleic acid system further comprises a third nucleic acid molecule encoding a third polypeptide of the protein complex of the present invention. In some embodiments, the nucleic acid system further comprises a fourth nucleic acid molecule encoding a fourth polypeptide of the protein complex of the present invention. In some embodiments, the first nucleic acid molecule encodes the first polypeptide of the present invention. In some embodiments, the second nucleic acid molecule encodes the second polypeptide of the present invention. In some embodiments, the third nucleic acid molecule encodes the third polypeptide of the present invention. In some embodiments, the fourth nucleic acid molecule encodes the fourth polypeptide.

[0273] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the nucleic acid molecule includes an open reading frame encoding a polypeptide chain. The expression of open reading frames in cells is well known to those skilled in the art. It can be carried out by transfection, viral infection, or direct modification of the cell's genome, among many other methods. Expression vectors are well known in the art, and any vector suitable for the target cell expressing the protein complex of the present invention may be used.

[0274] A vector nucleic acid sequence generally contains at least one origin of replication for intracellular proliferation and, optionally, further elements, such as heterologous polynucleotide sequences, expression regulatory elements (e.g., promoters, enhancers), selection markers (e.g., antibiotic resistance), and polyadenine sequences. In some embodiments, the vector includes a promoter. In some embodiments, the promoter is configured for expression in target cells expressing the protein complex of the present invention.

[0275] The vector may be a DNA plasmid delivered via a non-viral or viral method. The viral vector may be a retroviral vector, a herpesvirus vector, an adenovirus vector, an adeno-associated virus vector, or a poxvirus vector. The promoter may be active in mammalian cells. The promoter may be a viral promoter. The promoter may be active in bacterial cells. The promoter may be active in human cells. The promoter may be active in fibroblasts. As used herein, the term “promoter” refers to a group of transcriptional regulatory modules concentrated around the start site of RNA polymerase, i.e., RNA polymerase II. The promoter consists of individual functional modules, each consisting of approximately 7–20 bp of DNA and containing one or more recognition sites for transcription activators or repressor proteins.

[0276] In some embodiments, the open reading frame is operably linked to the promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that enables the expression of the nucleotide sequence (for example, in an in vitro transcription / translation system if the vector is introduced into a host cell, or in the host cell).

[0277] In some embodiments, the vector is introduced into cells by standard methods including electroporation (e.g., From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with a viral vector, or rapid ballistic transport by small particles having nucleic acids in or on the surface of a matrix of small beads or particles (Klein et al., Nature 327, 70-73 (1987)).

[0278] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize mRNA and the precursors of most snRNAs and microRNAs.

[0279] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, pCI, pCI, pMbac, pPbac, pBK-RSV, and pBK-CMV, pTRES, and derivatives thereof, all available from Invitrogen.

[0280] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used by the present invention. Examples of SV40 vectors include pSVT7 and pMT2. In some embodiments, examples of vectors derived from bovine papillomavirus include pBV-1MTHA, and examples of vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vectors that enable protein expression under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any other promoter that has been shown to be effective for expression in eukaryotic cells.

[0281] In some embodiments, recombinant viral vectors that offer advantages such as lateral transmission and target specificity are used for in vivo expression. In one embodiment, lateral transmission is a process inherent in the life cycle of, for example, retroviruses, in which a single infected cell produces many progeny virions that budding and infect neighboring cells. In one embodiment, this results in rapid infection of a wide area, most of which were not initially infected by the original viral particle. In one embodiment, a viral vector is produced that cannot spread laterally. In one embodiment, this feature may be useful when the desired objective is to introduce a specific gene into only a local number of target cells.

[0282] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, for information on the positive-negative selection method, please refer to U.S. Patent Nos. 5,464,764 and 5,487,992.

[0283] Beyond containing elements necessary for the transcription and translation of the inserted coding sequence (encoding the polypeptide), it will be understood that the expression constructs of the present invention may also include sequences manipulated to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0284] In some embodiments, the nucleic acid molecule is a single nucleic acid molecule. In some embodiments, the first and second nucleic acid molecules are different molecules. In some embodiments, the first and second nucleic acid molecules are the same molecule. In some embodiments, any two of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, any two of the first, second, third, and fourth nucleic acid molecules are the same molecule. In some embodiments, any three of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, the first, second, and third nucleic acid molecules are different molecules. In some embodiments, any three of the first, second, third, and fourth nucleic acid molecules are the same molecule. In some embodiments, all of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, all of the first, second, third, and fourth nucleic acid molecules are the same molecule.

[0285] Production method A method for producing a protein, in another embodiment, The objective is to obtain a first fragment of the extracellular domain of a first human receptor or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human receptor or an analog or derivative thereof, wherein the first and second human receptors are targets of a GD autoantibody, and the first fragment is linked to the second fragment to produce a single polypeptide chain. A method is provided that includes and thereby produces a protein.

[0286] A method for producing a protein, in another embodiment, The objective is to obtain a first fragment of the extracellular domain of a first human receptor or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human receptor or an analog or derivative thereof, wherein the first and second human receptors are targets of a GD autoantibody, the first fragment is linked to the second fragment to produce a single polypeptide chain, and the single polypeptide chain is linked to an effector portion that is not an unmodified Fc domain. A method is provided that includes and thereby produces a protein.

[0287] A method for producing a protein, in another embodiment, To obtain a first fragment of the extracellular domain of TSHR or its analogues or derivatives, and to shorten TSHR or its analogues or derivatives, wherein the shortening reduces the aggregation of the first fragment. A method is provided that includes and thereby produces a protein.

[0288] A method for producing a protein, in another embodiment, The method involves obtaining a first fragment of the extracellular domain of TSHR or its analogues or derivatives, and producing a shortened TSHR by shortening TSHR or its analogues or derivatives, wherein the shortening reduces aggregation of the first fragment and ligates the shortened TSHR to an effector portion that is not an unmodified Fc domain. A method is provided that includes and thereby produces a protein.

[0289] A method for producing a protein, in another embodiment, Obtaining a first fragment of the extracellular domain of TSHR or its analogues or derivatives, and inducing a mutation in TSHR or its analogues or derivatives such that the mutation reduces or induces aggregation of the first fragment. A method is provided that includes and thereby produces a protein.

[0290] A method for producing a protein, in another embodiment, The method involves obtaining a first fragment of the extracellular domain of TSHR or an analog or derivative, inducing a mutation in TSHR or an analog or derivative to produce mutant TSHR, wherein the mutation reduces the aggregation of the first fragment, and ligating the mutant TSHR to an effector portion that is not an unmodified Fc domain. A method is provided that includes and thereby produces a protein.

[0291] A method for producing a protein, in another embodiment, a. Obtaining a fragment of the extracellular domain of TSHR or its analogue or derivative, b. Producing a mutant fragment by generating at least one mutation in the fragment, c. Measuring the solubility, aggregation, or both of the mutant fragments. d. Select at least one mutant fragment that increases solubility, decreases aggregation, or both, compared to the obtained fragment. A method is provided that includes and thereby produces a protein.

[0292] A method for producing a protein, in another embodiment, a. Obtaining a fragment of the extracellular domain of TSHR or its analogue or derivative, b. Producing a mutant fragment by generating at least one mutation in the fragment, c. Measuring the solubility, aggregation, or both of the mutant fragments. d. Select at least one mutant fragment that increases solubility, decreases aggregation, or both, compared to the obtained fragment. e. Concatenating at least one selected mutant fragment to an effector portion that is not an unmodified Fc domain, A method is provided that includes and thereby produces a protein.

[0293] A method for producing a protein complex, in another embodiment, To obtain a first fragment of a first protein target of a GD autoantibody or an analog or derivative thereof, and a second fragment of a second protein target of a GD autoantibody or an analog or derivative thereof, to ligate the first fragment to a first dimerization domain to produce a first polypeptide, and to ligate the second fragment to a second dimerization domain to produce a second polypeptide chain, A method is provided for producing a protein complex by including [a certain substance].

[0294] A method for producing a protein complex, in another embodiment, Obtaining a first fragment of a first protein target or analog or derivative of a GD autoantibody and a second fragment of a second protein target or analog or derivative of a GD autoantibody; ligating the first fragment to a first dimerization domain to produce a first polypeptide; ligating the second fragment to a second dimerization domain to produce a second polypeptide chain; and ligating the first polypeptide, the second polypeptide, or both to an effector portion that is not an unmodified Fc domain. A method is provided for producing a protein complex by including [a certain substance].

[0295] A method for producing a protein, in another embodiment, The method involves culturing host cells containing one or more vectors that include nucleic acid sequences encoding a single polypeptide chain, wherein the single polypeptide chain is i. Obtaining a first fragment of the extracellular domain of a first human receptor or its analog or derivative, and a second fragment of the extracellular domain of a second human receptor or its analog or derivative, wherein the first and second human receptors are targets of GD autoantibodies and are different proteins. ii. Linking the first fragment to the second fragment to produce a single polypeptide chain, A method is provided for producing a protein, which involves culturing, and thereby producing a protein.

[0296] A method for producing a protein, in another embodiment, The method involves culturing host cells containing one or more vectors that include nucleic acid sequences encoding a single polypeptide chain, wherein the single polypeptide chain is i. Obtaining a first fragment of the extracellular domain of a first human receptor or its analog or derivative, and a second fragment of the extracellular domain of a second human receptor or its analog or derivative, wherein the first and second human receptors are targets of GD autoantibodies and are different proteins. ii. Linking the first fragment to the second fragment to produce a single polypeptide chain, iii. Linking a single polypeptide chain to an effector portion that is not an unmodified Fc domain, A method is provided for producing a protein, which involves culturing, and thereby producing a protein.

