Improved IG-like fusion proteins for treating graves disease

Abstract

Polypeptides comprising a fragment of an N-terminal extracellular domain (ECD) of TSHR wherein said ECD consists of SEQ ID NO: 1 and comprising at least one mutation selected from P7S, H12L, RISE, V19L, T36L, S64Y, I65F, V83I, R92K, Y96S, Y96E, Y96R, D131Q, D131N, V149K, S171D, G174A, V195D, K198Q, L210Y, V218I, I233K, I233K and V149K, are provided. Compositions comprising the polypeptide, and nucleic acid molecules encoding the polypeptide are also provided as are methods of treating Graves' Disease.

Description

IMPROVED IG-LIKE FUSION PROTEINS FOR TREATING GRAVES DISEASE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 717,441, filed on November 7, 2024, and U.S. Provisional Patent Application No.63 / 871,026, filed on August 27, 2025, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (CNPY-P-010-PCT.xml; Size: 255,454 bytes; and Date of Creation: October 27, 2025) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of fusion protein generation and Graves’ disease (GD) treatment.BACKGROUND OF THE INVENTION

[0004] Graves’ disease is an autoimmune disease that primarily affects the thyroid gland, manifesting as hyperthyroidism, diffuse goiter, thyroid eye disease (Graves' orbitopathy) (GO), and occasionally a dermopathy referred to as pretibial or localized myxedema (PTM). Graves' disease is the most common cause of hyperthyroidism (60-80% of all cases) and occurs at all ages but is most common in people ages 20 to 50 years, and even more so in women. In addition, there is a strong genetic background component linked to Graves' disease, as well as environmental factors including pregnancy (mainly postpartum), iodine excess, infections, emotional stress and smoking.

[0005] Auto-antibodies, primarily thyroid stimulating immunoglobulin (TSI), also known as Thyroid stimulating hormone Receptor Antibodies (TRAbs), bind to thyroid-stimulating hormone receptor (TSHR) on the thyroid cell membrane and stimulate the action of the thyroid-stimulating hormone (TSH), resulting in both thyroid hormone synthesis and thyroid gland growth, ultimately causing hyperthyroidism.

[0006] Symptoms of hyperthyroidism may include insomnia, hand tremor, hyperactivity, hair loss, excessive sweating, heat intolerance and weight loss despite increased appetite. Further signs are most commonly a diffusely enlarged non-tender thyroid, lid lag, excessive lacrimation (due to GO), heart arrhythmias and hypertension. Thyrotoxic patients may experience behavioral and personality changes, such as psychosis, agitation, and depression. In milder hyperthyroidism, patients may experience less overt manifestations, for example anxiety, restlessness, irritability and emotional lability.

[0007] There is currently no cure available for Graves' disease and current treatments are therefore directed towards targeting the presenting symptoms. Initially, symptomatic patients with cardiac involvement should be started on beta-adrenergic blockers such as Atenolol, specifically those with a heart rate above 90 beats / min, a history of cardiovascular disease and elderly patients. There are three main treatment modalities to reduce thyroid hormone synthesis: oral antithyroid drugs (ATDs) / Thionamides, radioactive iodine (RAI) and thyroidectomy. The latter two approaches eventually lead to patients becoming hypothyroid and result in lifetime supplementation of thyroid hormones.

[0008] The primary aim of ATD treatment is to achieve normalization of thyroid hormone production and induce remission of disease; occasionally it may be to prepare patients for radio-iodine ablation or surgery. ATD therapy such as Methimazole (MMI), or a derivative called Carbimazole, and Propylthiouracil (PTU) block thyroid hormone synthesis and are associated with some rare side-effects such as agranulocytosis, hepatotoxicity, and pancreatitis. While ATDs do control the symptoms, they do not cure the disease and hence relapses are common, with a remission rate of about 50-60%.

[0009] Due to the varying success of each treatment option, patients are often subjected to more than one approach if the first attempted treatment does not prove entirely successful. The risk of relapse or subsequent hypothyroidism is substantial and the general efficacy of available treatments for Graves' disease is less than desired.

[0010] As a result, there is an exigency for alternative and safe therapies that provide sufficient, long lasting effects. Specifically, there is a large unmet need for new therapies that target the TSH autoantibodies that cause Grave’s disease. Beyond this, 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 are greatly needed.

[0011] There does not currently exist any treatment that targets the cause of GD; neither the autoreactive antibodies nor the B cells that produce the antibodies. Improved therapeutic modalities that target the mechanistic causes of GD are greatly needed.SUMMARY OF THE INVENTION

[0012] The present invention provides polypeptides comprising a fragment of an N-terminal extracellular domain (ECD) of TSHR wherein the ECD consists of SEQ ID NO: 1 and comprising at least one mutation selected from P7S, H12L, R18L, V19L, T36L, S64Y, I65F, V83I, R92K, Y96S, Y96E, Y96R, D131Q, D131N, V149K, C156S, C156A, S171D, G174A, V195D, K198Q, L210Y, V218I, I233K, I233K and V149K, W238R, W238L, F249Y, L250I, F249Y and L250I, and equivalent mutations. Compositions comprising the polypeptide, and nucleic acid molecules encoding the polypeptide are also provided as are methods of treating Graves’ Disease.

[0013] According to first aspect, there is provided a polypeptide comprising a fragment of an N-terminal extracellular domain (ECD) of Thyroid Stimulating Hormone Receptor (TSHR) and at least one mutation; wherein the ECD consists of SEQ ID NO: 1 and the at least one mutation is selected from P7S, H12L, R18L, V19L, T36L, S64Y, I65F, V83I, R92K, Y96S, Y96E, Y96R, D131Q, D131N, V149K,, S171D, G174A, V195D, K198Q, L210Y, V218I, I233K, and I233K and V149K, and any combination thereof.

[0014] According to another aspect, there is provided a polypeptide comprising a fragment of an N-terminal extracellular domain (ECD) of Thyroid Stimulating Hormone Receptor (TSHR) and at least one mutation; wherein the ECD consists of SEQ ID NO: 1 and the at least one mutation is selected from D131Q and D131N, wherein the numbering is with respect to SEQ ID NO: 1.

[0015] According to another aspect, there is provided a polypeptide comprising a fragment of an N-terminal extracellular domain (ECD) of Thyroid Stimulating Hormone Receptor (TSHR) and at least one mutation; wherein the ECD consists of SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1 and which binds anti-TSHR autoantibodies and the at least one mutation is selected from D131Q and D131N, wherein the numbering is with respect to SEQ ID NO: 1.

[0016] According to some embodiments, the at least one mutation is selected from D131Q and D131N.

[0017] According to some embodiments, the at least one mutation is D131Q.

[0018] According to some embodiments, the polypeptide comprises a deletion of a C-peptide region of the N-terminal extracellular domain of TSHR.

[0019] According to some embodiments, the deletion is a deletion of amino acids 261-326 of SEQ ID NO: 1.

[0020] According to some embodiments, the fragment is SEQ ID NO: 5 and the mutation is within SEQ ID NO: 5.

[0021] According to some embodiments, the fragment is SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5 and which binds anti-TSHR autoantibodies and the mutation is within SEQ ID NO: 5 or the sequence with at least 85% sequence identity to SEQ ID NO: 5.

[0022] According to some embodiments, the fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 101-119.

[0023] According to some embodiments, the fragment comprising at least one mutation comprises or consists of SEQ ID NO: 110.

[0024] According to some embodiments, the deletion is deletion of amino acids 242-326 of SEQ ID NO: 1, and the fragment is SEQ ID NO: 3 and the mutation is within SEQ ID NO: 3.

[0025] According to some embodiments, the deletion is deletion of amino acids 242-326 of SEQ ID NO: 1, and the fragment is SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3 and which binds anti-TSHR autoantibodies and the mutation iswithin SEQ ID NO: 3 or the sequence with at least 85% sequence identity to SEQ ID NO: 3.

[0026] According to some embodiments, the at least one mutation is D131Q.

[0027] According to some embodiments, the polypeptide further comprises at least one additional mutation selected from the group consisting of I65F, G174A and V195D.

[0028] According to some embodiments, the polypeptide comprises D131Q and I65F.

[0029] According to some embodiments, the polypeptide further comprises at least a third mutation selected from G174A and V195D.

[0030] According to some embodiments, the fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 156 and 158-177.

[0031] According to some embodiments, the fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 156, 159, 161, 169, 171-172, 174-175 and 177.

[0032] According to some embodiments, the at least one mutation is D131N.

[0033] According to some embodiments, the polypeptide further comprises at least one additional mutation selected from the group consisting of I65F, G174A and V195D.

[0034] According to some embodiments, the polypeptide comprises D131Q and I65F.

[0035] According to some embodiments, the polypeptide further comprises at least a third mutation selected from G174A and V195D.

[0036] According to some embodiments, the fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 157 and 195-202.

[0037] According to some embodiments, the polypeptide further comprises at least one mutation in a ligand binding domain of the TSHR, wherein the mutation decreases binding of the N-terminal extracellular domain of TSHR to TSH.

[0038] According to some embodiments, the mutation is mutation of K163, E231 or both within SEQ ID NO: 1.

[0039] According to some embodiments, K163 is mutated to alanine, E231 is mutated to lysine or both.

[0040] According to some embodiments, the polypeptide further comprises an Fc domain of a human antibody heavy chain, optionally wherein the fragment comprising at least one mutation is linked to the Fc domain by an amino acid linker.

[0041] According to some embodiments, the Fc domain comprises a S267E mutation, optionally wherein the Fc domain comprises of consists of SEQ ID NO: 178, 189 or 193.

[0042] According to some embodiments, the Fc domain further comprises a L328F mutation, optionally wherein the Fc domain comprises or consists of SEQ ID NO: 56, 188 or 194.

[0043] According to some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153, 155 and 203-213.

[0044] According to some embodiments, the polypeptide further comprises an effector moiety wherein the effector moiety is not an Fc domain, optionally wherein the effector moiety is conjugated to the polypeptide by a linker.

[0045] According to some embodiments, the effector moiety is selected from an amatoxin / amanitin, an anthracycline, an anthramycin-based dimer, a calicheamicin, camptothecin or an analog thereof, a duocarmycin, triptolide and a tubulin inhibitor.

[0046] According to some embodiments, the effector moiety is selected from: alpha-amanitin, PNU- 159682, tesirine, deruxtecan (Dxd), mertansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and a combination thereof.

[0047] According to some embodiments, the effector moiety is MMAE.

[0048] According to another aspect, there is provided a composition, comprising:a. a first polypeptide comprising a polypeptide of the invention; and b. a second polypeptide comprising a polypeptide of the invention.

[0049] According to some embodiments, the first polypeptide and the second polypeptide comprise different fragments of TSHR, at least one different mutation or both.

[0050] According to another aspect, there is provided a pharmaceutical composition comprising a polypeptide of the invention or a composition of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0051] According to another aspect, there is provided a method of treating Graves’ disease (GD) in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the invention, thereby treating GD.

[0052] According to some embodiments, the method further comprises reducing in the subject the levels of circulating antibodies against TSHR before the administering.

[0053] According to another aspect, there is provided a nucleic acid molecule encoding a polypeptide of the invention.

[0054] According to another aspect, there is provided a nucleic acid system comprising a nucleic acid molecule, wherein a first nucleic acid molecule encodes the first polypeptide of a composition of the invention and a second nucleic acid molecule encodes the second polypeptide of a composition of the invention.

[0055] According to another aspect, there is provided a method of determining suitability of a subject in need thereof to be treated by a method of the invention, the method comprising receiving a sample from the subject, contacting the sample with a polypeptide of the invention or a composition of the invention and determining binding of autoantibodies within the sample to the polypeptide or the composition, wherein binding of autoantibodies to the polypeptide or the composition indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.

[0056] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since 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 DESCRIPTION OF THE DRAWINGS

[0057] Figure 1: Diagrams of an embodiment of four-chain therapeutic agent of the invention.

[0058] Figures 2A-2M: Diagrams of possible embodiments of the two-chain therapeutic agent of the invention: (2A) shows general embodiments of a molecule with two heavy chains for treating GD, (2B) shows embodiments in which at least one of the CHI, CH2 or CH3 domains has been excluded, (2C) shows an embodiment in which the two protein variants are the same, (2D) shows an embodiment in which the two protein variants are different, (2E) shows an embodiment in which the two protein variants are different and the molecule does not contain a CH1 domain, (2F) shows an embodiment in which the two protein variants are different and the molecule does not contain a CHI domain or a hinge domain, (2G) shows a general embodiment in which two tandem variants are included in each heavy chain and connected via a linker, (2H) shows the tandem variant configuration in which all the subunits are the same, (2I) shows the tandem variant configuration in which all the subunits are the same without the CH1 domain, (2J) shows the tandem variant configuration in which each heavy chain contains the same two different variants, (2K) shows the tandem variant configuration in which each heavy chain contains the same two different variants without a CHI domain, (2L) shows the tandem variant configuration in which the two heavy chains contain different variants that are not the same, and (2M) shows the tandem variant configuration in which the two heavy chains contain different variants that are not the same without the CH1 domain.

[0059] Figure 3: Diagram of a generic embodiment of the four-chain therapeutic agent of the invention.

[0060] Figures 4A-4B: (4A) Line graph of depletion rate of GD patient serum (n=7) after 1 hour incubation with various concentrations of CRD- 1100 and control molecule CRD-981.(4B) Bar graph of depletion rate of GD patient serum (n=4) after 1 hour incubation with various molecules of the invention.

[0061] Figure 5: Bar graph of relative binding of three TSHR antibodies to various molecules of the invention. Binding is given as compared to binding to CRD- 1105 and calculated by:> „.... O. D of CRD-XXXOD in M-22 ELISA plate- O. D of blank (no CRD treated well) CRD-1105 o. D of CRD-1105 OD in M-22 ELISA plate - - O. D of blank (no CRD treated well) for antibody M-22 and was calculated using the same equation for K1-18 and K1-70 abs.

[0062] Figures 6A-6B: (6A) Bar graph of the increase in MFI over background for CRD-1100 binding to TSHR-51 hybridomas cells and lack of binding to an irrelevant hybridoma.(6B) Line graph of CRD-1100 binding to TSHR-51 hybridoma cells at various concentrations. Irrelevant molecule CRD-981 is used as a control.

[0063] Figures 7A-7H: (7A, 7C, 7E, 7G) Bar graphs of depletion rate of GD patient serum (n=4) after 1 hour incubation with various molecules of the invention containing a D151Q mutation. (7B, 7D, 7F, 7H) Bar graphs of binding of various molecules of the invention containing a D151Q mutation to antibody M22. Irrelevant construct CRD-999 is used as a negative control.

[0064] Figures 8A-8F: (8A, 8C, 8E) Bar graphs of depletion rate of GD patient serum (n=4) after 1 hour incubation with various molecules of the invention containing a double mutation without D151Q. (8B, 8D, 8F) Bar graphs of binding of various molecules of the invention containing a double mutation without D151Q to antibody M22. Irrelevant construct CRD-999 is used as a negative control.

[0065] Figures 9A-9F: (9A, 9C, 9E) Bar graphs of depletion rate of GD patient serum (n=4) after 1 hour incubation with various molecules of the invention containing a triple or quadrupole mutation. (9B, 9D, 9F) Bar graphs of binding of various molecules of the invention containing a triple or quadruple mutation to antibody M22. Irrelevant construct CRD-999 is used as a negative control.

[0066] Figure 10: Bar graph of depletion rate of GD patient serum (n=8) after 1 hour incubation with various single, double, and triple mutant molecules of the invention.

[0067] Figures 11A-11F: (11A) Line graph of depletion rate of GD patient serum (n=28) after 1 hour incubation with D151Q and I85F molecules of the invention. (11B) Bar graph of depletion rates from 11 A. (11C-D) (11C) Line graph and (11D) bar graph of TSHR stimulation after contact with M22 activating antibody and various molecules of the invention. Irrelevant construct CRD-999 is used as a negative control. (11E-F) Bar graph of TSHR stimulation after contact with patient sera (n=3) and various molecules of the invention at (11E) 200 nM and (11F) 8 nM.

[0068] Figures 12A-12G: (12A) Bar graph of depletion rate of GD patient serum (n=4) after 1 hour incubation with D151N molecules of the invention and their D151Q counterparts.(12B-H) Line graphs of TSHR stimulation after contact with M22 (Fig. 12B-D) or Kl-18 (Fig. 12E-G) activating antibody and (12B, 12E) CRD-1260, (12C, 12F) CRD-1261 or (12D, 12G) CRD-1262. Their D151Q counterparts are also shown, as is irrelevant construct CRD-999 which is used as a negative control.

[0069] Figures 13A-13B: Line graphs of binding to CD32b for (13A) double mutant molecules and (13B) triple mutant molecules of the invention.

[0070] Figures 14A-14J: (14A-B) (14A) Bar graph and (14B) line graphs of binding of various molecules to six different anti-TSHR hybridomas (TSHR-51, -202, -178, -116, -60 and -126) or a negative control hybridoma (G-50). (14C-J) (14C, 14E, 14G, 141) Line graphs and (14D, 14F, 14H, 14J) bar graphs of cell killing of anti-TSHR hybridomas (14C-D) TSHR-51, (14E-F) TSHR-178, (14G-H) TSHR-126 and (14I-J) TSHR-110 in the presence of various concentrations of CRD-1162 and a non-specific control molecule conjugated to MMAE as well as unconjugated CRD-1162. The table at the bottom summarizes the results.

[0071] Figure 15: Table of CRD-1206, CRD-1222 and CRD-1225 purity following pooling and dialysis.DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention, in some embodiments, provides polypeptides comprising a fragment of an N-terminal extracellular domain (ECD) of TSHR comprising at least one mutation. Compositions comprising the polypeptide are also provided. Pharmaceutical compositions comprising the polypeptide or composition of the invention are also provided as are and nucleic acid molecules encoding the polypeptide and nucleic acid systems encoding the composition. Methods of treating Graves’ Disease and selecting subjects suitable for treatment are also provided.

[0073] By a first aspect, there is provided a composition comprising a fragment of thyroid stimulating hormone receptor (TSHR) comprising at least one mutation.

[0074] By another aspect, there is provided a protein comprising a fragment of thyroid stimulating hormone receptor (TSHR) comprising at least one mutation.

[0075] By another aspect, there is provided a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a polypeptide of the invention and a second polypeptide chain comprising a polypeptide of the invention.

[0076] In some embodiments, the composition comprises a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a fragment of TSHR comprising at least one mutation and a second polypeptide chain comprising a fragment of TSHR comprising at least one mutation. In some embodiments, the composition comprises a protein complex of the invention. In some embodiments, the composition comprises a protein of the invention. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is a fusion protein.

[0077] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating into 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 residue is an artificial chemical analogue of a corresponding naturally occurring amino acid. In some embodiments, the peptide is not a cyclic peptide. In some embodiments, the fragment is not a cyclic peptide. In some embodiments, the extracellular domain is not a cyclic peptide.

[0078] In some embodiments, the protein complex is an immunoglobulin (Ig)-like complex. In some embodiments, the protein complex comprises an Ig-like scaffold. In some embodiments, the protein complex comprises an Ig-like backbone. In some embodiments, the protein complex is an Ig Fc-fusion complex. In some embodiments, the composition is devoid of an antibody variable domain. In some embodiments, the protein complex is devoid of an antibody variable domain. In some embodiments, the composition is devoid of a variable domain. In some embodiments, the protein complex is devoid of a variable domain. In some embodiments, the first chain is devoid of a variable domain. In some embodiments,the second chain is devoid of 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 a therapeutic method. In some embodiments, the protein complex is for use in a therapeutic method. In some embodiments, the composition is for use in production of a medicament. In some embodiments, the protein complex is for use in the production of a medicament. In some embodiments, the composition is for use in treating Graves’ Disease (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 in GD. In some embodiments, the protein complex is for use in determining the autoantibody titer in GD.

[0079] As used herein, the term “polypeptide chain” refers to a polymer of amino acids linked by peptide bonds from an amino terminus (N-terminus) to a carboxyl terminus (C-terminus). In some embodiments, the polypeptide chain is a recombinant polypeptide. In some embodiments, a polypeptide chain comprises 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 separate embodiment of the invention. In some embodiments, the polypeptide chain comprises at least 290 amino acids. In some embodiments, the polypeptide chain comprises at least 300 amino acids. In some embodiments, a polypeptide chain comprises at most 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 separate embodiment of the invention.

[0080] As used herein, the term “recombinant polypeptide” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring. In some embodiments, the protein complex is not naturally occurring. In some embodiments, the polypeptide chain is not naturally occurring. In some embodiments, the recombinant polypeptide is a synthetic polypeptide. The term “recombinant DNA” refers to DNA molecules formed by laboratory methods. Generally, this recombinant DNA is in the form of a vector, plasmid or virus used to express the recombinant protein in a cell. Production of recombinant proteins by cellularexpression is well known in the art and any method of recombinant protein expression may be used to produce the polypeptide of the invention. Cell free expression systems for recombinant protein production may also be employed.

[0081] The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and / or translation of said gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or translation of RNA into a precursor or mature protein (polypeptide). In some embodiments, a nucleic acid molecule of the invention is expressed in a cell to produce a polypeptide of the invention. In some embodiments, a nucleic acid complex of the invention is expressed in a cell to produce a protein complex of the invention. In some embodiments, the RNA is a vector.

[0082] Expression of a DNA sequence or an RNA within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the DNA sequence is in an expression vector such as plasmid or viral vector. In some embodiments, a Kozak sequence is inserted upper stream of the transcription initiating codon. In some embodiments, the Kozak sequence enhances the amount of protein expressed.

[0083] 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.Proteins

[0084] In some embodiments, the TSHR is a mammalian TSHR. In some embodiments, the mammal is a human. In some embodiments, the TSHR is an GD-associated protein. In someembodiments, the TSHR is a synthetic TSHR. In some embodiments, the TSHR is a target of GD autoantibodies. In some embodiments, TSHR is the extracellular domain of TSHR.

[0085] In some embodiments, the fragment comprises an extracellular domain (ECD) of TSHR. In some embodiments, the extracellular domain is the N-terminal ECD. In some embodiments, the fragment comprises a fragment of an extracellular domain of TSHR. In some embodiments, the fragment consists of the extracellular domain of a fragment thereof. In some embodiments, the fragment consists of an extracellular domain of TSHR. In some embodiments, the fragment consists of a fragment of an extracellular domain of TSHR. In some embodiments, the fragment comprises a transmembrane domain of TSHR. In some embodiments, the fragment is devoid of a transmembrane domain of TSHR. In some embodiments, the fragment is devoid of an intracellular domain of TSHR. In some embodiments, the chain is devoid of a transmembrane domain. In some embodiments, the chain is devoid of an intracellular domain. In some embodiments, the fragment includes mutations in TSHR. In some embodiments, the protein is devoid of any portion of TSHR other than the ECD. In some embodiments, the protein is devoid of the TSHR transmembrane domain. In some embodiments, the protein is devoid of the TSHR intracellular domain.

[0086] In some embodiments, the fragment comprises at least 5 amino acids of the protein. In some embodiments, the fragment comprises at least 10 amino acids of the protein. In some embodiments, the fragment comprises 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 separate embodiment of the invention. In some embodiments, the fragment comprises at least 290 amino acids. In some embodiments, the fragment comprises at least 300 amino acids. In some embodiments, amino acids of the protein are consecutive amino acids of the protein. In some embodiments, the fragment comprises less than 100% of the protein. In some embodiments, the fragment comprises less than 100% of an extracellular domain of the protein. In some embodiments, the fragment comprises less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50% of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, the fragment comprises less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50% of an extracellular domain of the protein.Each possibility represents a separate embodiment of the invention. In some embodiments, the fragment comprises between 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 separate embodiment of the invention. In some embodiments, the fragment comprises between 290 and 350 amino acids. In some embodiments, the fragment comprises between 290 and 310 amino acids. In some embodiments, the fragment comprises between 5-50 amino acids. In some embodiments, a fragment comprises at most 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, 310, 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 separate embodiment of the invention.

[0087] 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 comprises increased solubility as compared to a region of the protein excluded from the chain. In some embodiments, a region of the protein is replaced by a region of protein that is not the protein. In some embodiments, the replacement region comprises increased solubility as compared to the region of the protein that has been replaced. In some embodiments, the replacement region comprises increased protein stability as compared to the region of the protein that has been replaced.

[0088] In some embodiments, the protein is a target of antibodies. As used herein, the term “antibody” includes all classes of IgA, IgD, IgE, IgG and IgM and also includes all subclasses thereof. In some embodiments, the antibody is a circulating antibody. In some embodiments, the antibody is a naturally occurring antibody. In some embodiments, the antibodies are autoantibodies.

