Ig-like fusion proteins for treatment of myasthenia gravis

Abstract

Provided are compositions comprising: a fragment of a first human acetylcholine receptor, or an analog or derivative thereof; and an effector portion that is not an Fc domain. Also provided are pharmaceutical compositions comprising the compositions, nucleic acid systems encoding polypeptides of the compositions, and methods of treating and determining suitability of treatment using the compositions; and a method of producing the composition.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 432,239, filed on December 13, 2022, and International Patent Application No. PCT / IL2022 / 051321, all of which are incorporated herein by reference in their entireties. Electronic Sequence Listing Reference

[0002] The contents of the electronic sequence listing (CNPY-P-002-PCT1.xml; size: 178,081 bytes; and creation date: December 10, 2023) are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of fusion protein generation and myasthenia gravis treatment. Background Art

[0004] Myasthenia gravis (MG) is the most common neuromuscular transmission disorder. The age of onset is bimodal, with the first peak occurring in the second and third decades (predominantly in women) and the second peak occurring in the sixth to eighth decades (predominantly in men). MG is an autoimmune disease characterized by skeletal muscle weakness due to disruption of the neuromuscular junction. Approximately 85% of MG patients have acetylcholine receptor (AChR)-specific antibodies in their serum, which act as AChR antagonists, causing receptor aggregation and internalization, and recruitment of complement, followed by tissue damage. Each patient has a mixture of different anti-AChR antibodies. Some AChR antibody-positive MG patients also have thymic abnormalities, with approximately two-thirds having hyperplasia and 10% having thymomas. The main clinical feature of MG is fluctuating skeletal muscle weakness, often accompanied by true muscle fatigue.

[0005] Myasthenia gravis has two clinical forms: ocular and generalized. In the ocular form, weakness is limited to the eyelids and extraocular muscles. In the generalized form, weakness may also affect the eye muscles, but it also involves a variable combination of ocular, limb, and respiratory muscles. Transient worsening of symptoms may occur as a result of infection, surgery, pregnancy, childbirth, medication, tapering of immunosuppressive medications, or as part of the natural progression of the disease. Respiratory muscle involvement is the most severe symptom of myasthenia gravis and can lead to respiratory insufficiency and impending respiratory failure, known as a "myasthenic crisis." A long list of medications must be avoided in patients with MG, including fluoroquinolones, aminoglycosides, magnesium sulfate, hydroxychloroquine, penicillamine, and botulinum toxin. Beta-blockers, procainamide, quinidine, and quinine should also be avoided if possible. Treatments for MG include acetylcholinesterase inhibitors (pyridostigmine), chronic immunosuppressive therapy, rapid and transient immunomodulatory therapies (e.g., plasma exchange and intravenous immunoglobulin - IVIG), and thymectomy. The goal of treatment is to minimize the patient's symptoms while minimizing drug-related side effects; however, there is no effective cure. Initial symptomatic treatment for patients with MG is based on acetylcholinesterase inhibitors (e.g., pyridostigmine). The cholinergic adverse effects of pyridostigmine can be dose-limiting in many patients and include abdominal cramps and diarrhea. Most patients with generalized MG require additional treatment with glucocorticoids and / or other immunosuppressive drugs, although this is second-line therapy. Therapeutic plasma exchange (plasmapheresis) and IVIG have rapid effects but are short-lived. International patent application WO2012141026 teaches AChR-α extracellular domain-Fc fusion proteins for the treatment of MG. New methods for treating MG, particularly those with long-lasting effects, are highly desirable. In particular, there is a need for therapies that target the autoantibodies that cause MG, and in addition, there is a great need for drugs that can directly target the autoreactive B cells / plasma cells that are the source of these autoantibodies and potentially cure these conditions. Summary of the Invention

[0006] The present invention provides a composition comprising a fragment of a first human acetylcholine receptor subunit and a fragment of a second human acetylcholine receptor subunit and an effector moiety that is not an unmodified Fc domain.

[0007] According to a first aspect, a composition is provided comprising: a fragment of a first human acetylcholine receptor subunit, or an analog or derivative thereof; a fragment of a second human acetylcholine receptor subunit, or an analog or derivative thereof; and an effector moiety, wherein the first and second subunits are different subunits and wherein the effector moiety is not an Fc domain.

[0008] According to a first aspect, a composition is provided comprising: a fragment of a first human acetylcholine receptor subunit, or an analog or derivative thereof; a fragment of a second human acetylcholine receptor subunit, or an analog or derivative thereof; and an effector moiety, wherein the first and second subunits are different subunits and wherein the effector moiety is not an unmodified Fc domain.

[0009] According to some embodiments, the fragment is a fragment of the extracellular domain of the acetylcholine receptor subunit.

[0010] According to some embodiments, the first and second acetylcholine receptor subunits are selected from acetylcholine receptor subunit alpha (ACHRA), acetylcholine receptor subunit beta (ACHRB), acetylcholine receptor subunit gamma (ACHRG), acetylcholine receptor subunit delta (ACHRD) and acetylcholine receptor subunit epsilon (ACHRE).

[0011] According to some embodiments, the effector moiety is capable of inducing death in a cell that binds either of the fragments.

[0012] According to some embodiments, the effector portion is selected from an Fc domain comprising at least one mutation that increases ADCC, amatoxin / amanitin, anthracycline, anthramycin-based dimer, calicheamicin, camptothecin or an analog thereof, duocarmycin, triptolide, and a tubulin inhibitor.

[0013] According to some embodiments, the effector moiety is selected from the group consisting of α-amanitin, PNU-159682, tesirine, deruxtecan (Dxd), mertansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0014] According to some embodiments, the effector portion is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141, wherein the Fc domain comprises a plurality of mutations within SEQ ID NO: 12 or SEQ ID NO: 141 selected from the group consisting of L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, and G16A / A110L / I112E.

[0015] According to some embodiments, the composition comprises a protein complex comprising: a. a first polypeptide chain comprising said fragment of a first human acetylcholine receptor subunit or an analog or derivative thereof and a first dimerization domain; and b. a second polypeptide chain comprising the fragment of a second human acetylcholine receptor subunit or an analog or derivative thereof and a second dimerization domain; wherein the first and second dimerization domains are configured to dimerize with each other.

[0016] According to some embodiments, the dimerization comprises forming a covalent bond between the first dimerization domain and the second dimerization domain.

[0017] According to some embodiments, the protein complex comprises an immunoglobulin scaffold.

[0018] According to some embodiments, a. the first dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin, and the second dimerization domain comprises a second hinge domain of a heavy chain, and the first and second dimerization domains dimerize via a disulfide bond; or b. The first and second dimerization domains each comprise a domain selected from the group consisting of the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and dimerize through a disulfide bond, and wherein the first and second dimerization domains do not comprise the CH1 domain and the CL domain at the same time.

[0019] According to some embodiments, the fragment of the first, second or both polypeptide chains and the dimerization domain are separated by a linker.

[0020] According to some embodiments, the first polypeptide chain, the second polypeptide chain, or both further comprise the effector moiety.

[0021] According to some embodiments, the effector moiety is linked to the first polypeptide chain, the second polypeptide chain, or both via a covalent bond.

[0022] According to some embodiments, the first polypeptide chain comprises a first CH3 domain of a heavy chain of an immunoglobulin, a first CH2 domain of a heavy chain of an immunoglobulin, or both and the second polypeptide chain comprises a second CH3 domain of a heavy chain of an immunoglobulin, a second CH2 domain of a heavy chain of an immunoglobulin, or both, and further comprises an effector portion that is not an Fc domain.

[0023] According to some embodiments, the first CH3 domain, the first CH2 domain, or both comprise at least a first mutation, and the second CH3 domain, the second CH2 domain, or both comprise at least a second mutation, and wherein the mutations allow heterodimerization of the first and second polypeptide chains and inhibit homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

[0024] According to some embodiments, the first mutation is selected from the mutations provided in Table 1, and the second mutation is provided in Table 1 and is a corresponding mutation of the first mutation.

[0025] According to some embodiments, the Fc region of the first, second or both polypeptide chains is separated from the fragment or the dimerization domain by a linker.

[0026] According to some embodiments, the Fc is derived from IgG2 or IgG4 or comprises at least one mutation that reduces effector function.

[0027] According to some embodiments, the dimerization domain of the first, second, or both polypeptide chains is at the C-terminus of the fragment or at the N-terminus of the fragment.

[0028] According to some embodiments, the composition is free of antibody variable domains.

[0029] According to some embodiments, the composition further includes a third polypeptide, which includes a fragment of a third human acetylcholine receptor subunit or an analog or derivative thereof and a third dimerization domain, wherein the first polypeptide further includes a fourth dimerization domain, and the third and fourth dimerization domains are capable of dimerizing with each other.

[0030] According to some embodiments, a. the third dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin, and the fourth dimerization domain comprises a second hinge domain of a heavy chain, and the first and second dimerization domains dimerize via a disulfide bond; or b. The third and fourth dimerization domains each comprise a domain selected from the group consisting of the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and dimerize through a disulfide bond, and wherein the first and third polypeptides do not comprise the CH1 domain and the CL domain at the same time.

[0031] According to some embodiments, the composition further includes a fourth polypeptide, which includes a fragment of a fourth human acetylcholine receptor subunit or an analog or derivative thereof and a fifth dimerization domain, wherein the second polypeptide further includes a sixth dimerization domain, and the fifth and sixth dimerization domains are capable of dimerizing with each other.

[0032] According to some embodiments, a. the fifth dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin, and the sixth dimerization domain comprises a second hinge domain of a heavy chain, and the first and second dimerization domains dimerize via a disulfide bond; or b. The fifth and sixth dimerization domains each comprise a domain selected from the group consisting of the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and dimerize through a disulfide bond, and wherein the first and third polypeptides do not comprise the CH1 domain and the CL domain at the same time.

[0033] According to some embodiments, the composition comprises a single polypeptide chain comprising: the fragment of a first human acetylcholine receptor subunit, or an analog or derivative thereof; and the fragment of a second human acetylcholine receptor subunit, or an analog or derivative thereof.

[0034] According to some embodiments, the single polypeptide chain further comprises: a fragment of a third human acetylcholine receptor subunit, or an analog or derivative thereof; and optionally a fragment of a fourth human acetylcholine receptor subunit, or an analog or derivative thereof.

[0035] According to some embodiments, the fragments are separated by an amino acid linker, optionally wherein the linker is a flexible GS linker, or wherein the linker is a rigid linker.

[0036] According to some embodiments, the polypeptide chain further comprises an Fc region of a human antibody heavy chain, and the second polypeptide chain comprises a third human acetylcholine receptor subunit or an analog or derivative thereof and an Fc region of a human antibody heavy chain, optionally wherein the second polypeptide chain further comprises a fourth human acetylcholine receptor subunit.

[0037] According to some embodiments, the composition comprises a second polypeptide chain comprising a third human acetylcholine receptor subunit or an analog or derivative thereof, optionally wherein the second polypeptide chain further comprises a fourth human acetylcholine receptor subunit.

[0038] According to some embodiments, the effector moiety is linked to the fragment via a linker.

[0039] According to some embodiments, the complex comprises at least one amino acid sequence selected from SEQ ID NOs: 64 to 69 or a derivative thereof having at least 80% identity thereto.

[0040] According to some embodiments, at least one of the fragments comprises a mutation that increases the stability or solubility of the fragment.

[0041] According to some embodiments, the mutation comprises replacing the cys loop within the acetylcholine receptor subunit with CDVSGVDTESGATNC (SEQ ID NO: 44).

[0042] According to some embodiments, the acetylcholine receptor subunit is selected from: an α subunit comprising the amino acid sequence provided in SEQ ID NO: 131, a β subunit comprising the amino acid sequence provided in SEQ ID NO: 132, a γ subunit comprising the amino acid sequence provided in SEQ ID NO: 133, a δ subunit comprising the amino acid sequence provided in SEQ ID NO: 134, and an ε subunit comprising the amino acid sequence provided in SEQ ID NO: 135.

[0043] According to some embodiments, the analog or derivative comprises at least 85% identity to said human protein.

[0044] According to some embodiments, the fragment comprises at least 20 consecutive amino acids from the protein.

[0045] According to some embodiments, the fragment comprises at least one B cell receptor (BCR)-specific epitope target of the autoantibody.

[0046] According to some embodiments, the fragment comprises at least one mutation that reduces aggregation of the fragment.

[0047] According to some embodiments, the fragment is selected from: a. A fragment of ACHRA and comprising a mutation selected from the group consisting of: deletion of N141; F100G; W149R; V155A; Y93F; Y93H; Y93R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 1 or the AChRa with increased solubility comprising SEQ ID NO: 131; b. A fragment of ACHRG and comprising a mutation selected from the group consisting of: M84S; Y105E; Y117E; Y117R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 6 or the solubility-enhanced AChRa comprising SEQ ID NO: 133; and c. A fragment of ACHRD and comprising a mutation selected from the group consisting of: C108A; C108I; Y119R; deletion of N141; L151E; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 8 or the AChRa with increased solubility comprising SEQ ID NO: 134.

[0048] According to some embodiments, the first polypeptide chain and the second polypeptide chain are selected from: SEQ ID NO:92 and SEQ ID NO:93; SEQ ID NO:95 and SEQ ID NO:96; SEQ ID NO:97 and SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, SEQ ID NO:92 and SEQ ID NO:102; SEQ ID NO:103 and SEQ ID NO:100; SEQ ID NO:105 and SEQ ID NO:130; SEQ ID NO:105 and SEQ ID NO:106; and SEQ ID NO:105 and SEQ ID NO:107.

[0049] According to some embodiments, the single polypeptide chain is selected from the group consisting of: SEQ ID NO: 94, SEQ ID NO: 104, and SEQ ID NOs: 108-129.

[0050] According to another aspect, a polypeptide is provided comprising: a fragment of a first human acetylcholine receptor subunit, the fragment comprising at least one mutation that reduces aggregation of the fragment; and an effector portion that is not an Fc domain, wherein the fragment is selected from the group consisting of: a. A fragment of ACHRA and comprising a mutation selected from the group consisting of: deletion of N141; F100G; W149R; V155A; Y93F; Y93H; Y93R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 1 or the AChRa with increased solubility comprising SEQ ID NO: 131; b. A fragment of ACHRG and comprising a mutation selected from the group consisting of: M84S; Y105E; Y117E; Y117R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 6 or the solubility-enhanced AChRa comprising SEQ ID NO: 133; and c. A fragment of ACHRD and comprising a mutation selected from the group consisting of: C108A; C108I; Y119R; deletion of N141; L151E; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 8 or the AChRa with increased solubility comprising SEQ ID NO: 134.

[0051] According to some embodiments, the polypeptide further comprises replacing a cys loop within an acetylcholine receptor subunit with CDVSGVDTESGATNC (SEQ ID NO:44), and wherein the subunit is ACHRA and the cys loop consists of CEIIVTHFPFDEQNC (SEQ ID NO:39), the subunit is ACHRG and the cys loop consists of CSISVTYFPFDWQNC (SEQ ID NO:41), or the subunit is ACHRD and the cys loop consists of CPISVTYFPFDWQNC (SEQ ID NO:42).

[0052] According to some embodiments, the polypeptide further comprises a second fragment of a second acetylcholine receptor subunit, the second fragment being connected to the first fragment via an amino acid linker; and optionally further comprises a fragment from a third, fourth or fifth acetylcholine receptor subunit.

[0053] According to some embodiments, the polypeptide further comprises an Fc region of a human antibody heavy chain, optionally wherein the Fc region is separated from the fragment by an amino acid linker.

[0054] According to some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs: 72-91.

[0055] According to some embodiments, the effector moiety is selected from an Fc domain comprising at least one mutation that increases ADCC, amatoxin / amanitin, anthracyclines, anthramycin-based dimers, calicheamicin, camptothecin or an analog thereof, duocarmycin, triptolide, and a tubulin inhibitor.

[0056] According to some embodiments, the effector moiety is selected from the group consisting of α-amanitin, PNU-159682, ticillin, Dxd, maytansine, MMAE, MMAF, and combinations thereof.

[0057] According to some embodiments, the effector portion is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141, wherein the Fc domain comprises a plurality of mutations within SEQ ID NO: 12 or SEQ ID NO: 141 selected from the group consisting of L15V / F23L / R72P / Y80L / P176L and S19D / A110L / I112E, G16A / A110L / I112E.

[0058] According to another aspect, a pharmaceutical composition is provided, which comprises a composition of the present invention or a polypeptide of the present invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0059] According to some embodiments, the pharmaceutical composition is formulated for systemic administration to a subject.

[0060] According to another aspect, a method of treating myasthenia gravis in a subject in need thereof is provided, the method comprising administering to the subject a composition of the present invention, a polypeptide of the present invention, or a pharmaceutical composition of the present invention, thereby treating myasthenia gravis.

[0061] According to some embodiments, the method further comprises reducing the level of circulating antibodies to at least the first human acetylcholine receptor subunit in the subject prior to said administering.

[0062] According to some embodiments, the method further comprises reducing the level of circulating antibodies in the subject against a human acetylcholine receptor subunit within a protein complex comprising the first human acetylcholine receptor subunit or the second human acetylcholine receptor subunit.

[0063] According to some embodiments, the treatment comprises reducing the concentration of circulating autoantibodies to the human acetylcholine receptor subunits.

[0064] According to some embodiments, the treatment comprises killing B cells that produce the autoantibody.

[0065] According to some embodiments, the B cells are autoreactive B cells that produce autoantibodies against a fragment of the composition or polypeptide.

[0066] According to another aspect, a nucleic acid system is provided, comprising nucleic acid molecules, wherein a first nucleic acid molecule encodes the first polypeptide chain of the composition of the present invention and a second nucleic acid molecule encodes the second polypeptide chain of the composition of the present invention, or the nucleic acid molecules encode a single polypeptide chain of the composition of the present invention or a polypeptide of the present invention.

[0067] According to some embodiments, the nucleic acid system further comprises: a third nucleic acid molecule encoding the third polypeptide chain of the composition of the present invention; and a fourth nucleic acid molecule encoding the fourth polypeptide chain of any one of the compositions of the present invention.

[0068] According to another aspect, a method of producing a composition of the invention or a polypeptide of the invention is provided, the method comprising expressing a nucleic acid system of the invention in a cell, wherein the nucleic acid system is configured to produce the encoded polypeptide in the cell, thereby producing a composition of the invention or a polypeptide of the invention.

[0069] According to another aspect, there is provided a method of producing a protein, the method comprising: obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit, or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different subunits, linking the first fragment to the second fragment to produce a single polypeptide chain, and linking the single polypeptide chain to an effector moiety that is not an Fc domain; or Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single polypeptide chain and linking the single polypeptide chain to an effector moiety that is not an Fc domain, wherein the single polypeptide chain is produced by: i. obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different subunits; and ii. joining the first fragment to the second fragment to produce a single polypeptide chain; This produces protein.

[0070] According to another aspect, there is provided a method of producing a protein complex, the method comprising: obtaining a first fragment of an extracellular domain of a first human acetylcholine receptor subunit, or an analog or derivative thereof, and a second fragment of an extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different proteins; linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain, wherein the first and second dimerization domains are capable of dimerizing with each other; and contacting the first polypeptide and the second polypeptide under conditions sufficient to induce dimerization; and linking the first polypeptide chain, the second polypeptide chain, or both to an effector moiety that is not an Fc domain; or Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding at least two polypeptide chains, and linking at least one of the at least two polypeptide chains to an effector moiety that is not an Fc domain, wherein the two polypeptide chains are produced by: i. obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different proteins; and ii. linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain, wherein the first and second dimerization domains are capable of dimerizing with each other; This creates a protein complex.

[0071] According to some embodiments, the protein is a single polypeptide chain of the composition of the present invention.

[0072] According to some embodiments, the protein complex is a protein complex of the composition of the present invention.

[0073] According to some embodiments, the method further comprises a. linking a third dimerization domain to the first dimerization domain or first fragment within the first polypeptide chain; obtaining a third fragment of the extracellular domain of a third human acetylcholine receptor subunit, or an analog or derivative thereof, and linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain, wherein the third dimerization domain and the fourth dimerization domain are capable of dimerizing with each other; and contacting the first, second, and third polypeptides under conditions sufficient to induce dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a third polypeptide chain, wherein the third polypeptide chain is produced by: i. obtaining a third fragment of the extracellular domain of a third human acetylcholine receptor subunit or an analog or derivative thereof; and ii. linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain; wherein the first polypeptide chain further comprises a third dimerization domain, and wherein the third dimerization domain and the fourth dimerization domain are capable of dimerizing with each other.

[0074] According to some embodiments, the method further comprises a. linking a sixth dimerization domain to the second dimerization domain or second fragment within the second polypeptide chain; obtaining a fourth fragment of the extracellular domain of a fourth human acetylcholine receptor subunit, or an analog or derivative thereof, and linking the fourth fragment to a fifth dimerization domain to produce a fourth polypeptide chain, wherein the fifth dimerization domain and the sixth dimerization domain are capable of dimerizing with each other; and contacting the first, second, third, and fourth polypeptides under conditions sufficient to induce dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a fourth polypeptide chain, wherein the fourth polypeptide chain is produced by: i. obtaining a fourth fragment of the extracellular domain of a fourth human acetylcholine receptor subunit or an analog or derivative thereof; and ii. linking the fourth fragment to a fifth dimerization domain to produce a fourth polypeptide chain; wherein the second polypeptide chain further comprises a sixth dimerization domain, and wherein the fifth dimerization domain and the sixth dimerization domain are capable of dimerizing with each other.

[0075] According to another aspect, there is provided a method of producing a polypeptide, the method comprising: a. obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit or an analog or derivative thereof; b. generating at least one mutation in the first fragment that reduces aggregation of the first fragment to produce a mutated first fragment; and c. linking the mutated first fragment to an effector portion that is not an Fc domain; Thus, a polypeptide is produced.

[0076] According to some embodiments, the analog or derivative comprises at least 85% identity to said human protein.

[0077] According to some embodiments, the effector portion is selected from an Fc domain comprising at least one mutation that increases ADCC, an amatoxin / amanitin, an anthracycline, an anthracycline-based dimer, an auristatin, a calicheamicin, a camptothecin or an analog thereof, a duocarmycin, a triptolide, and a tubulin inhibitor.

[0078] According to some embodiments, the effector moiety is selected from the group consisting of amatoxin / amanitin, anthracycline, anthramycin-based dimer, auristatin, calicheamicin, camptothecin or an analog thereof, duocarmycin, triptolide, and a tubulin inhibitor.

[0079] According to some embodiments, the effector moiety is selected from the group consisting of α-amanitin, PNU-159682, ticillin, delutecan (Dxd), maytansine, MMAE, MMAF, and combinations thereof.

[0080] According to some embodiments, the effector portion is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141, wherein the Fc domain comprises a plurality of mutations within SEQ ID NO: 12 or SEQ ID NO: 141 selected from the group consisting of L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, and G16A / A110L / I112E.

[0081] According to another aspect, there is provided a protein complex or protein produced by the method of the present invention.

[0082] According to another aspect, a method for determining the suitability of a subject in need thereof to be treated by the methods of the present invention is provided, the method comprising receiving a sample from the subject, contacting the sample with a composition of the present invention or a polypeptide of the present invention, and determining binding of autoantibodies to acetylcholine receptor subunits in the sample to the composition or the polypeptide, wherein binding of autoantibodies to the composition indicates that the subject is suitable for treatment by the methods of the present invention, thereby determining the suitability of the subject to be treated.

[0083] According to some embodiments, binding of at least 20% of the autoantibodies to AChR in the sample to the composition or polypeptide indicates that the subject is suitable for treatment by the methods of the present invention.

[0084] Other embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figures 1A-1G : (1A) Bar graph of the results of % depletion measured in an anti-AChR serological ELISA assay using α1ECD-depleted and non-depleted serum. The figure shows the results of 335 randomly selected MG patients, where the y-axis shows % depletion (which is defined as [100% - {anti-AChR concentration (previously depleted by α-ECD) / anti-AChR concentration (untreated)}) and the x-axis represents the different patient samples. (1B-1D) Line graphs showing dose-dependent depletion in (1B) an exemplary highly depleted sample, (1C) an exemplary moderately depleted sample, and (1D) an exemplary non-depleted sample. The y-axis represents the concentration of free anti-AChR antibodies in the sample in nM, while the x-axis shows the concentration of α-ECD used for the depleted sample in nM. (1E) Bar graph of total antibody concentration in the serum samples used in 1A. The gray line indicates the clinical cutoff of the disease. (1F) Diagram of the method for determining the percentage of AChR fragment-specific antibodies present in a sample using a depletion assay. (1G) Dot plot showing the correlation between % alpha binding and anti-AChR titer.

[0086] Figures 2A-2B: (2A) Contour plot showing the relative percentage of anti-AChR antibodies detected against each AChR subunit (ε / δ / γ / β / α) in each serum sample from 41 MG patients. (2B) Bar graph showing the percentage of blockade of various subunits or subunit combinations in serum samples from 80 randomly tested MG patients. In addition to the average % depletion, the graph also shows the percentage of patients with at least 50% or at least 75% depletion of each subunit or combination.

[0087] Figures 3A-3J Figures depicting five possible embodiments of the four-chain therapeutic agents of the invention: (3A) shows a general embodiment of a molecule for treating AChR-positive MG, (3B) shows an embodiment in which each of the four chains comprises a different protein fragment, (3C) shows an embodiment in which all four protein fragments are identical, (3D) shows an embodiment in which the two heavy chains are identical and the two light chains are identical, (3E) shows an embodiment in which the two heavy chains are different and the two light chains are identical, and (3F) shows an embodiment in which the two heavy chains comprise the same protein fragment and the two light chains comprise different protein fragments. (3G) Line graph of clinical scores in rats vaccinated with anti-AChR antibodies and then treated with various doses of the molecules of the invention. (3H) Kaplan Meier survival curves for rats vaccinated with anti-AChR antibodies and then treated with various doses of the molecules of the invention. (3I) Line graph of clinical scores in rats vaccinated with anti-AChR antibodies and then treated with the molecules of the invention. (3J) Kaplan Meier survival curves for rats vaccinated with anti-AChR antibodies and then treated with the molecules of the invention or NS-Fcs.

[0088] Figures 4A-4SFigures of possible embodiments of the dual-chain therapeutic agents of the present invention: (4A) shows a general embodiment of a molecule containing two heavy chains for treating AChR-positive MG, (4B) shows an embodiment in which at least one of the CH1, CH2, or CH3 domains is excluded, (4C) shows an embodiment in which the two protein fragments are identical, (4D) shows an embodiment in which the two protein fragments are different, (4E) shows a general embodiment in which two tandem fragments are included in each heavy chain, (4F) shows a tandem fragment configuration in which all subunits are identical, (4G) shows a tandem fragment configuration in which each heavy chain contains two identical different fragments, (4H) shows a tandem fragment configuration in which the two heavy chains contain different fragments that are not identical, (4I) shows a general embodiment in which two tandem fragments are included in one heavy chain and the other heavy chain contains only one fragment, (4J) shows a general embodiment in which two tandem fragments are included in one heavy chain and the other heavy chain contains no fragments, (4K) shows a tandem configuration in which all three subunits are identical, (4L) shows a tandem configuration in which two of the three subunits are identical, (4M) shows a tandem configuration in which all three subunits are different, (4N) shows a general embodiment in which three tandem fragments are included in each heavy chain, (4O) shows a tandem configuration in which all six subunits are identical, (4P) shows a tandem configuration in which all three subunits on a chain are different but both chains are identical, (4Q) shows a tandem configuration in which all three subunits on a chain are different and both chains are different, (4R) shows a general embodiment in which three tandem fragments are included in one heavy chain and the other heavy chain includes 2, 1 or 0 fragments, and (4S) shows a general embodiment in which a molecule contains one heavy chain and one light chain.