[0297] A method for producing a protein complex, in another embodiment, The method involves culturing host cells containing one or more vectors comprising nucleic acid sequences encoding at least two polypeptide chains, wherein the two polypeptide chains are i. Obtaining a first fragment of a first protein target of a GD autoantibody or an analog or derivative thereof, and a second fragment of a second protein target of a GD autoantibody or an analog or derivative thereof, and ii. To produce a first polypeptide chain by linking the first fragment to the first dimerization domain, and to produce a second polypeptide chain by linking the second fragment to the second dimerization domain. A method is provided for producing a protein complex, which involves culturing the substance produced by the substance.

[0298] A method for producing a protein complex, in another embodiment, The method involves culturing host cells containing one or more vectors comprising nucleic acid sequences encoding at least two polypeptide chains, wherein the two polypeptide chains are i. Obtain a first fragment of the first protein target of the GD autoantibody or an analog or derivative thereof, and a second fragment of the second protein target of the GD autoantibody or an analog or derivative thereof. ii. To produce a first polypeptide chain by linking the first fragment to the first dimerization domain, and to produce a second polypeptide chain by linking the second fragment to the second dimerization domain, and iii. Linking the first polypeptide chain, the second polypeptide chain, or both, to an effector portion that is not an unmodified Fc domain. A method is provided for producing a protein complex, which involves culturing the substance produced by the substance.

[0299] In some embodiments, the protein is the protein of the present invention. In some embodiments, the protein is a polypeptide. In some embodiments, the abbreviation is the abbreviation of the present invention. In some embodiments, the protein is the polypeptide of the present invention. In some embodiments, the mutation is the mutation of the present invention. In some embodiments, the protein complex is the protein complex of the present invention. In some embodiments, the protein composition is the composition of the present invention. In some embodiments, the protein is the protein of the present invention. In some embodiments, the protein is the polypeptide chain of the present invention. In some embodiments, the fragment is the fragment of the present invention. In some embodiments, the derivative is the derivative of the present invention. In some embodiments, the analog is an analog of the present invention. In some embodiments, the dimerizing domain is the dimerizing domain of the present invention. In some embodiments, the composition, protein complex, protein, fragment, analog, derivative, or dimerizing domain is as described above. In some embodiments, the method further comprises linking a protein, polypeptide, or protein complex to an effector moiety. In some embodiments, the effector moiety is not an Fc domain. In some embodiments, the effector moiety does not contain an Fc moiety. In some embodiments, the effector moiety is not an unmodified Fc domain. In some embodiments, the effector portion is an Fc domain containing at least one mutation that increases ADCC.

[0300] In some embodiments, the protein is a human protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the first and second proteins are the same protein. In some embodiments, the first and second proteins are different proteins. In some embodiments, the first and second proteins are targets of GD autoantibodies. In some embodiments, the first and second proteins are targets of GD-associated autoantibodies. In some embodiments, GD is characterized by autoantibodies against the first and second proteins. In some embodiments, the protein is a receptor and the fragment is a fragment of the extracellular domain. In some embodiments, the fragment contains a fragment of the extracellular domain. In some embodiments, the fragment consists of an extracellular domain.

[0301] In some embodiments, the first and second dimerization domains can dimerize with each other. In some embodiments, the first and second dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first and second polypeptides. In some embodiments, contacting comprises incubating the polypeptides together. In some embodiments, contacting is intracellular. In some embodiments, contacting is in vitro. In some embodiments, contacting is under conditions sufficient to allow dimerization. In some embodiments, enabling is induction. In some embodiments, the conditions are sufficient to allow dimerization of the polypeptides. In some embodiments, the conditions are physiological conditions.

[0302] In some embodiments, the method further includes inserting a third dimerization domain into the first polypeptide. In some embodiments, insertion is ligation. In some embodiments, insertion is inserting a nucleic acid sequence encoding the third dimerization domain into a nucleic acid molecule or vector encoding the first polypeptide. In some embodiments, ligation is ligation of the third dimerization domain to the first dimerization domain. In some embodiments, ligation is ligation of the third dimerization domain to the first fragment.

[0303] In some embodiments, the method further comprises obtaining a third fragment of a third protein target of a GD autoantibody or an analog or derivative thereof, and ligating it to a fourth dimerization domain to produce a third polypeptide chain. In some embodiments, the third and fourth dimerization domains can dimerize with each other. In some embodiments, the third and fourth dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first, second, and third polypeptide chains. In some embodiments, the method further comprises expressing a nucleic acid sequence encoding the third polypeptide chain in a host cell. In some embodiments, the third polypeptide chain is produced by obtaining a third fragment of a third protein and ligating it to a fourth dimerization domain to produce the third polypeptide chain. In some embodiments, the method comprises expressing the first, second, and third polypeptide chains in a cell.

[0304] In some embodiments, the method further includes inserting a fifth dimerization domain into a second polypeptide. In some embodiments, insertion is ligation. In some embodiments, insertion is inserting a nucleic acid sequence encoding the fifth dimerization domain into a nucleic acid molecule or vector encoding the second polypeptide. In some embodiments, ligation is ligation of the fifth dimerization domain to the second dimerization domain. In some embodiments, ligation is ligation of the fifth dimerization domain to the second fragment.

[0305] In some embodiments, the method further comprises obtaining a fourth fragment of a fourth protein target of a GD autoantibody or an analog or derivative thereof, and ligating it to a sixth dimerization domain to produce a fourth polypeptide chain. In some embodiments, the fifth and sixth dimerization domains can dimerize with each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first, second, third, and fourth polypeptide chains. In some embodiments, the method further comprises expressing a nucleic acid sequence encoding the fourth polypeptide chain in a host cell. In some embodiments, the fourth polypeptide chain is produced by obtaining a fourth fragment of a fourth protein and ligating it to a sixth dimerization domain to produce a fourth polypeptide chain. In some embodiments, the method comprises expressing the first, second, third, and fourth polypeptide chains intracellularly.

[0306] In some embodiments, the method further includes inserting the Fc region into a first strand. In some embodiments, the method further includes inserting the Fc region into a second strand. In some embodiments, the method further includes inserting the Fc region into a third strand. In some embodiments, the method further includes inserting the Fc region into a fourth strand. In some embodiments, the method further includes inserting a portion of the Fc region into the first strand and a portion of the Fc region into the second strand, with the interface of the two portions producing a complete Fc region. In some embodiments, the Fc region is not an unmodified Fc region. In some embodiments, the Fc region includes at least one mutation that increases ADCC.

[0307] In some embodiments, the Fc region is inserted into the C-terminus of the dimerization domain. In some embodiments, the Fc region is inserted into the C-terminus of the fragment. In some embodiments, the Fc region is inserted into the N-terminus of the dimerization domain. In some embodiments, the Fc region is inserted into the N-terminus of the fragment. In some embodiments, the fragment is inserted into or ligated to the C-terminus of the dimerization domain. In some embodiments, the fragment is inserted into or ligated to the N-terminus of the dimerization domain.

[0308] In some embodiments, the method further includes inserting a linker between at least two portions of a polypeptide chain. In some embodiments, the linker is inserted between a fragment and a dimerization domain. In some embodiments, the linker is inserted between a fragment and an Fc region. In some embodiments, the linker is inserted between an Fc region and a dimerization domain. In some embodiments, the linker is inserted between a dimerization domain and another dimerization domain. In some embodiments, the linker is inserted between a fragment and another fragment. In some embodiments, the linker is inserted between a fragment of a first protein and a fragment of a second protein.

[0309] In some embodiments, the method further comprises introducing at least one mutation into the protein. In some embodiments, the mutation occurs in the fragment. In some embodiments, the mutation occurs in the extracellular domain. In some embodiments, the mutation occurs in the cadherin domain of the fragment. In some embodiments, the method further comprises shortening the protein. In some embodiments, the method further comprises shortening the fragment. In some embodiments, the method further comprises shortening the extracellular domain. In some embodiments, the shortening removes at least one extracellular functional domain. In some embodiments, the shortening removes at least one cadherin domain.

[0310] In some embodiments, the method further includes measuring protein aggregation. In some embodiments, the method further includes measuring the aggregation of mutant proteins. In some embodiments, the method further includes measuring the aggregation of truncated proteins. In some embodiments, the method further includes selecting proteins with low aggregation. In some embodiments, low aggregation is aggregation below a predetermined threshold. In some embodiments, low aggregation is substantial aggregation. In some embodiments, the method further includes selecting mutant proteins with reduced aggregation. In some embodiments, the method further includes selecting truncated proteins with reduced aggregation. In some embodiments, reduced is when compared to a non-mutant or non-truncated protein. In some embodiments, reduced is when compared to a control protein. In some embodiments, reduced is when compared to a WT extracellular domain. In some embodiments, reduced includes a reduction of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100%. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the reduction includes a reduction of at least 50%. In some embodiments, the reduction includes a reduction of at least 25%. Methods for measuring aggregation are well known in the art, and any such method, including those provided herein, may be used. In some embodiments, aggregation is measured under non-reducing conditions.