[0089] As used herein, the term “autoantibodies” refers to antibodies generated by a subject’s own immune system against at least one of the subject’s own proteins. In some embodiments, an autoantibody is an autoreactive antibody. In some embodiments, autoantibodies target self-antigens. Self-antigens are also known as autoantigens. In some embodiments, the autoantibodies are associated with GD. In some embodiments, theautoantibodies characterize GD. In some embodiments, the autoantibodies are autoantibodies of GD. In some embodiments, autoantibodies are generated by auto-reactive B cells. In some embodiments, the protein is an antigen of the antibodies. In some embodiments, the fragment comprises an antigen of the antibodies. In some embodiments, the fragment comprises at least one antigen of the antibodies. In some embodiments, the fragment comprises at least two antigens of the antibodies. In some embodiments, the fragment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigens of the antibodies. Each possibility represents a separate embodiment of the invention. In some embodiments, an antigen of the antibodies is an autoantigen. In some embodiments, the antigen is an epitope. In some embodiments, the antigen includes at least one epitope. In some embodiments, an epitope comprises at least 5 amino acids. In some embodiments, an epitope comprises 5-6 amino acids. In some embodiments, an epitope comprises 5-10 amino acids. In some embodiments, an epitope is a simple epitope. In some embodiments, a simple epitope is a linear epitope. In some embodiments, an 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 sections of amino acids that combine to form an epitope. In some embodiments, a linker sequence is between the two sections of the epitope.

[0090] In some embodiments, the TSHR is an analog of TSHR. As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to the sequence of the protein but in which one or more residues have been conservatively substituted with a functionally similar residue. In some embodiments, an analog displays similar functionality to the original protein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention. In some embodiments, the substitution is outside of an antigenic region of the protein. In some embodiments, the substitution is outside an epitope of the antibodies. In some embodiments, the analog is still a target of the antibodies. In some embodiments, the analog retains bindingof autoantibodies. An analog may have deletions or mutations that result in an amino acids sequence that is different than the canonical amino acid sequence of protein. Further, an analog may be analogous to a fragment of the protein, however, in such a case the fragment must comprise at least 50 consecutive amino acids of protein or at least one epitope of the antibodies. In some embodiments, an analog is an analog to the canonical sequence of the protein.

[0091] In some embodiments, an analog to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, an analog to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, an analog to the protein comprises an amino acid sequence with at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the analog is still able to bind GD autoantibodies. In some embodiments, the analog is still able to sequester GD autoantibodies. In some embodiments, the analog is still able to treat GD. In some embodiments, the analog comprises at least one substitution. In some embodiments, an analog comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. Each possibility represents a separate embodiment of the invention. In some embodiments, substitution is a mutation of the canonical sequence.

[0092] The term “derivative” as used herein, refers to any polypeptide that is based off the protein and still comprises retains binding of the antibodies. A derivative is not merely a fragment of the protein, nor does it have amino acids replaced or removed (an analog), rather it may have additional modification made to the protein, such as post-translational modification. Further, a derivative may be a derivative of a fragment of the protein, however, in such a case the fragment must comprise at least 50 consecutive amino acids of the protein or at least one epitope of the antibodies. In some embodiments, the derivative is a derivative of a canonical sequence of the protein.

[0093] In some embodiments, a derivative to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, a derivative to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, a derivative to the protein comprises an amino acid sequence with at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the derivative is still able to bind GD autoantibodies. In some embodiments, the derivative is still able to sequester GD autoantibodies. In some embodiments, the derivative is still able to treat GD. In some embodiments, a derivative is the protein or fragment with a mutation.

[0094] As used herein, the term "variant" includes any peptide having an amino acid sequence substantially identical to the sequence of the protein but in which one or more residues have been substituted or in which one or more of the residues have been mutated. In some embodiments, the mutation in the variant is a non-conservative mutation.

[0095] Canonical amino acid sequences of known proteins are well known in the art. They can be found in a variety of databases, including UniProt, NCBI, and the UCSC Genome Browser. Any sequence accepted as a canonical sequence may be employed. For a nonlimiting 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, its canonical amino acid sequence can be found inNP_000360, NP_001018046 and NP001136098 and UniProt number Pl 6473. In some embodiments, a canonical sequence is a sequence identical to the sequence present in at least 50, 60, 70, 75, 80, 90, 95, 97, or 99 percent of a population. Each possibility represents a separate embodiment of the invention. In some embodiments, a canonical sequence is a sequence identical to the most prevalent sequence present in a population. In some embodiments, the population is a disease population. In some embodiments, the population is a population with the autoimmune disease.

[0096] In some embodiments, a canonical amino acid sequence of the N-terminal extracellular domain of TSHR comprises or consists of GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNIS RIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGL KMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGY AFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLK ELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQ SLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEI IGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMG (SEQ ID NO: 1). In some embodiments, the extracellular domain is devoid of 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). In some embodiments, embodiments, the extracellular domain comprises or consists of SEQ ID NO: 1 or a sequence with at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with identity to SEQ ID NO: 1 still comprises the at least one mutation. In some embodiments, the sequence with identity to SEQ ID NO: 1 retains autoantibody binding. Methods of testing autoantibody binding are well known in the art and several are provided hereinbelow. In some embodiments, the autoantibody is selected from the group consisting of. M22, KI-70, Kl-18, and patient serum autoantibodies. In some embodiments, the patients are GD patients. In some embodiments, the extracellular domain comprises or consists of SEQ ID NO: 1 or a sequence with at least 85% identity to SEQ ID NO: 1 and which binds a Graves Disease autoantibody. In some embodiments, the extracellular domain comprises or consists of SEQ ID NO: 1 or a sequence with at least 95% identity to SEQ ID NO: 1 and which binds a Graves’ Disease autoantibody. In some embodiments, a Graves’ Disease autoantibody is selected from the group consisting of: M22, KI-70; Kl-18, and Graves’ Disease patient serum autoantibodies.

[0097] In some embodiments, a canonical amino acid sequence of the N-terminal extracellular domain of TSHR comprises or consists of MRPADLLQLVLLLDLPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTL KLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGL CNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPS LLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAF KNQKKIRGILESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSK FQDTHNNAHYYVFFEEQEDEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVC TPKSDEFNPCEDIMG (SEQ ID NO: 2). In some embodiments, the extracellular domain is devoid of 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 MRARPRPRPLWATVL ALGAL AGVGVG (SEQ ID NO: 16). In some embodiments, the TSHR signal peptide is used.

[0098] 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 single peptide is of an antibody heavy chain. In some embodiments, the signal peptide is of an 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 comprises 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 comprises 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 comprises of 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 comprises or consists of MGWSCIILFLVATATGVHS (SEQ ID NO: 20). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 20.

[0099] 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: I. In some embodiments, the derivative is a variant of SEQ ID NO: 1. In some embodiments, the protein is a variant of the N-terminal extracellular domain of TSHR. In some embodiments, the protein is a variant of SEQ ID NO: 1. In some embodiments, the derivative or variant comprises a deletion of a C-peptide region of the N-terminal extracellular domain of TSHR. In some embodiments, deletion of a C-peptide region increases the solubility of theextracellular domain of TSHR. In some embodiments, deletion of a C-peptide region increases expression of the extracellular domain of TSHR. In some embodiments, the C- peptide region comprises amino acids 297-346 of SEQ ID NO: 1. In some embodiments, the C-peptide region comprises 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 comprises the amino acid sequence ALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGF (SEQ ID NO: 58). In some embodiments, the C-Peptide region consists of SEQ ID NO: 58. In some embodiments, a peptide with a deletion of the C-Peptide region comprises GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNIS RIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGL KMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGY AFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHL ELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQ SLRQRKSVNGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMG (SEQ ID NO: 13). In some embodiments, the peptide of the invention comprises SEQ ID NO: 13. In some embodiments, the peptide with a deletion of the C-peptide region consists of SEQ ID NO: 13. In some embodiments, the peptide of the invention consists of SEQ ID NO: 13.

[0100] In some embodiments, the fragment is a variant fragment. In some embodiments, a variant comprises at least one mutation. In some embodiments, the variant is a truncation of SEQ ID NO: 1. In some embodiments, the variant is a truncation of the extracellular domain of TSHR. In some embodiments, the mutation is a point mutation. In some embodiments, the truncation is a C -terminal truncation. In some embodiments, the variant is an N-terminal fragment of SEQ ID NO: 1. In some embodiments, the variant has a deletion of a C -terminal fragment of SEQ ID NO: 1. In some embodiments, the variant is a derivative of the truncation.

[0101] In some embodiments, the fragment comprises a deletion of amino acids 317-393 of SEQ ID NO: 1. In some embodiments, the fragment comprises C-terminal truncation starting from amino acid 317 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 position in SEQ ID NO: 2. Theposition in SEQ ID NO: 2 can be arrived at by adding 20 bases to the position in SEQ ID NO: I (owing to the addition of the 20-mer signal peptide in SEQ ID NO: 2). In some embodiments, N-terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNIS RIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGL KMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGY AFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLK ELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQ SLRQRKSVN (SEQ ID NO: 14) or a variant thereof. In some embodiments, the N-terminal fragment consists of SEQ ID NO: 14 or a variant thereof. In some embodiments, the polypeptide comprises SEQ ID NO: 14. In some embodiments, the polypeptide consists of SEQ ID NO: 14.

[0102] In some embodiments, the fragment comprises a deletion of amino acids 242-373 of SEQ ID NO: 1. In some embodiments, the fragment comprises a C -terminal truncation starting from amino acid 242 to the C-terminus of SEQ ID NO: 1. In some embodiments, the N-terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNIS RIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGL KMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGY AFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLK ELIARNTWTLK (SEQ ID NO: 3) or a variant thereof. In some embodiments, the N- terminal fragment consists of SEQ ID NO: 3 or a variant thereof. In some embodiments, the fragment comprises or consists of amino acids 1-241 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises SEQ ID NO: 3 or a variant thereof. In some embodiments, the polypeptide consists of SEQ ID NO: 3 or a variant thereof.

[0103] In some embodiments, the fragment comprises a deletion of amino acids 244-373 of SEQ ID NO: 1. In some embodiments, the fragment comprises a C-terminal truncation starting from amino acid 244 to the C-terminus of SEQ ID NO: 1. In some embodiments, the N-terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNIS RIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGL KMFPDLTKVYSTD1FFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLK ELIARNTWTLKKL (SEQ ID NO: 4) or a variant thereof. In some embodiments, the N-terminal fragment consists of SEQ ID NO: 4 or a variant thereof. In some embodiments, the fragment comprises or consists of amino acids 1-243 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises SEQ ID NO: 4 or a variant thereof. In some embodiments, the polypeptide consists of SEQ ID NO: 4 or a variant thereof.

[0104] In some embodiments, the fragment comprises a deletion of amino acids 261-373 of SEQ ID NO: 1. In some embodiments, the fragment comprises C-terminal truncation starting from amino acid 261 to the C-terminus of SEQ ID NO: 1. In some embodiments, the N- temiinal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNIS RIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGL KMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGY AFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLK ELIARNTWTLKKLPLSLSFLHLTRADLSYP (SEQ ID NO: 5) or a variant thereof. In some embodiments, the N-terminal fragment consists of SEQ ID NO: 5 or a variant thereof. In some embodiments, the fragment comprises or consists of amino acids 1-261 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises SEQ ID NO: 5 or a variant thereof. In some embodiments, the polypeptide consists of SEQ ID NO: 5 or a variant thereof. In some embodiments, embodiments, the extracellular domain comprises or consists of SEQ ID NO: 5 or a sequence with at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 5. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with identity to SEQ ID NO: 5 still comprises the at least one mutation. In some embodiments, the sequence with identity to SEQ ID NO: 5 retains autoantibody binding. Methods of testing autoantibody binding are well known in the art and several are provided hereinbelow. In some embodiments, the autoantibody is selected from the group consisting of: M22, KI-70; Kl-18, and patient serum autoantibodies. In some embodiments, the patients are GD patients. In some embodiments, the extracellular domain comprises or consists of SEQ ID NO: 5 or a sequence with at least 85% identity to SEQ ID NO: 5 and which binds a Graves Disease autoantibody. In some embodiments, the extracellular domain comprises or consists of SEQ ID NO: 5 or a sequence with at least 95% identity to SEQ ID NO: 5 and which binds a Graves’ Disease autoantibody. In someembodiments, a Graves’ Disease autoantibody is selected from the group consisting of: M22, KI-70; Kl-18, and Graves’ Disease patient serum autoantibodies.

[0105] In some embodiments, the fragment comprises a deletion of amino acids 247-373 of SEQ ID NO: 1. In some embodiments, the fragment comprises C -terminal truncation starting from amino acid 247 to the C-terminus of SEQ ID NO: I. In some embodiments, the N- terminal fragment comprises the amino acid sequence GMGCSSPPCECHQEEDFRVTCKDIQRH’SLPPSTQTLKLIETHLRTIPSHAFSNLPNIS RIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGL KMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGY AFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLK ELIARNTWTLKKLPLS (SEQ ID NO: 12) or a variant thereof. In some embodiments, the N-terniinal fragment consists of SEQ ID NO: 12 or a variant thereof. In some embodiments, the fragment comprises or consists of amino acids 1-246 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises SEQ ID NO: 12 or a variant thereof. In some embodiments, the polypeptide consists of SEQ ID NO: 12 or a variant thereof.

[0106] In some embodiments, the first protein and the second protein are the same protein. In some embodiments, the first and second proteins are from the same proteins, and the fragments are different fragments. In some embodiments, the fragments are different fragments. In some embodiments, the fragments comprise or consist of different sequences. In some embodiments, the first and second proteins are different proteins. In some embodiments, the first and second proteins are the same proteins. In some embodiments, the first and second fragments are on the said polypeptide chain.

[0107] In some embodiments, the protein or fragment comprises a mutation. In some embodiments, the variant of the extracellular domain comprises a mutation. In some embodiments, the mutation decreases aggregation. In some embodiments, the mutation decreases cleavage of the molecule. In some embodiments, the protein or fragment comprises a mutation that decreases aggregation. In some embodiments, aggregation is aggregation of the protein. In some embodiments, aggregation is aggregation of the fragment. In some embodiments, aggregation is aggregation of the extracellular domain. In some embodiments, aggregation is aggregation of the complex. In some embodiments, aggregation is aggregation of the polypeptide. In some embodiments, decreasing aggregationcomprises increasing solubility. In some embodiments, decreasing aggregation comprises increasing stability. In some embodiments, the protein or fragment comprises a mutation that increases solubility. In some embodiments, the protein or fragment comprises a mutation that increases stability of the protein or fragment. In some embodiments, the protein is the polypeptide. In some embodiments, the mutation is an insertion. In some embodiments, the protein is a surface protein and comprises a mutation that increases solubility. In some embodiments, the fragment is an extracellular domain of a surface protein and comprises an insertion that increases solubility. In some embodiments, the fragment is an extracellular domain of a surface protein and comprises a deletion that increases solubility.

[0108] In some embodiments, the mutation is selected from mutation of phenylalanine 7 (P7), mutation of histidine 12 (H12), mutation of arginine 18 (R18), mutation of valine 19 (V19), mutation of threonine 36 (T36), mutation of serine 64 (S64), mutation of isoleucine 65 (165), mutation of valine 83 (V83), mutation of arginine 92 (R92), mutation of tyrosine 96 (Y96), mutation of aspartic acid 131 (D131), mutation of valine 149 (V149), mutation of cysteine 156 (C156), mutation of serine 171 (S171), mutation of glycine 174 (G174), mutation of valine 195 (V195), mutation of lysine 198 (K198), mutation of leucine 210 (L210), mutation of valine 218 (V218), mutation of isoleucine 233 (1233), mutation of tryptophan 238 (W238), mutation of phenylalanine 249 (F249) and mutation of leucine 250 (L250). In some embodiments, the mutation is within SEQ ID NO: 1. In some embodiments, the mutation is selected from mutation of P7, mutation of H12, mutation of R18, mutation of V19, mutation of T36, mutation of S64, mutation of 165, mutation of V83, mutation of R92, mutation of Y96, mutation of D131, mutation of C156, mutation of S171, mutation of G174, mutation of Cl 56, mutation of S171, mutation of G174, mutation of V195, mutation of K198, mutation of L210, mutation of V218, mutation of W238 and mutation of F249. In some embodiments, the mutation is selected from mutation of P7, mutation of H12, mutation of R18, mutation of V19, mutation of T36, mutation of S64, mutation of 165, mutation of V83, mutation of R92, mutation of Y96, mutation of D131, mutation of V149, mutation of S171, mutation of G174, mutation of V195, mutation of K198, mutation of L210, and mutation of V218. In some embodiments, the mutation is selected from 185, D151, G194 and V215. In some embodiments, the mutation is within SEQ ID NO: 1. In some embodiments, the numbering is with respect to SEQ ID NO: 1. In some embodiments, the mutation is within SEQ ID NO: 3. In some embodiments, the numbering is with respect toSEQ ID NO: 3. In some embodiments, the mutation in SEQ ID NO: 3 is not mutation of F249 or F250. In some embodiments, the mutation is within SEQ ID NO: 4. In some embodiments, the numbering is with respect to SEQ ID NO: 4. In some embodiments, the mutation in SEQ ID NO: 4 is not mutation of F249 or L250. It will be understood that SEQ ID NO: 3 and 4 contain only 240 and 242 amino acids, respectively, and therefore there are no F249 and L250 in these amino acid sequences. In some embodiments, the mutation is within SEQ ID NO: 5. In some embodiments, the numbering is with respect to SEQ ID NO: 5.

[0109] It will be understood that SEQ ID NO: 1 is identical to SEQ ID NO: 2 except for the first 20 amino acids of SEQ ID NO: 2. As such, all of the mutations recited herein also are envisioned in SEQ ID NO: 2, but then the number given would be increased by 20. For example, P7 in SEQ ID NO: 1 would be P27 is SEQ ID NO: 2.

[0110] In some embodiments, the mutation is mutation of H12. In some embodiments, the mutation is mutation of R18. In some embodiments, the mutation is mutation of V19. In some embodiments, the mutation is mutation of T36. In some embodiments, the mutation is mutation of S64. In some embodiments, the mutation is mutation of I65. In some embodiments, the mutation is mutation of V83. In some embodiments, the mutation is mutation of R92. In some embodiments, the mutation is mutation of Y96. In some embodiments, the mutation is mutation of aspartic acid D131. In some embodiments, the mutation is mutation of V149. In some embodiments, the mutation is mutation of C156. In some embodiments, the mutation is mutation of S 171. In some embodiments, the mutation is mutation of G174. In some embodiments, the mutation is mutation of VI 95. In some embodiments, the mutation is mutation of K198. In some embodiments, the mutation is mutation of L210. In some embodiments, the mutation is mutation of V218. In some embodiments, the mutation is mutation of 1233. In some embodiments, the mutation is mutation of W238. In some embodiments, the mutation is mutation of F249. In some embodiments, the mutation is mutation of L250. In some embodiments, the mutation is mutation of 1233 and V149. In some embodiments, the mutation is mutation of F249 and L250.

[0111] In some embodiments, P7 is mutated to serine (P7S). In some embodiments, P7 is mutated to serine or an equivalent amino acid. Serine is a small polar amino acid and isknown to be structurally / functionally similar to alanine, valine, glycine and asparagine. In some embodiments, P7 is mutated to serine, alanine, valine, glycine or asparagine (P7S / A / V / G / N). In some embodiments, P7 is not mutated to threonine. In some embodiments, the mutation is P7A. In some embodiments, the mutation is P7V. In some embodiments, the mutation is P7G. In some embodiments, the mutation is P7N.

[0112] In some embodiments, H12 is mutated to leucine (H12L). In some embodiments, H12 is mutated to leucine or an equivalent amino acid. Leucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar to valine, methionine and phenylalanine. In some embodiments, H12 is mutated to leucine, valine, methionine or phenylalanine (H12L / V / M / F). In some embodiments, H12 is not mutated to isoleucine. In some embodiments, the mutation is H12V. In some embodiments, the mutation is H12M. In some embodiments, the mutation is H12F.

[0113] In some embodiments, R18 is mutated to leucine (R18L). In some embodiments, R18 is mutated to leucine or an equivalent amino acid. Leucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar to valine, methionine and phenylalanine. In some embodiments, R18 is mutated to leucine, valine, methionine or phenylalanine (R18L / V / M / F). In some embodiments, R18 is not mutated to isoleucine. In some embodiments, the mutation is R18V. In some embodiments, the mutation is R18M. In some embodiments, the mutation is R18F.

[0114] In some embodiments, V19 is mutated to leucine (V19L). In some embodiments, V19 is mutated to leucine or an equivalent amino acid. Leucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar to methionine and phenylalanine. In some embodiments, V19 is mutated to leucine, methionine or phenylalanine (V19L / M / F). In some embodiments, V19 is not mutated to isoleucine. In some embodiments, the mutation is V19M. In some embodiments, the mutation is V19F.

[0115] In some embodiments, T36 is mutated to leucine (T36L). In some embodiments, T36 is mutated to leucine or an equivalent amino acid. Leucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar to valine, methionine and phenylalanine. In some embodiments, T36 is mutated to leucine, valine, methionine or phenylalanine (T36L / V / M / F). In some embodiments, T36 is not mutated to isoleucine. Insome embodiments, the mutation is T36V. In some embodiments, the mutation is T36M. In some embodiments, the mutation is T36F.

[0116] In some embodiments, S64 is mutated to tyrosine (S64Y). In some embodiments, S64 is mutated to tyrosine or an equivalent amino acid. Tyrosine is an aromatic amino acid and is known to be structurally / functionally similar to histidine and tryptophan. In some embodiments, S64 is mutated to tyrosine, histidine or tryptophan (S64Y / H / W). In some embodiments, S64 is not mutated to phenylalanine. In some embodiments, the mutation is S64H. In some embodiments, the mutation is S64W.

[0117] In some embodiments, 165 is mutated to phenylalanine (I65F). In some embodiments, 165 is mutated to phenylalanine or an equivalent amino acid. Phenylalanine is and aromatic and hydrophobic amino acid and is known to be structurally / functionally similar to leucine and tryptophan. In some embodiments, 165 is mutated to phenylalanine, leucine or tryptophan (I65F / L / W). In some embodiments, 165 is not mutated to tyrosine. In some embodiments, the mutation is I65W. In some embodiments, the mutation is I65L.

[0118] In some embodiments, V83 is mutated to isoleucine (V83I). In some embodiments, V83 is mutated to isoleucine or an equivalent amino acid. Isoleucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar to methionine. In some embodiments, V83 is mutated to isoleucine or methionine (V83I / M). In some embodiments, V83 is not mutated to leucine. In some embodiments, the mutation is V83M.

[0119] In some embodiments, R92 is mutated to lysine (R92K). In some embodiments, R92 is mutated to lysine or an equivalent amino acid. Lysine is a basic and positively charged amino acid and is known to be structurally / functionally similar to histidine and glutamine. In some embodiments, R92 is mutated to lysine, histidine or glutamine (R92K / H / Q). In some embodiments, R92 is not mutated to proline. In some embodiments, the mutation is R92H. In some embodiments, the mutation is R92Q.

[0120] In some embodiments, Y96 is mutated to serine (Y96S). In some embodiments, Y96 is mutated to serine or an equivalent amino acid. Serine is a small polar amino acid and is known to be structurally / functionally similar to alanine, valine, glycine and asparagine. In some embodiments, Y96 is mutated to serine, alanine, valine, glycine or asparagine (Y96S / A / V / G / N). In some embodiments, Y96 is not mutated to phenylalanine. In someembodiments, the mutation is Y96A. In some embodiments, the mutation is Y96V. In some embodiments, the mutation is Y96G. In some embodiments, the mutation is Y96N.

[0121] In some embodiments, Y96 is mutated to glutamic acid (Y96E). In some embodiments, Y96 is mutated to glutamic acid or an equivalent amino acid. Glutamic acid is a negatively charged amino acid and is known to be structurally / functionally similar to aspartic acid and glutamine. In some embodiments, Y96 is mutated to glutamic acid, aspartic acid or glutamine (Y96E / D / Q). In some embodiments, Y96 is not mutated to phenylalanine. In some embodiments, the mutation is Y96D. In some embodiments, the mutation is Y96Q.

[0122] In some embodiments, Y96 is mutated to arginine (Y96R). In some embodiments, Y96 is mutated to arginine or an equivalent amino acid. Arginine is a basic and positively charged amino acid and is known to be structurally / functionally similar to lysine, histidine and glutamine. In some embodiments, Y96 is mutated to arginine, lysine, histidine or glutamine (Y96R / K / H / Q). In some embodiments, Y96 is not mutated to phenylalanine. In some embodiments, the mutation is Y96K. In some embodiments, the mutation is Y96H. In some embodiments, the mutation is Y96Q.

[0123] In some embodiments, D131 is mutated to glutamine (D131Q). In some embodiments, D131 is mutated to glutamine or an equivalent amino acid. Glutamine is a polar amide and is known to be structurally / functionally similar to asparagine. In some embodiments, D131 is mutated to glutamine, or asparagine (D131Q / N). In some embodiments, D131 is not mutated to glutamic acid. In some embodiments, D131 is mutated to asparagine (D131N).

[0124] In some embodiments, V149 is mutated to lysine (V149K). In some embodiments, V149 is mutated to lysine or an equivalent amino acid. Lysine is a basic and positively charged amino acid and is known to be structurally / functionally similar to histidine and glutamine. In some embodiments, V149 is mutated to lysine, histidine or glutamine (V149K / H / Q). In some embodiments, V149 is not mutated to arginine. In some embodiments, the mutation is V149H. In some embodiments, the mutation is V149Q.

