EP4633660A1

EP4633660A1 - Ig-like fusion proteins for treating myasthenia gravis

Info

Publication number: EP4633660A1
Application number: EP23902953.1A
Authority: EP
Inventors: Kfir Oved; Galit Denkberg; Sharon REEF; Yelena PINZUR; Inbar ARMAN ZELMAN
Original assignee: Canopy Immuno Therapeutics Ltd
Current assignee: Canopy Immuno Therapeutics Ltd
Priority date: 2022-12-13
Filing date: 2023-12-13
Publication date: 2025-10-22
Also published as: WO2024127398A1; CN120500348A

Abstract

Compositions comprising a fragment of a first human acetylcholine receptor subunit or an analog or derivative thereof, and an effector moiety that is not an Fc domain are provided. Pharmaceutical compositions comprising the composition, nucleic acid systems encoding the polypeptides of the composition and methods of treatment and determining suitability for treatment using the composition; as well as methods of producing the composition are also provided.

Description

IG-LIKE FUSION PROTEINS FOR TREATING MYASTHENIA GRAVIS

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/432,239, and of International Patent Application No. PCT/IL2022/051321, both filed on December 13, 2022, all of which are hereby incorporated by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[002] The contents of the electronic sequence listing (CNPY-P-002-PCTl.xml; Size: 178,081 bytes; and Date of Creation: December 10, 2023) is herein incorporated by reference in its entirety.

FIELD OF INVENTION

[003] The present invention is in the field of fusion protein generation and myasthenia gravis treatment.

BACKGROUND OF THE INVENTION

[004] Myasthenia gravis (MG) is the most common neuromuscular transmission disorder. The age of onset is bimodal with a first peak in the second and third decades (female predominance) and a second peak in the sixth to eighth decade (male predominance). MG is an autoimmune disease characterized by weakness of skeletal muscles due to disruption of neuromuscular junction function. -85% of MG patients have acetylcholine receptor (AChR) specific antibodies in their serum, which act as AChR antagonists, cause receptor clustering and internalization, and recruit complement which subsequently cause tissue damage. Individual patients have a mix of different antibodies to the AChR. Some patients with AChR antibody-positive MG also have thymic abnormalities with roughly two thirds with hyperplasia, and 10% with thymoma. The predominant clinical feature of MG is fluctuating skeletal muscle weakness, often with true muscle fatigue.

[005] There are two clinical forms of myasthenia gravis: ocular and generalized. In ocular myasthenia, the weakness is limited to the eyelids and extraocular muscles. In generalized disease, the weakness may also affect ocular muscles, but it also involves a variable combination of bulbar, limb, and respiratory muscles. Transient worsening of symptoms can be triggered by infection, surgery, pregnancy, childbirth, medications, tapering of immunosuppressive medications, or as part of the natural progression of the disease. Respiratory muscles involvement is the most serious symptom of myasthenia gravis, that might lead to a respiratory insufficiency and pending respiratory failure, called "myasthenic crisis". There is also a long list of drugs that must be avoided in MG patients including fluoroquinolone, aminoglycoside, magnesium sulfate, hydroxychloroquine, penicillamine, and botulinum toxin. Beta blockers, procainamide, quinidine, and quinine should also be avoided when possible. The therapies for MG include acetylcholinesterase inhibitor (pyridostigmine), chronic immunosuppressive therapies, rapid and transient immunomodulatory therapies (e.g. plasma exchange and intravenous immune globulin - IVIG), and thymectomy. The treatment goal is to allow the patient minimal symptoms with minimal drug related side effects; however, no cure is available. Initial symptomatic therapy in patients with MG is based on acetylcholinesterase inhibitor (e.g., pyridostigmine). Cholinergic adverse effects of pyridostigmine can be dose-limiting in many patients and comprise abdominal cramping and diarrhea. Most patients with generalized MG require additional therapy with glucocorticoids and/or other immunosuppressive drugs, though this is a second line therapy. Therapeutic plasma exchange (plasmapheresis) and IVIG have a prompt effect but a short duration. International Patent Application W02012141026 teaches AChR-alpha extracellular domain-Fc fusion proteins for treating MG. New methods of treating MG, especially ones that have a long-lasting effect, are greatly needed. Specifically, therapies that target the autoantibodies that cause MG are needed, and beyond this, drugs that can directly target the autoreactive B cells/plasma cells that are the source of these autoantibodies and potentially cure the conditions are greatly needed. SUMMARY OF THE INVENTION

[006] The present invention provides compositions 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.

[007] According to a first aspect, there is provided 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.

[008] According to a first aspect, there is provided 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 unmodified Fc domain.

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

[010] 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).

[Oi l] According to some embodiments, the effector moiety capable of inducing death in a cell binding either of the fragments.

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

[013] According to some embodiments, the effector moiety is selected from: alpha- amanitin, PNU- 159682, tesirine, deruxtecan (Dxd), mertansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and a combination thereof. [014] According to some embodiments, the effector moiety is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141 comprising a plurality of mutations selected from: L15V/F23L/R72P/Y80L/P176L, S19D/A110L/I112E, and G16A/A110L/I112E within the SEQ ID NO: 12 or SEQ ID NO: 141.

[015] According to some embodiments, the composition comprises a protein complex comprising a. a first polypeptide chain comprising the 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 second dimerization domain; wherein the first and second dimerization domains are configured to dimerize with each other.

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

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

[018] 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 the second dimerization domains dimerizes by a disulfide bond; or b. the first and second dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein the first and second dimerization domains do not both comprise the CHI domain or the CL domain.

[019] According to some embodiments, the fragment and the dimerization domain of the first, second or both polypeptide chains are separated by a linker. [020] According to some embodiments, the first polypeptide chain, the second polypeptide chain or both further comprise the effector moiety.

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

[022] 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 comprise an effector moiety that is not an Fc domain.

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

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

[025] 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.

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

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

[028] According to some embodiments, the composition is devoid of an antibody variable domain.

[029] According to some embodiments, the composition further comprises 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 to each other. [030] 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 the second dimerization domains dimerizes by a disulfide bond; or b. the third and fourth dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein the first and third polypeptides do not both comprise the CHI domain or the CL domain.

[031] According to some embodiments, the composition further comprises 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 six dimerization domains are capable of dimerizing to each other.

[032] 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 the second dimerization domains dimerizes by a disulfide bond; or b. the fifth and six dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein the first and third polypeptides do not both comprise the CHI domain or the CL domain.

[033] 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. [034] 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.

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

[036] According to some embodiments, the polypeptide chain further comprises an Fc region of a human antibody heavy chain and a 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.

[037] According to some embodiments, the composition comprises a second polypeptide chain comprises 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.

[038] According to some embodiments, the effector moiety is linked to the fragments by a linker.

[039] According to some embodiments, the complex comprises at least one amino acid sequence selected from SEQ ID NO: 64 to 69 or a derivative thereof comprising at least 80% identity thereto.

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

[041] According to some embodiments, the mutation comprises replacement of a cys loop within an acetylcholine receptor subunit with CDVSGVDTESGATNC (SEQ ID NO: 44).

[042] According to some embodiments, the acetylcholine receptor subunit is selected from: 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.

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

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

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

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

[047] According to some embodiments, the fragment is selected from: a. a fragment of ACHRA and comprises a mutation selected from: deletion of N141, F100G, W149R, V155A, Y93F, Y93H, Y93R and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 1 or a AChRa with increased solubility comprising SEQ ID NO: 131; b. a fragment of ACHRG and comprises a mutation selected from: M84S, Y105E, Y117E, Y117R, and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 6 or a AChRa with increased solubility comprising SEQ ID NO: 133; and c. a fragment of ACHRD and comprises a mutation selected from: C108A, C108I, Y 119R, deletion of N141, L151E and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 8 or a AChRa with increased solubility comprising SEQ ID NO: 134.

[048] 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. [049] According to some embodiments, the single polypeptide chain is selected from: SEQ ID NO: 94, SEQ ID NO: 104, and SEQ ID NO: 108-129.

[050] According to another aspect, there is provided a polypeptide comprising a fragment of a first human acetylcholine receptor subunit comprising at least one mutation that decreases aggregation of the fragment, and an effector moiety that is not an Fc domain, wherein the fragment is selected from: a. a fragment of ACHRA and comprises a mutation selected from: deletion of N141, F100G, W149R, V155A, Y93F, Y93H, Y93R and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 1 or a AChRa with increased solubility comprising SEQ ID NO: 131; b. a fragment of ACHRG and comprises a mutation selected from: M84S, Y105E, Y117E, Y117R, and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 6 or a AChRa with increased solubility comprising SEQ ID NO: 133; and c. a fragment of ACHRD and comprises a mutation selected from: C108A, C108I, Y 119R, deletion of N141, L151E and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 8 or a AChRa with increased solubility comprising SEQ ID NO: 134.

[051] According to some embodiments, the polypeptide further comprises replacement of 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).

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

[053] 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. [054] According to some embodiments, the polypeptide comprises a sequence selected from SEQ ID NO: 72-91.

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

[056] According to some embodiments, the effector moiety is selected from alpha- amanitin, PNU- 159682, tesirine, Dxd, mertansine, MMAE, MMAF and a combination thereof.

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

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

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

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

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

[062] According to some embodiments, the method further comprises reducing in the subject the levels of circulating antibodies against a human acetylcholine receptor subunit within a protein complex comprising the first human acetylcholine receptor subunit or the second human acetylcholine receptor subunit. [063] According to some embodiments, the treating comprises decreasing the concentration of circulating autoantibodies against the human acetylcholine receptor subunits.

[064] According to some embodiments, the treating comprises killing B cells producing the autoantibodies.

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

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

[067] According to some embodiments, the nucleic acid system further comprises a third nucleic acid molecule encoding the third polypeptide chain of a composition of the invention, a fourth nucleic acid molecule encoding the fourth polypeptide chain of a composition of any of the invention.

[068] According to another aspect, there is provided a method of producing a composition of the invention or the polypeptide of the invention, 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 the polypeptide of the invention.

[069] According to another aspect, there is provided a method for producing a protein 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 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 culturing 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 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 analog or derivative thereof, wherein the first and second human acetylcholine receptor subunit are different subunits; and ii. linking the first fragment to the second fragment to produce a single polypeptide chain; thereby producing a protein.

[070] According to another aspect, there is provided a method for 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 analog or derivative thereof, wherein the first and second human acetylcholine receptor subunit 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 the dimerization and linking the first polypeptide chain, the second polypeptide chain or both to an effector moiety that is not an Fc domain; or culturing 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 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 analog or derivative thereof, wherein the first and second human acetylcholine receptor subunit 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; thereby producing a protein complex.

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

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

[073] 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 an 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 to each other; and contacting the first, second, and third polypeptides under conditions sufficient to induce the dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a third polypeptide chain produced by: i. obtaining a third fragment of an 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 or capable of dimerizing to each other.

[074] 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 an 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 to each other; and contacting the first, second, third and fourth polypeptides under conditions sufficient to induce the dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a fourth polypeptide chain produced by: i. obtaining a fourth fragment of an 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 or capable of dimerizing to each other.

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

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

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

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

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

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

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

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

[083] According to some embodiments, binding of at least 20% of autoantibodies against AChR in the sample to the composition or polypeptide indicates the subject is suitable to be treated by a method of the invention.

[084] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[085] Figures 1A-G: (1A) A bar graph of the % depletion results as measured in an anti- AChR serology ELISA assay with and without serum depletion with Alphal ECD. The graph shows the results of 335 randomly selected MG patients where the y-axis shows the % depletion (defined as [100% - {anti- AChR cone (with prior alpha-ECD depletion) / anti- AChR cone (untreated)}] and the x-axis represent the different patient samples. (1B-D) Line graphs showing dose-dependent depletion in (IB) an exemplary highly depleted sample, (1C) an exemplary moderately depleted sample and (ID) and exemplary non-depleted sample. The y-axis represents the concentration of the free anti-AChR antibody in the sample in nM, and the x-axis shows the concentration of Alpha-ECD used for depleting the sample in nM. (IE) A bar graph of total antibody concentration in the serum samples used in 1A. Grey line indicates the clinical cutoff for disease. (IF) Diagram of the method of determining the percentage of AChR fragment- specific antibodies present in a sample by using a depletion assay. (1G) Dot plot showing the correlation between % Alpha binding and Anti- AChR titer. [086] Figures 2A-B: (2A) A contour plot showing for each serum sample of 41 MG patients the percentage of the relative anti-AchR antibodies that was detected against each AChR subunit (s/6/y/p/a). (2B) A bar graph of the percent blocking for various subunits or combinations of subunits in the serum samples from 80 randomly tested MG patients. In addition to the average % depletion, the graph also shows the percent of patients that would have at least 50% or at least 75% depletion in each subunit or combination thereof.

[087] Figures 3A-J: Diagrams of five possible embodiments of the four-chain therapeutic agent of the invention: (3A) shows a general embodiment of a molecule for treating AChR positive MG, (3B) shows an embodiment in which the four chains each comprise a different protein fragment, (3C) shows an embodiment in which the four protein fragments are all the same, (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 contain the same protein fragment and the two light chains contain different protein fragments. (3G) Line graph of clinical score in rats inoculated with anti- AChR antibodies and then treated with various doses of the molecule of the invention. (3H) Kaplan Meier survival curve of rats inoculated with anti-AChR antibodies and then treated with various doses of the molecule of the invention. (31) Line graph of clinical score in rats inoculated with anti-AChR antibodies and then treated with the molecule of the invention or two non-specific (NS) Fes. (3J) Kaplan Meier survival curve of rats inoculated with anti- AChR antibodies and then treated with the molecule of the invention or the NS-Fcs.

[088] Figures 4A-S: Diagrams of possible embodiments of the two-chain therapeutic agent of the invention: (4A) shows a general embodiment of a molecule with two heavy chains for treating AChR positive MG, (4B) shows embodiments in which at least one of the CHI, CH2 or CH3 domains has been excluded, (4C) shows an embodiment in which the two protein fragments are the same, (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 the tandem fragment configuration in which all the subunits are the same, (4G) shows the tandem fragment configuration in which each heavy chain contains the same two different fragments, (4H) shows the tandem fragment configuration in which the two have chains contain different fragments that are not the same, (41) shows a general embodiment in which two tandem fragments are included in one heavy chain while the other includes only one fragment, (4J) shows a general embodiment in which two tandem fragments are included in one heavy chain while the other is devoid of a fragment, (4K) shows the tandem configuration in which all three subunits are the same, (4L) shows the tandem configuration in which two of the three subunits are the same, (4M) shows the tandem configuration in which all three of the subunits are different, (4N) shows a general embodiment in which three tandem fragments are included in each heavy chain, (40) shows the tandem configuration in which all six subunits are the same, (4P) shows the tandem configuration in which all three subunits on a chain are different but the two chains are the same, (4Q) shows the tandem configuration in which all three subunits on a chain are different and the two chains are different, (4R) shows a general embodiments 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 of a molecule with one heavy chain and one light chain.

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

[090] Figure 6: Diagram of an embodiment of a four-chain therapeutic agent for treating AChR positive MG of the invention similar to those shown in Figure 3 but in which the four chains each comprise a different protein fragment and a different immunoglobulin scaffold which promotes formation of the four-chain molecule.

[091] Figures 7A-B: (7A-B) Diagrams of generic embodiments of the four-chain therapeutic agent of the invention: (7A) shows a generic embodiment of four chains in which two contain chains contain two dimerization domain and two chains contain a single dimerization domain, and (7B) shows an embodiment with optional linkers separating the various domains and fragments.

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

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

[094] Figures 10A-D: 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.

[095] Figure 11: Bar graph of fluorescent increase indicating binding of ECD tetramers to hybridoma cells. MFI fold change from background values were calculated by dividing ECD tetramer binding MFI of any hybridoma by the negative control background MFI. Hybridoma 204-4 was used as a negative control and tetramers CRD-233 and CRD-242 were also used as negative controls.

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

[097] Figure 13A-C: Bar graphs of binding of an alpha-gamma combination molecule tetramers to various B cell hybridoma cell lines: (13A) binding of CRD-506 to anti-alpha hybridoma and anti-gamma hybridoma, (13B) binding of CRD-509 and CRD-600 to antialpha hybridoma and (13C) binding of CRD-509 and CRD-600 to anti-gamma hybridoma and to negative control hybridomas.

[098] Figure 14: Bar graph of binding of various alpha subunit containing molecules to an anti-alpha hybridoma.

[099] Figures 15A-C: Bar graphs of binding of (15A) an alpha-delta molecule, (15B) a gamma-delta molecule, and (15C) an alpha-gamma molecule with an IgG4 Fc to various hybridomas. [0100] Figure 16A-S: (16A-F) Diagrams of possible generic embodiments of the four-chain therapeutic agent of the invention: (16A) shows a generic embodiment of four chains in which two contain chains contain two dimerization domain an effector domain and two chains contain a single dimerization domain and no effector domains, (16B) shows an embodiment in which the chains with only a single dimerization domain contain the effector domain, (16C) shows embodiments with only a single effector domain, (16D) shows an embodiment with optional linkers separating 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 a radio-labeled material and (16F) shows embodiments that include the cytotoxic molecule but lack the effector domain. (16G) Diagrams of dimeric heavy chains each containing two AchR regions and a cytotoxic moiety linked to various regions. (16H) Diagrams of dimeric heavy chain/light chain molecules with a cytotoxic moiety linked to various regions. (161) Diagrams of embodiments of the conjugate of the invention comprising a cytotoxic moiety. (16J-S) Diagrams of possible generic embodiments of the single-chain therapeutic agent of the invention: (16J) the molecule of 8A, (16K) the molecule of 8B, (16L) the molecule of 8C, (16M) the molecule of 8D, and (16N) the molecules of 8E with an effector moiety; (160) the molecule of 8A, (16P) the molecule of 8B, (16Q) the molecule of 8C, (16R) the molecule of 8D, and (16S) the molecules of 8E with a cytotoxic moiety.

[0101] Figures 17A-B: Line graphs of anti-AChR titers in EAMG rats treated with (17A) vehicle only or (17B) a tetrabody with 2 alpha and 2 beta subunits and an Fc domain.

[0102] Figures 18A-E: (18A-C) Histograms of dose dependent binding of B2A2 conjugated to (18A) alpha-amanitin, (18B) Dxd, and (18C) tesirine to an anti-AChRa hybridoma cell line. Non-conjugated B2A2 is shown as control. (18D) Histogram of dose dependent binding of heavy chain heterodimer containing alpha and gamma subunits to TIB -185 anti-AChRa expressing cells and control cells expressing an irrelevant antibody. (18E) Histogram of dose dependent binding of A2G2 heavy chain dimer in which each heavy chain contains a tandem of the alpha and gamma subunits to TIB -185 anti-AChRa expressing cells and control cells expressing an irrelevant antibody.

[0103] Figures 19A-E: (19A-B) Various hybridomas expressing BCR against (19A) AChR alpha (a-18-C5-F6, TIB175 and a-192) and (19B) AChR gamma (g-63-E6-A10, g-50-Hl- E2 and g-66) were incubated in the presence of 32nM, l lnM or 4nM of Molecules 1-4. CRD-509 and CRD-269 were used as controls. Binding was detected with PE-conjugated anti-human pAb and each hybridoma was also stained with PE-anti-RAT BCR. Y-axis represents the MFI value of each sample, divided by the PE anti-RAT BCR MFI value of the tested hybridoma. The presented values for each molecule represent the average calculated value of all three tested alpha hybridomas or gamma hybridomas. (19C-D) Death rate of hybridoma cells for (19C) the anti-AChR gamma hybridomas g-50-Hl-E2 (left) and g-66 (right) and (19D) the anti-AChR alpha hybridoma a-18-C5-F6 when cultured in the presence of PBMCs and Molecules 1, 2, 4 and 5. Addition of CRD-509 was used as a control (percent increase is relative to culture of hybridoma cells and PBMCs). (19E) Live cell count of anti-AChR alpha or gamma hybridomas in the presence of increasing concentrations of CRD-509 or molecule 3 and complement.

[0104] Figures 20A-C: Bar charts of (20A) B2A2-PNU glyco-site conjugated, (20B) B2A2- tesirine cysteine conjugated IgG-like molecules and (20C) A2G2-PNU glyco-site conjugated tandem AChR fragment molecules killing of TIB- 175 AChR- Alpha specific hybridoma cells as compared with control hybridoma cells.

[0105] Figures 21A-C: (21A) Bar chart of CRD-600 and CRD-600-tesirine binding to gamma-specific hybridoma g-66. A delta- specific hybridoma (delta) and an irrelevant hybridoma (204-4) were used as negative controls. (21B-C) Line graphs of the percent increase in cell killing over the unconjugated molecule of (21B) CRD-600-tesirine and (21C) an irrelevant Ig-like molecule conjugated to tesirine. CRD-600-tesirine’s binding and killing was specific to the gamma hybridoma and tesirine background killing, as shown by the irrelevant Ig-like molecule was non-existent.

[0106] Figures 22A-M: Bar charts of binding of molecules of the invention to various hybridomas. Binding of (22A) CRD-213 -tesirine to alpha- and beta- specific hybridomas, (22B) CRD-506-tesirine to alpha- specific hybridomas, (22C) CRD-506-tesirine to gammaspecific hybridomas, (22D) CRD-506-a-amanitin to alpha- and gamma- specific hybridomas, (22E) CRD-506-PNU to alpha- specific hybridomas, (22F) CRD-506-PNU to gamma-specific hybridomas, (22G) CRD-509-tesirine, CRD-509-a-amanitin and CRD-509- PNU to gamma-specific hybridomas, (22H) CRD-509-tesirine, CRD-509-a-amanitin and CRD-509-PNU to alpha- specific hybridomas, (221) CRD-509-MMAE and CRD-509- MMAF to alpha- specific hybridomas, (22J) CRD-509-MMAE and CRD-509-MMAF to gamma-specific hybridomas, (22K) CRD-586-PNU to gamma- and delta- specific hybridomas, (22L) CRD-586-tesirine to gamma- and delta- specific hybridomas, and (22M) CRD-586-a-amanitin to gamma- and delta- specific hybridomas is shown.

[0107] Figure 23: Bar chart of cell internalization of CRD-509.

[0108] Figures 24A-L: (24A-B) Bar charts of cell death induced by CRD-213 conjugated to (24A) tesirine and (24B) DM-1. (24C) Line graph of percent cytotoxicity induced in antigamma hybridoma cells (Anti-AchR hyb) and an irrelevant hybridoma (204-4) co-cultured together with increasing concentrations of CRD-509 unconjugated, CRD-509-MMAE and CRD-509-MMAF.(24D-H) Bar charts of percentage of live cells coming from two hybridoma cocultures treated with (24D) CRD-213 -tesirine, (24E) CRD-506-tesirine, (24F) CRD-509-a-amanitin, (24G) CRD-509-PNU, and (24H) CRD-509-tesirine. (24I-J) Line graphs of cell death induced by (241) CRD-506-tesirine and (24J) CRD-213-tesirine. (24K) Bar charts of cell death induced by CRD-586 or irrelevant fusion molecule conjugated to tesirine and (24L) Bar charts of cell death induced by CRD-586 conjugated to a-amanitin.