[0311] In some embodiments, the method further includes measuring the solubility, aggregation, or both of the obtained fragments. In some embodiments, increased means significantly increased. In some embodiments, decreased means decreased. In some embodiments, decreased means significantly decreased. In some embodiments, significant means statistically significant.

[0312] In some embodiments, the method further includes measuring the binding of an autoantibody to TSHR to a protein. In some embodiments, the method further includes measuring the binding of an autoantibody to TSHR to a mutant protein. In some embodiments, the method further includes measuring the binding of an autoantibody to TSHR to a fragment. In some embodiments, the autoantibody is in serum. In some embodiments, the autoantibody is in blood. In some embodiments, the measurement is to measure the binding in serum. In some embodiments, the measurement is to measure the binding in blood. In some embodiments, the serum or blood is derived from a subject suffering from GD. In some embodiments, the binding is depletion. In some embodiments, the measurement is to measure the depletion of an autoantibody from serum / blood by a protein. In some embodiments, the protein is conjugated to an artificial scaffold. In some embodiments, the conjugation is immobilized. In some embodiments, the artificial scaffold is beads. In some embodiments, the beads are paramagnetic beads. In some embodiments, the beads are Sepharose beads. In some embodiments, the beads are avidin beads. In some embodiments, avidin is streptavidin.

[0313] In some embodiments, a fragment is selected that binds to at least a predetermined threshold of the autoantibody. In some embodiments, the method further includes selecting a fragment that binds to at least a predetermined threshold of the autoantibody. In some embodiments, the threshold is a threshold percentage of the autoantibody. In some embodiments, the threshold is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99%. Each possibility represents a distinct embodiment of the invention. In some embodiments, the threshold is at least 20%. In some embodiments, the threshold is at least 40%. In some embodiments, the threshold is at least 50%. In some embodiments, the threshold is at least 70%. In some embodiments, the threshold is at least 75%. In some embodiments, the threshold is at least 80%.

[0314] In some embodiments, the protein is for use in the method of the present invention. In some embodiments, the polypeptide is for use in the method of the present invention. In some embodiments, the protein complex is for use in the method of the present invention. In some embodiments, the method is a therapeutic method. In some embodiments, the method is a diagnostic method. In some embodiments, the method is a treatment method. In some embodiments, the method is a method for determining suitability for treatment.

[0315] In another embodiment, a protein complex produced by the method of the present invention is provided.

[0316] In another aspect, a protein produced by the method of the present invention is provided.

[0317] In another embodiment, a composition produced by the method of the present invention is provided.

[0318] Patient Selection In another aspect, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising: receiving a sample from the object; contacting the sample with a composition of the present invention; and determining the binding of antibodies in the sample to the composition, wherein the binding of antibodies to the composition indicates that the object is suitable for treatment by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0319] In another aspect, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising: receiving a sample from the object; contacting the sample with a protein complex of the present invention; and determining the binding of an antibody in the sample to the protein complex, wherein the binding of the antibody to the protein complex indicates that the object is suitable to be treated by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0320] In another aspect, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising: receiving a sample from the object; contacting the sample with the protein or polypeptide of the present invention; and determining the binding of an antibody in the sample to the protein, wherein the binding of the antibody to the protein or polypeptide indicates that the object is suitable to be treated by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0321] In some embodiments, the subject is one who needs it. In some embodiments, the subject is one of the above-described subjects. In some embodiments, the subject has GD. In some embodiments, the subject is known to be positive for autoantibodies associated with GD. In some embodiments, the subject is serologically positive. In some embodiments, the subject is serologically negative. In some embodiments, the subject is naive to treatment. In some embodiments, the treatment is treatment for GD. In some embodiments, the subject has been treated and is experiencing a relapse.

[0322] In some embodiments, the method includes obtaining a sample from a subject. In some embodiments, the sample includes tissue. In some embodiments, the sample is biopsy material. In some embodiments, the sample is a body fluid. In some embodiments, the body fluid is blood. In some embodiments, the body fluid is serum. In some embodiments, the body fluid is plasma. In some embodiments, the body fluid is a fluid containing antibodies. In some embodiments, the body fluid is selected from at least one of blood, serum, plasma, intestinal fluid, saliva, tumor fluid, urine, interstitial fluid, cerebrospinal fluid, and feces.

[0323] In some embodiments, contact is incubation. In some embodiments, contact is carried out under conditions sufficient for the antibody to bind to the protein complex. In some embodiments, the conditions include sufficient time for the antibody to bind to the protein complex. In some embodiments, the conditions include physiological conditions. In some embodiments, the protein complex is added to the sample. In some embodiments, the protein complex is dissolved in body fluids. In some embodiments, the antibody is an autoantibody. In some embodiments, the antibody is an antibody against a protein.

[0324] In some embodiments, the composition further comprises a detectable portion. In some embodiments, the protein complex further comprises a detectable portion. In some embodiments, the protein further comprises a detectable portion. In some embodiments, the method further comprises contacting the composition, complex and / or protein with a peptide containing the detectable portion. In some embodiments, the peptide is configured to bind to the composition, protein and / or complex. In some embodiments, the peptide is specific to the composition, protein and / or complex. As used herein, the term “specific binding” means binding to a specific molecule while excluding other molecules. In some embodiments, the peptide is specific to the composition, protein and / or complex by excluding other proteins in the sample. In some embodiments, the peptide is specific to the composition, protein and / or complex by excluding naturally occurring antibodies in the sample. In some embodiments, the peptide is specific to the composition, protein and / or complex by excluding antibodies in the sample. In some embodiments, determining binding includes detecting the portion. In some embodiments, determining includes isolating the protein complex. In some embodiments, determining includes eluting antibodies from the complex. Methods for protein identification are well known in the art, and any such method may be used. Examples of such methods include protein sequencing by Western blotting, ELISA, FACS analysis, and mass spectrometry. In some embodiments, sequencing involves ELISA. In some embodiments, the ELISA is a competitive ELISA. In some embodiments, the competitive ELISA involves competition with an antibody. In some embodiments, the antibody is a disease-related antibody.

[0325] In some embodiments, binding is positive binding. In some embodiments, binding is binding exceeding a predetermined threshold. In some embodiments, binding is specific binding. In some embodiments, binding is binding to at least one of the fragments of the protein complex. In some embodiments, binding is binding to at least two of the fragments of the protein complex. In some embodiments, binding is binding to at least three of the fragments of the protein complex. In some embodiments, binding is binding to at least four of the fragments of the protein complex. In some embodiments, binding is binding to at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% of the antibody in the sample. Each possibility represents a distinct embodiment of the invention. In some embodiments, binding is binding to at least 50% of the antibody in the sample. In some embodiments, binding is binding to at least 70% of the antibody in the sample. In some embodiments, binding is binding to at least 75% of the antibody in the sample. In some embodiments, binding refers to the binding of at least 80% of the antibodies in the sample. In some embodiments, the percentage of antibodies is the percentage of autoantibodies. In some embodiments, the percentage of antibodies is the percentage of antibodies against proteins. In some embodiments, the percentage of antibodies is the percentage of disease-related antibodies.

[0326] As used herein, the term "approximately," when combined with a value, refers to a range of plus or minus 10% of the given value. For example, a length of approximately 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0327] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless otherwise explicitly indicated in the context. Thus, for example, a reference to “polynucleotide” includes multiple such polynucleotides, and a reference to “polypeptide” includes one or more polypeptides and their equivalents known to those skilled in the art, and so on. It should be further noted that the claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as an antecedent for the use of exclusive technical terms such as “alone,” “only,” etc., in relation to the enumeration of elements of the claims or the use of “negative” limitations.

[0328] Where conventions similar to “at least one of A, B, and C, etc.” are used, such configurations are generally intended to be understood by those skilled in the art (for example, “a system having at least one of A, B, and C” could mean a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will further be understood by those skilled in the art that substantially any disjunctive word and / or phrase presenting two or more alternative terms should be understood as construing the possibility of including one of the terms, either of the terms, or both of the terms, whether in the specification, claims, or drawings. For example, the phrase “A or B” is understood to include the possibilities of “A” or “B” or “A and B”.

[0329] For clarity, certain features of the Invention described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, various features of the Invention described in the context of a single embodiment for brevity may be provided individually or in any preferred partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if each and all combinations were individually and explicitly disclosed. In addition, all partial combinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if each and all such partial combinations were individually and explicitly disclosed herein.

[0330] Further objectives, advantages, and novel features of the present invention will become apparent to those skilled in the art by considering the following examples, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the present invention described above herein and claimed in the following claims section will find experimental support in the following examples.

[0331] The various embodiments and aspects of the invention described above and claimed in the following claims section will find experimental support in the following examples. [Examples]

[0332] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, immunological, microbiological, and recombinant DNA techniques. Such techniques are well described in the literature. For example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); Methodology described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook," Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology," Volumes I-III, Coligan, JE, ed. (1994); Stites et al.See (eds), "Basic and Clinical Immunology" (Volume 8), Appleton & Lange, Norwalk, CT (1994); and Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996). All of these are incorporated by reference. Other general references are provided throughout this document.

[0333] Example 1 Autoantibodies against TSHR are a major cause of gynecomastiasis (GD). Anti-TSHR antibodies act as thyroid-stimulating immunoglobulins (TSIs), and their stimulation of the thyroid gland leads to the production of excessive amounts of thyroid hormones, resulting in hyperthyroidism. Therefore, therapeutic agents targeting TSHR autoantibodies have been developed. TSHR has a large N-terminal extracellular domain containing four distinct extracellular domains: nine leucine-rich repeat (LRR) domains and three extracellular loops. The large N-terminal extracellular domain was tested.