[0125] In some embodiments, C156 is mutated to serine (C156S). In some embodiments, C156 is mutated to serine or an equivalent amino acid. Serine is a small polar amino acid and is known to be structurally / functionally similar to alanine, valine, glycine and asparagine. In some embodiments, C156 is mutated to serine, alanine, valine, glycine or asparagine(C156S / A / V / G / N). In some embodiments, the mutation is C156A. In some embodiments, the mutation is Cl 56V. In some embodiments, the mutation is C156G. In some embodiments, the mutation is C156N.

[0126] In some embodiments, C156 is mutated to alanine (C156A). In some embodiments, C156 is mutated to alanine or an equivalent amino acid. Alanine is a small, non-polar amino acid and is known to be structurally / functionally similar to glycine, serine, threonine and valine. In some embodiments, C156 is mutated to alanine, serine, threonine or valine (C156A / G / S / T / V). In some embodiments, the mutation is C156T. In some embodiments, the mutation is Cl 56V. In some embodiments, the mutation is C156G.

[0127] In some embodiments, S171 is mutated to aspartic acid (S171D). In some embodiments, S171 is mutated to aspartic acid or an equivalent amino acid. Aspartic acid is a negatively charged amino acid and is known to be structurally / functionally similar to asparagine. In some embodiments, S171 is mutated to aspartic acid or asparagine (S171D / N). In some embodiments, S171 is not mutated to glutamic acid. In some embodiments, the mutation is S171N.

[0128] In some embodiments, G174 is mutated to alanine (G174A). In some embodiments, G174 is mutated to alanine or an equivalent amino acid. Alanine is a small, non-polar amino acid and is known to be structurally / functionally similar to serine, threonine and valine. In some embodiments, G174 is mutated to alanine, serine, threonine or valine (G174A / S / T / V). In some embodiments, G174 is not mutated to proline. In some embodiments, the mutation is G174T. In some embodiments, the mutation is G174V. In some embodiments, the mutation is G174S.

[0129] In some embodiments, V195 is mutated to aspartic acid (V195D). In some embodiments, V195 is mutated to aspartic acid or an equivalent amino acid. Aspartic acid is a negatively charged amino acid and is known to be structurally / functionally similar to asparagine. In some embodiments, V195 is mutated to aspartic acid or asparagine (V195D / N). In some embodiments, V195 is not mutated to glutamic acid. In some embodiments, the mutation is V195N.

[0130] In some embodiments, K198 is mutated to glutamine (K198Q). In some embodiments, K198 is mutated to glutamine or an equivalent amino acid. Glutamine is a polar amide and is known to be structurally / functionally similar to glutamic acid andhistidine. In some embodiments, K198 is mutated to glutamine, glutamic acid or histidine (K198Q / E / H). In some embodiments, K198 is not mutated to arginine. In some embodiments, K198 is mutated to glutamic acid (K198E). In some embodiments, the mutation is K198H.

[0131] In some embodiments, L210 is mutated to tyrosine (L210Y). In some embodiments, L210 is mutated to tyrosine or an equivalent amino acid. Tyrosine is an aromatic amino acid and is known to be structurally / functionally similar to histidine and tryptophan. In some embodiments, L210 is mutated to tyrosine, histidine or tryptophan (L210Y / H / W). In some embodiments, L210 is not mutated to phenylalanine. In some embodiments, the mutation is L210H. In some embodiments, the mutation is L210W.

[0132] In some embodiments, V218 is mutated to isoleucine (V218I). In some embodiments, V218 is mutated to isoleucine or an equivalent amino acid. Isoleucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar to methionine. In some embodiments, V218 is mutated to isoleucine or methionine (V218I / M). In some embodiments, V218 is not mutated to leucine. In some embodiments, the mutation is V218M.

[0133] In some embodiments, 1233 is mutated to lysine (I233K). In some embodiments, 1233 is mutated to lysine or an equivalent amino acid. Lysine is a basic and positively charged amino acid and is known to be structurally / functionally similar to histidine and glutamine. In some embodiments, 1233 is mutated to lysine, histidine or glutamine (I233K / H / Q). In some embodiments, 1233 is not mutated to arginine. In some embodiments, the mutation is I233H. In some embodiments, the mutation is I233Q.

[0134] In some embodiments, W238 is mutated to arginine (W238R). In some embodiments, W238 is mutated to arginine or an equivalent amino acid. Arginine is a basic and positively charged amino acid and is known to be structurally / functionally similar to lysine, histidine and glutamine. In some embodiments, W238 is mutated to arginine, lysine, histidine or glutamine (W238R / K / H / Q). In some embodiments, the mutation is W238K. In some embodiments, the mutation is W238H. In some embodiments, the mutation is W238Q.

[0135] In some embodiments, W238 is mutated to arginine leucine (W238L). In some embodiments, W238 is mutated to leucine or an equivalent amino acid. Leucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar tovaline, methionine and phenylalanine. In some embodiments, W238 is mutated to leucine, valine, methionine or phenylalanine (W238L / V / M / F). In some embodiments, W238 is not mutated to isoleucine. In some embodiments, the mutation is W238V. In some embodiments, the mutation is W238M. In some embodiments, the mutation is W238F.

[0136] In some embodiments, F249 is mutated to tyrosine (F249Y). In some embodiments, F249 is mutated to tyrosine or an equivalent amino acid. Tyrosine is an aromatic amino acid and is known to be structurally / functionally similar to histidine and tryptophan. In some embodiments, F249 is mutated to tyrosine, histidine or tryptophan (F249Y / H / W). In some embodiments, F249 is not mutated to phenylalanine. In some embodiments, the mutation is F249H. In some embodiments, the mutation is F249W.

[0137] In some embodiments, L250 is mutated to isoleucine (L250I). In some embodiments, L250 is mutated to isoleucine or an equivalent amino acid. Isoleucine is a hydrophobic aliphatic amino acid and is known to be structurally / functionally similar to valine and methionine In some embodiments, L250 is mutated to isoleucine, valine or methionine (L250W / M). In some embodiments, the mutation is L250M. In some embodiments, the mutation is L250V.

[0138] In some embodiments, the at least one mutation is 1233K and V149K. In some embodiments, the at least one mutation is I233K or mutation of 1233 to an amino acid equivalent to K and V149K. In some embodiments, the at least one mutation is I233K and V149K or mutation of V149 to an amino acid equivalent to K. In some embodiments, the at least one mutation is I233K or mutation of 1233 to an amino acid equivalent to K and V149K or mutation of V149 to an amino acid equivalent to K.

[0139] In some embodiments, the at least one mutation is F249Y and L250I. In some embodiments, the at least one mutation is F249Y or mutation of F249 to an amino acid equivalent to Y and L250I. In some embodiments, the at least one mutation is F249Y and L250I or mutation of L250 to an amino acid equivalent to I. In some embodiments, the at least one mutation is F249Y or mutation of F249 to an amino acid equivalent to Y and L250I or mutation of L250 to an amino acid equivalent to I.

[0140] In some embodiments, the mutation is a plurality of mutation. In some embodiments, the plurality of mutation is at least 2, 3, 4, 5, 6, 7, 8 or 9 mutations. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality of mutation isselected from mutation of P7, H12, R18, V19, T36, S64, 165, V83, R92, Y96, Y96, Y96, D131, V149, C156, C156, S171, G174, V195, K198, L210, V218, 1233K, W238, F249 and L250. In some embodiments, the plurality of mutation is selected from mutation of P7, H12, R18, V19, T36, S64, 165, V83, R92, Y96, Y96, Y96, D131, V149K, V149, S171, G174, V195, K198, L210, V218, and I233K. In some embodiments, the plurality of mutation is selected from P7S, H12L, R18L, V19L, T36L, S64Y, I65F, V83I, R92K, Y96S, Y96E, Y96R, D131Q, D131N, V149K, C156S, C156A, S171D, G174A, V195D, K198Q, L210Y, V218I, I233K, I233K and V149K, W238R, W238L, F249Y, L250I and F249Y and L250I, and any combination thereof. In some embodiments, the plurality of mutation is selected from P7S, H12L, R18L, V19L, T36L, S64Y, I65F, V83I, R92K, Y96S, Y96E, Y96R, D131Q, D131N, V149K, S171D, G174A, V195D, K198Q, L210Y, V218I, I233K, and 1233K and V149K and any combination thereof. It will be understood that various combinations of the mutations recited herein are considered. Combinations with synergistically enhanced efficacy are envisioned. In some embodiments, a combination of mutations further comprises V149R, I233R or both.

[0141] In some embodiments, the at least one mutation is D131Q. In some embodiments, the D131Q mutation is within SEQ ID NO: 3. In some embodiments, the at least one mutation is D131Q and at least one additional mutation. In some embodiments, the at least one mutation is D131N. In some embodiments, the D13 IN mutation is within SEQ ID NO: 3. In some embodiments, the at least one mutation is D131N and at least one additional mutation.

[0142] In some embodiments, the at least one additional mutation is selected from the group consisting of I65F, G174A and V195D. In some embodiments, the at least one additional mutation is I65F. In some embodiments, the at least one additional mutation is G174A. In some embodiments, the at least one additional mutation is V195D. In some embodiments, the plurality of mutations comprises D131Q and I65F. In some embodiments, the plurality of mutations is D131Q and I65F. In some embodiments, the plurality of mutations comprises D13 IQ and I65F and at least one third mutation. In some embodiments, the at least one third mutation is selected from G174A and V195D. In some embodiments, the at least one third mutation is G174A. In some embodiments, the at least one third mutation is V195D. In some embodiments, the plurality of mutations is D13 IQ, I65F and G174A. In some embodiments, the plurality of mutations is D131Q, I65F and V195D. In some embodiments, the pluralityof mutations is D131Q, I65F, G174A and V195D. In some embodiments, the at least one mutation is D131Q, I65F, G174A and V195D.

[0143] In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 101. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 101. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 102. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 102. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 103. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 103. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 104. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 104. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 105. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 105. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 106. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 106. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 107. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 107. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 108. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 108. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 109. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 109. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 110. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 110. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 111. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 111. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 112. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 112. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 113. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 113. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 114. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 114. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 115. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO:115. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 116. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 116. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 117. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 117. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 118. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 118. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 119. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 119. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 120. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 120. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 121. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 121. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 122. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 122. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 123. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 123. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 124. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 124. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 125. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 125. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 126. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 126. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 127. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 127. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 195. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 195. It will be noted that SEQ ID NO: 101-126 and 195 contain only the fragment from 21-261 from TSHR with various mutations. The 261 and 263 fragments have both been tested and were functional. Thus, though the sequences of SEQ ID NO: 101-126 are given with just 241 amino acids, it will be understood that the sequences with an additional “KL” (thus corresponding to amino acids 21-263 of TSHR) are also considered and are covered as part of the invention. In some embodiments, the fragment consists of amino acids 21-261 withthe recited mutation / s. In some embodiments, the fragment consists of amino acids 21-263 with the recited mutation / s. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-119. In some embodiments, the fragment comprising at least one mutation consist of a sequence selected from SEQ ID NO: 101-119. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-119 and 195. In some embodiments, the fragment comprising at least one mutation consist of a sequence selected from SEQ ID NO: 101-119 and 195. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-127. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 101-127. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-127 and 195. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 101-127 and 195.

[0144] In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 156. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 156. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 157. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 157. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 159. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 159. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 161. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 161. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 163. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 163. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 165. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 165. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 167. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 167. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 169. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 169. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 171. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO:171. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 172. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 172. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 174. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 174. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 175. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 175. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 177. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 177. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 195. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 195. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 196. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 196. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 197. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 197. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 198. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 198. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 199. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 199. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 200. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 200. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 201. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 201. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 202. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 202. In some embodiments, the fragment comprising a mutation comprises SEQ ID NO: 203. In some embodiments, the fragment comprising a mutation consists of SEQ ID NO: 203. It will be noted that SEQ ID NO: 156-157, 159, 161, 163, 165, 167, 1691 171-172, 174-175, 177 and 195-203 contain only the fragment from 21-261 from TSHR with various mutations. The 261 and 263 fragments have both been tested and were functional. Thus, though the sequences of SEQ ID NO: 156-157, 159, 161, 163, 165, 167, 1691 171-172, 174-175, 177 and 195-203 are given with just 241 amino acids, it will be understood that the sequences with an additional “KL” (thus corresponding to amino acids 21-263 of TSHR) arealso considered and are covered as part of the invention. In some embodiments, the fragment consists of amino acids 21-261 with the recited mutations. In some embodiments, the fragment consists of amino acids 21-263 with the recited mutations.

[0145] In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 156-177. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 156-177. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-119 and 156-177. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 101-119 and 156-177. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-127 and 156-177. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 101-127 and 156-177. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 156-177 and 195-203. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 156-177 and 195-203. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-119, 156-177 and 195-203. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 101-119, 156-177 and 195-203. In some embodiments, the fragment comprising at least one mutation comprises a sequence selected from SEQ ID NO: 101-127, 156-177 and 195-203. In some embodiments, the fragment comprising at least one mutation consists of a sequence selected from SEQ ID NO: 101-127, 156-177 and 195-203.

[0146] In some embodiments, the fragment comprises 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 comprises at least one mutation in a ligand binding domain. In some embodiments, a variant of the fragment comprises at least one mutation in a ligand binding domain. In some embodiments, the at least one mutation decreases binding to TSH. In some embodiments, decreases is abrogates. In some embodiments, decreasing comprises a decrease 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 separate embodiment of the invention. In some embodiments, thedecrease is a decrease of at least 50%. In some embodiments, the decrease is a decrease of at least 90%. In some embodiments, binding is binding of TSHR to TSH. In some embodiments, binding is binding of the N-terminal extracellular domain of TSHR to TSH. In some embodiments, binding is binding of the peptide to TSH. In some embodiments, binding is binding of the fragment to TSH. In some embodiments, the mutation that increases solubility also decreases ligand binding.

[0147] In some embodiments, the polypeptide further comprises at least one mutation selected from mutation of lysine 163 (K163) and mutation of glutamic acid 231 (E231). In some embodiments, the mutation is within any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the numbering is with respect to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the further at least one mutation is selected from mutation of KI 83 and mutation of E251 within SEQ ID NO: 2. It will be understood that K163 of SEQ ID NO: 1 and KI 83 of SEQ ID NO: 2 are the same reside just as E231 of SEQ ID NO: 1 and E251 of SEQ ID NO: 2 are the same residue. KI 83 and E231 are known in the art to be important for ligand binding and are evolutionarily conserved residues. Mutation of either of these resides, especially a mutation that abrogates charge or alters charge will result in reduced binding to TSH. In some embodiments, the further mutation is mutation of K163 of SEQ ID NO: 1. In some embodiments, the further mutation is mutation of E231 of SEQ ID NO: 1.

[0148] In some embodiments, KI 63 is mutated to a non-positively charged amino acid. In some embodiments, K163 is mutated to a non-charged amino acid. In some embodiments, K163 is mutated to a negatively charged amino acid. In some embodiments, K163 is mutated to alanine (K163A). In some embodiments, K163 is mutated to alanine or an amino acid equivalent to alanine. Alanine is a small, non-polar amino acid and is known to be structurally / functionally similar to serine, threonine, glycine and valine. In some embodiments, K163 is mutated to alanine, serine, threonine, glycine or valine (K163A / S / T / G / V). In some embodiments, K163 is not mutated to arginine. In some embodiments, the mutation is K163T. In some embodiments, the mutation is K163V. In some embodiments, the mutation is K163G. In some embodiments, the mutation is K163S. In some embodiments, the further at least one mutation is K163A within SEQ ID NO: 1, 3, 4 or 5. In some embodiments, the further at least one mutation is K163A or a mutation of KI 63 to an amino acid equivalent to A within SEQ ID NO: 1, 3, 4 or 5.

[0149] 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 (E231A). In some embodiments, E231 is mutated to alanine or an amino acid equivalent to alanine. Alanine is a small, non-polar amino acid and is known to be structurally / functionally similar to serine, threonine, glycine and valine. In some embodiments, E231 is mutated to alanine, serine, threonine, glycine or valine (E231A / S / T / G / V). In some embodiments, E231 is not mutated to cysteine. In some embodiments, the mutation is E23 IT. In some embodiments, the mutation is E23 IV. In some embodiments, the mutation is E231G. In some embodiments, the mutation is E23 IS. In some embodiments, the further at least one mutation is E231 A within SEQ ID NO: 1, 3, 4 or 5. In some embodiments, the further at least one mutation is E231A or a mutation of E231 to an amino acid equivalent to A within SEQ ID NO: 1, 3, 4 or 5.

[0150] In some embodiments, E231 is mutated to a lysine or arginine (E231K / R). In some embodiments, E231 is mutated to a lysine (E23 IK). Lysine is a basic and positively charged amino acid and is known to be structurally / functionally similar to arginine, histidine and glutamine. In some embodiments, E231 is mutated to lysine, arginine, histidine or glutamine (E231K / R / H / Q). In some embodiments, E231is not mutated to cysteine. In some embodiments, the mutation is E231R. In some embodiments, the mutation is E231H. In some embodiments, the mutation is E231Q. In some embodiments, the at least one mutation is E231K within SEQ ID NO: 1, 3,4 or 5. In some embodiments, the at least one mutation is E23 IK or mutation of E231 to an amino acid equivalent to K within SEQ ID NO: 1, 3,4 or 5.

[0151] It will be understood by a skilled artisan that as the protein complex of the invention is meant to bind antibodies and B cells it would be advantageous not to bind to itself or to other copies of the therapeutic molecule. As such, mutations and truncations that decrease aggregation but do not interfere with autoantibody binding are advantageous. Similarly, if the fragment comprises a complete ligand binding domain then it may be advantageous to abrogate ligand binding. The endogenous ligand may be present in circulation and binding of the ligand may sequester it and keep it from reaching its intended target receptor. In some embodiments, the fragment comprises a truncation of the extracellular domain. In some embodiments, the fragment consists of a truncation of the extracellular domain. In someembodiments, the truncation lacks at least one extracellular functional domain. In some embodiments, the truncation lacks at least two extracellular functional domains.

[0152] In some embodiments, a derivative is a derivative of the fragment. In some embodiments, the derivative comprises at least 85% identity to the fragment and does not further comprise a stretch of amino acids homologous / identical to a sequence from TSHR. In some embodiments, the derivative comprises the at least one mutation. Thus, it will be understood that a sequence with sequence identity to a truncation is not a sequence which is not truncated. In some embodiments, the derivative comprises at least one mutation. In some embodiments, the derivative comprises at least 85% identity to SEQ ID NO: 1. In some embodiments, the derivative comprises at least 85% identity to SEQ ID NO: 2. In some embodiments, the derivative comprises at least 85% identity to SEQ ID NO: 3. n some embodiments, the derivative comprises at least 85% identity to SEQ ID NO: 4. n some embodiments, the derivative comprises at least 85% identity to SEQ ID NO: 5.Dimerization domains

[0153] In some embodiments, dimerization domains are capable of dimerizing with each other. In some embodiments, the first dimerization domain is capable of dimerization with the second dimerization domain. In some embodiments, the first and second dimerization domains are capable of dimerizing with each other. In some embodiments, capable of dimerizing is configured to dimerize. In some embodiments, dimerization is under physiological conditions. In some embodiments, dimerization is within a bodily fluid. In some embodiments, the bodily fluid is blood. In some embodiments, the bodily fluid is plasma. In some embodiments, the bodily fluid is serum. In some embodiments, dimerization is within a subject. In some embodiments, dimerization is in vivo. In some embodiments, dimerization is in vitro.

[0154] As used herein, the term “dimerization domain” refers to an amino acid sequence that upon contacting another amino acid sequence (the other dimerization domain) binds to it to form a dimer. Dimerization domains are well known in the art, as many protein sequences are known to bind to each other. In some embodiments, dimerization comprises formation of a covalent bond between the dimerization domains. In some embodiments, dimerization comprises electrostatic binding. In some embodiments, dimerization does not comprise electrostatic binding. In some embodiments, dimerization is reversible. In someembodiments, dimerization is irreversible. In some embodiments, dimerization comprises a bond forming between the dimerization 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 dimerization domain include the hinge domain of antibody heavy chains, the CH1 / CL domains of antibody heavy / light chains, and the ECD domains of TCR alpha / beta to name but a few. Additionally, the upper hinge domain can be engineered with cysteine substitutions / mutations to serine in order to prevent dimerization. In some embodiments, the dimerization domain comprises or consists of the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 21).

[0155] In some embodiments, the dimerization domain comprises or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, an Ig hinge domain is a heavy chain hinge domain. In some embodiments, the Ig is a human Ig. In some embodiments, the immunoglobulin is elected from IgA, IgD, IgE, IgG and IgM. In some embodiments, the immunoglobulin is IgG. In some embodiments, the IgG is IgGl. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is selected from IgGl and IgG3. In some embodiments, the IgG is IgG4. In some embodiments, the first and second dimerization domains are both 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 separate embodiment of the invention.

[0156] In some embodiments, the hinge domain comprises 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 IgGl hinge comprises or consists of SEQ ID NO: 22. In some embodiments, the hinge domain comprises 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 comprises or consists of SEQ ID NO: 23. In some embodiments, the hinge domain comprises 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 comprises or consists of SEQ ID NO: 24. In some embodiments, the hinge domaincomprises the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC PRCPAPELLGGP (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 comprises or consists of SEQ ID NO: 25. In some embodiments, the hinge domain comprises the amino acid sequence EPKSSDKTHTCPPCP (SEQ ID NO: 21). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the hinge domain comprises the amino acid sequence EPKSSDKTHTCPPCP APELLGGP (SEQ ID NO: 180). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 180. In some embodiments, the IgGl hinge comprises or consists of SEQ ID NO: 180. In some embodiments, the hinge domain comprises a CPXCP (SEQ ID NO: 26) motif. In some embodiments, the 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 comprises EPKSCDKTHTCPPCP (SEQ ID NO: 29). It will thus be understood that the hinge region can be considered to end after the CPXCP motif.

[0157] In some embodiments, the dimerization domain comprises or consists of an Ig CHI domain. In some embodiments, the dimerization domain comprises or consists of an Ig heavy chain CHI domain. In some embodiments, the dimerization domain comprises or consists of an Ig light chain. In some embodiments, the dimerization domain comprises 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 CHI domain of the Ig heavy chain dimerizes with the light chain CL domain. In some embodiments, the first dimerization domain comprises or consists of a CHI domain, and the second dimerization domain comprises or consists of a CL domain. In some embodiments, the first and second dimerization domains both comprise a hinge domain. In some embodiments, the first and second dimerization domains do not both comprise a CHI domain. In some embodiments, the first and second dimerization domains do not both comprise a CL domain. In some the first and second polypeptide chains do not both comprise a CHI domain. In some the first and second polypeptide chains do not both comprise a CL domain.

[0158] In some embodiments, an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 30). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 30. In some embodiments, SEQ ID NO: 30 is the IgGl CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 31). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 31. In some embodiments, SEQ ID NO: 31 is the IgG2 CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 32). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 32. In some embodiments, SEQ ID NO: 32 is the IgG3 CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEGDTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 33 In some embodiments, an Ig CHI domain consists of SEQ ID NO: 33. In some embodiments, SEQ ID NO: 33 is the IgG4 CHI domain.

[0159] In some embodiments, an Ig CL Kappa domain comprises of the amino acid sequence AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSGDTKSFNRGEC (SEQ ID NO: 34). In some embodiments, an Ig CL Kappa domain consists of SEQ ID NO: 34. In some embodiments, an Ig CL Lambda domain comprises of the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSGDKAGVET TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 35). In some embodiments, an Ig CL Lambda domain consists of SEQ ID NO: 35.

[0160] 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 a Fc domain of an antibody heavy chain. In some embodiments, the Fc domain is a Fcdomain of a human antibody heavy chain. In some embodiments, an Fc domain is an IgGl Fc domain. In some embodiments, an Fc domain is an IgGl heavy chain Fc domain. In some embodiments, an Fc domain comprises the constant region of an antibody heavy chain. In some embodiments, an Fc domain comprises a CHI, hinge, CH2 and CH3 domain. In some embodiments, an Fc domain comprises a hinge CH2 and CH3 domain. In some embodiments, an Fc domain comprises a CH2 and CH3 domain.

[0161] In some embodiments, the composition comprises an Fc region. In some embodiments, the dimerization domain is an Fc domain. Herein the terms Fc region and Fc domain are used interchangeably. In some embodiments, an Fc is an unmodified Fc. In some embodiments, an Fc is an unmutated Fc. In some embodiments, an Fc is a naturally occurring Fc. In some embodiments, the Fc is not a naturally occurring Fc. In some embodiments, an Fc is a human Fc. In some embodiments, a superior Fc is an Fc comprising at least one mutation that increases ADCC In some embodiments, an Fc region is an Fc domain. In some embodiments, an Fc region is an Fc fragment. In some embodiments, the first polypeptide chain comprises an Fc region. In some embodiments, the second polypeptide chain comprises an Fc region. In some embodiments, both the first and second polypeptide chains comprise an Fc region. In some embodiments, the Fc region is an 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, the IgG is selected from IgGl, IgG2, IgG3 and IgG4. In some embodiments, the IgG is selected from IgGl and IgG3. In some embodiments, the IgG is IgGl. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is IgG4.