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

[0110] Figures 26A-B: (26A) Bar graph of anti-AChR alpha/gamma autoreactive antibody titers in naive C57B16 mice. (26B) Bar graph of autoreactive antibody titers in mice 14 days after immunization with AChR-alpah and AChR-gamma ECDs. Only CRD-509-tesirine reduced titers indicating that autoreactive B cells present before the immunization were killed.

DETAILED DESCRIPTION OF THE INVENTION

[0111] The present invention, in some embodiments, provides compositions comprising a fragment of a first human receptor target of myasthenia gravis autoantibodies or an analog or derivative thereof and a fragment of a second human protein receptor of myasthenia gravis autoantibodies or an analog or derivative thereof. Protein complexes comprising at least two polypeptide chains wherein a first chain comprises a fragment of a first human protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and a first dimerization domain and a second chain comprises a fragment of a second human protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and a second dimerization domain capable of dimerizing with the first dimerization domain are also provided. The present invention further concerns pharmaceutical composition comprising the compositions and/or protein complexes, nucleic acids encoding the polypeptides of the compositions and/or protein complexes and methods of treatment and determining suitability for treatment using the compositions and/or protein complexes; as well as methods of producing the compositions and/or protein complexes.

[0112] By a first aspect, there is provided a protein comprising a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof.

[0113] By another aspect, there is provided a composition comprising a fragment of a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and a fragment of a second protein target of myasthenia gravis autoantibodies or an analog or derivative thereof.

[0114] By another aspect, there is provided a protein comprising a fragment of a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and a fragment of a second protein target of myasthenia gravis autoantibodies or an analog or derivative thereof.

[0115] By another aspect, there is provided a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a fragment of a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and a first dimerization domain and a second polypeptide chain comprising a fragment of a second protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and second dimerization domain.

[0116] By another aspect, there is provided a fusion protein or protein conjugate comprising a fragment of a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and an effector moiety.

[0117] In some embodiments, the protein is for use in treating myasthenia gravis. In some embodiments, the protein is a fusion protein. In some embodiments, the protein is a fusion protein of the invention. In some embodiments, the polypeptide is for use in treating myasthenia gravis. In some embodiments, the polypeptide chain is for use in treating myasthenia gravis. In some embodiments, the protein complex is for use in treating myasthenia gravis. In some embodiments, the composition is for use in treating 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, the 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 myasthenia gravis autoantibodies or an analog or derivative thereof and a first dimerization domain and a second polypeptide chain comprising a fragment of a second protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and second dimerization domain. In some embodiments, the composition comprises a protein complex of the invention. In some embodiments, the composition comprises a protein of the invention. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is a fusion protein.

[0119] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating into cells. In one embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[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 is devoid of an antibody variable domain. In some embodiments, the protein complex is devoid of an antibody variable domain. In some embodiments, the composition is devoid of a variable domain. In some embodiments, the protein complex is devoid of a variable domain. In some embodiments, the first chain is devoid of a variable domain. In some embodiments, the second chain is devoid of a variable domain. In some embodiments, the protein complex is a multi-chain complex. In some embodiments, the composition is a therapeutic composition. In some embodiments, the protein complex is a therapeutic complex. In some embodiments, the composition is for use in a therapeutic method. In some embodiments, the protein complex is for use in a therapeutic method. In some embodiments, the composition is for use in production of a medicament. In some embodiments, the protein complex is for use in the production of a medicament. In some embodiments, the composition is for use in treating myasthenia gravis. In some embodiments, the protein complex is for use in treating myasthenia gravis. In some embodiments, the protein complex is for use in diagnosing myasthenia gravis. In some embodiments, the protein complex is for use in determining appropriate treatment in myasthenia gravis. In some embodiments, the protein complex is for use in characterizing the serological response in myasthenia gravis. In some embodiments, the protein complex is for use in determining the AChR antibody titer in myasthenia gravis.

[0121] As used herein, the term “polypeptide chain” refers to a polymer of amino acids linked by peptide bonds from an amino terminus (N-terminus) to a carboxyl terminus (C- terminus). In some embodiments, the polypeptide chain is a recombinant polypeptide. In some embodiments, a polypeptide chain comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, a polypeptide chain comprises at most 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 amino acids. Each possibility represents a separate embodiment of the invention.

[0122] As used herein, the term “recombinant polypeptide” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring. In some embodiments, the protein complex is not naturally occurring. In some embodiments, the polypeptide chain is not naturally occurring. In some embodiments, the recombinant polypeptide is a synthetic polypeptide. The term “recombinant DNA” refers to DNA molecules formed by laboratory methods. Generally, this recombinant DNA is in the form of a vector, plasmid or virus used to express the recombinant protein in a cell. Production of recombinant proteins by cellular expression is well known in the art and any method of recombinant protein expression may be used to produce the polypeptide of the invention. Cell free expression systems for recombinant protein production may also be employed.

[0123] The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide). In some embodiments, a nucleic acid molecule of the invention is expressed in a cell to produce a polypeptide of the invention. In some embodiments, a nucleic acid complex of the invention is expressed in a cell to produce a protein complex of the invention. In some embodiments, the RNA is a vector.

[0124] Expressing a DNA sequence or an RNA within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the DNA sequence is in an expression vector such as plasmid or viral vector. In some embodiments, a Kozak sequence is inserted upper stream of the transcription initiating codon. In some embodiments, the Kozak sequence enhances the amount of protein expresses.

[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 the same. In some embodiments, the polypeptide chains are different. In some embodiments, at least two of the polypeptide chains are the same. In some embodiments, at least two of the polypeptide chains are different.

[0126] It will be understood by a skilled artisan that the ECDs of the various subunits interact to form the full receptor and thus even without a dimerization domain can form a protein complex. 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.

Proteins

[0127] In some embodiments, the protein is a mammalian protein. In some embodiments, the mammal is a human. In some embodiments, the protein is a transmembrane protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a subunit 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 capable of transducing a signal to the cytoplasm of the cell. In some embodiments, a receptor comprises a ligand binding domain. In some embodiments, a receptor comprises a transmembrane domain. In some embodiments, a receptor comprises an intracellular domain.

[0129] In some embodiments, the fragment comprises an extracellular domain (ECD) of the 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, a variant is a mutant. In some embodiments, a variant comprises a replacement of a portion of the extracellular domain. In some embodiments, the fragment comprises a fragment of an extracellular domain of the protein. In some embodiments, the fragment consists of an extracellular domain of the protein. In some embodiments, the fragment consists of a fragment of an extracellular domain of the protein. In some embodiments, the fragment comprises a transmembrane domain of the protein. In some embodiments, the fragment is devoid of a transmembrane domain of the protein. In some embodiments, the fragment is devoid of an intracellular domain of the protein. In some embodiments, the chain is devoid of a transmembrane domain. In some embodiments, the chain is devoid of an intracellular domain. In some embodiments, the fragment includes a sequence from a homologous human protein. In some embodiments, the fragment includes a sequence from a homologous non-human protein. In some embodiments, the fragment includes mutations in the human protein.

[0130] In some embodiments, the fragment comprises at least 5 amino acids of the protein. In some embodiments, the fragment comprises at least 10 amino acids of the protein. In some embodiments, the fragment comprises at least 5, 10 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, amino acids of the protein are consecutive amino acids of the protein. In some embodiments, the fragment comprises less than 100% of the protein. In some embodiments, the fragment comprises less than 100% of an extracellular domain of the protein. In some embodiments, the fragment comprises less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50% of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, the fragment comprises less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50% of an extracellular domain of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, the fragment comprises between 5-500, 5-250, 5-100, 5-50, 10-500, 10-250, 10-100, 10-50, 20-500, 20-250, 20-200, 20-50, 25-500, 25-250, 25-100, 25-50, 50-500, 50-250, 50-100, 100-500, or 100-250 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, 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, a 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 invention. In some embodiments, a variant comprises at least 85% homology or identity. In some embodiments, a variant comprises at least 90% homology or identity. In some embodiments, a variant comprises at least 92% homology or identity. In some embodiments, a variant comprises at least 95% homology or identity. In some embodiments, a variant comprises at least 97% homology or identity. In some embodiments, a variant comprises at least 99% homology or identity. In some embodiments, a 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 as compared to a region of the protein excluded from the chain. In some embodiments, a region of the protein is replaced by a region of protein that is not the protein. In some embodiments, the replacement region comprises increased solubility as compared to the region of the protein that has been replaced. In some embodiments, the replacement region comprises increased protein stability as compared to the region of the protein that has been replaced.

[0133] In some embodiments, the protein is a target of antibodies. As used herein, the term “antibody” includes all classes of IgA, IgD, IgE, IgG and IgM and also includes all subclasses thereof. In some embodiments, the antibody is a circulating antibody. In some embodiments, the antibody is a naturally occurring antibody. In some embodiments, the antibodies are autoantibodies.

[0134] As used herein, the term “autoantibodies” refers to antibodies generated by a subject’s own immune system against at least one of the subject’s own proteins. In some embodiments, an autoantibody is an autoreactive antibody. In some embodiments, autoantibodies target self-antigens. Self-antigens are also known as autoantigens. In some embodiments, the autoantibodies are associated with myasthenia gravis. In some embodiments, the autoantibodies characterize myasthenia gravis. In some embodiments, the autoantibodies are autoantibodies of myasthenia gravis. In some embodiments, autoantibodies are generated by auto-reactive B cells. In some embodiments, the protein is an antigen of the antibodies. In some embodiments, the fragment comprises an antigen of the antibodies. In some embodiments, the fragment comprises at least one antigen of the antibodies. In some embodiments, the fragment comprises at least two antigens of the antibodies. In some embodiments, the fragment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigens of the antibodies. Each possibility represents a separate embodiment of the invention. In some embodiments, an antigen of the antibodies is an autoantigen. In some embodiments, the antigen is an epitope. In some embodiments, the antigen includes at least one epitope. In some embodiments, an epitope comprises at least 5 amino acids. In some embodiments, an epitope comprises 5-6 amino acids. In some embodiments, an epitope comprises 5-10 amino acids. In some embodiments, an epitope is a simple epitope. In some embodiments, a simple epitope is a linear epitope. In some embodiments, an epitope is a complex epitope. In some embodiments, a complex epitope is a 3D epitope. In some embodiments, a complex epitope is a discontinuous epitope. In some embodiments, a discontinuous epitope comprises at least two discontinuous sections of amino acids that combine to form an epitope. In some embodiments, a linker sequence is between the two sections of the epitope.

[0135] As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to the sequence of the protein but in which one or more residues have been conservatively substituted with a functionally similar residue. In some embodiments, an analog displays similar functionality to the original protein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention. In some embodiments, the substitution is outside of an antigenic region of the protein. In some embodiments, the substitution is outside an epitope of the antibodies. In some embodiments, the analog is still a target of the antibodies. In some embodiments, the analog retains binding of autoantibodies. An analog may have deletions or mutations that result in an amino acids sequence that is different than the canonical amino acid sequence of protein. Further, an analog may be analogous to a fragment of the protein, however, in such a case the fragment must comprise at least 50 consecutive amino acids of protein or at least one epitope of the antibodies. In some embodiments, an analog is an analog to the canonical sequence of the protein. [0136] In some embodiments, an analog to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, an analog to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, the analog comprises at least one substitution. In some embodiments, an analog comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. Each possibility represents a separate embodiment of the invention. In some embodiments, substitution is a mutation of the canonical sequence.

[0137] The term “derivative” as used herein, refers to any polypeptide that is based off the protein and still comprises retains binding of the antibodies. A derivative is not merely a fragment of the protein, nor does it have amino acids replaced or removed (an analog), rather it may have additional modification made to the protein, such as post-translational modification. Further, a derivative may be a derivative of a fragment of the protein, however, in such a case the fragment must comprise at least 50 consecutive amino acids of the protein or at least one epitope of the antibodies. In some embodiments, the derivative is a derivative of a canonical sequence of the protein.

[0138] In some embodiments, a derivative to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention. In some embodiments, a derivative to the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a separate embodiment of the invention.

[0139] Canonical amino acid sequences of known proteins are well known in the art. They can be found in a variety of databases, including UniProt, NCBI, and the UCSC Genome Browser. Any sequence accepted as a canonical sequence may be employed. For a non- limiting example, human acetylcholine receptor subunit alpha is encoded by the CHRNA1 gene, its canonical nucleic acid sequence can be found in Entrez gene 1134, its canonical protein coding mRNA sequence can be found in NM_001039523 and NM_000079, its canonical amino acid sequence can be found in NP_000070 and NP_031415 and UniProt number P02708. In some embodiments, a canonical sequence is a sequence identical to the sequence present in at least 50, 60, 70, 75, 80, 90, 95, 97, or 99 percent of a population. Each possibility represents a separate embodiment of the invention. In some embodiments, a canonical sequence is a sequence identical to the most prevalent sequence present in a population. In some embodiments, the population is a disease population. In some embodiments, the population is a population with the autoimmune disease.

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

[0141] In some embodiments, the protein is the acetylcholine receptor alpha subunit (AChRa). In some embodiments, 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, AChRa is identified by UniProt ID P02708. In some embodiments, AChRa is identified by UniProt ID P02708.1 or P02708.2. In some embodiments, AChRa is identified by UniProt ID P02708.1. In some embodiments, AChRa is identified by UniProt ID P02708.2. In some embodiments, CHRNA1 comprises or consists of the nucleotide sequence provided in NM_001039523 or NM_000079. In some embodiments, AChRa comprises or consists of an amino acid sequence provided in NP_000070 or NP_001034612. In some embodiments, a canonical amino acid sequence of an extracellular domain of AChRa comprises or consists of SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDL SNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLP (SEQ ID NO: 1). In some embodiments, a canonical amino acid sequence of an extracellular domain of AChRa comprises or consists of

SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQGDMVDLPRPSCVTLGVPLFSHLQNEQWVDYNLKWNPDDYGGVKKIHIPSEKI WRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNC SMKEGTWTYDGSVVAINPESDQPDESNFMESGEWVIKESRGWKHSVTYSCCPDTP YEDITYHFVMQREP (SEQ ID NO: 2). In some embodiments, the extracellular domain is devoid of a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the AChRa signal peptide comprises or consists of MEPWPEEEEFSECSAGEVEG (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, AChRb is encoded by the gene CHRNB 1. In some embodiments, the AChRb is human AChRb. In some embodiments, CHRNB1 is identified by Entrez gene #1140. In some embodiments, AChRb is identified by UniProt ID Pl 1230. In some embodiments, CHRNB 1 comprises or consists of the nucleotide sequence provided in NM_000747. In some embodiments, AChRb comprises or consists of an amino acid sequence provided in NP_000738. In some embodiments, a canonical amino acid sequence of an extracellular domain of AChRb comprises or consists of SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYL DLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVS SDGSVRWQPPGIYRSSCSIQVTYFPFDWQNCTMVFSSYSYDSSEVSLQTGLGPDGQ GHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKP (SEQ ID NO: 4). In some embodiments, the extracellular domain is devoid of a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the AChRb signal peptide comprises or consists of MTPGALLMLLGALGAPLAPGVRG (SEQ ID NO: 5).

[0143] In some embodiments, the protein is the acetylcholine receptor gamma subunit (AChRg). In some embodiments, 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, AChRg comprises or consists of an amino acid sequence provided in NP_005190. In some embodiments, a canonical amino acid sequence of an extracellular domain of AChRg comprises or consists of RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVW IEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNV LVSPDGCIYWLPPAIFRSACSISVTYFPFDWQNCSLIFQSQTYSTNEIDLQLSQEDGQ TIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKP (SEQ ID NO: 6). In some embodiments, the extracellular domain is devoid of a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the AChRg signal peptide comprises or consists of MHGGQGPLLLLLLLAVCLGAQG (SEQ ID NO: 7).

[0144] In some embodiments, the protein is the acetylcholine receptor delta subunit (AChRd). In some embodiments, 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, AChRd comprises or consists of an amino acid sequence provided in NP_000742, NP_001243586, NP_001298124 or NP_001298125. In some embodiments, a canonical amino acid sequence of an extracellular domain of AChRd comprises or consists of

LNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNV WIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVL V YH YGF V YWLPPAIFRS S CPIS VTYFPFDWQNCSLKFSSLKYTAKEITLSLKQD AKE NRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSPSRQDITFYLIIRRKP (SEQ ID NO: 8). In some embodiments, the extracellular domain is devoid of a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the AChRd signal peptide comprises or consists of MEGPVLTLGLLAALAVCGSWG (SEQ ID NO: 9).

[0145] In some embodiments, the protein is the acetylcholine receptor epsilon subunit (AChRe). In some embodiments, 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, AChRe is identified by UniProt ID Q04844. In some embodiments, CHRNE comprises or consists of the nucleotide sequence provided in NM_000080. In some embodiments, AChRe comprises or consists of an amino acid sequence provided in NP_000071. In some embodiments, a canonical amino acid sequence of an extracellular domain of AChRe comprises or consists of KNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGI DWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLVYEG GSVTWLPPAIYRSVCAVEVTYFPFDWQNCSLIFRSQTYNAEEVEFTFAVDNDGKTI NKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRKP (SEQ ID NO: 10). In some embodiments, the extracellular domain is devoid of a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the 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 single peptide is of an antibody heavy chain. In some embodiments, the signal peptide is of an antibody light chain. In some embodiments, the signal peptide is of the Kappa light chain. In some embodiments, the signal peptide is of the Lambda light chain. In some embodiments, the heavy chain signal peptide comprises MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 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 the 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 proteins, and the fragments are different fragments. In some embodiments, the fragments are different fragments. In some embodiments, the fragments comprise or consist of different sequences. In some embodiments, the first and second proteins are different proteins.

[0148] In some embodiments, the protein or fragment comprises a mutation that increases solubility. In some embodiments, the protein or fragment comprises a mutation that increases stability of the protein or fragment. In some embodiments, the mutation is an insertion. In some embodiments, the protein is a surface protein and comprises a mutation that increases solubility. In some embodiments, the fragment is an extracellular domain of a surface protein and comprises an insertion that increases solubility. In some embodiments, the insertion is in place of a region of the protein or fragment. In some embodiments, a loop of the protein is replaced with a loop with higher solubility. In some embodiments, the loop of AChRa comprises or consists of CEIIVTHFPFDEQNC (SEQ ID NO: 39). In some embodiments, the loop of AChRb comprises or consists of CSIQVTYFPFDWQNC (SEQ ID NO: 40). In some embodiments, the loop of AChRg comprises or consists of CSISVTYFPFDWQNC (SEQ ID NO: 41). In some embodiments, the loop of AChRd comprises 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 loop with higher solubility comprises or consists of CDVSGVDTESGATNC (SEQ ID NO: 44). In some embodiments, the insert of higher solubility comprises or consists of SEQ ID NO: 44. In some embodiments, the insert of higher solubility comprises DVSGVDTESGAT (SEQ ID NO: 63). In some embodiments, 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 mutation of at least two of V8E, W 149R and V155A. In some embodiments, the mutation is mutation of all three of V8E, W149R and V155A. In some embodiments, AChRa comprises the mutation. In some embodiments, a non- AChRa acetylcholine receptor subunit comprises parallel mutations. In some embodiments, parallel mutation are mutations to amino acids with homology.

[0149] In some embodiments, the alpha subunit extracellular domain into which mutations are made comprises or consists of

SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCDVSGVDTESGATNCSMKLGTWTYDGSVVAINPESDQP DLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLP (SEQ ID NO: 131). In some embodiments, the beta subunit extracellular domain into which mutations are made comprises or consists of

SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYL DLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVS SDGSVRWQPPGIYRSSCDVSGVDTESGATNCTMVFSSYSYDSSEVSLQTGLGPDG QGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKP (SEQ ID NO: 132). In some embodiments, the gamma subunit extracellular domain into which mutations are made comprises or consists of RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVW IEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNV LVSPDGCIYWLPPAIFRSACDVSGVDTESGATNCSLIFQSQTYSTNEIDLQLSQEDG QTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKP (SEQ ID NO: 133). In some embodiments, the delta subunit extracellular domain into which mutations are made comprises or consists of LNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNV WIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVL VYHYGFVYWLPPAIFRSSCDVSGVDTESGATNCSLKFSSLKYTAKEITLSLKQDAK ENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSPSRQDITFYLIIRRKP (SEQ ID NO: 134). In some embodiments, the epsilon subunit extracellular domain into which mutations are made comprises or consists of KNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGI DWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLVYEG GSVTWLPPAIYRSVCDVSGVDTESGATNCSLIFRSQTYNAEEVEFTFAVDNDGKTI NKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRKP (SEQ ID NO: 135).

[0150] By another aspect, there is provided a protein comprises any one of SEQ ID NO: 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 NO: 131-135. In some embodiments, a complex of the invention comprises a first chain comprising a sequence selected from SEQ ID NO: 131-135. In some embodiments, a complex of the invention comprises a second chain comprising a sequence selected from SEQ ID NO: 131-135. In some embodiments, a complex of the invention comprises a third chain comprising a sequence selected from SEQ ID NO: 131-135. In some embodiments, a complex of the invention comprises a fourth chain comprising a sequence selected from SEQ ID NO: 131- 135. In some embodiments, the extracellular domain fragment is selected from SEQ ID NO: 131-135. In some embodiments, the extracellular domain variant is selected from SEQ ID NO: 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. It will be understood by a skilled artisan that as the protein complex of the invention is meant to bind antibodies and B cells it would be advantageous not to bind the endogenous ligand present in the subject and thus leaves normal ligand levels available to bind to the endogenous receptor. In some embodiments, the protein is AChRa and the mutation is 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 tyrosine 190 of SEQ ID NO: 1 to phenylalanine. In some embodiments, the protein is AChRa and the mutation is tyrosine 215 of SEQ ID NO: 2 to phenylalanine.

[0153] By another aspect, there is provided a protein comprising an extracellular domain of an acetylcholine receptor subunit comprising at least one mutation that decreases aggregation.