[0089] Figures 5A-5D : Figures of four possible embodiments of the three-chain therapeutic agents of the present invention: (5A) shows a general embodiment of a molecule containing two heavy chains and one light chain, (5B) shows an embodiment in which the three protein fragments are identical, (5C) shows an embodiment in which each protein fragment is different, and (5D) shows an embodiment in which two of the protein fragments are identical and the third protein fragment is different.

[0090] Figure 6 : A diagram similar to that shown in FIG3 of an embodiment of a four-chain therapeutic agent of the present invention for treating AChR-positive MG, but wherein each of the four chains comprises a different protein fragment and a different immunoglobulin scaffold that promotes the formation of the four-chain molecule.

[0091] Figures 7A-7B : (7A-7B) Figures of general embodiments of four-chain therapeutic agents of the present invention: (7A) shows a general embodiment of four chains, wherein two chains contain two dimerization domains and two chains contain a single dimerization domain, and (7B) shows an embodiment containing optional linkers separating various domains and fragments.

[0092] Figures 8A-8E : Figures of single-chain therapeutic agents of the present invention: (8A) shows an embodiment of a single-chain molecule comprising fragments from two different AChR subunits, (8B) shows an embodiment of a single-chain molecule comprising fragments from three different AChR subunits, (8C) shows an embodiment of a single-chain molecule comprising fragments from four different AChR subunits, (8D) shows an embodiment of a single-chain molecule comprising fragments from two different AChR subunits and a heavy chain constant region, and (8E) shows a single-chain molecule of 8A-8D containing an amino acid (AA) linker separating the various domains.

[0093] Figure 9 : Bar graph of the average percent depletion of AChR-specific autoantibodies from MG sera exposed to various molecules of the invention. Unrelated extracellular domain constructs (CRD-239 and CRD-241) were used as negative controls. Each molecule was exposed to at least 17 different patient serum samples.

[0094] Figures 10A-10D : Dot plots comparing the depletion rates produced by pairs of molecules: (10A) CRD-101 and CRD-269, (10B) CRD-101 and CRD-642, (10C) CRD-104 and CRD-391, and (10D) CRD-103 and CRD-382.

[0095] Figure 11 Bar graph indicating the increase in fluorescence upon ECD tetramer binding to hybridoma cells. The MFI fold change relative to background was calculated by dividing the ECD tetramer binding MFI for any hybridoma by the negative control background MFI. Hybridoma 204-4 served as a negative control, as did tetramers CRD-233 and CRD-242.

[0096] Figure 12 : Histogram of ECD tetramer binding to a negative control B cell hybridoma line.

[0097] Figures 13A-13C : Bar graph of binding of α-γ combination molecule tetramers to various B cell hybridoma cell lines: (13A) binding of CRD-506 to anti-α hybridoma and anti-γ hybridoma, (13B) binding of CRD-509 and CRD-600 to anti-α hybridoma, and (13C) binding of CRD-509 and CRD-600 to anti-γ hybridoma and to negative control hybridoma.

[0098] Figure 14 : Bar graph of binding of various α-subunit-containing molecules to anti-α hybridomas.

[0099] Figures 15A-15C: Bar graphs showing the binding of (15A) α-δ molecules, (15B) γ-δ molecules, and (15C) IgG4 Fc-containing α-γ molecules to various hybridomas.

[0100] Figures 16A-16S : (16A-16F) Diagrams of possible general embodiments of four-chain therapeutics of the present invention: (16A) shows a general embodiment of four chains, wherein two chains comprise two dimerization domains and one effector domain, two chains comprise a single dimerization domain and no effector domain, (16B) shows an embodiment in which a chain having only a single dimerization domain comprises an effector domain, (16C) shows an embodiment having only a single effector domain, (16D) shows an embodiment in which an optional linker separates the various domains and fragments, (16E) shows 16A with a chemical bond or linker between the EF and a cytotoxic molecule (CM), such as a drug or radiolabeled substance, and (16F) shows an embodiment that includes a cytotoxic molecule but lacks an effector domain. (16G) Diagrams of dimeric heavy chains each comprising two AChR regions and a cytotoxic moiety linked to each region. (16H) Diagram of a dimeric heavy chain / light chain molecule in which the cytotoxic moiety is linked to each region. (16I) Diagram of an embodiment of a conjugate of the invention comprising a cytotoxic moiety. (16J-16S) Diagrams of possible general embodiments of single-chain therapeutics of the invention: (16J) molecule of 8A, (16K) molecule of 8B, (16L) molecule of 8C, (16M) molecule of 8D, and (16N) molecule of 8E with an effector moiety; (16O) molecule of 8A, (16P) molecule of 8B, (16Q) molecule of 8C, (16R) molecule of 8D, and (16S) molecule of 8E with a cytotoxic moiety.

[0101] Figures 17A-17B : Line graph of anti-AChR titers in EAMG rats treated with (17A) vehicle alone or with (17B) a tetrabody containing two α subunits and two β subunits and one Fc domain.

[0102] Figures 18A-18E: (18A-18C) Histograms of dose-dependent binding of B2A2 conjugated to (18A) α-amanitin, (18B) Dxd, and (18C) ticillin to anti-AChRα hybridoma cell lines. Unconjugated B2A2 is shown as a control. (18D) Histograms of dose-dependent binding of heavy chain heterodimers containing α and γ subunits to cells expressing TIB-185 anti-AChRα and control cells expressing an irrelevant antibody. (18E) Histograms of dose-dependent binding of A2G2 heavy chain dimers, each containing tandem α and γ subunits, to cells expressing TIB-185 anti-AChRα and control cells expressing an irrelevant antibody.

[0103] Figures 19A-19E : (19A-19B) Individual hybridomas expressing BCRs against (19A) AChRα (a-18-C5-F6, TIB175, and a-192) and (19B) AChRγ (g-63-E6-A10, g-50-H1-E2, and g-66) were incubated in the presence of 32 nM, 11 nM, or 4 nM molecules 1-4. CRD-509 and CRD-269 were used as controls. Binding was detected using PE-conjugated anti-human pAbs, and each hybridoma was also stained with PE-anti-RAT BCR. The Y axis represents the MFI value for each sample divided by the PE anti-RAT BCR MFI value of the test hybridoma. The displayed value for each molecule represents the calculated average of all three test α hybridomas or γ hybridomas. (19C-19D) Hybridoma cell death rates of (19C) anti-AChRγ hybridomas g-50-H1-E2 (left) and g-66 (right) and (19D) anti-AChRα hybridoma a-18-C5-F6 when cultured in the presence of PBMCs and molecules 1, 2, 4, and 5. Addition of CRD-509 served as a control (percent increase is relative to the culture of hybridoma cells and PBMCs). (19E) Viable cell counts of anti-AChRα or γ hybridomas in the presence of CRD-509 or molecule 3 and increasing concentrations of complement.

[0104] Figures 20A-20C : Bar graph showing that (20A) B2A2-PNU glyco-site conjugated IgG-like molecules, (20B) B2A2-ticillin cysteine conjugated IgG-like molecules and (20C) A2G2-PNU glyco-site conjugated tandem AChR fragment molecules kill TIB-175 AChR-α specific hybridoma cells compared with control hybridoma cells.

[0105] Figures 21A-21C: (21A) Bar graph of binding of CRD-600 and CRD-600-ticillin to the gamma-specific hybridoma g-66. A delta-specific hybridoma (δ) and an irrelevant hybridoma (204-4) were used as negative controls. (21B-21C) Line graphs of the percent increase in cell killing compared to (21B) unconjugated molecules of CRD-600-ticillin and (21C) irrelevant Ig-like molecules conjugated to ticillin. Binding and killing by CRD-600-ticillin were specific for the gamma hybridoma and ticillin background killing, as shown by the absence of irrelevant Ig-like molecules.

[0106] Figures 22A-22M : Bar graphs showing binding of molecules of the invention to various hybridomas. Shown are (22A) binding of CRD-213-ticillin to α- and β-specific hybridomas, (22B) binding of CRD-506-ticillin to α-specific hybridomas, (22C) binding of CRD-506-ticillin to γ-specific hybridomas, (22D) binding of CRD-506-α-amanitin to α- and γ-specific hybridomas, (22E) binding of CRD-506-PNU to α-specific hybridomas, (22F) binding of CRD-506-PNU to γ-specific hybridomas, (22G) binding of CRD-509-ticillin, CRD-509-α-amanitin, and CRD-509-PNU to γ-specific hybridomas, (22H) Binding of CRD-509-ticillin, CRD-509-α-amanitin, and CRD-509-PNU to α-specific hybridomas, (22I) Binding of CRD-509-MMAE and CRD-509-MMAF to α-specific hybridomas, (22J) Binding of CRD-509-MMAE and CRD-509-MMAF to γ-specific hybridomas, (22K) Binding of CRD-586-PNU to γ- and δ-specific hybridomas, (22L) Binding of CRD-586-ticillin to γ- and δ-specific hybridomas, and (22M) Binding of CRD-586-α-amanitin to γ- and δ-specific hybridomas.

[0107] Figure 23 : Bar graph of cellular internalization of CRD-509.

[0108] Figures 24A-24L: (24A-24B) Bar graphs of cell death induced by CRD-213 conjugated to (24A) tesicillin and (24B) DM-1. (24C) Line graphs of the percentage of cytotoxicity induced by anti-γ hybridoma cells (anti-AchR hyb) and an irrelevant hybridoma (204-4) when co-cultured with increasing concentrations of unconjugated CRD-509, CRD-509-MMAE, and CRD-509-MMAF. (24D-24H) Bar graphs of the percentage of viable cells from co-cultures of two hybridomas treated with (24D) CRD-213-tesicillin, (24E) CRD-506-tesicillin, (24F) CRD-509-α-amanitin, (24G) CRD-509-PNU, and (24H) CRD-509-tesicillin. (24I-24J) Line graphs of cell death induced by (24I) CRD-506-ticillin and (24J) CRD-213-ticillin. (24K) Bar graph of cell death induced by CRD-586 or an irrelevant fusion molecule conjugated to ticillin, and (24L) Bar graph of cell death induced by CRD-586 conjugated to α-amanitin.

[0109] Figure 25 : Line graph of relative EAMG compared to time point zero in rats treated with CRD-586-ticillin or unconjugated CRD-586.

[0110] Figures 26A-26B : (26A) Bar graph of anti-AChRα / γ autoreactive antibody titers in naive C57B16 mice. (26B) Bar graph of autoreactive antibody titers in mice 14 days after immunization with AChR-α ECD and AChR-γ ECD. Only CRD-509-ticillin reduced the titers, indicating that autoreactive B cells present before immunization were killed. DETAILED DESCRIPTION

[0111] The present invention provides, in some embodiments, a composition comprising: a fragment of a first human receptor target of an autoantibody to myasthenia gravis, or an analog or derivative thereof; and a fragment of a second human protein receptor of an autoantibody to myasthenia gravis, or an analog or derivative thereof. A protein complex is also provided, comprising at least two polypeptide chains, wherein the first chain comprises a fragment of a first human protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, and a first dimerization domain, and the second chain comprises a fragment of a second human protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, and a second dimerization domain capable of dimerizing with the first dimerization domain. The present invention also relates to pharmaceutical compositions comprising compositions and / or protein complexes, nucleic acids encoding polypeptides of compositions and / or protein complexes, and methods of treating and determining suitability of treatment using compositions and / or protein complexes; and methods of producing compositions and / or protein complexes.

[0112] In a first aspect, a protein is provided that includes a first protein target of a myasthenia gravis autoantibody or an analog or derivative thereof.

[0113] In another aspect, a composition is provided that includes: a fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; and a fragment of a second protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof.

[0114] In another aspect, a protein is provided that includes: a fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; and a fragment of a second protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof.

[0115] In another aspect, a protein complex is provided that includes at least two polypeptide chains, wherein a first polypeptide chain includes a fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a first dimerization domain, and a second polypeptide chain includes a fragment of a second protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a second dimerization domain.

[0116] In another aspect, a fusion protein or protein conjugate is provided that includes: a fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; and an effector moiety.

[0117] In some embodiments, the protein is used to treat myasthenia gravis. In some embodiments, the protein is a fusion protein. In some embodiments, the protein is a fusion protein of the present invention. In some embodiments, the polypeptide is used to treat myasthenia gravis. In some embodiments, the polypeptide chain is used to treat myasthenia gravis. In some embodiments, the protein complex is used to treat myasthenia gravis. In some embodiments, the composition is used to treat myasthenia gravis. In some embodiments, the protein is a therapeutic agent. In some embodiments, the polypeptide is a therapeutic agent. In some embodiments, the polypeptide chain is a therapeutic agent. In some embodiments, the protein complex is a therapeutic agent.

[0118] In some embodiments, a composition comprises a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a first dimerization domain, and a second polypeptide chain comprises a fragment of a second protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a second dimerization domain. In some embodiments, the composition comprises a protein complex of the present invention. In some embodiments, the composition comprises a protein of the present invention. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is a fusion protein.

[0119] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to polymers of amino acid residues. In another embodiment, the terms "peptide," "polypeptide," and "protein" as used herein encompass natural peptides, peptidomimetics (generally including non-peptide bonds or other synthetic modifications), and peptide analogs peptidomimetics and hemi-peptidomimetics, or any combination thereof. In another embodiment, the peptides, polypeptides, and proteins described have modifications that make them more stable or more able to penetrate cells in vivo. In one embodiment, the terms "peptide," "polypeptide," and "protein" are applicable to naturally occurring amino acid polymers. In another embodiment, the terms "peptide," "polypeptide," and "protein" are applicable to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0120] 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 does not contain an antibody variable domain. In some embodiments, the protein complex does not contain an antibody variable domain. In some embodiments, the composition does not contain a variable domain. In some embodiments, the protein complex does not contain a variable domain. In some embodiments, the first chain does not contain a variable domain. In some embodiments, the second chain does not contain 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 the 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 the treatment of myasthenia gravis. In some embodiments, the protein complex is for use in the treatment of myasthenia gravis. In some embodiments, the protein complex is for use in the diagnosis of myasthenia gravis. In some embodiments, the protein complex is for use in determining appropriate treatment for myasthenia gravis. In some embodiments, the protein complex is used to characterize the serological response in myasthenia gravis. In some embodiments, the protein complex is used to determine AChR antibody titers in myasthenia gravis.

[0121] As used herein, the term "polypeptide chain" refers to a polymer of amino acids connected from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) by a peptide bond. In some embodiments, the polypeptide chain is a recombinant polypeptide. In some embodiments, the polypeptide chain includes 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. Every possibility represents a separate embodiment of the present invention. 4250, 4500, 4750, or 5000 amino acids. Each possibility represents a separate embodiment of the invention.

[0122] As used herein, the term "recombinant polypeptide" refers to a protein that is encoded by recombinant DNA and, therefore, is 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 a DNA molecule formed by laboratory methods. Typically, this recombinant DNA is in the form of a vector, plasmid, or virus used to express the recombinant protein in a cell. The production of recombinant proteins by cellular expression is well known in the art, and any recombinant protein expression method can be used to produce the polypeptides of the present invention. Cell-free expression systems for recombinant protein production can also be used.

[0123] As used herein, the term "expression" refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. Thus, expression of a nucleic acid molecule can refer to the transcription of a nucleic acid fragment (e.g., transcription to produce mRNA or other functional RNA) and / or the 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.

[0124] The expression of DNA sequences or RNA in cells is well known to those skilled in the art. Among various methods, this can be done by transfection, viral infection, or directly altering the genome of the cell. In some embodiments, the DNA sequence is in an expression vector such as a plasmid or viral vector. In some embodiments, a Kozak sequence is inserted upstream of the transcription initiation codon. In some embodiments, the Kozak sequence enhances the amount of protein expression.

[0125] 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 identical. In some embodiments, the polypeptide chains are different. In some embodiments, at least two of the polypeptide chains are identical. In some embodiments, at least two of the polypeptide chains are different.

[0126] Those skilled in the art will appreciate that the ECDs of the various subunits interact to form a complete receptor, and thus, even without a dimerization domain, a protein complex can be formed. In some embodiments, the protein complex comprises at least two proteins. In some embodiments, the protein complex comprises at least three proteins. In some embodiments, the protein complex comprises at least four proteins. protein

[0127] In some embodiments, the protein is a mammalian protein. In some embodiments, the mammal is human. In some embodiments, the protein is a transmembrane protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a subunit in a receptor. In some embodiments, the protein is a cell surface protein. In some embodiments, the cell surface protein is an integral membrane protein. In some embodiments, the cell surface protein is a plasma membrane embedded protein. In some embodiments, the cell surface protein is a membrane anchored protein. In some embodiments, the protein is a myasthenia gravis-associated protein. In some embodiments, the protein is a synthetic protein. In some embodiments, the protein is a naturally occurring protein. In some embodiments, the protein is a target of myasthenia gravis autoantibodies. In some embodiments, the protein is selected from AChRa, AChRb, AChRg, AChRd, and AChRe.

[0128] As used herein, the term "receptor" refers to a protein expressed on the surface of a cell that is capable of binding a ligand. In some embodiments, a receptor is a protein that is capable of transmitting a signal to the cytoplasm of a cell. In some embodiments, a receptor includes a ligand binding domain. In some embodiments, a receptor includes a transmembrane domain. In some embodiments, a receptor includes an intracellular domain.

[0129] In some embodiments, the fragment comprises the extracellular domain (ECD) of a protein. In some embodiments, the fragment comprises the entire extracellular domain or a variant thereof. In some embodiments, the fragment consists of the entire extracellular domain or a variant thereof. In some embodiments, the variant is a mutant. In some embodiments, the variant comprises a replacement of a portion of the extracellular domain. In some embodiments, the fragment comprises a fragment of the extracellular domain of a protein. In some embodiments, the fragment consists of the extracellular domain of a protein. In some embodiments, the fragment consists of a fragment of the extracellular domain of a protein. In some embodiments, the fragment comprises the transmembrane domain of a protein. In some embodiments, the fragment does not contain the transmembrane domain of a protein. In some embodiments, the fragment does not contain the intracellular domain of a protein. In some embodiments, the chain does not contain the transmembrane domain. In some embodiments, the chain does not contain the intracellular domain. In some embodiments, the fragment comprises a sequence from a homologous human protein. In some embodiments, the fragment comprises a sequence from a homologous non-human protein. In some embodiments, the fragment comprises a mutation in a human protein.

[0130] In some embodiments, a fragment comprises at least 5 amino acids of a protein. In some embodiments, a fragment comprises at least 10 amino acids of a protein. In some embodiments, a fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the amino acids of a protein are contiguous amino acids of the protein. In some embodiments, a fragment comprises less than 100% of the protein. In some embodiments, a fragment comprises less than 100% of the extracellular domain of a protein. In some embodiments, a 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 present invention. In some embodiments, the fragment comprises less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50% of the extracellular domain of the protein. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fragment comprises 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 present invention. 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.

[0131] In some embodiments, the variant comprises at least 70, 75, 80, 85, 90, 92, 95, 97, or 99% homology or identity. Each possibility represents a separate embodiment of the present invention. In some embodiments, the variant comprises at least 85% homology or identity. In some embodiments, the variant comprises at least 90% homology or identity. In some embodiments, the variant comprises at least 92% homology or identity. In some embodiments, the variant comprises at least 95% homology or identity. In some embodiments, the variant comprises at least 97% homology or identity. In some embodiments, the variant comprises at least 99% homology or identity. In some embodiments, the variant is a mutant.

[0132] 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 an amino acid linker. In some embodiments, the linker comprises at least one amino acid. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises increased solubility compared to the protein region excluded from the chain. In some embodiments, the protein region is replaced with a protein region that is not the protein. In some embodiments, the replacement region comprises increased solubility compared to the replaced protein region. In some embodiments, the replacement region comprises increased protein stability compared to the replaced protein region.

[0133] In some embodiments, the protein is the target of an antibody. 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 antibody is an autoantibody.

[0134] As used herein, the term "autoantibody" refers to an antibody produced by a subject's own immune system against at least one of the subject's own proteins. In some embodiments, the autoantibody is an autoreactive antibody. In some embodiments, the autoantibody targets self-antigens. Autoantigens are also called autoantigens. In some embodiments, the autoantibody is associated with myasthenia gravis. In some embodiments, the autoantibody characterizes myasthenia gravis. In some embodiments, the autoantibody is an autoantibody to myasthenia gravis. In some embodiments, the autoantibody is produced by autoreactive B cells. In some embodiments, the protein is an antigen of the antibody. In some embodiments, the fragment comprises an antigen of the antibody. In some embodiments, the fragment comprises at least one antigen of the antibody. In some embodiments, the fragment comprises at least two antigens of the antibody. In some embodiments, the fragment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigens of the antibody. Each possibility represents a separate embodiment of the present invention. In some embodiments, the antigen of the antibody is a self-antigen. In some embodiments, the antigen is an epitope. In some embodiments, the antigen comprises at least one epitope. In some embodiments, the epitope comprises at least 5 amino acids. In some embodiments, the epitope comprises 5-6 amino acids. In some embodiments, the epitope comprises 5-10 amino acids. In some embodiments, the epitope is a simple epitope. In some embodiments, a simple epitope is a linear epitope. In some embodiments, the epitope is a complex epitope. In some embodiments, a complex epitope is a 3D epitope. In some embodiments, a complex epitope is a discontinuous epitope. In some embodiments, a discontinuous epitope comprises at least two discontinuous segments of amino acids that combine to form the epitope. In some embodiments, a linker sequence is between the two segments of the epitope.

[0135] As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to that of a protein, but in which one or more residues have been conservatively substituted with functionally similar residues. In some embodiments, the analog exhibits functionality similar to that of the original protein. Examples of conservative substitutions include: a non-polar (hydrophobic) residue such as isoleucine, valine, leucine, or methionine replacing another residue; a polar (hydrophilic) residue replacing another residue, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; a basic residue such as lysine, arginine, or histidine replacing another residue; or an acidic residue such as aspartic acid or glutamic acid replacing another residue. Each possibility represents a separate embodiment of the present invention. In some embodiments, the substitution is outside the antigenic region of the protein. In some embodiments, the substitution is outside the epitope of the antibody. In some embodiments, the analog remains a target of the antibody. In some embodiments, the analog retains the binding of its own antibody. The analog may have a deletion or mutation that results in an amino acid sequence that is different from the typical amino acid sequence of the protein. Furthermore, an analog may also resemble a fragment of a protein, however, in this case the fragment must include at least 50 consecutive amino acids of the protein or at least one epitope of an antibody.In some embodiments, an analog is an analog of a typical sequence of a protein.

[0136] In some embodiments, an analog of a protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the present invention. In some embodiments, an analog of a protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the present invention. In some embodiments, an 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 present invention. In some embodiments, a substitution is a mutation of the canonical sequence.

[0137] As used herein, the term "derivative" refers to any polypeptide that is based on a protein and still includes and retains the binding of an antibody. A derivative is not merely a fragment of a protein, nor does it have amino acids replaced or removed (analogs). Rather, it may have additional modifications to the protein, such as post-translational modifications. In addition, a derivative can also be a derivative of a fragment of a protein. However, in this case, the fragment must include at least 50 consecutive amino acids of the protein or at least one epitope of the antibody. In some embodiments, a derivative is a derivative of the typical sequence of a protein.

[0138] In some embodiments, a derivative of a protein comprises an amino acid sequence that is 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% homologous to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the present invention. In some embodiments, a derivative of a protein comprises an amino acid sequence that is 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% homologous to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the present invention.

[0139] Typical amino acid sequences of known proteins are well known in the art. They can be found in various databases, including UniProt, NCBI, and the UCSC genome browser. Any sequence accepted as a typical sequence can be used. As a non-limiting example, human acetylcholine receptor subunit α is encoded by the CHRNA1 gene, whose typical nucleic acid sequence can be found in Entrez gene 1134, its typical protein-coding mRNA sequence can be found in NM_001039523 and NM_000079, and its typical amino acid sequence can be found in NP_000070 and NP_031415 and UniProt numbering P02708. In some embodiments, a typical sequence is a sequence identical to a sequence present in at least 50, 60, 70, 75, 80, 90, 95, 97, or 99% of a population. Each possibility represents a separate embodiment of the present invention. In some embodiments, a typical sequence is a sequence identical to the most common sequence present in a population. In some embodiments, a population is a disease population. In some embodiments, a population is a population suffering from an autoimmune disease.

[0140] In some embodiments, the protein is an acetylcholine receptor (AChR). In some embodiments, the protein is an acetylcholine receptor subunit. In some embodiments, the subunit is an alpha subunit. In some embodiments, the subunit is a beta subunit. In some embodiments, the subunit is a gamma subunit. In some embodiments, the subunit is a delta subunit. In some embodiments, the subunit is an epsilon subunit. In some embodiments, the subunit is selected from the group consisting of alpha, beta, gamma, delta, and epsilon subunits.

[0141] In some embodiments, the protein is the acetylcholine receptor alpha subunit (AChRa). In some embodiments, the AChRa is encoded by the gene CHRNA1. In some embodiments, the AChRa is human AChRa. In some embodiments, CHRNA1 is identified by Entrez gene #1134. In some embodiments, the AChRa is identified by UniProt ID P02708. In some embodiments, the AChRa is identified by UniProt ID P02708.1 or P02708.2. In some embodiments, the AChRa is identified by UniProt ID P02708.1. In some embodiments, the AChRa is identified by UniProt ID P02708.2. In some embodiments, the CHRNA1 comprises or consists of the nucleotide sequence provided in NM_001039523 or NM_000079. In some embodiments, the AChRa comprises or consists of the amino acid sequence provided in NP_000070 or NP_001034612. In some embodiments, a representative amino acid sequence of the extracellular domain of AChRa comprises or consists of: SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLP (SEQ ID NO: 1). In some embodiments, the typical amino acid sequence of the AChRa extracellular domain comprises or consists of the following: SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQGDMVDLPRPSCVTLGVPLFSHLQNEQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLP (SEQ ID NO: 2). In some embodiments, the extracellular domain does not contain a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide.In some embodiments, the AChRa signal peptide comprises or consists of: MEPWPLLLLFSLCSAGLVLG (SEQ ID NO: 3). In some embodiments, the AChRa signal peptide comprises or consists of: MFMCLEGGEKNLTVLVSSAVSAGLVLG (SEQ ID NO: 61).

[0142] In some embodiments, the protein is the acetylcholine receptor beta subunit (AChRb). In some embodiments, the AChRb is encoded by the gene CHRNB1. In some embodiments, the AChRb is human AChRb. In some embodiments, CHRNB1 is identified by Entrez gene #1140. In some embodiments, the AChRb is identified by UniProt ID P11230. In some embodiments, CHRNB1 comprises or consists of the nucleotide sequence provided in NM_000747. In some embodiments, the AChRb comprises or consists of the amino acid sequence provided in NP_000738. In some embodiments, the typical amino acid sequence of the extracellular domain of AChRb includes or consists of the following: SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYLDLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVSSDGSVRWQPPGIYRSSCSIQVTYFPFDWQNCTMVFSSYSYDSSEVSLQTGLGPDGQGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKP (SEQ ID NO: 4). In some embodiments, the extracellular domain does not contain a signal peptide. In some embodiments, the extracellular domain also includes a signal peptide. In some embodiments, the AChRb signal peptide includes or consists of the following: MTPGALLMLLGALGAPLAPGVRG (SEQ ID NO: 5).