[0162] In some embodiments, the Fc region is capable of inducing a cytotoxic effect. In some embodiments, the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CI< VSNI< ALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 61). In some embodiments, the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 62). In some embodiments, the Fc domain comprises EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 181). It will be understood that SEQ ID NO: 62 and SEQ ID NO: 181 contain 5 additional N-terminal amino acids as compared to SEQ ID NO: 61. As such, while numbering herein is given with respect to SEQ ID NO: 61 the numbering for SEQ ID NO: 62 and 181 can be found by adding 5. In some embodiments, the Fc region is capable of inducing a cytotoxic effect. In some embodiments, the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CI< VSNI< ALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 63). In some embodiments, the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 64). In some embodiments, the Fc domain comprises EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN K ALP APIEKTISK AKGQPREPQ VYTLPP SREEMTKNQ VSLTCL VKGF YP SDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 182). It will be understood that SEQ ID NO: 64 and SEQ ID NO: 182 contain 5 additional N-terminal amino acids as compared to SEQ ID NO: 63. As such, while numbering herein is given with respect to SEQ ID NO: 63 (or SEQ ID NO: 61 which is equivalent) the numbering for SEQ ID NO: 64 and 182 can be found by adding 5. SEQ ID NO: 61 and SEQ ID NO: 63 differ by two amino acids. The two sequences can be interchanged and when mutations are given with respect to SEQ ID NO: 61 it will beunderstood that they apply also to SEQ ID NO: 63 and vice-versa. So too SEQ ID NO: 62 and SEQ ID NO: 64 also differ by only two amino acids and these two sequences can be interchanged. SEQ ID NO: 181 and 182 are identical to SEQ ID NO: 62 and 64, respectively, but with the first cysteine of the hinge converted to a serine. These sequences can all be interchanged.

[0163] In some embodiments, the Fc domain consists of SEQ ID NO: 61. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 61. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 61. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 62. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 62. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 62. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 63. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 63. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 63. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 64. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 64. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 64. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 181. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 181. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 181. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 182. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 182. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 182. Each possibility represents a separate embodiment of the invention.

[0164] In some embodiments, the Fc region is capable of inducing a cytotoxic effect. In some embodiments, the Fc region is configured to induce a cytotoxic effect. In some embodiments, the cytotoxic effect is against a target cell. In some embodiments, the cytotoxic effect is upon binding. In some embodiments, the cytotoxic effect is against a cell bound by the protein complex. In some embodiments, the cytotoxic effect is against a cell binding the protein complex. In some embodiments, the cytotoxic effect is mediated by immune cell binding to the Fc region. In some embodiments, the cytotoxic effect is mediated by immune cell activation by the Fc region. In some embodiments, the cytotoxic effect is mediated by immune cell recruitment by the Fc region. 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 Fc region induces antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the Fc region induces complement-dependent cytotoxicity (CDC).

[0165] In some embodiments, the Fc region comprises an Ig CH2 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH2 domain. In some embodiments, the Fc region comprises an Ig CH3 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH3 domain. In some embodiments, the Fc region comprises or consists of both an Ig CH2 domain and Ig CH3 domain. In some embodiments, the Fc region comprises or consists of both an Ig heavy chain CH2 and an Ig heavy chain CH3 domain. In some embodiments, the first chain comprises a first portion of an Fc region and the second chain comprises a second portion of the Fc region. In some embodiments, the first portion comprises a CH2 domain, a CH3 domain or both. In some embodiments, the second portion comprises a CH2 domain, a CH3 domain or both. In some embodiments, interface of the first portion of an Fc region and the second portion of an Fc region produces a functional Fc region. In some embodiments, interface comprises contact. In some embodiments, interface comprises adjacent positioning. In some embodiments, interface comprises formation of the protein complex of the invention. In some embodiments, interface comprises 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.

[0166] In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (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 IgGl CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (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, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (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, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPRE EQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (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.

[0167] In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP GDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 40). In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPG DLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 IgGl CH3 domain. In some embodiments, SEQ ID NO: 41 is the IgGl CH3 domain. In some embodiments, the SEQ ID NO: 40 sequence is the sequence found predominantly is humans of European and American descent. In some embodiments, SEQ ID NO: 41 is the sequence found predominantly in humans of Asian descent. In someembodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP GDLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(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, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPP MLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (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.

[0168] In some embodiments, the Fc comprises a mutation. In some embodiments, a CH3 domain comprises a mutation. In some embodiments, the first CH3 domain comprises a first mutation. In some embodiments, the second CH3 domain comprises a second mutation. In some embodiments, a CH2 domain comprises a mutation. In some embodiments, the first CH2 domain comprises a first mutation. In some embodiments, the second CH2 domain comprises a second mutation. In some embodiments, the CH2 and CH3 domains both comprise mutations. In some embodiments, the first CH2 domain and first CH3 domains each comprise a first mutation. In some embodiments, the second CH2 domain and the second CH3 domain each comprise a second mutation. In some embodiments, the mutations inhibit homodimerization of the first polypeptide chain. In some embodiments, the first mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the mutations inhibit homodimerization of the second polypeptide chain. In some embodiments, the second mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the mutations permit heterodimerization. In some embodiments, the mutations permit heterodimerization of the first and second chains. In some embodiments, permitting is promoting. In some embodiments, permitting is enhancing.

[0169] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutations or alterations may be used for constructing the polypeptides of the invention. In some embodiments, a region from an IgG is replaced with a region from an IgA. In some embodiments, a region from a TCRa is inserted into the first CH3 domain and a region from TCRb is inserted in to the second CH3 domain. In some embodiments, the mutation is insertion of a region from a TCR. In some embodiments, the TCR is selected from TCRa and TCRb. In some embodiments, the mutation is insertion of a region from a different Ig. Examples of these mutations can be found in Table 1. In some embodiments, the mutation is selected from a mutation in Table 1. In some embodiments, the first mutation is selected from a group of mutations provided in a row and the second column of Table 1 and the second mutation is the group of mutations provided in that same row of Table 1 in the third column. The mutations in Table 1 are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in other IgGs. In some embodiments, the first mutation is T366Y, and the second mutation is Y407T. In some embodiments, the first mutation is S354C and T366W and the second mutation is Y349C, T366S, L368A, and Y407V. In some embodiments, the first mutation is S364H and F405A and the second mutation is Y349T and T392F. In some embodiments, the first mutation is T350V, L351Y, F405A, and Y407V and the second mutation is T350V, T366L, K392L, and T394W. In some embodiments, the first mutation is K392D, and K409D and the second mutation is E356K, and D399K. In some embodiments, the first mutation is D221E, P228E, and L368E and the second mutation is D221R, P228R, and K409R. In some embodiments, the first mutation is K360E, and K409W and the second mutation is Q347R, D399V, and F405T. In some embodiments, the first mutation is K360E, K409W, and Y349C and the second mutation is Q347R, D399V, F405T, and S354C. In some embodiments, the first mutation is F405L and the second mutation is K409R. In some embodiments, the first mutation is K360D, D399M, and Y407A and the second mutation is E345R, Q347R, T366V, and K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, and K409V and the second mutation is 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, and Y349D and R355D. In some embodiments, the first mutation is T366K and C351K and the second mutation isselected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, and Y349D and R355D. In some embodiments, the first mutation is L351D and L368E and the second mutation is L351K and T366K. In some embodiments, the first mutation is L368D and K370S and the second mutation is E357Q and S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A and Y407V. In some embodiments, the Ig is IgG2, and the first mutation is C223E, P228E, and L368E and the second mutation is C223R, E225R, P228R, and K409R. In some embodiments, the first mutation is S354C or T366W and the second mutation is Y349C, T366S, L368A, or Y407V. In some embodiments, the first mutation is S364H or F405 A 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, the 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 comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP GDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 45). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP GDLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 46). In some embodiments, the CH3 domain comprises or consists ofGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTP GDLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 47). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP GDLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 48). In some embodiments, a first chain comprises SEQ ID NO: 59 and a second chain comprises SEQ ID NO: 60. In some embodiments, SEQ ID NO: 59 and SEQ ID NO: 60 heterodimerize and reduce homodimerization. In some embodiments, a chain is a polypeptide.

[0170] Table 1: Mutations for enhancing heterodimerization and inhibiting homodimerization of CH3 domains.Strategy CH3 domain Chain 1 CH3 domain Chain 2 1 Knobs-into-holes (Y-T) T366Y Y407T2 Knobs-into-holes (CW- S354C, T366W Y349C, T366S, L368A, CSAV) Y407V3 HA-TF S364H, F405A Y349T, T394F 4 ZW1 (VYAV-VLLW) T350V, L351Y, F405A, T350V, T366L, K392L,Y407V T394W5 CH3 charge pairs (DD- K392D, K409D E356K, D399K KK)6 Hinge / CH3 charge (EEE- D221E, P228E, L368E D221R, P228R, K409R RRR)7 EW-RVT K360E, K409W, Q347R, D399V, F405T 8 EW-RVTS-S K360E, K409W, Y349C Q347R, D399V, F405T,S354C9 (L-R) F405L K409R10 7.8.60 (DMA-RRVV) K360D, D399M, E345R, Q347R, T366V,Y407A K409V11 20.8.34 (SYMV-GDQA) Y349S, K370Y, E356G, E357D, S364Q,T366M, K409V Y407A12 Electrostatic steering 366K or 366K+ C351K C351D or E or D at 349, effects 368, 349, or 349 +355 13 “DEKK” L351D and L368E L351K and T366K 14 XmAb L368D / K370S E357Q / S364K15 KiH T366W T366S / L368A / Y407V 16 IgG2 hinge / CH3 charge IgG2: C223E, P228E, IgG2: C223R, E225R, (EEE-RRRR) L368E P228R, K409R 17 SEEDbody IgG / A chimera IgG / A chimera 18 BEAT residues from TCRa residues from TCRb interface interface

[0171] In some embodiments, the Fc domain comprises at least one mutation that increases effector function. In some embodiments, the Fc domain comprises at least one mutation that increases CDC, ADCC or both. In some embodiments, the Fc domain comprises at least one mutation that increases CDC. In some embodiments, the Fc domain comprises at least one mutation that increases ADCC. In some embodiments, the Fc domain comprises at least one mutation that increases antibody effector function. In some embodiments, the Fc domain comprises at least one mutation that increases antibody stability. In some embodiments, stability is half-life. In some embodiments, half-life is circulation half-life. In some embodiments, half-life is half-life in blood. In some embodiments, blood is serum.

[0172] In some embodiments, the mutation reduces effector function. In some embodiments, effector function comprises ADCC, CDC or both. In some embodiments, reduced effector function comprises reduced cytotoxicity. In some embodiments, reduces is abolishes. In some embodiments, the Fc is from IgGl or IgG3 and the mutation reduces effector function. In some embodiments, the Fc is from IgGl and comprises 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. Examples of such mutations can be found in Saunders, 2019, “Conceptual approaches to modulating antibody effector functions and circulation half-life” Front Immunol., Jun 7; 10: 1296, herein incorporated by reference in its entirety.

[0173] It will be known by a skilled artisan that IgG2 and IgG4 possess greatly reduced effector function and are not generally cytotoxic in nature. Additionally, mutations such as S228P and L235E in IgG4 are known to reduce effector function even more. Further,mutations that reduce the cytotoxicity / effector function of IgGl and IgG3 are well known in the art. In some embodiments, the IgG comprises at least one mutation. In some embodiments, the mutation is a plurality of mutations. In some embodiments, the mutation decreases cytotoxicity. In some embodiments, the mutation increases stability. In some embodiments, the mutation decreases aggregation. In some embodiments, the Fc domain comprises at least one mutation that decreases antibody effector function. In some embodiments, the Fc domain comprises at least one mutation that decreases ADCC. In some embodiments, the at least one mutation that decreases ADCC is a LALA mutation. As used herein, the LALA mutation refers to mutation of two successive leucine residues to alanine residues. In some embodiments, the LALA mutation is within the hinge domain. In some embodiments, the hinge domain is the hinge domain of IgGl. In some embodiments, the LALA mutation is mutation of L19 and L20 of SEQ ID NO: 22 to A19 and A20. In some embodiments, a LALA mutation hinge comprises an L19A and an L20A mutation of SEQ ID NO: 22. In some embodiments, the Fc domain comprises a hinge domain comprising EPKSCDKTHTCPPCPAPEAA (SEQ ID NO: 49). In some embodiments, an Fc domain comprising a LALA mutation comprises SEQ ID NO: 49. In some embodiments, the Fc domain comprises a hinge domain consisting of SEQ ID NO: 49. In some embodiments, a LALA mutated hinge domain consists of SEQ ID NO: 49. In some embodiments, the Fc domain comprises a hinge domain comprising EPKSSDKTHTCPPCPAPEAA (SEQ ID NO: 183). In some embodiments, an Fc domain comprising a LALA mutation comprises SEQ ID NO: 183. In some embodiments, the Fc domain comprises a hinge domain consisting of SEQ ID NO: 183. In some embodiments, a LALA mutated hinge domain consists of SEQ ID NO: 183. In some embodiments, the LALA mutation is a L234A and L235A mutation of the Fc. In some embodiments, the at least one mutation that decreases ADCC is a N297A mutation. In some embodiments, the N297A mutation is within the CH2 domain. In some embodiments, the N297A mutation is mutation of asparagine 59 of SEQ ID NO: 36 to alanine. In some embodiments, an N297A mutated CH2 domain comprises an N59A mutation of SEQ ID NO: 36. In some embodiments, the Fc domain comprises a CH2 domain comprising SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 50). In some embodiments, the Fc domain comprises a CH2 domain consisting of SEQID NO: 50. In some embodiments, the N297A mutated CH2 domain consists of SEQ ID NO: 50.

[0174] In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the LALA mutations. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the PG-LALA mutations. In some embodiments, the mutation is mutation of proline 329 of the IgGl human heavy chain to glycine (P329G). In some embodiments, the P to G mutation is mutation of P109 of SEQ ID NO: 61 to G. In some embodiments, the mutation is mutation of leucine 234 of the IgGl human heavy chain to alanine (L234A). In some embodiments, the L to A mutation is mutation of L14 of SEQ ID NO: 61 to A. In some embodiments, the mutation is mutation of leucine 235 of the IgGl human heavy chain to alanine (L235A). In some embodiments, the L to A mutation is mutation of LI 5 of SEQ ID NO: 61 to A. In some embodiments, the plurality of mutation comprises P109G, L14A and L15A of SEQ ID NO: 61. In some embodiments, the plurality of mutation comprises L14A and LI 5 A of SEQ ID NO: 61. In some embodiments, the plurality of mutation comprises P329G, L234A and L235A of the IgGl human heavy chain. In some embodiments, the plurality of mutation comprises L234A and L235A of the IgGl human heavy chain. It will be understood by a skilled artisan that parallel mutation can also be performed in the IgG3 heavy chain or the heavy chains of non-human IgGls. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the YTE mutations. In some embodiments, the mutation is mutation of methionine 252 of the IgGl human heavy chain to tyrosine (M252Y). In some embodiments, the M to Y mutation is mutation of M32 of SEQ ID NO: 61 to Y. In some embodiments, the mutation is mutation of serine 254 of the IgGl human heavy chain to threonine (S254T). In some embodiments, the S to T mutation is mutation of S34 of SEQ ID NO: 61 to T. In some embodiments, the mutation is mutation of threonine 256 of the IgGl human heavy chain to glutamic acid (T256E). In some embodiments, the T to E mutation is mutation of T36 of SEQ ID NO: 61 to E. In some embodiments, the plurality of mutation comprises M32Y, S34T and T36E of SEQ ID NO: 61. In some embodiments, the plurality of mutation comprises M252Y, S254T and T256E of the IgGl human heavy chain. In some embodiments, the mutation is mutation of asparagine 297 of the IgGl human heavy chain (N297). In some embodiments, the asparagine is mutated to alanine (N297A). In some embodiments, the asparagine is mutatedto glutamine (N297Q). In some embodiments, the asparagine is N77 of SEQ ID NO: 61 (N77A orN77Q).

[0175] In some embodiments, the mutation increases the half-life of the molecule, peptide, polypeptide or protein complex. In some embodiments, a mutation that increases half-life is a mutation that increases binding to the neonatal Fc receptor (FcRn). In some embodiments, a mutation that increases binding to FcRn is selected from the mutations provided in Table 4. In some embodiments, the mutation is mutation of asparagine 434 to histidine (N434H). In some embodiments, an N434H mutated Fc domain comprises an N214H mutation of SEQ ID NO: 61 or 65. In some embodiments, the mutation is mutation of valine 308 to proline (V308P). In some embodiments, an H435A mutated Fc domain comprises an H215A mutation of SEQ ID NO: 61 or 65. In some embodiments, the mutation attenuates binding to FcRN. In some embodiments, the mutation that attenuates binding is mutation of histidine 435 to alanine (H435A). In some embodiments, an H435A mutated Fc domain comprises an H215 A mutation of SEQ ID NO: 61 or 65. In some embodiments, the mutation that increases binding to FcRn is a plurality of mutations. In some embodiments, the plurality comprises or consists of mutation of methionine 252 to tyrosine (M252Y), mutations of serine 254 to threonine and mutation of threonine 256 to glutamic acid (T256E) (also termed YTE). In some embodiments, an M252Y / S254T / T256E mutated Fc domain comprises an M32Y, S34T and T36E mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of methionine 428 to leucine (M428L) and mutation of asparagine 434 to serine (N434S) (also termed LS). In some embodiments, an M428L / N434S mutated Fc domain comprises an M208L and N214S mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of M428L and mutation of asparagine 434 to alanine (N434A) (also termed LA). In some embodiments, an M428L / N434A mutated Fc domain comprises an M208L and N214A mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of threonine 250 to glutamine (T250Q) and mutation of methionine 428 to leucine (M428L) (also termed QL). In some embodiments, an T250Q / M428L mutated Fc domain comprises an T30Q and M208L mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of histidine 433 to lysine (H433K) and mutation of asparagine 434 to phenylalanine (N434F). In some embodiments, an H433K / N434F mutated Fc domain comprises an H213K and N214F mutation of SEQ ID NO: 61 or 65. In someembodiments, the plurality comprises or consists of M252Y, S254T, T256E, H433K and N434F. In some embodiments, anM252Y / S254T / T256E / H433K / N434F mutated Fc domain comprises an M32Y, S34T, T35E, H213K and N214F mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of threonine 307 to alanine (T307A), mutation of glutamic acid 380 to alanine (E380A) and mutation of asparagine 434 to alanine (N434A). In some embodiments, an T307A / E380A / N434A mutated Fc domain comprises an T87A, E160A and N214A mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of methionine 252 to tyrosine (M252Y), mutation of valine 308 to proline (V308P) and mutation of asparagine 343 to tyrosine (N343Y). In some embodiments, an M252Y / V308P / N343Y mutated Fc domain comprises an M32Y, V88P and N123Y mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of M252Y, mutation of valine 308 to proline (V308P) and mutation of asparagine 434 to tyrosine (N434Y). In some embodiments, an M252Y / V308P / N434Y mutated Fc domain comprises an M32Y, V88P and N214Y mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of histidine 258 to aspartic acid (H258D), mutation of threonine 307 to glutamine (T307Q) and mutation of alanine 378 to valine (A378V). In some embodiments, an H258D / T307Q / A378V mutated Fc domain comprises an H38D, T87Q and A158V mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of leucine 309 to aspartic acid (L309D), mutation of glutamine 311 to histidine (Q311H) and mutation of asparagine 434 to serine (N434S). In some embodiments, an L309D / Q311H / N434S mutated Fc domain comprises an L89D, Q91H and N214S mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality that attenuates binding comprises or consists of mutation of isoleucine 253 to alanine (1253 A), H435A and mutation of histidine 436 to alanine (H436A). In some embodiments, an I253A / H435A / H436A mutated Fc domain comprises an I33A, H215A and H216A mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality that attenuates binding comprises or consists of 1253 A, mutation of histidine 310 to alanine (H310A) and H435A. In some embodiments, an I253A / H310A / H435A mutated Fc domain comprises an I33A, H90A and H215A mutation of SEQ ID NO: 61 or 65.

[0176] Table 4: Mutations influencing FcRn bindingMutation Description M252Y / S254T / T256E (YTE) Increased in 10-fold FcRn binding at pH 6.0 in comparison to WT hlgGl Increase half-lifeM428L / N434S (LS) Increased in 11 -fold FcRn binding at pH 6.0 in comparison to WT hlgGl, Increase half-lifeM428L / N434A (LA) increase binding affinity to FcRn under pH 6.0 and prolong serum half-lifeT250Q / M428L (QL) increase binding affinity to FcRn under pH 6.0 and prolong serum half-lifeN434H prolong the half-life by enhancing FcRn binding M252Y / S254T / T256E (YTE) + increase binding affinity to FcRn under H433K / N434F pH 6.0 and 7.4H433K / N434F increase binding affinity to FcRn under pH 6.0 and 7.4 T307A / E380A / N434A increase binding affinity to FcRn under pH 6.0 and 7.4 prolong the half-life by enhancing FcRn binding M252Y / V308P / N343Y increase binding affinity to FcRn under pH 6.0 and 7.4 prolong the half-life by enhancing FcRn bindingV308P increase binding affinity to FcRn under pH 6.0 and prolong serum half-life M252Y / V308P / N434Y increase binding affinity to FcRn under pH 6.0 and 7.4H258D / T307Q / A378V increase binding affinity to FcRn under pH 6.0 and prolong serum half-life L309D / Q311H / N434S increase binding affinity to FcRn under pH 6.0 and prolong serum half-life H435A attenuate binding to FcRn at pH = 6.0 I253A, H435A, H436A disable binding to FcRn I253A / H310A / H435A reduced FcRn binding

[0177] In some embodiments, the mutation is a mutation that decreases binding to an Fc receptor. In some embodiments, the Fc receptor is FcyR. In some embodiments, FcyR is FcyRI. In some embodiments, the mutation is a mutation that decreases binding to Clq. In some embodiments, a mutation that decreases binding to Fc receptor decreases ADCC. In some embodiments, the mutation is mutation of N297. As N-glycans are linked to N297 its mutation abrogates the glycosylation of this residue. In some embodiments, mutation of N297 is mutation to alanine (N297A). In some embodiments, mutation of N297 is mutation to glutamine (N297Q). In some embodiments, mutation of N297 is mutation to glycine (N297G). In some embodiments, an N297A mutated CH2 domain comprises an N59A mutation of SEQ ID NO: 36. In some embodiments, an N297A mutated Fc domain comprises an N77A mutation of SEQ ID NO: 61 or 65. In some embodiments, an N297Q mutated CH2 domain comprises an N59Q mutation of SEQ ID NO: 36. In some embodiments, an N297Q mutated Fc domain comprises an N77Q mutation of SEQ ID NO: 61 or 65. In some embodiments, an N297G mutated CH2 domain comprises an N59G mutation of SEQ ID NO: 36. In some embodiments, an N297G mutated Fc domain comprises an N77G mutation of SEQ ID NO: 61 or 65. In some embodiments, the mutation is a plurality of mutations that decrease binding to an Fc receptor. In some embodiments, the plurality comprises or consists of glycine 236 to arginine (G236R) and mutation of leucine 328 to arginine (L328R). In some embodiments, an G236R / L328R mutated Fc comprises a hinge domain comprising a G21R mutation of SEQ ID NO: 22 and a CH2 domain comprising a L90R mutation of SEQ ID NO: 36. In some embodiments, a G236R / L328R mutated Fc domain comprises an G16R and L108R mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of serine 298 to glycine (S298G) and mutation of threonine 299 to alanine (T299A). In some embodiments, an S298G / T299Amutated CH2 domain comprises a S60G and T61A mutation of SEQ ID NO: 36. In some embodiments, a S298G / T299A mutated Fc domain comprises an S78G and T79A mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of leucine 234 to phenylalanine (L234F), leucine 235 to glutamic acid (L235E) and mutation of aspartic acid 265 to alanine (D265A). In some embodiments, an L234F / L235E / D265A mutated Fc comprises a hinge domain comprising a L19F and L20E mutation of SEQ ID NO: 22 and a CH2 domain comprising a D27A mutation of SEQ ID NO: 36. In some embodiments, a L234F / L235E / D265A mutated Fc domain comprises an L14F, L15E and D45A mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of leucine 234 to alanine (L234A), leucine 235 to alanine (L235A) and mutation of proline 329 to glycine (P329G). In some embodiments, an L234A / L235A / P329G mutated Fc comprises a hinge domain comprising a L19A and L20A mutation of SEQ ID NO: 22 and a CH2 domain comprising a P91G mutation of SEQ ID NO: 36. In some embodiments, a L234A / L235A / P329G mutated Fc domain comprises an L14A, L15A and P109G mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of L234F, L235E and mutation of proline 331 to serine (P331S). In some embodiments, an L234F / L235E / P331S mutated Fc comprises a hinge domain comprising a L19F and L20E mutation of SEQ ID NO: 22 and a CH2 domain comprising a P93S mutation of SEQ ID NO: 36. In some embodiments, a L234F / L235E / P331S mutated Fc domain comprises an L14F, L15E and P111S mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of leucine 235 to alanine (L235A), glycine 237 to alanine (G237A) and mutation of glutamic acid 318 to alanine (E318A). In some embodiments, an L235A / G237A / E318A mutated Fc comprises a hinge domain comprising a L20A and G22A mutation of SEQ ID NO: 22 and a CH2 domain comprising a E80A mutation of SEQ ID NO: 36. In some embodiments, a L235A / G237A / E318A mutated Fc domain comprises an L15A, G17A and E98A mutation of SEQ ID NO: 61 or 65.