[0334] The complete N-terminal extracellular domain of TSHR (SEQ ID NO: 1) is not expressed as well as the soluble protein. Therefore, a variant of this domain containing a deletion of amino acids 297-346 of SEQ ID NO: 1 (equivalent to amino acids 317-366 of SEQ ID NO: 2, which contains the TSHR signal peptide) was used as a bait for TSHR autoantibodies.

[0335] Long-term remission in GD patients requires the removal of a significant proportion of autoreactive B cells that produce an autoantibody pool. While simply removing autoantibodies from circulation is potentially effective in treating GD symptoms, repeated treatments will be necessary for the remainder of the subject's life, as long-lived B cells continue to produce new autoantibodies. Importantly, B cells that produce autoantibodies express B cell receptors (BCRs), which are the same membrane-bound form of these autoantibodies, on their surface. This makes it possible to target the B cells themselves with therapeutic agents containing autoantibody BCR-specific epitopes. By linking the target epitope to the Fc region of the antibody heavy chain, the therapeutic agent can direct the specific death of autoantibody-producing B cells. This approach is also robust against the potential avoidance of specific subpopulations that can occur when using drugs that target specific differentiation markers on the cell surface (e.g., CD19, CD38, BCMA), as all cells possessing autoreactive BCRs are targeted regardless of their differentiation state. This approach is also beneficial for protecting and preserving non-autoreactivity, including protective (e.g., antiviral, antibacterial) subpopulations that are damaged by treatments targeting nonspecific differentiation markers (e.g., CD20, CD38, BCMA), regardless of whether they possess an autoreactive BCR.

[0336] Figure 1A shows one embodiment of the therapeutic agent of the present invention. The immunoglobulin (Ig)-like protein complex 101 comprises four polypeptide chains: two heavy-chain-like polypeptides 110 and two light-chain-like polypeptides 120. Chain 110 can be dimerized via disulfide bonds between them. Furthermore, chain 110 may contain any or all of the CH3 domain 111, CH2 domain 112, hinge region 113, and CH1 domain 114. In this embodiment, the CH3 domain 111, CH2 domain 112, and hinge region 113 all contain disulfide bonds and act as dimerizing domains, but other dimerizing domains can also be used. These domains are well known in the art and can be selected from, for example, any of the human (or non-human) IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD domains. Those skilled in the art will understand that the Fc portions of IgG1 and IgG3 incorporated into chain 110 enable the molecule to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). Chain 120 can dimerize with chain 110 via disulfide bonds found in the CH1 domain 114 and CL domain 124.

[0337] Unlike naturally occurring or synthetic antibodies, chains 110 and 120 lack a variable region. As a substitute for the variable region, each chain contains a fragment or derivative 130 from the N-terminal extracellular portion of the human thyroid-stimulating hormone receptor (TSHR). Each chain can be generated to contain the same or a different fragment / derivative of TSHR. In fact, as shown in Figure 1B, the two heavy chains 115 and 116 can be manipulated separately such that chain 115 contains, for example, a fragment of the extracellular domain of TSHR (TSHR Frag) 131, and chain 116 contains another fragment (same or different) 132. The same applies to light chains 125 and 126, which can contain, for example, a fragment of the extracellular domain of TSHR (TSHR Frag) 133 and another fragment (same or different) 134, respectively. This fragment could be, for example, SEQ ID NO: 3, which contains a deletion of the C-peptide region, or any other fragment. Therefore, therapeutic molecules can be designed using four copies of a protein (TSHRN-terminal extracellular domain 135) or fragment (Figure 1C), two copies each of two different proteins or fragments (Figure 1D), one copy each of four different protein fragments or domains (Figure 1B), or any other combination thereof. In fact, molecules are modular enough to be manipulated with three copies of one protein / domain and one copy of another protein / domain, or two copies of one protein / domain and one copy of two other proteins / domains. Figure 1E shows an embodiment in which the two light chains are identical but the two heavy chains are different. Figure 1F also shows an embodiment in which the two heavy chains are identical but the two light chains are different. Importantly, therapeutic molecules can be manipulated to include any combination of different domains of two proteins that can bind to autoantibodies from a wide variety of patients, not just some. Those skilled in the art will understand that any chain can contain any protein, fragment, domain, or variant, and that the chain-subunit combinations shown in Figures 1A to 1F are illustrative and not limiting.

[0338] Figures 2A–2N illustrate several embodiments of the present invention in which only two chains are combined. In Figures 2A–2F, protein complex 201 comprises two polypeptide chains, specifically two heavy chains. Heavy chains 215 and 216 may optionally contain hinge domains 213, CH2 212, CH3 211, and / or CH1 214 domains. In this embodiment, the heavy chain hinges 213, CH2 212, and CH3 211 all dimerize via disulfide bonds. Only one of these three domains is required for dimerization, although other dimerizing domains are also conceivable. It includes the N-terminal extracellular domain of TSHR or a fragment or derivative thereof 230. Protein complex 201 is also conceivable as only a fragment of the TSHR extracellular domain, such as TSHR Fragment (Frag) 231. Protein complex 201 is also conceivable that has two distinct fragments, either identical or different (e.g., fragment 231 on one chain and the extracellular domain 235 on the other chain). These fragments represent any fragment of the extracellular domain of any protein that can be used in any combination.

[0339] Figure 2B shows molecules lacking CH2 domain 212, CH3 domain 211, or CH1 domain 214. Molecules lacking both hinge 213 and CH1 domain 214 are also shown. Combinations lacking two of these domains, with or without hinge 213, are also conceivable (Figure 2B). As an alternative to the variable region, each chain has a fragment or derivative 230 of the N-terminal extracellular domain of TSHR. Although not illustrated, it will be understood by those skilled in the art that any fragment or derivative from the N-terminal extracellular portion of TSHR can be used. Each chain can be generated to have the same protein, fragment or derivative / mutant (Figure 2C) or different proteins, fragments or derivatives / mutants (Figure 2D). For ease of use, fragments of the N-terminal extracellular domain of TSHR235 and the N-terminal extracellular domain of TSHR231 are shown, but it will be understood that any fragment, derivative or mutant of these domains can also be used. When two different subunits are used, it is advantageous to design the molecule so that primarily heterodimers of 215 and 216 are formed, rather than homodimers. The same applies to the heterodimer formation of chains 115 and 116 in Figure 1. The same applies to Figure 2A. There are many techniques known in the art for designing mutations in the CH3 / CH2 domain, such as Knobs-into-Holes and DuoBodies, which inhibit homodimerization and promote heterodimerization. Any such technique may be used. Removal of CH1 by direct conjugation to the hinge region (Figure 2E) or further removal of the hinge by direct conjugation to CH2 (Figure 2F) is also possible. For all figures, if an extracellular domain is shown, it will be understood that it is intended to include all fragments, variants, and mutations of that extracellular domain.

[0340] Figure 2G shows an alternative configuration containing two heavy chains. Instead of containing a single fragment 230 as a substitute for the variable region, two tandem fragments 230 are used. These fragments may be separated by an optional linker 290. This configuration is structurally similar to a single-chain antibody in which the heavy chain variable domain and light chain variable domain reside on a single peptide, and is essentially equivalent to the molecule shown in Figure 1D. Heavy chains 215 and 216 can optionally contain hinge 213, CH2 212, CH3 211, and / or CH1 214 domains. Dimerization is as described above. For simplicity, examples containing all three CH domains are shown, as well as examples lacking the CH1 domain. Molecules lacking the hinge, CH2, or CH3 domains, or any two-thirds of these domains, are also conceivable (as long as at least one dimerization region is retained). Fragment 230 is derived from the N-terminal extracellular domain of TSHR and may contain the entire extracellular domain, only a part of it, or a variant or derivative. Therefore, repeats of two identical proteins / fragments can be inserted into a single strand (Figures 2H-2I, two TSHR fragments 231), or two different proteins / fragments can be combined on a single strand (Figures 2J-2K, TSHR fragment 231 and the complete TSHR N-terminal extracellular domain 235, or two different TSHR fragments 231 and 234). Naturally, the heavy chains do not need to be identical, as various techniques can be used to promote heterodimerization rather than homodimerization (Figures 2L-2M, different TSHR fragments on each chain). As mentioned above, the CH1 domain 214 may be included (Figures 2H, 2J, 2L) or excluded (Figures 2I, 2K, 2M), and the same applies to hinge 213, CH2 212 and / or CH3 211, as long as one dimerized domain (e.g., hinge, CH2, CH3) remains.

[0341] In Figure 2N, the protein complex 201 comprises two polypeptide chains, specifically a heavy chain 215 and a light chain 220. In such embodiments, the dimerization domains are the CH1 domain 214 and the CL domain 224. The heavy chain 215 may optionally include a CH3 domain 211, a CH2 domain 212, and / or a hinge region 213. The absence of the hinge / CH2 / CH3 domain is one option to exclude homodimerization of the two heavy chains 215. Alternatively, a cysteine ​​substitution / mutation (e.g., for serine or glutamine) may be introduced into the hinge, or one of the CH2 / CH3 region mutations that promote heterodimerization and inhibit homodimerization may be used. As an alternative to the variable region, each chain has a fragment 230 from the N-terminal extracellular portion of TSHR, or any fragment or variant / derivative thereof.