[0143] In some embodiments, the protein is the acetylcholine receptor gamma subunit (AChRg). In some embodiments, the AChRg is encoded by the gene CHRNG. In some embodiments, the AChRg is human AChRg. In some embodiments, CHRNG is identified by Entrez gene #1146. In some embodiments, AChRg is identified by UniProt ID P07510. In some embodiments, CHRNG comprises or consists of the nucleotide sequence provided in NM_005199. In some embodiments, the AChRg comprises or consists of the amino acid sequence provided in NP_005190. In some embodiments, the typical amino acid sequence of the extracellular domain of AChRg includes or consists of the following: RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNVLVSPDGCIYWLPPAIFRSACSISVTYFPFDWQNCSLIFQSQTYSTNEIDLQLSQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKP (SEQ ID NO: 6). In some embodiments, the extracellular domain does not contain a signal peptide. In some embodiments, the extracellular domain also includes a signal peptide. In some embodiments, the AChRg signal peptide includes or consists of the following: MHGGQGPLLLLLLLAVCLGAQG (SEQ ID NO: 7).

[0144] In some embodiments, the protein is an acetylcholine receptor delta subunit (AChRd). In some embodiments, the AChRd is encoded by the gene CHRND. In some embodiments, the AChRd is human AChRd. In some embodiments, CHRND is identified by Entrez gene #1144. In some embodiments, AChRd is identified by UniProt ID Q07001. In some embodiments, CHRND comprises or consists of the nucleotide sequence provided in NM_000751, NM_001256657, NM_001311195, or NM_001311196. In some embodiments, the AChRd comprises or consists of the amino acid sequence provided in NP_000742, NP_001243586, NP_001298124, or NP_001298125. In some embodiments, the typical amino acid sequence of the extracellular domain of AChRd includes or consists of: LNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNVWIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVLVYHYGFVYWLPPAIFRSSCPISVTYFPFDWQNCSLKFSSLKYTAKEITLSLKQDAKENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSPSRQDITFYLIIRRKP (SEQ ID NO: 8). In some embodiments, the extracellular domain does not contain a signal peptide. In some embodiments, the extracellular domain also includes a signal peptide. In some embodiments, the AChRd signal peptide includes or consists of: MEGPVLTLGLLAALAVCGSWG (SEQ ID NO: 9).

[0145] In some embodiments, the protein is the acetylcholine receptor epsilon subunit (AChRe). In some embodiments, the AChRe is encoded by the gene CHRNE. In some embodiments, the AChRe is human AChRe. In some embodiments, CHRNE is identified by Entrez gene #1145. In some embodiments, the AChRe is identified by UniProt ID Q04844. In some embodiments, the CHRNE comprises or consists of the nucleotide sequence provided in NM_000080. In some embodiments, the AChRe comprises or consists of the amino acid sequence provided in NP_000071. In some embodiments, the representative amino acid sequence of the extracellular domain of AChRe comprises or consists of: KNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGIDWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLVYEGGSVTWLPPAIYRSVCAVEVTYFPFDWQNCSLIFRSQTYNAEEVEFTFAVDNDGKTINKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRKP (SEQ ID NO: 10). In some embodiments, the extracellular domain does not contain a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the AChRe signal peptide comprises or consists of: MARAPLGVLLLLGLLGRGVG (SEQ ID NO: 11).

[0146] In some embodiments, the signal peptide is a signal peptide of an antibody chain. In some embodiments, the signal peptide is a signal peptide of an antibody heavy chain. In some embodiments, the signal peptide is a signal peptide of an antibody light chain. In some embodiments, the signal peptide is a signal peptide of a kappa light chain. In some embodiments, the signal peptide is a signal peptide of a lambda light chain. In some embodiments, the heavy chain signal peptide comprises MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 70). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 70. In some embodiments, the heavy chain signal peptide comprises MEFGLSWLFLVAILKGVQC (SEQ ID NO: 14). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 14. In some embodiments, the light chain signal peptide comprises MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 71). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 71. In some embodiments, the heavy chain signal peptide is a mouse heavy chain signal peptide and comprises MGWSCIILFLVATATGVHS (SEQ ID NO: 15). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO:15.

[0147] In some embodiments, the first protein and the second protein are the same protein. In some embodiments, the first and second proteins are the same protein, 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.

[0148] In some embodiments, the protein or fragment includes a mutation that increases solubility. In some embodiments, the protein or fragment includes a mutation that increases stability of the protein or fragment. In some embodiments, the mutation is an insertion. In some embodiments, the protein is a surface protein and includes a mutation that increases solubility. In some embodiments, the fragment is the extracellular domain of a surface protein and includes an insertion that increases solubility. In some embodiments, the insertion replaces a region of the protein or fragment. In some embodiments, a loop of the protein is replaced with a loop that has higher solubility. In some embodiments, the loop of AChRa includes or consists of: CEIIVTHFPFDEQNC (SEQ ID NO:39). In some embodiments, the loop of AChRb includes or consists of: CSIQVTYFPFDWQNC (SEQ ID NO:40). In some embodiments, the loop of AChRg includes or consists of: CSISVTYFPFDWQNC (SEQ ID NO:41). In some embodiments, the loop of AChRd includes or consists of: CPISVTYFPFDWQNC (SEQ ID NO:42). In some embodiments, the loop of AChRe comprises or consists of: CAVEVTYFPFDWQNC (SEQ ID NO: 43). In some embodiments, the more soluble loop comprises or consists of: CDVSGVDTESGATNC (SEQ ID NO: 44). In some embodiments, the more soluble insert comprises or consists of: SEQ ID NO: 44. In some embodiments, the more soluble insert comprises or consists of: DVSGVDTESGAT (SEQ ID NO: 63). In some embodiments, the AChR is mutated to increase solubility and stability. In some embodiments, the mutation is selected from V8E, W149R, and V155A. In some embodiments, the mutation is a mutation of at least two of V8E, W149R, and V155A. In some embodiments, the mutation is a mutation of all three of V8E, W149R, and V155A. In some embodiments, the AChRa comprises mutations. In some embodiments, the non-AChRa acetylcholine receptor subunit comprises parallel mutations. In some embodiments, parallel mutations are mutations to amino acids that have homology.

[0149] In some embodiments, the mutated alpha subunit extracellular domain comprises or consists of SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCDVSGVDTESGATNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLP (SEQ ID NO: 131). In some embodiments, the mutated beta subunit extracellular domain comprises or consists of SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYLDLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVSSDGSVRWQPPGIYRSSCDVSGVDTESGATNCTMVFSSYSYDSSEVSLQTGLGPDGQGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKP (SEQ ID NO: 132). In some embodiments, the mutated gamma subunit extracellular domain comprises or consists of RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNVLVSPDGCIYWLPPAIFRSACDVSGVDTESGATNCSLIFQSQTYSTNEIDLQLSQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKP (SEQ ID NO: 133).In some embodiments, the delta subunit extracellular domain that is mutated comprises or consists of LNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNVWIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVLVYHYGFVYWLPPAIFRSSCDVSGVDTESGATNCSLKFSSLKYTAKEITLSLKQDAKENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSPSRQDITFYLIIRRKP (SEQ ID NO: 134). In some embodiments, the epsilon subunit extracellular domain that is mutated comprises or consists of KNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGIDWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLVYEGGSVTWLPPAIYRSVCDVSGVDTESGATNCSLIFRSQTYNAEEVEFTFAVDNDGKTINKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRKP (SEQ ID NO: 135).

[0150] In another aspect, a protein is provided that includes any one of SEQ ID NOs: 131-135.

[0151] In some embodiments, the protein comprises SEQ ID NO: 131. In some embodiments, the protein comprises SEQ ID NO: 132. In some embodiments, the protein comprises SEQ ID NO: 133. In some embodiments, the protein comprises SEQ ID NO: 134. In some embodiments, the protein comprises SEQ ID NO: 135. In some embodiments, the protein comprises a plurality of sequences selected from SEQ ID NOs: 131-135. In some embodiments, the complex of the invention comprises a first chain comprising a sequence selected from SEQ ID NOs: 131-135. In some embodiments, the complex of the invention comprises a second chain comprising a sequence selected from SEQ ID NOs: 131-135. In some embodiments, the complex of the invention comprises a third chain comprising a sequence selected from SEQ ID NOs: 131-135. In some embodiments, the complex of the invention comprises a fourth chain comprising a sequence selected from SEQ ID NOs: 131-135. In some embodiments, the extracellular domain fragment is selected from SEQ ID NOs: 131-135. In some embodiments, the extracellular domain variant is selected from SEQ ID NOs: 131-135.

[0152] In some embodiments, the fragment comprises a ligand binding domain and further comprises a mutation that inhibits ligand binding. In some embodiments, the mutation is in the ligand binding domain. One skilled in the art will appreciate that since the protein complexes of the present invention are intended to bind antibodies and B cells, it will be advantageous not to bind endogenous ligands present in the subject, thereby leaving normal ligand levels available for binding to endogenous receptors. In some embodiments, the protein is AChRa and the mutation is a mutation of tyrosine 190 of SEQ ID NO: 1 or tyrosine 215 of SEQ ID NO: 2 to phenylalanine. In some embodiments, the protein is AChRa and the mutation is a mutation of tyrosine 190 of SEQ ID NO: 1 to phenylalanine. In some embodiments, the protein is AChRa and the mutation is a mutation of tyrosine 215 of SEQ ID NO: 2 to phenylalanine.

[0153] In another aspect, a protein is provided that includes an extracellular domain of an acetylcholine receptor subunit, the extracellular domain of the acetylcholine receptor subunit including at least one mutation that reduces aggregation.

[0154] In some embodiments, the fragment comprises a mutation that reduces aggregation. In some embodiments, aggregation comprises self-dimerization. In some embodiments, aggregation comprises multimerization. In some embodiments, the extracellular domain of the acetylcholine receptor subunit is a protein. In some embodiments, the mutation is a plurality of mutations. In some embodiments, the plurality is two. In some embodiments, the plurality is three.

[0155] In some embodiments, the protein is AChR a and the mutation is a deletion of N141. In some embodiments, N141 is within SEQ ID NO: 1. In some embodiments, N141 is within SEQ ID NO: 131. In some embodiments, the protein is AChR a and the mutation is a mutation of phenylalanine 100. In some embodiments, phenylalanine 100 is mutated to glycine (F100G). In some embodiments, phenylalanine 100 is mutated to tyrosine (F100Y). In some embodiments, phenylalanine 100 is mutated to isoleucine (F100I). In some embodiments, F100 is within SEQ ID NO: 1. In some embodiments, F100 is within SEQ ID NO: 131. In some embodiments, the protein is AChR a and the mutation is a mutation of tryptophan 149. In some embodiments, tryptophan 149 is mutated to a charged amino acid. In some embodiments, tryptophan 149 is mutated to a negatively charged amino acid. In some embodiments, tryptophan 149 is mutated to glutamic acid (W149E). In some embodiments, tryptophan 149 is mutated to aspartic acid (W149D). In some embodiments, tryptophan 149 is mutated to a positively charged amino acid. In some embodiments, tryptophan 149 is mutated to lysine (W149K). In some embodiments, tryptophan 149 is mutated to arginine (W149R). In some embodiments, tryptophan 149 is mutated to histidine (W149H). In some embodiments, tryptophan 149 is mutated to glutamine (W149Q). In some embodiments, W149 is within SEQ ID NO: 1. In some embodiments, W149 is within SEQ ID NO: 131. In some embodiments, the protein is AChR a and the mutation is a mutation of valine 155. In some embodiments, valine 155 is mutated to alanine (V155A). In some embodiments, valine 155 is mutated to isoleucine (V155I). In some embodiments, valine 155 is mutated to leucine (V155L). In some embodiments, V155 is within SEQ ID NO: 1. In some embodiments, V155 is within SEQ ID NO: 131. In some embodiments, the protein is AChRa and the mutation is a mutation of tyrosine 93. In some embodiments, the mutation of tyrosine 93 reduces the α-gamma interaction. In some embodiments, tyrosine 93 is mutated to any amino acid that reduces the α and gamma interaction. In some embodiments, tyrosine 93 is mutated to phenylalanine (Y93F). In some embodiments, tyrosine 93 is mutated to a positively charged amino acid. In some embodiments, tyrosine 93 is mutated to histidine (Y93H). In some embodiments, tyrosine 93 is mutated to arginine (Y93R).In some embodiments, tyrosine 93 is mutated to lysine (Y93K). In some embodiments, Y93 is within SEQ ID NO: 1. In some embodiments, Y93 is within SEQ ID NO: 131.

[0156] In some embodiments, the mutation reduces protein oxidation. In some embodiments, the protein is AChRg, and the mutation is a mutation at methionine 84. The mutation is generated, at least in part, to reduce methionine oxidation and extend shelf life. Mutations in any amino acid can produce this result. In some embodiments, the mutation at methionine 84 reduces α-gamma interaction. In some embodiments, methionine 84 is mutated to any amino acid that reduces α-gamma interaction. In some embodiments, methionine 84 is deleted. In some embodiments, methionine 84 is mutated to serine (M84S). In some embodiments, M84 is within SEQ ID NO:6. In some embodiments, M84 is within SEQ ID NO:133. In some embodiments, the protein is AChRg, and the mutation is a mutation at tyrosine 105. In some embodiments, the mutation at tyrosine 105 reduces α-gamma interaction. In some embodiments, tyrosine 105 is mutated to any amino acid that reduces α-gamma interaction. In some embodiments, tyrosine 105 is mutated to a charged amino acid. In some embodiments, tyrosine 105 is mutated to a negatively charged amino acid. In some embodiments, tyrosine 105 is mutated to glutamic acid (Y105E). In some embodiments, tyrosine 105 is mutated to aspartic acid (Y105D). In some embodiments, tyrosine 105 is mutated to a positively charged amino acid. In some embodiments, tyrosine 105 is mutated to arginine (Y105R). In some embodiments, tyrosine 105 is mutated to lysine (Y105K). In some embodiments, tyrosine 105 is mutated to histidine (Y105H). In some embodiments, Y105 is within SEQ ID NO:6. In some embodiments, Y105 is within SEQ ID NO:133. In some embodiments, the protein is AChRg, and the mutation is a mutation of tyrosine 117. In some embodiments, the mutation of tyrosine 117 reduces α-gamma interaction. In some embodiments, tyrosine 117 is mutated to any amino acid that reduces α and gamma interaction. In some embodiments, tyrosine 117 is mutated to a charged amino acid. In some embodiments, tyrosine 117 is mutated to a negatively charged amino acid. In some embodiments, tyrosine 117 is mutated to glutamic acid (Y117E). In some embodiments, tyrosine 117 is mutated to aspartic acid (Y117D). In some embodiments, tyrosine 117 is mutated to a positively charged amino acid. In some embodiments, tyrosine 117 is mutated to arginine (Y117R). In some embodiments, tyrosine 117 is mutated to lysine (Y117K). In some embodiments, tyrosine 117 is mutated to histidine (Y117H). In some embodiments, Y117 is within SEQ ID NO:6.In some embodiments, Y117 is within SEQ ID NO: 133. In some embodiments, the mutation is multiple mutations and includes at least two mutations selected from the group consisting of M84, Y105, and Y117. In some embodiments, the mutation is multiple mutations and includes at least two mutations selected from the group consisting of M84S, Y105E, and Y117E. In some embodiments, the mutation is multiple mutations and includes at least two mutations selected from the group consisting of M84S, Y105E, and Y117R. In some embodiments, the mutation is multiple mutations and includes all of M84S, Y105E, and Y117R. In some embodiments, the mutation is multiple mutations and includes all of M84S, Y105E, and Y117R. In some embodiments, the mutation is two mutations and includes M84S and Y105E. In some embodiments, the mutation is two mutations and includes Y117E and Y105E. In some embodiments, the mutation is two mutations and includes Y117R and Y105E.

[0157] In some embodiments, the protein is AChRd, and the mutation is a mutation of cysteine 108. In some embodiments, cysteine 108 is mutated to any other amino acid. In some embodiments, cysteine 108 is deleted. Those skilled in the art will appreciate that it is desirable to reduce aggregation by removing free cysteines that may have formed disulfide bonds. Therefore, any mutation is possible. In some embodiments, cysteine 108 is mutated to alanine (C108A). In some embodiments, cysteine 108 is mutated to isoleucine (C108I). In some embodiments, C108 is within SEQ ID NO: 8. In some embodiments, C108 is within SEQ ID NO: 134. In some embodiments, the protein is AChRd, and the mutation is a mutation of tyrosine 119. In some embodiments, the mutation of tyrosine 119 reduces delta hydrophobicity. In some embodiments, tyrosine 119 is mutated to any amino acid that reduces delta hydrophobicity. In some embodiments, tyrosine 119 is mutated to a positively charged amino acid. In some embodiments, tyrosine 119 is mutated to arginine (Y119R). In some embodiments, tyrosine 119 is mutated to lysine (Y119K). In some embodiments, tyrosine 119 is mutated to histidine (Y119H). In some embodiments, tyrosine 119 is mutated to a negatively charged amino acid. In some embodiments, tyrosine 119 is mutated to glutamic acid (Y119E). In some embodiments, tyrosine 119 is mutated to aspartic acid (Y119D). In some embodiments, Y119 is within SEQ ID NO:8. In some embodiments, Y119 is within SEQ ID NO:134. In some embodiments, the protein is AChRα and the mutation is a deletion of N141. In some embodiments, N141 is within SEQ ID NO:8. In some embodiments, N141 is within SEQ ID NO:134. In some embodiments, the protein is AChRd and the mutation is a mutation of leucine 151. In some embodiments, the mutation of leucine 151 reduces subunit interactions. In some embodiments, Leucine 151 is mutated to any amino acid that reduces subunit interactions. In some embodiments, Leucine 151 is deleted. In some embodiments, Leucine 151 is mutated to a charged amino acid. In some embodiments, Leucine 151 is mutated to a positively charged amino acid. In some embodiments, Leucine 151 is mutated to a non-positively charged amino acid. In some embodiments, Leucine 151 is mutated to any amino acid other than arginine and histidine. In some embodiments, Leucine 151 is mutated to a negatively charged amino acid. In some embodiments, Leucine 151 is mutated to glutamic acid (L151E). In some embodiments, Leucine 151 is mutated to aspartic acid (L151D).In some embodiments, L151 is within SEQ ID NO: 8. In some embodiments, L151 is within SEQ ID NO: 134. In some embodiments, the mutation is a plurality of mutations selected from the group consisting of: mutations of C108, Y119, and L151; and a deletion of N141. In some embodiments, the mutation is a plurality of mutations selected from the group consisting of: mutations of C108A, Y119R, and L151E; and a deletion of N141. In some embodiments, the mutation is two mutations and includes C108A and Y119R. In some embodiments, the mutation is two mutations and includes C108A and L151E. In some embodiments, the mutation is two mutations and includes C108A; and a deletion of N141. In some embodiments, the mutation is three mutations and includes C108A, Y119R, and L151E. In some embodiments, the mutation is three mutations and includes C108A, Y119R, and L151E. Dimerization domain

[0158] In some embodiments, the dimerization domains are capable of dimerizing with each other. In some embodiments, the first dimerization domain is capable of dimerizing with the second dimerization domain. In some embodiments, the first and second dimerization domains are capable of dimerizing with each other. In some embodiments, the dimerization is configured to dimerize. In some embodiments, the dimerization occurs under physiological conditions. In some embodiments, the dimerization occurs in a body fluid. In some embodiments, the body fluid is blood. In some embodiments, the body fluid is plasma. In some embodiments, the body fluid is serum. In some embodiments, the dimerization occurs in a subject. In some embodiments, the dimerization occurs in vivo. In some embodiments, the dimerization occurs in vitro.

[0159] As used herein, the term "dimerization domain" refers to an amino acid sequence that, when in contact with another amino acid sequence (another dimerization domain), binds to it to form a dimer. Dimerization domains are well known in the art, as multiple protein sequences are known to bind to each other. In some embodiments, dimerization comprises forming 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 some embodiments, dimerization is irreversible. In some embodiments, dimerization comprises a bond formed 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 domains include the hinge domain of an antibody heavy chain, the CH1 / CL domain of an antibody heavy / light chain, and the ECD domain of TCR α / β. In addition, the upper hinge domain can be modified by cysteine substitution / mutation of serine to prevent dimerization. In some embodiments, the dimerization domain comprises or consists of: EPKSSDKTHTCPPCP (SEQ ID NO: 63).

[0160] In some embodiments, the dimerization domain comprises or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, the Ig hinge domain is a heavy chain hinge domain. In some embodiments, the Ig is a human Ig. In some embodiments, the immunoglobulin is selected from IgA, IgD, IgE, IgG, and IgM. In some embodiments, the immunoglobulin is an IgG. In some embodiments, the IgG is IgG1. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is selected from IgG1 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 present invention.

[0161] In some embodiments, the hinge domain comprises the amino acid sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 16). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IgG1 hinge comprises or consists of SEQ ID NO: 16. In some embodiments, the hinge domain comprises the amino acid sequence EPKCCVECPPCPAPPAAAP (SEQ ID NO: 17). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the IgG2 hinge comprises or consists of SEQ ID NO: 17. In some embodiments, the hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFLGGP (SEQ ID NO: 18). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the IgG4 hinge comprises or consists of SEQ ID NO: 18. In some embodiments, the hinge domain comprises the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAP ELLGGP (SEQ ID NO: 19). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the IgG3 hinge comprises or consists of SEQ ID NO: 19. In some embodiments, the hinge domain comprises the CPXCP (SEQ ID NO: 20) motif. In some embodiments, X in SEQ ID NO: 20 is selected from P and R. In some embodiments, SEQ ID NO: 20 is CPPCP (SEQ ID NO: 21). In some embodiments, SEQ ID NO: 20 is CPRCP (SEQ ID NO: 22). In some embodiments, the hinge domain comprises EPKSCDKTHTCPPCP (SEQ ID NO: 37). Thus, it will be understood that the hinge region can be considered to end after the CPXCP motif.

[0162] 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 CLκ domain. In some embodiments, the CL domain is a CLλ domain. It is known in the art that the CHI domain of an Ig heavy chain dimerizes with the CL domain of a light chain. 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, both the first and second dimerization domains include a hinge domain. In some embodiments, the first and second dimerization domains do not both include a CHI domain. In some embodiments, both the first and second dimerization domains do not both include a CL domain. In some embodiments, the first and second polypeptide chains do not both comprise a CH1 domain. In some embodiments, the first and second polypeptide chains do not both comprise a CL domain.

[0163] In some embodiments, the Ig CHI domain comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 23). In some embodiments, the Ig CHI domain consists of SEQ ID NO: 23. In some embodiments, SEQ ID NO: 23 is an IgG1 CHI domain. In some embodiments, the Ig CHI domain comprises the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 24). In some embodiments, the Ig CHI domain consists of SEQ ID NO: 24. In some embodiments, SEQ ID NO: 24 is an IgG2 CHI domain. In some embodiments, the Ig CHI domain comprises the amino acid sequence ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 25). In some embodiments, the Ig CHI domain consists of SEQ ID NO: 25. In some embodiments, SEQ ID NO: 25 is an IgG3 CHI domain. In some embodiments, the Ig CHI domain comprises the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 26). In some embodiments, the Ig CHI domain consists of SEQ ID NO: 26. In some embodiments, SEQ ID NO: 26 is an IgG4 CHI domain.

[0164] In some embodiments, the Ig CLκ domain comprises the amino acid sequence AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 27). In some embodiments, the Ig CLκ domain consists of SEQ ID NO: 27. In some embodiments, the Ig CLλ domain comprises the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 28). In some embodiments, the Ig CLλ domain consists of SEQ ID NO: 28.

[0165] Effector part

[0166] In some embodiments, the composition includes an effector portion. In some embodiments, the first polypeptide chain includes an effector portion. In some embodiments, the second polypeptide chain includes an effector portion. In some embodiments, both the first and second polypeptide chains include an effector portion. As used herein, the term "moiety" refers to a portion of a molecule that may include an entire functional group or parts of a functional group as a substructure. The term "part" may also refer to a portion of a molecule that exhibits a set of specific chemical and / or pharmacological properties similar to a corresponding molecule. As used herein, the term "effector portion" refers to a molecule or a fragment of a molecule that exerts a cytotoxic effect. In some embodiments, the effector portion is an effector molecule.

[0167] 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, the death is cell death. In some embodiments, the death is apoptosis. In some embodiments, the death is necrosis. In some embodiments, the death is cell-mediated death. In some embodiments, the death is phagocytosis. In some embodiments, the cytotoxic effect is directed to the target cell. In some embodiments, the death occurs in the target cell. In some embodiments, the cytotoxic effect occurs after binding. In certain embodiments, the death occurs after binding. In some embodiments, the cytotoxic effect is directed to the target cell bound by the composition. In some embodiments, the death is the death of the target cell bound by the composition. In some embodiments, the cytotoxic effect is directed to a cell bound by the protein complex. In some embodiments, the cytotoxic effect is directed to a cell bound by the protein complex. In some embodiments, the death is the death of the cell bound by the protein complex. In some embodiments, the death is the death of the cell bound by the protein complex. In some embodiments, the cytotoxic effect is a direct effect. In some embodiments, the cytotoxic effect is indirect. In some embodiments, the binding composition is a binding fragment. In some embodiments, the binding protein complex is a binding fragment. In some embodiments, the fragment is at least one of the fragments. In some embodiments, the fragment is one of the fragments. In some embodiments, the fragment is both of the fragments.

[0168] 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 a chemotherapeutic agent. In some embodiments, the effector moiety is an anticancer agent. In some embodiments, the effector moiety is an engager. In some embodiments, the engager binds to cytotoxic cells. In some embodiments, binding to cytotoxic cells is recruiting cytotoxic cells. In some embodiments, binding is being bound.

[0169] In some embodiments, the effector portion recruits a cytotoxic agent. In some embodiments, the cytotoxic agent is a cytotoxic cell. In some embodiments, the cytotoxic cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is a CD8 positive T cell. In some embodiments, the effector portion triggers antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the effector portion triggers complement-dependent cytotoxicity (CDC).

[0170] In some embodiments, the effector moiety binds to a receptor on the cell surface of a cytotoxic cell. Examples of receptors include, but are not limited to, CD3, CD8, CD56, CD14, and CD16. In some embodiments, the receptor is a marker for a 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 to CD3. In some embodiments, the conjugate is an agent that binds to CD3. In some embodiments, CD3 is human CD3. In some embodiments, the agent that binds to CD3 is an anti-CD3 antibody or an antigen-binding fragment thereof. In some embodiments, the receptor is CD16. In some embodiments, the effector moiety is an agent that binds to CD16. In some embodiments, the conjugate is an agent that binds to CD16. In some embodiments, CD16 is human CD16. In some embodiments, the agent that binds to CD16 is an anti-CD16 antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a single-domain antibody. In some embodiments, the antibody or 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 can be used. For example, the anti-human CD3 scFv known as OKT3 can be used as an agent. In some embodiments, the cytotoxic moiety is selected from α-amanitin, a radioactive moiety, and an anti-CD3 binding agent. Other examples of human anti-CD3 antibodies include: Muromonab (trade name Orthoclone OKT3), a murine anti-human CD3 monoclonal antibody (DrugBank Accession No. DB00075); Teplizumab, a humanized form of the murine OKT3 anti-CD3 monoclonal antibody (DrugBank Accession No. DB06606); UCHT1, a murine anti-human CD3 monoclonal antibody; UCHT1 variant-9, a humanized form of the UCHT1 clone; and the bispecific CD19-CD3 Blinatumomab (DrugBank Accession No. DB09052). Examples of human anti-CD16 include: AFM13, a bispecific tetravalent innate cell engager / adapter targeting CD30 on tumor cells and CD16A on NK cells and macrophages (Innate Cell Engager, ); and GTB-3550 (CD16 / IL-15 / CD33), a trispecific killer cell engager.