[0178] In some embodiments, the Fc is modified to decrease binding to Fc receptor. In some embodiments, the modification is removal of glycosylation. In some embodiments, Fc glycosylation is removed enzymatically. In some embodiments, enzymatic de-glycosylation is performed with a deglycosylase. In some embodiments, enzymatic de-glycosylation is performed with a cleavase that cleaves sugars. Examples of enzymes for de-glycosylationinclude but are not limited to Peptide-N-Glycosidase F (PNGase) and Endoglycosidase H (Endo H). Kits for de-glycosylation are also commercially available.

[0179] In some embodiments, the mutation is a mutation that increases binding to an 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 mutation of serine 267 to glutamic acid (S267E). In some embodiments, an S267E mutated CH2 domain comprises an S29E mutation of SEQ ID NO: 36. In some embodiments, an S267E mutated Fc domain comprises an S47E mutation of SEQ ID NO: 61 or 65. In some embodiments, the SE Fc comprises or consists of SEQ ID NO: 178. In some embodiments, the mutation is mutations of proline 238 to aspartic acid (P238D). In some embodiments, a P238D mutated hinge domain comprises an P23D mutation of SEQ ID NO: 22. In some embodiments, aP238D mutatedFc domain comprises anP18D mutation of SEQ ID NO: 61 or 65. In some embodiments, the mutation is a plurality of mutations that increase binding to an Fc receptor. In some embodiments, the plurality comprises or consists of S267E and mutation of leucine 328 to phenylalanine (L328F) (also termed SELF). In some embodiments, an S267E / L328F mutated CH2 domain comprises an S29E and L90F mutation of SEQ ID NO: 36. In some embodiments, an S267E / L328F mutated Fc domain comprises an S47E and L108F mutation of SEQ ID NO: 61 or 65. In some embodiments, the SELF Fc comprises or consists of SEQ ID NO: 56. In some embodiments, the SELF Fc comprises or consists of SEQ ID NO: 188. In some embodiments, the SELF Fc comprises or consists of SEQ ID NO: 56 or 188. In some embodiments, the plurality comprises or consists of S267E and mutation of histidine 268 to phenylalanine (H268F) and mutation of serine 324 to threonine (S324T) (also termed EFT). In some embodiments, an S267E / H268F / S324T mutated CH2 domain comprises an S29E, H30F and S86T mutation of SEQ ID NO: 36. In some embodiments, an S267E / H268F / S324T mutated Fc domain comprises an S47E, H48F and S104T mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of glycine 237 to aspartic acid (G237D), P238D, proline 271 to glycine (P271G) and mutation of alanine 330 to arginine (A330R) (also termed V9). In some embodiments, a G237D / P238D / P271G / A330R mutated polypeptide comprises a mutated hinge domain comprising a G22D and P23D mutation of SEQ ID NO: 22 and a mutated CH2 domain comprising a P33G and A92R mutation of SEQ ID NO: 36. In some embodiments, a G237D / P238D / P271G / A330R mutated Fc domaincomprises a G17D, P18D, P51G and A110R mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of G237D, P238D, histidine 268 to aspartic acid (H268D), P271G and A330R (also termed V11). In some embodiments, a G237D / P238D / H268D / P271G / A330R mutated polypeptide comprises a mutated hinge domain comprising a G22D and P23D mutation of SEQ ID NO: 22 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 36. In some embodiments, a G237D / P238D / H268D / P271G / A330R mutated Fc domain comprises a G17D, P18D, H48D, P51G and A110R mutation of SEQ ID NO: 61 or 65. In some embodiments, the plurality comprises or consists of mutation of glutamic acid 233 to aspartic acid (E233D), G237D, P238D, H268D, P271G and A330R (also termed V12). In some embodiments, a E233D / G237D / P238D / H268D / P271G / A330R mutated polypeptide comprises a mutated hinge domain comprising a E18D, G22D and P23D mutation of SEQ ID NO: 22 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 36. In some embodiments, a E233D / G237D / P238D / H268D / P271G / A330R mutated Fc domain comprises a E13D, G17D, P18D, H48D, P51G and A110R mutation of SEQ ID NO: 61 or 65.

[0180] The S267E mutation was found to enhance affinity toward the inhibitory FcγRIIB and also toward the activating FcγRIIa. The SELF mutations in hlgGl resulted in a substantial 430-fold increase in the binding toward FcyRIIB, with minimal alterations in binding to FcγRI and FcγRIIA-H131 in comparison to human WT IgGl. The EFT mutation was found to increase FcγRIIB binding by 18-fold in comparison to human WT IgGl. EFT also increased CDC, ADCC and antibody-dependent cellular phagocytosis (ADCP) activity via the enhancement of C1q and activator FcG receptors binding. In some embodiments, a mutation that increases ADCC is the EFT plurality of mutations. P238D demonstrated enhanced binding to FcγRIIB with about 4.3-fold increased affinity in comparison to WT human IgGl. P238D also significantly reduces the binding toward all other activating FcG receptors. V9 significantly enhanced the affinity of antibodies toward hFcγRIIB, by approximately a 32-fold change in comparison to WT IgGl. V9 also was found to reduce the affinity toward hFcγRIIA R131 allele by about 3 -fold in comparison to WT IgGl. VI 1 was found to significantly enhance the affinity of antibodies for hFcγRIIB by approximately 96-fold, while reducing the affinity toward hFcγRIIA R131 by about 3-fold in comparison to human WT IgGl. V12 demonstrated significant enhancement of binding toward FcγRIIB,with 217-fold change in comparison to human WT IgGl. V12 mutations also show no detectable binding toward FcγRIIIA allotypes, reduced FcγRI binding (0.061-fold change relative to WT IgGl) and FcγRIIA-H131 (0.068-fold change relative to WT IgG1). It should be noted that V12 slightly improves the binding toward FcγRIIA-R131, with a 2-fold binding increase in compared to WT hlgGl.

[0181] Mutations that produce the above recited functions are well known in the art and any such mutation can be used. Examples of such mutations can be found at least in K. O. Saunders, 2019, “Conceptual approaches to modulating antibody effector functions and circulation half-life”, Front, Immunol., 2019 Jun 7; 10: 1296, herein incorporated 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 a skilled artisan that parallel mutation can also be performed in the IgG3 heavy chain or the heavy chains of non -human IgGls. It will be understood that the number given herein is in reference to a full-length IgG including the variable domains. The numbers can be shifted to correspond to the positions of these amino acids within just the Fc portion of the IgG.

[0182] In some embodiments, the mutation increases effector function. In some embodiments, the mutation increases ADCC. In some embodiments, the mutation is not a mutation that increases CDC. In some embodiments, the mutation increases ADCC and not CDC. It will be understood by a skilled artisan that while the unmodified Fc is not sufficiently cytotoxic to overcome the booster effect produced by the molecules of the invention, an Fc comprising a mutation that increases ADCC is. In some embodiments, effector function comprises ADCC. In some embodiments, effector function comprises ADCC and not CDC. In some embodiments, increased effector function comprises increased cytotoxicity. In some embodiments, the Fc is from IgGl or IgG3 and the mutation increases effector function. In some embodiments, the Fc is from IgGl and comprises 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), 2020 Dec; 9(4): 64, herein incorporated by reference in its entirety.

[0183]

[0183] In some embodiments, a mutation that increases ADCC is a plurality of mutations that increase ADCC. In some embodiments, the plurality of mutations comprises mutation of leucine 235 to valine (L235V), phenylalanine 243 to leucine (F243L), arginine 292 to proline (R292P), tyrosine 300 to leucine (Y300L) and proline 296 to leucine (P396L) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of leucine 15 to valine (L15V), phenylalanine 23 to leucine (F23L), arginine 72 to proline (R72P), tyrosine 80 to leucine (Y80L) and proline 176 to leucine (P176L) within SEQ ID NO: 61. In some embodiments, the plurality of mutations comprises mutation of serine 239 to aspartic acid (S239D) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of serine 19 to aspartic acid (S19D) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 61. In some embodiments, the S239D / I332E mutations also increase ADCP. In some embodiments, the plurality of mutations comprises mutation of serine 239 to aspartic acid (S239D), alanine 330 to leucine (A330L) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of serine 19 to aspartic acid (S19D), alanine 110 to leucine (A110L) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 61. In some embodiments, the S239D / A330L / I332E mutations also increase ADCP. In some embodiments, the plurality of mutations comprises mutation of glycine 236 to alanine (G236A), alanine 330 to leucine (A330L) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glycine 16 to alanine (G16A), alanine 110 to leucine (A110L) and isoleucine 112 to glutamic acid (Il 12E) within SEQ ID NO: 61. In some embodiments, the plurality of mutations comprises mutation of serine 298 to alanine (S298A), glutamic acid 333 to alanine (E333A), and lysine 334 to alanine (K334A) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of mutation of serine 78 to alanine (S78A), glutamic acid 113 to alanine (E113A), and lysine 114 to alanine (K114A) within SEQ ID NO: 61. In some embodiments, the plurality of mutations comprises mutation of proline 247 to isoleucine (P247I), and alanine 339 to glutamine (A339Q) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of mutation of proline 27 to isoleucine (P27I), and alanine 119 to glutamine (A119Q) within SEQ ID NO: 61. In some embodiments, the plurality of mutations comprises mutation of glycine 236 to alanine (G236A), serine 239 to aspartic acid (S239D) and isoleucine 332 to glutamic acid(I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glycine 16 to alanine (G16A), serine 19 to aspartic acid (S19D) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 61. In some embodiments, the G236A / S239D / I332E mutations also increase ADCP. In some embodiments, the plurality of mutations comprises mutation of lysine 234 to tyrosine (L234Y), lysine 235 to glutamine (L235Q), glycine 236 to tryptophan (G236W), serine 239 to methionine (S239M), histidine 268 to aspartic acid (H268D), aspartic acid 270 to glutamic acid (D270E) and serine 298 to alanine (S298A) within a first heavy chain of human IgGl and mutation of aspartic acid 270 to glutamic acid (D270E), lysine 326 to aspartic acid (K26D), alanine 330 to methionine (A330M) and lysine 334 to glutamic acid (K334E) within the second heavy chain of IgGl. In some embodiments, the plurality of mutations comprises mutation of 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) within a first chain of SEQ ID NO: 61 and 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) within a second chain of SEQ ID NO: 61. It will be understood that all of the above recited mutations given with respect to SEQ ID NO: 61 also apply to SEQ ID NO: 63. Indeed, they also apply to SEQ ID NO: 62 and SEQ ID NO: 64, but all numbering given hereinabove must be increased by 5 for these sequences.

[0184] In some embodiments, the Fc domain with increased ADCC comprises L15V / F23L / R72P / Y80L / P176L mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 61 and SEQ ID NO: 63. In some embodiments, the Fc domain is selected from SEQ ID NO: 62 and SEQ ID NO: 64. In some embodiments, the Fc domain is selected from SEQ ID NO: 181 and SEQ ID NO: 182. In some embodiments, the Fc domain with increased ADCC comprises EPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 65). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 65. In some embodiments, the Fc comprising theL15V / F23L / R72P / Y80L / P176L mutations is SEQ ID NO: 65. 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: 65 and comprises L15V / F23L / R72P / Y80L / P176L mutations. In some embodiments, the Fc domain with increased ADCC comprises EPKSSDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 184). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 184. In some embodiments, the Fc comprising the L15V / F23L / R72P / Y80L / P176L mutations is SEQ ID NO: 184. 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: 184 and comprises L15V / F23L / R72P / Y80L / P176L mutations.

[0185] In some embodiments, the Fc domain with increased ADCC comprises S19D / A110L / I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 61 and SEQ ID NO: 63. In some embodiments, the Fc domain is selected from SEQ ID NO: 62 and SEQ ID NO: 64. In some embodiments, the Fc domain is selected from SEQ ID NO: 181 and SEQ ID NO: 182. In some embodiments, the Fc domain with increased ADCC comprises EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 66). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 66. In some embodiments, the Fc comprising the S19D / A110L / I112E mutations is SEQ ID NO: 66. 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: 66 and comprises S19D / A110L / I112E mutations. In some embodiments, the Fc domain with increased ADCC comprises EPKSSDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 185). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 185. In some embodiments, the Fc comprising the S19D / A110L / I112E mutations is SEQ ID NO: 185. 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: 185 and comprises S19D / A110L / I112E mutations.

[0186] In some embodiments, the Fc domain with increased ADCC comprises G16A / A110L / I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 61 and SEQ ID NO: 63. In some embodiments, the Fc domain is selected from SEQ ID NO: 62 and SEQ ID NO: 64. In some embodiments, the Fc domain is selected from SEQ ID NO: 181 and SEQ ID NO: 182. In some embodiments, the Fc domain with increased ADCC comprises EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 68). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 68. In some embodiments, the Fc comprising the G16A / A110L / I112E mutations is SEQ ID NO: 68. 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: 68 and comprises G16A / A110L / I112E mutations. In some embodiments, the Fc domain with increased ADCC comprises EPKSSDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 186). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 186. In some embodiments, the Fc comprising the G16A / A110L / I112E mutations is SEQ ID NO: 186. 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: 186 and comprises G16A / A110L / I112E mutations.

[0187] In some embodiments, the mutation increases CDC. In some embodiments, a plurality of mutation increases CDC. In some embodiments, the plurality of mutations comprises mutation of glycine 236 to alanine (G236A), serine 267 to glutamic acid (S267E), histidine 268 for phenylamine (H268F), serine 324 to threonine (S324T) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glycine 16 to alanine (G16A), serine 47 to glutamic acid (S47E), histidine 48 for phenylamine (H48F), serine 104 to threonine (S104T) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 61. It will be understood that all of the above recited mutations given with respect to SEQ ID NO: 61 also apply to SEQ ID NO: 63. Indeed, they also apply to SEQ ID NO: 62 and SEQ ID NO: 64, but all numbering given hereinabove must be increased by 5 for these sequences.

[0188] In some embodiments, the Fc domain with increased CDC comprises G16A / S47E / H48F / S104T / I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 61 and SEQ ID NO: 63. In some embodiments, the Fc domain is selected from SEQ ID NO: 62 and SEQ ID NO: 64. In some embodiments, the Fc domain is selected from SEQ ID NO: 181 and SEQ ID NO: 182. In some embodiments, the Fc domain with increased CDC comprises EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV TNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67). In some embodiments, the Fc domain with increased CDC consists of SEQ ID NO: 67. In some embodiments, the Fc comprising the G16A / S47E / H48F / S104T / I112E mutations is SEQ ID NO: 67. 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: 67 and comprises G16A / S47E / H48F / S104T / I112E mutations. In some embodiments, the Fc domain with increased CDC comprises EPKSSDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV TNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 187). In some embodiments, the Fc domain withincreased CDC consists of SEQ ID NO: 187. In some embodiments, the Fc comprising the G16A / S47E / H48F / S104T / I112E mutations is SEQ ID NO: 187. 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: 187 and comprises G16A / S47E / H48F / S104T / I112E mutations.

[0189] In some embodiments, the Fc domain is selected from SEQ ID NO: 65-68. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 65-68. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 65-68. In some embodiments, the Fc domain is selected from SEQ ID NO: 65-68 and 184-187. In some embodiments, theFc domain comprises any one of SEQ ID NO: 65-68 and 184-187. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 65-68 and 184-187. In some embodiments, the Fc domain is selected from SEQ ID NO: 61-68. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 61-68. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 61-68. In some embodiments, the Fc domain is selected from SEQ ID NO: 61-68 and 184-187. In some embodiments, theFc domain comprises any one of SEQ ID NO: 61-68 and 184-187. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 61-68 and 184-187. In some embodiments, the Fc domain comprises at least 75, 80, 85, 90, 92, 95, 97 or 99% identity to any one of SEQ ID NO: 65-68 and 184-187 and retains increased ADCC as compared to a control Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 56, and 65-68. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, and 65-68. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, and 65-68. In some embodiments, the Fc domain is selected from SEQ ID NO: 56, 65-68, and 184-188. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, 65-68, and 184-188. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, 65-68, and 184-188. In some embodiments, theFc domain is selected from SEQ ID NO: 56, 65, 68 and 188. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, 65, 68 and 188. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, 65, 68 and 188. In some embodiments, the Fc domain comprises at least 75, 80, 85, 90, 92, 95, 97 or 99% identity to any one of SEQ ID NO: 56, 65, 68 and 188 and retains increased ADCC as compared to a control Fc domain. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain is selected from SEQ ID NO: 56, 65, 68 184, 187 and 188. In some embodiments, the Fcdomain comprises any one of SEQ ID NO: 56, 65, 68, 184, 187 and 188. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, 65, 68, 184, 187 and 188. In some embodiments, the Fc domain comprises at least 75, 80, 85, 90, 92, 95, 97 or 99% identity to any one of SEQ ID NO: 56, 65, 68, 184, 187 and 188 and retains increased ADCC as compared to a control Fc domain. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain is selected from SEQ ID NO: 56, 65-68, 178, 184-189, and 193-194. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, 65-68, 178, 184-189 and 193-194. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, 65-68, 178, 184-189 and 193-194. In some embodiments, the Fc domain is selected from SEQ ID NO: 56, 65-68, 178, and 184-189. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, 65-68, 178, and 184-189. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, 65-68, 178 and 184-189. In some embodiments, the Fc domain is selected from SEQ ID NO: 56, 65, 68, 178, 184 and 187-189. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, 65, 68, 178, 184 and 187-189. In some embodiments, theFc domain consists of any one of SEQ ID NO: 56, 65, 68, 178, 184 and 187-189. In some embodiments, the Fc domain comprises at least 75, 80, 85, 90, 92, 95, 97 or 99% identity to any one of SEQ ID NO: 56, 65, 68, 178, 184 and 187-189 and retains increased ADCC as compared to a control Fc domain. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, 178, 188 and 189. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, 178, 188 and 189. In some embodiments, the Fc domain comprises at least 75, 80, 85, 90, 92, 95, 97 or 99% identity to any one of SEQ ID NO: 56, 178, 188 and 189 and retains increased Fc receptor binding as compared to a control Fc domain. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain comprises any one of SEQ ID NO: 56, 178, 188, 189, 193 or 194. In some embodiments, the Fc domain consists of any one of SEQ ID NO: 56, 178, 188, 189, 193 or 194. In some embodiments, the Fc domain comprises at least 75, 80, 85, 90, 92, 95, 97 or 99% identity to any one of SEQ ID NO: 56, 178, 188, 189, 193 or 194 and retains increased Fc receptor binding as compared to a control Fc domain. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc receptor is FcyR. In some embodiments, FcyR is FcyRII. In some embodiments, FcyRII is FcyRIIb. In someembodiments, the control Fc domain is an unmodified Fc domain. In some embodiments, unmodified Fc is an Fc found in nature. In some embodiments, unmodified Fc is a human Fc found in nature.

[0190] In some embodiments, the Fc is modified to increase ADCC. In some embodiments, the modification is removal of fucosylation. In some embodiments, Fc fucosylation is removed enzymatically. In some embodiments, the Fc is afucosylated. In some embodiments, the method comprises performing afucosylation of the molecule. In some embodiments, the molecules of the invention are produced in a cell line engineered to produce afucosylated molecules.

[0191] In some embodiments, the mutation increases CDC. In some embodiments, a plurality of mutations increases CDC. In some embodiments, the plurality of mutations comprises mutation of glycine 236 to alanine (G236A), serine 267 to glutamic acid (S267E), histidine 268 for phenylamine (H268F), serine 324 to threonine (S324T) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glycine 16 to alanine (G16A), serine 47 to glutamic acid (S47E), histidine 48 for phenylamine (H48F), serine 104 to threonine (S104T) and isoleucine 112 to glutamic acid (Il 12E) within SEQ ID NO: 61. In some embodiments, the plurality of mutation comprises mutation of lysine 326 to tryptophan (K326W) and glutamic acid 333 to serine (E333S) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of lysine 106 to tryptophan (K106W) and glutamic acid 113 to serine (El 13S) within SEQ ID NO: 61. In some embodiments, the plurality of mutation comprises mutation of glutamic acid 345 to arginine (E345R), glutamic acid 430 to glycine (E430G) and serine 440 to tyrosine (S440Y) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glutamic acid 125 to arginine (E125R), glutamic acid 210 to glycine (E210G) and serine 220 to tyrosine (S220Y) within SEQ ID NO: 61. It will be understood that all of the above recited mutations given with respect to SEQ ID NO: 61 also apply to SEQ ID NO: 63. Indeed, they also apply to SEQ ID NO: 62 and SEQ ID NO: 64, but all numbering given hereinabove must be increased by 5 for these sequences.Effector moiety

[0192] In some embodiments, the composition comprises an effector moiety. In some embodiments, the effector moiety is not an Fc region. In some embodiments, the effectormoiety does not comprise an Fc region. In some embodiments, the composition comprises an effector moiety that is not an Fc region. In some embodiments, the composition comprises an effector moiety other than an Fc region. In some embodiments, the composition is devoid of an Fc region. In some embodiments, the composition comprises an Fc region that is the dimerization domain and an effector moiety that does not comprises an Fc domain. In some embodiments, the protein comprises an effector moiety that is not an Fc region. In some embodiments, the protein comprises an effector moiety other than an Fc region. In some embodiments, the protein is devoid of an Fc region. In some embodiments, the protein comprises an Fc region that is the dimerization domain and is further conjugated to an effector moiety. In some embodiments, the engager is an Fc region. In some embodiments, the engager is not an Fc region. In some embodiments, the composition comprises an effector moiety that is superior at killing as compared to an Fc. In some embodiments, superior at killing is superior at killing B cells.

[0193] In some embodiments, the first polypeptide chain comprises an effector moiety. In some embodiments, the second polypeptide chain comprises an effector moiety. In some embodiments, both the first and second polypeptide chains comprise an effector moiety. The term "moiety", as used herein, relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures. The term "moiety" may also refer to part of a molecule that exhibits a particular set of chemical and / or pharmacologic characteristics which are similar to the corresponding molecule. As used herein, the term “effector moiety” refers to a molecule or fragment of a molecule that carriers out a cytotoxic effect. In some embodiments, an effector moiety is an effector molecule.

[0194] In some embodiments, the effector moiety is capable of inducing a cytotoxic effect. In some embodiments, the effector moiety is configured to induce a cytotoxic effect. In some embodiments, the effector moiety is capable of inducing death. In some embodiments, the effector moiety 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 against a target cell. In some embodiments, death is in a target cell. In some embodiments, the cytotoxic effect is upon binding. In some embodiments, death is upon binding. In some embodiments, the cytotoxic effect is against a target cell binding the composition. In some embodiments, the death is death of a target cellbinding the composition. In some embodiments, the cytotoxic effect is against a cell bound by the protein complex. In some embodiments, the cytotoxic effect is against a cell binding the protein complex. In some embodiments, the death is death of a cell bound by the protein complex. In some embodiments, the death is death of a cell binding the 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 the composition is binding the fragments. In some embodiments, binding the protein complex is binding the fragments. In some embodiments, the fragments are at least one of the fragments. In some embodiments, the fragments are one of the fragments. In some embodiments, the fragments are both of the fragments.

[0195] In some embodiments, the effector moiety is a cytotoxic moiety. In some embodiments, the effector moiety is a toxin. In some embodiments, the effector moiety is a poison. In some embodiments, the effector moiety is chemotherapeutic. In some embodiments, the effector moiety is an anticancer agent. In some embodiments, the effector moiety is an engager. In some embodiments, an engager binds a cytotoxic cell. In some embodiments, binding a cytotoxic cell is recruiting a cytotoxic cell. In some embodiments, binds is bound by.

[0196] In some embodiments, the effector moiety 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 moiety induces antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the effector moiety induces complement-dependent cytotoxicity (CDC).