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

[0155] In some embodiments, the protein is AChRa and the mutation is 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 AChRa and the mutation is 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 AChRa and the mutation is 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 AChRa and the mutation is 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 mutation of tyrosine 93. In some embodiments, mutation of tyrosine 93 decreases alpha-gamma interactions. In some embodiments, the tyrosine 93 is mutated to any amino acid that decreases alpha to 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 oxidation of the protein. In some embodiments, the protein is AChRg and the mutation is mutation of methionine 84. The mutation is made, at least in part, to decrease methionine oxidation and increase shelf life. Mutation to any amino acid will produce this result. In some embodiments, mutation of methionine 84 reduces alpha-gamma interaction. In some embodiments, the methionine 84 is mutated to any amino acid that decreases alpha to 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 mutation of tyrosine 105. In some embodiments, mutation of tyrosine 105 reduces alpha-gamma interaction. In some embodiments, the tyrosine 105 is mutated to any amino acid that decreases alpha to 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 (Y 105K). 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 mutation of tyrosine 117. In some embodiments, mutation of tyrosine 117 reduces alpha-gamma interaction. In some embodiments, the tyrosine 117 is mutated to any amino acid that decreases alpha to 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 (Y 117K). 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 a plurality of mutations and comprises at least two mutations selected from mutation of M84, Y105 and Y 117. In some embodiments, the mutation is a plurality of mutations and comprises at least two mutations selected from M84S, Y 105E and Y 117E. In some embodiments, the mutation is a plurality of mutations and comprises at least two mutations selected from M84S, Y 105E and Y 117R. In some embodiments, the mutation is a plurality of mutations and comprises at all of M84S, Y105E and Y117E. In some embodiments, the mutation is a plurality of mutations and comprises all of M84S, Y105E and Y117R In some embodiments, the mutation is two mutations and comprises M84S and Y105E. In some embodiments, the mutation is two mutations and comprises Y117E and Y105E. In some embodiments, the mutation is two mutations and comprises Y 117R and Y 105E.

[0157] In some embodiments, the protein is AChRd and the mutation is mutation of cysteine 108. In some embodiments, cysteine 108 is mutated to any other amino acid. In some embodiments, cysteine 108 is deleted. It will be understood by a skilled artisan that the desire is to decrease aggregation by removing a free cysteine that could have formed a disulfide bond. As such 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 mutation of tyrosine 119. In some embodiments, mutation of tyrosine 119 reduces delta hydrophobicity. In some embodiments, the tyrosine 119 is mutated to any amino acid that decreases 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 (Y 119E). In some embodiments, tyrosine 119 is mutated to aspartic acid (Y 119D). 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 AChRa and the mutation is 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 mutation of leucine 151. In some embodiments, mutation of leucine 151 reduces subunit interaction. In some embodiments, the leucine 151 is mutated to any amino acid that decreases subunit interaction. 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 an amino acid that is not positively charged. 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 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 mutation of C108, Y119, L151 and deletion of N141. In some embodiments, the mutation is a plurality of mutations selected from C 108A, Y 119R, L151E and deletion of N141. In some embodiments, the mutation is two mutations and comprises C108A and Y119R. In some embodiments, the mutation is two mutations and comprises C108A and L151E. In some embodiments, the mutation is two mutations and comprises C108A and deletion of N141. In some embodiments, the mutation is three mutations and comprises C108A, Y 119R and L151E. In some embodiments, the mutation is three mutations and comprises Cl 08 A, Y119R and deletion of N 141.

Dimerization domains

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

[0159] As used herein, the term “dimerization domain” refers to an amino acid sequence that upon contacting another amino acid sequence (the other dimerization domain) binds to it to form a dimer. Dimerization domains are well known in the art, as many protein sequences are known to bind to each other. In some embodiments, dimerization comprises formation of a covalent bond between the dimerization domains. In some embodiments, dimerization comprises electrostatic binding. In some embodiments, dimerization does not comprise electrostatic binding. In some embodiments, dimerization is reversible. In some embodiments, dimerization is irreversible. In some embodiments, dimerization comprises a bond forming between the dimerization domains. In some embodiments, the bond is a chemical bond. In some embodiments, the bond is a disulfide bond. In some embodiments, the bond is a peptide bond. Examples of dimerization domain include the hinge domain of antibody heavy chains, the CH1/CL domains of antibody heavy/light chains, and the ECD domains of TCR alpha/beta to name but a few. Additionally, the upper hinge domain can be engineered with cysteine substitutions/mutations to serine in order to prevent dimerization. In some embodiments, the dimerization domain comprises or consists of the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 63).

[0160] In some embodiments, the dimerization domain comprises or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, an Ig hinge domain is a heavy chain hinge domain. In some embodiments, the Ig is a human Ig. In some embodiments, the immunoglobulin is selected from IgA, IgD, IgE, IgG and IgM. In some embodiments, the immunoglobulin is IgG. In some embodiments, the IgG is IgGl. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is selected from IgGl and IgG3. In some embodiments, the IgG is IgG4. In some embodiments, the first and second dimerization domains are both Ig hinge domains. In some embodiments, the first and second dimerization domains are identical. In some embodiments, the first and second dimerization domains are at least 95% identical. In some embodiments, the first and second dimerization domains are at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% identical. Each possibility represents a separate embodiment of the invention.

[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 IgGl 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

ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC PRCPAPELLGGP (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 a CPXCP (SEQ ID NO: 20) motif. In some embodiments, the 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). It will thus 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 CL kappa domain. In some embodiments, the CL domain is a CL lambda domain. It is well known in the art that the CHI domain of the Ig heavy chain dimerizes with the light chain CL domain. In some embodiments, the first dimerization domain comprises or consists of a CHI domain, and the second dimerization domain comprises or consists of a CL domain. In some embodiments, the first and second dimerization domains both comprise a hinge domain. In some embodiments, the first and second dimerization domains do not both comprise a CHI domain. In some embodiments, the first and second dimerization domains do not both comprise a CL domain. In some the first and second polypeptide chains do not both comprise a CHI domain. In some the first and second polypeptide chains do not both comprise a CL domain.

[0163] In some embodiments, an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 23). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 23. In some embodiments, SEQ ID NO: 23 is the IgGl CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequence

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 24). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 24. In some embodiments, SEQ ID NO: 24 is the IgG2 CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequence

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV EQSSGEYSESSVVTVPSSSEGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 25). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 25. In some embodiments, SEQ ID NO: 25 is the IgG3 CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequence

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 26). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 26. In some embodiments, SEQ ID NO: 26 is the IgG4 CHI domain.

[0164] In some embodiments, an Ig CL Kappa domain comprises of the amino acid sequence AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 27). In some embodiments, an Ig CL Kappa domain consists of SEQ ID NO: 27. In some embodiments, an Ig CL Lambda domain comprises of the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVET TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 28). In some embodiments, an Ig CL Lambda domain consists of SEQ ID NO: 28.

[0165] Effector moiety

[0166] In some embodiments, the composition comprises an effector moiety. In some embodiments, the first polypeptide chain comprises an effector moiety. In some embodiments, the second polypeptide chain comprises an effector moiety. In some embodiments, both the first and second polypeptide chains comprise an effector moiety. The term "moiety", as used herein, relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures. The term "moiety" may also refer to part of a molecule that exhibits a particular set of chemical and/or pharmacologic characteristics which are similar to the corresponding molecule. As used herein, the term “effector moiety” refers to a molecule or fragment of a molecule that carriers out a cytotoxic effect. In some embodiments, an effector moiety is an effector molecule. [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, death is cell death. In some embodiments, death is apoptosis. In some embodiments, death is necrosis. In some embodiments, death is cell mediated death. In some embodiments, death is phagocytosis. In some embodiments, the cytotoxic effect is against a target cell. In some embodiments, death is in a target cell. In some embodiments, the cytotoxic effect is upon binding. In some embodiments, death is upon binding. In some embodiments, the cytotoxic effect is against a target cell binding the composition. In some embodiments, the death is death of a target cell binding the composition. In some embodiments, the cytotoxic effect is against a cell bound by the protein complex. In some embodiments, the cytotoxic effect is against a cell binding the protein complex. In some embodiments, the death is death of a cell bound by the protein complex. In some embodiments, the death is death of a cell binding the protein complex. In some embodiments, the cytotoxic effect is a direct effect. In some embodiments, the cytotoxic effect is an indirect effect. In some embodiments, binding the composition is binding the fragments. In some embodiments, binding the protein complex is binding the fragments. In some embodiments, the fragments are at least one of the fragments. In some embodiments, the fragments are one of the fragments. In some embodiments, the fragments are both of the fragments.

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

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

[0170] In some embodiments, the effector moiety binds a receptor on a cell surface of the cytotoxic cell. Examples of receptors include, but are not limited to CD3, CD8, CD56, CD14 and CD16. In some embodiments, the receptor is a marker of the cytotoxic cell. In some embodiments, the receptor is unique to the cytotoxic cell. In some embodiments, the receptor is CD3. In some embodiments, the effector moiety is an agent that binds CD3. In some embodiments, the engager is an agent that binds CD3. In some embodiments, CD3 is human CD3. In some embodiments, the agent that binds CD3 is an anti-CD3 antibody or antigen binding fragment thereof. In some embodiments, the receptor is CD 16. In some embodiments, the effector moiety is an agent that binds CD16. In some embodiments, the engager is an agent that binds CD16. In some embodiments, CD16 is human CD16. In some embodiments, the agent that binds CD16 is an anti-CD16 antibody or antigen binding fragment thereof. In some embodiments, the antibody of antigen binding fragment thereof is a single chain antibody. In some embodiments, the antibody of antigen binding fragment thereof is a single domain antibody. In some embodiments, the antibody of antigen binding fragment thereof is a single chain variable fragment (scFv). Anti-CD3 agents are well known in the art and any such binding agent may be used. For example, the anti-human CD3 scFv known as OKT3 may be used as the agent. In some embodiments, the cytotoxic moiety is selected from alpha-amanitin, a radioactive moiety and an anti-CD3 binding agent. Other example of human anti-CD3 antibodies include: Muromonab (trade name Orthoclone OKT3), a murine monoclonal anti-human CD3 antibody (DrugBank Accession Number DB00075); Teplizumab, a humanized version of the murine OKT3 anti-CD3 monoclonal antibody (DrugBank Accession Number DB06606); UCHT1, a murine monoclonal antihuman CD3 antibody; UCHT1 variant-9, a humanized version of the UCHT1 clone and the bi-specific CD19-CD3 Blinatumomab (DrugBank Accession Number DB09052). Examples of human anti-CD16 include: AFM13, a bispecific tetravalent Innate Cell Engager (ICE®) targeting CD30 on tumor cells and CD16A on NK cells and macrophages and GTB-3550 (CD16/IL-15/CD33) a tri- specific killer cell engager.

[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 is 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 is devoid of 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 is devoid of an Fc region. In some embodiments, the engager is an Fc region. In some embodiments, the engager is not an Fc region. In some embodiments, the composition comprises an effector moiety that is superior at killing as compared to an Fc. In some embodiments, superior at killing is superior at killing B cells. In some embodiments, an Fc is an unmodified Fc. In some embodiments, an Fc is an unmutated Fc. In some embodiments, an Fc is a naturally occurring Fc. In some embodiments, an Fc is a human Fc. In some embodiments, a superior Fc is an Fc comprising at least one mutation that increases ADCC. In some embodiments, an Fc region is an Fc domain. In some embodiments, an Fc region is an Fc fragment. In some embodiments, the first polypeptide chain comprises an Fc region. In some embodiments, the second polypeptide chain comprises an Fc region. In some embodiments, both the first and second polypeptide chains comprise an Fc region. In some embodiments, the Fc region is an Fc region of an antibody heavy chain. In some embodiments, the antibody heavy chain is a human antibody heavy chain. In some embodiments, the heavy chain is an IgG heavy chain. In some embodiments, the IgG is selected from IgGl, IgG2, IgG3 and IgG4. In some embodiments, the IgG is selected from IgGl and IgG3. In some embodiments, the IgG is IgGl. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is IgG4.

[0172] In some embodiments, the Fc region is capable of inducing a cytotoxic effect. In some embodiments, the Fc domain comprises

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 12). In some embodiments, the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 140). It will be understood that SEQ ID NO: 140 contains 5 additional N-terminal amino acids as compared to SEQ ID NO: 12. As such, while number herein is given with respect to SEQ ID NO: 12 the numbering for SEQ ID NO: 140 can be found by adding 5. In some embodiments, the Fc region is capable of inducing a cytotoxic effect. In some embodiments, the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 141). In some embodiments, the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 142). It will be understood that SEQ ID NO: 142 contains 5 additional N-terminal amino acids as compared to SEQ ID NO: 141. As such, while number herein is given with respect to SEQ ID NO: 141 (or SEQ ID NO: 12 which is equivalent) the numbering for SEQ ID NO: 142 can be found by adding 5. SEQ ID NO: 12 and SEQ ID NO: 141 differ by two amino acids. The two sequences can be interchanged and when mutations are given with respect to SEQ ID NO: 12 it will be understood that they apply also to SEQ ID NO: 141 and vice-versa. So too SEQ ID NO: 140 and SEQ ID NO: 142 also differ by only two amino acids and these two sequences can be interchanged.

[0173] In some embodiments, the Fc domain consists of SEQ ID NO: 12. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 12. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 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 IgGl comprises or consists of SEQ ID NO: 140. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 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 IgGl comprises or consists of SEQ ID NO: 141. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 141. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 142. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 142. In some embodiments, the Fc domain comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 142. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc region is configured to induce a cytotoxic effect. In some embodiments, the cytotoxic effect is against a target cell. In some embodiments, the cytotoxic effect is upon binding. In some embodiments, the cytotoxic effect is against a cell bound by the protein complex. In some embodiments, the cytotoxic effect is against a cell binding the protein complex. In some embodiments, the cytotoxic effect is mediated by immune cell binding to the Fc region. In some embodiments, the cytotoxic effect is mediated by immune cell activation by the Fc region. In some embodiments, the cytotoxic effect is mediated by immune cell recruitment by the Fc region. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is a CD8 positive T cell. In some embodiments, the Fc region induces antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the Fc region induces complement-dependent cytotoxicity (CDC).

[0174] In some embodiments, the Fc region comprises an Ig CH2 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH2 domain. In some embodiments, the Fc region comprises an Ig CH3 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH3 domain. In some embodiments, the Fc region comprises or consists of both an Ig CH2 domain and Ig CH3 domain. In some embodiments, the Fc region comprises or consists of both an Ig heavy chain CH2 and an Ig heavy chain CH3 domain. In some embodiments, the first chain comprises a first portion of an Fc region and the second chain comprises a second portion of the Fc region. In some embodiments, the first portion comprises a CH2 domain, a CH3 domain or both. In some embodiments, the second portion comprises a CH2 domain, a CH3 domain or both. In some embodiments, interface of the first portion of an Fc region and the second portion of an Fc region produces a functional Fc region. In some embodiments, interface comprises contact. In some embodiments, interface comprises adjacent positioning. In some embodiments, interface comprises formation of the protein complex of the invention. In some embodiments, interface comprises dimerization of the first and second dimerization domains. In some embodiments, the CH2 domain is an Ig CH2 domain. In some embodiments the CH2 domain is a heavy chain CH2 domain. In some embodiments, the CH3 domain is an Ig CH3 domain. In some embodiments, the CH3 domain is a heavy chain CH3 domain.

[0175] In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 29). In some embodiments, a CH2 domain comprises the amino acid sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAK (SEQ ID NO: 13). In some embodiments, the CH2 domain consists of SEQ ID NO: 29. In some embodiments, SEQ ID NO: 29 is the IgGl CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO:

30). In some embodiments, the CH2 domain consists of SEQ ID NO: 30. In some embodiments, SEQ ID NO: 30 is the IgG2 CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequence

SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO:

31). In some embodiments, the CH2 domain consists of SEQ ID NO: 31. In some embodiments, SEQ ID NO: 31 is the IgG4 CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequence

SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPRE EQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO:

32). In some embodiments, the CH2 domain consists of SEQ ID NO: 32. In some embodiments, SEQ ID NO: 32 is the IgG3 CH2 domain. [0176] In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33). In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 the IgGl CH3 domain. In some embodiments, SEQ ID NO: 62 is the IgGl CH3 domain. In some embodiments, the SEQ ID NO: 33 sequence is the sequence found predominantly is humans of European and American descent. In some embodiments, SEQ ID NO: 62 is the sequence found predominantly in humans of Asian descent. In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34). In some embodiments, the CH3 domain consists of SEQ ID NO: 34. In some embodiments, SEQ ID NO: 34 is the IgG2 CH3 domain. In some embodiments, a CH3 domain comprises the amino acid sequence

GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 35). In some embodiments, the CH3 domain consists of SEQ ID NO: 35. In some embodiments, SEQ ID NO: 35 is the IgG4 CH3 domain. In some embodiments, a CH3 domain comprises the amino acid sequence

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPP MLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 36). In some embodiments, the CH3 domain consists of SEQ ID NO: 36. In some embodiments, SEQ ID NO: 36 is the IgG3 CH3 domain.

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

[0178] Mutations that promote heavy chain heterodimerization and/or inhibit homodimerization are well known in the art. Any such mutations or alterations may be used for constructing the polypeptides of the invention. In some embodiments, a region from an IgG is replaced with a region from an IgA. In some embodiments, a region from a TCRa is inserted into the first CH3 domain and a region from TCRb is inserted in to the second CH3 domain. In some embodiments, the mutation is insertion of a region from a TCR. In some embodiments, the TCR is selected from TCRa and TCRb. In some embodiments, the mutation is insertion of a region from a different Ig. Examples of these mutations can be found in Table 1. In some embodiments, the mutation is selected from a mutation in Table 1. In some embodiments, the first mutation is selected from a group of mutation provided in a row and the second column of Table 1 and the second mutation is the group of mutations provided in that same row of Table 1 in the third column. The mutations in Table 1 are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in other IgGs. In some embodiments, the first mutation is T366Y, and the second mutation is Y407T. In some embodiments, the first mutation is S354C and T366W and the second mutation is Y349C, T366S, L368A, and Y407V. In some embodiments, the first mutation is S364H and F405A and the second mutation is Y349T and T392F. In some embodiments, the first mutation is T350V, E351Y, F405A, and Y407V and the second mutation is T350V, T366E, K392E, 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 GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 46). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTEPPSREEMTKNQVSESCAVKGFYPSDIAVEWESNGQPENNYKTTPP VEDSDGSFFEVSKETVDKSRWQQGNVFSCSVMHEAEHNHYTQKSESESPGK (SEQ ID NO: 47). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTEPPSREEMTKNQVSEYCEVKGFYPSDIAVEWESNGQPENNYKTTP PVEDSDGSFFEYSKETVDKSRWQQGNVFSCSVMHEAEHNHYTQKSESESPGK (SEQ ID NO: 48). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 49).

[0179] Table 1: Mutations for enhancing heterodimerization and inhibiting homodimerization of CH3 domains.

[0180] In some embodiments, the mutation reduces effector function. In some embodiments, effector function comprises ADCC, CDC or both. In some embodiments, reduced effector function comprises reduced cytotoxicity. In some embodiments, reduces is abolishes. In some embodiments, the Fc is from IgGl or IgG3 and the mutation reduces effector function. In some embodiments, the Fc is from IgGl and comprises at least one mutation that reduces effector function. Mutations that reduce effector function are well known in the art and any such mutation can be used. Examples of such mutations can be found in Saunders, 2019, “Conceptual approaches to modulating antibody effector functions and circulation half-life” Front Immunol., Jun 7; 10: 1296, herein incorporated by reference in its entirety.

[0181] It will be known by a skilled artisan that IgG2 and IgG4 possess greatly reduced effector function and are not generally cytotoxic in nature. Additionally, mutations such as S228P and L235E in IgG4 are known to reduce effector function even more. Further, mutations that reduce the cytotoxicity /effector function of IgGl and IgG3 are well known in the art. In some embodiments, the IgG comprises at least one mutation. In some embodiments, the mutation is a plurality of mutations. In some embodiments, the mutation decreases cytotoxicity. In some embodiments, the mutation increases stability. In some embodiments, the mutation decreases aggregation. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the LALA mutations. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the PG-LALA mutations. In some embodiments, the mutation is mutation of proline 329 of the IgGl human heavy chain to glycine (P329G). In some embodiments, the P to G mutation is mutation of P109 of SEQ ID NO: 12 to G. In some embodiments, the mutation is mutation of leucine 234 of the IgGl human heavy chain to alanine (L234A). In some embodiments, the L to A mutation is mutation of L14 of SEQ ID NO: 12 to A. In some embodiments, the mutation is mutation of leucine 235 of the IgGl human heavy chain to alanine (L235A). In some embodiments, the L to A mutation is mutation of L15 of SEQ ID NO: 12 to A. In some embodiments, the plurality of mutation comprises P109G, L14A and L15A of SEQ ID NO: 12. In some embodiments, the plurality of mutation comprises L14A and L15A of SEQ ID NO: 12. In some embodiments, the plurality of mutation comprises P329G, L234A and L235A of the IgGl human heavy chain. In some embodiments, the plurality of mutation comprises L234A and L235A of the IgGl human heavy chain. It will be understood by a skilled artisan that parallel mutation can also be performed in the IgG3 heavy chain or the heavy chains of nonhuman IgGls. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the YTE mutations. In some embodiments, the mutation is mutation of methionine 252 of the IgGl human heavy chain to tyrosine (M252Y). In some embodiments, the M to Y mutation is mutation of M32 of SEQ ID NO: 12 to Y. In some embodiments, the mutation is mutation of serine 254 of the IgGl human heavy chain to threonine (S254T). In some embodiments, the S to T mutation is mutation of S34 of SEQ ID NO: 12 to T. In some embodiments, the mutation is mutation of threonine 256 of the IgGl human heavy chain to glutamic acid (T256E). In some embodiments, the T to E mutation is mutation of T36 of SEQ ID NO: 12 to E. In some embodiments, the plurality of mutation comprises M32Y, S34T and T36E of SEQ ID NO: 12. In some embodiments, the plurality of mutation comprises M252Y, S254T and T256E of the IgGl human heavy chain. In some embodiments, the mutation is mutation of asparagine 297 of the IgGl human heavy chain (N297). In some embodiments, the asparagine is mutated to alanine (N297A). In some embodiments, the asparagine is mutated to glutamine (N297Q). In some embodiments, the asparagine is N77 of SEQ ID NO: 12 (N77A or N77Q).