[0171] In some embodiments, the composition comprises an Fc region. In some embodiments, the effector moiety is not an Fc region. In some embodiments, "not an Fc region" means "not an unmodified 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 does not contain an Fc region. 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 does not contain an Fc region. In some embodiments, the conjugate is an Fc region. In some embodiments, the conjugate is not an Fc region. In some embodiments, the composition comprises an effector moiety that is superior to an Fc in killing. In some embodiments, superior killing is superior in killing B cells. In some embodiments, the Fc is an unmodified Fc. In some embodiments, the Fc is an unmutated Fc. In some embodiments, the Fc is a naturally occurring Fc. In some embodiments, the Fc is a human Fc. In some embodiments, the superior Fc is an Fc comprising at least one mutation that increases ADCC. In some embodiments, the Fc region is an Fc domain. In some embodiments, the Fc region is an Fc fragment. In some embodiments, the first polypeptide chain 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 Fc regions. In some embodiments, the Fc region is the Fc region of an antibody heavy chain. In some embodiments, the antibody heavy chain is a human antibody heavy chain. In some embodiments, the heavy chain is an IgG heavy chain. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG is selected from IgG1 and IgG3. In some embodiments, the IgG is IgG1. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is IgG4.

[0172] In some embodiments, the Fc region is capable of inducing cytotoxicity. In some embodiments, the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12). In some embodiments, the Fc domain includes EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:140). It should be understood that SEQ ID NO: 140 includes five additional N-terminal amino acids compared to SEQ ID NO: 12. Thus, while numbers herein are given with respect to SEQ ID NO: 12, the numbering of SEQ ID NO: 140 can be obtained by adding 5. In some embodiments, the Fc domain is capable of eliciting a cytotoxic effect. In some embodiments, the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 141).In some embodiments, the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 142). It will be appreciated that SEQ ID NO: 142 includes five additional N-terminal amino acids compared to SEQ ID NO: 141. Thus, although numbers are given herein with respect to SEQ ID NO: 141 (or equivalent SEQ ID NO: 12), the numbering of SEQ ID NO: 142 can be obtained by adding 5. SEQ ID NO: 12 and SEQ ID NO: 141 differ by two amino acids. The two sequences are interchangeable, and when mutations are given with respect to SEQ ID NO: 12, it should be understood that they also apply to SEQ ID NO: 141, and vice versa. Similarly, SEQ ID NO: 140 and SEQ ID NO: 142 also differ by only two amino acids, and the two sequences are interchangeable.

[0173] In some embodiments, the Fc domain consists of SEQ ID NO: 12. In some embodiments, the Fc domain of IgG1 comprises or consists of SEQ ID NO: 12. In some embodiments, the Fc domain comprises or consists of a sequence that is at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homologous to SEQ ID NO: 12. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 140. In some embodiments, the Fc domain of IgG1 comprises or consists of SEQ ID NO: 140. In some embodiments, the Fc domain comprises or consists of a sequence that is at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homologous to SEQ ID NO: 140. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 141. In some embodiments, the Fc domain of IgG1 comprises or consists of SEQ ID NO: 141. In some embodiments, the Fc domain comprises or consists of a sequence that is at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homologous to SEQ ID NO: 141. Each possibility represents a separate embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 142. In some embodiments, the Fc domain of IgG1 comprises or consists of SEQ ID NO: 142. In some embodiments, the Fc domain comprises or consists of a sequence that is at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homologous to SEQ ID NO: 142. Each possibility represents a separate embodiment of the present invention. In some embodiments, the Fc region is configured to elicit a cytotoxic effect. In some embodiments, the cytotoxic effect is directed against a target cell. In some embodiments, the cytotoxic effect is post-binding. In some embodiments, the cytotoxic effect is directed against cells bound by the protein complex. In some embodiments, the cytotoxic effect is directed against cells bound to 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 caused by the Fc region. In some embodiments, the cytotoxic effect is mediated by immune cell recruitment caused 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 triggers antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region triggers complement-dependent cytotoxicity (CDC).

[0174] In some embodiments, the Fc region comprises an Ig C2 domain. In some embodiments, the Fc region comprises an Ig heavy chain C2 domain. In some embodiments, the Fc region comprises an Ig C3 domain. In some embodiments, the Fc region comprises an Ig heavy chain C3 domain. In some embodiments, the Fc region comprises or consists of both an Ig C2 domain and an Ig C3 domain. In some embodiments, the Fc region comprises or consists of both an Ig heavy chain C2 domain and an Ig heavy chain C3 domain. In some embodiments, the first chain comprises a first portion of the Fc region, and the second chain comprises a second portion of the Fc region. In some embodiments, the first portion comprises a C2 domain, a C3 domain, or both. In some embodiments, the second portion comprises a C2 domain, a C3 domain, or both. In some embodiments, the interface between the first portion of the Fc region and the second portion of the Fc region creates a functional Fc region. In some embodiments, the interface comprises contact. In some embodiments, the interface comprises adjacent positioning. In some embodiments, the interface comprises formation of a protein complex of the present invention. In some embodiments, the interface comprises dimerization of a first and a second dimerization domain. 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.

[0175] In some embodiments, the CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 29). In some embodiments, the CH2 domain comprises the amino acid sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 13). In some embodiments, the CH2 domain consists of SEQ ID NO: 29. In some embodiments, SEQ ID NO: 29 is an IgG1 CH2 domain. In some embodiments, the CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO: 30). In some embodiments, the CH2 domain consists of SEQ ID NO: 30. In some embodiments, SEQ ID NO: 30 is an IgG2 CH2 domain. In some embodiments, the CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 31). In some embodiments, the CH2 domain consists of SEQ ID NO: 31. In some embodiments, SEQ ID NO: 31 is an IgG4 CH2 domain. In some embodiments, the CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNS TFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO: 32). In some embodiments, the CH2 domain consists of SEQ ID NO: 32. In some embodiments, SEQ ID NO: 32 is an IgG3 CH2 domain.

[0176] In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33). In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62). In some embodiments, the CH3 domain consists of SEQ ID NO: 33. In some embodiments, the CH3 domain consists of SEQ ID NO: 62. In some embodiments, SEQ ID NO: 33 is an IgG1 CH3 domain. In some embodiments, SEQ ID NO: 62 is an IgG1 CH3 domain. In some embodiments, the sequence of SEQ ID NO:33 is a sequence primarily found in humans of European and American descent. In some embodiments, SEQ ID NO:62 is a sequence primarily found in humans of Asian descent. In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:34). In some embodiments, the CH3 domain consists of SEQ ID NO:34. In some embodiments, SEQ ID NO:34 is an IgG2 CH3 domain. In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 35). In some embodiments, the CH3 domain consists of SEQ ID NO: 35. In some embodiments, SEQ ID NO: 35 is an IgG4 CH3 domain.In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 36). In some embodiments, the CH3 domain consists of SEQ ID NO: 36. In some embodiments, SEQ ID NO: 36 is an IgG3 CH3 domain.

[0177] In some embodiments, the Fc domain comprises a mutation. In some embodiments, the 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, the 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, both the CH2 and CH3 domains comprise mutations. In some embodiments, the first CH2 domain and the first CH3 domain 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 mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the first mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the second mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the mutation permits heterodimerization. In some embodiments, the mutation permits heterodimerization of the first and second chains. In certain embodiments, permitting is promoting. In some embodiments, permitting is enhancing.

[0178] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutation or alteration can be used to construct the polypeptides of the present invention. In some embodiments, a region from IgG is replaced with a region from IgA. In some embodiments, a region from TCRα is inserted into the first CH3 domain, and a region from TCRb is inserted into the second CH3 domain. In some embodiments, the mutation is an insertion of a region from a TCR. In some embodiments, the TCR is selected from TCRα and TCRb. In some embodiments, the mutation is an insertion of a region from a different Ig. Examples of such mutations can be found in Table 1. In some embodiments, the mutation is selected from the mutations in Table 1. In some embodiments, the first mutation is selected from the group of mutations provided in the row and second column of Table 1, and the second mutation is the group of mutations provided in the same row and third column of Table 1. Unless otherwise indicated, the mutations in Table 1 are provided with Kabat numbering for IgG1; corresponding mutations can be generated in other IgGs, and in particular, 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 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 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 F405A, and the second mutation is Y349T or T392F. In some embodiments, the first mutation is T350V, L351Y, F405A, or Y407V, and the second mutation is T350V, T366L, K392L, or T394W. In some embodiments, the first mutation is K392D or K409D, and the second mutation is E356K or D399K. In some embodiments, the first mutation is D221E, P228E, or L368E, and the second mutation is D221R, P228R, or K409R. In some embodiments, the first mutation is K360E or K409W, and the second mutation is Q347R, D399V, or F405T. In some embodiments, the first mutation is K360E, K409W, or Y349C, and the second mutation is Q347R, D399V, F405T, or S354C. In some embodiments, the first mutation is K360D, D399M, or Y407A, and the second mutation is E345R, Q347R, T366V, or K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, or K409V, and the second mutation is E356G, E357D, S364Q, or Y407A.In some embodiments, the first mutation is L351D or L368E, and the second mutation is L351K or T366K. In some embodiments, the first mutation is L368D or K370S, and the second mutation is E357Q or S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A, or Y407V. In some embodiments, 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: GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 46). In some embodiments, the CH3 domain comprises or consists of: GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47). In some embodiments, the CH3 domain comprises or consists of: GQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 48). In some embodiments, the CH3 domain comprises or consists of: GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 49).

[0179] Table 1: Mutations that enhance CH3 domain heterodimerization and inhibit homodimerization.

[0180] In some embodiments, the mutation reduces effector function. In some embodiments, the effector function includes ADCC, CDC, or both. In some embodiments, the reduced effector function includes reduced cytotoxicity. In some embodiments, reduction is abolition. In some embodiments, Fc is from IgG1 or IgG3, and the mutation reduces effector function. In some embodiments, Fc is from IgG1 and includes at least one mutation that reduces effector function. Mutations that reduce effector function are well known in the art, and any such mutation can be used. 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 (incorporated herein by reference in its entirety).

[0181] Those skilled in the art will appreciate that IgG2 and IgG4 have greatly reduced effector function and are essentially non-cytotoxic overall. Furthermore, it is known that mutations such as S228P and L235E in IgG4 can reduce effector function even further. Furthermore, mutations that reduce cytotoxicity / effector function in IgG1 and IgG3 are also well known in the art. In some embodiments, the IgG comprises at least one mutation. In some embodiments, the mutation is multiple mutations. In some embodiments, the mutation reduces cytotoxicity. In some embodiments, the mutation increases stability. In some embodiments, the mutation reduces aggregation. In some embodiments, the multiple mutations that reduce cytotoxicity include a LALA mutation. In some embodiments, the multiple mutations that reduce cytotoxicity include a PG-LALA mutation. In some embodiments, the mutation is a proline 329 to glycine mutation (P329G) in the IgG1 human heavy chain. In some embodiments, the P to G mutation is a P109 to G mutation in SEQ ID NO: 12. In some embodiments, the mutation is a leucine 234 to alanine mutation (L234A) in the IgG1 human heavy chain. In some embodiments, the L to A mutation is a mutation of L14 to A of SEQ ID NO: 12. In some embodiments, the mutation is a mutation of leucine 235 to alanine (L235A) of the IgG1 human heavy chain. In some embodiments, the L to A mutation is a mutation of L15 to A of SEQ ID NO: 12. In some embodiments, the multiple mutations include P109G, L14A, and L15A of SEQ ID NO: 12. In some embodiments, the multiple mutations include L14A and L15A of SEQ ID NO: 12. In some embodiments, the multiple mutations include P329G, L234A, and L235A of the IgG1 human heavy chain. In some embodiments, the multiple mutations include L234A and L235A of the IgG1 human heavy chain. Those skilled in the art will appreciate that parallel mutations can also be performed in the IgG3 heavy chain or the heavy chain of non-human IgG1. In some embodiments, the multiple mutations that reduce cytotoxicity include YTE mutations. In some embodiments, the mutation is a methionine 252 to tyrosine mutation (M252Y) of the IgG1 human heavy chain. In some embodiments, the M to Y mutation is a M32 to Y mutation of SEQ ID NO: 12. In some embodiments, the mutation is a serine 254 to threonine mutation (S254T) of the IgG1 human heavy chain. In some embodiments, the S to T mutation is a S34 to T mutation of SEQ ID NO: 12. In some embodiments, the mutation is a threonine 256 to glutamic acid mutation (T256E) of the IgG1 human heavy chain. In some embodiments, the T to E mutation is a T36 to E mutation of SEQ ID NO: 12.In some embodiments, the plurality of mutations comprises M32Y, S34T and T36E of SEQ ID NO: 12. In some embodiments, the plurality of mutations comprises M252Y, S254T and T256E of the IgG1 human heavy chain. In some embodiments, the mutation is a mutation of asparagine 297 (N297) of the IgG1 human heavy chain. In some embodiments, the asparagine is mutated to alanine (N297A). In some embodiments, the asparagine is mutated to glutamine (N297Q). In some embodiments, the asparagine is N77 (N77A or N77Q) of SEQ ID NO: 12.

[0182] In some embodiments, the mutation increases the half-life of a molecule, peptide, polypeptide, or protein complex. In some embodiments, the mutation that increases half-life is a mutation that increases binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutation that increases binding to FcRn is selected from the mutations provided in Table 8. In some embodiments, the mutation is a mutation from asparagine 434 to histidine (N434H). In some embodiments, the Fc domain of the N434H mutation includes the N214H mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation is a mutation from valine 308 to proline (V308P). In some embodiments, the Fc domain of the H435A mutation includes the H215A mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation weakens binding to FcRn. In some embodiments, the mutation that weakens binding is a mutation from histidine 435 to alanine (H435A). In some embodiments, the Fc domain of the H435A mutation comprises the H215A mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation that increases binding to FcRn is a plurality of mutations. In some embodiments, the plurality of mutations comprises or consists of: a mutation of methionine 252 to tyrosine (M252Y), a mutation of serine 234 to threonine, and a mutation of threonine 256 to glutamate (T256E) (also referred to as YTE). In some embodiments, the Fc domain of the M252Y / S254T / T256E mutation comprises the M32Y, S34T, and T35E mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality of mutations comprises or consists of: a mutation of methionine 428 to leucine (M428L) and a mutation of asparagine 434 to serine (N434S) (also referred to as LS). In some embodiments, the M428L / N434S mutant Fc domain comprises the M208L and N214S mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of: M428L and a mutation of asparagine 434 to alanine (N434A) (also referred to as LA). In some embodiments, the M428L / N434A mutant Fc domain comprises the M208L and N214A mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of a mutation of threonine 250 to glutamine (T250Q) and a mutation of methionine 428 to leucine (M428L) (also referred to as QL). In some embodiments, the T250Q / M428L mutant Fc domain comprises the T30Q and M208L mutations of SEQ ID NO: 12 or 141.In some embodiments, the plurality comprises or consists of: a histidine 433 to lysine mutation (H433K) and an asparagine 434 to phenylalanine mutation (N434F). In some embodiments, the H433K / N434F mutant Fc domain comprises the H213K and N214F mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of: M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the M252Y / S254T / T256E / H433K / N434F mutant Fc domain comprises the M32Y, S34T, T35E, H213K, and N214F mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of a mutation of threonine 307 to alanine (T307A), a mutation of glutamate 380 to alanine (E380A), and a mutation of asparagine 434 to alanine (N434A). In some embodiments, the Fc domain of the T307A / E380A / N434A mutation comprises the T87A, E160A, and N214A mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of a mutation of methionine 252 to tyrosine (M252Y), a mutation of valine 308 to proline (V308P), and a mutation of asparagine 343 to tyrosine (N343Y). In some embodiments, the Fc domain of the M252Y / V308P / N343Y mutation comprises the M32Y, V88P, and N123Y mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of: M252Y, a mutation of valine 308 to proline (V308P), and a mutation of asparagine 434 to tyrosine (N434Y). In some embodiments, the Fc domain of the M252Y / V308P / N434Y mutation comprises the M32Y, V88P, and N214Y mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of: a mutation of histidine 258 to aspartic acid (H258D), a mutation of threonine 307 to glutamine (T307Q), and a mutation of alanine 378 to valine (A378V). In some embodiments, the Fc domain of the H258D / T307Q / A378V mutation comprises the H38D, T87Q, and A158V mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of a leucine 309 to aspartic acid mutation (L309D), a glutamine 311 to histidine mutation (Q311H), and an asparagine 434 to serine mutation (N434S).In some embodiments, the L309D / Q311H / N434S mutant Fc domain comprises the L89D, Q91H, and N214A mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality of residues that reduce binding comprises or consists of: an isoleucine 253 to alanine mutation (I253A), H435A, and a histidine 436 to alanine mutation (H436A). In some embodiments, the I253A / H435A / H436A mutant Fc domain comprises the I33A, H215A, and H216A mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality of residues that reduce binding comprises or consists of: I253A, a histidine 310 to alanine mutation (H310A), and H435A. In some embodiments, the I253A / H310A / H435A mutated Fc domain comprises the I33A, H90A, and H215A mutations of SEQ ID NO: 12 or 141.

[0183] Table 8: Mutations affecting FcRn binding

[0184] In some embodiments, the mutation is a mutation that reduces binding to an Fc receptor. In some embodiments, the Fc receptor is an FcγR. In some embodiments, the FcγR is an FcγRI. In some embodiments, the mutation is a mutation that reduces binding to C1q. In some embodiments, the mutation that reduces binding to an Fc receptor reduces ADCC. In some embodiments, the mutation is a mutation of N297. Because the N-glycan is attached to N297, the mutation eliminates glycosylation of this residue. In some embodiments, the mutation of N297 is a mutation to alanine (N297A). In some embodiments, the mutation of N297 is a mutation to glutamine (N297Q). In some embodiments, the mutation of N297 is a mutation to glycine (N297G). In some embodiments, the CH2 domain with the N297A mutation includes the N59A mutation of SEQ ID NO: 29. In some embodiments, the Fc domain with the N297A mutation includes the N77A mutation of SEQ ID NO: 12 or 141. In some embodiments, the CH2 domain of the N297Q mutation includes the N59Q mutation of SEQ ID NO: 29. In some embodiments, the Fc domain of the N297Q mutation includes the N77Q mutation of SEQ ID NO: 12 or 141. In some embodiments, the CH2 domain of the N297G mutation includes the N59G mutation of SEQ ID NO: 29. In some embodiments, the Fc domain of the N297G mutation includes the N77G mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation is a plurality of mutations that reduce binding to Fc receptors. In some embodiments, the plurality includes or consists of: a mutation of glycine 236 to arginine (G236R) and a mutation of leucine 328 to arginine (L328R). In some embodiments, the G236R / L328R mutated Fc comprises: a hinge domain comprising the G21R mutation of SEQ ID NO: 16; and a CH2 domain comprising the L90R mutation of SEQ ID NO: 29. In some embodiments, the G236R / L328R mutated Fc domain comprises the G16R and L108R mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of: a mutation of serine 298 to glycine (S298G) and a mutation of threonine 299 to alanine (T299A). In some embodiments, the S298G / T299A mutated CH2 domain comprises the S60G and T61A mutations of SEQ ID NO: 29. In some embodiments, the S298G / T299A mutated Fc domain comprises the S78G and T79A mutations of SEQ ID NO: 12 or 141.In some embodiments, the plurality comprises or consists of a leucine 234 to phenylalanine mutation (L234F), a leucine 235 to glutamic acid mutation (L235E), and an aspartic acid 265 to arginine mutation (D265A). In some embodiments, the L234F / L235E / D265A mutant Fc comprises a hinge domain comprising the L19F and L20E mutations of SEQ ID NO: 16, and a CH2 domain comprising the D27A mutation of SEQ ID NO: 19. In some embodiments, the L234F / L235E / D265A mutant Fc domain comprises the L14F, L15E, and D45A mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of a leucine 234 to alanine mutation (L234A), a leucine 235 to alanine mutation (L235A), and a proline 329 to glycine mutation (P329G). In some embodiments, the L234A / L235A / P329G mutant Fc comprises a hinge domain comprising the L19A and L20A mutations of SEQ ID NO: 16; and a CH2 domain comprising the P91G mutation of SEQ ID NO: 29. In some embodiments, the L234A / L235A / P329G mutant Fc domain comprises the L14A, L15A, and P109G mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of L234F, L235E, and a proline 331 to serine mutation (P331S). In some embodiments, the L234F / L235E / P331S mutant Fc comprises: a hinge domain comprising the L19F and L20E mutations of SEQ ID NO: 16; and a CH2 domain comprising the P93S mutation of SEQ ID NO: 29. In some embodiments, the L234F / L235E / P331S mutant Fc domain comprises the L14F, L15E, and P111S mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of: a leucine 235 to alanine mutation (L235A), a glycine 237 to alanine mutation (G237A), and a glutamic acid 318 to alanine mutation (E318A). In some embodiments, the L235A / G237A / E318A mutant Fc comprises: a hinge domain comprising the L20A and G22A mutations of SEQ ID NO: 16; and a CH2 domain comprising the E80A mutation of SEQ ID NO: 29. In some embodiments, the L235A / G237A / E318A mutant Fc domain comprises the L15A, G17A, and E98A mutations of SEQ ID NO: 12 or 141.

[0185] In some embodiments, the Fc is modified to reduce binding to Fc receptors. In some embodiments, the modification is removal of glycosylation. In some embodiments, Fc glycosylation is enzymatically removed. In some embodiments, enzymatic deglycosylation is performed using a deglycosylase. In some embodiments, enzymatic deglycosylation is performed using a lyase that cleaves sugars. Examples of enzymes used for deglycosylation include, but are not limited to, peptide-N-glycosidase F (PNGase) and endoglycosidase H (Endo H). Kits for deglycosylation are also commercially available.

[0186] 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 a mutation from serine 267 to glutamate (S267E). In some embodiments, the CH2 domain of the S267E mutation includes the S29E mutation of SEQ ID NO:29. In some embodiments, the Fc domain of the S267E mutation includes the S47E mutation of SEQ ID NO:12 or 141. In some embodiments, the mutation is a mutation from proline 238 to aspartic acid (P238D). In some embodiments, the hinge domain of the P238D mutation includes the P23D mutation of SEQ ID NO:16. In some embodiments, the Fc domain of the P238D mutation includes the P18D mutation of SEQ ID NO:12 or 141. In some embodiments, the mutation is a plurality of mutations that increase binding to Fc receptors. In some embodiments, the plurality comprises or consists of: S267E and a mutation of leucine 328 to phenylalanine (L328F) (also referred to as SELF). In some embodiments, the CH2 domain of the S267E / L328F mutation comprises the S29E and L90F mutations of SEQ ID NO: 29. In some embodiments, the Fc domain of the S267E / L328F mutation comprises the S47E and L108F mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of: S267E and a mutation of histidine 268 to phenylalanine (H268F) and a mutation of serine 324 to threonine (S324T) (also referred to as EFT). In some embodiments, the CH2 domain of the S267E / H268F / S324T mutation comprises the S29E, H30F, and S86T mutations of SEQ ID NO: 29. In some embodiments, the Fc domain of the S267E / H268F / S324T mutation comprises the S47E, H48F, and S104T mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises a glycine 237 to aspartic acid mutation (G237D), P238D, a proline 271 to glycine mutation (P271G), and an alanine 330 to arginine mutation (A330R) (also known as V9). In some embodiments, the G237D / P238D / P271G / A330R mutant polypeptide comprises: a mutated hinge domain comprising the G22D and P23D mutations of SEQ ID NO: 16; and a mutated CH2 domain comprising the P33G and A92R mutations of SEQ ID NO: 29.In some embodiments, the G237D / P238D / P271G / A330R mutant Fc domain comprises the G17D, P18D, P51G, and A110R mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of G237D, P238D, a histidine 268 to aspartic acid mutation (H268D), P271G, and A330R (also known as V11). In some embodiments, the G237D / P238D / H268D / P271G / A330R mutant polypeptide comprises: a mutant hinge domain comprising the G22D and P23D mutations of SEQ ID NO: 16; and a mutant CH2 domain comprising the H30D, P33G, and A92R mutations of SEQ ID NO: 29. In some embodiments, the G237D / P238D / H268D / P271G / A330R mutant Fc domain comprises the G17D, P18D, H48D, P51G, and A110R mutations of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of a glutamic acid 233 to aspartic acid mutation (E233D), G237D, P238D, H268D, P271G, and A330R (also known as V12). In some embodiments, the E233D / G237D / P238D / H268D / P271G / A330R mutant polypeptide comprises: a mutated hinge domain comprising the E18D, G22D, and P23D mutations of SEQ ID NO: 16; and a mutated CH2 domain comprising the H30D, P33G, and A92R mutations of SEQ ID NO: 29. In some embodiments, the E233D / G237D / P238D / H268D / P271G / A330R mutant Fc domain comprises the E13D, G17D, P18D, H48D, P51G, and A110R mutations of SEQ ID NO: 12 or 141.

[0187] The S267E mutation was found to enhance affinity for both inhibitory FcγRIIB and activating FcγRIIa. Compared to human WT IgG1, the SELF mutation of hIgG1 resulted in a substantial 430-fold increase in binding to FcγRIIB, while minimal changes in binding to FcγRI and FcγRIIA-H131. The EFT mutation was found to increase FcγRIIB binding by 18-fold compared to human WT IgG1. EFT also increases CDC, ADCC, and antibody-dependent cellular phagocytosis (ADCP) activity by enhancing C1q and activator FcG receptor binding. In some embodiments, the mutation that increases ADCC is an EFT multiple mutation. P238D showed enhanced binding to FcγRIIB, with an affinity increase of approximately 4.3-fold compared to WT human IgG1. P238D also significantly reduced binding to all other activating Fcg receptors. V9 significantly enhanced the antibody's affinity for hFcγRIIB, with a change of approximately 32-fold compared to WT IgG1. It was also found that V9 reduced the affinity for the hFcγRIIAR131 allele by about 3 times compared to WT IgG1. It was found that V11 significantly enhanced the affinity of the antibody for hFcγRIIB by about 96 times compared to human WT IgG1, while reducing the affinity for hFcγRIIAR131 by about 3 times. V12 showed a significant enhancement in binding to FcγRIIB, with a 217-fold change compared to human WT IgG1. The V12 mutation also showed no detectable binding to the FcγRIIIA allotype, with reduced FcγRI binding (0.061-fold change relative to WT IgG1) and FcγRIIA-H131 (0.068-fold change relative to wt IgG1). It should be noted that V12 slightly improved binding to FcγRIIA-R131, with a 2-fold increase in binding compared to WT hIgG1.

[0188] The mutations that produce the above functions are well known in the art, and any such mutations can be used. Examples of such mutations can be found at least in KO Saunders, 2019, "Conceptual approaches to modulating antibody effector functions and circulation half-life", Front, Immunol., 2019 Jun 7; 10: 1296 (which is incorporated herein by reference in its entirety). Table 1 of Saunders provides Fc modifications that enhance antibody effector functions. Table 2 of Saunders provides Fc modifications that improve antibody circulation half-life. Table 3 of Saunders provides Fc modifications that inhibit antibody effector functions. It will be understood by those skilled in the art that parallel mutations can also be carried out in the heavy chain of IgG3 heavy chains or non-human IgG1. It will be understood that the numbers given herein refer to full-length IgG including variable domains. These numbers can be shifted so as to correspond to the positions of these amino acids only in the Fc portion of IgG.