[0197] In some embodiments, the effector moiety binds a receptor on a cell surface of the 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 the cytotoxic cell. In some embodiments, the receptor is unique to the cytotoxic cell. In some embodiments, the receptor is CD3. In some embodiments, the effector moiety is an agent that binds CD3. In some embodiments, the engager is an agent that binds CD3. In some embodiments, CD3 is humanCD3. In some embodiments, the agent that binds CD3 is an anti-CD3 antibody or antigen binding fragment thereof. In some embodiments, the receptor is CD 16. In some embodiments, the effector moiety is an agent that binds CD 16. In some embodiments, the engager is an agent that binds CD 16. In some embodiments, CD 16 is human CD 16. In some embodiments, the agent that binds CD16 is an anti-CD16 antibody or antigen binding fragment thereof. In some embodiments, the antibody of antigen binding fragment thereof is a single chain antibody. In some embodiments, the antibody of antigen binding fragment thereof is a single domain antibody. In some embodiments, the antibody of antigen binding fragment thereof is a single chain variable fragment (scFv). Anti-CD3 agents are well known in the art and any such binding agent may be used. For example, the anti-human CD3 scFv known as 0KT3 may be used as the agent. In some embodiments, the cytotoxic moiety is selected from alpha-amanitin, a radioactive moiety and an anti-CD3 binding agent. Other example of human anti-CD3 antibodies include: Muromonab (trade name Orthoclone 0KT3), a murine monoclonal anti-human CD3 antibody (DrugBank Accession Number DB00075); Teplizumab, a humanized version of the murine 0KT3 anti-CD3 monoclonal antibody (DrugBank Accession Number DB06606); UCHT1, a murine monoclonal antihuman CD3 antibody; UCHT1 variant-9, a humanized version of the UCHT1 clone and the bi-specific CD19-CD3 Blinatumomab (DrugBank Accession Number DB09052). Examples of human anti-CD16 include: AFM13, a bispecific tetravalent Innate Cell Engager (ICE®) targeting CD30 on tumor cells and CD16A on NK cells and macrophages and GTB-3550 (CD16 / IL-15 / CD33) a tri-specific killer cell engager.

[0198] In some embodiments, the effector moiety 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 moiety is cytotoxic. In some embodiments, the effector moiety is radioactive. In some embodiments, the effector moiety is a radioactive moiety. In some embodiments, effector moiety is a radioactive label. In some embodiments, the effector moiety is a chemotherapeutic. In some embodiments, the effector moiety is not a chemotherapeutic. In some embodiments, the effector moiety is toxic to a cell that is not replicating. In some embodiments, toxic is lethal. In some embodiments, the effector moiety is sufficient to killa cell. Drug conjugation, and particularly drug conjugation to an antibody backbone, are well known in the art and any method of conjugation may be used.

[0199] In some embodiments, the effector moiety is an amatoxin. In some embodiments, the effector moiety is an amanitin. Amatoxins are a group of toxic compounds found in poisonous mushrooms. These are made up of eight amino acid residues arranged in a macrobicyclic motif and inhibit RNA polymerase. Amatoxins are also known as amanitins. In some embodiments, the amanitin is selected from alpha-amanitin, beta-amanitin, gamma-amanitin, epsilon-amanitin, amanullin, amanullinic acid, amaninamide, amanin and proamanullin. In some embodiments, the amanitin is alpha-amanitin. In some embodiments, the effector moiety is alpha-amanitin.

[0200] In some embodiments, the chemotherapeutic is an anthracycline. In some embodiments, the effector moiety is an anthracycline. Anthracyclines are a class of drugs extracted from streptomyces bacterium 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, ladirubicin and idarubicin. In some embodiments, the anthracycline is PNU-159682.

[0201] In some embodiments, the chemotherapeutic is an anthramycin-based dimer. In some embodiments, the anthramycin-based dimer is a pyrrolobenzodiazepine (PBD). In some embodiments, the chemotherapeutic is PBD. In some embodiments, the anthramycin-based dimer is an indolinobenzodiazepine dimer (IGN). In some embodiments, the chemotherapeutic is a pyrridinobenzodiazepine (PDD). In some embodiments, the anthramycin-based dimer is PDD. In some embodiments, the effector moiety is a PBD. In some embodiments, the effector moiety is a PDD. PBDs and PDDs are families of DNA minor-grove binding agents that inhibit DNA and RNA synthesis. In some embodiments, the PBD is a PBD dimer. Examples of PBDs and PDDs include, but are not limited to anthramycin, SJG-136, NS 694501 and FGX2-62. In some embodiments, the 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 based dimer. In some embodiments, the PBD is tesirine (SG3249). In some embodiments, tesirine is SG3199. In some embodiments, the chemotherapeutic is SG3249. In some embodiments, the chemotherapeutic is SG3199. Insome embodiments, the effector moiety comprises tesirine. In some embodiments, the effector moiety consists of tesirine.

[0202] In some embodiments, the chemotherapeutic is a calicheamicin. In some embodiments, the effector moiety is a calicheamicin. Calicheamicins are a class of antibiotics derived from bacterium micromonospora echinospora that bind the DNA minor groove and cause strand scission. Examples of calicheamicins include but are not limited to calicheamicin gamma 1, esperamicin and ozogamicin.

[0203] In some embodiments, the chemotherapeutic is camptothecin or an analog thereof. In some embodiments, the effector moiety is camptothecin or an analog thereof. In some embodiments, the effector moiety is camptothecin. Examples of analogs of camptothecin include, but are not limited to exatecan, SN-38, and deruxtecan (Dxd). In some embodiments, the camptothecin analog is Dxd. In some embodiments, the chemotherapeutic is Dxd. In some embodiments, the effector moiety is Dxd.

[0204] In some embodiments, the chemotherapeutic is a duocarmycin. In some embodiments, the effector moiety is a duocarmycin. Duocarmycins are small molecules isolated from streptomyces bacteria that bind the DNA minor groove and alkylate adenine bases. Examples of duocarmycins include, but are not limited to duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin TM, duocarmycin MA and CC-1065.

[0205] In some embodiments, the chemotherapeutic is triptolide. In some embodiments, the effector moiety is triptolide.

[0206] In some embodiments, the effector moiety is a tubulin inhibitor. In some embodiments, the effector moiety is a maytansinoid. In some embodiments, the maytansinoid is a thiol containing maytansinoid. Mayttansinoids or maytansine are known to be tubulin inhibitors that inhibit the assembly of microtubules by binding tubulin att the rhizoxin binding site. In some embodiments, the maytansinoid is mertansine (DM-1). In some embodiments, mertansine is emtansine. In some embodiments, the tubulin inhibitor is an auristatin. In some embodiments, the auristatin is selected from Monomethyl auristatin E (MMAE) and Monomethyl auristatin F (MMAF). In some embodiments, the tubulin inhibitor is a tubulysin. In some embodiments, the tubulysin is tubulysin A. In some embodiments, the auristatin is MMAE. In some embodiments, the auristatin is MMAF. Insome embodiments, the effector moiety is MMAE. In some embodiments, the effector moiety is MMAF.

[0207] In some embodiments, the effector moiety is a combination of moieties. In some embodiments, the effector moiety is a plurality of effector moieties. In some embodiments, the effector moiety is a combination of cytotoxic moieties. In some embodiments, the effector moiety comprises at least two cytotoxic moieties selected from the group consisting of: an amatoxin, an anthracy cline, a pyrrol obenzodiazepine, a calicheamicin, a camptothecin, a duocarmycin, a triptolide, and a tubulin inhibitor. In some embodiments, the effector moiety comprises at least two cytotoxic moieties selected from the group consisting of: an amatoxin, an anthracy cline, a pyrrol obenzodiazepine, a calicheamicin, a camptothecin, a duocarmycin, a triptolide, and a maytansinoid.Third and fourth chains

[0208] In some embodiments, the protein complex further comprises a third polypeptide chain. In some embodiments, the third polypeptide chain comprises a third fragment of a protein target of GD autoantibodies. In some embodiments, the third fragment is different than the first fragment. In some embodiments, the third fragment is different than 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 as is the same sequence. In some embodiments, different is a different sequence.

[0209] 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 are capable of dimerizing to each other. In some embodiments, the third and fourth dimerization domains are configured to dimerizing to each other. In some embodiments, the third dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the third dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, configured to dimerize is capable of dimerizing. In some embodiments, the third and fourthdimerization domains are different than 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.

[0210] In some embodiments, the protein complex further comprises a fourth polypeptide chain. In some embodiments, the fourth polypeptide chain comprises a fourth fragment of a protein target of GD autoantibodies. In some embodiments, the fourth fragment is different than the first fragment. In some embodiments, the fourth fragment is different than the second fragment. In some embodiments, the fourth fragment is different than 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 as is the same sequence. In some embodiments, different is a different sequence. In some embodiments, different is from a different protein. In some embodiments, different is from the same protein but comprising a different sequence. In some embodiments, different is from the same protein but from a different region 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 complex in mammals. In some embodiments, the protein complex is a complex in humans.

[0211] 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 are capable of dimerizing to each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerizing to each other. In some embodiments, the fifth dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize to the third dimerization domain. In some embodiments, the fifth dimerizationdomain is not configured to dimerize to the fourth dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the third dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the fourth dimerization domain. In some embodiments, the fifth and sixth dimerization domains are different than the first and second dimerization domains. In some embodiments, the fifth and sixth dimerization domains are different than 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, the first polypeptide and second polypeptide do not both comprise a CHI domain. In some embodiments, first polypeptide and second polypeptide both comprise a CHI domain, first polypeptide and second polypeptide both comprise a CL domain. In some embodiments, first polypeptide and second polypeptide do not both comprise a CL domain. In some embodiments, the first polypeptide comprises a CHI domain, and the second polypeptide comprises a CL domain. In some embodiments, the third polypeptide comprises a CL domain and the fourth polypeptide comprise a CHI domain. In some embodiments, the first polypeptide comprises a CL domain and the second polypeptide comprises a CHI domain. In some embodiments, the third polypeptide comprises a CHI domain, and the fourth polypeptide comprise a CL domain.

[0212] In some embodiments, the third and fourth dimerization domains comprises mutations that permit 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 comprises mutations that permit 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 comprises mutations that permit dimerization of the fifth and sixth dimerization domains and inhibitdimerization of the fifth dimerization domain to the third, fourth or both dimerization domains. In some embodiments, the fifth and sixth dimerization domains comprises mutations that permit dimerization of the fifth and sixth dimerization domains and inhibit dimerization of the sixth dimerization domain to the third, sixth or both dimerization domains.

[0213] In some embodiment, the polypeptide comprises a linker. In some embodiments, the composition comprises a linker. In some embodiments, a fragment is separated from the Fc region by a linker. In some embodiments, separated is linked. In some embodiments, the effector moiety is attached by a linker. In some embodiments, the effector moiety is attached to the fragment by a linker. In some embodiments, the effector moiety is attached to the Fc by a linker.

[0214] In some embodiments, the fragment and the 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 moiety is attached by a linker. In some embodiments, the effector moiety and 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. Linkers are well known in the art and any linker may be used.

[0215] In some embodiments, a linker is a chemical linker. In some embodiments, 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 dibenzocyclooctyne 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 dimethylethylenediamine (DMEDA) linker. In some embodiments, the linker is a 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, is a disulfide linker. In some embodiments, thelinker is a thiol linker. In some embodiments, the linker isa azide linker. In some embodiments, the linker is a maleimide (Mai) linker. In some embodiments, the Mai linker is a maleimidocaproyl linker. In some embodiments, the Mai linker is a Mal-C6 linker. In some embodiments, the Mai 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-labile linker. In some embodiments, the linker is a cleavable linker. In some embodiments, cleavable is protease cleavable. In some embodiments, a cleavable linker is a glutathione cleavable linker. In some embodiments, the linker is a non-cleavable 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 (me) linker. In some embodiments, the linker comprises me. 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 -MM AE linker. In some embodiments, the linker is a mc-VC-PABC-MMAE linker. In some embodiments, the linker is a 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 Val-Cit-PAB linkers, Phe-Lys(Trt)-PAB linkers, and Ala-Ala-Asn-PAB linkers. In some embodiments, the linker is a mix 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 fragment and the dimerization domains are linked by a non-cleavable linker. In some embodiments, the fragment and the dimerization domains are linked by a cleavable linker. In some embodiments, the effector moiety is linked by a cleavable linker. In some embodiments, the effector moiety is linked by a non-cleavable linker.

[0216] In some embodiments, conjugated is linked. In some embodiments, conjugation is via a bond. 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 moiety is conjugated by a linker.

[0217] In some embodiments, conjugating is conjugating of an amino acid linker, moiety or both and comprises extension of the amino acid sequence of a chain of the agent of the invention. It will be understood that a nucleic acid molecule encoding the agent of the invention can be modified to include the coding sequence for the linker, moiety or both and thus upon translation the full conjugate will be produced. In some embodiments, the conjugate is a fusion protein. Methods of linking and conjugating moieties 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 of linking and conjugating to an IgG scaffold are also well known. Methods of linking / conjugating include, but are not limited to, native cysteine reduction (including native hinge reduction, also referred to herein as native cysteine conjugation), engineered cysteine reduction, disulphide bridging, lysine conjugation, and enzymatic conjugation. Examples of enzymatic conjugation include, but are not limited to: Click chemistry, sortase assisted-SMAC technology, transglutaminase addition of amine azide, and glycan remodeling.

[0218] For example, native cysteine conjugation can be performed as follows. The CRD protein was reduced using TCEP and incubated at 37°C for 90 minutes. Subsequently, DMA and the linker-payload were added, followed by a 2-hour incubation at room temperature. Finally, the conjugated materials were purified by Size-Exclusion Chromatography.

[0219] In some embodiments, the conjugation is site-specific conjugation. In some embodiments, the conjugation is not random conjugation. In some embodiments, the conjugation or linking is to the IgG backbone. In some embodiments, the conjugation or linking is not to a TSHR fragment. In some embodiments, the conjugation or linking does not interfere with antibody binding to a TSHR fragment. In some embodiments, the antibody is an autoantibody. In some embodiments, the conjugation or linking is to a dimerization domain. In some embodiments, the conjugation or linking is to the hinge region. In some embodiments, the conjugation or linking is to a CH2 region. In some embodiments, the conjugation or linking is to a CH3 region. In some embodiments, the conjugation or linking is to a CHI region. In some embodiments, the conjugation or linking is to a CL region. In some embodiments, the linking or conjugating is to a native amino acid residue. In some embodiments, the linking or conjugating is to an engineered amino acid residue. In some embodiments, the residue is a cysteine. Examples of engineered cysteines include, but are not limited to A231C, S239C, N325C, L328C, D265C, and S442C of the heavy chain ofIgG. In some embodiments, the residue is a lysine. In some embodiments, the residue is an asparagine. In some embodiments, glycan remodeling is used to link to an asparagine. In some embodiments, the asparagine is N297 of the heavy chain of IgG. In some embodiments, the residue is a glutamine. In some embodiments, N297 is converted, engineered, or mutated to glutamine (N297Q). In some embodiments, the glutamine is Q295 of the heavy chain of IgG. An example of an engineered glutamine includes but is not limited to Q297. The cites are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in other IgGs. In some embodiments, the linking or conjugating is to a C- or N-terminus of a chain of the agent of the invention. In some embodiments, the linking or conjugating is to a C-terminus. In some embodiments, the linking or conjugating is to an N-terminus. In some embodiments, the terminus is a terminus of the heavy chain. In some embodiments, the terminus is a terminus of the light chain. In some embodiments, the conjugation or linking is to a plurality of sites.

[0220] In some embodiments, the linker is of a sufficient length to inhibit steric hindrance between different sections of the chain. In some embodiments, the linker is of a sufficient length to inhibit steric hindrance between different sections of the conjugate. In some embodiments, the linker is of a sufficient length to allow binding of an antibody to the fragment without steric hindrance from another section of the chain. In some embodiments, the linker is of a sufficient length to allow binding of an antibody to the fragment without steric hindrance from another section of the conjugate. In some embodiments, the linker is of a sufficient length to allow binding of a cell to the fragment without steric hindrance from another section of the chain. In some embodiments, the linker is of a sufficient length to allow binding of a cell to the fragment without steric hindrance from another section 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 acids in length. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker is at least 1 amino acid in length. In some embodiments, the linker is at least 5 amino acids in length. In some embodiments, the linker is at least 10 amino acids in length. In some embodiments, the linker is at least 15 amino acids in length. In some embodiments, the linker is at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 amino acids in length. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker is at most 10 amino acids in length. In some embodiments, the linker is at most 20 amino acids in length. In someembodiments, the linker is at most 50 amino acids in length. In some embodiments, the linker is at most 100 amino acids in length.

[0221] In some embodiments, the linker comprises GGS. In some embodiments, the linker comprises EPKSC (SEQ ID NO: 214). In some embodiments, the linker comprises GGSEPKSC (SEQ ID NO: 215). In some embodiments, the linker comprises EPKSS (SEQ ID NO: 130). In some embodiments, the linker comprises GGSEPKSS (SEQ ID NO: 179). In some embodiments, the linker comprises GGGGS (SEQ ID NO: 7). In some embodiments, the linker consists of SEQ ID NO: 7. In some embodiments, the linker comprises (GGGGS)n wherein n is an integer. In some embodiments, the linker consists of (GGGGS)n wherein n is an integer. In some embodiments, the linker comprises GGGS (SEQ ID NO: 6). In some embodiments, the linker consists of SEQ ID NO: 6. In some embodiments, the linker comprises (GGGS)n wherein n is an integer. In some embodiments, the linker consists of (GGGS)n wherein n is an integer. In some embodiments, the linker comprises or consists of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 52). In some embodiments, the linker comprises GSAGSAAGSGEF (SEQ ID NO: 51). In some embodiments, the linker comprises or consists of (GGGS)nGS wherein 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 comprises SEQ ID NO: 8. In some embodiments, the linker consists of SEQ ID NO: 8.

[0222] In some embodiments, the linker is a rigid linker. In some embodiments, the linker comprises EAAAK (SEQ ID NO: 11). In some embodiments, the linker consists of SEQ ID NO: 11. In some embodiments, the rigid linker comprises or consists of SEQ ID NO: 11. In some embodiments, the linker comprises (EAAAK)n wherein n is an integer. In some embodiments, the linker consists of (EAAAK)n wherein n is an integer. In some embodiments, the linker comprises SEQ ID NO: 9. In some embodiments, the linker consists of SEQ ID NO: 9. In some embodiments, the linker comprises A(EAAAK)nA wherein n is an integer. In some embodiments, the linker consists of A(EAAAK)nA wherein n is an integer. In some embodiments, the linker comprises AEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 10). In some embodiments, the linker consists of SEQ ID NO: 10. In some embodiments, the linker comprises a sequence selected from SEQ ID NO: 6-11 and 51-52.In some embodiments, the linker consists of a sequence selected from SEQ ID NO: 6-11 and 51-52.

[0223] In some embodiments, the dimerization domain is C-terminal to the fragment. In some embodiments, the fragment is C-terminal to the dimerization domain. In some embodiments, the Fc region is C-terminal to the fragment. In some embodiments, the fragment is C-terminal to the Fc region. In some embodiments, the dimerization domain is C-terminal to the Fc region. In some embodiments, the Fc region is C-terminal to the dimerization domain. In some embodiments, the dimerization domain is N-terminal to the fragment. In some embodiments, the fragment is N-terminal to the dimerization domain. In some embodiments, the Fc region is N-terminal to the fragment. In some embodiments, the fragment is N-terminal to the Fc region. In some embodiments, the dimerization domain is N-terminal to the Fc region. In some embodiments, the Fc region is N-terminal to the dimerization domain.

[0224] In some embodiments, the epitope spans at least two fragments. In some embodiments, the epitope spans the first and second fragments. In some embodiments, the epitope spans the first and third fragments. In some embodiments, the epitope spans the first and fourth fragments. In some embodiments, the epitope spans the second and third fragments. In some embodiments, the epitope spans the second and fourth fragments. In some embodiments, the epitope spans the third and fourth fragments. In some embodiments, the epitope spans two proteins. In some embodiments, the epitope spans two proteins in a protein complex. In some embodiments, the epitope spans three fragments. In some embodiments, the epitope spans three proteins. In some embodiments, the epitope spans four fragments. In some embodiments, the epitope spans four proteins. In some embodiments, the epitope is a complex epitope. In some embodiments, the epitope is a B cell receptor (BCR)-specific epitope.

[0225] In some embodiments, all fragments are from TSHR. In some embodiments, a fragment from TSHR is mutated. In some embodiments, the complex comprises a TSHR truncation. In some embodiments, the complex comprises a TSHR mutation. In some embodiments, the mutation is a point mutation. In some embodiments, the mutation is a deletion.

[0226] In some embodiments, the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 53). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 53. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 53. In some embodiments, the first polypeptide comprises a fragment linked to EPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 190). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 190. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 190.

[0227] In some embodiments, the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPITPLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 54). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 54. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 54. In some embodiments, the first polypeptide comprises a fragment linked to EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPITPLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 191). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 191. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 191.

[0228] In some embodiments, the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 55). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 55. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 55. In some embodiments, the first polypeptide comprises a fragment linked to EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 192). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 192. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 192.

[0229] In some embodiments, the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 56). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 56. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 56. In some embodiments, the first polypeptide comprises a fragment linked to EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 188). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 188. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 188. In some embodiments,the first polypeptide comprises a fragment linked to DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFN WYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CI< VSNI< AFP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 193). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 193. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 193.

[0230] In some embodiments, the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 178). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 178. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 178. In some embodiments, the first polypeptide comprises a fragment linked to EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPE VI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 189). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 189. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 189. In some embodiments, the first polypeptide comprises a fragment linked to DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFN WYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGI< EYI< CT< VSNI< ALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 194). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 194. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 194.

[0231] In some embodiments, the first polypeptide comprises a fragment linked to SEQ ID NO: 59. In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 59. In some embodiments, the first and second polypeptides both comprises a fragment linked to SEQ ID NO: 59. In some embodiments, the first polypeptide comprises a fragment linked to SEQ ID NO: 60. In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 60. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 60. In some embodiments, the first polypeptide comprises a fragment linked to SEQ ID NO: 56, 178, 188 or 189. In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 56, 178, 188 or 189. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 56, 178, 188 or 189. In some embodiments, the first polypeptide comprises a fragment linked to SEQ ID NO: 56, 178, 188, 189, 193 or 194. In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 56, 178, 188, 189, 193 or 194. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 56, 178, 188, 189, 193 or 194.

[0232] In some embodiments, the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 57). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 57. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 57. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 57.

[0233] In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 53. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 54. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 55. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 56. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 57. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 178. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 188. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 189. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 193. In some embodiments, the third polypeptide comprises afragment linked to SEQ ID NO: 194. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 53. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 54. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 55. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 56. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 57. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 178. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 188. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 189. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 193. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 194.

[0234] In some embodiments, a polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 69-100. In some embodiments, a polypeptide chain consists of an amino acid sequence selected from SEQ ID NO: 69-100. In some embodiments, a polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 71-97. In some embodiments, a polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 71-97 and 99-100. In some embodiments, a polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 133-135, 137, 139, 141, 143, 145, 147, 149-150,152-153 and 155. In some embodiments, a polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 71-97, 99-100, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155. In some embodiments, a polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 71-89. In some embodiments, a polypeptide chain consists of an amino acid sequence selected from SEQ ID NO: 71-97 and 99-100. In some embodiments, a polypeptide chain consists of an amino acid sequence selected from SEQ ID NO: 133-135, 137, 139, 141, 143, 145, 147, 149-150,152-153 and 155. In some embodiments, a polypeptide chain consists of an amino acid sequence selected from SEQ ID NO: 71-97, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155. In some embodiments, a polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 71-97, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153. 155 and 204-214. In some embodiments, a polypeptide chain consists of an amino acid sequence selected from SEQ ID NO: 71-97, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153. 155 and 204-214. In some embodiments, a polypeptide chain consists of an amino acid sequence selected from SEQ ID NO: 71-89. In some embodiments, a polypeptide chain comprises or consists of an amino acid sequence provided in SEQ ID NO: 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155 or an analog or derivative thereof with at least 85% identity and comprising the at least one mutation. In some embodiments, a polypeptide chain comprises or consists of an amino acid sequence provided in SEQ ID NO: 71-97, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155 or an analog or derivative thereof with at least 85% identity and comprising the at least one mutation. In some embodiments, a polypeptide chain comprises or consists of an amino acid sequence provided in SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155 or an analog or derivative thereof with at least 85% identity and comprising the at least one mutation. In some embodiments, a polypeptide chain comprises or consists of an amino acid sequence provided in SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153, 155 and 204-214 or an analog or derivative thereof with at least 85% identity and comprising the at least one mutation. In some embodiments, a polypeptide chain comprises or consists of an amino acid sequence provided in SEQ ID NO: 71-89 or an analog or derivative thereof with at least 85% identity and comprising the at least one mutation. In some embodiments, a 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: 69-100. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153, 155 and 204-214. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-89. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-97. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-97 and 99-100. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-97, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155. In some embodiments, the complexcomprises or consists of two polypeptide chains selected from SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153, 155 and 204-214. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-89. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-97 or an analog or derivative thereof comprising at least 85% identity and the at least one mutation. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-89 or an analog or derivative thereof comprising at least 85% identity and the at least one mutation. In some embodiments, the two chains are the same chain. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153 and 155 or an analog or derivative thereof comprising at least 85% identity and the at least one mutation. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-97, 133-135, 137, 139, 141, 143, 145, 147, 149-150 152-153 and 155 or an analog or derivative thereof comprising at least 85% identity and the at least one mutation. In some embodiments, the two chains are different chains. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150 152-153 and 155 or an analog or derivative thereof comprising at least 85% identity and the at least one mutation. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150 152-153, 155 and 204-214 or an analog or derivative thereof comprising at least 85% identity and the at least one mutation. In some embodiments, the two chains are different chains.