[0182] In some embodiments, the mutation increases the half-life of the molecule, peptide, polypeptide or protein complex. In some embodiments, a mutation that increases half-life is a mutation that increases binding to the neonatal Fc receptor (FcRn). In some embodiments, a mutation that increases binding to FcRn is selected from the mutations provided in Table 8. In some embodiments, the mutation is mutation of asparagine 434 to histidine (N434H). In some embodiments, an N434H mutated Fc domain comprises an N214H mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation is mutation of valine 308 to proline (V3O8P). In some embodiments, an H435A mutated Fc domain comprises an H215A mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation attenuates binding to FcRN. In some embodiments, the mutation that attenuates binding is mutation of histidine 435 to alanine (H435A). In some embodiments, an H435A mutated Fc domain comprises an 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 comprises or consists of mutation of methionine 252 to tyrosine (M252Y), mutations of serine 234 to threonine and mutation of threonine 256 to glutamic acid (T256E) (also termed YTE). In some embodiments, an M252Y/S254T/T256E mutated Fc domain comprises an M32Y, S34T and T35E mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of methionine 428 to leucine (M428E) and mutation of asparagine 434 to serine (N434S) (also termed ES). In some embodiments, an M428E/N434S mutated Fc domain comprises an M208E and N214S mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of M428E and mutation of asparagine 434 to alanine (N434A) (also termed LA). In some embodiments, an M428L/N434A mutated Fc domain comprises an M208L and N214A mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of threonine 250 to glutamine (T250Q) and mutation of methionine 428 to leucine (M428L) (also termed QL). In some embodiments, an T250Q/M428L mutated Fc domain comprises an T30Q and M208L mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of histidine 433 to lysine (H433K) and mutation of asparagine 434 to phenylalanine (N434F). In some embodiments, an H433K/N434F mutated Fc domain comprises an H213K and N214F mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of M252Y, S254T, T256E, H433K and N434F. In some embodiments, an M252Y/S254T/T256E/H433K/N434F mutated Fc domain comprises an M32Y, S34T, T35E, H213K and N214F mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of threonine 307 to alanine (T307A), mutation of glutamic acid 380 to alanine (E38OA) and mutation of asparagine 434 to alanine (N434A). In some embodiments, an T307A/E380A/N434A mutated Fc domain comprises an T87A, E160A and N214A mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of methionine 252 to tyrosine (M252Y), mutation of valine 308 to protein (V3O8P) and mutation of asparagine 343 to tyrosine (N343Y). In some embodiments, an M252Y/V308P/N343Y mutated Fc domain comprises an M32Y, V88P and N123Y mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of M252Y, mutation of valine 308 to proline (V3O8P) and mutation of asparagine 434 to tyrosine (N434Y). In some embodiments, an M252Y/V308P/N434Y mutated Fc domain comprises an M32Y, V88P and N214Y mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of histidine 258 to aspartic acid (H258D), mutation of threonine 307 to glutamine (T307Q) and mutation of alanine 378 to valine (A378V). In some embodiments, an H258D/T307Q/A378V mutated Fc domain comprises an H38D, T87Q and A158V mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of leucine 309 to aspartic acid (E309D), mutation of glutamine 311 to histidine (Q311H) and mutation of asparagine 434 to serine (N434S). In some embodiments, an E309D/Q311H/N434S mutated Fc domain comprises an E89D, Q91H and N214A mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality that attenuates binding comprises or consists of mutation of isoleucine 253 to alanine (I253A), H435A and mutation of histidine 436 to alanine (H436A). In some embodiments, an I253A/H435A/H436A mutated Fc domain comprises an I33A, H215A and H216A mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality that attenuates binding comprises or consists of 1253 A, mutation of histidine 310 to alanine (H310A) and H435A. In some embodiments, an I253A/H310A/H435A mutated Fc domain comprises an 133 A, H90A and H215A mutation of SEQ ID NO: 12 or 141.

[0183] Table 8: Mutations influencing FcRn binding

[0184] In some embodiments, the mutation is a mutation that decreases binding to an Fc receptor. In some embodiments, the Fc receptor is FcyR. In some embodiments, FcyR is FcyRI. In some embodiments, the mutation is a mutation that decreases binding to Clq. In some embodiments, a mutation that decreases binding to Fc receptor decreases ADCC. In some embodiments, the mutation is mutation of N297. As N-glycans are linked to N297 its mutation abrogates the glycosylation of this residue. In some embodiments, mutation of N297 is mutation to alanine (N297A). In some embodiments, mutation of N297 is mutation to glutamine (N297Q). In some embodiments, mutation of N297 is mutation to glycine (N297G). In some embodiments, an N297A mutated CH2 domain comprises an N59A mutation of SEQ ID NO: 29. In some embodiments, an N297A mutated Fc domain comprises an N77A mutation of SEQ ID NO: 12 or 141. In some embodiments, an N297Q mutated CH2 domain comprises an N59Q mutation of SEQ ID NO: 29. In some embodiments, an N297Q mutated Fc domain comprises an N77Q mutation of SEQ ID NO: 12 or 141. In some embodiments, an N297G mutated CH2 domain comprises an N59G mutation of SEQ ID NO: 29. In some embodiments, an N297G mutated Fc domain comprises an N77G mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation is a plurality of mutations that decrease binding to an Fc receptor. In some embodiments, the plurality comprises or consists of glycine 236 to arginine (G236R) and mutation of leucine 328 to arginine (L328R). In some embodiments, an G236R/L328R mutated Fc comprises a hinge domain comprising a G21R mutation of SEQ ID NO: 16 and a CH2 domain comprising a L90R mutation of SEQ ID NO: 29. In some embodiments, a G236R/L328R mutated Fc domain comprises an G16R and L108R mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of serine 298 to glycine (S298G) and mutation of threonine 299 to alanine (T299A). In some embodiments, an S298G/T299A mutated CH2 domain comprises a S60G and T61A mutation of SEQ ID NO: 29. In some embodiments, a S298G/T299A mutated Fc domain comprises an S78G and T79A mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of leucine 234 to phenylalanine (L234F), leucine 235 to glutamic acid (L235E) and mutation of aspartic acid 265 to arginine (D265A). In some embodiments, an L234F/L235E/D265A mutated Fc comprises a hinge domain comprising a E19F and E20E mutation of SEQ ID NO: 16 and a CH2 domain comprising a D27A mutation of SEQ ID NO: 19. In some embodiments, a L234F/L235E/D265A mutated Fc domain comprises an L14F, L15E and D45A mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of leucine 234 to alanine (L234A), leucine 235 to alanine (L235A) and mutation of proline 329 to glycine (P329G). In some embodiments, an L234A/L235A/P329G mutated Fc comprises a hinge domain comprising a L19A and L20A mutation of SEQ ID NO: 16 and a CH2 domain comprising a P91G mutation of SEQ ID NO: 29. In some embodiments, a L234A/L235A/P329G mutated Fc domain comprises an L14A, L15A and P109G mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of L234F, L235E and mutation of proline 331 to serine (P331S). In some embodiments, an L234F/L235E/P331S mutated Fc comprises a hinge domain comprising a L19F and L20E mutation of SEQ ID NO: 16 and a CH2 domain comprising a P93S mutation of SEQ ID NO: 29. In some embodiments, a L234F/L235E/P331S mutated Fc domain comprises an L14F, L15E and PH IS mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of leucine 235 to alanine (L235A), glycine 237 to alanine (G237A) and mutation of glutamic acid 318 to alanine (E318A). In some embodiments, an L235A/G237A/E318A mutated Fc comprises a hinge domain comprising a L20A and G22A mutation of SEQ ID NO: 16 and a CH2 domain comprising a E80A mutation of SEQ ID NO: 29. In some embodiments, a L235A/G237A/E318A mutated Fc domain comprises an L15A, G17A and E98A mutation of SEQ ID NO: 12 or 141.

[0185] In some embodiments, the Fc is modified to decrease binding to Fc receptor. In some embodiments, the modification is removal of glycosylation. In some embodiments, Fc glycosylation is removed enzymatically. In some embodiments, enzymatic de-glycosylation is performed with a deglycosylase. In some embodiments, enzymatic de-glycosylation is performed with a cleavase that cleaves sugars. Examples of enzymes for de-glycosylation include but are not limited to Peptide-N-Glycosidase F (PNGase) and Endoglycosidase H (Endo H). Kits for de-glycosylation 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 FcyRI, FcyRIIA, FcyRIIIA, and FcyRIIIB. In some embodiments, the Fc receptor is FcyRI. In some embodiments, the mutation is mutation of serine 267 to glutamic acid (S267E). In some embodiments, an S267E mutated CH2 domain comprises an S29E mutation of SEQ ID NO: 29. In some embodiments, an S267E mutated Fc domain comprises an S47E mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation is mutations of proline 238 to aspartic acid (P238D). In some embodiments, a P238D mutated hinge domain comprises an P23D mutation of SEQ ID NO: 16. In some embodiments, a P238D mutated Fc domain comprises an P18D mutation of SEQ ID NO: 12 or 141. In some embodiments, the mutation is a plurality of mutations that increase binding to an Fc receptor. In some embodiments, the plurality comprises or consists of S267E and mutation of leucine 328 to phenylalanine (L328F) (also termed SELF). In some embodiments, an S267E/L328F mutated CH2 domain comprises an S29E and L90F mutation of SEQ ID NO: 29. In some embodiments, an S267E/L328F mutated Fc domain comprises an S47E and L108F mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of S267E and mutation of histidine 268 to phenylalanine (H268F) and mutation of serine 324 to threonine (S324T) (also termed EFT). In some embodiments, an S267E/H268F/S324T mutated CH2 domain comprises an S29E, H30F and S86T mutation of SEQ ID NO: 29. In some embodiments, an S267E/H268F/S324T mutated Fc domain comprises an S47E, H48F and S104T mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of glycine 237 to aspartic acid (G237D), P238D, proline 271 to glycine (P271G) and mutation of alanine 330 to arginine (A33OR) (also termed V9). In some embodiments, a G237D/P238D/P271G/A330R mutated polypeptide comprises a mutated hinge domain comprising a G22D and P23D mutation of SEQ ID NO: 16 and a mutated CH2 domain comprising a P33G and A92R mutation of SEQ ID NO: 29. In some embodiments, a G237D/P238D/P271G/A330R mutated Fc domain comprises a G17D, P18D, P51G and A110R mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of G237D, P238D, histidine 268 to aspartic acid (H268D), P271G and A33OR (also termed VI 1). In some embodiments, a G237D/P238D/H268D/P271G/A330R mutated polypeptide comprises a mutated hinge domain comprising a G22D and P23D mutation of SEQ ID NO: 16 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 29. In some embodiments, a G237D/P238D/H268D/P271G/A330R mutated Fc domain comprises a G17D, P18D, H48D, P51G and A110R mutation of SEQ ID NO: 12 or 141. In some embodiments, the plurality comprises or consists of mutation of glutamic acid 233 to aspartic acid (E233D), G237D, P238D, H268D, P271G and A33OR (also termed V12). In some embodiments, a E233D/G237D/P238D/H268D/P271G/A330R mutated polypeptide comprises a mutated hinge domain comprising a E18D, G22D and P23D mutation of SEQ ID NO: 16 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 29. In some embodiments, a E233D/G237D/P238D/H268D/P271G/A330R mutated Fc domain comprises a E13D, G17D, P18D, H48D, P51G and A110R mutation of SEQ ID NO: 12 or 141.

[0187] The S267E mutation was found to enhance affinity toward the inhibitory FcyRIIB and also toward the activating FcyRIIa. The SELF mutations in hlgGl resulted in a substantial 430-fold increase in the binding toward FcyRIIB, with minimal alterations in binding to FcyRI and FcyRIIA-H131 in comparison to human WT IgGl. The EFT mutation was found to increase FcyRIIB binding by 18-fold in comparison to human WT IgGl. EFT also increased CDC, ADCC and antibody-dependent cellular phagocytosis (ADCP) activity via the enhancement of Clq and activator FcG receptors binding. In some embodiments, a mutation that increases ADCC is the EFT plurality of mutations. P238D demonstrated enhanced binding to FcyRIIB with about 4.3-fold increased affinity in comparison to WT human IgGl. P238D also significantly reduces the binding toward all other activating Fcg receptors. V9 significantly enhanced the affinity of antibodies toward hFcyRIIB, by approximately a 32-fold change in comparison to WT IgGl. V9 also was found to reduce the affinity toward hFcyRIIA R131 allele by about 3-fold in comparison to WT IgGl. Vl l was found to significantly enhance the affinity of antibodies for hFcyRIIB by approximately 96-fold, while reducing the affinity toward hFcyRIIA R131 by about 3-fold in comparison to human WT IgGl . V12 demonstrated significant enhancement of binding toward FcyRIIB, with 217-fold change in comparison to human WT IgGl. V12 mutations also show no detectable binding toward FcyRIIIA allotypes, reduced FcyRI binding (0.061-fold change relative to WT IgGl) and FcyRIIA-H131 (0.068-fold change relative to wt IgGl). It should be noted that V12 slightly improves the binding toward FcyRIIA-R131, with a 2-fold binding increase in compared to WT hlgGl.

[0188] Mutations that produce the above recited functions are well known in the art and any such mutation can be used. Examples of such mutations can be found at least in K. O. Saunders, 2019, “Conceptual approaches to modulating antibody effector functions and circulation half-life”, Front, Immunol., 2019 Jun 7; 10: 1296, herein incorporated by reference in its entirety. Table 1 of Saunders provides Fc modifications that enhance antibody effector function. Table 2 of Saunders provides Fc modifications that improve antibody circulation half-life. Table 3 of Saunders provides Fc modifications that inhibit antibody effector function. It will be understood by a skilled artisan that parallel mutation can also be performed in the IgG3 heavy chain or the heavy chains of non-human IgGls. It will be understood that the number given herein is in reference to a full-length IgG including the variable domains. The numbers can be shifted to correspond to the positions of these amino acids within just the Fc portion of the IgG.

[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 and not CDC. It will be understood by a skilled artisan that while the unmodified Fc is not sufficiently cytotoxic to overcome the booster effect produced by the molecules of the invention, an Fc comprising a mutation that increases ADCC is. In some embodiments, effector function comprises ADCC. In some embodiments, effector function comprises ADCC and not CDC. In some embodiments, increased effector function comprises increased cytotoxicity. In some embodiments, the Fc is from IgGl or IgG3 and the mutation increases effector function. In some embodiments, the Fc is from IgGl and comprises at least one mutation that increases effector function. Mutations that increase effector function are well known in the art and any such mutation can be used. Examples of such mutations can be found in Liu, 2020, “Fc-engineering for modulated effector functions-improving antibodies for cancer treatment” Antibodies (Basel), 2020 Dec; 9(4): 64, herein incorporated by reference in its entirety.

[0190] In some embodiments, a mutation that increases ADCC is a plurality of mutations that increase ADCC. In some embodiments, the plurality of mutations comprises mutation of leucine 235 to valine (L235V), phenylalanine 243 to leucine (F243L), arginine 292 to proline (R292P), tyrosine 300 to leucine (Y300L) and proline 296 to leucine (P396L) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of leucine 15 to valine (L15V), phenylalanine 23 to leucine (F23L), arginine 72 to proline (R72P), tyrosine 80 to leucine (Y80L) and proline 176 to leucine (P176L) within SEQ ID NO: 12. In some embodiments, the plurality of mutations comprises mutation of serine 239 to aspartic acid (S239D) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of serine 19 to aspartic acid (S19D) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 12. In some embodiments, the S239D/I332E mutations also increase ADCP. In some embodiments, the plurality of mutations comprises mutation of serine 239 to aspartic acid (S239D), alanine 330 to leucine (A33OL) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of serine 19 to aspartic acid (S19D), alanine 110 to leucine (A110L) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 12. In some embodiments, the S239D/A330L/I332E mutations also increase ADCP. In some embodiments, the plurality of mutations comprises mutation of glycine 236 to alanine (G236A), alanine 330 to leucine (A33OL) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glycine 16 to alanine (G16A), alanine 110 to leucine (A110L) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 12. In some embodiments, the plurality of mutations comprises mutation of serine 298 to alanine (S298A), glutamic acid 333 to alanine (E333A), and lysine 334 to alanine (K334A) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of mutation of serine 78 to alanine (S78A), glutamic acid 113 to alanine (E113A), and lysine 114 to alanine (K114A) within SEQ ID NO: 12. In some embodiments, the plurality of mutations comprises mutation of proline 247 to isoleucine (P247I), and alanine 339 to glutamine (A339Q) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of mutation of proline 27 to isoleucine (P27I), and alanine 119 to glutamine (A119Q) within SEQ ID NO: 12. In some embodiments, the plurality of mutations comprises mutation of glycine 236 to alanine (G236A), serine 239 to aspartic acid (S239D) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glycine 16 to alanine (G16A), serine 19 to aspartic acid (S19D) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 12. In some embodiments, the G236A/S239D/I332E mutations also increase ADCP. In some embodiments, the plurality of mutations comprises mutation of lysine 234 to tyrosine (L234Y), lysine 235 to glutamine (L235Q), glycine 236 to tryptophan (G236W), serine 239 to methionine (S239M), histidine 268 to aspartic acid (H268D), aspartic acid 270 to glutamic acid (D270E) and serine 298 to alanine (S298A) within a first heavy chain of human IgGl and mutation of aspartic acid 270 to glutamic acid (D270E), lysine 326 to aspartic acid (K26D), alanine 330 to methionine (A33OM) and lysine 334 to glutamic acid (K334E) within the second heavy chain of IgGl. In some embodiments, the plurality of mutations comprises mutation of mutation of lysine 14 to tyrosine (L14Y), lysine 15 to glutamine (L15Q), glycine 16 to tryptophan (G16W), serine 19 to methionine (S19M), histidine 48 to aspartic acid (H48D), aspartic acid 50 to glutamic acid (D50E) and serine 78 to alanine (S78A) within a first chain of SEQ ID NO: 12 and mutation of aspartic acid 50 to glutamic acid (D50E), lysine 326 to aspartic acid (K106D), alanine 110 to methionine (A110M) and lysine 114 to glutamic acid (K114E) within a second chain of SEQ ID NO: 12. It will be understood that all of the above recited mutations given with respect to 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 numbering given hereinabove must be increased by 5 for these sequences.

[0191] In some embodiments, the Fc domain with increased ADCC comprises L15V/F23L/R72P/Y80L/P176L mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 12 and SEQ ID NO: 141. In some embodiments, the Fc domain with increased ADCC comprises EPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 143). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 143. In some embodiments, the Fc comprising the L15V/F23L/R72P/Y80L/P176L mutations is SEQ ID NO: 143. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 143 and comprises L15V/F23L/R72P/Y80L/P176L mutations. [0192] In some embodiments, the Fc domain with increased ADCC comprises S19D/A110L/I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 12 and SEQ ID NO: 141. In some embodiments, the Fc domain with increased ADCC comprises

EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 144). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 144. In some embodiments, the Fc comprising the S19D/A110L/I112E mutations is SEQ ID NO: 144. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 144 and comprises S19D/A110L/I112E mutations.

[0193] In some embodiments, the Fc domain with increased CDC comprises G16A/S47E/H48F/S 104T/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 Fc domain with increased CDC comprises EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVT NKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO: 145). In some embodiments, the Fc domain with increased CDC consists of SEQ ID NO: 145. In some embodiments, the Fc comprising the G16A/S47E/H48F/S104T/I112E mutations is SEQ ID NO: 145. In some embodiments, the Fc domain with increased CDC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 145 and comprises G16A/S47E/H48F/S104T/I112E mutations.

[0194] In some embodiments, the Fc domain with increased ADCC comprises G16A/A110L/I112E mutations within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 12 and SEQ ID NO: 141. In some embodiments, the Fc domain with increased ADCC comprises

EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 146). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 146. In some embodiments, the Fc comprising the G16A/A110L/I112E mutations is SEQ ID NO: 146. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 146 and comprises G16A/A110L/I112E mutations.

[0195] In some embodiments, the effector domain is selected from SEQ ID NO: 143-146. In some embodiments, the effector domain comprises any one of SEQ ID NO: 143-146. In some embodiments, the effector domain consists of any one of SEQ ID NO: 143-146. In some embodiments, the effector domain is selected from SEQ ID NO: 143, 144 and 146. In some embodiments, the effector domain comprises any one of SEQ ID NO: 143, 144 and 146. In some embodiments, the effector domain consists of any one of SEQ ID NO: 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 NO: 143, 144 and 146 and retains increased ADCC as compared to a control Fc domain. In some embodiments, the control Fc domain is an unmodified Fc domain. In some embodiments, unmodified Fc is an Fc found in nature. In some embodiments, unmodified Fc is a human Fc found in nature.

[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 removed enzymatically. In some embodiments, the Fc is afucosylated. In some embodiments, the method comprises performing afucosylation of the molecule. In some embodiments, the molecules of the invention are produced in a cell line engineered to produce afucosylated molecules.

[0197] In some embodiments, the mutation increases CDC. In some embodiments, a plurality of mutations increases CDC. In some embodiments, the plurality of mutations comprises mutation of glycine 236 to alanine (G236A), serine 267 to glutamic acid (S267E), histidine 268 for phenylamine (H268F), serine 324 to threonine (S324T) and isoleucine 332 to glutamic acid (I332E) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glycine 16 to alanine (G16A), serine 47 to glutamic acid (S47E), histidine 48 for phenylamine (H48F), serine 104 to threonine (S 104T) and isoleucine 112 to glutamic acid (Il 12E) within SEQ ID NO: 12. In some embodiments, the plurality of mutation comprises mutation of lysine 326 to tryptophan (K326W) and glutamic acid 333 to serine (E333S) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of lysine 106 to tryptophan (K106W) and glutamic acid 113 to serine (El 13S) within SEQ ID NO: 12. In some embodiments, the plurality of mutation comprises mutation of glutamic acid 345 to arginine (E345R), glutamic acid 430 to glycine (E430G) and serine 440 to tyrosine (S440Y) within human IgGl. In some embodiments, the plurality of mutations comprises mutation of glutamic acid 125 to arginine (E125R), glutamic acid 210 to glycine (E210G) and serine 220 to tyrosine (S220Y) within SEQ ID NO: 12. It will be understood that all of the above recited mutations given with respect to 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 numbering given hereinabove 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, effector moiety is a radioactive label. In some embodiments, the effector moiety is a chemotherapeutic. In some embodiments, the effector moiety is not a chemotherapeutic. In some embodiments, the effector moiety is toxic to a cell that is not replicating. In some embodiments, toxic is lethal. In some embodiments, the effector moiety is sufficient to kill a cell. Drug conjugation, and particularly drug conjugation to an antibody backbone, are well known in the art and any method of conjugation may be used.

[0199] In some embodiments, the effector moiety is an amatoxin. In some embodiments, the effector moiety is an amanitin. Amatoxins are a group of toxic compounds found in poisonous mushrooms. These are made up of eight amino acid residues arranged in a macrobicyclic motif and inhibit RNA polymerase. Amatoxins are also known as amanitins. In some embodiments, the amanitin is selected from alpha-amanitin, beta-amanitin, gamma- amanitin, epsilon-amanitin, amanullin, amanullinic acid, amaninamide, amanin and proamanullin. In some embodiments, the amanitin is alpha-amanitin. In some embodiments, the effector moiety is alpha-amanitin. [0200] In some embodiments, the chemotherapeutic is an anthracy cline. In some embodiments, the effector moiety is an anthracy cline. Anthracyclines are a class of drugs extracted from streptomyces bacterium that intercalate into DNA and cause cytotoxicity primarily by inhibiting topoisomerase. Examples of anthracyclines include, but are not limited to doxorubicin, daunorubicin, epirubicin, nemorubicin, PNU-159682, ladirubicin and idarubicin. In some embodiments, the anthracycline is PNU-159682.