[0189] 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 but not CDC. Those skilled in the art will appreciate that while the cytotoxicity of an unmodified Fc is insufficient to overcome the potentiation effect produced by the molecules of the present invention, the cytotoxicity of an Fc comprising a mutation that increases ADCC is sufficient to overcome this. In some embodiments, the effector function comprises ADCC. In some embodiments, the effector function comprises ADCC but not CDC. In some embodiments, the increased effector function comprises increased cytotoxicity. In some embodiments, the Fc is from IgG1 or IgG3 and the mutation increases effector function. In some embodiments, the Fc is from IgG1 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 may 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 (which is incorporated herein by reference in its entirety).

[0190] In some embodiments, the mutation that increases ADCC is a plurality of mutations that increase ADCC. In some embodiments, the plurality of mutations include a mutation of leucine 235 to valine (L235V), a mutation of phenylalanine 243 to leucine (F243L), a mutation of arginine 292 to proline (R292P), a mutation of tyrosine 300 to leucine (Y300L), and a mutation of proline 296 to leucine (P396L) in human IgG1. In some embodiments, the plurality of mutations include a mutation of leucine 15 to valine (L15V), a mutation of phenylalanine 23 to leucine (F23L), a mutation of arginine 72 to proline (R72P), a mutation of tyrosine 80 to leucine (Y80L), and a mutation of proline 176 to leucine (P176L) in SEQ ID NO: 12. In some embodiments, the multiple mutations include a mutation of serine 239 to aspartic acid (S239D) and a mutation of isoleucine 332 to glutamic acid (I332E) in human IgG1. In some embodiments, the multiple mutations include a mutation of serine 19 to aspartic acid (S19D) and a mutation of isoleucine 112 to glutamic acid (I112E) in SEQ ID NO: 12. In some embodiments, the S239D / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include a mutation of serine 239 to aspartic acid (S239D), a mutation of alanine 330 to leucine (A330L), and a mutation of isoleucine 332 to glutamic acid (I332E) in human IgG1. In some embodiments, the multiple mutations include a mutation of serine 19 to aspartic acid (S19D), a mutation of alanine 110 to leucine (A110L), and a mutation of isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 12. In some embodiments, the S239D / A330L / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include a mutation of glycine 236 to alanine (G236A), a mutation of alanine 330 to leucine (A330L), and a mutation of isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include a mutation of glycine 16 to alanine (G16A), a mutation of alanine 110 to leucine (A110L), and a mutation of isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 12. In some embodiments, the plurality of mutations includes a serine 298 to alanine mutation (S298A), a glutamate 333 to alanine mutation (E333A), and a lysine 334 to alanine mutation (K334A) in human IgG1.In some embodiments, the multiple mutations include a mutation of serine 78 to alanine (S78A), a mutation of glutamate 113 to alanine (E113A), and a mutation of lysine 114 to alanine (K114A) in SEQ ID NO: 12. In some embodiments, the multiple mutations include a mutation of proline 247 to isoleucine (P247I) and a mutation of alanine 339 to glutamine (A339Q) in human IgG1. In some embodiments, the multiple mutations include a mutation of proline 27 to isoleucine (P27I) and a mutation of alanine 119 to glutamine (A119Q) in SEQ ID NO: 12. In some embodiments, the multiple mutations include a mutation of glycine 236 to alanine (G236A), a mutation of serine 239 to aspartic acid (S239D), and a mutation of isoleucine 332 to glutamic acid (I332E) in human IgG1. In some embodiments, the plurality of mutations includes a glycine 16 to alanine mutation (G16A), a serine 19 to aspartic acid mutation (S19D), and an isoleucine 112 to glutamic acid mutation (I112E) within SEQ ID NO: 12. In some embodiments, the G236A / S239D / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include a lysine 234 to tyrosine mutation (L234Y), a lysine 235 to glutamine mutation (L235Q), a glycine 236 to tryptophan mutation (G236W), a serine 239 to methionine mutation (S239M), a histidine 268 to aspartic acid mutation (H268D), an aspartic acid 270 to glutamate mutation (D270E), and a serine 298 to alanine mutation (S298A) in the first heavy chain of human IgG1, as well as an aspartic acid 270 to glutamate mutation (D270E), a lysine 326 to aspartic acid mutation (K26D), an alanine 330 to methionine mutation (A330M), and a lysine 334 to glutamate mutation (K334E) in the second heavy chain of IgG1. In some embodiments, the multiple mutations include a lysine 14 to tyrosine mutation (L14Y), a lysine 15 to glutamine mutation (L15Q), a glycine 16 to tryptophan mutation (G16W), a serine 19 to methionine mutation (S19M), a histidine 48 to aspartic acid mutation (H48D), an aspartic acid 50 to glutamate mutation (D50E), and a serine 78 to alanine mutation (S78A) within the first chain of SEQ ID NO: 12, and an aspartic acid 50 to glutamate mutation (D50E), a lysine 326 to aspartic acid mutation (K106D), an alanine 110 to methionine mutation (A110M), and a lysine 114 to glutamate mutation (K114E) within the second chain of SEQ ID NO: 12.It will be understood that all of the mutations given above for SEQ ID NO: 12 also apply to SEQ ID NO: 141. Indeed, they also apply to SEQ ID NO: 140 and SEQ ID NO: 142, but all numbers given above must be increased by 5 for these sequences.

[0191] In some embodiments, the ADCC-enhancing Fc domain comprises L15V / F23L / R72P / Y80L / P176L mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 12 and SEQ ID NO: 141. In some embodiments, the ADCC-enhancing Fc domain comprises EPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 143). In some embodiments, the ADCC-enhancing Fc domain consists of SEQ ID NO: 143. In some embodiments, the Fc comprising L15V / F23L / R72P / Y80L / P176L mutations is SEQ ID NO: 143. In some embodiments, the ADCC-enhancing Fc domain has at least 75, 80, 85, 90, 92, 95, 97, or 99% identity to SEQ ID NO: 143 and comprises L15V / F23L / R72P / Y80L / P176L mutations.

[0192] In some embodiments, the ADCC-enhancing Fc domain comprises S19D / A110L / I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 12 and SEQ ID NO: 141. In some embodiments, the ADCC-enhancing Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 144). In some embodiments, the ADCC-enhancing Fc domain consists of SEQ ID NO: 144. In some embodiments, the Fc comprising S19D / A110L / I112E mutations is SEQ ID NO: 144. In some embodiments, the ADCC-enhancing Fc domain has at least 75, 80, 85, 90, 92, 95, 97, or 99% identity to SEQ ID NO: 144 and comprises S19D / A110L / I112E mutations.

[0193] In some embodiments, the CDC-added Fc domain comprises G16A / S47E / H48F / S104T / I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 12 and SEQ ID NO: 141. In some embodiments, the CDC-added Fc domain comprises EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVTNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 145). In some embodiments, the CDC increased Fc domain consists of SEQ ID NO: 145. In some embodiments, the Fc comprising G16A / S47E / H48F / S104T / I112E mutations is SEQ ID NO: 145. In some embodiments, the CDC increased Fc domain has at least 75, 80, 85, 90, 92, 95, 97, or 99% identity to SEQ ID NO: 145 and comprises G16A / S47E / H48F / S104T / I112E mutations.

[0194] In some embodiments, the ADCC-enhancing Fc domain comprises G16A / A110L / I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 141. In some embodiments, the ADCC-enhancing Fc domain comprises EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 146). In some embodiments, the ADCC-enhancing Fc domain consists of SEQ ID NO: 146. In some embodiments, the Fc comprising G16A / A110L / I112E mutations is SEQ ID NO: 146. In some embodiments, the ADCC-enhancing Fc domain is at least 75, 80, 85, 90, 92, 95, 97, or 99% identical to SEQ ID NO: 146 and comprises G16A / A110L / I112E mutations.

[0195] In some embodiments, the effector domain is selected from the group consisting of SEQ ID NOs: 143-146. In some embodiments, the effector domain comprises any one of SEQ ID NOs: 143-146. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 143-146. In some embodiments, the effector domain is selected from the group consisting of SEQ ID NOs: 143, 144, and 146. In some embodiments, the effector domain comprises any one of SEQ ID NOs: 143, 144, and 146. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 143, 144, and 146. In some embodiments, the effector domain comprises at least 75, 80, 85, 90, 92, 95, 97, or 99% identity to any one of SEQ ID NOs: 143, 144, and 146 and retains increased ADCC compared to a control Fc domain. In some embodiments, the control Fc domain is an unmodified Fc domain. In some embodiments, the unmodified Fc is an Fc that occurs in nature. In some embodiments, the unmodified Fc is a human Fc that occurs in nature.

[0196] 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 enzymatically removed. In some embodiments, the Fc is non-fucosylated. In some embodiments, the method comprises performing non-fucosylation of the molecule. In some embodiments, the molecules of the invention are produced in a cell line engineered to produce non-fucosylated molecules.

[0197] In some embodiments, the mutation increases CDC. In some embodiments, multiple mutations increase CDC. In some embodiments, multiple mutations include a mutation of glycine 236 to alanine (G236A), a mutation of serine 267 to glutamate (S267E), a mutation of histidine 268 to aniline (H268F), a mutation of serine 324 to threonine (S324T), and a mutation of isoleucine 332 to glutamate (I332E) in human IgG1. In some embodiments, multiple mutations include a mutation of glycine 16 to alanine (G16A), a mutation of serine 47 to glutamate (S47E), a mutation of histidine 48 to aniline (H48F), a mutation of serine 104 to threonine (S104T), and a mutation of isoleucine 112 to glutamate (I112E) in SEQ ID NO: 12. In some embodiments, the multiple mutations include a lysine 326 to tryptophan mutation (K326W) and a glutamate 333 to serine mutation (E333S) in human IgG1. In some embodiments, the multiple mutations include a lysine 106 to tryptophan mutation (K106W) and a glutamate 113 to serine mutation (E113S) in SEQ ID NO: 12. In some embodiments, the multiple mutations include a glutamate 345 to arginine mutation (E345R), a glutamate 430 to glycine mutation (E430G), and a serine 440 to tyrosine mutation (S440Y) in human IgG1. In some embodiments, the multiple mutations include a glutamate 125 to arginine mutation (E125R), a glutamate 210 to glycine mutation (E210G), and a serine 220 to tyrosine mutation (S220Y) in SEQ ID NO: 12. It will be understood that all of the mutations given above for SEQ ID NO: 12 also apply to SEQ ID NO: 141. Indeed, they also apply to SEQ ID NO: 140 and SEQ ID NO: 142, but all numbers given above must be increased by 5 for these sequences.

[0198] In some embodiments, the effector moiety is a drug. In some embodiments, the protein is an AChR ECD drug conjugate. In some embodiments, the protein is an AChR-Fc drug conjugate. In some embodiments, the complex is an AChR ECD drug conjugate. In some embodiments, the complex is an AChR-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, the effector moiety is a radiolabeled drug. In some embodiments, the effector moiety is a chemotherapeutic agent. In some embodiments, the effector moiety is not a chemotherapeutic agent. In some embodiments, the effector moiety is toxic to non-replicating cells. In some embodiments, the toxicity is lethal. In some embodiments, the effector moiety is sufficient to kill cells. Drug conjugation, particularly conjugation of drugs to antibody backbones, is well known in the art, and any conjugation method can be used.

[0199] In some embodiments, the effector moiety is an amatoxin. In some embodiments, the effector moiety is an amatoxin. Amatoxins are a group of toxic compounds found in poisonous mushrooms. They are composed of eight amino acid residues arranged in a large bicyclic motif and inhibit RNA polymerase. Amatoxins are also known as amatoxins. In some embodiments, the amatoxin is selected from α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanullin, amanullinic acid, amaninamide, amanin, and proamanullin. In some embodiments, the amatoxin is α-amanitin. In some embodiments, the effector moiety is α-amanitin.

[0200] In some embodiments, the chemotherapeutic agent is an anthracycline. In some embodiments, the effector moiety is an anthracycline. Anthracyclines are a class of drugs extracted from Streptomyces bacteria that primarily intercalate into DNA and cause cytotoxicity by inhibiting topoisomerases. 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 agent is an anthramycin-based dimer. In some embodiments, the anthramycin-based dimer is a pyrrolobenzodiazepine (pyrrolobenzodiazepine, PBD). In some embodiments, the chemotherapeutic agent is PBD. In some embodiments, the anthramycin-based dimer is an indolin-benzodiazepine In some embodiments, the chemotherapeutic agent is a pyridobenzodiazepine dimer. (pyrridinobenzodiazepine, PDD). In some embodiments, the anthramycin-based dimer is PDD. In some embodiments, the effector moiety is PBD. In some embodiments, the effector moiety is PDD. PBD and PDD are a family of DNA minor groove binders that inhibit DNA and RNA synthesis. In some embodiments, PBD is a PBD dimer. Examples of PBD and PDD include, but are not limited to, anthramycin, SJG-136, NS 694501, and FGX2-62. In some embodiments, PBD is anthramycin. In some embodiments, the effector moiety is anthramycin. In some embodiments, anthramycin is anthramycin-methyl-ether (AME). In some embodiments, anthramycin is anthramycin-based dimer. In some embodiments, PBD is tescilin (SG3249). In some embodiments, tescilin is SG3199. In some embodiments, the chemotherapeutic agent is SG3249. In some embodiments, the chemotherapeutic agent is SG3199.

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

[0203] In some embodiments, the chemotherapeutic agent 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 delutec (Dxd). In some embodiments, the camptothecin analog is Dxd. In some embodiments, the chemotherapeutic agent is Dxd. In some embodiments, the effector moiety is Dxd.

[0204] In some embodiments, the chemotherapeutic agent is a duocarmycin. In some embodiments, the effector moiety is a duocarmycin. Duocarmycins are small molecules isolated from Streptomyces bacteria that bind to the minor groove of DNA and alkylate adenine bases. Examples of duocarmycins include, but are not limited to, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin™, duocarmycin MA, and CC-1065.

[0205] In some embodiments, the chemotherapeutic agent 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. Maytansinoids or maytansines are known to be tubulin inhibitors that inhibit the assembly of microtubules by binding to tubulin at the rhizoxin binding site. In some embodiments, the maytansinoid is maytansine (DM-1). In some embodiments, maytansine is emtansine. In some embodiments, the tubulin inhibitor is 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 tubulysin. In some embodiments, the tubulysin is tubulysin A. In some embodiments, the auristatin is MMAE. In some embodiments, the auristatin is MMAF. In some 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 multiple 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 amatoxins, anthracyclines, pyrrolobenzodiazepines, In some embodiments, the effector moiety comprises at least two cytotoxic moieties selected from the group consisting of amatoxins, anthracyclines, pyrrolobenzodiazepines, and cytotoxic agents. calicheamicin, camptothecin, duocarmycin, triptolide, and maytansinoids. 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 the protein target of the myasthenia gravis autoantibody. In some embodiments, the third fragment is different from the first fragment. In some embodiments, the third fragment is different from the second fragment. In some embodiments, the third fragment is identical to the first fragment. In some embodiments, the first fragment is identical to the second fragment. In some embodiments, the third fragment is identical to the first and second fragments. In some embodiments, identical refers to identical sequences. In some embodiments, different refers to different sequences.

[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 with each other. In some embodiments, the third and fourth dimerization domains are configured to dimerize with each other. In some embodiments, the third dimerization domain is not configured to dimerize with the first dimerization domain. In some embodiments, the third dimerization domain is not configured to dimerize with the second dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize with the first dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize with the second dimerization domain. In some embodiments, being configured to dimerize means being capable of dimerizing. In some embodiments, the third and fourth dimerization domains are different from the first and second dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, and the third and fourth dimerization domains are CH1 / CL domains. In some embodiments, the first and second dimerization domains are CH1 / CL domains, and the third and fourth dimerization domains are hinge domains.

[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 the protein target of the myasthenia gravis autoantibody. In some embodiments, the fourth fragment is different from the first fragment. In some embodiments, the fourth fragment is different from the second fragment. In some embodiments, the fourth fragment is different from the third fragment. In some embodiments, the fourth fragment is identical to the first fragment. In some embodiments, the fourth fragment is identical to the second fragment. In some embodiments, the fourth fragment is identical to the third fragment. In some embodiments, the fourth fragment is identical to the first, second, and third fragments. In some embodiments, the first, second, and third fragments are all identical. In some embodiments, the first, second, third, and fourth fragments are all different. In some embodiments, the identical sequences are identical. In some embodiments, the different sequences are different. In some embodiments, the different sequences are from different proteins. In some embodiments, the different sequences are from the same protein but include different sequences. In some embodiments, the different sequences are from the same protein but from different regions of the protein. In some embodiments, at least two of the first, second, third, and fourth proteins are part of a single protein complex. In some embodiments, the protein complex is a complex in a mammal. In some embodiments, the protein complex is a complex in a human.

[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 with each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerize with each other. In some embodiments, the fifth dimerization domain is not configured to dimerize with the first dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize with the second dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize with the third dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize with the fourth dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize with the first dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize with the second dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize with the third dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize with the fourth dimerization domain. In some embodiments, the fifth and sixth dimerization domains are different from the first and second dimerization domains. In some embodiments, the fifth and sixth dimerization domains are different from the third and fourth dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, the third and fourth dimerization domains are CH1 / CL domains, and the fifth and sixth dimerization domains are CH1 / CL domains. In some embodiments, the first and second dimerization domains are CH1 / CL domains, the third and fourth dimerization domains are hinge domains, and the fifth and sixth dimerization domains are hinge domains. In some embodiments, the first polypeptide and the second polypeptide do not both include a CH1 domain. In some embodiments, the first polypeptide and the second polypeptide both include a CH1 domain. The first polypeptide and the second polypeptide both include a CL domain. In some embodiments, the first polypeptide and the second polypeptide do not both include a CL domain. In some embodiments, the first polypeptide includes a CH1 domain, and the second polypeptide includes a CL domain. In some embodiments, the third polypeptide comprises a CL domain and the fourth polypeptide comprises a CH1 domain. In some embodiments, the first polypeptide comprises a CL domain and the second polypeptide comprises a CH1 domain. In some embodiments, the third polypeptide comprises a CH1 domain and the fourth polypeptide comprises a CL domain.

[0212] In some embodiments, the third and fourth dimerization domains include mutations that allow dimerization of the third and fourth dimerization domains and inhibit dimerization of the third dimerization domain with the fifth, sixth, or both dimerization domains. In some embodiments, the third and fourth dimerization domains include mutations that allow dimerization of the third and fourth dimerization domains and inhibit dimerization of the fourth dimerization domain with the fifth, sixth, or both dimerization domains. In some embodiments, the fifth and sixth dimerization domains include mutations that allow dimerization of the fifth and sixth dimerization domains and inhibit dimerization of the fifth dimerization domain with the third, fourth, or both dimerization domains. In some embodiments, the fifth and sixth dimerization domains include mutations that allow dimerization of the fifth and sixth dimerization domains and inhibit dimerization of the sixth dimerization domain with the third, sixth, or both dimerization domains. Optional configuration

[0213] In some embodiments, the composition includes a polypeptide chain comprising: a fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; and a fragment of a second protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof. In some embodiments, the polypeptide chain is a single polypeptide chain. In some embodiments, the single chain comprises a fragment of the first protein and a fragment of the second protein. In some embodiments, the polypeptide chain further comprises a fragment of a third protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof. In some embodiments, the polypeptide chain further comprises a fragment of a fourth protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof. In some embodiments, the polypeptide chain further comprises an Fc region.

[0214] In some embodiments, a fragment of a first protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, is separated from a fragment of a second protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, by a linker. In some embodiments, a fragment of a third protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, is separated from a fragment of a first or second protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, by a linker. In some embodiments, a fragment of a fourth protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, is separated from a fragment of a first, second, or third protein target of an autoantibody to myasthenia gravis, or an analog or derivative thereof, by a linker. In some embodiments, the fragment is separated from the Fc region by a linker. In some embodiments, the fragment is separated from the effector portion by a linker.

[0215] 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 dimerization domain and the effector portion are separated by a linker. In some embodiments, the fragment and the Fc region are separated by a linker. In some embodiments, the fragment and the effector portion 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 can be used. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is a polyethylene glycol (PEG) linker. In some embodiments, the PEG linker is a Gly 3-PEG-azide linker. In some embodiments, the linker is a dibenzocyclooctyne (DBCO) linker. In some embodiments, the DBCO linker is a DBCO-C6 linker. In some embodiments, the DBCO linker is a DBCO-Gly5-EDA linker. In some embodiments, the linker is a dimethylethylenediamine (DMEDA) linker. In some embodiments, the linker is an N-dimethylethylenediamine (DMAE) linker. In some embodiments, the linker is a glutathione linker. In some embodiments, the linker is a CLICK linker. In some embodiments, the CLICK linker is a CLICK-DBCO linker. In some embodiments, the CLICK linker is a CLICK azide linker. In some embodiments, is a disulfide linker. In some embodiments, the linker is a thiol linker. In some embodiments, the linker is an azide linker. In some embodiments, the linker is a maleimide (Mal) linker. In some embodiments, the Mal linker is a maleimidocaproyl linker. In some embodiments, the Mal linker is a Mal-C6 linker. In some embodiments, the Mal linker is a Mal-Gly5-EDA linker. In some embodiments, the linker is a lysine linker. In some embodiments, the linker is an asparagine linker. In some embodiments, the linker is an acid-labile linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the 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 butyric acid linkers. In some embodiments, the linker is a p-aminobenzyl (PAB) linker. In some embodiments, the linker is a p-aminocarbamate (PABC) linker. In some embodiments, the linker is a maleimidocaproyl (mc) linker. In some embodiments, the linker comprises mc. In some embodiments, the linker is a Val-Cit-PAB linker. In some embodiments, the linker is a Val-Cit-PABC linker. In some embodiments, the linker is a Val-Cit-PAB-MMAE linker. In some embodiments, the linker is an mc-VC-PABC-MMAE linker. In some embodiments, the linker is an mc-MMAF linker. In some embodiments, the linker is a monomethyl auristatin E (MMAE) linker. Examples of peptide linkers include, but are not limited to, Val-Cit-PAB linkers, Phe-Lys(Trt)-PAB linkers, and Ala-Ala-Asn-PAB linkers. In some embodiments, the linker is a mixture of linkers. In some embodiments, the linker is a DBCO-PEG linker. In some embodiments, the linker is a PBCO-PEG-DMEDA linker. In some embodiments, the linker is a DBCO-PEG-VC-PAB-DMEDA linker. In some embodiments, VC in the linker is replaced by EVC. In some embodiments, VC in the linker is replaced by EVA. In some embodiments, the fragment and the dimerization domain are connected by a non-cleavable linker. In some embodiments, the fragment and the dimerization domain are connected by a cleavable linker. In some embodiments, the effector moiety is connected by a cleavable linker. In some embodiments, the effector moiety is connected by a non-cleavable linker.

[0216] In some embodiments, the conjugation is a connection. In some embodiments, the conjugation is through a bond. In some embodiments, the conjugate is directly conjugated. In some embodiments, the conjugate is conjugated through a linker.

[0217] In some embodiments, the conjugation is an amino acid linker, a portion, or both, and includes an extension of the amino acid sequence of the chain of the agent of the present invention. It should be understood that the nucleic acid molecule encoding the agent of the present invention can be modified to include the coding sequence of the linker, a portion, or both, and thus a complete conjugate will be produced when translated. In some embodiments, the conjugate is a fusion protein. Methods for connecting and conjugating the various parts are well known in the art, and any such method can be used. In some embodiments, the method is a combination of at least two methods. Specifically, methods for connecting and conjugating to IgG scaffolds are also well known. The methods of connection / conjugation include, but are not limited to, native cysteine reduction (including native hinge reduction, also referred to herein as native cysteine conjugation), engineered cysteine reduction, disulfide 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 amineazide, and glycan remodeling.

[0218] In some embodiments, the conjugation is site-specific. In some embodiments, the conjugation is not random. In some embodiments, the conjugation or connection is to an IgG backbone. In some embodiments, the conjugation or connection is not to an AChR fragment. In some embodiments, the conjugation or connection does not interfere with the binding of the antibody to the AChR fragment. In some embodiments, the antibody is an autoantibody. In some embodiments, the conjugation or connection is to a dimerization domain. In some embodiments, the conjugation or connection is to a hinge region. In some embodiments, the conjugation or connection is to a CH2 region. In some embodiments, the conjugation or connection is to a CH3 region. In some embodiments, the conjugation or connection is to a CH1 region. In some embodiments, the conjugation or connection is to a CL region. In some embodiments, the connection or conjugation is to a natural amino acid residue. In some embodiments, the connection or conjugation is to an engineered amino acid residue. In some embodiments, the residue is cysteine. Examples of engineered cysteine residues include, but are not limited to, A231C, S239C, N325C, L328C, D265C, and S442C of the heavy chain of IgG. In some embodiments, the residue is lysine. In some embodiments, the residue is asparagine. In some embodiments, glycan remodeling is used for attachment to asparagine. In some embodiments, the asparagine is N297 of the heavy chain of IgG. In some embodiments, the residue is 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. Examples of engineered glutamine residues include, but are not limited to, Q297. Unless otherwise specified, these sites are provided with the Kabat numbering of IgG1; corresponding mutations may be made in other IgGs, and in particular in other IgGs. In some embodiments, the attachment or conjugation is to the C- or N-terminus of the chain of the agent of the present invention. In some embodiments, the attachment or conjugation is to the C-terminus. In some embodiments, the connection or conjugation is to the N-terminus. In some embodiments, the terminus is the terminus of the heavy chain. In some embodiments, the terminus is the terminus of the light chain. In some embodiments, the conjugation or connection is to multiple sites.

[0219] In some embodiments, the length of the linker is sufficient to suppress steric hindrance between different segments of the chain. In some embodiments, the length of the linker is sufficient to suppress steric hindrance between different segments of the conjugate. In some embodiments, the length of the linker is sufficient to allow the antibody to bind to the fragment without steric hindrance from other segments of the chain. In some embodiments, the length of the linker is sufficient to allow the antibody to bind to the fragment without steric hindrance from other segments of the conjugate. In some embodiments, the length of the linker is sufficient to allow cells to bind to the fragment without steric hindrance from other segments of the chain. In some embodiments, the length of the linker is sufficient to allow cells to bind to the fragment without steric hindrance from other segments of the conjugate. In some embodiments, the length of the linker is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the length of the linker is at least 1 amino acid. In some embodiments, the length of the linker is at least 5 amino acids. In some embodiments, the length of the linker is at least 10 amino acids. In some embodiments, the length of the connector is at least 15 amino acids. In some embodiments, the length of the connector is at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the length of the connector is at most 10 amino acids. In some embodiments, the length of the connector is at most 20 amino acids. In some embodiments, the length of the connector is at most 50 amino acids. In some embodiments, the length of the connector is at most 100 amino acids.