[0235] In some embodiments, the polypeptide comprises SEQ ID NO: 69. In some embodiments, the polypeptide consists of SEQ ID NO: 69. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 69. In some embodiments, the polypeptide comprises SEQ ID NO: 70. In some embodiments, the polypeptide consists of SEQ ID NO: 70. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 70. In some embodiments, the polypeptide comprises SEQ ID NO: 71. In some embodiments, the polypeptide consists of SEQ ID NO: 71. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 71. In some embodiments, the polypeptide comprises SEQ ID NO: 72. In some embodiments, the polypeptide consistsof SEQ ID NO: 72. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 72. In some embodiments, the polypeptide comprises SEQ ID NO: 73. In some embodiments, the polypeptide consists of SEQ ID NO: 73. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 73. In some embodiments, the polypeptide comprises SEQ ID NO: 74. In some embodiments, the polypeptide consists of SEQ ID NO: 74. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 74. In some embodiments, the polypeptide comprises SEQ ID NO: 75. In some embodiments, the polypeptide consists of SEQ ID NO: 75. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 75. In some embodiments, the polypeptide comprises SEQ ID NO: 76. In some embodiments, the polypeptide consists of SEQ ID NO: 76. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 76. In some embodiments, the polypeptide comprises SEQ ID NO: 77. In some embodiments, the polypeptide consists of SEQ ID NO: 77. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 77. In some embodiments, the polypeptide comprises SEQ ID NO: 78. In some embodiments, the polypeptide consists of SEQ ID NO: 78. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 78. In some embodiments, the polypeptide comprises SEQ ID NO: 79. In some embodiments, the polypeptide consists of SEQ ID NO: 79. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 79. In some embodiments, the polypeptide comprises SEQ ID NO: 80. In some embodiments, the polypeptide consists of SEQ ID NO: 80. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 80. In some embodiments, the polypeptide comprises SEQ ID NO: 81. In some embodiments, the polypeptide consists of SEQ ID NO: 81. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 81. In some embodiments, the polypeptide comprises SEQ ID NO: 82. In some embodiments, the polypeptide consists of SEQ ID NO: 82. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 82. In some embodiments, the polypeptide comprises SEQ ID NO: 83. In some embodiments, the polypeptide consists of SEQ ID NO: 83. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 83. In some embodiments, the polypeptide comprises SEQ ID NO: 84. In some embodiments, the polypeptide consists of SEQ ID NO: 84. In some embodiments, the first or second polypeptide comprises or consistsof SEQ ID NO: 84. In some embodiments, the polypeptide comprises SEQ ID NO: 85. In some embodiments, the polypeptide consists of SEQ ID NO: 85. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 85. In some embodiments, the polypeptide comprises SEQ ID NO: 86. In some embodiments, the polypeptide consists of SEQ ID NO: 86. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 86. In some embodiments, the polypeptide comprises SEQ ID NO: 87. In some embodiments, the polypeptide consists of SEQ ID NO: 87. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 87. In some embodiments, the polypeptide comprises SEQ ID NO: 88. In some embodiments, the polypeptide consists of SEQ ID NO: 88. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 88. In some embodiments, the polypeptide comprises SEQ ID NO: 89. In some embodiments, the polypeptide consists of SEQ ID NO: 89. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 89. In some embodiments, the polypeptide comprises SEQ ID NO: 90. In some embodiments, the polypeptide consists of SEQ ID NO: 90. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 90. In some embodiments, the polypeptide comprises SEQ ID NO: 91. In some embodiments, the polypeptide consists of SEQ ID NO: 91. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 91. In some embodiments, the polypeptide comprises SEQ ID NO: 92. In some embodiments, the polypeptide consists of SEQ ID NO: 92. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 92. In some embodiments, the polypeptide comprises SEQ ID NO: 93. In some embodiments, the polypeptide consists of SEQ ID NO: 93. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 93. In some embodiments, the polypeptide comprises SEQ ID NO: 94. In some embodiments, the polypeptide consists of SEQ ID NO: 94. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 94. In some embodiments, the polypeptide comprises SEQ ID NO: 95. In some embodiments, the polypeptide consists of SEQ ID NO: 95. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 95. In some embodiments, the polypeptide comprises SEQ ID NO: 96. In some embodiments, the polypeptide consists of SEQ ID NO: 96. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 96. In some embodiments, the polypeptide comprises SEQ ID NO: 97. In someembodiments, the polypeptide consists of SEQ ID NO: 97. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 97. In some embodiments, the polypeptide comprises SEQ ID NO: 98. In some embodiments, the polypeptide consists of SEQ ID NO: 98. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 98. In some embodiments, the polypeptide comprises SEQ ID NO: 133. In some embodiments, the polypeptide consists of SEQ ID NO: 133. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 133. In some embodiments, the polypeptide comprises SEQ ID NO: 134. In some embodiments, the polypeptide consists of SEQ ID NO: 134. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 134. In some embodiments, the polypeptide comprises SEQ ID NO: 135. In some embodiments, the polypeptide consists of SEQ ID NO: 135. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 135. In some embodiments, the polypeptide comprises SEQ ID NO: 137. In some embodiments, the polypeptide consists of SEQ ID NO: 137. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 137. In some embodiments, the polypeptide comprises SEQ ID NO: 139. In some embodiments, the polypeptide consists of SEQ ID NO: 139. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 139. In some embodiments, the polypeptide comprises SEQ ID NO: 141. In some embodiments, the polypeptide consists of SEQ ID NO: 141. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 141. In some embodiments, the polypeptide comprises SEQ ID NO: 143. In some embodiments, the polypeptide consists of SEQ ID NO: 143. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 143. In some embodiments, the polypeptide comprises SEQ ID NO: 145. In some embodiments, the polypeptide consists of SEQ ID NO: 145. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 145. In some embodiments, the polypeptide comprises SEQ ID NO: 147. In some embodiments, the polypeptide consists of SEQ ID NO: 147. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 147. In some embodiments, the polypeptide comprises SEQ ID NO: 149. In some embodiments, the polypeptide consists of SEQ ID NO: 149. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 149. In some embodiments, the polypeptide comprises SEQ ID NO: 150. In some embodiments, thepolypeptide consists of SEQ ID NO: 150. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 150. In some embodiments, the polypeptide comprises SEQ ID NO: 152. In some embodiments, the polypeptide consists of SEQ ID NO: 152. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 152. In some embodiments, the polypeptide comprises SEQ ID NO: 153. In some embodiments, the polypeptide consists of SEQ ID NO: 153. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 153. In some embodiments, the polypeptide comprises SEQ ID NO: 155. In some embodiments, the polypeptide consists of SEQ ID NO: 155. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 155. In some embodiments, the polypeptide comprises SEQ ID NO: 204. In some embodiments, the polypeptide consists of SEQ ID NO: 204. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 204. In some embodiments, the polypeptide comprises SEQ ID NO: 205. In some embodiments, the polypeptide consists of SEQ ID NO: 205. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 205. In some embodiments, the polypeptide comprises SEQ ID NO: 206. In some embodiments, the polypeptide consists of SEQ ID NO: 206. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 206. In some embodiments, the polypeptide comprises SEQ ID NO: 207. In some embodiments, the polypeptide consists of SEQ ID NO: 207. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 207. In some embodiments, the polypeptide comprises SEQ ID NO: 208. In some embodiments, the polypeptide consists of SEQ ID NO: 208. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 208. In some embodiments, the polypeptide comprises SEQ ID NO: 209. In some embodiments, the polypeptide consists of SEQ ID NO: 209. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 209. In some embodiments, the polypeptide comprises SEQ ID NO: 210. In some embodiments, the polypeptide consists of SEQ ID NO: 210. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 210. In some embodiments, the polypeptide comprises SEQ ID NO: 211. In some embodiments, the polypeptide consists of SEQ ID NO: 211. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 211. In some embodiments, the polypeptide comprises SEQ ID NO: 212. In some embodiments, thepolypeptide consists of SEQ ID NO: 212. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 212. In some embodiments, the polypeptide comprises SEQ ID NO: 213. In some embodiments, the polypeptide consists of SEQ ID NO: 213. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 213. In some embodiments, the polypeptide comprises SEQ ID NO: 214. In some embodiments, the polypeptide consists of SEQ ID NO: 214. In some embodiments, the first or second polypeptide comprises or consists of SEQ ID NO: 214.

[0236] In some embodiments, the polypeptide comprises or consists of CRD-1100. In some embodiments, the complex comprises or consists of CRD-1101. In some embodiments, the complex comprises or consists of CRD-1102. In some embodiments, the complex comprises or consists of CRD-1103. In some embodiments, the complex comprises or consists of CRD-1104. In some embodiments, the complex comprises or consists of CRD-1105. In some embodiments, the polypeptide comprises or consists of CRD-1106. In some embodiments, the complex comprises or consists of CRD-1107. In some embodiments, the complex comprises or consists of CRD-1108. In some embodiments, the complex comprises or consists of CRD-1109. In some embodiments, the complex comprises or consists of CRD-1110. In some embodiments, the complex comprises or consists of CRD-1111. In some embodiments, the polypeptide comprises or consists of CRD-1112. In some embodiments, the complex comprises or consists of CRD-1113. In some embodiments, the complex comprises or consists of CRD-1114. In some embodiments, the complex comprises or consists of CRD-1115. In some embodiments, the complex comprises or consists of CRD-1116. In some embodiments, the complex comprises or consists of CRD-1117. In some embodiments, the polypeptide comprises or consists of CRD-1118. In some embodiments, the complex comprises or consists of CRD-1119. In some embodiments, the complex comprises or consists of CRD-1120. In some embodiments, the complex comprises or consists of CRD-1121. In some embodiments, the complex comprises or consists of CRD-1122. In some embodiments, the complex comprises or consists of CRD-1123. In some embodiments, the polypeptide comprises or consists of CRD-1124. In some embodiments, the complex comprises or consists of CRD-1125. In some embodiments, the complex comprises or consists of CRD-1126. In some embodiments, the complex comprises or consists of CRD-1127. In some embodiments, the complex comprises or consists of CRD-1128. In some embodiments, the complex comprises or consists of CRD-1129. In someembodiments, the complex comprises or consists of CRD-1130. In some embodiments, the complex comprises or consists of CRD-1131. In some embodiments, the complex comprises or consists of CRD-1132. In some embodiments, the complex comprises or consists of CRD-1133. In some embodiments, the complex comprises or consists of CRD-1204. In some embodiments, the complex comprises or consists of CRD-1202. In some embodiments, the complex comprises or consists of CRD-1206. In some embodiments, the complex comprises or consists of CRD-1207. In some embodiments, the complex comprises or consists of CRD-1208. In some embodiments, the complex comprises or consists of CRD-1209. In some embodiments, the complex comprises or consists of CRD-1211. In some embodiments, the complex comprises or consists of CRD-1213. In some embodiments, the complex comprises or consists of CRD-1215. In some embodiments, the complex comprises or consists of CRD-1217. In some embodiments, the complex comprises or consists of CRD-1222. In some embodiments, the complex comprises or consists of CRD-1224. In some embodiments, the complex comprises or consists of CRD-1225. In some embodiments, the complex comprises or consists of CRD-1227. In some embodiments, the complex comprises or consists of CRD-1219. In some embodiments, the complex comprises or consists of CRD-1221. In some embodiments, the complex comprises or consists of CRD-1260. In some embodiments, the complex comprises or consists of CRD-1261. In some embodiments, the complex comprises or consists of CRD-1262. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 70% identity to a sequence provided herein. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 75% identity to a sequence provided herein. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 80% identity to a sequence provided herein. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 85% identity to a sequence provided herein. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 90% identity to a sequence provided herein. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 95% identity to a sequence provided herein. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 97% identity to a sequence provided herein. In some embodiments, a polypeptide chain comprises or consists of a sequence with at least 99% identity to a sequence provided herein.Pharmaceutical compositions

[0237] By another aspect, there is provided a pharmaceutical composition comprising a protein or polypeptide of the invention.

[0238] By another aspect, there is provided a pharmaceutical composition comprising a protein complex of the invention.

[0239] By another aspect, there is provided a pharmaceutical composition comprises a composition of the invention.

[0240] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant. As used herein, the term “carrier,” “adjuvant” or “excipient” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, 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 solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplatedherein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N. J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U. S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's 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 Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U. S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0241] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0242] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the protein complex of the invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the conjugateof the invention. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In some embodiments, a therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition. In some embodiments, an effective amount is an amount sufficient to treat at least one symptom of a disease. In some embodiments, the disease is GD. 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 autoantibody against TSHR.

[0243] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition or method herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life. Treatment of GD is well known in the art and may include any acceptable measure for assessing improvement of a GD symptom. This may include, Rituximab, steroids, steroid-sparing immunosuppressants (such as azathioprine, mycophenolate and cyclophosphamide) dapsone, intravenous immunoglobulin (IVIG) and the like. Treatment may include improved quality of life, suppression of blister formation, reduction of autoantibodies and killing of autoreactive B cells.

[0244] 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.

[0245] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Othersuitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal. In some embodiments, the administering is intravenous administering. In some embodiments, the administering is topical administration. In some embodiments, the administering is selected from oral, intravenous, intramuscular, intraperitoneal, intertumoral, topical, or subdermal administration. In some embodiments, administering is administering to a site of disease.

[0246] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.Methods of treatment

[0247] By another aspect, there is provided a method of treating GD in a subject in need thereof, the method comprising administering to the subject a protein or polypeptide of the invention, thereby treating GD in a subject.

[0248] By another aspect, there is provided a method of treating GD in a subject in need thereof, the method comprising administering to the subject a protein complex of the invention, thereby treating GD in a subject.

[0249] By another aspect, there is provided a method of treating GD in a subject in need thereof, the method comprising administering to the subject a composition of the invention, thereby treating GD in a subject.

[0250] In some embodiments, the administering is administering a pharmaceutical composition of the invention. In some embodiments, GD is characterized by antibodies against the protein. In some embodiments, the protein is a target of GD antibodies. It will be understood by the skilled artisan that a protein complex will be designed with fragments of proteins which are targeted by GD antibodies in the subject. In some embodiments, antibodies are autoantibodies. In some embodiments, the disease is GD and the autoantibodies are against TSHR.

[0251] In some embodiments, treating comprises lowering antibody concentration. In some embodiments, treating comprises lower antibody number. In some embodiments, antibody concentration is circulating antibody concentration. In some embodiments, treating comprises depleting antibodies. In some embodiments, treating comprises killing B cells. Insome embodiments, the B cell are autoreactive B cells. In some embodiments, killing B cells is specific B cell killing. In some embodiments, treating comprises killing B cells that produce the antibodies. In some embodiments, treating comprises killing B cells that produce the antibodies and the not substantially killing other B cells. In some embodiments, treating comprises killing B cell that produce antibodies against the protein complex. In some embodiments, treating comprises killing B cell that produce antibodies against the fragment. In some embodiments, treating comprises killing B cell that produce antibodies against a fragment of the protein complex.

[0252] In some embodiments, lowering antibodies comprises binding antibodies. In some embodiments, lowering is removing at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% of the antibodies. Each possibility represents a separate embodiment of the invention. In some embodiments, lowering is removing at least 80% of the antibodies. In some embodiments, antibodies are autoantibodies. In some embodiments, antibodies in antibodies in the subject. In some embodiments, antibodies are circulating antibodies. In some embodiments, autoantibodies are autoantibodies against the protein or fragment. In some embodiments, autoantibodies are cytotoxic autoantibodies. In some embodiments, autoantibodies comprise IgGl autoantibodies. In some embodiments, autoantibodies comprise IgG3. In some embodiments, autoantibodies comprise IgGl and IgG3 autoantibodies. In some embodiments, autoantibodies comprise IgGl, IgG2 and IgG3 autoantibodies. In some embodiments, autoantibodies comprise IgGl, IgG3 and IgG4 autoantibodies. In some embodiments, autoantibodies comprise IgGl, IgG2, IgG3 and IgG4 autoantibodies. In some embodiments, lowering is removing at least 25% of the antibodies. In some embodiments, lowering is removing at least 50% of the antibodies. In some embodiments, lowering is removing at least 70% of the antibodies. In some embodiments, lowering is removing at least 75% of the antibodies. In some embodiments, lowering is removing at least 80% of the antibodies. In some embodiments, percent of the antibodies is percent of the autoantibodies. In some embodiments, percent of the antibodies is percent of the antibodies against the protein or fragment. In some embodiments, percent of the antibodies is percent of the antibodies associated with the disease.

[0253] In some embodiments, the method further comprises reducing antibodies in the subject. In some embodiments, the reducing is before the administering. In some embodiments, the reducing antibodies is reducing circulating antibodies. In someembodiments, the antibodies are autoantibodies. In some embodiments, the antibodies are against a protein. In some embodiments, the antibodies are against the protein that the fragment is from. In some embodiments, the antibodies are against the protein that at least one of the fragments is from. In some embodiments, the reducing is reducing antibodies against all proteins that at least one of the fragments are from. In some embodiments, the antibodies are against the protein complex. Methods of reducing antibodies are well known in the art and include, for example, plasmapheresis, intravenous Ig (IVIg), antibody filtering, and B cell targeting therapies, any of which may be employed. In some embodiments, the method comprises plasmapheresis of the antibodies before administering. In some embodiments, the method comprises administering a B cell targeting therapy before administering the therapeutic of the invention. In some embodiments, a B cell targeting therapy is an anti-B cell therapy. In some embodiments, the B cell targeting therapy is B cell lethal therapy. In some embodiments, the B cell targeting therapy is a pan B cell therapy. In some embodiments, the B cell targeting therapy is not a targeted therapy. As used herein, a “targeted B cell therapy” is a therapy that targets only specific B cell clones that produce specific antibodies. In some embodiments, an anti-B cell therapy is an anti-B cell antibody. B cell targeting antibodies are known in the art and include for non-limiting example, 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, tositumomab, and ublituximab. In some embodiments, the B cell targeting therapy is rituximab.Nucleic acids

[0254] By another aspect, there is provided a nucleic acid system comprising at least two nucleic acid molecules, wherein a first nucleic acid molecule encodes the first polypeptide chain of a protein complex of the invention and a second nucleic acid molecules encodes the second polypeptide chain of the protein complex of the invention.

[0255] By another aspect, there is provided a nucleic acid system comprising at least two nucleic acid molecules, wherein a first nucleic acid molecule encodes a first polypeptide chain comprising a fragment of a first human protein target of GD autoantibodies or an analog or derivative thereof and a first dimerization domain and a second nucleic acid molecule encodes a second polypeptide chain comprising a fragment of a second humanprotein target of GD autoantibodies or an analog or derivative thereof and second dimerization domain.

[0256] By another aspect, there is provided a nucleic acid molecule encoding a protein of the invention.

[0257] By another aspect, there is provided a nucleic acid molecule encoding a polypeptide chain of a composition of the invention.

[0258] By another aspect, there is provided a nucleic acid molecule encoding a composition of the invention.

[0259] By another aspect, there is provided a nucleic acid molecule encoding a fragment of a first protein target of GD autoantibodies or an analog or derivative thereof and fragment of a second human protein target of GD autoantibodies or an analog or derivative thereof.

[0260] In some embodiments, the nucleic acid system further comprises a third nucleic acid molecule that encodes a third polypeptide of the protein complex of the invention. In some embodiments, the nucleic acid system further comprises a fourth nucleic acid molecule that encodes a fourth polypeptide of the protein complex of the invention. In some embodiments, a first nucleic acid molecule encodes the first polypeptide of the invention. In some embodiments, a second nucleic acid molecule encodes the second polypeptide of the invention. In some embodiments, a third nucleic acid molecule encodes the third polypeptide of the invention. In some embodiments, a fourth nucleic acid molecule encodes the fourth polypeptide.

[0261] 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 comprises an open reading frame encoding the polypeptide chain. Expressing of an open reading frame within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. Expression vectors are well known in the art and any vector compatible with a target cell in which the protein complex of the invention is being expressed may be used.

[0262] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectablemarker (e.g., antibiotic resistance), poly-Adenine sequence. In some embodiments, the vector comprises a promoter. In some embodiments, the promoter is configured for expression in a target cell in which the protein complex of the invention is being expressed.

[0263] The vector may be a DNA plasmid delivered via non -viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoter may be active in mammalian cells. The promoters 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. The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0264] In some embodiments, the open reading frame is operably linked to a promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0265] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and / or the like.

[0266] 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 precursors of mRNA and most snRNA and microRNA.

[0267] 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, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0268] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0269] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0270] 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), in 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 at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U. S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0271] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0272] 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 molecule 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.Patient selection

[0273] By another aspect, there is provided a method of determining suitability of a subject to be treated by a method of the invention, the method comprising receiving a sample from the subject, contacting the sample with a composition of the invention and determining binding of antibodies within the sample to the composition, wherein binding of the antibodies to the composition indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.

[0274] By another aspect, there is provided a method of determining suitability of a subject to be treated by a method of the invention, the method comprising receiving a sample from the subject, contacting the sample with a protein complex of the invention and determining binding of antibodies within the sample to the protein complex, wherein binding of the antibodies to the protein complex indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.

[0275] By another aspect, there is provided a method of determining suitability of a subject to be treated by a method of the invention, the method comprising receiving a sample fromthe subject, contacting the sample with a protein or polypeptide of the invention and determining binding of antibodies within the sample to the protein, wherein binding of the antibodies to the protein or polypeptide indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.

[0276] In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject is a subject such as described hereinabove. In some embodiments, the subject suffers from GD. In some embodiments, the subject is known to be positive for autoantibodies associated with GD. In some embodiments, the subject is seropositive. In some embodiments, the subject is seronegative. In some embodiments, the subject is naive to treatment. In some embodiments, the treatment is treatment for GD. In some embodiments, the subject has received treatment and has relapsed.

[0277] In some embodiments, the method comprises obtaining the sample from the subject. In some embodiments, the sample comprises tissue. In some embodiments, the sample is a biopsy. In some embodiments, the sample is a bodily fluid. In some embodiments, the bodily fluid is blood. In some embodiments, the bodily fluid is serum. In some embodiments, the bodily fluid is plasma. In some embodiments, the bodily fluid is a fluid that comprises antibodies. In some embodiments, the bodily fluid is selected from at least one of: blood, serum, plasma, intestinal fluid, saliva, tumor fluid, urine, interstitial fluid, cerebral spinal fluid and stool.

[0278] In some embodiments, contacting is incubating. In some embodiments, contacting is under conditions sufficient for binding of antibodies to the protein complex. In some embodiments, conditions comprise a time sufficient for binding of antibodies to the protein complex. In some embodiments, conditions comprise physiological conditions. In some embodiments, the protein complex is added to the sample. In some embodiments, the protein complex is dissolved in the bodily fluid. In some embodiments, the antibodies are autoantibodies. In some embodiments, the antibodies are antibodies against a protein.

[0279] In some embodiments, the composition further comprises a detectable moiety. In some embodiments, the protein complex further comprises a detectable moiety. In some embodiments, the protein further comprises a detectable moiety. In some embodiments, the method further comprises contacting the composition, complex and / or protein with a peptide comprising a detectable moiety. In some embodiments, the peptide is configured to bind thecomposition, 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” refers to binding to a specific molecule to the exclusion of other molecules. In some embodiments, the peptide is specific to the composition, protein and / or complex to the exclusion of other proteins in the sample. In some embodiments, the peptide is specific to the composition, protein and / or complex to the exclusion of naturally occurring antibodies in the sample. In some embodiments, the peptide is specific to the composition, protein and / or complex to the exclusion of the antibodies in the sample. In some embodiments, the determining binding comprises detecting the moiety. In some embodiments, the determining comprises isolating the protein complex. In some embodiments, the determining comprises eluting antibodies from the complex. Methods of protein identification are well known in the art and any such method may be used. Examples of such method include western blotting, ELISA, FACS analysis and protein sequencing, such as by mass spectrometry. In some embodiments, the determining comprises ELISA. In some embodiments, the ELISA is a competitive ELISA. In some embodiments, the competitive ELISA comprises competition with antibodies. In some embodiments, the antibodies are antibodies associated with the disease.

[0280] In some embodiments, binding is positive binding. In some embodiments, binding is binding above 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 of at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% of the antibodies in the sample. Each possibility represents a separate embodiment of the invention. In some embodiments, binding is binding of at least 50% of the antibodies in the sample. In some embodiments, binding is binding of at least 70% of the antibodies in the sample. In some embodiments, binding is binding of at least 75% of the antibodies in the sample. In some embodiments, binding is binding of at least 80% of the antibodies in the sample. In some embodiments, percent of the antibodies is percent of the autoantibodies. In some embodiments, percent of the antibodies is percent of the antibodies against the protein. In some embodiments, percent of the antibodies is percent of the antibodies associated with the disease.

[0281] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0282] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0283] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase " A or B" will be understood to include the possibilities of " A" or " B" or " A and B."