[0342] The creation of a protein complex 301 having three chains, heavy chain 315, heavy chain 316, and light chain 320, is also envisioned (Figures 3A-3D). Figure 3A shows one possible embodiment in which heavy chain 316 contains a CL domain 364 as a substitute for the CH1 domain. The above method can be used to ensure the 315 / 316 heterodimer. Heavy chains 315 and 316 may optionally contain a CH3 domain 311, a CH2 domain 312, and / or a hinge region 313, or may use different dimerization domains. The CL domain 324 in light chain 320 can only dimerize with the CH1 domain 314 in heavy chain 315. As a substitute for the variable region, each chain has a fragment, variant, or derivative 330 from the N-terminal extracellular portion of TSHR. The three chains can all contain the same protein (e.g., TSHR extracellular domain 335, Figure 3B), the three chains can all contain different fragments (Figure 3C), or the three chains can contain two different fragments, one of which is repeated (Figure 3D). Figure 3D may also have two identical fragments as either a light chain or a heavy chain, and thus it will be understood by those skilled in the art that this embodiment has three different configurations.

[0343] This configuration, using one of the heavy chains containing a CL domain as a substitute for the CH1 domain, can also enable the formation of protein complexes with four different fragments. Similar to the protein complex in Figure 1B, the protein complex 401 shown in Figure 4 has four different fragments on each chain. In this embodiment, fragments 431, 434, 432, and 433 are used, but those skilled in the art will understand that these refer to any four fragments. Embodiments can also be envisioned in which different fragments from the same protein may be on different chains. In this embodiment, the second light chain 426 contains a CH1 domain 474 so that it can dimerize with the CL domain 464 of the heavy chain 416. The heavy chain 415 contains a CH1 domain 414, and the light chain 425 contains a CL domain 424. This allows chain 425 to dimerize with chain 415 alone, and chain 426 to dimerize with chain 416 alone. As described above, heterodimerization of chains 415 and 416 can be promoted using mutations in the arbitrarily selected CH2 domain 412 and CH3 domain 413. The hinge region 413, CH2 domain 412, and CH3 domain 411 are all used here as dimerization domains between the two heavy chains, but any dimerization domain (other than CH1 / CL) can be used.

[0344] In the embodiments described above, an immunoglobulin skeleton is shown and described, but it will be understood by those skilled in the art that similar molecules can be generated by selecting other dimerization domains. Figures 5A and 5B show a typical protein complex 501. In Figure 5A, the first chain 515 contains a first dimerization domain (DD1) 563 which can specifically dimerize with a second dimerization domain (DD2) 573 of the second chain 516. Chain 515 further contains a third dimerization domain (DD3) 514 which can specifically dimerize with a fourth dimerization domain (DD4) 524 of the third chain 525. Chain 516 further contains a fifth dimerization domain (DD5) 564 which can specifically dimerize with a sixth dimerization domain (DD6) 574 of the fourth chain 526. Each of the four chains also contains a fragment 530 of a human protein target of a GD autoantibody. These could all be the same fragment with the same amino acid sequence, or they could be different sequences, variants / derivatives, proteins, or fragments.

[0345] Figure 5B shows an alternative embodiment of Figure 5A in which each distinct domain is separated by a linker. Those skilled in the art will understand that all of these linkers are optional and that combinations of linkers are possible. It will be further understood that the configuration of Figure 5B can also be used with linkers between any or all of the various domains / fragments. Similarly, linkers can be inserted between the CH1 domain, hinge region, CH2 domain, CH3 domain and / or TSHR fragment. Thus, the linkers shown in Figure 5B can be extrapolated to the same positions in immunoglobulin backbone molecules (Figures 1A-4).

[0346] Figures 6A to 6F illustrate single-strand embodiments of the present invention. Figure 6A shows a single-strand fusion protein 601 containing the TSHRN-terminal extracellular domain 635 and the TSHR extracellular domain fragment 631. Figure 6B shows a single-strand containing only the TSHR fragment. It will be understood that any fragment of the N-terminal extracellular domain may be used. It will be further understood that any permutation of Figures 6A and 6B may be generated such that two fragments from different parts of the protein are in the same single-strand. Two different derivatives / mutants may also be used. Figures 6C and 6D show similar embodiments but contain three and four fragments from different proteins, respectively. It will be understood that any fragment or N-terminal extracellular domain may be used. As shown in Figure 6E, the single-strand may also contain at least the CH3 domain 611 or CH2 domain 612, and optionally, a heavy-chain constant region having the CH1 domain 614, hinge region 613 and / or CH2 domain 612 or CH3 domain 611. Figure 6F shows an embodiment in which the CH1 domain has been removed. Finally, an amino acid linker can be used to separate any of the single-stranded domains. Figure 6G shows embodiments in which two, three, or four fragments are all separated by linker 690, as well as embodiments in which linker 690 separates the C-terminal fragment 635 from either the CH1 domain 614 or the CH2 domain 612. It is also shown that a linker replaces the hinge region, but this is not necessary, and it will be understood that the hinge can be held by a linker that ligates the C-terminal fragment 635 to the hinge region. Linkers separating the CH1 domain 614, the hinge region 613, the CH2 domain 612, and the CH3 domain 611 are not shown, but it will be understood by those skilled in the art that any or all of these domains can be separated by linkers. Furthermore, it will be understood that these various linkers can all contain the same sequence or can be made from different amino acid sequences.

[0347] Example 2 The complete N-terminal extracellular domain of TSHR (SEQ ID NO: 2, lacking the signal peptide) is known to be poorly expressed as a soluble protein. Therefore, a variant of this domain (SEQ ID NO: 3, CRD-238) in which amino acids 297-346 of SEQ ID NO: 2 (corresponding to amino acids 317-366 of the full-length protein) was removed was transiently expressed in CHO cells on a 250 ml scale (Figure 7A). For easier protein purification, molecules were generated using 8×His tags and AVI tags, and the tags were isolated from the C-terminus of the extracellular domain using a GGGS linker (SEQ ID NO: 4) (SEQ ID NO: 5, CRD-238).

[0348] Further molecules were also generated in CHO cells in which the N-terminal extracellular domain of TSHR was combined with the CH1-CH2-CH3 domain derived from the IgG1 heavy chain (SEQ ID NO: 6, CRD-240) (Figure 7B). The C-terminus of the extracellular domain was linked to CH1 using the (GGGGS)3 linker (SEQ ID NO: 7, repeated three times for SEQ ID NO: 8). Similar molecules were also generated that contained small extracellular domain variants but fused only the IgG1 hinge and the CH2-CH3 domain (SEQ ID NO: 9, CRD-239) with the same linker (Figure 7C). All molecules were also given heterologous N-terminal signal peptides (SEQ ID NO: 19).

[0349] The molecules produced are summarized in Table 2, which provides the identifiers used herein, the expected molecular weight (MW), expected isoelectric point (pI, (M-1*cm-1)), expected extinction coefficient (EC), and actual yield for each molecule. All molecules were expressed at their expected molecular weights when observed by SDS-PAGE.

[0350] [Table 3]

[0351] Example 3 Next, the binding capacities of various molecules to actual pathogenic antibodies were determined in serum samples from GD patients. To perform this antibody depletion assay, CRD-239 and CRD-240 were biotinylated in the Fc region and conjugated onto avidin-coated Sepharose beads. Eighty-five human serum samples (titers approximately 3–30 RU / mL) positive for anti-TSHR IgG antibodies were incubated with the bead-conjugated CRD-239 molecules for 1 hour, and then the beads were isolated by centrifugation (3000 g, 2 min). Autoantibody titers were determined using ELISA both before and after incubation. Each serum sample was incubated separately with CRD-239. CRD-239 was able to deplete over 80% of TSHR autoantibodies in over 75% of GD patient serum samples (64 out of 85 samples) (Figure 8A). The median depletion rate was 88.4%.

[0352] Nine serum samples were also incubated with CRD-240, and their binding affinity to autoantibodies was calculated. CRD-239 had a higher binding affinity (lower Kd) than CRD-240 in eight of the nine samples tested, and generally resulted in insufficient binding (Figure 8B). The median binding Kd for CRD-239 was 56 nM, compared to 267 nM for CRD-240. When an equal amount of CRD-240 was loaded onto beads and added to the samples, CH1-containing molecules showed significantly inferior depletion, with 75% depletion in samples far less than 80%.

[0353] Several additional linkers were tested to fuse the N-terminal extracellular domain of the TSHR mutant to the IgG1 hinge, CH2, and CH3 regions. The following molecules were produced in CHO cells: CRD-285 (SEQ ID NO: 10) without a linker between the TSHR mutant and the hinge region, CRD-286 (SEQ ID NO: 11) with the EAAAKEAAAK (SEQ ID NO: 14) linker, and CRD-287 (SEQ ID NO: 12) with the AEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 15) linker (Figure 7C). A mutant of CRD-239 containing the S381C mutation (which is S293C in IgG itself) within the CH2 region (CRD-527, SEQ ID NO: 13) was also generated. All proteins were well expressed, and in fact, all molecules lacking the CH1 domain showed much better expression than CRD-240. The molecules are summarized in Table 2.