[0220] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a GS linker. In some embodiments, the linker is a glycine-serine containing linker. In some embodiments, the linker consists of glycine and serine residues. In some embodiments, the linker comprises GGGS (SEQ ID NO: 38). In some embodiments, the linker comprises GGGGS (SEQ ID NO: 136). In some embodiments, the linker consists of SEQ ID NO: 38. In some embodiments, the linker consists of SEQ ID NO: 136. In some embodiments, the linker comprises (GGGS)n, where n is an integer. In some embodiments, the linker comprises (GGGGS)n, where n is an integer. In some embodiments, the linker consists of (GGGS)n, where n is an integer. In some embodiments, the linker consists of (GGGS)n, where n is an integer. In some embodiments, the linker consists of (GGGGS)n, where n is an integer. In some embodiments, the linker consists of GSAGSAAGSGEF (SEQ ID NO: 45). 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, 5, and 6. Each possibility represents a separate embodiment of the present invention. In some embodiments, n is 6. In some embodiments, the linker is a rigid linker. In some embodiments, the rigid linker comprises EAAAK (SEQ ID NO: 137). In some embodiments, the rigid linker consists of SEQ ID NO: 137. In some embodiments, the rigid linker comprises (EAAAK)n, wherein n is an integer. In some embodiments, the rigid linker consists of (EAAAK)n, wherein n is an integer. In some embodiments, the rigid linker comprises (EAAAK)nGS, wherein n is an integer. In some embodiments, the rigid linker consists of (EAAAK)nGS, wherein n is an integer. In some embodiments, the rigid linker comprises (EAAAK)nGGS, wherein n is an integer. In some embodiments, the rigid linker consists of (EAAAK)nGGS, wherein n is an integer. In some embodiments, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each possibility represents a separate embodiment of the present invention. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0221] In some embodiments, the dimerization domain is at the C-terminus of the fragment. In some embodiments, the fragment is at the C-terminus of the dimerization domain. In some embodiments, the Fc region is at the C-terminus of the fragment. In some embodiments, the fragment is at the C-terminus of the Fc region. In some embodiments, the dimerization domain is at the C-terminus of the Fc region. In some embodiments, the Fc region is at the C-terminus of the dimerization domain. In some embodiments, the dimerization domain is at the N-terminus of the fragment. In some embodiments, the fragment is at the N-terminus of the dimerization domain. In some embodiments, the Fc region is at the N-terminus of the fragment. In some embodiments, the fragment is at the N-terminus of the Fc region. In some embodiments, the dimerization domain is at the N-terminus of the Fc region. In some embodiments, the Fc region is at the N-terminus of the dimerization domain.

[0222] 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 composite epitope. In some embodiments, the epitope is a B cell receptor (BCR) specific epitope.

[0223] In some embodiments, all three fragments are from AChRa. In some embodiments, all three fragments are from AChRb. In some embodiments, all three fragments are from AChRg. In some embodiments, all three fragments are from AChRd. In some embodiments, all three fragments are from AChRe. In some embodiments, the three fragments are selected from AChRa, AChRb, AChRg, AChRd, and AChRe. In some embodiments, the three fragments comprise two different proteins from AChRa, AChRb, AChRg, AChRd, and AChRe. In some embodiments, the three fragments comprise three different proteins from AChRa, AChRb, AChRg, AChRd, and AChRe.

[0224] In some embodiments, all four segments are derived from AChRa. In some embodiments, all four segments are derived from AChRb. In some embodiments, all four segments are derived from AChRg. In some embodiments, all four segments are derived from AChRd. In some embodiments, all four segments are derived from AChRe. In some embodiments, the four segments are selected from AChRa, AChRb, AChRg, AChRd, and AChRe. In some embodiments, the four segments comprise two different proteins from AChRa, AChRb, AChRg, AChRd, and AChRe. In some embodiments, the four segments comprise three different proteins from AChRa, AChRb, AChRg, AChRd, and AChRe. In some embodiments, the four segments comprise four different proteins from AChRa, AChRb, AChRg, AChRd, and AChRe.

[0225] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 50). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 50. In some embodiments, the first and second polypeptides both include a fragment linked to SEQ ID NO:50.

[0226] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 51. In some embodiments, the first and second polypeptides both include a fragment linked to SEQ ID NO:51.

[0227] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 52). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 52. In some embodiments, the first and second polypeptides both include a fragment linked to SEQ ID NO:52.

[0228] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 include a fragment linked to SEQ ID NO:53.

[0229] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 include a fragment linked to SEQ ID NO:54.

[0230] In some embodiments, the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 include a fragment linked to SEQ ID NO:55.

[0231] In some embodiments, the first polypeptide comprises a fragment linked to GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 include a fragment linked to SEQ ID NO:56.

[0232] In some embodiments, the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 57). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 57. In some embodiments, the first and second polypeptides both include a fragment linked to SEQ ID NO:57.

[0233] In some embodiments, the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 58). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 58. In some embodiments, the first and second polypeptides both include a fragment linked to SEQ ID NO:58.

[0234] In some embodiments, the first polypeptide comprises a fragment linked to GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 include a fragment linked to SEQ ID NO:59.

[0235] In some embodiments, the first polypeptide comprises a fragment linked to GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 include a fragment linked to SEQ ID NO:60.

[0236] In some embodiments, the third polypeptide comprises a fragment connected to SEQ ID NO:23. In some embodiments, the third polypeptide comprises a fragment connected to SEQ ID NO:24. In some embodiments, the third polypeptide comprises a fragment connected to SEQ ID NO:25. In some embodiments, the third polypeptide comprises a fragment connected to SEQ ID NO:26. In some embodiments, the third polypeptide comprises a fragment connected to SEQ ID NO:27. In some embodiments, the third polypeptide comprises a fragment connected to SEQ ID NO:28. In some embodiments, the fourth polypeptide comprises a fragment connected to SEQ ID NO:23. In some embodiments, the fourth polypeptide comprises a fragment connected to SEQ ID NO:24. In some embodiments, the fourth polypeptide comprises a fragment connected to SEQ ID NO:25. In some embodiments, the fourth polypeptide comprises a fragment connected to SEQ ID NO:26. In some embodiments, the fourth polypeptide comprises a fragment connected to SEQ ID NO:27. In some embodiments, the fourth polypeptide comprises a fragment connected to SEQ ID NO:28.

[0237] In some embodiments, the polypeptide chain comprises a fragment of AChRa comprising a mutation that increases solubility and connected to a light chain CLκ domain via a linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCDVSGVDTESGATNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPGGGGSGGGGSGGGGSAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 64).

[0238] In some embodiments, the polypeptide chain comprises: a fragment of AChR a comprising a mutation that increases solubility and connected by a linker to a heavy chain comprising a CH2 and CH3 domain, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 92. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 95. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 97. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 98. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 99. It will be understood that unless otherwise expressly stated, the above-mentioned CH2 and CH3 domains and all other CH2 / CH3 domains are derived from IgG1.

[0239] In some embodiments, the polypeptide chain comprises: a fragment of AChRb comprising a mutation that increases solubility and connected via a linker to a heavy chain comprising CH1, CH2, and CH3 domains. In some embodiments, the polypeptide comprises or consists of the amino acid sequence (SEQ ID NO: 65).

[0240] In some embodiments, the polypeptide chain comprises: a fragment of AChRb comprising a mutation that increases solubility and connected via a linker to a heavy chain comprising CH1, hinge, CH2, and CH3 domains. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 65.

[0241] In some embodiments, the protein complex comprises two polypeptides, each comprising SEQ ID NO: 65. In some embodiments, the protein complex comprises two polypeptides, each consisting of SEQ ID NO: 65. In some embodiments, the protein complex comprises: a first polypeptide chain comprising or consisting of SEQ ID NO: 65; and a second polypeptide chain comprising or consisting of SEQ ID NO: 64. In some embodiments, the protein complex further comprises: a third polypeptide chain comprising or consisting of SEQ ID NO: 65. In some embodiments, the protein complex further comprises: a fourth polypeptide chain comprising or consisting of SEQ ID NO: 64.

[0242] In some embodiments, the polypeptide chain comprises: a fragment of an AChRb comprising a solubility-enhancing mutation connected to a heavy chain comprising a CH1, hinge, CH2, and CH3 domains via a linker, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the mutation that inhibits homodimerization is T366W. In some embodiments, the polypeptide comprises or consists of the following: the amino acid sequence (SEQ ID NO: 66).

[0243] In some embodiments, the polypeptide chain comprises: a fragment of AChRg comprising a mutation that increases solubility and connected by a linker to a heavy chain comprising a CH1, hinge, CH2, and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the mutation that inhibits homodimerization is T366S, L368A, and Y407V. In some embodiments, the polypeptide comprises or consists of the following: amino acid sequence (SEQ ID NO: 67).

[0244] In some embodiments, the polypeptide chain comprises: a fragment of AChRg comprising a mutation that increases solubility and at least one mutation that reduces aggregation, connected via a linker to a heavy chain comprising CH2 and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 93. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 100.

[0245] In some embodiments, the polypeptide chain comprises: a fragment of AChRd comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, connected via a linker to a heavy chain comprising CH2 and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:98. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:102. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:103. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:106. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:107. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:130. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO:92; and a second chain comprising or consisting of SEQ ID NO:93. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 92; and a second chain comprising or consisting of SEQ ID NO: 102. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 103; and a second chain comprising or consisting of SEQ ID NO: 102. One skilled in the art will appreciate that in this embodiment, the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S / L368A / Y407V mutations, but the mutations can be switched to opposite chains and the molecule will still be operable.

[0246] In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a solubility-enhancing mutation, linked to an AChRg via a GS linker; the AChRg comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, linked to a heavy chain comprising a CH2 and CH3 domain via a linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 94. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 104.

[0247] In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a mutation that increases solubility, linked to an AChRg via a GS linker, wherein the AChRg comprises a mutation that increases solubility and at least one mutation that reduces aggregation, and is linked via a linker to a heavy chain comprising CH2 and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 95. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 105.

[0248] In some embodiments, the polypeptide chain comprises: a fragment of AChR a comprising a mutation that increases solubility, linked to an AChR d via a GS linker, the AChR d comprising a mutation that increases solubility and at least one mutation that decreases aggregation, and linked via a linker to a heavy chain comprising a CH2 and CH3 domain, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 96. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 95; and a second chain comprising or consisting of SEQ ID NO: 96. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 106. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 107. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 105; and a second chain comprising or consisting of SEQ ID NO: 106. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 105; and a second chain comprising or consisting of SEQ ID NO: 107. One skilled in the art will appreciate that in this embodiment, the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S / L368A / Y407V mutations, but the mutations can be switched to opposite chains and the molecule still be operable.

[0249] In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a solubility-enhancing mutation and an aggregation-reducing mutation, linked to AChRg via a GS linker, the AChRg comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, and linked to a heavy chain comprising CH2 and CH3 domains via a linker, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 97. In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a solubility-enhancing mutation and an aggregation-reducing mutation, linked to AChRg via a GS linker, the AChRg comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, and linked to a heavy chain comprising CH2 and CH3 domains via a linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 114.

[0250] In some embodiments, the polypeptide chain comprises: a fragment of AChRd comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, connected via a linker to a heavy chain comprising CH2 and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:99. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO:97; and a second chain comprising or consisting of SEQ ID NO:98. Those skilled in the art will appreciate that in this embodiment, the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S / L368A / Y407V mutations, but the mutations can be switched to the opposite chain and the molecule will still be operable.

[0251] In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a solubility-enhancing mutation and an aggregation-reducing mutation, linked to an AChRd via a GS linker, wherein the AChRd comprises a solubility-enhancing mutation and at least one aggregation-reducing mutation, and is linked via a linker to a heavy chain comprising a CH2 and CH3 domain, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 99. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 106. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 107.

[0252] In some embodiments, the polypeptide chain comprises: a fragment of AChRg comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, connected by a linker to a heavy chain comprising CH2 and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 100. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 99; and a second chain comprising or consisting of SEQ ID NO: 100. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 92; and a second chain comprising or consisting of SEQ ID NO: 102. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 103; and a second chain comprising or consisting of SEQ ID NO: 100. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 105; and a second chain comprising or consisting of SEQ ID NO: 130. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 105; and a second chain comprising or consisting of SEQ ID NO: 106. In some embodiments, the composition comprises: a first chain comprising or consisting of SEQ ID NO: 105; and a second chain comprising or consisting of SEQ ID NO: 107. One skilled in the art will appreciate that in this embodiment, the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S / L368A / Y407V mutations, but the mutations can be switched to opposite chains and the molecule will still be operable.

[0253] In some embodiments, the polypeptide chain comprises: a fragment of an AChRg comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, linked via a GS linker to an AChRα comprising a solubility-enhancing mutation, linked via a GS linker to an AChRd comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, linked via a GS linker to a heavy chain comprising a CH2 and CH3 domains. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 108. In some embodiments, the polypeptide chain comprises: a fragment of an AChRg comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, linked via a GS linker to an AChRα comprising a solubility-enhancing mutation, linked via a GS linker to an AChRg comprising a solubility-enhancing mutation and at least one aggregation-reducing mutation, linked via a GS linker to a heavy chain comprising a CH2 and CH3 domains. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 109. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 116.

[0254] In some embodiments, the polypeptide chain comprises: a fragment of an AChRg comprising a mutation that increases solubility and at least one mutation that decreases aggregation, linked to an AChRa comprising a mutation that increases solubility and a mutation that decreases aggregation, via a GS linker, the AChRa linked to an AChRg comprising a mutation that increases solubility and at least one mutation that decreases aggregation, via a GS linker, and linked to a heavy chain comprising a CH2 and CH3 domain via a linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 115.

[0255] In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a solubility-increasing mutation and a solubility-reducing mutation, linked via a GS linker to an AChRg comprising a solubility-increasing mutation and at least one aggregation-reducing mutation, and linked via a linker to a heavy chain comprising CH2 and CH3 domains. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 110. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 111. Those skilled in the art will appreciate that, where the tandem subunits are separated by a linker, the order of the subunits can be as described above or can be reversed.

[0256] In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a mutation that increases solubility, linked to an AChRg comprising a mutation that increases solubility and at least one mutation that reduces aggregation, via a GS linker, and linked to a heavy chain comprising the CH2 and CH3 domains from IgG4 via a linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 112. In some embodiments, in any of SEQ ID NOs: 92-111, 114-116, and 124-126, the CH2 and CH3 domains from IgG1 are replaced with the CH2 and CH3 domains from IgG4. It will be understood that any mutations present that reduce homodimerization will be conservative and also present in the IgG4 CH3.

[0257] In some embodiments, the polypeptide chain comprises: a fragment of AChRα comprising a mutation that increases solubility, linked to an AChRg comprising a mutation that increases solubility and at least one mutation that decreases aggregation, via a GS linker, and linked to a heavy chain comprising CH2 and CH3 comprising mutations that decrease effector function. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 113. In some embodiments, in any of SEQ ID NOs: 92-111, 114-116, and 124-126, CH2 and CH3 may comprise mutations that decrease effector function.

[0258] It will be understood that although specific linkers are provided in the above molecules, any linker can be used. In some embodiments, any flexible linker can be used. In some embodiments, the linker is a (GGGGS)6 linker. In some embodiments, the linker between two subunits is a (GGGGS)6 linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 117. In some embodiments, the linker is a (GGGGS)3 linker. In some embodiments, the linker of the CH2 domain is a (GGGGS)3 linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 118. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 127. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 128. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 129. In some embodiments, the linker is a (GGGGS)6GS linker. In some embodiments, the linker between the two subunits is a (GGGGS)6GS linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 119. In some embodiments, any flexible linker can be used. In some embodiments, the linker is a (GGGGS)5 linker. In some embodiments, the linker between the two subunits is a (GGGGS)5 linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 120.

[0259] In some embodiments, the connector is a rigid connector. In some embodiments, the connector between the two subunits is a rigid connector. In some embodiments, the connector is a (EAAAK)2GGS connector. In some embodiments, the connector between the two subunits is a (EAAAK)2GGS connector. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 121. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 122. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 123.

[0260] In some embodiments, the polypeptide chain comprises CH2 and CH3 domains, which are linked to a fragment of AChRg via a GS linker, the fragment of AChRg comprising a mutation that increases solubility and at least one mutation that reduces aggregation and linked to an AChRa comprising a mutation that increases solubility and at least one mutation that reduces aggregation via a GS linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 124. In some embodiments, the polypeptide chain comprises CH2 and CH3 domains, which are linked to a fragment of AChRa comprising a mutation that increases solubility and at least one mutation that reduces aggregation via a GS linker and linked to an AChRg comprising a mutation that increases solubility and at least one mutation that reduces aggregation via a GS linker. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 125.

[0261] In some embodiments, the protein complex comprises: a first polypeptide comprising or consisting of SEQ ID NO: 66; and a second polypeptide comprising or consisting of SEQ ID NO: 67. One skilled in the art will appreciate that in this embodiment, the polypeptide comprising AChRb comprises the T366W mutation and the polypeptide comprising AChRg comprises T366S / L368A / Y407V, but the mutations can be switched to opposite chains and the molecule still be operable (see, e.g., SEQ ID NO: 69). In some embodiments, the protein complex comprises: a first polypeptide chain comprising or consisting of SEQ ID NO: 66; and a second polypeptide chain comprising or consisting of SEQ ID NO: 64. In some embodiments, the protein complex comprises: a first polypeptide chain comprising or consisting of SEQ ID NO: 67; and a second polypeptide chain comprising or consisting of SEQ ID NO: 64. In some embodiments, the protein complex further comprises: a first polypeptide comprising or consisting of SEQ ID NO: 66; a second polypeptide comprising or consisting of SEQ ID NO: 67; and a third polypeptide chain comprising or consisting of SEQ ID NO: 64. In some embodiments, the protein complex further comprises: a fourth polypeptide chain comprising or consisting of SEQ ID NO: 64.

[0262] In some embodiments, the polypeptide chain comprises: a fragment of AChRe comprising a mutation that increases solubility and connected by a linker to a heavy chain comprising a CH1, hinge, CH2, and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the mutation that inhibits homodimerization is T366S, L368A, and Y407V. In some embodiments, the polypeptide comprises or consists of the following: amino acid sequence (SEQ ID NO: 68).

[0263] In some embodiments, the protein complex comprises: a first polypeptide comprising or consisting of SEQ ID NO:66; and a second polypeptide comprising or consisting of SEQ ID NO:68. One skilled in the art will appreciate that in this embodiment, the polypeptide comprising AChRb comprises the T366W mutation and the polypeptide comprising AChRe comprises T366S / L368A / Y407V, but the mutations can be switched to opposite chains and the molecule still be operable. In some embodiments, the protein complex comprises: a first polypeptide chain comprising or consisting of SEQ ID NO:68; and a second polypeptide chain comprising or consisting of SEQ ID NO:64. In some embodiments, the protein complex further comprises: a first polypeptide comprising or consisting of SEQ ID NO:66; a second polypeptide comprising or consisting of SEQ ID NO:68; and a third polypeptide chain comprising or consisting of SEQ ID NO:64. In some embodiments, the protein complex further comprises: a fourth polypeptide chain comprising or consisting of SEQ ID NO:64.

[0264] In some embodiments, the polypeptide chain comprises: a fragment of AChRg comprising a solubility-enhancing mutation and connected via a linker to a heavy chain comprising a CH1, hinge, CH2, and CH3 domains, wherein the CH3 domain comprises a mutation that inhibits homodimerization of the polypeptide chain. In some embodiments, the mutation that inhibits homodimerization is T366W. In some embodiments, the polypeptide comprises or consists of the following: amino acid sequence (SEQ ID NO: 69).

[0265] In some embodiments, the protein complex comprises: a first polypeptide comprising or consisting of SEQ ID NO:67; and a second polypeptide comprising or consisting of SEQ ID NO:69. Those skilled in the art will appreciate that such a molecule can also be prepared using a CH3 domain without a mutation that inhibits homodimerization. In this case, only a single polypeptide chain would be required, as it would homodimerize. This polypeptide would be similar to SEQ ID NO:65, but would include an AChRg fragment instead of an AChRb fragment.

[0266] In some embodiments, the protein complex comprises: a first polypeptide comprising or consisting of SEQ ID NO:69; and a second polypeptide comprising or consisting of SEQ ID NO:68. One skilled in the art will appreciate that in this embodiment, the polypeptide comprising AChRg comprises the T366W mutation and the polypeptide comprising AChRe comprises T366S / L368A / Y407V, but the mutations can be switched to opposite chains and the molecule still be operable. In some embodiments, the protein complex comprises: a first polypeptide chain comprising or consisting of SEQ ID NO:69; and a second polypeptide chain comprising or consisting of SEQ ID NO:64. In some embodiments, the protein complex further comprises: a first polypeptide comprising or consisting of SEQ ID NO:69; a second polypeptide comprising or consisting of SEQ ID NO:68; and a third polypeptide chain comprising or consisting of SEQ ID NO:64. In some embodiments, the protein complex further comprises: a fourth polypeptide chain comprising or consisting of SEQ ID NO:64.

[0267] In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 70% identical to a sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 75% identical to a sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 80% identical to a sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 85% identical to a sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 90% identical to a sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 95% identical to a sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 97% identical to a sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence that is at least 99% identical to a sequence provided herein.

[0268] In some embodiments, the protein is selected from any one of SEQ ID NOs: 72-91. In some embodiments, the polypeptide is selected from any one of SEQ ID NOs: 72-91. In some embodiments, the polypeptide is selected from any one of SEQ ID NOs: 92-100 and 102-130. In some embodiments, the polypeptide is selected from any one of SEQ ID NOs: 72-100 and 102-130. In some embodiments, the polypeptide is selected from any one of SEQ ID NOs: 94, 104, and 108-129. Pharmaceutical composition

[0269] In another aspect, a pharmaceutical composition is provided, which comprises a protein of the present invention.

[0270] In another aspect, a pharmaceutical composition is provided comprising a polypeptide chain of the invention.

[0271] In another aspect, a pharmaceutical composition is provided, which comprises a protein complex of the present invention.

[0272] In another aspect, a pharmaceutical composition is provided, comprising a composition of the present invention.

[0273] In some embodiments, the pharmaceutical composition includes 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 an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can be used 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 carboxymethylcellulose, ethylcellulose 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, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions and phosphate buffered saline, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can be used as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein.In this regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, such as those described in The Merck Index, 13th edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th edition (2004); and "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (the entire contents of which are hereby incorporated by reference in their entirety). Examples of pharmaceutically acceptable excipients, carriers, and diluents for use 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 herein by reference in its entirety). The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, slow-release particles, and other vehicles that increase the half-life of the peptide or polypeptide in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes used with the peptides described herein are formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is generally determined by considerations such as liposome size and stability in blood.For example, Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, reviews various methods that can be used to prepare liposomes, and see also US Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0274] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0275] In some embodiments, the pharmaceutical composition is used to treat myasthenia gravis. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a protein complex of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a conjugate of the present invention. The term "therapeutically effective amount" refers to an amount of a drug that is effective to treat a disease or disorder in a mammal. In some embodiments, a therapeutically effective amount is an amount that is effective to achieve the desired therapeutic or preventive result at the necessary dosage and for the necessary period of time. The exact dosage form and regimen will be determined by the physician based on the patient's condition. In some embodiments, an effective amount is an amount sufficient to treat at least one symptom of the disease. In some embodiments, the disease is myasthenia gravis. In some embodiments, myasthenia gravis is characterized by autoantibodies to the protein.

[0276] As used herein, the terms "treatment" or "treating" of a disease, disorder, or condition encompass alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, the compositions or methods useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of the symptoms associated therewith, or improve the quality of life of the patient or subject. Treatments for myasthenia gravis are well known in the art and may include any acceptable measure for assessing improvement in the symptoms of myasthenia gravis. This may include: improved muscle control; reduced muscle drooping, lapping, or heaviness; improved breathing; reduced autoantibody titers; improved synaptic function; or any other improvement measures.

[0277] 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.

[0278] As used herein, the terms "administering," "administration," and similar terms refer to any method of delivering a composition comprising an active agent to a subject in a manner that provides a therapeutic effect in reasonable medical practice. 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. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal. In some embodiments, administration is intravenous administration. In some embodiments, administration is selected from oral, intravenous, intramuscular, intraperitoneal, intertumoral, topical, or subcutaneous administration. In some embodiments, administration is administration to the site of disease.

[0279] 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.

[0280] In some embodiments, the compositions are formulated to increase the hydrophilicity of the molecules of the invention. In some embodiments, the compositions are formulated to reduce aggregation of the molecules of the invention. Formulations that increase hydrophilicity are well known in the art, and any such formulation can be used. For example, a hydrophilic carrier or polymer (e.g., PEG) can be added to the formulation to increase hydrophilicity and reduce aggregation. Treatment

[0281] In another aspect, a method of treating myasthenia gravis in a subject in need thereof is provided, the method comprising administering to the subject a protein of the invention, thereby treating myasthenia gravis in the subject.

[0282] In another aspect, a method of treating myasthenia gravis in a subject in need thereof is provided, the method comprising administering to the subject a polypeptide chain of the invention, thereby treating myasthenia gravis in the subject.

[0283] In another aspect, a method of treating myasthenia gravis in a subject in need thereof is provided, the method comprising administering to the subject a protein complex of the invention, thereby treating myasthenia gravis in the subject.

[0284] In another aspect, provided is a method of treating myasthenia gravis in a subject in need thereof, the method comprising administering to the subject a composition of the present invention, thereby treating myasthenia gravis in the subject.

[0285] In some embodiments, administration is administration of a pharmaceutical composition of the present invention. In some embodiments, myasthenia gravis is characterized by antibodies directed against a protein. In some embodiments, the protein is the target of the myasthenia gravis antibody. One skilled in the art will appreciate that the protein complex will be designed using fragments of the protein targeted by the myasthenia gravis antibody in the subject. In some embodiments, the antibody is an autoantibody.

[0286] In some embodiments, treatment comprises reducing the concentration of an antibody. In some embodiments, treatment comprises reducing the number of antibodies. In some embodiments, the antibody concentration is the circulating antibody concentration. In some embodiments, treatment comprises depleting the antibody. In some embodiments, treatment comprises sequestering the antibody. In some embodiments, binding of the antibody to the molecules of the invention results in sequestration of the antibody. In some embodiments, treatment comprises killing B cells. In some embodiments, the B cells are autoreactive B cells. In some embodiments, killing of B cells is specific B cell killing. In some embodiments, treatment comprises killing B cells that produce the antibody. In some embodiments, treatment comprises killing B cells that produce the antibody without substantially killing other B cells. In some embodiments, treatment comprises killing B cells that produce antibodies to a protein complex. In some embodiments, treatment comprises killing B cells that produce antibodies to a fragment. In some embodiments, treatment comprises killing B cells that produce antibodies to a fragment of a protein complex.

[0287] In some embodiments, reducing antibodies comprises binding antibodies. In some embodiments, reducing is the removal of at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% of antibodies. Each possibility represents a separate embodiment of the present invention. In some embodiments, the antibodies are autoantibodies. In some embodiments, the antibodies are antibodies in the subject. In some embodiments, the antibodies are circulating antibodies. In some embodiments, the autoantibodies are autoantibodies to a protein or fragment. In some embodiments, the autoantibodies are cytotoxic autoantibodies. In some embodiments, the autoantibodies comprise IgG1 autoantibodies. In some embodiments, the autoantibodies comprise IgG3. In some embodiments, the autoantibodies comprise IgG1 and IgG3 autoantibodies. In some embodiments, the autoantibodies comprise IgG1, IgG2, and IgG3 autoantibodies. In some embodiments, the autoantibodies comprise IgG1, IgG3, and IgG4 autoantibodies. In some embodiments, the autoantibodies comprise IgG1, IgG2, IgG3, and IgG4 autoantibodies. In some embodiments, reducing is the removal of at least 25% of antibodies. In some embodiments, the reduction is the removal of at least 50% of antibodies. In some embodiments, the reduction is the removal of at least 70% of antibodies. In some embodiments, the reduction is the removal of at least 75% of antibodies. In some embodiments, the percentage of antibodies is the percentage of autoantibodies. In some embodiments, the percentage of antibodies is the percentage of antibodies directed against a protein or fragment. In some embodiments, the percentage of antibodies is the percentage of antibodies associated with a disease.