[0284] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0285] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0286] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0287] 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 thoroughly explained in the literature. See, for example, " Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); " Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., 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); methodologies as set forth in U. S. Pat. 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, J. E., ed. (1994); " Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley -Liss, N. Y. (1994), Third Edition; " Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), " Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), " Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Example 1:

[0288] Autoantibodies to TSHR are the primary cause of GD. Anti-TSHR antibodies act as thyroid-stimulating immunoglobulins (TSIs) and their stimulation of the thyroid gland causes it to produce excessive amounts of thyroid hormones leading to hyperthyroidism. Therapeutic agents were therefore developed that target TSHR autoantibodies. TSHR has 4 different extracellular regions: a large N-terminal extracellular region that includes nine leucine-rich repeats (LRR) domains and three extracellular loops. The large N-terminal extracellular domain was tested.

[0289] The full N-terminal extracellular domain of TSHR (SEQ ID NO: 1) does not express well as a soluble protein. As such, fragments of this domain comprising C-terminal deletions from amino acids 262, 264 or 281 and on of SEQ ID NO: 1 and lacking the N-terminal signal peptide were used for constructing superior autoantibody capturing molecules.

[0290] Long term remission for GD patients would need to remove a significant proportion of autoreactive B cells which produce the autoantibody pool. Simply removing the autoantibodies from circulation, while potentially effective in treating the symptoms of GD, would require repeated treatments for the rest of the subject’s life as the long-lived B cells would perpetually continue to make new autoantibodies. Importantly, the B cells that produced the autoantibodies express a B-cell receptor (BCR) on their surfaces which is an identical membrane-bound form of these autoantibodies. This allows the B cells themselves to be targeted by a therapeutic that contains the autoantibody BCR-specific epitope(s). By coupling the target epitope to the Fc region of the antibody heavy chain, a therapeutic can direct specific killing of autoantibody producing B cells. This approach is also robust to potential evasion of specific subpopulations, which occurs when using agents that are targeting specific differentiation markers on the cell surface (e.g., CD 19, CD38, BCMA), as every cell carrying the autoreactive BCR will be targeted regardless of its differentiation state. This approach is also beneficial in protecting and preserving non-autoreactive, including protective (e.g., anti-viral, anti-bacterial) subpopulations, which are damaged by treatments that are targeting nonspecific differentiation markers (e.g., CD20, CD38, BCMA) regardless of whether or not they are carrying an autoreactive BCR.

[0291] Figure 1A shows one embodiment of the therapeutic agent of the invention. Immunoglobulin (Ig)-like protein complex 101 comprises four polypeptide chains: twoheavy-chain-like polypeptides 110 and two light-chain-like polypeptides 120. Chains 110 are able to dimerize via disulfide bonds between them. Further, chains 110 may comprise any or all of CH3 domain 111, CH2 domain 112, hinge region 113 and CHI domain 114. In this embodiment, the CH3 domain 111, CH2 domain 112, and hinge region 113 all comprise disulfide bonds and act as dimerization domains, though use of other dimerization domains is also possible. These domains are well known in the art and can be selected from any of human (or non-human) IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD domains for example. A skilled artisan will appreciate that the Fc portion of IgGl and IgG3 incorporated into chain 110 will allow the molecule to induce antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Chains 120 are able to dimerize with chains 110 via disulfide bonds found in CHI domain 114 and CL domain 124.

[0292] Chains 110 and 120 are devoid of variable regions, unlike naturally occurring or manmade antibodies. In place of the variable region each chain has 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 have the same fragment or derivative of TSHR or different fragments / derivatives. Indeed, the two heavy chains can be engineered separately such that the two chains have different fragments of TSHR. The same is true for the light chains. Thus, the therapeutic molecule can be designed with four copies of a TSHR variant of the invention, two copies each of two variants, or one copy each of four different variants or any other combination thereof. Indeed, the molecule is sufficiently modular that it could be engineered with three copies of one protein / domain and one copy of another, or two copies of one protein / domain and one copy of two others. Importantly, the therapeutic molecule can be engineered to comprise any combination of the various domains of the two proteins which will be able to bind autoantibodies from a wide variety of patients and not just some patients. It will be understood by a skilled artisan that any chain can include any protein, fragment, domain or variant.

[0293] Figures 2A-2M show some embodiments of the invention in which only two chains are combined. In Figures 2A-2F, protein complex 201 comprises 2 polypeptide chains which specifically are two heavy chains. Heavy chains 215 and 216 can optionally include a hinge domain 213, CH2212, CH3 211 and / or CHI 214 domain. In this embodiment, the heavy chain hinge 213, CH2212, and CH3 211 all dimerize via disulfide bonds. Only one of these 3 domains is needed for dimerization and other dimerization domains are alsoenvisioned. The N-terminal extracellular domain of TSHR or a fragment or derivative thereof 230 is included. Protein complex 201 is also envisioned with only a fragment of the TSHR extracellular domain, such as fragment 231 of TSHR. A protein complex 201 with two different fragments either being the same or different (such as fragment 231 on one chain and extracellular domain 235 on the other) is also envisioned. These fragments represent any fragment of the extracellular domain of either protein which may be used in any combination.

[0294] Figure 2B shows the molecule without CH2 domain 212, CH3 domain 211 or CHI domain 214. Molecules lacking both hinge 213 and CHI domain 214 are also depicted. Combinations lacking two of these domains are also envisioned, with or without hinge 213 (Fig. 2B). In place of the variable region each chain has a variant 230 of the N-terminal extracellular domain of TSHR. Although it is not depicted, it will be understood by a skilled artisan that any variant from the N-terminal extracellular portion of TSHR can be used. Each chain can be generated to have the same variant (Fig. 2C) or different (Fig. 2D). For ease the N-terminal extracellular domain of TSHR 235 and a fragment of the N-terminal extracellular domain of TSHR 231 are depicted, but it will be understood that any fragments, derivatives or variants of these domains can also be used. When two different subunits are employed, it is advantageous to design the molecule such that predominantly heterodimers of 215 and 216 are formed and not homodimers. The same applies to Figure 2A. There are numerous technologies known in the art for designing mutations in the CH3 / CH2 domains, such as Knobs-in-Holes, DuoBodies, etc., that inhibit homodimerization and promote heterodimerization. Any such technology may be employed. Removal of the CHI with direct conjugation to the hinge region (Fig. 2E) or additional removal of the hinge with direct conjugation to the CH2 (Fig. 2F) is also possible. It will be understood that for all figures, when an extracellular domain is depicted, it is intended also include all fragments, variant and mutants of that extracellular domain.

[0295] In Figure 2G alternative configurations comprising two heavy chains are shown. Instead of containing a single variant 230 in place of the variable region, two tandem variants 230 are used. These fragments may be separated by optional linker 290. This configuration is similar in structure to a single chain antibody in which the heavy and light chain variable domains are on a single peptide and is essentially equivalent to the molecule shown in Figure ID. Heavy chains 215 and 216 can optionally include a hinge 213, CH2212, CH3211 and / orCHI 214 domain. Dimerization is as described above. For simplicity an example containing all three CH domains is shown as is an example lacking the CHI domain. Molecules lacking the hinge, CH2 or CH3 domain or lacking any two / three of these domains are also envisioned (so long as at least one region of dimerization is retained). It will be understood that variant 230 are from the N-terminal extracellular domain of TSHR and can include variants of N-terminal fragments of the domain. Thus, a repeat of two of the same protein / variants can be inserted on a single chain (Fig. 2H-2I, two TSHR fragments 231) or two different proteins / variants can be combined on one chain. Of course, the heavy chains need not be identical as various technologies may be used to favor heterodimerization over homodimerization (Fig. 2L-2M, different TSHR fragments on each chain). As before, CHI domain 214 can be included (Fig. 2H, 2J, 2L) or excluded (Fig. 21, 2K, 2M) and the same is true for hinge 213, CH2212 and / or CH3 211 so long as one dimerization domain (e.g., hinge, CH2, CH3) remains.

[0296] The creation of a protein complex which has three chains, a first heavy chain, a second heavy chain and a light chain is also envisioned. In one possible embodiment the heavy chain comprises a CL domain in place of a CHI domain. The hereinabove described methods of ensuring a heterodimer can be employed. Heavy chains may optionally include a CH3 domain, CH2 domain and / or a hinge region or may employ a different dimerization domain. The CL domain within the light chain can only dimerize with the CHI domain in one of the heavy chains. In place of the variable region each chain has a variant of the N-terminal extracellular portion of TSHR. The three chains can all contain the same protein, all three chains can contain different variants, or the three chains can contain two different fragments in which one is repeated. This configuration, with one of the heavy chains comprising a CL domain in place of a CHI domain can also allow for the formation of the protein complex with four different fragments.

[0297] In the above-described embodiments, an immunoglobulin backbone is depicted and described, but it will be understood by a skilled artisan that by selecting other dimerization domains similar molecules can be generated. Figure 3 shows a generic protein complex 301.In Figure 3 the first chain 315 contains a first dimerization domain (DD1) 363 which can dimerize specifically with a second dimerization domain (DD2) 373 of second chain 316.Chain 315 further comprises a third dimerization domain (DD3) 314 which can dimerize specifically with a fourth dimerization domain (DD4) 324 of third chain 325. Chain 316further comprises a fifth dimerization domain (DD5) 364 which can dimerize specifically with a sixth dimerization domain (DD6) 374 of fourth chain 326. Each of the four chains also comprises a variant 330 of a human protein target of GD autoantibodies (TSHR). These can all be the same fragment with the same amino acid sequence, or they can be different sequences or variants / derivatives or the proteins or fragments.

[0298] It will be understood that each distinct domain can also be separated by a linker. It will be understood by a skilled artisan that all of these linkers are optional, and that combination of linkers is envisioned. It will be further understood that various configurations could employ linkers between any or all of the various domains / fragments. Similarly, linkers can be inserted between the CHI domain, hinge region, CH2 domain, CH3 domain and / or the TSHR fragments. This applies to all configurations in Figures 1 and 2.

[0299] Single chain embodiments of the invention are also envisioned. Two TSHR N-terminal extracellular domain variants can be combined. The single chain can also contain a heavy chain constant region with at least a CH3 domain or CH2 domain and optionally a CHI domain, and / or hinge region. Finally, amino acid linkers can be used to separate any of the domains of the single chain.Example 2:

[0300] The full N-terminal extracellular domain of TSHR (SEQ ID NO: 2, lacking signal peptide) is known not to express well as a soluble protein. Therefore, three fragments of this domain containing only amino acids 21-261 (SEQ ID NO: 3), amino acids 21-263 (SEQ ID NO: 4) or amino acids 21-280 (SEQ ID NO: 5) (N-terminal portions of the extracellular domain without the signal peptide) were generated. Additionally, a variety of mutations, including ones within the ligand binding pocket and ones designed to reduce intra-molecule aggregation or cleavage, were generated in these fragments. A slightly different fragment from amino acids 21-266 (SEQ ID NO: 12) was also generated. The molecules were variant extracellular domains with a chimeric signal peptide (SEQ ID NO: 20) fused to an Fc fragment bearing the SELF mutations (S267E and L328F) which increase binding to FCγRIIB and are summarized in Table 2.

[0301] Table 2: Short ECD and mutated ECD moleculesSEQ ID NO: CRD# Molecule Description Yield (mg / L) %Monomer (SEC-HPLC) 69 CRD- 1100 TSHR 21-261-(GGGGS)3-CH2-CH3 SELF 200 16 70 CRD-1105 TSHR 21-263-(GGGGS)3-CH2-CH3 SELF 241 25 71 CRD- 1106 TSHR 21-263 [P27S]-(GGGGS)3-CH2-CH3 SELF 309 22TSHR 21-263 [H32L]-(GGGGS)3-CH2-CH372 CRD- 1107 295 28SELF73 CRD-1108 TSHR 21-263 [R38L]-(GGGGS)3-CH2-CH3 SELF 325 37 74 CRD- 1109 TSHR 21-263 [V39L]-(GGGGS)3-CH2-CH3 SELF 151 11 75 CRD-1110 TSHR 21-263 [T56L]-(GGGGS)3-CH2-CH3 SELF 168 9 76 CRD-1111 TSHR 21-263 [S84Y]-(GGGGS)3-CH2-CH3 SELF 247 37 77 CRD-1112 TSHR 21-263 [I85F]-(GGGGS)3-CH2-CH3 SELF 374 42TSHR 21-263 [V103I]-(GGGGS)3-CH2-CH378 CRD-1113 28SELF 223TSHR 21-263 [R112K]-(GGGGS)3-CH2-CH379 CRD-1114 28SELF 238TSHR 21-263 [D151Q]-(GGGGS)3-CH2-CH380 CRD-1115 55SELF 319TSHR 21-263 [V169K]-(GGGGS)3-CH2-CH381 CRD-1116 43SELF 294TSHR 21-263 [S191D]-(GGGGS)3-CH2-CH382 CRD-1117 20SELF 274TSHR 21-263 [G194A]-(GGGGS)3-CH2-CH383 CRD-1118 41SELF 336TSHR 21-263 [V215D]-(GGGGS)3-CH2-CH384 CRD-1119 32SELF 345TSHR 21-263 [K218Q]-(GGGGS)3-CH2-CH385 CRD- 1120 26SELF 313TSHR 21-263 [L230Y]-(GGGGS)3-CH2-CH386 CRD-1121 37SELF 306 TSHR 21-263 [V238I]-(GGGGS)3-CH2-CH387 CRD- 1122 35SELF 318 TSHR 21-263 [I253K]-(GGGGS)3-CH2-CH388 CRD- 1123 36SELF 310 TSHR 21-263 [I253K, V169K]-(GGGGS)3-CH2- 89 CRD- 1124 37CH3 SELF 270 TSHR 21-263 [Y116S]-(GGGGS)3-CH2-CH390 CRD-1125 18SELF 122 TSHR 21-263 [Y116E]-(GGGGS)3-CH2-CH391 CRD- 1126 17SELF 107 TSHR 21-263 [Y116R]-(GGGGS)3-CH2-CH392 CRD- 1127 21SELF 158 TSHR 21-263 [C176S]-(GGGGS)3-CH2-CH393 CRD-1128 22SELF 187 TSHR 21-263 [C176A]-(GGGGS)3-CH2-CH394 CRD- 1129 15SELF 210 TSHR 21-263 [W258R]-(GGGGS)3-CH2-CH395 CRD-1130 15SELF 189 TSHR 21-263 [W258L]-(GGGGS)3-CH2-CH396 CRD-1131 17SELF 165 TSHR 21-280 [F269Y, L270I]-(GGGGS)3-CH2- 97 CRD-1132 21CH3 SELF 19598 CRD-1133 TSHR 21-266-(GGGGS)3-CH2-CH3 SELF 237 34TSHR 21-263 [I253R]-(GGGGS)3-CH2-CH399 CRD-1134 48SELF 306 TSHR 21-263 [I253R, V169R]-(GGGGS)3-CH2- 100 CRD-1135 36CH3 SELF 254

[0302] All of these additional variant molecules expressed well; however, it was notable that several (CRDs 1106-1108, 1112 and 1115-1124) showed improved expression as compared to the parent molecule (CRD- 1105) without any mutations. Improved expressionas compared to a previously identified mutant TSHR, CRD-1135, was also observed. Only 25% of the unmutated molecule was present in the form of a monomer indicating a great deal of intermolecular interaction and agglomeration. Several of the mutations were able to reduce aggregation and resulted in a greater monomer percentage (CRDs 1108, 1111-1112, 1115-1116, 1118-1119, 1121-1124, and 1133). In particular, CRD-1108, CRD-1112, CRD-1115, CRD-1116, CRD-1118, CRD-1119, CRD-1121, CRD-1122, CRD-1123 and CRD-1124 showed both increased yield and reduced aggregation.

[0303] The selected variant molecules were also tested for their ability to deplete autoantibodies from Grave’s disease patients’ serum. 7 serum samples from GD patients were incubated with varying concentrations of CRD-1100 (1176, 392, 130, 43, 14 and 0 nM) for 1 hour and then anti-TSHR titer was measured by ELISA. Depletion rate was calculated as 100*(1-(depleted serum titer / original titer)). A control molecule (CRD-981) with a fragment from an irrelevant protein was used as a negative control and produced no depletion at the highest concentration. An 80% depletion rate was observed even at a low concentration of only 14 nM and essentially complete depletion was observed at all higher concentrations (Fig. 4A).

[0304] CRD-1106, CRD-1107, CRD-1108, CRD-1111, CRD-1112, CRD-1113, CRD-1114, CRD-1115, CRD-1116, CRD-1118, CRD-1119, CRD-1120, CRD-1122, CRD-1123 and CRD-1124 were also tested (569 nM) for their ability to deplete autoantibodies from patient serum. CRD-1106, CRD-1107, CDR-1111, CRD-1112, CRD-1113, CRD-1114, CRD-1115, CRD-1116, CRD-1118, CRD-1119, CRD-1122, CRD-1123, and CRD-1124 all produced comparable depletion to CRD-1100 (Fig. 4B). This indicates that the mutations in these variant molecules do not adversely affect autoantibody binding. CRD-1108 and CRD-1120 produced very high depletion that was almost comparable to CRD-1100 (within the error), while CRD-1117 and CRD-1121 produced significantly reduced depletion although it was still greater than 50%. CRD-1109 and CRD-1110 produced poor depletion that was less than 40%, indicating that these two mutations alter (either directly or through altered protein conformation) a target epitope of autoantibodies. All molecules other than CRD-1109, CRD-1110, CRD-1117 and CRD-1121 are thus considered highly effective at reducing TSHR autoantibody levels and treat GD, and these other 4 molecules are considered able to reduce autoantibody levels but at reduced efficiency / capability.

[0305] Antibody M-22 is a commercially available human monoclonal antibody that targets TSHR. M-22 is a thyroid stimulating autoantibody that mimics TSH binding. It was isolated from GD patient lymphocytes (see Furmaniak et al., “Mechanisms of action of TSHR autoantibodies”, Horm Metab Res. 2015, Sep;47(10):735-52 the contents of which are hereby incorporated herein in their entirety). Similarly, antibody Kl-18 is another known stimulating antibody. A third antibody, KI -70, binds TSHR with very high affinity (4 × 1010l / mol) but is a blocking antibody and not a stimulating antibody. Binding of M-22, Kl-18 and Kl-70 to the molecules of the invention was tested by ELISA. The plate was coated with 10 ug / ml antibody, in lOOul / well over night at 4° C. Following washing, the plates were blocked in the presence of PBS-2% BSA 250ul / well for Ih RT, washed again and then incubated with various biotinylated forms molecules of the invention (0.5 nM). Standard ELISA detection was performed (Fig. 5). Increased binding of the antibodies as compared to CRD- 1105 was calculated and it was found that many of the molecules showed superior binding as compared to CRD-1105. In particular, CRD-1111, CRD-1112, CRD-1115, CRD-1116, CRD-1123 and CRD-1124 showed greatly increased antibody binding for all three antibodies as compared to CRD-1105. CRD-1118 and 1119 showed increased binding for M-22 but not for the other 2 antibodies.Example 3:

[0306] Having established that the new molecules can sequester autoantibodies, it was next tested if they could bind to autoreactive B cells. B cell hybridoma cell line TSHR-51, which expresses anti...

Claims

CLAIMS:

1. A polypeptide comprising a fragment of an N-terminal extracellular domain (ECD) of Thyroid Stimulating Hormone Receptor (TSHR) and at least one mutation; wherein said ECD of TSHR consists of SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1 and which binds anti-TSHR autoantibodies and said at least one mutation is D131Q or D131N, wherein said numbering is with respect to SEQ ID NO: 1.

2. The polypeptide of claim 1, further comprising at least one mutation selected from P7S, H12L, R18L, V19L, T36L, S64Y, I65F, V83I, R92K, Y96S, Y96E, Y96R, V149K, S171D, G174A, V195D, K198Q, L210Y, V218I, I233K, and I233K and V149K, and any combination thereof.

3. The polypeptide of claim 1 or 2, comprising a deletion of a C-peptide region of said N-terminal extracellular domain of TSHR.

4. The polypeptide of claim 3, wherein said deletion is a deletion of amino acids 261- 326 of SEQ ID NO: 1.

5. The polypeptide of any one of claims 1 to 4, wherein said fragment is SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5 and which binds anti-TSHR autoantibodies and said mutation is within SEQ ID NO: 5 or said sequence with at least 85% sequence identity to SEQ ID NO: 5.

6. The polypeptide of claim 5, wherein said fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 101-119.

7. The polypeptide of claim 6, wherein said fragment comprising at least one mutation comprises or consists of SEQ ID NO: 110.

8. The polypeptide of claim 3, wherein said deletion is deletion of amino acids 242-326 of SEQ ID NO: 1, and said fragment is SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3 and which binds anti-TSHR autoantibodies and said mutation is within SEQ ID NO: 3 or said sequence with at least 85% sequence identity to SEQ ID NO: 3.

9. The polypeptide of claim 1 or 8, wherein said at least one mutation is D131Q.

10. The polypeptide of claim 9, further comprising at least one additional mutation selected from the group consisting of I65F, G174A and V195D.

11. The polypeptide of claim 10, comprising D131Q and I65F.

12. The polypeptide of claim 11, further comprising at least a third mutation selected from G174A and V195D.

13. The polypeptide of any one of claims 8 to 12, wherein said fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 156 and 158-177.

14. The polypeptide of any one of claims 9 to 12, wherein said fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 156, 159, 161, 169, 171-172, 174-175 and 177.

15. The polypeptide of claim 1 or 8, wherein said at least one mutation is D131N.

16. The polypeptide of claim 15, further comprising at least one additional mutation selected from the group consisting of I65F, G174A and V195D.

17. The polypeptide of claim 16, comprising D131N and I65F.

18. The polypeptide of claim 17, further comprising at least a third mutation selected from G174A and V195D.

19. The polypeptide of any one of claims 14 to 18, wherein said fragment comprising at least one mutation comprises or consists of a sequence selected from SEQ ID NO: 157 and 195-203.

20. The polypeptide of any one of claims 1 to 19, further comprising at least one mutation in a ligand binding domain of said TSHR, wherein said mutation decreases binding of said N-terminal extracellular domain of TSHR to TSH.

21. The polypeptide of claim 20, wherein said mutation is mutation of K163, E231 or both within SEQ ID NO: 1.

22. The polypeptide of claim 20 or 21, wherein K163 is mutated to alanine, E231 is mutated to lysine or both.

23. The polypeptide of any one of claims 1 to 22, further comprising an Fc domain of a human antibody heavy chain, optionally wherein said fragment comprising at least one mutation is linked to said Fc domain by an amino acid linker.

24. The polypeptide of claim 23, wherein said Fc domain comprises a S267E mutation, optionally wherein said Fc domain comprises of consists of SEQ ID NO: 178, 189 or 193.

25. The polypeptide of claim 24, wherein said Fc domain further comprises a L328F mutation, optionally wherein said Fc domain comprises or consists of SEQ ID NO: 56, 188 or 194.

26. The polypeptide of claim 24 or 25, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 71-89, 133-135, 137, 139, 141, 143, 145, 147, 149-150, 152-153, 155 and 204-214.

27. The polypeptide of any one of claims 1 to 26, further comprising an effector moiety wherein said effector moiety is not an Fc domain, optionally wherein said effector moiety is conjugated to said polypeptide by a linker.

28. The polypeptide of claim 27, wherein said effector moiety is selected from an amatoxin / amanitin, an anthracycline, an anthramycin-based dimer, a calicheamicin, camptothecin or an analog thereof, a duocarmycin, triptolide and a tubulin inhibitor.

29. The polypeptide of claim 27 or 28, wherein said effector moiety is selected from:alpha-amanitin, PNU-159682, tesirine, deruxtecan (Dxd), mertansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and a combination thereof.

30. The polypeptide of claim 29, wherein said effector moiety is MMAE.

31. A composition, comprising:a. a first polypeptide comprising a polypeptide of any one of claims 1 to 30; and b. a second polypeptide comprising a polypeptide of any one of claims 1 to 30.

32. The composition of claim 25, wherein said first polypeptide and said second polypeptide comprise different fragments of TSHR, at least one different mutation or both.

33. A pharmaceutical composition comprising a polypeptide of any one of claims 1 to 30 or a composition of claim 31 or 32 and a pharmaceutically acceptable carrier, excipient or adjuvant.

34. A method of treating Graves’ disease (GD) in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition of claim 33, thereby treating GD.

35. The method of claim 34, further comprising reducing in said subject the levels of circulating antibodies against TSHR before said administering.

36. A nucleic acid molecule encoding a polypeptide of any one of claims 1 to 30.

37. A nucleic acid system comprising a nucleic acid molecule, wherein a first nucleic acid molecule encodes said first polypeptide of a composition of claim 31 or 32 and a second nucleic acid molecule encodes said second polypeptide of a composition of claim 31 or 32.

38. A method of determining suitability of a subject in need thereof to be treated by a method of claim 34 or 35, the method comprising receiving a sample from the subject, contacting said sample with a polypeptide of any one of claims 1 to 30 or a composition of claim 31 or 32 and determining binding of autoantibodies within said sample to said polypeptide or said composition, wherein binding of autoantibodies to said polypeptide or said composition indicates said subject is suitable to be treated by a method of claim 34 or 35, thereby determining suitability of the subject to be treated.

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