[0354] Example 4 To test the molecule's ability to target autoreactive B cells themselves, rather than simply capturing autoantibodies, CRD-239 was incubated with various hybridomas (see Table 3), one of which produced anti-TSHR antibodies and the other producing antibodies against unrelated proteins. Incubation was 60 minutes at 4 degrees Celsius. After incubation, cells were washed twice with FACS buffer (DPBS containing 1% FBS) and then incubated with an anti-human IgGFc region fluorophore conjugate antibody. After this second incubation, cells were washed twice again and analyzed by flow cytometry (CytoFlex by Beckman-Coulter). Cells incubated with the secondary antibody alone were used as a negative control. The mean fluorescence intensity (MFI) multiplier change from background values ​​was calculated. CRD-239 strongly bound to hybridomas expressing BCR against TSHR, with a 22-fold change compared to background MFI, while no binding to control hybridomas was observed (Figure 9). This data demonstrates that the molecule of the present invention can target B cells and provide permanent remission of GD, not merely a temporary reduction in autoantibody levels.

[0355] [Table 4]

[0356] Example 5 To test the ability of molecules to be specifically internalized by autoreactive B cells, hybridoma B cell lines TSHR-51, which express BCR for TSHR, and hybridoma B cell line g-66, which expresses BCR for the acetylcholine receptor (AChR), were incubated for 5 hours with Zenon® pHrodo® iFL-labeled TSHR-FC fusion molecule, CRD-239, or a negative control molecule (AChR alpha and gamma subunit fusion molecule, CRD-509). After incubation under standard culture conditions, cells were washed with buffer (DPBS 1% FBS) and analyzed using flow cytometry (CytoFlex, Beckman Coulter) to detect molecular internalization. The CRD-specific internalization ratio was calculated by dividing the MFI of hybridomas incubated with pHrodo-labeled CRD by the MFI of the same hybridomas incubated with pHrodo alone (=basal internalization background): Internalization ratio = (MFI[hybridoma X + pHrodo-labeled CRD] / MFI[hybridoma X + pHrodo]-1)*100%. The internalization ratio was normalized to BCR expression intensity by considering the hybridoma with the highest BCR expression as 100% BCR expression and dividing the internalization ratio by the percentage of BCR expression: Internalization ratio normalized to BCR expression = Internalization ratio of hybridoma "X" / (BCR FC hybridoma "X" / highest BCR FC among hybridomas).

[0357] An internalization ratio of 23.5% for CRD-239 was observed in TSHR hybridoma (TSHR-51), and no nonspecific internalization of CRD-509 was observed (Figure 10). An internalization ratio of 43.5% for CRD-509 was observed in AChR hybridoma (g-66), and no internalization of CRD-239 was observed.

[0358] Example 6 Next, we generated even shorter TSHR extracellular domain variants. Instead of simply removing amino acids 317–366 of the extracellular main, the new shorter variants contained only amino acids 21–316, or, as even shorter variants, contained only amino acids 21–280 or even 21–261 (the N-terminal portion of the extracellular domain without the signal peptide) or 1–261 (the N-terminal portion containing the endogenous TSHR signal peptide). Furthermore, we generated several mutations, including those within the ligand-binding pocket and those designed to reduce intramolecular aggregation or cleavage. Mutations were generated in short, intermediate, and long variants of the extracellular domain. The molecules are mutant extracellular domains with chimeric signal peptides (SEQ ID NO: 20, or the endogenous signal peptide in CRD-1044) fused to the Fc fragment as described above, summarized in Table 5.

[0359] [Table 5]

[0360] However, it is noteworthy that most of the new mutant molecules were well expressed, and the removal of the first 32 amino acids (CRD-1034) significantly reduced the yield, as did the mutations of the four initial cysteines within the molecule (C24, C29, C31, and C41). Cysteine ​​can form disulfide bridges between molecules, but it can also play a role in proper folding / tertiary structure. Disulfide bridges are known to form between C283 and C398 and between C284 and C408. Therefore, it was quite surprising that while mutations in the initial cysteine ​​were detrimental, some mutations in the later cysteine ​​(C283V, C284V, C301V, C390V, C398V, C408V) were not detrimental to the yield. GQE367-369 for NET mutations are known in the art to reduce receptor cleavage, and these mutations were also found to negatively affect the yield. Lysine 313 was also found to be a potential cleavage site, and mutation to alanine neutralized the cleavage, blocking it without adversely impacting the yield.

[0361] Molecules that yielded high yields were also tested for their ability to deplete autoantibodies from the serum of Graves' disease patients. As before, 20 serum samples from GD patients were incubated with 2.94 mM of various molecules of the present invention for 1 hour, and then anti-TSHR titers were measured by ELISA. The depletion rate was calculated as 100*(1-depleted serum titer / original titer). All of the molecules tested performed as well as CRD-239 (Figure 11), indicating that shorter shortenings did not significantly remove autoantibody epitope targets. Furthermore, mutations that inactivated ligand binding and reduced aggregation did not adversely affect autoantibody binding. In fact, some molecules performed even better than CRD-239, which produced very close to 100% depletion. Therefore, all of these molecules were considered effective in reducing TSHR autoantibody levels and treating GD.

[0362] Example 7 Having established that the novel molecule could capture autoantibodies, we then tested whether it could bind to autoreactive B cells. B cell hybridoma cell lines TSHR-51 expressing anti-TSHR BCR and an unrelated hybridoma (5H10) cell line were cultured with the novel molecule (concentration 32 nM). The unrelated molecule that binds to 5H10 cells was used as a control along with the secondary antibody alone. All molecules were highly specific to TSHR-51 cells, and all bound at least as well as CRD-527, with the exception of CRD-1038 (Figure 12A).

[0363] Next, we tested whether the molecule could kill autoreactive B cells. Removal of autoreactive B cells could potentially result in a much more persistent treatment of GD. To induce sufficient B cell death, CRD-527 was conjugated to the pyrrolobenzodiazepine (PBD) chemotherapy tesirin by using Fc-native cysteine ​​conjugation. Hybridomas expressing BCR against TSHR were incubated for 48 hours with either tesirin-conjugated CRD-527, naked CRD-527, or an unrelated molecule (CRD-509) conjugated to tesirin. Neither naked CRD-527 nor the negative control molecule resulted in significant B cell death. In contrast, CRD-527-tesirin resulted in dose-dependent cell death, killing approximately 90% of B cells at 2.5 nm (the highest concentration tested) (Figure 12B). Therefore, tesirin is a very potent effector component, and its conjugation can be used to kill autoreactive B cells when conjugated to the Ig-like molecules of the present invention. Conjugation to tesirin is superior to the use of Fc alone in inducing B cell death. It will be understood that tesirin conjugation to any of the molecules of the present invention has the same effect and increases B cell death.

[0364] Other effector molecules are tested. These include alpha-amanitin, tesirin, Dxd, PNU-159682, MMAE, MMAF, and the anti-TSHR molecules of the present invention conjugated to triptolide. All show superior killing than that produced by the unmodified Fc domain. Fc domains with mutations that increase ADCC are also tested. The above-described Fc mutations are generated in Fc, and killing is tested in anti-TSHR hybridomas. The killing is specific to these hybridomas but not to hybridomas against other targets, and the killing is superior to that produced by the unmodified Fc.

[0365] Autoreactive B cells are well established to be found in naive / healthy mice, particularly in inbred lines (e.g., Ding and Yan, "Regulation of autoreactive B cells: checkpoints and activation," Arch.Immunol.Ther.Exp., 2007, 55, 83-89; Wang et al., "The naive B cell repertoire predisposes to antigen-induced systemic lupus erythematosus," J Immunol. May 1, 2003; 170(9):4826-32; and Fereidan-Esfahani et al., "IgM natural autoantibodies in physiology and the treatment of disease," Methods Mol Biol. 2019:1904:53-81). To confirm this, blood was collected from 6-8 week old naive C57Bl6 inbred female mice and an ELISA immunoassay was performed to measure anti-TSHR antibody titers. All mice were found to be positive for the antibody. The presence of these autoreactive antibodies indicates the existence of potentially autoreactive B cells even before immunization with TSHR fragments.

[0366] To test the ability of the molecule of the present invention to kill these potentially autoreactive B cells, 6-8 week old female C57BL6 mice were intravenously immunized with the naked TSHR-Fc molecule of the present invention or the drug-conjugated TSHR-Fc molecule of the present invention (at doses of 0.5 mg / kg or higher) twice a week for a total of 6-8 injections. Subcutaneous injection was also tested. Negative control groups included mice treated with PBS and mice administered with an unrelated Ig-like molecule conjugated to the drug. Serum samples were isolated during the experiment and autoantibody titers were assessed. At the end of the immunization period, relative antibody titers against TSHR were compared using a TSHR-specific immunoassay (ELISA). Post-immunization, animals immunized with naked TSHR-Fc showed an increase in anti-TSHR titer, while animals immunized with drug-conjugated TSHR-Fc did not show an increase in titer. This demonstrates that the drug-conjugated molecule of the present invention can kill GD autoreactive B cells and treat this disease.

[0367] GD is also induced in mice by subcutaneous injection of TSHR ECD fragments. The ability to treat GD in this organism with the molecule of the present invention is confirmed. Serum is obtained and antibody titer levels are monitored. The molecule of the present invention not only kills target B cells but also reduces circulating antibody levels.

[0368] Various molecules of the present invention are tested in vivo. All of these molecules are found to effectively treat GD in vivo, kill autoreactive B cells, and reduce autoantibody titer levels. All tested effector molecules are found to be superior to Fc.