[0288] In some embodiments, the method further comprises reducing antibodies in the subject. In some embodiments, the reduction occurs prior to administration. In some embodiments, the reduction of antibodies involves reducing circulating antibodies. In some embodiments, the antibodies are autoantibodies. In some embodiments, the antibodies are directed against a protein. In some embodiments, the antibodies are directed against a protein, and fragments thereof are derived from the protein. In some embodiments, the antibodies are directed against a protein, and at least one of the fragments is derived from the protein. In some embodiments, the reduction involves reducing antibodies against all proteins, and at least one of the fragments is derived from these proteins. In some embodiments, the antibodies are directed against a protein complex. Methods for reducing antibodies are well known in the art and include, for example, plasma exchange, intravenous Ig (IVIg), antibody filtration, and B cell-targeted therapy, any of which may be used. In some embodiments, the method comprises plasma exchange prior to administration of the antibodies. In some embodiments, the method comprises administering a B cell-targeted therapy prior to administering the therapeutic agent of the present invention. In some embodiments, the B cell-targeted therapy is an anti-B cell therapy. In some embodiments, the B cell-targeted therapy is a B cell-lethal therapy. In some embodiments, the B cell-targeted therapy is a pan-B cell therapy. In some embodiments, the B cell-targeted therapy is not a targeted therapy. As used herein, "targeted B cell therapy" is a therapy that targets only specific B cell clones that produce specific antibodies. In some embodiments, anti-B cell therapy is an anti-B cell antibody. B cell targeting antibodies are known in the art and include, but are not limited to, for 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, B cell targeted therapy is rituximab. Nucleic Acids

[0289] In another aspect, a nucleic acid molecule is provided that encodes a protein of the present invention.

[0290] In another aspect, a nucleic acid system is provided that includes at least two nucleic acid molecules, wherein the first nucleic acid molecule encodes a first polypeptide chain of a protein complex of the invention and the second nucleic acid molecule encodes a second polypeptide chain of a protein complex of the invention.

[0291] In another aspect, a nucleic acid system is provided comprising at least two nucleic acid molecules, wherein the first nucleic acid molecule encodes: a first polypeptide chain comprising a fragment of a first human protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a first dimerization domain; and the second nucleic acid molecule encodes: a second polypeptide chain comprising a fragment of a second human protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a second dimerization domain.

[0292] In another aspect, a nucleic acid molecule is provided that encodes a polypeptide chain of a composition of the present invention.

[0293] In another aspect, nucleic acid molecules are provided that encode the compositions of the invention.

[0294] In another aspect, nucleic acid molecules are provided that encode: a fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; and a fragment of a second human protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof.

[0295] In some embodiments, the nucleic acid molecules are used to treat myasthenia gravis. In some embodiments, the nucleic acid systems are used to treat myasthenia gravis.

[0296] In some embodiments, the nucleic acid system further comprises a third nucleic acid molecule encoding a third polypeptide of the protein complex of the present invention. In some embodiments, the nucleic acid system further comprises a fourth nucleic acid molecule encoding a fourth polypeptide of the protein complex of the present invention. In some embodiments, the first nucleic acid molecule encodes a first polypeptide of the present invention. In some embodiments, the second nucleic acid molecule encodes a second polypeptide of the present invention. In some embodiments, the third nucleic acid molecule encodes a third polypeptide of the present invention. In some embodiments, the fourth nucleic acid molecule encodes a fourth polypeptide.

[0297] 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 a polypeptide chain. Expression of open reading frames in cells is well known to those skilled in the art. This can be accomplished by transfection, viral infection, or direct alteration of the cell's genome, among other methods. Expression vectors are well known in the art, and any vector compatible with the target cell in which the protein complex of the invention is to be expressed can be used.

[0298] Vector nucleic acid sequences generally contain at least one origin of replication for propagation in cells and optionally additional elements such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), selectable markers (e.g., antibiotic resistance), polyadenylation sequences. In some embodiments, the vector includes a promoter. In some embodiments, the promoter is configured for expression in the target cell in which the protein complex of the invention is expressed.

[0299] The vector can be a DNA plasmid delivered via a non-viral method or via a viral method. The viral vector can be a retroviral vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector or a poxvirus vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter. The promoter can be active in bacterial cells. The promoter can be active in human cells. The promoter can be active in fibroblasts. As used herein, the term "promoter" refers to a group of transcription control modules that gather around the start site of RNA polymerase (i.e., RNA polymerase II). The promoter is composed of discrete functional modules, each of which is composed of approximately 7-20bp of DNA and comprises recognition sites for one or more transcriptional activators or repressors.

[0300] In some embodiments, the open reading frame is operably linked to a promoter. The term "operably linked" refers to a nucleotide sequence of interest that is linked to one or more regulatory elements in a manner that allows 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 a host cell).

[0301] 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 with a viral vector, high-speed ballistic penetration by small particles having nucleic acid within or on the surface of a bead or particle array (Klein et al., Nature 327, 70-73 (1987)), and / or the like.

[0302] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme present in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0303] In some embodiments, mammalian expression vectors include but are not limited to: pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which can be purchased from Invitrogen; pCI, which can be purchased from Promega; pMbac, pPbac, pBK-RSV and pBK-CMV, which can be purchased from Strategene; pTRES, which can be purchased from Clontech, and derivatives thereof.

[0304] In some embodiments, the present invention uses expression vectors containing regulatory elements from eukaryotic viruses (such as retroviruses). SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and vectors that allow protein expression under the guidance of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that show efficient expression in eukaryotic cells.

[0305] In some embodiments, the recombinant viral vector of the advantage such as lateral infection and target specificity is provided to be used for expression in vivo.In one embodiment, lateral infection is intrinsic in the life cycle of for example retrovirus, and is the process that single infected cell produces a plurality of progeny virions, and this progeny virion sprouts and infects adjacent cell.In one embodiment, the result is that large tracts of land is infected rapidly, and wherein major part is not infected by original virus particle at first.In one embodiment, produce the viral vector that can not lateral spread.In one embodiment, if the purpose of expectation is only to import specific gene into the target cell of local quantity, then this characteristic can be useful.

[0306] 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. Also, see US Patent Nos. 5,464,764 and 5,487,992 for positive and negative selection methods.

[0307] In one embodiment, a plant expression vector is used. In one embodiment, expression of the polypeptide coding sequence is driven by multiple promoters. In some embodiments, viral promoters are used, such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter of TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)]. In another embodiment, a plant promoter is used, such as the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224: 838-843 (1984)] or a heat shock promoter, such as soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6: 559-565 (1986)]. In one embodiment, the construct is introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and other techniques known to those skilled in the art. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY Part VIII, pp 421-463 (1988)]. Other expression systems known in the art, such as insect and mammalian host cell systems, may also be used with the present invention.

[0308] It will be appreciated that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (which encodes a polypeptide), the expression constructs of the present invention may also include sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0309] In some embodiments, the nucleic acid molecule is a single nucleic acid molecule. In some embodiments, the first and second nucleic acid molecules are different molecules. In some embodiments, the first and second nucleic acid molecules are the same molecule. In some embodiments, any two of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, any two of the first, second, third, and fourth nucleic acid molecules are the same molecule. In some embodiments, any three of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, the first, second, third, and fourth 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, the first, second, third, and fourth nucleic acid molecules are all different molecules. In some embodiments, the first, second, third, and fourth nucleic acid molecules are all the same molecule. Generation method

[0310] In another aspect, the invention provides a method of producing a protein, comprising: obtaining a first fragment of the extracellular domain of a first human protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; and linking the first fragment to an effector portion that is not an Fc domain.

[0311] In another aspect, a method of producing a protein is provided, comprising: obtaining a first fragment of the extracellular domain of a first human protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; and linking the first fragment to an effector moiety that is not an unmodified Fc domain.

[0312] In another aspect, a method of producing a protein is provided, the method comprising: obtaining a first fragment of an extracellular domain of a first human receptor, or an analog or derivative thereof, and a second fragment of an extracellular domain of a second human receptor, or an analog or derivative thereof, wherein the first and second human receptors are targets of myasthenia gravis autoantibodies and are different proteins; and linking the first fragment to the second fragment to produce a single polypeptide chain and further linking the polypeptide chain to an effector moiety that is not an Fc domain; This produces protein.

[0313] In another aspect, a method of producing a protein is provided, the method comprising: obtaining a first fragment of an extracellular domain of a first human receptor, or an analog or derivative thereof, and a second fragment of an extracellular domain of a second human receptor, or an analog or derivative thereof, wherein the first and second human receptors are targets of myasthenia gravis autoantibodies and are different proteins; and linking the first fragment to the second fragment to produce a single polypeptide chain and further linking the polypeptide chain to an effector moiety that is not an unmodified Fc domain; This produces protein.

[0314] In another aspect, a method of producing a protein is provided, the method comprising: obtaining a first fragment of a human receptor target for a myasthenia gravis autoantibody and generating at least one mutation in the first fragment that reduces aggregation of the first fragment to produce a mutant fragment, and linking the mutant fragment to an effector moiety that is not an Fc domain; This produces protein.

[0315] In another aspect, a method of producing a protein is provided, the method comprising: obtaining a first fragment of a human receptor target for a myasthenia gravis autoantibody and generating at least one mutation in the first fragment that reduces aggregation of the first fragment to produce a mutant fragment, and linking the mutant fragment to an effector moiety that is not an unmodified Fc domain; This produces protein.

[0316] In another aspect, a method of producing a protein complex is provided, the method comprising: Obtaining a first fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a second fragment of a second protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; linking the first fragment to a first dimerization domain to produce a first polypeptide; and linking the second fragment to a second dimerization domain to produce a second polypeptide chain; and linking the first polypeptide chain, the second polypeptide chain, or both to an effector moiety that is not an Fc domain; This creates a protein complex.

[0317] In another aspect, a method of producing a protein complex is provided, the method comprising: Obtaining a first fragment of a first protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof, and a second fragment of a second protein target of a myasthenia gravis autoantibody, or an analog or derivative thereof; linking the first fragment to a first dimerization domain to produce a first polypeptide; linking the second fragment to a second dimerization domain to produce a second polypeptide chain; and linking the first polypeptide chain, the second polypeptide chain, or both to an effector moiety that is not an unmodified Fc domain; This creates a protein complex.

[0318] In another aspect, a method of producing a protein is provided, the method comprising: Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single polypeptide chain, wherein the single polypeptide chain is produced by: i. obtaining a first fragment of the human receptor target of myasthenia gravis autoantibody; and ii. generating at least one mutation in the first fragment that reduces aggregation of the first fragment; This produces protein.

[0319] In another aspect, a method of producing a protein is provided, the method comprising: Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single polypeptide chain, wherein the single polypeptide chain is produced by: i. obtaining a first fragment of the extracellular domain of a first human receptor, or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human receptor, or an analog or derivative thereof, wherein the first human receptor and the second human receptor are targets of myasthenia gravis autoantibodies and are different proteins; and ii. joining the first fragment to the second fragment to produce a single polypeptide chain; This produces protein.

[0320] In another aspect, a method of producing a protein complex is provided, the method comprising: Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding at least two polypeptide chains, wherein the two polypeptide chains are produced by: i. obtaining a first fragment of a first protein target of a myasthenia gravis autoantibody or an analog or derivative thereof, and a second fragment of a second protein target of a myasthenia gravis autoantibody or an analog or derivative thereof; and ii. linking the first fragment to a first dimerization domain to produce a first polypeptide chain, and linking the second fragment to a second dimerization domain to produce a second polypeptide chain; This creates a protein complex.

[0321] In some embodiments, the protein is a polypeptide. In some embodiments, the protein complex is a protein complex of the present invention. In some embodiments, the protein composition is a composition of the present invention. In some embodiments, the protein is a protein of the present invention. In some embodiments, the protein is a polypeptide chain of the present invention. In some embodiments, the fragment is a fragment of the present invention. In some embodiments, the derivative is a derivative of the present invention. In some embodiments, the analog is an analog of the present invention. In some embodiments, the dimerization domain is a dimerization domain of the present invention. In some embodiments, the composition, protein complex, protein, fragment, analog, derivative, or dimerization domain is as described above. In some embodiments, the method further comprises linking the protein, polypeptide, or protein complex to an effector moiety. In some embodiments, the effector moiety is not an Fc domain. In some embodiments, the effector moiety does not include an Fc moiety. In some embodiments, the effector moiety is not an unmodified Fc domain. In some embodiments, the effector moiety is an Fc domain comprising at least one mutation that increases ADCC.

[0322] In some embodiments, the protein is a human protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the first and second proteins are the same protein. In some embodiments, the first and second proteins are different proteins. In some embodiments, the first and second proteins are targets of myasthenia gravis autoantibodies. In some embodiments, the first and second proteins are targets of autoantibodies associated with myasthenia gravis. In some embodiments, myasthenia gravis is characterized by autoantibodies to the first and second proteins. In some embodiments, the protein is a receptor, and the fragment is a fragment of the extracellular domain. In some embodiments, the fragment includes a fragment of the extracellular domain. In some embodiments, the fragment consists of the extracellular domain.

[0323] In some embodiments, the first and second dimerization domains are capable of dimerizing with each other. In some embodiments, the first and second dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first and second polypeptides. In some embodiments, the contacting comprises incubating the polypeptides together. In some embodiments, the contacting is in a cell. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is under conditions sufficient to allow dimerization. In some embodiments, the allowing is priming. In some embodiments, the conditions are sufficient to allow dimerization of the polypeptides. In some embodiments, the conditions are physiological conditions.

[0324] In some embodiments, the method further comprises inserting a third dimerization domain into the first polypeptide. In some embodiments, the insertion is ligation. In some embodiments, the insertion is inserting a nucleic acid sequence encoding the third dimerization domain into a nucleic acid molecule or vector encoding the first polypeptide. In some embodiments, the ligation is ligating the third dimerization domain to the first dimerization domain. In some embodiments, the ligation is ligating the third dimerization domain to the first fragment.

[0325] In some embodiments, the method further comprises: obtaining a third fragment of a third protein target of the myasthenia gravis autoantibody, or an analog or derivative thereof; and linking it to a fourth dimerization domain to produce a third polypeptide chain. In some embodiments, the third and fourth dimerization domains are capable of dimerizing with each other. In some embodiments, the third and fourth dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first, second, and third polypeptide chains. In some embodiments, the method further comprises expressing a nucleic acid sequence encoding the third polypeptide chain in a host cell. In some embodiments, the third polypeptide chain is produced by obtaining a third fragment of a third protein and linking it to a fourth dimerization domain to produce the third polypeptide chain. In some embodiments, the method comprises expressing the first, second, and third polypeptide chains in a cell.

[0326] In some embodiments, the method further comprises inserting the fifth dimerization domain into the second polypeptide. In some embodiments, the insertion is ligation. In some embodiments, the insertion is inserting a nucleic acid sequence encoding the fifth dimerization domain into a nucleic acid molecule or vector encoding the second polypeptide. In some embodiments, the ligation is ligating the fifth dimerization domain to the second dimerization domain. In some embodiments, the ligation is ligating the fifth dimerization domain to the second fragment.

[0327] In some embodiments, the method further comprises: obtaining a fourth fragment of a fourth protein target of the myasthenia gravis autoantibody, or an analog or derivative thereof; and linking it to a sixth dimerization domain to produce a fourth polypeptide chain. In some embodiments, the fifth and sixth dimerization domains are capable of dimerizing with each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first, second, third, and fourth polypeptide chains. In some embodiments, the method further comprises expressing a nucleic acid sequence encoding the fourth polypeptide chain in a host cell. In some embodiments, the fourth polypeptide chain is obtained by obtaining a fourth fragment of a fourth protein and linking it to a sixth dimerization domain to produce the fourth polypeptide chain. In some embodiments, the method comprises expressing the first, second, third, and fourth polypeptide chains in a cell.

[0328] In some embodiments, the method further comprises inserting the Fc region into the first chain. In some embodiments, the method further comprises inserting the Fc region into the second chain. In some embodiments, the method further comprises inserting the Fc region into the third chain. In some embodiments, the method further comprises inserting the Fc region into the fourth chain. In some embodiments, the method further comprises inserting a portion of the Fc region into the first chain and a portion of the Fc region into the second chain, wherein the interface of the two portions creates a complete Fc region.

[0329] In some embodiments, the Fc region is inserted at the C-terminus of the dimerization domain. In some embodiments, the Fc region is inserted at the C-terminus of the fragment. In some embodiments, the Fc region is inserted at the N-terminus of the dimerization domain. In some embodiments, the Fc region is inserted at the N-terminus of the fragment. In some embodiments, the fragment is inserted or linked at the C-terminus of the dimerization domain. In some embodiments, the fragment is inserted or linked at the N-terminus of the dimerization domain.

[0330] In some embodiments, the method further comprises inserting a linker between at least two segments of the polypeptide chain. In some embodiments, the linker is inserted between the fragment and the dimerization domain. In some embodiments, the linker is inserted between the fragment and the Fc region. In some embodiments, the linker is inserted between the Fc region and the dimerization domain. In some embodiments, the linker is inserted between the dimerization domain and another dimerization domain. In some embodiments, the linker is inserted between the fragment and another fragment. In some embodiments, the linker is inserted between a fragment of a first protein and a fragment of a second protein.

[0331] In some embodiments, the method further comprises generating at least one mutation in the fragment that increases the solubility of the fragment. In some embodiments, the method further comprises measuring the solubility of the mutant fragment. In some embodiments, the method further comprises selecting a mutant fragment with increased solubility. In some embodiments, the increase is an increase of at least a predetermined threshold value. In some embodiments, the increase is a significant increase. In some embodiments, the increase is an increase of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase is an increase of at least 25%. In some embodiments, the increase is an increase of at least 50%.

[0332] In some embodiments, the method further comprises generating at least one mutation in the fragment that reduces the aggregation of the fragment. In some embodiments, the method further comprises measuring the aggregation of the mutant fragment. In some embodiments, the method further comprises selecting a mutant fragment with reduced solubility. In some embodiments, reducing is reducing by at least a predetermined threshold value. In some embodiments, reducing is significantly reducing. In some embodiments, reducing is reducing by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500%. Each possibility represents a separate embodiment of the present invention. In some embodiments, reducing is reducing by at least 25%. In some embodiments, reducing is reducing by at least 50%.

[0333] In some embodiments, the method further includes confirming that at least one mutation does not significantly reduce binding of the first fragment to an autoantibody directed against the first fragment. In some embodiments, the method further measures binding of the autoantibody to the mutated first fragment. In some embodiments, the method includes selecting a mutated fragment that exhibits substantially the same or greater binding of an autoantibody. In some embodiments, the autoantibody is a myasthenia gravis autoantibody. In some embodiments, the autoantibody is an autoantibody present in a subject with myasthenia gravis. In some embodiments, confirming or measuring includes contacting the mutated fragment with a sample from a subject with myasthenia gravis and measuring binding of the autoantibody in the sample to the mutated fragment. In some embodiments, the sample is blood. In some embodiments, the sample is serum. In some embodiments, the sample includes isolated autoantibodies. In some embodiments, confirming or measuring includes contacting the unmutated fragment with a sample from a subject with myasthenia gravis and measuring binding of the autoantibody in the sample to the mutated fragment. In some embodiments, confirming or measuring includes comparing binding of the unmutated fragment to the mutated fragment and selecting the mutated fragment that does not exhibit substantially less binding of the autoantibody. In some embodiments, substantially less means significantly less. In some embodiments, substantially less means less. In some embodiments, substantially less is more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% less. Each possibility represents a separate embodiment of the present invention. In some embodiments, substantially less is more than 10% less.

[0334] In another aspect, a protein complex is provided, which is produced by the method of the present invention.

[0335] In another aspect, a protein is provided that is produced by the method of the present invention.

[0336] In another aspect, compositions are provided that are produced by the methods of the present invention. Patient selection

[0337] In another aspect, a method of determining the suitability of a subject to be treated by the methods of the invention is provided, the method comprising receiving a sample from the subject, contacting the sample with a composition of the invention, and determining binding of antibodies in the sample to the composition, wherein binding of the antibodies to the composition indicates that the subject is suitable for treatment by the methods of the invention, thereby determining the suitability of the subject to be treated.

[0338] In another aspect, a method of determining the suitability of a subject to be treated by the methods of the invention is provided, the method comprising receiving a sample from the subject, contacting the sample with a protein complex of the invention, and determining binding of antibodies in the sample to the protein complex, wherein binding of the antibodies to the protein complex indicates that the subject is suitable for treatment by the methods of the invention, thereby determining the suitability of the subject to be treated.

[0339] In another aspect, a method for determining the suitability of a subject to be treated by the methods of the invention is provided, the method comprising receiving a sample from the subject, contacting the sample with a protein of the invention, and determining binding of antibodies in the sample to the protein, wherein binding of the antibodies to the protein indicates that the subject is suitable for treatment by the methods of the invention, thereby determining the suitability of the subject to be treated.

[0340] In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject is a subject such as described above. In some embodiments, the subject has myasthenia gravis. In some embodiments, the subject is known to be positive for autoantibodies associated with myasthenia gravis. In some embodiments, the subject is seropositive. In some embodiments, the subject is seronegative. In some embodiments, the subject is treatment naive. In some embodiments, the treatment is for myasthenia gravis. In some embodiments, the subject has received treatment and has relapsed.

[0341] In some embodiments, the method comprises obtaining a sample from a subject. In some embodiments, the sample comprises tissue. In some embodiments, the sample is a biopsy. In some embodiments, the sample is a body fluid. In some embodiments, the body fluid is blood. In some embodiments, the body fluid is serum. In some embodiments, the body fluid is plasma. In some embodiments, the body fluid is a fluid comprising antibodies. In some embodiments, the body fluid is selected from at least one of the following: blood, serum, plasma, intestinal fluid, saliva, tumor fluid, urine, interstitial fluid, cerebrospinal fluid, and feces.

[0342] In some embodiments, the autoantibodies are myasthenia gravis autoantibodies. In some embodiments, the autoantibodies are directed against AChR. In some embodiments, the autoantibodies are directed against AChR subunits. In some embodiments, the autoantibodies are directed against AChR subunits. In some embodiments, the autoantibodies are pathological autoantibodies. In some embodiments, the autoantibodies are disease-causing autoantibodies.

[0343] In some embodiments, the contacting is incubation. In some embodiments, the contacting is under conditions sufficient for the antibody to bind to the protein complex. In some embodiments, the conditions include a time sufficient for the antibody to bind to the protein complex. In some embodiments, the conditions include physiological conditions. In some embodiments, the protein complex is added to a sample. In some embodiments, the protein complex is dissolved in a body fluid. In some embodiments, the antibody is an autoantibody. In some embodiments, the antibody is an antibody directed against a protein.

[0344] In some embodiments, at least a threshold amount of antibody binding to a protein or protein complex indicates that a subject is suitable for treatment. In some embodiments, more than a threshold amount of antibody binding to a protein or protein complex indicates that a subject is suitable for treatment. In some embodiments, the amount of antibody is the number of antibodies. In some embodiments, the amount of antibody is the percentage of antibodies. In some embodiments, the percentage is the percentage of antibodies in a sample. In some embodiments, the threshold is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% of the antibodies in the sample. Each possibility represents a separate embodiment of the present invention. In some embodiments, the threshold is 20%. In some embodiments, the threshold is 25%. In some embodiments, the threshold is 50%. In some embodiments, the threshold is 70%. In some embodiments, the threshold is 75%.

[0345] 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 to the composition, protein, and / or complex. In some embodiments, the peptide is specific for 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 for the composition, protein, and / or complex to the exclusion of other proteins in the sample. In some embodiments, the peptide is specific for the composition, protein, and / or complex to the exclusion of naturally occurring antibodies in the sample. In some embodiments, the peptide is specific for the composition, protein, and / or complex to the exclusion of antibodies in the sample. In some embodiments, determining binding comprises a detection moiety. In some embodiments, determining comprises separating the protein complex. In some embodiments, determining comprises eluting antibodies from the complex. Methods for protein identification are well known in the art, and any such method may be used. The example of such method comprises Western blotting, ELISA, FACS analysis and protein sequencing (such as by mass spectrometry).In some embodiments, determining comprises ELISA.In some embodiments, ELISA is a competitive ELISA.In some embodiments, competitive ELISA comprises competition with antibody.In some embodiments, antibody is the antibody relevant to disease.

[0346] 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 fragment of a protein complex. In some embodiments, binding is binding to at least two fragments of a protein complex. In some embodiments, binding is binding to at least three fragments of a protein complex. In some embodiments, binding is binding to at least four fragments of a protein complex. In some embodiments, at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% of the antibodies in a sample bind. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 50% of the antibodies in a sample bind. In some embodiments, at least 70% of the antibodies in a sample bind. In some embodiments, at least 75% of the antibodies in a sample bind. In some embodiments, the percentage of antibodies is the percentage of autoantibodies. In some embodiments, the percentage of antibodies is the percentage of antibodies directed against a protein. In some embodiments, the percentage of antibodies is the percentage of antibodies associated with a disease.

[0347] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm +- 100 nm.

[0348] It should be 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 a "polypeptide" includes one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely" and "only" in connection with a limitation of claim elements, or for the use of a "negative" limitation.

[0349] In those instances where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended to be in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One skilled in the art would further understand that virtually any disjunctive conjunction and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one, either, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0350] It should be understood that certain features of the present invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all subcombinations of various embodiments and elements thereof are also specifically encompassed by the present invention and disclosed herein, just as each such subcombination is individually and explicitly disclosed herein.

[0351] Other objects, advantages and novel features of the present invention will become apparent to those 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 described above and as claimed in the claims section below, is experimentally supported in the following examples.

[0352] 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. Example

[0353] In general, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Vols. I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology", Vols. I-III Coligan JE, 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:

[0354] 80% to 90% of myasthenia gravis (MG) patients have been found to have autoantibodies against the acetylcholine receptor (AChR). However, the AChR complex is composed of five subunits (α1, β1, γ, δ, and ε). Diagnostic evaluations of MG patients do not typically distinguish between antibodies directed against one subunit or another. However, most antigen-specific attempts to generate MG therapeutics have focused on the α subunit of the AChR (AChRa) and therapeutics targeting autoantibodies against this molecule.

[0355] To determine the percentage of the myasthenia gravis population that actually has resistance to AChRα1, serum samples were collected from 335 AChR seropositive MG patients. The samples were tested in a direct ELISA assay using the cis-loop modified AChRα1 extracellular domain (ECD, SEQ ID NO: 131) as a decoy molecule (an example of this procedure is described in Figure 1F In this assay, the intact AChR complex with all its subunits is used as bait to bind autoantibodies in serum. Binding is determined in the presence or absence of increasing concentrations of solid-phase-bound AChRα1, and the percent reduction in binding is measured for each sample ( Figure 1A The reduction in AChR receptor binding was proportional to the concentration of autoantibodies against AChRα1 present. Surprisingly, although some subjects had very high levels of inhibition, indicating the presence of autoantibodies primarily against AChRα1 ( Figure 1A , the leftmost sample; and Figure 1B ), but other subjects showed only moderate levels of inhibition, indicating that most autoantibodies were not directed against the α subunit ( Figure 1C ), and some subjects did not have substantial suppression, indicating that although they were positive for autoantibodies to AChR, no more than 10% of their autoantibodies were to AChRα1 ( Figure 1D Importantly, when measuring total AChR binding ( Figure 1E), there was no correlation between the total antibody concentration and the percentage of anti-AChRα1 antibodies ( Figure 1G ), several samples with the highest total antibody titers had low or absent anti-AChRa1 autoantibodies. Example 2:

[0356] To better understand the autoantibody repertoire of most MG patients, we analyzed the data presented in Zisimopoulou et al., 2008, “Antigen-specific apheresis of human anti-acetylcholine receptor autoantibodies from myasthenia gravis patients' sera using Escherichia coli-expressed receptor domains.” We plotted the contributions of autoantibodies to individual AChR subunits to the total anti-AChR autoantibody repertoire for the 41 patient samples tested ( Figure 2A ). Thus, although there are many subjects with autoantibodies to the α subunit, many other subjects also have autoantibodies primarily to other subunits, and in fact the vast majority of subjects have a combination of autoantibodies targeting different subunits. Therefore, a graph is drawn to show the percentage of subjects that will obtain at least 50% or 75% inhibition by contact with a single AChR subunit or combination of subunits ( Figure 2B Surprisingly, the α subunit alone, or indeed any subunit alone, rarely produced 75% blockade in any patient tested. In fact, the α subunit alone produced greater than 50% inhibition in only about 20% of patients.