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

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

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

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

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

[0206] In some embodiments, the effector moiety is a tubulin inhibitor. In some embodiments, the effector moiety is a maytansinoid. In some embodiments, the maytansinoid is a thiol containing maytansinoid. Mayttansinoids or maytansine are known to be tubulin inhibitors that inhibit the assembly of microtubules by binding tubulin att the rhizoxin binding site. In some embodiments, the maytansinoid is mertansine (DM-1). In some embodiments, mertansine is emtansine. In some embodiments, the tubulin inhibitor is an auristatin. In some embodiments, the auristatin is selected from Monomethyl auristatin E (MMAE) and Monomethyl auristatin F (MMAF). In some embodiments, the tubulin inhibitor is a tubulysin. In some embodiments, the tubulysin is tubulysin A. In some embodiments, the auristatin is MMAE. In some embodiments, the auristatin is MMAF. 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 moieties. In some embodiments, the effector moiety is a plurality of effector moieties. In some embodiments, the effector moiety is a combination of cytotoxic moieties. In some embodiments, the effector moiety comprises at least two cytotoxic moieties selected from the group consisting of: an amatoxin, an anthracycline, a pyrrolobenzodiazepine, a calicheamicin, a camptothecin, a duocarmycin, a triptolide, and a tubulin inhibitor. In some embodiments, the effector moiety comprises at least two cytotoxic moieties selected from the group consisting of: an amatoxin, an anthracycline, a pyrrolobenzodiazepine, a calicheamicin, a camptothecin, a duocarmycin, a triptolide, and a maytansinoid. Third and fourth chains

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

[0209] In some embodiments, the third polypeptide further comprises a third dimerization domain. In some embodiments, the first polypeptide further comprises a fourth dimerization domain. In some embodiments, the third and fourth dimerization domains are capable of dimerizing to each other. In some embodiments, the third and fourth dimerization domains are configured to dimerizing to each other. In some embodiments, the third dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the third dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, configured to dimerize is capable of dimerizing. In some embodiments, the third and fourth dimerization domains are different than the first and second dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains and the third and fourth dimerization domains are CH1/CL domains. In some embodiments, the first and second dimerization domains are CH1/CL domains and the third and fourth dimerization domains are hinge domains.

[0210] In some embodiments, the protein complex further comprises a fourth polypeptide chain. In some embodiments, the fourth polypeptide chain comprises a fourth fragment of a protein target of myasthenia gravis autoantibodies. In some embodiments, the fourth fragment is different than the first fragment. In some embodiments, the fourth fragment is different than the second fragment. In some embodiments, the fourth fragment is different than the third fragment. In some embodiments, the fourth fragment is the same as the first fragment. In some embodiments, the fourth fragment is the same as the second fragment. In some embodiments, the fourth fragment is the same as the third fragment. In some embodiments, the fourth fragment is the same as the first, second and third fragments. In some embodiments, the first, second, and third fragments are all the same. In some embodiments, the first, second, third and fourth fragments are all different. In some embodiments, the same as is the same sequence. In some embodiments, different is a different sequence. In some embodiments, different is from a different protein. In some embodiments, different is from the same protein but comprising a different sequence. In some embodiments, different is from the same protein but from a different region of the protein. In some embodiments, at least two of the first, second, third and fourth proteins are part of a single protein complex. In some embodiments, the protein complex is a complex in mammals. In some embodiments, the protein complex is a complex in humans.

[0211] In some embodiments, the fourth polypeptide further comprises a fifth dimerization domain. In some embodiments, the second polypeptide further comprises a sixth dimerization domain. In some embodiments, the fifth and sixth dimerization domains are capable of dimerizing to each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerizing to each other. In some embodiments, the fifth dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize to the third dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize to the fourth dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the first dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the second dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the third dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize to the fourth dimerization domain. In some embodiments, the fifth and sixth dimerization domains are different than the first and second dimerization domains. In some embodiments, the fifth and sixth dimerization domains are different than the third and fourth dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, the third and fourth dimerization domains are CH1/CL domains and the fifth and sixth dimerization domains are CH1/CL domains. In some embodiments, the first and second dimerization domains are CH1/CL domains, the third and fourth dimerization domains are hinge domains and the fifth and sixth dimerization domains are hinge domains. In some embodiments, the first polypeptide and second polypeptide do not both comprise a CHI domain. In some embodiments, first polypeptide and second polypeptide both comprise a CHI domain, first polypeptide and second polypeptide both comprise a CL domain. In some embodiments, first polypeptide and second polypeptide do not both comprise a CL domain. In some embodiments, the first polypeptide comprises a CHI domain, and the second polypeptide comprises a CL domain. In some embodiments, the third polypeptide comprises a CL domain and the fourth polypeptide comprise a CHI domain. In some embodiments, the first polypeptide comprises a CL domain and the second polypeptide comprises a CHI domain. In some embodiments, the third polypeptide comprises a CHI domain, and the fourth polypeptide comprise a CL domain.

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

Alternative configurations

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

[0214] In some embodiments, the fragment of the first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof is separated from the fragment of the second protein target of myasthenia gravis autoantibodies or an analog or derivative thereof by a linker. In some embodiments, the fragment of the third protein target of myasthenia gravis autoantibodies or an analog or derivative thereof is separated from the fragment of the first or the second protein target of myasthenia gravis autoantibodies or an analog or derivative thereof by a linker. In some embodiments, the fragment of the fourth protein target of myasthenia gravis autoantibodies or an analog or derivative thereof is separated from the fragment of the first, the second or the third protein target of myasthenia gravis autoantibodies or an analog or derivative thereof by a linker. In some embodiments, a fragment is separated from the Fc region by a linker. In some embodiments, a fragment is separated from an effector moiety 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 effector moiety 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 moiety are separated by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a bond. In some embodiments, the bond is a peptide bond. In some embodiments, the bond is an amino acid bond. In some embodiments, the linker is a flexible linker. Linkers are well known in the art and any linker may be used. In some embodiments, a linker is a chemical linker. In some embodiments, chemical linker is a polyethylene glycol (PEG) linker. In some embodiments, the PEG linker is a Gly3-PEG-azide linker. In some embodiments, the linker is a dibenzocyclooctyne group (DBCO) linker. In some embodiments, the DBCO linker is a DBCO-C6 linker. In some embodiments, the DBCO linker is a DBCO-Gly5-EDA linker. In some embodiments, the linker is dimethylethylenediamine (DMEDA) linker. In some embodiments, the linker is a N- dimethylethylenediamine (DMAE) linker. In some embodiments, the linker is a glutathione linker. In some embodiments, the linker is a CLICK linker. In some embodiments, the CLICK linker is a CLICK-DBCO linker. In some embodiments, the CLICK linker is a CLICK azide linker. In some embodiments, is a disulfide linker. In some embodiments, the linker is a thiol linker. In some embodiments, the linker isa azide linker. In some embodiments, the linker is a maleimide (Mai) linker. In some embodiments, the Mai linker is a maleimidocaproyl linker. In some embodiments, the Mai linker is a Mal-C6 linker. In some embodiments, the Mai linker is a Mal-Gly5-EDA linker. In some embodiments, the linker is a lysine linker. In some embodiments, the linker is an asparagine linker. In some embodiments, the linker is an acid-labile linker. In some embodiments, the linker is a cleavable linker. In some embodiments, cleavable is protease cleavable. In some embodiments, a cleavable linker is a glutathione cleavable linker. In some embodiments, the linker is a non-cleavable linker. Other examples of linkers include for example SPDB linkers, SMCC linkers, MCC linkers, and butanoic acid linkers. In some embodiments, the linker is a p-aminobenzyl (PAB) linker. In some embodiments, the linker is a p- aminocarbamate (PABC) linker. In some embodiments, the linker is a Maleimidocaproyl (me) linker. In some embodiments, the linker comprises me. In some embodiments, the linker is a Val-Cit-PAB linker. In some embodiments, the linker is a Val-Cit-PABC linker. In some embodiments, the linker is a Val-Cit-PAB -MM AE linker. In some embodiments, the linker is a mc-VC-PABC-MMAE linker. In some embodiments, the linker is a mc- MMAF linker. In some embodiments, the linker is a monomethyl auristatin E (MMAE) linker. Examples of peptide linkers include, but are not limited to Val-Cit-PAB linkers, Phe- Lys(Trt)-PAB linkers, and Ala-Ala-Asn-PAB linkers. In some embodiments, the linker is a mix of linkers. In some embodiments, the linker is a DBCO-PEG linker. In some embodiments, the linker is a PBCO-PEG-DMEDA linker. In some embodiments, the linker is a DB CO-PEG- VC-PAB -DMEDA linker. In some embodiments, VC in the linker is replaced with EVC. In some embodiments, VC in the linker is replaced with EVA. In some embodiments, the fragment and the dimerization domains are linked by a non-cleavable linker. In some embodiments, the fragment and the dimerization domains are linked by a cleavable linker. In some embodiments, the effector moiety is linked by a cleavable linker. In some embodiments, the effector moiety is linked by a non-cleavable linker.

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

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

[0218] In some embodiments, the conjugation is site-specific conjugation. In some embodiments, the conjugation is not random conjugation. In some embodiments, the conjugation or linking is to the IgG backbone. In some embodiments, the conjugation or linking is not to an AchR fragment. In some embodiments, the conjugation or linking does not interfere with antibody binding to an AchR fragment. In some embodiments, the antibody is an autoantibody. In some embodiments, the conjugation or linking is to a dimerization domain. In some embodiments, the conjugation or linking is to the hinge region. In some embodiments, the conjugation or linking is to a CH2 region. In some embodiments, the conjugation or linking is to a CH3 region. In some embodiments, the conjugation or linking is to a CHI region. In some embodiments, the conjugation or linking is to a CL region. In some embodiments, the linking or conjugating is to a native amino acid residue. In some embodiments, the linking or conjugating is to an engineered amino acid residue. In some embodiments, the residue is a cysteine. Examples of engineered cysteines include, but are not limited to A231C, S239C, N325C, L328C, D265C, and S442C of the heavy chain of IgG. In some embodiments, the residue is a lysine. In some embodiments, the residue is an asparagine. In some embodiments, glycan remodeling is used to link to an asparagine. In some embodiments, the asparagine is N297 of the heavy chain of IgG. In some embodiments, the residue is a glutamine. In some embodiments, N297 is converted, engineered, or mutated to glutamine (N297Q). In some embodiments, the glutamine is Q295 of the heavy chain of IgG. An example of an engineered glutamine includes but is not limited to Q297. The cites are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in other IgGs. In some embodiments, the linking or conjugating is to a C- or N-terminus of a chain of the agent of the invention. In some embodiments, the linking or conjugating is to a C-terminus. In some embodiments, the linking or conjugating is to an N-terminus. In some embodiments, the terminus is a terminus of the heavy chain. In some embodiments, the terminus is a terminus of the light chain. In some embodiments, the conjugation or linking is to a plurality of cites.

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

[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 wherein n is an integer. In some embodiments, the linker comprises (GGGGS)n wherein n is an integer. In some embodiments, the linker consists of (GGGS)n wherein n is an integer. In some embodiments, the linker consists of (GGGGS)n wherein n is an integer. In some embodiments, the linker comprises 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 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 where n is an integer. In some embodiments, the rigid linker consists of (EAAAK)n where n is an integer. In some embodiments, the rigid linker comprises (EAAAK)nGS where n is an integer. In some embodiments, the rigid linker consists of (EAAAK)nGS where n is an integer. In some embodiments, the rigid linker comprises (EAAAK)nGGS where n is an integer. In some embodiments, the rigid linker consists of (EAAAK)nGGS where 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 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 C-terminal to the fragment. In some embodiments, the fragment is C-terminal to the dimerization domain. In some embodiments, the Fc region is C-terminal to the fragment. In some embodiments, the fragment is C-terminal to the Fc region. In some embodiments, the dimerization domain is C-terminal to the Fc region. In some embodiments, the Fc region is C-terminal to the dimerization domain. In some embodiments, the dimerization domain is N-terminal to the fragment. In some embodiments, the fragment is N-terminal to the dimerization domain. In some embodiments, the Fc region is N-terminal to the fragment. In some embodiments, the fragment is N-terminal to the Fc region. In some embodiments, the dimerization domain is N-terminal to the Fc region. In some embodiments, the Fc region is N-terminal to the dimerization domain.

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

[0225] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 comprises a fragment linked to SEQ ID NO: 50.

[0226] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 comprises a fragment linked to SEQ ID NO: 51.

[0227] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 comprises a fragment linked to SEQ ID NO: 52.

[0228] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 comprises a fragment linked to SEQ ID NO: 53.

[0229] In some embodiments, the first polypeptide comprises a fragment linked to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 comprises a fragment linked to SEQ ID NO: 54.

[0230] In some embodiments, the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 comprises a fragment linked to SEQ ID NO: 55.

[0231] In some embodiments, the first polypeptide comprises a fragment linked to GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVET TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (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 comprises a fragment linked to SEQ ID NO: 56.

[0232] In some embodiments, the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 comprises a fragment linked to SEQ ID NO: 57.

[0233] In some embodiments, the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (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 comprises a fragment linked to SEQ ID NO: 58.

[0234] In some embodiments, the first polypeptide comprises a fragment linked to GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVET TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 59). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 59. In some embodiments, the first and second polypeptides both comprises a fragment linked to SEQ ID NO: 59.

[0235] In some embodiments, the first polypeptide comprises a fragment linked to GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVET TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (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 comprises a fragment linked to SEQ ID NO: 60.

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

[0237] In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility and linked via a linker to a light chain CL kappa domain. In some embodiments, the polypeptide comprises or consists of the amino acid sequence

SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCDVSGVDTESGATNCSMKLGTWTYDGSVVAINPESDQP DLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPGGGGSGGG GSGGGGSAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 64).

[0238] In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility and 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: 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 the above described CH2 and CH3 domains and all other CH2/CH3 domains unless explicitly stated otherwise are from IgGl.

[0239] In some embodiments, a polypeptide chain comprises a fragment of AChRb comprising a mutation to increase solubility and linked via a linker to a heavy chain comprising CHI, CH2 and CH3 domains. In some embodiments, the polypeptide comprises or consists of the amino acid sequence

SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYL DLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVS SDGSVRWQPPGIYRSSCDVSGVDTESGATNCTMVFSSYSYDSSEVSLQTGLGPDG QGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKPGG GGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 65).

[0240] In some embodiments, a polypeptide chain comprises a fragment of AChRb comprising a mutation to increase solubility and linked via a linker to a heavy chain comprising CHI, hinge, CH2 and CH3 domains. In some embodiments, the polypeptide comprises or consists of the amino acid sequence 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 of consisting of SEQ ID NO: 64.

[0242] In some embodiments, a polypeptide chain comprises a fragment of AChRb comprising a mutation to increase solubility and linked via a linker to a heavy chain comprising CHI, 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 amino acid sequence SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYL DLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVS SDGSVRWQPPGIYRSSCDVSGVDTESGATNCTMVFSSYSYDSSEVSLQTGLGPDG QGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKPGG GGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 66).

[0243] In some embodiments, a polypeptide chain comprises a fragment of AChRg comprising a mutation to increase solubility and linked via a linker to a heavy chain comprising CHI, 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 amino acid sequence RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVW IEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNV LVSPDGCIYWLPPAIFRSACDVSGVDTESGATNCSLIFQSQTYSTNEIDLQLSQEDG QTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKPGG GGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 67).

[0244] In some embodiments, a polypeptide chain comprises a fragment of AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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: 93. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 100.

[0245] In some embodiments, a polypeptide chain comprises a fragment of AChRd comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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: 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. It will be understood by a skilled artisan that in this embodiment the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S/L368A/Y407V mutations, but that the mutations could be switched to the opposite chains and the molecule would still be operable.

[0246] In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation 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: 94. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 104. [0247] In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility linked via a GS linker to AChRd comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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: 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. It will be understood by a skilled artisan that in this embodiment the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S/L368A/Y407V mutations, but that the mutations could be switched to the opposite chains and the molecule would still be operable.

[0249] In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility and a mutation to decrease aggregation linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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: 97. In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility and a mutation to decrease aggregation linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation 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: 114.

[0250] In some embodiments, a polypeptide chain comprises a fragment of AChRd comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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: 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. It will be understood by a skilled artisan that in this embodiment the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S/L368A/Y407V mutations, but that the mutations could be switched to the opposite chains and the molecule would still be operable.

[0251] In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility and a mutation to decrease aggregation linked via a GS linker to AChRd comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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: 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, a polypeptide chain comprises a fragment of AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation and 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: 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. It will be understood by a skilled artisan that in this embodiment the first polypeptide comprises the T366W mutation and the second polypeptide comprises the T366S/L368A/Y407V mutations, but that the mutations could be switched to the opposite chains and the molecule would still be operable.

[0253] In some embodiments, a polypeptide chain comprises a fragment of AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation linked via a GS linker to AChRa comprising a mutation to increase solubility linked via a GS linker to AChRd comprising a mutation to increase solubility and at least one mutation to decrease aggregation 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: 108. In some embodiments, a polypeptide chain comprises a fragment of AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation linked via a GS linker to AChRa comprising a mutation to increase solubility linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation 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: 109. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 116. [0254] In some embodiments, a polypeptide chain comprises a fragment of AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation linked via a GS linker to AChRa comprising a mutation to increase solubility and a mutation to decrease aggregation linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation 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: 115.

[0255] In some embodiments, a polypeptide chain comprises a fragment of AChRa comprising a mutation to increase solubility and a mutation to decrease solubility linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation 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. It will be understood by a skilled artisan that in cases of tandem subunits separated by a linker the order of the subunits can be as recited hereinabove or can be switched.

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

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

[0258] It will be understood that though specific linkers are provided in the above-described 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 to 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 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 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 linker is a rigid linker. In some embodiments, the linker between the two subunits is a rigid linker. In some embodiments, the linker is a (EAAAK)2GGS linker. In some embodiments, the linker between two subunits is a (EAAAK)2GGS linker. 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, a polypeptide chain comprises a CH2 and CH3 domain is linked via a GS linker to a fragment of AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation and linked via a GS linker to AChRa comprising a mutation to increase solubility and at least one mutation to decrease aggregation. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 124. In some embodiments, a polypeptide chain comprises a CH2 and CH3 domain is linked via a GS linker to a fragment of AChRa comprising a mutation to increase solubility and at least one mutation to decrease aggregation and linked via a GS linker to AChRg comprising a mutation to increase solubility and at least one mutation to decrease aggregation. 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. It will be understood by a skilled artisan that in this embodiment the polypeptide comprising AChRb contains the T366W mutation and the polypeptide comprising AChRg contains T366S/L368A/Y407V, but that the mutations could be switched to the opposite chains and the molecule would still be operable (see for example 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 of consisting of SEQ ID NO: 64.

[0262] In some embodiments, a polypeptide chain comprises a fragment of AChRe comprising a mutation to increase solubility and linked via a linker to a heavy chain comprising CHI, 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 amino acid sequence KNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGI DWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLVYEG GSVTWLPPAIYRSVCDVSGVDTESGATNCSLIFRSQTYNAEEVEFTFAVDNDGKTI NKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRKPGGGGSG GGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (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. It will be understood by a skilled artisan that in this embodiment the polypeptide comprising AChRb contains the T366W mutation and the polypeptide comprising AChRe contains T366S/L368A/Y407V, but that the mutations could be switched to the opposite chains and the molecule would 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 of consisting of SEQ ID NO: 64.

[0264] In some embodiments, a polypeptide chain comprises a fragment of AChRg comprising a mutation to increase solubility and linked via a linker to a heavy chain comprising CHI, 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 amino acid sequence RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVW IEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNV LVSPDGCIYWLPPAIFRSACDVSGVDTESGATNCSLIFQSQTYSTNEIDLQLSQEDG QTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKPGG GGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (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. It will be understood by a skilled artisan that such a molecule could also be made using CH3 domains without the mutations that that inhibit homodimerization. In such a case only a single polypeptide chain would be needed as it would homodimerize. This polypeptide would be similar to SEQ ID NO: 65 but would include the AChRg fragment in place of the 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. It will be understood by a skilled artisan that in this embodiment the polypeptide comprising AChRg contains the T366W mutation and the polypeptide comprising AChRe contains T366S/L368A/Y407V, but that the mutations could be switched to the opposite chains and the molecule would 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 of consisting of SEQ ID NO: 64.

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

[0268] In some embodiments, the protein is selected from any one of SEQ ID NO: 72-91. In some embodiments, the polypeptide is selected from any one of SEQ ID NO: 72-91. In some embodiments, the polypeptide is selected from any one of SEQ ID NO: 92-100 and 102-130. In some embodiments, the polypeptide is selected from any one of SEQ ID NO: 72-100 and 102-130. In some embodiments, the polypeptide is selected from any one of SEQ ID NO: 94, 104, and 108-129.

Pharmaceutical compositions

[0269] By another aspect, there is provided a pharmaceutical composition comprising a protein of the invention.

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

[0271] By another aspect, there is provided a pharmaceutical composition comprising a protein complex of the invention.

[0272] By another aspect, there is provided a pharmaceutical composition comprises a composition of the invention.

[0273] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant. As used herein, the term “carrier,” “adjuvant” or “excipient” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non- toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0274] The carrier 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 for use in treating myasthenia gravis. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the protein complex of the invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the conjugate of the invention. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In some embodiments, a therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition. In some embodiments, an effective amount is an amount sufficient to treat at least one symptom of a disease. In some embodiments, the disease is myasthenia gravis. In some embodiments, myasthenia gravis is characterized by autoantibodies against the protein.

[0276] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition or method herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life. Treatment of myasthenia gravis is well known in the art and may include any acceptable measure for assessing improvement of a myasthenia gravis symptom. This may include, improved muscle control, reduced muscle drooping, lapping or heaviness, improved breathing, reduced autoantibody titer, improved synapsis function or any other measure of improvement.

[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 like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal. In some embodiments, the administering is intravenous administering. In some embodiments, the administering is selected from oral, intravenous, intramuscular, intraperitoneal, intertumoral, topical, or subdermal administration. In some embodiments, administering is administering to a 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 composition is formulated to increase the hydrophilicity of the molecule of the invention. In some embodiments, the composition is formulated to decrease aggregation of the molecule of the invention. Formulations that increase hydrophilicity are well known in the art and any such formulation may be used. For example, a hydrophilic carrier or polymer (e.g., PEG) may be added to the formulation to increase hydrophilicity and decrease aggregation.

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

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

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

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

[0285] In some embodiments, the administering is administering a pharmaceutical composition of the invention. In some embodiments, myasthenia gravis is characterized by antibodies against the protein. In some embodiments, the protein is a target of myasthenia gravis antibodies. It will be understood by the skilled artisan that a protein complex will be designed with fragments of proteins which are targeted by myasthenia gravis antibodies in the subject. In some embodiments, antibodies are autoantibodies.