[0369] While the present invention has been described in conjunction with its specific embodiments, it will be apparent that many alternative, modified, and variant forms are obvious to those skilled in the art. Therefore, it is intended to encompass all such alternative, modified, and variant forms that fall within the spirit and broad scope of the appended claims.

Claims

1. A polypeptide comprising a fragment of the N-terminal extracellular domain of a thyroid-stimulating hormone receptor (TSHR) and at least one mutation that increases the solubility of the fragment, decreases the aggregation of the fragment, or both.

2. The polypeptide according to claim 1, comprising a deletion of the C-peptide region of the N-terminal extracellular domain of the TSHR.

3. The polypeptide according to claim 1 or 2, wherein the N-terminal extracellular domain of TSHR comprises the amino acid sequence provided in SEQ ID NO:

1.

4. The polypeptide according to any one of claims 1 to 3, wherein the mutation is a deletion of amino acids 297 to 346 of SEQ ID NO:

1.

5. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide includes or consists of Sequence ID No.

3.

6. The polypeptide according to any one of claims 1 to 4, wherein the mutation is a deletion of amino acids 297 to 393 of SEQ ID NO:

1.

7. The polypeptide according to any one of claims 1 to 4 and 6, wherein the polypeptide includes or consists of Sequence ID No.

83.

8. The polypeptide according to any one of claims 1 to 4, wherein the mutation is a deletion of amino acids 261 to 393 of SEQ ID NO:

1.

9. The polypeptide according to any one of claims 1 to 4, 6, and 8, wherein the polypeptide comprises or consists of Sequence ID No.

84.

10. The polypeptide according to any one of claims 1 to 4, wherein the mutation is a deletion of amino acids 242 to 393 of SEQ ID NO:

1.

11. The polypeptide according to any one of claims 1 to 4, 6, 8, and 10, wherein the polypeptide includes or consists of Sequence ID No.

85.

12. The aforementioned at least one mutation is a. Mutation of C263 to valine and mutation of C264 to valine, b. Mutation of K293 to alanine, c. Mutation of G347 to asparagine, mutation of Q348 to glutamate, and mutation of E349 to threonine, d. Mutations to valine at C263, C264, C281, G347, Q348, glutamine, E349, threonine, C370, C378, and C388, and e. Combinations of those, A polypeptide according to any one of claims 3 to 11, wherein the amino acid position is selected from and the amino acid position is relative to SEQ ID NO:

1.

13. The polypeptide according to any one of claims 1 to 12, further comprising at least one mutation in the ligand-binding domain of the TSHR, wherein the mutation reduces the binding of the N-terminal extracellular domain of the TSHR to TSH.

14. The polypeptide according to claim 13, wherein the mutation is the K163, E231, or both mutations in SEQ ID NO:

1.

15. The polypeptide according to claim 13 or 14, wherein K163 is mutated to alanine, E231 is mutated to lysine, or both.

16. The polypeptide according to any one of claims 1 to 4, 6, 8, 10, 13, and 14, wherein the polypeptide includes or consists of sequences selected from sequence numbers 86 to 88.

17. The polypeptide according to any one of claims 1 to 16, further comprising an effects pedal section.

18. The polypeptide according to claim 17, wherein the effector portion includes the Fc domain of a human antibody heavy chain.

19. The polypeptide according to any one of claims 1 to 18, wherein the polypeptide comprises or consists of sequences selected from sequence numbers 6, 9 to 13 and 71 to 82.

20. The polypeptide according to claim 17, wherein the effector portion is not an Fc domain.

21. The polypeptide according to claim 17 or 18, wherein the effector portion comprises an Fc domain having at least one mutation that increases antibody-dependent cell-mediated cytotoxicity (ADCC).

22. The polypeptide according to any one of claims 17 to 21, wherein the effector portion can induce cell death of cells bound to the fragment.

23. The polypeptide according to any one of claims 17 to 22, wherein the effector portion is selected from an Fc domain containing at least one mutation that increases ADCC, amatoxin / amanitin, anthracycline, anthramycin dimer, calicheamycin, camptothecin or its analogues, duocalmycin, triptolide, and a tubulin inhibitor.

24. The polypeptide according to any one of claims 17 to 23, wherein the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

25. The polypeptide according to claim 24, wherein the effector portion is tesirin.

26. The polypeptide according to any one of claims 17 to 25, wherein the effector portion includes an Fc domain containing SEQ ID NO: 61 or SEQ ID NO: 63, and the Fc domain contains a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 61 or SEQ ID NO:

63.

27. The polypeptide according to any one of claims 17 to 26, wherein the effector portion is conjugated to the polypeptide by a linker.

28. a. A first polypeptide comprising a fragment of the N-terminal extracellular domain of a thyroid-stimulating hormone receptor (TSHR) or an analog or derivative thereof, and a first dimerization domain, b. A second polypeptide comprising a fragment of the N-terminal extracellular domain of TSHR or an analog or derivative and a second dimerization domain, A composition comprising the first and second dimerizing domains, wherein the first and second dimerizing domains are configured to dimerize with each other.

29. The composition according to claim 28, wherein the dimerization includes forming a covalent bond between the first dimerization domain and the second dimerization domain.

30. The composition according to claim 28 or 29, wherein the protein complex comprises an immunoglobulin scaffold.

31. a. The first dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the second dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains dimerize via a disulfide bond, or b. The first and second dimerization domains each include a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, wherein neither the first nor the second dimerization domain contains the CH1 domain or the CL domain. The composition according to any one of claims 28 to 30.

32. The composition according to any one of claims 28 to 31, wherein the fragments and dimerization domains of the first, second, or both polypeptide chains are separated by a linker.

33. The composition according to any one of claims 28 to 32, wherein the first dimerization domain and the second dimerization domain each comprise the Fc region of a human antibody heavy chain.

34. The composition according to claim 33, wherein the Fc region can induce cytotoxicity against cells bound to the protein complex.

35. The composition according to claim 33 or 34, wherein the first polypeptide chain comprises a first CH3 domain of an immunoglobulin heavy chain, a first CH2 domain of an immunoglobulin heavy chain, or both, and the second polypeptide chain comprises a second CH3 domain of an immunoglobulin heavy chain, a second CH2 domain of an immunoglobulin heavy chain, or both.

36. The composition according to claim 35, wherein the first CH3 domain comprises at least a first mutation, and the second CH3 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and the second polypeptide chain.

37. The composition according to claim 35 or 36, wherein the first CH2 domain comprises at least a first mutation, and the second CH2 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and the second polypeptide chain.

38. The composition according to claim 36 or 37, wherein the first mutation is selected from the mutations provided in Table 1, and the second mutation is a mutation provided in Table 1 that corresponds to the first mutation.

39. The composition according to claim 38, wherein the first mutation is a T366W mutation in the CH3 domain, and the second mutation is a combination of a T366S mutation, an L368A mutation, and a Y407V mutation.

40. The composition according to any one of claims 31 to 39, wherein the Fc region of the first, second, or both of the polypeptide chains is separated from the fragment or the dimerization domain by a linker.

41. The composition according to any one of claims 33 to 40, wherein the Fc region comprises at least one mutation that increases ADCC or CDC.

42. The composition according to claim 41, wherein the Fc region is an Fc region containing sequence number 61 or sequence number 63, and comprises a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E and G16A / S47E / H48F / S104T / I112E in sequence number 61 or sequence number 63.

43. The composition according to any one of claims 33 to 42, wherein the dimerization domain of the first, second, or both of the polypeptide chains is C-terminus with respect to the fragment and N-terminus with respect to the Fc region.

44. The composition according to any one of claims 28 to 43, which lacks an antibody variable domain.

45. The composition according to any one of claims 28 to 43, wherein the first polypeptide chain and the second polypeptide chain each contain or consist of the polypeptides described in any one of claims 18 to 19 and 21 to 27.

46. The composition according to any one of claims 28 to 45, further comprising a fragment of TSHR or an analog or derivative thereof and a third polypeptide comprising a third dimerizing domain, wherein the first polypeptide further comprises a fourth dimerizing domain, and the third and fourth dimerizing domains are dimerizable to each other.

47. a. The third dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the fourth dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains dimerize via disulfide bonds, or b. The third and fourth dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, wherein neither the first nor the third polypeptide contains the CH1 domain or the CL domain. The composition according to claim 46.

48. The composition according to claim 46 or 47, further comprising a fragment of TSHR or an analog or derivative thereof and a fourth polypeptide comprising a fifth dimerization domain, wherein the second polypeptide further comprises a sixth dimerization domain, and the fifth and sixth dimerization domains are dimerizable to each other.

49. a. The fifth dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the sixth dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains dimerize via disulfide bonds, or b. The composition according to claim 48, wherein the fifth and sixth dimerizing domains each comprise a domain selected from the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and are dimerized by a disulfide bond, and neither the first nor the third polypeptide contains the CH1 domain or the CL domain.

50. The composition according to any one of claims 31 to 49, wherein both the first polypeptide and the second polypeptide do not contain a CH1 domain, or both contain a CL domain.

51. The composition according to any one of claims 46 to 50, wherein the third and fourth dimerizing domains or the fifth and sixth dimerizing domains include mutations that enable dimerization of the third and fourth dimerizing domains and dimerization of the fifth and sixth dimerizing domains, and that inhibit dimerization of the third dimerizing domain to the fifth or sixth dimerizing domain and dimerization of the sixth dimerizing domain to the third or fourth dimerizing domain.