[0357] For a MG therapeutic to treat at least 50% of the target population and neutralize more than 50%, ideally more than 75%, of the autoantibodies, a combination of α / β / γ / δ / ε would be required ( Figure 2B While any potential reduction in autoantibody levels is beneficial, creating a treatment that can achieve a substantial reduction and be effective for the majority of the MG population will require targeting multiple AChR subunits. This could also be achieved through dual and triple combinations of these subunits. Example 3:

[0358] Long-term remission in MG patients requires the removal of a substantial portion of the autoreactive B cells that produce the autoantibody pool. While simply removing autoantibodies from the circulation may be effective in treating MG symptoms, repeated treatments are required for the rest of the patient's life, as long-lived B cells will continue to produce new autoantibodies in perpetuity. Importantly, autoantibody-producing B cells express the same B cell receptor (BCR) on their surface as the autoantibodies. This allows the B cells themselves to be targeted by therapeutic agents containing BCR- (and autoantibody-) specific epitopes. By coupling the target epitope to the Fc region of the antibody heavy chain, therapeutic agents can directly and specifically kill autoantibody-producing B cells. This approach is also robust against the potential escape of specific subpopulations—which occurs when using agents that target specific differentiation markers on the cell surface (e.g., CD19, CD38, BCMA)—because every cell carrying an autoreactive BCR will be targeted, regardless of its differentiation state. This approach may also be beneficial in protecting and preserving non-self-reactive subsets that are harmed by therapies targeting nonspecific differentiation markers (e.g., CD19, CD38, BCMA) regardless of whether they carry self-reactive BCRs.

[0359] Figure 3A One embodiment of the therapeutic agent of the present invention is shown. Immunoglobulin (Ig) sample protein complex 101 includes four polypeptide chains: two heavy chain sample polypeptides 110 and two light chain sample polypeptides 120. Chain 110 can be dimerized by the disulfide bond between them. In addition, chain 110 can also include any one or all of CH3 domain 111, CH2 domain 112, hinge area 113 and CH1 domain 114. In this embodiment, hinge area 113 includes a disulfide bond and serves as a dimerization domain, but it is also possible to use other dimerization domains. These domains are well known in the art and can be selected from any one of, for example, human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD domains. It will be understood by those skilled in the art that the Fc part of the IgG1 and IgG3 incorporated into chain 110 will allow the molecule to trigger antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Chain 120 is capable of dimerizing with chain 110 via disulfide bonds present in the CH1 domain 114 and the CL domain 124 .

[0360] Chains 110 and 120 do not contain variable regions, unlike naturally occurring or artificial antibodies. Instead of variable regions, each chain has a fragment 130 from the extracellular portion of the human acetylcholine receptor. Each chain generated can have the same AChR subunits or different subunits. In practice, Figure 3BAs shown, the two heavy chains 115 and 116 can be individually engineered so that chain 115 comprises, for example, a β subunit 131, while chain 116 comprises a γ subunit 132. The same is true for light chains 125 and 126, which can, for example, comprise α 133 and ε 134 subunits. Thus, a therapeutic molecule can be designed with four copies of a single subunit ( Figure 3C ), two copies of each of the two different subunits ( Figure 3D ), or one copy of each of the four different subunits ( Figure 3B ), or any other combination thereof. In fact, the molecule is modular enough that it can be engineered to have three copies of one subunit and one copy of another, or two copies of one subunit and one copy of the other two subunits. Figure 3E An embodiment is shown in which the two light chains are identical but the two heavy chains are different. Figure 3F An embodiment is shown in which the two heavy chains are identical and the two light chains are different. Importantly, the therapeutic molecule can be engineered to include four of the five different AChR subunits, which, as described above, can induce greater than 50% inhibition in at least 50% of AChR-positive patients and at least 40% inhibition in all patients tested. It will be understood by those skilled in the art that any chain can include any subunit, and Figures 3A-3F The combinations of chains and subunits depicted are illustrative only and not limiting.

[0361] Figures 4A-4F Some embodiments of the invention are shown in which only two chains are combined. Figures 4A-4E In the embodiment, protein complex 201 includes two polypeptide chains, specifically two heavy chains. Heavy chains 215 and 216 may optionally include CH2 212, CH3 211, and / or CH1 214 domains. In this embodiment, the dimerization domain is a heavy chain hinge 213 that dimerizes via disulfide bonds, but other dimerization domains are also contemplated. Figure 4B Molecules are shown without the CH2 domain 212 or the CH3 domain 211 or the CH1 domain 214. Combinations lacking two of these domains are also contemplated ( Figure 4B ). Instead of the variable region, each chain has a fragment 230 from the extracellular portion of the human acetylcholine receptor. Each chain generated can have the same AChR subunit ( Figure 4C ) or different subunits ( Figure 4D When two different subunits are used, it is advantageous to design the molecule so that it forms primarily heterodimers of 215 and 216 rather than homodimers. This is also true for the formation of heterodimers of chains 115 and 116 in Figure 3. Various techniques are known in the art for designing mutations in CH3 / CH2 domains, such as knob-in-hole structures and DuoBodies, which inhibit homodimerization and promote heterodimerization. Any such technique can be used.

[0362] exist Figure 4E In FIG, an alternative configuration comprising two heavy chains is shown. Instead of including a single variable region, two tandem segments 230 are used. These segments may be separated by an optional linker 290. This configuration is structurally similar to a single-chain antibody in which the variable heavy and light chains are located on a single peptide and is essentially equivalent to Figure 3D The molecules shown. The heavy chains 215 and 216 may optionally include CH2 212, CH3 211 and / or CH1 214 domains. In this embodiment, the dimerization domain is a heavy chain hinge 213 that dimerizes via disulfide bonds, but other dimerization domains are also contemplated. For simplicity, examples are shown that include all three CH domains, as well as examples that lack the CH1 domain. Molecules that lack the CH2 or CH3 domains or any two of these domains are also contemplated. It will be understood that the fragment 230 can be from any AChR subunit. Thus, two repeats of the same subunit can be inserted into a single chain ( Figure 4F , two AChRg subunits 232), or two different subunits can be combined in one chain ( Figure 4G , one AChRg subunit 232, o...

Claims

1. A composition comprising: a fragment of a first human acetylcholine receptor subunit, or an analog or derivative thereof; a fragment of a second human acetylcholine receptor subunit, or an analog or derivative thereof; and an effector moiety, wherein the first and second subunits are different subunits, and wherein the effector moiety is not an Fc domain or is an Fc domain that includes at least one mutation that increases antibody-dependent cellular cytotoxicity (ADCC).

2. The composition of claim 1, wherein the fragment is a fragment of the extracellular domain of the acetylcholine receptor subunit.

3. The composition of claim 1 or 2, wherein the first and second acetylcholine receptor subunits are selected from the group consisting of acetylcholine receptor subunit alpha (ACHRA), acetylcholine receptor subunit beta (ACHRB), acetylcholine receptor subunit gamma (ACHRG), acetylcholine receptor subunit delta (ACHRD) and acetylcholine receptor subunit epsilon (ACHRE).

4. The composition of any one of claims 1 to 3, wherein the effector moiety is capable of inducing death in a cell that binds any one of the fragments.

5. The composition of claim 4, wherein the effector moiety is selected from an Fc domain comprising at least one mutation that increases ADCC, amatoxin / amanitin, anthracyclines, anthramycin-based dimers, calicheamicin, camptothecin or an analog thereof, duocarmycin, triptolide, and a tubulin inhibitor.

6. The composition of any one of claims 1 to 5, wherein the effector moiety is selected from the group consisting of α-amanitin, PNU-159682, ticillin, delutecan (Dxd), maytansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

7. The composition of any one of claims 1 to 5, wherein the effector moiety is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141, wherein the Fc domain comprises a plurality of mutations within SEQ ID NO: 12 or SEQ ID NO: 141 selected from the group consisting of L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, and G16A / A110L / I112E.

8. The composition according to any one of claims 1 to 7, comprising a protein complex comprising: a. a first polypeptide chain comprising said fragment of a first human acetylcholine receptor subunit or an analog or derivative thereof and a first dimerization domain; and b. a second polypeptide chain comprising the fragment of a second human acetylcholine receptor subunit or an analog or derivative thereof and a second dimerization domain; wherein the first and second dimerization domains are configured to dimerize with each other.

9. The composition of claim 8, wherein the dimerization comprises forming a covalent bond between the first dimerization domain and the second dimerization domain.

10. The composition of claim 8 or 9, wherein the protein complex comprises an immunoglobulin scaffold.

11. The composition according to any one of claims 8 to 10, wherein a. the first dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin, and the second dimerization domain comprises a second hinge domain of a heavy chain, and the first and second dimerization domains dimerize via a disulfide bond; or b. The first and second dimerization domains each comprise a domain selected from the group consisting of the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and dimerize through a disulfide bond, and wherein the first and second dimerization domains do not comprise the CH1 domain and the CL domain at the same time.

12. The composition of any one of claims 8 to 10, wherein the fragment of the first, second or both polypeptide chains and the dimerization domain are separated by a linker.

13. The composition of any one of claims 8 to 12, wherein the first polypeptide chain, the second polypeptide chain, or both further comprises the effector moiety.

14. The composition of claim 13, wherein the effector moiety is linked to the first polypeptide chain, the second polypeptide chain, or both via a covalent bond.

15. The composition of claim 13 or 14, wherein the first polypeptide chain comprises a first CH3 domain of a heavy chain of an immunoglobulin, a first CH2 domain of a heavy chain of an immunoglobulin, or both and the second polypeptide chain comprises a second CH3 domain of a heavy chain of an immunoglobulin, a second CH2 domain of a heavy chain of an immunoglobulin, or both, and further comprises an effector portion that is not an Fc domain or comprises at least one mutation that increases ADCC within the first CH3 domain, the first CH2 domain, the second CH3 domain, the second CH2 domain, or a combination thereof.

16. The composition of claim 15, wherein the first CH3 domain, the first CH2 domain, or both comprise at least a first mutation, and the second CH3 domain, the second CH2 domain, or both comprise at least a second mutation, and wherein the mutations allow heterodimerization of the first and second polypeptide chains and inhibit homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

17. The composition of claim 16, wherein the first mutation is selected from the mutations provided in Table 1, and the second mutation is provided in Table 1 and is a corresponding mutation of the first mutation.

18. The composition of any one of claims 13 to 17, wherein the Fc region of the first, second or both polypeptide chains is separated from the fragment or the dimerization domain by a linker.

19. The composition of any one of claims 13 to 18, wherein the Fc is derived from IgG2 or IgG4 or comprises at least one mutation that reduces effector function.

20. The composition of any one of claims 13 to 19, wherein the dimerization domain of the first, second or both polypeptide chains is at the C-terminus of the fragment or at the N-terminus of the fragment.

21. The composition of any one of claims 1 to 20, which does not contain antibody variable domains.

22. The composition of any one of claims 8 to 21, further comprising a third polypeptide comprising a fragment of a third human acetylcholine receptor subunit or an analog or derivative thereof and a third dimerization domain, wherein the first polypeptide further comprises a fourth dimerization domain, and the third and fourth dimerization domains are capable of dimerizing with each other.

23. The composition of claim 22, wherein a. the third dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin, and the fourth dimerization domain comprises a second hinge domain of a heavy chain, and the first and second dimerization domains dimerize via a disulfide bond; or b. The third and fourth dimerization domains each comprise a domain selected from the group consisting of the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and dimerize through a disulfide bond, and wherein the first and third polypeptides do not comprise the CH1 domain and the CL domain at the same time.

24. The composition of claim 22 or 23, further comprising a fourth polypeptide comprising a fragment of a fourth human acetylcholine receptor subunit or an analog or derivative thereof and a fifth dimerization domain, wherein the second polypeptide further comprises a sixth dimerization domain, and the fifth and sixth dimerization domains are capable of dimerizing with each other.

25. The composition of claim 24, wherein a. the fifth dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin, and the sixth dimerization domain comprises a second hinge domain of a heavy chain, and the first and second dimerization domains dimerize via a disulfide bond; or b. The fifth and sixth dimerization domains each comprise a domain selected from the group consisting of the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and dimerize through a disulfide bond, and wherein the first and third polypeptides do not comprise the CH1 domain and the CL domain at the same time.

26. The composition of any one of claims 1 to 25, comprising a single polypeptide chain comprising: said fragment of the first human acetylcholine receptor subunit or an analog or derivative thereof; and said fragment of a second human acetylcholine receptor subunit or an analog or derivative thereof.

27. The composition of claim 26, wherein the single polypeptide chain further comprises: a fragment of a third human acetylcholine receptor subunit, or an analog or derivative thereof; and optionally a fragment of a fourth human acetylcholine receptor subunit, or an analog or derivative thereof.

28. The composition of claim 26 or 27, wherein the fragments are separated by an amino acid linker, optionally wherein the linker is a flexible GS linker, or wherein the linker is a rigid linker.

29. The composition of any one of claims 26 to 28, wherein the polypeptide chain further comprises an Fc region of a human antibody heavy chain, and the second polypeptide chain comprises a third human acetylcholine receptor subunit or an analog or derivative thereof and an Fc region of a human antibody heavy chain, optionally wherein the second polypeptide chain further comprises a fourth human acetylcholine receptor subunit.

30. The composition of any one of claims 26 to 29, further comprising a second polypeptide chain comprising a third human acetylcholine receptor subunit or an analog or derivative thereof, optionally wherein the second polypeptide chain further comprises a fourth human acetylcholine receptor subunit.

31. The composition of any one of claims 5 to 30, wherein the effector moiety is linked to the fragment via a linker.

32. The composition of any one of claims 1 to 31, wherein the complex comprises at least one amino acid sequence selected from SEQ ID NOs: 64 to 69 or a derivative thereof that is at least 80% identical thereto.

33. The composition of any one of claims 1 to 32, wherein at least one of the fragments comprises a mutation that increases the stability or solubility of the fragment.

34. The composition of claim 33, wherein the mutation comprises replacing a cys loop within an acetylcholine receptor subunit with CDVSGVDTESGATNC (SEQ ID NO: 44).

35. The composition of claim 33 or 34, wherein the acetylcholine receptor subunit is selected from the group consisting of an alpha subunit comprising the amino acid sequence provided in SEQ ID NO: 131, a beta subunit comprising the amino acid sequence provided in SEQ ID NO: 132, a gamma subunit comprising the amino acid sequence provided in SEQ ID NO: 133, a delta subunit comprising the amino acid sequence provided in SEQ ID NO: 134, and an epsilon subunit comprising the amino acid sequence provided in SEQ ID NO:

135.

36. The composition of any one of claims 1 to 35, wherein the analog or derivative thereof comprises at least 85% identity to the human protein.

37. The composition of any one of claims 1 to 36, wherein the fragment comprises at least 20 consecutive amino acids from the protein.

38. The composition of any one of claims 1 to 37, wherein the fragment comprises at least one B cell receptor (BCR) specific epitope target of the autoantibody.

39. The composition of any one of claims 1 to 38, wherein the fragment comprises at least one mutation that reduces aggregation of the fragment.

40. The composition of claim 39, wherein the fragment is selected from the group consisting of: a. A fragment of ACHRA and comprising a mutation selected from the group consisting of: deletion of N141; F100G; W149R; V155A; Y93F; Y93H; Y93R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 1 or the AChRa with increased solubility comprising SEQ ID NO: 131; b. A fragment of ACHRG and comprising a mutation selected from the group consisting of: M84S; Y105E; Y117E; Y117R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 6 or the solubility-enhanced AChRa comprising SEQ ID NO: 133; and c. A fragment of ACHRD and comprising a mutation selected from the group consisting of: C108A; C108I; Y119R; deletion of N141; L151E; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 8 or the AChRa with increased solubility comprising SEQ ID NO:

134.

41. The composition of any one of claims 8 to 40, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of: SEQ ID NO: 92 and SEQ ID NO: 93; SEQ ID NO: 95 and SEQ ID NO: 96; SEQ ID NO: 97 and SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, SEQ ID NO: 92 and SEQ ID NO: 102; SEQ ID NO: 103 and SEQ ID NO: 100; SEQ ID NO: 105 and SEQ ID NO: 130; SEQ ID NO: 105 and SEQ ID NO: 106; and SEQ ID NO: 105 and SEQ ID NO:

107.

42. The composition of any one of claims 26 to 40, wherein the single polypeptide chain is selected from the group consisting of: SEQ ID NO: 94, SEQ ID NO: 104, and SEQ ID NOs: 108-129.

43. A polypeptide comprising: A fragment of a first human acetylcholine receptor subunit, said fragment comprising at least one mutation that reduces aggregation of said fragment; and an effector portion that is not an Fc domain or is an Fc domain that comprises at least one mutation that increases ADCC, wherein said fragment is selected from the group consisting of: a. A fragment of ACHRA and comprising a mutation selected from the group consisting of: deletion of N141; F100G; W149R; V155A; Y93F; Y93H; Y93R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 1 or the AChRa with increased solubility comprising SEQ ID NO: 131; b. A fragment of ACHRG and comprising a mutation selected from the group consisting of: M84S; Y105E; Y117E; Y117R; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 6 or the solubility-enhanced AChRa comprising SEQ ID NO: 133; and c. A fragment of ACHRD and comprising a mutation selected from the group consisting of: C108A; C108I; Y119R; deletion of N141; L151E; and combinations thereof within the wild-type AChRa comprising SEQ ID NO: 8 or the AChRa with increased solubility comprising SEQ ID NO:

134.

44. The polypeptide of claim 43, further comprising replacing a cys loop within an acetylcholine receptor subunit with CDVSGVDTESGATNC (SEQ ID NO: 44), and wherein the subunit is ACHRA and the cys loop consists of CEIIVTHFPFDEQNC (SEQ ID NO: 39), the subunit is ACHRG and the cys loop consists of CSISVTYFPFDWQNC (SEQ ID NO: 41), or the subunit is ACHRD and the cys loop consists of CPISVTYFPFDWQNC (SEQ ID NO: 42).

45. The polypeptide of claim 43 or 44, further comprising a second fragment of a second acetylcholine receptor subunit, the second fragment being connected to the first fragment by an amino acid linker; and optionally further comprising a fragment from a third, fourth, or fifth acetylcholine receptor subunit.

46. The polypeptide of any one of claims 43 to 45, further comprising an Fc region of a human antibody heavy chain, optionally wherein the Fc region is separated from the fragment by an amino acid linker.

47. A polypeptide according to any one of claims 43 to 46 comprising a sequence selected from the group consisting of SEQ ID NOs: 72-91.

48. The polypeptide of any one of claims 43 to 47, wherein the effector moiety is selected from an Fc domain comprising at least one mutation that increases ADCC, an amatoxin / amanitin, an anthracycline, an anthracycline-based dimer, calicheamicin, camptothecin or an analog thereof, duocarmycin, triptolide, and a tubulin inhibitor.

49. The polypeptide of any one of claims 43 to 48, wherein the effector moiety is selected from the group consisting of α-amanitin, PNU-159682, ticillin, Dxd, maytansine, MMAE, MMAF, and combinations thereof.

50. The polypeptide of any one of claims 43 to 48, wherein the effector moiety is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141, wherein the Fc domain comprises a plurality of mutations within SEQ ID NO: 12 or SEQ ID NO: 141 selected from the group consisting of: L15V / F23L / R72P / Y80L / P176L and S19D / A110L / I112E, G16A / A110L / I112E.

51. A pharmaceutical composition comprising a composition according to any one of claims 1 to 42 or a polypeptide according to any one of claims 43 to 50 and a pharmaceutically acceptable carrier, excipient or adjuvant; optionally wherein the pharmaceutical composition is formulated for systemic administration to a subject.

52. A method of treating myasthenia gravis in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1 to 42, the polypeptide of any one of claims 43 to 50, or the pharmaceutical composition of claim 51, thereby treating myasthenia gravis.

53. The method of claim 52, further comprising reducing the level of circulating antibodies to at least the first human acetylcholine receptor subunit in the subject prior to said administering.

54. The method of claim 52 or 53, further comprising reducing the level of circulating antibodies in the subject to human acetylcholine receptor subunits within a protein complex comprising the first human acetylcholine receptor subunit or the second human acetylcholine receptor subunit.

55. The method of any one of claims 52 to 54, wherein the treatment comprises reducing the concentration of circulating autoantibodies to the human acetylcholine receptor subunits.

56. The method of any one of claims 52 to 55, wherein the treatment comprises killing B cells that produce the autoantibody.

57. The method of claim 55, wherein the B cell is an autoreactive B cell that produces autoantibodies against a fragment of the composition or polypeptide.

58. A nucleic acid system comprising nucleic acid molecules, wherein a first nucleic acid molecule encodes the first polypeptide chain of the composition according to any one of claims 8 to 25 and 31 to 42 and a second nucleic acid molecule encodes the second polypeptide chain of the composition according to any one of claims 8 to 25 and 4 to 42, or the nucleic acid molecules encode a single polypeptide chain of the composition according to any one of claims 26 to 42 or a polypeptide according to any one of claims 43 to 50.

59. The nucleic acid system of claim 58, further comprising: a third nucleic acid molecule encoding the third polypeptide chain of the composition of any one of claims 22 to 25 and 31 to 42; A fourth nucleic acid molecule encoding the fourth polypeptide chain of the composition of any one of claims 24 or 25 and 31 to 42; or both.

60. A method of producing a composition according to any one of claims 1 to 42 or a polypeptide according to any one of claims 43 to 50, the method comprising expressing the nucleic acid system according to claim 58 or 59 in a cell, wherein the nucleic acid system is configured to produce the encoded polypeptide in the cell, thereby producing a composition according to any one of claims 1 to 42 or a polypeptide according to any one of claims 43 to 50.

61. A method of producing a protein, the method comprising: obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit, or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different subunits, linking the first fragment to the second fragment to produce a single polypeptide chain, and linking the single polypeptide chain to an effector moiety that is not an Fc domain or an Fc domain that includes at least one mutation that increases ADCC; or Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single polypeptide chain and linking the single polypeptide chain to an effector moiety that is not an Fc domain or an Fc domain that comprises at least one mutation that increases ADCC, wherein the single polypeptide chain is produced by: i. obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit, or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different subunits; and ii. joining the first fragment to the second fragment to produce a single polypeptide chain; This produces protein.

62. A method of producing a protein complex, the method comprising: obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit, or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different proteins, linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain, wherein the first and second dimerization domains are capable of dimerizing with each other, and contacting the first polypeptide and the second polypeptide under conditions sufficient to induce dimerization and linking the first polypeptide chain, the second polypeptide chain, or both to an effector moiety that is not an Fc domain or an Fc domain that includes at least one mutation that increases ADCC; or Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding at least two polypeptide chains, and linking at least one of the at least two polypeptide chains to an effector moiety that is not an Fc domain or an Fc domain that comprises at least one mutation that increases ADCC, wherein the two polypeptide chains are produced by: i. obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit, or an analog or derivative thereof, and a second fragment of the extracellular domain of a second human acetylcholine receptor subunit, or an analog or derivative thereof, wherein the first and second human acetylcholine receptor subunits are different proteins; and ii. linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain, wherein the first and second dimerization domains are capable of dimerizing with each other; This creates a protein complex.

63. The method of claim 61, wherein the protein is a single polypeptide chain of the composition of any one of claims 26 to 42.

64. The method of claim 62, wherein the protein complex is a protein complex of the composition of any one of claims 8 to 25 and 21 to 42.

65. The method of claim 61 or 64, further comprising a. linking a third dimerization domain to the first dimerization domain or first fragment within the first polypeptide chain; obtaining a third fragment of the extracellular domain of a third human acetylcholine receptor subunit, or an analog or derivative thereof, and linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain, wherein the third dimerization domain and the fourth dimerization domain are capable of dimerizing with each other; and contacting the first, second, and third polypeptides under conditions sufficient to induce dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a third polypeptide chain, wherein the third polypeptide chain is produced by: i. obtaining a third fragment of the extracellular domain of a third human acetylcholine receptor subunit or an analog or derivative thereof; and ii. linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain; wherein the first polypeptide chain further comprises a third dimerization domain, and wherein the third dimerization domain and the fourth dimerization domain are capable of dimerizing with each other.

66. The method of claim 65, further comprising a. linking a sixth dimerization domain to the second dimerization domain or second fragment within the second polypeptide chain; obtaining a fourth fragment of the extracellular domain of a fourth human acetylcholine receptor subunit, or an analog or derivative thereof, and linking the fourth fragment to a fifth dimerization domain to produce a fourth polypeptide chain, wherein the fifth dimerization domain and the sixth dimerization domain are capable of dimerizing with each other; and contacting the first, second, third, and fourth polypeptides under conditions sufficient to induce dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a fourth polypeptide chain, wherein the fourth polypeptide chain is produced by: i. obtaining a fourth fragment of the extracellular domain of a fourth human acetylcholine receptor subunit or an analog or derivative thereof; and ii. linking the fourth fragment to a fifth dimerization domain to produce a fourth polypeptide chain; wherein the second polypeptide chain further comprises a sixth dimerization domain, and wherein the fifth dimerization domain and the sixth dimerization domain are capable of dimerizing with each other.

67. A method for producing a polypeptide, the method comprising: a. obtaining a first fragment of the extracellular domain of a first human acetylcholine receptor subunit or an analog or derivative thereof; b. generating at least one mutation in the first fragment that reduces aggregation of the first fragment to produce a mutated first fragment; and c. linking the mutant first fragment to an effector moiety that is not an Fc domain or an Fc domain that includes at least one mutation that increases ADCC; Thus, a polypeptide is produced.

68. The method of any one of claims 61 to 67, wherein the analog or derivative thereof comprises at least 85% identity to the human protein.

69. The method of any one of claims 61 to 68, wherein the effector moiety is selected from an Fc domain comprising at least one mutation that increases ADCC, an amatoxin / amanitin, an anthracycline, an anthracycline-based dimer, an auristatin, a calicheamicin, a camptothecin or an analog thereof, a duocarmycin, triptolide, and a tubulin inhibitor.

70. The method of claim 69, wherein the effector moiety is selected from the group consisting of: α-amanitin, PNU-159682, ticillin, delutecan (Dxd), maytansine, MMAE, MMAF, and combinations thereof.

71. The method of claim 69, wherein the effector portion is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141, wherein the Fc domain comprises a plurality of mutations within SEQ ID NO: 12 or SEQ ID NO: 141 selected from the group consisting of: L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, and G16A / A110L / I112E.

72. A protein complex or protein produced by the method of any one of claims 59 to 68.

73. A method for determining the suitability of a subject in need thereof to be treated by the method of any one of claims 50 to 55, the method comprising receiving a sample from the subject, contacting the sample with a composition of any one of claims 1 to 41 or a polypeptide of any one of claims 42 to 48, and determining binding of autoantibodies to acetylcholine receptor subunits to the composition or the polypeptide within the sample, wherein binding of autoantibodies to the composition indicates that the subject is suitable for treatment by the method of any one of claims 50 to 55, thereby determining the suitability of the subject to be treated.

74. The method of claim 70, wherein binding of at least 20% of the autoantibodies to AChR in the sample to the composition or polypeptide indicates that the subject is suitable for treatment by the method of any one of claims 50 to 55.

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