[0286] In some embodiments, treating comprises lowering antibody concentration. In some embodiments, treating comprises lower antibody number. In some embodiments, antibody concentration is circulating antibody concentration. In some embodiments, treating comprises depleting antibodies. In some embodiments, treating comprises sequestering antibodies. In some embodiments, binding of the antibodies to the molecules of the invention result in sequestering of the antibodies. In some embodiments, treating comprises killing B cells. In some embodiments, the B cell are autoreactive B cells. In some embodiments, killing B cells is specific B cell killing. In some embodiments, treating comprises killing B cells that produce the antibodies. In some embodiments, treating comprises killing B cells that produce the antibodies and the not substantially killing other B cells. In some embodiments, treating comprises killing B cell that produce antibodies against the protein complex. In some embodiments, treating comprises killing B cell that produce antibodies against the fragment. In some embodiments, treating comprises killing B cell that produce antibodies against a fragment of the protein complex.

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

[0288] In some embodiments, the method further comprises reducing antibodies in the subject. In some embodiments, the reducing is before the administering. In some embodiments, the reducing antibodies is reducing circulating antibodies. In some embodiments, the antibodies are autoantibodies. In some embodiments, the antibodies are against a protein. In some embodiments, the antibodies are against the protein that the fragment is from. In some embodiments, the antibodies are against the protein that at least one of the fragments is from. In some embodiments, the reducing is reducing antibodies against all proteins that at least one of the fragments are from. In some embodiments, the antibodies are against the protein complex. Methods of reducing antibodies are well known in the art and include, for example, plasmapheresis, intravenous Ig (IVIg), antibody filtering, and B cell targeting therapies, any of which may be employed. In some embodiments, the method comprises plasmapheresis of the antibodies before administering. In some embodiments, the method comprises administering a B cell targeting therapy before administering the therapeutic of the invention. In some embodiments, a B cell targeting therapy is an anti-B cell therapy. In some embodiments, the B cell targeting therapy is B cell lethal therapy. In some embodiments, the B cell targeting therapy is a pan B cell therapy. In some embodiments, the B cell targeting therapy is not a targeted therapy. As used herein, a “targeted B cell therapy” is a therapy that targets only specific B cell clones that produce specific antibodies. In some embodiments, an anti-B cell therapy is an anti-B cell antibody. B cell targeting antibodies are known in the art and include for non-limiting example, anti- CD20 antibodies. Anti-CD20 therapeutic antibodies are well known in the art and include, but are not limited to rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tiuxetan, tositumomab, and ublituximab. In some embodiments, the B cell targeting therapy is rituximab.

Nucleic acids

[0289] By another aspect, there is provided a nucleic acid molecule encoding a protein of the invention.

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

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

[0292] By another aspect, there is provided a nucleic acid molecule encoding a polypeptide chain of a composition of the invention.

[0293] By another aspect, there is provided a nucleic acid molecule encoding a composition of the invention.

104

SUBSTITUTE SHEET (RULE 26) [0294] By another aspect, there is provided a nucleic acid molecule encoding a fragment of a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and fragment of a second human protein target of myasthenia gravis autoantibodies or an analog or derivative thereof.

[0295] In some embodiments, the nucleic acid molecule is for use in treating myasthenia gravis. In some embodiments, the nucleic acid system is for use in treating myasthenia gravis.

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

[0297] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, nucleic acid molecule comprises an open reading frame encoding the polypeptide chain. Expressing of an open reading frame within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. Expression vectors are well known in the art and any vector compatible with a target cell in which the protein complex of the invention is being expressed may be used.

[0298] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence. In some embodiments, the vector comprises a promoter. In some embodiments, the promoter is configured for expression in a target cell in which the protein complex of the invention is being expressed.

[0299] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoter may be active in mammalian cells. The promoters may be a viral promoter. The promoter may be active in bacterial cells. The promoter may be active in human cells. The promoter may be active in fibroblasts. The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0300] In some embodiments, the open reading frame is operably linked to a promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

[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 by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and/or the like.

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

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

[0304] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallo thionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0305] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[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. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0307] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0308] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

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

Methods of production

[0310] By another aspect, there is provided a method for producing a protein, the method comprising: obtaining a first fragment of an extracellular domain of a first human protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and linking the first fragment to an effector moiety that is not an Fc domain.

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

[0312] By another aspect, there is provided a method for producing a protein, 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 analog or derivative thereof, wherein the first and second human receptors are targets of myasthenia gravis autoantibodies and 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; thereby producing a protein.

[0313] By another aspect, there is provided a method for producing a protein, 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 analog or derivative thereof, wherein the first and second human receptors are targets of myasthenia gravis autoantibodies and 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; thereby producing a protein.

[0314] By another aspect, there is provided a method for producing a protein, the method comprising: obtaining a first fragment of a human receptor target of myasthenia gravis autoantibodies and generating in the first fragment at least one mutation that decreases 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; thereby producing a protein. [0315] By another aspect, there is provided a method for producing a protein, the method comprising: obtaining a first fragment of a human receptor target of myasthenia gravis autoantibodies and generating in the first fragment at least one mutation that decreases 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; thereby producing a protein.

[0316] By another aspect, there is provide a method for producing a protein complex, the method comprising: obtaining a first fragment of a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and a second fragment of a second protein target of myasthenia gravis autoantibodies 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 either the first polypeptide chain, the second polypeptide chain or both to an effector moiety that is not an Fc domain; thereby producing a protein complex.

[0317] By another aspect, there is provide a method for producing a protein complex, the method comprising: obtaining a first fragment of a first protein target of myasthenia gravis autoantibodies or an analog or derivative thereof and a second fragment of a second protein target of myasthenia gravis autoantibodies 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 either the first polypeptide chain, the second polypeptide chain or both to an effector moiety that is not an unmodified Fc domain; thereby producing a protein complex.

[0318] By another aspect, there is provided a method for producing a protein, the method comprising: culturing 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 a human receptor target of myasthenia gravis autoantibodies; and ii. generating in the first fragment at least one mutation that decreases aggregation of the first fragment; thereby producing a protein.

[0319] By another aspect, there is provided a method for producing a protein, the method comprising: culturing 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 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 analog or derivative thereof, wherein the first and second human receptors are targets of myasthenia gravis autoantibodies and are different proteins; and ii. linking the first fragment to the second fragment to produce a single polypeptide chain; thereby producing a protein.

[0320] By another aspect, there is provide a method for producing a protein complex, the method comprising: culturing 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 myasthenia gravis autoantibodies or an analog or derivative thereof and a second fragment of second protein target of myasthenia gravis autoantibodies or analog or derivative thereof; and

I l l 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; thereby producing a protein complex.

[0321] In some embodiments, the protein is a polypeptide. In some embodiments, the protein complex is a protein complex of the invention. In some embodiments, the protein composition is a composition of the invention. In some embodiments, the protein is a protein of the invention. In some embodiments, the protein is a polypeptide chain of the invention. In some embodiments, the fragment is a fragment of the invention. In some embodiments, the derivative is a derivative of the invention. In some embodiments, the analog is an analog of the invention. In some embodiments, the dimerization domain is a dimerization domain of the invention. In some embodiments, the composition, protein complex, protein, fragment, analog, derivative or dimerization domain is such as is described hereinabove. 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 comprise 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 protein are the same protein. In some embodiments, the first and second protein 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 against the first and second proteins. In some embodiments, the protein is a receptor, and the fragment is a fragment of the extracellular domain. In some embodiments, the fragment comprises 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 to 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, allowing is inducing. 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, inserting is linking. In some embodiments, inserting 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 linking is linking the third dimerization domain to the first dimerization domain. In some embodiments, the linking is linking 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 myasthenia gravis autoantibodies 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 dimerization to each other. In some embodiments, the third and fourth dimerization domains are configured to dimerize to 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 in the host cell a nucleic acid sequence encoding a third polypeptide chain. 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 a third polypeptide chain. In some embodiments, the method comprises expression the first, second and third polypeptide chains in a cell.

[0326] In some embodiments, the method further comprises inserting a fifth dimerization domain into the second polypeptide. In some embodiments, inserting is linking. In some embodiments, inserting 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 linking is linking the fifth dimerization domain to the second dimerization domain. In some embodiments, the linking is linking 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 myasthenia gravis autoantibodies 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 dimerization to each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerize to 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 in the host cell a nucleic acid sequence encoding a fourth polypeptide chain. In some embodiments, the fourth polypeptide chain is produced by obtaining a fourth fragment of a fourth protein and linking it to a sixth dimerization domain to produce a fourth polypeptide chain. In some embodiments, the method comprises expression the first, second, third and fourth polypeptide chains in a cell.

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

[0329] In some embodiments, an Fc region is inserted C-terminally to a dimerization domain. In some embodiments, an Fc region is inserted C-terminally to a fragment. In some embodiments, an Fc region is inserted N-terminally to a dimerization domain. In some embodiments, an Fc region is inserted N-terminally to a fragment. In some embodiments, a fragment is inserted or linked C-terminally to a dimerization domain. In some embodiments, a fragment is inserted or linked N-terminally to a dimerization domain.

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

[0331] In some embodiments, the method further comprises producing at least one mutation in the fragment that increases solubility of the fragment. In some embodiments, the method further comprises measuring solubility of the mutated fragment. In some embodiments, the method further comprises selecting a mutated fragment with increased solubility. In some embodiments, increasing is increasing by at least a predetermined threshold. In some embodiments, increasing is significantly increasing. In some embodiments, increasing is 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 invention. In some embodiments, increasing is by at least 25%. In some embodiments, increasing is by at least 50%.

[0332] In some embodiments, the method further comprises producing at least one mutation in the fragment that decreases aggregation of the fragment. In some embodiments, the method further comprises measuring aggregation of the mutated fragment. In some embodiments, the method further comprises selecting a mutated fragment with decreased solubility. In some embodiments, decreasing is decreasing by at least a predetermined threshold. In some embodiments, decreasing is significantly decreasing. In some embodiments, decreasing is 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 invention. In some embodiments, decreasing is by at least 25%. In some embodiments, decreasing is by at least 50%.

[0333] In some embodiments, the method further comprises confirming the at least one mutation does not substantially decrease binding of the first fragment to autoantibodies against the first fragment. In some embodiments, the method further measuring binding of autoantibodies to the mutated first fragment. In some embodiments, the method comprises selecting a mutated fragment comprising substantially the same or more autoantibody binding. In some embodiments, autoantibodies and myasthenia gravis autoantibodies. In some embodiments, the autoantibodies are autoantibodies found in myasthenia gravis subjects. In some embodiments, the confirming or measuring comprises contacting the mutated fragment with sample from a subject suffering from myasthenia gravis and measuring binding of autoantibodies in the sample to the mutated fragment. In some embodiments, the sample is blood. In some embodiments, the sample is sera. In some embodiments, the sample comprises isolated autoantibodies. In some embodiments, the confirming or measuring comprises contacting the unmutated fragment with sample from a subject suffering from myasthenia gravis and measuring binding of autoantibodies in the sample to the mutated fragment. In some embodiments, the confirming or measuring comprises comparing the binding of the unmutated fragment to the mutated fragment and selecting a mutated fragment that does not have substantially less autoantibody binding. In some embodiments, substantially less is significantly less. In some embodiments, substantially less is less. In some embodiments, substantially less is more than 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% less binding. Each possibility represents a separate embodiment of the invention. In some embodiments, substantially less is more 10% less binding.

[0334] By another aspect, there is provided a protein complex produced by a method of the invention.

[0335] By another aspect, there is provided a protein produced by a method of the invention.

[0336] By another aspect, there is provided a composition produced by a method of the invention.

Patient selection

[0337] By another aspect, there is provided a method of determining suitability of a subject to be treated by a method of the invention, the method comprising receiving a sample from the subject, contacting the sample with a composition of the invention and determining binding of antibodies within the sample to the composition, wherein binding of the antibodies to the composition indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.

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

[0339] By another aspect, there is provided a method of determining suitability of a subject to be treated by a method of the invention, the method comprising receiving a sample from the subject, contacting the sample with a protein of the invention and determining binding of antibodies within the sample to the protein, wherein binding of the antibodies to the protein indicates the subject is suitable to be treated by a method of the invention, thereby determining 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 hereinabove. In some embodiments, the subject suffers from 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 naive to treatment. In some embodiments, the treatment is treatment for myasthenia gravis. In some embodiments, the subject has received treatment and has relapsed.

[0341] In some embodiments, the method comprises obtaining the sample from the subject. In some embodiments, the sample comprises tissue. In some embodiments, the sample is a biopsy. In some embodiments, the sample is a bodily fluid. In some embodiments, the bodily fluid is blood. In some embodiments, the bodily fluid is serum. In some embodiments, the bodily fluid is plasma. In some embodiments, the bodily fluid is a fluid that comprises antibodies. In some embodiments, the bodily fluid is selected from at least one of: blood, serum, plasma, intestinal fluid, saliva, tumor fluid, urine, interstitial fluid, cerebral spinal fluid and stool.

[0342] In some embodiments, the autoantibodies are myasthenia gravis autoantibodies. In some embodiments, the autoantibodies are against AChR. In some embodiments, the autoantibodies are antibodies against AChR. In some embodiments, the autoantibodies are antibodies against an AChR subunit. In some embodiments, the autoantibodies are against an AChR subunit. In some embodiments, autoantibodies are pathologic autoantibodies. In some embodiments, the autoantibodies are disease causing autoantibodies [0343] In some embodiments, contacting is incubating. In some embodiments, contacting is under conditions sufficient for binding of antibodies to the protein complex. In some embodiments, conditions comprise a time sufficient for binding of antibodies to the protein complex. In some embodiments, conditions comprise physiological conditions. In some embodiments, the protein complex is added to the sample. In some embodiments, the protein complex is dissolved in the bodily fluid. In some embodiments, the antibodies are autoantibodies. In some embodiments, the antibodies are antibodies against a protein.

[0344] In some embodiments, binding of at least a threshold amount of antibodies to the protein or protein complex indicates the subject is suitable for treatment. In some embodiments, binding of more than a threshold amount of antibodies to the protein or protein complex indicates the subject is suitable for treatment. In some embodiments, the amount of antibodies is the number of antibodies. In some embodiments, the amount of antibodies is the percentage of antibodies. In some embodiments, the percentage is the percentage of antibodies in the sample. In some embodiments, the threshold is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% of antibodies in the sample. Each possibility represents a separate embodiment of the 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 the composition, protein and/or complex. In some embodiments, the peptide is specific to the composition, protein and/or complex. As used herein, the term “specific binding” refers to binding to a specific molecule to the exclusion of other molecules. In some embodiments, the peptide is specific to the composition, protein and/or complex to the exclusion of other proteins in the sample. In some embodiments, the peptide is specific to the composition, protein and/or complex to the exclusion of naturally occurring antibodies in the sample. In some embodiments, the peptide is specific to the composition, protein and/or complex to the exclusion of the antibodies in the sample. In some embodiments, the determining binding comprises detecting the moiety. In some embodiments, the determining comprises isolating the protein complex. In some embodiments, the determining comprises eluting antibodies from the complex. Methods of protein identification are well known in the art and any such method may be used. Examples of such method include western blotting, ELISA, FACS analysis and protein sequencing, such as by mass spectrometry. In some embodiments, the determining comprises ELISA. In some embodiments, the ELISA is a competitive ELISA. In some embodiments, the competitive ELISA comprises competition with antibodies. In some embodiments, the antibodies are antibodies associated with the disease.

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

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

[0348] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0349] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0350] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0351] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[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. EXAMPLES

[0353] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, immunological, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Example 1:

[0354] 80 to 90% of all Myasthenia Gravis (MG) patients are found to have autoantibodies against acetylcholine receptor (AChR). However, the acetylcholine receptor complex is made up of five subunits (alphal, betal, gamma, delta and epsilon). Diagnostic assessment of MG patients does not generally distinguish between antibodies against one subunit or the other. Nevertheless, most antigen specific attempts at generating MG therapeutics have focused on the alpha subunit of AChR (AChRa) and therapeutics that target autoantibodies against this molecule.

[0355] In order to determine the percentage of the Myasthenia Gravis population that actually has anti-AChRal, serum samples were collected from 335 AChR- seropositive MG patients. The samples were tested in a direct ELISA assay, using a cis loop modified AChRal extracellular domain (ECD, SEQ ID NO: 131) as a decoy depleting molecule (Example for the process is described in Fig. IF). For this assay, the full AChR complex, with all of its subunits, was used for bait to bind autoantibodies in the serum. This binding assay was performed with or without the presence of increasing concentration of solid phase bound AChRal and the percent reduction in binding was measured for each sample (Fig. 1A). The reduction in the AChR receptor binding is proportional to the concentration of autoantibodies present against AChRal. Surprisingly, though some subjects had very high levels of inhibition, indicating the presence of predominantly autoantibodies against AChRal (Fig. 1A, left-most samples; and Fig. IB), others showed only moderate levels of inhibition indicating that the majority of autoantibodies were not against the alpha subunit (Fig. 1C) and still others had no substantial inhibition indicating that though they were positive for autoantibodies against AChR, no more than 10% of their autoantibodies were against the AChRal (Fig. ID). Importantly, when total AChR binding was measured (Fig. IE) there was no correlation between the total antibody concentration and the percent of the antibodies that are anti-AChRal (Fig. 1G), several of the samples with the highest total antibody titer had low or absent anti-AChRal autoantibodies.

Example 2:

[0356] To better understand the autoantibody repertoire of most MG patients, an analysis was run on data provided 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”. The 41 patient samples that were tested were mapped based on the contribution of autoantibodies against each AChR subunit to the total anti- AChR autoantibody pool (Fig. 2A). As can be seen, though autoantibodies against the alpha subunit contributes to many subjects, many others have autoantibodies predominantly against other subunits, and indeed the vast majority have a combination of autoantibodies targeting different subunits. Therefore, a plot was created showing the percent of subjects that would have at least a 50% or 75% inhibition by contacting with either a single AChR subunit or a combination of subunits (Fig. 2B). Surprisingly, the alpha subunit alone, and indeed any of the subunits alone, would rarely produce 75% blocking in any of the tested patients. Indeed, the alpha subunit alone would only produce greater than 50% inhibition in about 20% of patients. [0357] In order for an MG therapeutic to be able to treat at least 50% of the target population and neutralize over 50% and ideally over 75% of the autoantibodies a combination of alpha/beta/gamma/delta/epsilon would be needed (Fig. 2B). While potentially any reduction in the levels of autoantibodies would be beneficial, to produce a treatment that could make a substantial reduction and be effective for a large percentage of the MG population, multiple AChR subunits need to be targeted. This can also be accomplished with double and triple combinations of these subunits.

Example 3:

[0358] Long term remission for MG patients would need to remove the majority of autoreactive B cells which produce the autoantibody pool. Simply removing the autoantibodies from circulation, while potentially effective in treating the symptoms of MG, would require repeated treatments for the rest of the subject’s life as the long-lived B cells would perpetually continue to make new autoantibodies. Importantly, the B cells that produced the autoantibodies express B-cell receptor (BCR) on their surfaces which is identical to the autoantibodies. This allows the B cells themselves to be targeted by a therapeutic that contains the BCR- (and autoantibody) specific epitope. By coupling the target epitope to the Fc region of the antibody heavy chain, a therapeutic can direct specific killing of autoantibody producing B cells. This approach is also robust to potential evasion of specific subpopulations, which occurs when using agents that are targeting specific differentiation markers on the cell surface (e.g., CD19, CD38, BCMA), as every cell carrying the autoreactive BCR will be targeted regardless of its differentiations state. This approach is also beneficial in protecting and preserving non-autoreactive subpopulations, which are damaged by treatments that is targeting nonspecific differentiation markers (e.g., CD19, CD38, BCMA) regardless of whether or not they are carrying an autoreactive BCR.

[0359] Figure 3A shows one embodiment of the therapeutic agent of the invention. Immunoglobulin (Ig)-like protein complex 101 comprises four polypeptide chains: two heavy-chain-like polypeptides 110 and two light-chain-like polypeptides 120. Chains 110 are able to dimerize via disulfide bonds between them. Further, chains 110 may comprise any or all of CH3 domain 111, CH2 domain 112, hinge region 113 and CHI domain 114. In this embodiment, the hinge region 113 comprises disulfide bonds and acts as the dimerization domain, though use of other dimerization domains is also possible. These domains are well known in the art and can be selected from any of human IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD domains for example. A skilled artisan will appreciate that the Fc portion of IgGl and IgG3 incorporated into chain 110 will allow the molecule to induce antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Chains 120 are able to dimerize with chains 110 via disulfide bonds found in CHI domain 114 and CL domain 124.

[0360] Chains 110 and 120 are devoid of variable regions, unlike naturally occurring or manmade antibodies. In place of the variable region each chain has a fragment 130 from the extracellular portion of the human acetylcholine receptor. Each chain can be generated to have the same AChR subunit or different subunits. Indeed, as shown in Figure 3B, the two heavy chains 115 and 116 can be engineered separately such that chain 115 contains, for example the beta subunit 131 and chain 116 contains the gamma subunit 132. The same is true for light chains 125 and 126, which, for example, can contain the alpha 133 and epsilon 134 subunits. Thus, the therapeutic molecule can be designed with four copies of a single subunit (Fig. 3C), two copies each of two different subunits (Fig. 3D), or one copy each of four different subunits (Fig. 3B) or any other combination thereof. Indeed, the molecule is sufficiently modular that it could be engineered with three copies of one subunit and one copy of another subunit, or two copies of one subunit and one copy of two other subunits. Figure 3E shows embodiments where the two light chains are identical, but the two heavy chains are different. And Figure 3F shows embodiments where the two heavy chains are identical, and the two light chains are different. Importantly, the therapeutic molecule can be engineered to comprise four of the five different AChR subunits, which as explained above can induce over 50% inhibition in at least 50% of AChR positive patients and can induce at least 40% inhibition in all tested patients. It will be understood by a skilled artisan that any chain can include any subunit, and the combinations of chains and subunit depicted in Figures 3A-F are meant only to be illustrative and not limiting.

[0361] Figures 4A-F show some embodiments of the invention in which only two chains are combined. In Figures 4A-E, protein complex 201 comprises 2 polypeptide chains which specifically are two heavy chains. Heavy chains 215 and 216 can optionally include a CH2 212, CH3 211 and/or CHI 214 domain. In this embodiment, the dimerization domain is the heavy chain hinge 213 which dimerizes via disulfide bonds, although other dimerization domains are also envisioned. Figure 4B shows the molecule without CH2 domain 212 or CH3 domain 211 or CHI domain 214. Combinations lacking two of these domains are also envisioned (Fig. 4B). In place of the variable region each chain has a fragment 230 from the extracellular portion of the human acetylcholine receptor. Each chain can be generated to have the same AChR subunit (Fig. 4C) or different subunits (Fig. 4D). When two different subunits are employed, it is advantageous to design the molecule such that predominantly heterodimers of 215 and 216 are formed and not homodimers. The same is true of forming heterodimers of chain 115 and chain 116 in Figure 3. There are numerous technologies known in the art for designing mutations in the CH3/CH2 domains, such as Knobs-in-Holes, DuoBodies, etc., that inhibit homodimerization and promote heterodimerization. Any such technology may be employed.