52. The composition according to any one of claims 28 to 51, wherein the fragment is a polypeptide according to any one of claims 1 to 17.

53. The composition according to any one of claims 28 to 52, wherein the first polypeptide chain and the second polypeptide chain comprise different TSHR fragments.

54. The composition according to any one of claims 28 to 53, wherein TSHR lacks its endogenous signal peptide and contains a heterologous signal peptide.

55. The composition according to any one of claims 28 to 54, wherein at least one of the fragments comprises a mutation that increases the solubility of the composition, decreases the aggregation of the composition, or both.

56. The composition according to any one of claims 28 to 55, wherein at least one of the fragments comprises a mutation in the ligand-binding domain of TSHR, the mutation reducing the binding of the N-terminal extracellular domain of TSHR to TSH.

57. The composition according to any one of claims 28 to 56, wherein the analog or derivative contains at least 85% identity with TSHR.

58. The composition according to any one of claims 28 to 57, wherein the fragment comprises at least 20 consecutive amino acid TSHRs.

59. The composition according to any one of claims 28 to 58, wherein the fragment comprises at least one B cell receptor (BCR) specific epitope target of an autoantibody.

60. The composition according to any one of claims 28 to 59, comprising a first or second polypeptide containing a sequence selected from sequence numbers 6, 9 to 13 and 71 to 82.

61. The composition according to any one of claims 31 to 60, wherein the hinge domain, the CH2 domain, or the CH3 domain contains at least one mutation that reduces ADCC.

62. The at least one mutation that reduces ADCC, a. Mutations in the hinge domain, including the L19A and L20A mutations of Sequence ID No. 22, and b. Mutations in the CH2 domain, including the N59A mutation of Sequence ID No. 36, A composition according to claim 61, selected from the following.

63. The composition according to any one of claims 28 to 62, further comprising at least one effector portion capable of inducing cell death of cells bound to the composition.

64. The composition according to claim 63, wherein the effector portion is not an Fc domain.

65. The composition according to claim 63 or 64, wherein the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

66. The composition according to claim 65, wherein the effector portion is tesirin.

67. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 27 or a composition according to any one of claims 28 to 66, and a pharmaceutically acceptable carrier, excipient, or adjuvant.

68. A method for treating Graves' disease (GD) in a subject requiring treatment for GD, the method comprising administering to the subject a composition comprising a fragment of the N-terminal extracellular domain of TSHR or an analog or derivative thereof, thereby treating GD.

69. The method according to claim 68, wherein the TSHR lacks a signal peptide and contains or comprises SEQ ID NO: 1, or the TSHR contains a signal peptide and contains or comprises SEQ ID NO: 2, or both.

70. The method according to claim 68 or 69, wherein the composition comprises the polypeptide described in any one of claims 1 to 27.

71. The method according to any one of claims 68 to 70, wherein the composition is the composition according to any one of claims 28 to 66.

72. The method according to any one of claims 68 to 71, wherein the composition is the pharmaceutical composition described in claim 67.

73. The method according to any one of claims 68 to 72, further comprising reducing the level of circulating antibodies against TSHR in the subject.

74. The method according to any one of claims 68 to 73, wherein the treatment comprises reducing the concentration of circulating autoantibodies against TSHR.

75. The method according to any one of claims 68 to 74, wherein the composition comprises an Fc region, and the treatment comprises killing B cells that produce anti-TSHR autoantibodies.

76. The method according to claim 75, wherein the B cells are autoreactive B cells that produce autoantibodies against the fragments of the composition.

77. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 27.

78. A nucleic acid system comprising a nucleic acid molecule, wherein the first nucleic acid molecule encodes the first polypeptide of the composition according to any one of claims 28 to 66, and the second nucleic acid molecule encodes the second polypeptide of the composition according to any one of claims 28 to 66.

79. The nucleic acid system according to claim 78, further comprising a third nucleic acid molecule encoding the third polypeptide of the composition according to any one of claims 46 to 66, a fourth nucleic acid molecule encoding the fourth polypeptide of the composition according to any one of claims 46 to 66, or both.

80. A method for producing a polypeptide according to any one of claims 1 to 27 or a composition according to any one of claims 28 to 66, comprising expressing a nucleic acid molecule according to claim 77 or a nucleic acid system according to claim 78 or 79 in a cell, wherein the nucleic acid system is configured to produce the encoded polypeptide in the cell, thereby producing a polypeptide according to any one of claims 1 to 27 or a composition according to any one of claims 28 to 66.

81. A method for producing proteins, To obtain a first fragment of the extracellular domain of TSHR or an analog or derivative thereof, and a second fragment of the extracellular domain of TSHR or an analog or derivative thereof, The first fragment is linked to a first dimerization domain to produce a first polypeptide chain, the second fragment is linked to a second dimerization domain to produce a second polypeptide chain, the first and second dimerization domains can dimerize with each other, and the first polypeptide and the second polypeptide are brought into contact under conditions sufficient to induce the dimerization, or The method involves culturing host cells containing one or more vectors comprising nucleic acid sequences encoding at least two polypeptide chains, wherein the two polypeptide chains are i. Obtaining a first fragment of the extracellular domain of TSHR or an analog or derivative thereof, and a second fragment of the extracellular domain of TSHR or an analog or derivative thereof, ii. The first fragment is linked to a first dimerization domain to produce a first polypeptide chain, and the second fragment is linked to a second dimerization domain to produce a second polypeptide chain, wherein the first and second dimerization domains are capable of dimerizing with each other. A method for producing a protein, comprising culturing, which is produced by [a certain method].

82. The method according to claim 81, wherein the protein complex is a protein complex of the composition according to any one of claims 28 to 66.

83. a. Linking a third dimerization domain to the first dimerization domain or first fragment within the first polypeptide chain to obtain a third fragment of the extracellular domain of TSHR or its analog or derivative, linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain, wherein the third dimerization domain and the fourth dimerization domain can dimerize with each other, and the first, second and third polypeptides are brought into contact under conditions sufficient to induce the dimerization, or b. In the host cells, i. To obtain a third fragment of TSHR or an analog or derivative thereof, and ii. Linking the third fragment to the fourth dimerization domain to produce a third polypeptide chain. The further comprising expressing a nucleic acid sequence encoding a third polypeptide chain produced by, The method according to claim 81 or 82, wherein the first polypeptide chain further comprises a third dimerization domain, and the third dimerization domain and the fourth dimerization domain can dimerize with each other.

84. a. Linking the sixth dimerization domain to the second dimerization domain or second fragment within the second polypeptide chain to obtain a fourth fragment of the extracellular domain of TSHR or its analogue or derivative, linking the fourth fragment to the fifth dimerization domain to produce a fourth polypeptide chain, wherein the fifth dimerization domain and the sixth dimerization domain can dimerize with each other, and the first, second, third and fourth polypeptides are brought into contact under conditions sufficient to induce the dimerization, or b. In the host cells, i. Obtain a fourth fragment of the extracellular domain of TSHR or its analogue or derivative, and ii. Linking the fourth fragment to the fifth dimerization domain to produce a fourth polypeptide chain. The further comprising expressing a nucleic acid sequence encoding a fourth polypeptide chain produced by, The method according to claim 83, wherein the second polypeptide chain further comprises a sixth dimerization domain, and the fifth dimerization domain and the sixth dimerization domain can dimerize with each other.

85. The method according to any one of claims 81 to 84, further comprising causing at least one mutation in the extracellular domain of the protein, or shortening the protein and removing a portion thereof.

86. The method according to claim 85, further comprising measuring the aggregation of the mutant protein or shortened protein, and selecting the mutant protein or shortened protein in which aggregation has decreased.

87. A method for producing proteins, a. Obtaining a fragment of the extracellular domain of TSHR or its analogues or derivatives, b. Producing a mutant fragment by generating at least one mutation in the aforementioned fragment, c. Measuring the solubility, aggregation, or both of the mutant fragments, d. Selecting at least one mutant fragment that increases solubility, decreases aggregation, or both, compared to the obtained fragment, A method that includes and thereby produces a protein.

88. The method according to any one of claims 80 to 87, further comprising linking an effector portion to at least one of the polypeptide chain or the mutant fragment, wherein the effector portion can kill a cell bound to the at least one polypeptide.

89. The method according to claim 88, wherein the effector portion is not in the Fc domain.

90. The method according to claim 88, wherein the effector portion includes the Fc domain of the antibody heavy chain.

91. The method according to any one of claims 88 to 90, wherein the effector portion comprises a molecule selected from an Fc domain having at least one mutation that increases ADCC or CDC, alpha-amanitin, PNU-159682, tecilin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

92. The method according to claim 91, wherein the effector portion is an Fc domain comprising at least one mutation that increases ADCC or CDC, comprising SEQ ID NO: 61 or SEQ ID NO: 63, wherein the Fc domain comprises a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 61 or SEQ ID NO:

63.

93. A protein produced by the method described in any one of claims 81 to 92.

94. A method for determining the suitability of an object to be treated by the method of any one of claims 68 to 76, the method comprising: receiving a sample from the object; contacting the sample with a protein according to any one of claims 1 to 27 and 93 or a composition according to any one of claims 28 to 66; and determining the binding of an autoantibody in the sample to the protein or the composition, wherein the binding of the autoantibody to the protein or the composition indicates that the object is suitable to be treated by the method of any one of claims 68 to 76, and the method for determining the suitability of the object to be treated by such method.

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