[0362] In Figure 4E alternative configurations comprising two heavy chains are shown. Instead of containing a single fragment 230 in place of the variable region, two tandem fragments 230 are used. These fragments may be separated by optional linker 290. This configuration is similar in structure to a single chain antibody in which the heavy and light chain variable are on a single peptide and is essentially equivalent to the molecule shown in Figure 3D. Heavy chains 215 and 216 can optionally include a CH2 212, CH3 211 and/or CHI 214 domain. In this embodiment, the dimerization domain is the heavy chain hinge 213 which dimerizes via disulfide bonds, although other dimerization domains are also envisioned. For simplicity an example containing all three CH domains is shown as is an example lacking the CHI domain. Molecules lacking the CH2 or CH3 domain or lacking any two of these domains are also envisioned. It will be understood that fragment 230 can be from any AChR subunit. Thus, a repeat of two of the same subunits can be inserted on a single chain (Fig. 4F, two AChRg subunits 232) or two different subunits can be combined on one chain (Fig. 4G, an AChRg subunit 232 and an AChRa subunit 233). Of course, the heavy chains need not be identical as various technologies may be used to favor heterodimerization over homodimerization (Fig. 4H, an AChRg subunit 232 and an AChRa subunit 233 on one chain and an AChRd subunit 234 and AChRb subunit 231 on the other chain).

[0363] Of course, if the two heavy chains are not identical the molecule can be designed with only one of the heavy chains containing two tandem fragments 230 and with the other heavy chain containing a single fragment 230 (Fig. 41) or no fragment (Fig. 4J). The same fragment could be included on both chains in all positions (Fig. 4K, two AChRg subunits 232 on one chain and a single AChRg subunits 232 on the other chain), included on both chains but with a different subunit in the tandem location (Fig. 4L, an AChRg subunit 232 and an AChRa subunit 233 on one chain and a single AChRg subunits 232 on the other chain), or all three fragments could be different (Fig. 4M, an AChRg subunit 232 and an AChRa subunit 233 on one chain and a single AChRb subunits 231 on the other chain).

[0364] In Figure 4N alternative configurations comprising two heavy chains are shown in which three tandem fragments 230 are used on both chains. These fragments may be separated by optional linker 290. As with two tandem fragments, configurations wherein the fragments are the same (Fig. 40), or different (Fig. 4P) are possible. Further, the two chains need not contain the same fragments (Fig. 4Q) or even the same number of fragments (Fig. 4R). It will be understood that though Figures 4F-R show molecules with only CH2 212 and CH3 213 domains they can also be constructed to include the CHI domain 214 or even only one of the domains (CHI, CH2 or CH3).

[0365] In Figure 4S, protein complex 201 comprises 2 polypeptide chains which specifically are a heavy chain 215 and a light chain 220. In such an embodiment the dimerization domains are CHI domain 214 and CL domain 224. Heavy chain 215 may optionally include CH3 domain 211, CH2 domain 212 and/or hinge region 213. Absence of the hinge domain is one option for eliminating homodimerization of two heavy chains 215. Alternatively, cysteine substitutions/mutations (to serine or glutamine for example) may be introduced into the hinge or one of the mutations in the CH2/CH3 regions that promote heterodimerization and inhibit homodimerization may be employed. In place of the variable region each chain has a fragment 230 from the extracellular portion of any of the human acetylcholine receptor subunits.

[0366] The creation of a protein complex 301, which has three chains, a heavy chain 315, a heavy chain 316 and a light chain 320 is also envisioned (Fig. 5A-D). Figure 5A shows one possible embodiment in which heavy chain 316 comprises a CL domain 364 in place of a CHI domain. The hereinabove described methods of ensuring a 315/316 heterodimer can be employed. Heavy chains 315 and 316 may optionally include CH3 domain 311, CH2 domain 312 and/or hinge region 313 or may employ a different dimerization domain. CL domain 324 within light chain 320 can only dimerize with CHI domain 314 within heavy chain 315. In place of the variable region each chain has a fragment 330 from the extracellular portion of any of the human acetylcholine receptor subunits. The three chains can all contain the same fragment (for example AChRg fragment 332, Fig. 5B), all three chains can contain different fragments (for example AChRb fragment 331, AChRg fragment 332 and AChRa fragment 333, Fig. 5C), or the three chains can contain two different fragments in which one is repeated (for example AChRg fragment 332 and AChRa fragment 333, Fig. 5D). It will be understood by a skilled artisan that Figure 5D could also have the two identical fragments as the light chain and either of the heavy chains, thus there are 3 different configurations to this embodiment.

[0367] This configuration, with one of the heavy chains comprising a CL domain in place of a CHI domain can also allow for the formation of the protein complex with four different fragments. Similar to the protein complex of Figure 3B, protein complex 401 depicted in Figure 6 has four different fragments on each chain. In this embodiment, the fragments AChRa 433, AChRb 431, AChRg 432 and AChRe 434 are employed but a skilled artisan will appreciate that any four fragments can be used. For the treatment of MG specifically any four of AChRa, AChRb, AChRg, AChRd, and AChRe, can be used. Embodiments are also envisioned in which different fragment from the same protein can be on different chains. In this embodiment, the second light chain 426 contains a CHI domain 474 so that it can dimerize with the CL domain 464 in heavy chain 416. Heavy chain 415 will contain a CHI domain 414 and light chain 425 will contain a CL domain 424. This ensures that chain 425 can dimerize only with chain 415 and chain 426 can dimerize only with chain 416. As described hereinabove, mutations in the optional CH2 domains 412 and the CH3 domains 413 can be employed to promote heterodimerization of chains 415 and 416. Hinge region 413 is used here as the dimerization domain between the two heavy chains, though any dimerization domain (other than CH1/CL) can be employed.

[0368] In the above-described embodiments, an immunoglobulin backbone is depicted and described, but it will be understood by a skilled artisan that by selecting other dimerization domains similar molecules. Figures 7A-D show a generic protein complex 501. In Figure 7A the first chain 515 contains a first dimerization domain (DD1) 563 which can dimerize specifically with a second dimerization domain (DD2) 573 of second chain 516. Chain 515 further comprises a third dimerization domain (DD3) 514 which can dimerize specifically with a fourth dimerization domain (DD4) 524 of third chain 525. Chain 516 further comprises a fifth dimerization domain (DD5) 564 which can dimerize specifically with a sixth dimerization domain (DD6) 574 of fourth chain 526. Each of the four chains also comprises a fragment 530 of a human protein target of myasthenia gravis autoantibodies. As discussed hereinabove these targets include AChRa, AChRb, AChRg, AChRd, and AChRe. These can be all the same fragment with the same amino acid sequence, or they can be different sequences (either from the same protein or from different proteins).

[0369] Figure 7B shows an alternative embodiment to Figure 7A in which each distinct domain is separated by a linker. It will be understood by a skilled artisan that all of these linkers are optional, and that combination of linkers is envisioned. It will be further understood that the configurations of Figures 7B also could employ linkers between any or all of the various domains/fragments.

[0370] In Figure 8A-E single chain embodiments of the invention are depicted. Figure 8A shows a single chain fusion protein 601 containing a fragment of a first acetylcholine receptor subunit (AChRa, 633) and a fragment of a second acetylcholine receptor subunit (AChRg, 632). It will of course be understood that any permutation of two different fragments can be used. Specifically fragments from two different proteins can be used. Figures 8B and 8C show a similar embodiment but containing 3 and 4 fragments from different proteins respectively. As shown in Figure 8D, the single chain can also contain a heavy chain constant region with at least a CH3 domain 611 and optionally CHI domain 614, hinge region 613 and/or CH2 domain 612. Finally, amino acid linkers can be used to separate any of the domains of the single chain. Figure 8E depicts embodiments, with 2, 3, or 4 fragments all separated by linkers 690 as well as an embodiment in which fragments 633 and 632 are separated by linkers 690 and a linker 690 also separates the C-terminal fragment 632 from CHI domain 614. Although, no linkers are depicted separating the CHI domain 614, the hinge region 613, the CH2 domain 612 and the CH3 domain 611, it will be understood by a skilled artisan that any or all of these domains could be separated by linkers. Further, it will be understood that these various linkers can all contain the same sequence or can be made of different amino acid sequences. Also, it will be understood that permutations with only 1 or all 5 fragments are envisioned.

[0371] Although not depicted in Figures 4-8, the acetylcholine receptor subunits may contain mutations. Such mutations may increase solubility, such as is described hereinabove. Such mutations may inhibit ligand binding, such as is described hereinabove. Such mutations may decrease aggregation, such as is described hereinabove. Any of the constructs described above and shown in Figures 4-8 may include such mutations in one or many of the fragments.

[0372] The various above-described molecules were generated in various forms. Loop replaced subunit ECDs (SEQ ID NO: 131-135) with increased solubility (as disclosed in Lazaridis et al., 2014, “Expression of human AChR extracellular domain mutants with improved characteristics”, Int J Biol Macromol. 2014 Feb;63:210-7, hereby incorporated by reference in its entirety) were used to generate these molecules as far greater yields of protein could be produced due to the increased solubility.

Example 4:

[0373] To further test the ability to treat a large proportion of the MG population, the in vitro depletion assay described herein above is repeated in a larger cohort. The optimal combination of AChR subunits from the five ECD-loop replaced-subunits: alpha- 1, beta-1, gamma, delta and epsilon is determined based on the ability to neutralize anti-AChR antibodies in at least 50% of the population. The following molecules (protein complex) of the invention: 1) al -CL / 01-CH1-CH2-CH3 (Fig 3D); 2) l-CL / 01-CH1-CH2-CH3 /y - CH1-CH2-CH3 (Fig 3E) or 3) al-CL / 01-CH1-CH2-CH3 /e -CH1-CH2-CH3 (Fig 3E) are then employed in the assay showing that it produces this elevated level of blocking.

Induction and Clinical Evaluation of MG Models in rat

[0374] Passive Transfer MG Model: Female Lewis rats, were intraperitoneally or subcutaneously injected with antibodies against AChR subunits in order to induce MG and were assessed for changes in EAMG clinical score by following the method of Losen et al. Experimental Neurology 270, 2015 herein incorporated by reference in its entirety. The molecule of the invention was intravenously or subcutaneously administered at different doses and at different time points after anti-AChR- subunit antibodies administration and the ability of the molecule of the invention to reduce the titer of autoreactive antibodies, lower the MG clinical score and increase the overall survival of the treatment group compared to the control is determined. In order to examine the alpha- 1 AChR subunit the molecule of the invention al-CL / 01-CH1-CH2-CH3 (Fig 3D) was intravenously administered at different doses and at different time points after mAb35 anti-alpha- 1 monoclonal antibody administration. Rats of group #1 (n=12) received PBS as a negative control. Rats of group #2 (n-10) received four doses of 10 mg/kg of the molecule of the invention. Doses were given at 4, 12, 24 and 32 hours after antibody administration. Rats of group #3 (n=6) received 1 dose of 40 mg/kg at 7 hours after antibody administration. Rats of group #4 (n=6) received 1 dose of 20 mg/kg at 7 hours after antibody administration. Rats of group #6 (n=6) received 1 dose of 6 mg/kg at 7 hours after antibody administration. Rats of group #5 (n=6) also received 1 dose of 20 mg/kg at 7 hours after antibody administration, but this administration was subcutaneous. Regardless of dose or route of administration, the molecule of the invention consistently lowered the MG clinical score of the antibody treated rats (Fig. 3G). Further, a dose dependency was observed as 1 dose of 20 or 40 mg/kg was slightly superior to a single dose of 6 mg/kg. Interestingly, 4 doses of 10 mg/kg showed the best clinical effect. The subcutaneous administration show showing an improvement over the PBS control has significantly inferior to the I.V. administration. The overall survival of the rats was also measured (Fig 3H). In the case of survival, the minor differences between treatments were not observable as all I.V. injections resulted in 100% survival after more than 5 days. Again, subcutaneous administration had a reduced effect.

[0375] Next, a comparison of a molecule of the invention (al-CL / 01-CH1-CH2-CH3) to the equivalent of IVIG was tested. The mechanism by which IVIG is likely to treat MG is by reduction of overall antibody production by saturating the body with non-specific antibodies. Further, the presence of saturating levels of Fc competes for Fc receptors all over the body. To mimic this, passive MG rats were treated with either 1 dose of 20 mg/kg of the molecule by intravenous administration, or with the same dose of two different irrelevant proteins each linked to Fc. These non-specific Fes are essentially equivalent to IVIG. As can be seen in Figure 31, as expected the molecule of the invention had a pronounced effect on clinical score (see also Fig. 3G). The non-specific Fc had a very mild positive effect, but one that was significantly worse than the specific molecule. Similar results were observed when mouse survival was observed (Fig. 3J), the molecule of the invention resulted in 100% survival, while the non-specific molecules produced only a modest improvement.

[0376] Analysis of affinity using Surface Plasmon Resonance (SPR): The affinity versus avidity of al -ECD monomer and al-CL / al-CHl-CH2-CH3 (Fig 3C) tetramer or al-CH2- CH3 (Fig 4B) dimer to mAb35 antibody is measured using SPR device. mAb35 antibody is fixed on a sensor chip as ligand. Different concentrations of al -monomer, al-CL / al-CHl- CH2-CH3 tetramer (Fig 3C) and al-CH2-CH3 dimer (Fig 4B) are reacted as analyte. Alternatively, al -monomer, l -CL / al-CHl-CH2-CH3 tetramer (Fig 3C) and/or al -CH2- CH3 dimer (Fig 4B) is fixed on a sensor chip as ligand Different concentrations of mAb35 antibody are reacted as analyte. The obtained sensor-gram is analyzed by the global fitting method using the SPR evaluation software. The ability of the above al -ECD dimer and tetramer molecules to increase the binding strength (avidity) to mAb35 antibody compared to the al -monomer (affinity) is determined.

Example 5: Mutations that decrease subunit aggregation

[0377] The ACHR subunits are meant to complex together to form the active receptor. However, it was observed that there is also a great deal of self-interaction resulting in high levels of aggregation when the subunits are each expressed individually. As such, mutations were generated in areas of the various subunits hypothesized to be responsible for the aggregation. In particular, free cytosines that can cause disulfide binding were abolished. Biinteraction surfaces on the subunits were disrupted and hydrophobic surfaces were made more hydrophilic. The mutants were designed to reduce aggregation while not significantly disrupting autoantibody epitopes and antibody binding. The loop replaced subunits were used as the basis for the mutations.

[0378] Tables 2-4 provide the mutations made in the alpha, gamma and delta subunits. The beta subunit did not show substantial aggregation and so mutants were not designed for it. The molecules were expressed by transient expression in CHO cells and the proteins were purified by affinity chromatography on a nickel-column. For purification purposes a rigid linker was added to the C-terminus of each subunit followed by a His-Avi tag (HHHHHHHHPGSGLNDIFEAQKIEWHE, SEQ ID NO: 138). Proteins were visualized on both reduced and non-reduced SDS page to visualize aggregation and western blot was used to confirm the bands were indeed the aggregates of the expressed subunits. Finally, the produced protein was examined by SEC-HPLC. As all the subunits (WT and mutants) were of the same size they should elute at the same time point. Monomers of the subunit would be expected to elute last. The area under the last peak was considered to represent monomeric subunits and from this the percentage of the molecule found in the monomeric form was calculated and is provided in the Tables.

[0379] Table 2: Alpha subunit mutants

[0380] Table 3: Gamma subunit mutants

[0381] Table 4: Delta subunit mutants

[0382] All the generated mutants, other than the M84S mutation in the gamma subunit, did indeed decrease aggregation and increased the amount of monomer produced. In many cases the total yield of protein produced was also greatly increased. While the M84S mutation did not decrease aggregation it also did not substantially increase it or affect yield and this mutation has the added benefit of preventing oxidation of the exposed methionine during the shelf-life of the molecule. Some of the delta mutations produced only a modest increase in monomer, however, it was noted that dimers were also increased over much higher molecular weight aggregates. This is also a beneficial outcome and dimers, though not as desired as monomers, are superior to large aggregates.

[0383] Next, the ability of the various molecules to deplete subunit specific anti-AChR IgG antibodies from human MG sera (titer > 0.5 nM) was tested. To this end avidin coated Sepharose beads were decorated with c-terminally biotinylated ECD containing molecules of the invention. MG serum samples were then incubated with or without the molecule coated beads (0.57 uM) for 1 hour at room temperature under shaking (400 RPM, orbital). Following centrifugation (3,200Xg for 5 minutes), the supernatant was collected and tested for anti-AChR IgG concentration using the Euroimmun ELISA. The depletion rate was calculated as above. Depletion with the mutated ECDs was compared to depletion with the WT counterparts.

[0384] Figure 9 summarizes the results of the depletion study by showing average depletion over many sera samples. CRD- 101 containing the replacement of the cys loop of the alpha subunit in order to increase solubility was as effective as the wild-type alpha subunit fused to an Fc fragment (CRD-269, SEQ ID NO: 101, parallel to the construct taught in International Patent Application W02012141026). CRD-642, which contains the alpha subunit with a W149R mutation (and the loop replacement which is included in all of the following molecules), was just as effective as CRD-101. CRD-382, which contains the triple mutation M84S/Y 105E/Y 117R in the gamma subunit, was just as effective as the wild-type gamma subunit (CRD103). Similarly, CRD-391, containing the delta subunit with a C108A mutation, was just as effective as the wild-type delta (CRD- 104).

[0385] When individual sera samples were examined, it was found that many of the mutant ECDs were actually superior to their unmutated counterparts. CRD-101 (Cys loop replaced alpha subunit) was directly compared to CRD-269 (WT alpha fused to Fc) and was found to produce -17% more depletion which as statistically significant (p< 0.0001) (Fig. 10A). The addition of the W149R mutation was found to increase depletion by -13% (p<0.0001) on top of the increase produced by replacement of the cys loop (Fig. 10B). The addition of the C108A mutation into the delta subunit increased depletion by -15% (p<0.0001) (Fig. 10C). The triple mutation in the gamma subunit, however, did not cause an increase in depletion, though it also did not decrease depletion (Fig. 10D).

Example 6: Ability of the ECDs to bind B cells [0386] Next, the ability of the molecules of the invention to bind to B cell hybridomas expressing MG autoantibodies was tested. Fluorescently labeled streptavidin molecules were incubated with the various mutated ECD molecules C-terminally tagged with biotin. Each streptavidin molecule binds four biotin molecules, hence the resultant molecules were actually ECD tetramers. Various hybridomas expressing BCR against different AChR subunits were cultured. Four of the hybridomas expressed anti-alpha subunit antibodies (Mab35, a-192, a-195 and a-198), one expressed anti-beta antibodies (b-73), two expressed anti-gamma antibodies (g-66 and g-67) and three expressed anti-delta antibodies (43-E4-B4, 69-G11-D5-F7 and 23-F3-H1). Various hybridomas producing antibodies to irrelevant targets were used as negative controls. Following incubation of the hybridoma cells with the ECD tetramers, cells were washed (DPBS+1% FBS) twice and analyzed by flow cytometry for fluorescence on the cell surface (CytoFlex, Beckman Coulter). As can be seen in Figure 11, each subunit tetramer specific bound to the hybridoma cells expressing antibodies against that tetramer. Non-specific binding to other hybridomas was similar to the binding to the negative control. This indicates that not only can the molecules of the invention bind MG autoantibodies in serum, but they can bind to (and kill) the B cells from which the autoantibodies originate without harming other B cells.

[0387] 17 different hybridoma lines that produced antibodies to irrelevant targets were tested to determine non-specific/background binding. These targets included: protein targes of other autoimmune diseases (3 hybridomas), Integrin beta 3 (2 hybridomas), pathogenic agents (9 hybridomas including viruses and bacteria) and 3 human surface proteins (3 hybridomas). Surprisingly it was found that the tetramer comprising the unmutated alpha subunit (CRD-101, however, containing the cys loop substitution) bound non- specifically to at least one negative control hybridoma (AP-3, antibodies against integrin beta 3, Fig. 12). CRD-642, however, did not show this non-specific binding and thus shows unexpectedly improved specificity.

Example 7: Molecules comprising combinations of ECDs

[0388] Next various combinations of the mutant extracellular domains were generated. Three different sets of combination molecules were generated using the CH2 and CH3 domains of the IgG Fc (WT IgGl was used unless explicitly stated otherwise). In the first batch (Table 5), a combination of heavy chains one with wild-type alpha subunit and the other with mutated gamma produced a surprisingly high yield. Placing the alpha subunit and mutant gamma on the same chain but separated by a GS flexible also produced good protein yield. A combination of a wild-type alpha on one chain with a mutant delta on another chain and a combination of a chain with mutant delta and a second chain with mutant gamma both produced surprisingly high yields. Triple combinations were also generated with mutant alpha, mutant gamma and mutant delta. Both of these molecules (one with delta on its own heavy chain and one with gamma on its own heavy chain) did not show poor yield. A sortase tag (LPETG, SEQ ID NO: 139) was added to the C-terminus of some of the chains.

[0389] Table 5: Combination molecules different heavy chains (via knob-in-holes modifications) and combinations on a single chain via linkers all produced sufficient yields. The final batch of molecules tested (Table 7) tested various other linkers (longer flexible linkers and rigid linkers) as well as changes in the Fc region such as that decrease cytotoxicity. Again, sufficient yields were produced. Thus, all the produced molecules are viable as therapeutic agents.

[0391] Table 6: Further combination molecules

[0392] Table 7: Combination molecules with altered linkers or Fc modifications

[0393] The molecules produced were tested for their depletion ability in the same assay as was performed on the ECDs alone. The double and triple subunit molecules appeared to produce close to an additive effect, indicating that all the subunits present are still binding their target autoantibodies when included in the molecule (Fig. 9).

[0394] The hybridoma binding assay was also performed using the combination molecules. In this assay, molecule binding on the hybridomas was detected by FACS using a PE labeled anti-human FC polyclonal antibody. CRD-506, comprising one copy of the cys loop replaced alpha subunit and one copy of a mutated gamma subunit (also loop replaced), was incubated with three anti-alpha hybridoma lines, one anti-beta line, one anti-gamma line and three negative control lines producing antibodies to irrelevant proteins. As can be seen in Figure 13A, the combination molecule strongly bound to all of the alpha and gamma hybridomas but not to the beta or negative control hybridomas. This demonstrates the specificity and functionality of the combination molecules.

[0395] Two other alpha-gamma molecules were also tested. CRD-509 comprises two heavy chains each with a tandem alpha and gamma subunit separated by a linker. The gamma subunit is double mutated. CRD-600 is a similar molecule, but with a triple mutation. Both molecules bound only to anti-alpha hybridoma cells and not a negative control hybridoma (Fig. 13B). Similarly, both molecules bound to anti-gamma hybridomas (Fig. 13C). Interestingly, 5 different negative control hybridoma lines were tested and while both molecules showed very little non-specific binding the triple mutant showed less than the double indicating an unexpected superiority. These results reinforce that combination of the ECDs does not abrogate their ability to bind, even when the different ECDs are on the same chain.

[0396] Several of the combined molecules were tested against a heavy chain homodimer containing only the wild-type alpha subunit (CRD-269). Unexpectedly, these molecules, although only having one copy of the alpha subunit actually were superior binders to the anti-alpha hybridoma (Fig. 14). This may be due to the replacement of the cys loop in the alpha subunit.

[0397] Testing of an alpha-delta double molecule (CRD-585) showed specific binding only to anti-alpha and anti-delta hybridomas (Fig. 15A). Similarly, a gamma-dela double molecule (CRD-586) showed specific binding only to anti-gamma and anti-delta hybridomas (Fig. 15B). Inclusion of an IgG4 Fc instead of an IgGl did not negatively impact binding (Fig. 15C). Similarly, mutations in the IgGl Fc that reduced effector function do not negatively impact binding.

Example 8: Effector moiety compositions

[0398] In the above-described embodiments, an immunoglobulin backbone is depicted and described, but it will be understood by a skilled artisan that by selecting other dimerization and effector domains parallel molecules can be generated. Figures 16A-D show a generic protein complex 701. In Figure 16A the first chain 715 contains a first dimerization domain (DD1) 763 which can dimerize specifically with a second dimerization domain (DD2) 773 of second chain 716. Both of these chains comprise an optional effector domain (EF) 711 which is cytotoxic. The EF 711 of chain 715 and the EF 711 of chain 716 can be the same molecule or different molecules. Chain 715 further comprises a third dimerization domain (DD3) 714 which can dimerize specifically with a fourth dimerization domain (DD4) 724 of third chain 725. Chain 716 further comprises a fifth dimerization domain (DD5) 764 which can dimerize specifically with a sixth dimerization domain (DD6) 774 of fourth chain 726. Each of the four chains also comprises a fragment 730 of a human protein target of myasthenia gravis autoantibodies. As discussed hereinabove these targets include AchRa, AchRb, AchRg, AchRd, and AchRe. These can be all the same fragment with the same amino acid sequence, or they can be different sequences (either from the same protein or from different proteins).

[0399] Figures 16B-C show alternative embodiments of the positioning of the EF 711. In Figure 16B each of chain 725 and 726 comprise an EF 711. Figure 16C shows two embodiments in which a single chain (either 716 or 726) contains an EF 511 , although it will be understood by a skilled artisan that just as easily chains 715 or 725 could have been designed to contain EF 711. Figure 16D shows an alternative embodiment to Figure 16A in which each distinct domain is separated by a linker. It will be understood by a skilled artisan that all of these linkers are optional, and that combination of linkers is envisioned. It will be further understood that the configurations of Figures 16B-C also could employ linkers between any or all of the various domains/fragments. Figure 16E shows an alternative embodiment, in which a cytotoxic molecule (CM, such as a toxin, radiolabeled molecule or poison) 791 is connect by a chemical bond or linker 790 to the EF 711.

[0400] It will be understood by a skilled artisan that when a cytotoxic molecule is employed the additional inclusion of an EF is not essential. As depicted in Figure 16F, the CM 791 can be linked to any of the dimerization domains 714, 724, 763, 764, 773 and 774. Similarly, more than one CM 791 can be employed. As shown the CM 791 can be attached to a single heavy chain or to both heavy chains, to a single light chain or both light chains or indeed could even be attached to one light chain and one heavy chain or to 3 chains or all 4 chains. Although, the figure depicts the use of only 1 or 2 CMs 791 it will be understood that the use of any number of CMs whether attached to a single location or attached to multiple locations on the agent 701 are envisioned. Further, the use of a linker 791 is depicted, but it will be understood that direct conjugation of CM 791 is also envisioned.

[0401] Figure 16G depicts embodiments in which two heavy chains comprising tandem AchR fragments are linked to a cytotoxic moiety. It will be understood that any of the agents 201 shown in Figure 4E-4R can be conjugated to CM 791 either directly or through a linker 790 to produce agent 701. As shown in Figure 16G, CM 791 can be linked to any of CHI 714, hinge 713, CH2712 and CH3 711. Similarly, attached of CM 791 can be to both chains or only to a single chain. The attached can be at multiple locations along the chain or only at a single location, for example a cytotoxic moiety attached at CHI and CH2, CHI and CH3, or CH2 and CH3. Similarly, any of these linkages can be only one or on both chains. Any possible permutation of CMs attached along the agent 701 are envisioned. Although Figure 16G depicts the molecules of Figure 4E with various CM attachments, it will be understood that CM attachment as shown in Figure 16G can be done as well as the molecules of Figures 4F-4R. It will further be understood that a CM can be attached to a single chain or to both chains, at one position or multiple positions. Figure 16H shows the attachment of CM 791 to a heavy chain/light chain dimer such as is depicted in Figure 4S. As before the CM 791 can be attached to the CHI 714, CH2 712 and CH3 711 domains, but also to the CL 724 domain. As before, any combination of 1 to 4, or more, CMs may be conjugated/linked. The inclusion of hinge region 713 is not essential and the alternative molecule of Figure 4S lacking a hinge may similarly be linked to CM 791.

[0402] Figure 161 shows embodiments of a conjugate of the invention in which the cytotoxic molecule 791 is conjugated directly to a fragment of one of the acetylcholine receptor subunits (AchRa 533, AchRb 531, AchRg 532, AchRd 535, and AchRe 534), optionally by a linker 790. It will be understood that fragments comprising mutations that increase solubility and/or decrease aggregation can be used.

[0403] Figures 16J-N show embodiments of the single chain agents of the invention shown in Figures 8A-E but comprising effector moiety EF 711. Further, it will be understood by a skilled artisan that that EF 711 can be replaced by cytotoxic molecule 791, or cytotoxic molecule 791 can be linked to EF 711. The cytotoxic molecule 791, whether linked to EF 711 or to one of the fragments 730 or any part of the single chain, can be linked via linker 790 (Fig. 16O-S).

Example 9: Analysis of the Fc domain as a cytotoxic moiety

[0404] The Fc domain of human antibodies is known to produce a cytotoxic affect by the induction of antibody dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Indeed, International Patent Application W02012/141026 teaches the treatment of myasthenia gravis with an AChR alpha subunit linked to the Fc domain (human heavy chain constant region, and specifically recites that ADCC induced by the Fc leads to B cell neutralization. To test this in vivo a mouse model of myasthenia gravis was generated.

[0405] Experimental Autoimmune Myasthenia Gravis (EAMG) is the active animal model of Myasthenia Gravis. It is induced by immunizing rodents with AChR (e.g., the alpha subunit ECD). The active immunization leads to the production of anti-AChR antibodies and disease pathology with muscle weakness occurs within 30-50 days after immunization. EAMG was induced in female Lewis rats by active immunization with a mix of alpha and beta ECDs in complete Freund’s adjuvant (CFA).

[0406] Serum was drawn at days 0 and 22 and anti-AChR antibody concentration was determined by ELISA. 7 rats then received (on day 28) intravenous injections of 0.5 mg/rat of CRD-213, a tetrabody with 2 heavy chains comprising the beta subunit ECD and 2 light chains comprising the alpha subunit ECD. Injections were continued every 24 hours for 5 consecutive days (days 28-32). Control animals received vehicle only (PBS). Serum was drawn on day 29, 32, 33, 41, and 47 (for surviving rats). As can be seen in Figure 17A, the control rats showed a gradual and steady increase in antibody levels. In contrast, treatment with the alpha/beta/Fc fusion protein rather than treating the rats actually made the disease worse (Fig. 17B). Administration of the molecule had a booster effect, simulating the B cells and greatly increasing the antibody levels. Antibody titer jumped to 300-500 nM just five days after treatment and stayed at this elevated level weeks afterward. In contrast, the control rats did not reach such high antibody titers even by the end of the experiment. It is thus apparent that the Fc domain is not a sufficiently strong effector moiety in vivo. Though it may induce some B cell death it is not enough to counter the booster effect of the ECD. It is thus clear that a much stronger effector moiety is needed in order to not only kill B cells but also counteract the booster effect.

Example 10: Generation of non-Fc cytotoxic moiety conjugates

[0407] Four conjugates comprising a non-Fc cytotoxic moiety were generated. An agent of the invention comprising two heavy chains each comprising an AchR beta subunit and two light chains each comprising an alpha subunit (hereinafter referred to as B2A2) was conjugated to the four different cytotoxic moieties: alpha-amanitin, tesirine, Dxd and PNU- 159682. Alpha-amanitin is an amatoxin. Tesirine is a pyrrolobenzodiazepine (PBD) chemotherapeutic. Dxd is an analog of the chemotherapeutic agent camptothecin. PNU is an anthracy cline type chemotherapeutic. In order to avoid disruption of binding to the AchR subunits, all cytotoxic moieties were site-specifically conjugated to the IgG scaffold. Alpha- amanitin, Dxd and tesirine were conjugated to native cysteines. PNU was glycol-conjugated to the CH2 domain. [0408] Native cysteine conjugation was performed as follows. The CRD protein was reduced using TCEP and incubated at 37°C for 90 minutes. Subsequently, DMA and the linker-payload were added, followed by a 2-hour incubation at room temperature. Finally, the conjugated materials were purified by Size-Exclusion Chromatography.

[0409] To test that binding to the AchR subunits was retained after cytotoxic moiety conjugation, a binding assay with a hybridoma cell line expressing Mab35, an anti-AchR alpha antibody, was performed. The hybridoma cells were mixed with increasing concentrations of the conjugates and then stained with a fluorescent anti-human Fab secondary antibody. FACS analysis was performed to confirm binding of the conjugates to the antibody expressing cells. All four conjugates were found to bind the cells in a dose dependent manner (Fig. 18A-C, data not shown for PNU conjugate).

[0410] Two other molecules were generated and tested although they lacked an additional cytotoxic moiety. The first was an AchRa and AchRg heavy chain dimer (see Fig. 4A for structure). The knobs-in -holes mutational approach was used to generate heavy chain heterodimers such that each molecule contained one AchRa subunit and one AchRg subunit. The second molecule was a heavy chain dimer in which tandem AchR fragments (AchRa and AchRg) were included in each heavy chain (see Fig. 4E for structure). Both these molecules also bound the anti-AchR antibody expressing hybridoma cells in a dose dependent manner and failed to bind control cells expressing an irrelevant antibody (Fig. 18D-E).

Example 11: Generation of Fc molecules with enhanced ADCC

[0411] Four conjugates comprising an AChRA ECD and an AChRG ECD were generated in which each also contains mutations of the Fc domain that are known to increase ADCC or CDC. Molecule 1 comprised the quintuple mutation L235V/F243L/R292P/Y300L/P396L known to increase ADCC. Molecule 2 comprised the triple mutation S239D/A330L/I332E known to increase ADCC. Molecule 3 comprises the quintuple mutation G236A/S267E/H268F/S324T/I332E known to increase CDC. Molecule 4 comprises the triple mutation G236A/A330L/I332E known to increase ADCC. CRD-509 and CRD-269 were used as controls. A fifth molecule bearing the LALA mutation that decreases ADCC was also included as a negative control. [0412] Hybridomas expressing B cell receptor (BCR) against AChR alpha or gamma subunits were incubated in the presence of the various molecules. Following incubation, cells were washed with FACS buffer (DPBS with 1% FBS) three times and incubated in the presence of PE-conjugated anti-human polyclonal Antibody (pAb). Washing was again repeated three times and the cells were analyzed by flow cytometry (CytoFlex, Beckman Coulter). Incubation with secondary antibody only was used to determine background. Incubation with PE-anti-Rat only, was used to assess BCR expression level on each hybridoma cell line and for each molecules MFI fold change was calculated as molecule MFI value divided by a-Rat BCR MFI value. As can be seen in Figure 19A, all of the mutants were able to bind to hybridomas expressing BCR against AChR alpha (a-18-C5-F6, TIB 175 and a- 192) in manner that was comparable to CRD-509 and CRD-269. The mutants were also able to bind to hybridomas expression BCR against AChR gamma (g-63-E6-A10, g-50-Hl-E2 and g-66) in a manner that was comparable to CRD-509 (Fig. 19B). As expected, CRD-269 did not bind these hybridomas.

[0413] The ability of the constructs with enhanced ADCC to kill B cells was directly tested. Two different hybridomas against the gamma subunit and one hybridoma against the alpha subunit were cultured with fresh human PBMCs (pre-stimulated with hIL-2 (20 U/ml) for ~24 hours) at a ratio of 1:4 (20k per well:80K per well). Molecules 1, 2 and 4 were also added, as was CRD-509 as a positive control (2 ug/ml for all molecules). A molecule comprising the LALA mutation in the Fc (Molecule 5) which decreases ADCC was also used as a negative control with the alpha hybridoma. Following a 4.5 hour incubation, propidium iodide was added and live/dead quantification by flow cytometry was immediately performed. Hybridoma cells were distinguished from PBMCs by size discrimination (forward scatter). The cytotoxicity rate was determined relative to the reference culture without addition of IgG-like therapeutic molecules. All the ADCC enhanced molecules produce superior B cell killing to the control molecules with unmodified Fc domains.

[0414] The unmodified Fc domain of CRD-509 produced an 18% increase in cytotoxicity against the first gamma hybridoma (g-50-Hl-E2) and about a 13% increase against the second gamma hybridoma (g-66) over the PBMCs alone. However, the three molecules with enhanced ADCC function produced even greater cytotoxicity (Fig. 19C). Molecule 1 produced an -26% increase against the first hybridoma as compared to the unmodified Fc molecule (CRD-509) and an -36% increase against the second hybridoma. Molecule 2 produced a 37% increase against the first hybridoma (as compared to CRD-509) and an -36% increase against the second hybridoma. Molecule 4 produced only a -16% increase against the first hybridoma (as compared to CRD-509) but a -79% increase against the second hybridoma. Similar enhanced killing was observed with the anti-AChR alpha hybridoma only with an even more pronounced effect (Fig. 19D). Molecule 1 produced 81% greater killing, Molecule 2 produced -114% greater killing and Molecule 4 produced 87% greater killing that the unmodified Fc molecule (CRD-509). Notably, Molecule 5 produced significantly less killing than the unmodified Fc as expected. Thus, all three molecules with enhanced ADCC produced enhanced killing of B cells and may be used as effector moieties as part of the molecules of the invention.

[0415] Having determined that the molecules of the invention can kill B cells via ADCC and that mutations that enhance ADCC will improve this effect, the ability of these molecules to induce cell death by CDC was investigated. Cells from two anti-AChR alpha hybridomas and two anti-AChR gamma hybridoma were incubated with increasing concentrations of CRD-509 or Molecule 3 (0.16-20 pg/mL) and cultured in RPMI medium supplemented with 33.3% guinea pig serum (to add sufficient complement). After a 3-hour incubation propidium iodine (PI) staining was carried out and cells were analyzed by flow cytometry. The addition of CRD molecules did not reduce the concentration of live cells at all as compared to culture of the cells with no molecule (Fig. 19E), indicating that the conjugates of the invention do not induce CDC. Thus, surprisingly, the AChR conjugates can induce killing by ADCC but not CDC and this allows for the inclusion of mutations that enhance ADCC, but mutations that enhance CDC would be of no effect.

Example 12: In-vitro cytotoxicity of PNU-159682 Glyco-connected to heavy chain-light chain tetrabody on Anti-Alpha Hybridoma cell line

[0416] An agent of the invention comprising two heavy chains each comprising an AchR beta subunit and two light chains each comprising an alpha subunit (hereinafter referred to as B2A2) was conjugated to the cytotoxic moiety PNU-159682 by using GlyCLICK ADC kit (GENOVIS) (see above). This produced site specific linkage at asparagine 297 of the heavy chains within the CH2 domain. [0417] The cytotoxic activity of the B2A2-PNU was determined on the TIB-175-Mab35 Anti-AchR Alpha Hybridoma cell line and the control JY-human EBV-transformed B cell line using an XTT -based assay. IxlO⁴ cells per well were seeded into 96-well V-shape plates and treated with different concentrations of B2A2-PNU or unconjugated B2A2 in RPMI medium supplemented with 10% human serum for 1 h at 37°C, 5% CO2 and 100% humidity. Cells were centrifuge at 750 g for 5 min, and supernatant was removed. Cells were resuspended with complete fresh medium, transferred to 96 well flat-plates and incubated for 72 hours at 37°C, 5% CO2 and 100% humidity. Cytotoxicity was determined by adding 50 pL of the calorimetric substrate Cell Proliferation Kit II XTT (Roche) to each well, and the plates were incubated for up to 6 hours under the same conditions. Substrate conversion by viable cells was confirmed by measuring the absorbance at 450 nm using a TECAN microplate reader. The experiments were carried out in triplicate. B2A2-PNU was highly effective at specific killing of Mab35 expressing cells with greater than 80% killing achieved at a concentration of 1 ug/ml (Fig. 20A). A low level of killing was observed in the control cells, although this was observed even without conjugation of the PNU moiety.

Example 13: In-vitro cytotoxicity of Tesirine conjugated B2A2 on Anti- Alpha Hybridoma cell line

[0418] B2A2 was also conjugated to Tesirine by using Fc-native cysteine conjugation (see above). The cytotoxic activity of the B2A2-tesirine conjugate was determined on the TIB- 175-Mab35 Anti- Alpha Hybridoma cell line and the control Cont-T5-51 hybridoma cell line using an XTT-based assay. The control cell line was modified to contain an antibody against an irrelevant target. 2xl0⁴ cells per well were seeded into 96-well V-shape plates and treated with different concentrations of B2A2-tesirine, or unconjugated B2A2 in RPMI medium supplemented with 1% rat serum for 2 hours at 37°C, 5% CO2 and 100% humidity. Cells were centrifuge at 750 g for 5 minutes, and supernatant was removed. Cells were resuspended with complete fresh medium, transferred to 96 well flat-plates and incubated for 72 hour at 37°C, 5% CO2 and 100% humidity. Cytotoxicity was determined by adding 50 pL of the calorimetric substrate Cell Proliferation Kit II XTT (Roche) to each well, and the plates were incubated for up to 6 h under the same conditions. Substrate conversion by viable cells was confirmed by measuring the absorbance at 450 nm using a TECAN microplate reader. The experiments were carried out in triplicate. B2A2-tesirine was highly effective at specific killing of Mab35 expressing cells with nearly 60% killing achieved with almost no non-specific killing in not Mab35 expressing cells (Fig. 20B).

Example 14: In-vitro cytotoxicity of PNU conjugated A2G2 tandem fragment heavy chain on Anti- Alpha Hybridoma cell line

[0419] A second PNU conjugate molecule was also created. In this case only heavy chains were employed, and each heavy chain comprised tandem AchR subunits: an alpha subunit and a gamma subunit (A2G2). The site-specific GlyCLICK conjugation to asparagine 297 was again used.

[0420] The A2G2 molecule was again tested for killing of TIB-175-Mab35 Anti- Alpha Hybridoma cells as performed before. Three unrelated antibodies were also expressed in cells and were used as control. The A2G2 molecule produced a very high level of specific killing with nearly 100% of anti-AChRa expressing cells killed at a A2G2 concentration of only 0.67 ug/ml (Fig. 20C). Some very low non-specific killing was observed at this concentration, but at a 1/3 concentration (0.22 ug/ml) nonspecific killing was abrogated and specific killing of 40% of anti-AChRa expressing cells was still observed. This indicates that the tandem subunit structure is functional for cell binding and cell killing.

Example 15: In-vitro cytotoxicity of Tesirine conjugated G2 on Anti-Gamma Hybridoma cell line

[0421] A second Tesirine conjugate molecule was also created. In this case only heavy chains were employed, and each heavy chain comprised a gamma subunit (G2, CRD-600). (204-4) in the co-culture was observed (Fig. 21B) and an irrelevant Ig-like fusion protein conjugated to tesirine produced no killing of either cell line (Fig. 21C). Thus, tesirine is a highly effective effector moiety whose conjugation can be used to increase the B cell killing capacity of Ig-like molecules of the invention beyond the insufficient killing produced merely by an unmodified Fc.

Example 16: In-vitro analysis of complex molecule binding

[0422] Next several of the tandem complexes of the invention were conjugated to drugs and their binding to B cell hybridomas was examined. Tesirine, alpha- amanitin or triptolide was linked via a cleavable linker to CRD-213, CRD-506 or CRD-509. Tubulin inhibitors MMAE and MMAF were also linked to CRD-509; MMAE with a cleavable linker and MMAF with a non-cleavable linker. Tesirine and alpha- amanitin conjugation was either performed by native cystine conjugation or by transglutaminase conjugation. Triptolide conjugation was carried out by Click chemistry (Glycoconnect conjugation). Tesirine was also conjugated to an irrelevant Fc fusion protein as a negative control. MMAE and MMAF conjugation was carried out by native cysteine conjugation.

[0423] CRD-213 -tesirine was incubated with alpha- specific hybridomas, beta-specific hybridoma, gamma- specific hybridomas and hybridomas producing antibodies to an irrelevant protein. Following incubation cells were washed (DPBS, 1% FBS) and stained with PE-conjugated anti-human polyclonal antibody. Antibody alone was used as a negative control. The MFI fold change or shift (%) over the background was used as the measure of binding. CRD-213 -tesirine bound strongly to both alpha and beta expressing hybridomas as expected, but not to gamma expressing hybridomas or hybridomas producing an irrelevant antibody (Fig. 22A). CRD-506-tesirine (alpha and gamma ECDs) was also incubated with various hybridomas and as expected bound only to alpha- (Fig. 22B) and gamma- specific hybridomas (Fig. 22C). This demonstrates that tesirine conjugation does not interfere with autoantibody recognition of the ECDs.

[0424] The same experiments were performed with CRD-506-a-amanitin (Fig. 22D), CRD- 506-PNU (Fig. 22E-F), CRD-509-tesirine, CRD-509-a-amanitin and CRD-509-PNU (Fig. 22G-H) and CRD-509-MMAE and CRD-509-MMAF (Fig. 221- J). For MMAE and MMAF experiments, PE-anti Rat was used to measure BCR expression levels in each hybridoma and binding of the molecules of the invention was provided as MFI fold change over BCR expression. In all cases the conjugation of the drug did not interfere with recognition of the ECD by the hybridomas. Importantly, when tesirine was conjugated to an irrelevant fusion protein (irrelevant ECD fused to Fc) non-specific binding by tesirine was not observed. This demonstrates that the molecules of the invention are highly specific and will not bind indiscriminately even to non-pathological B cell populations.

[0425] Finally, CRD-586 was conjugated to the three drugs. However, in this case PNU was linked via a non-cleavable linker (DBCO-PEG(4u)-DMEDA). Regardless of the type of linker used CRD-586 still bound to gamma and delta hybridomas as expected (Fig. 22K). The tesirine conjugated (Fig. 22L) and a-amanitin conjugated (Fig. 22M) constructs also bound specifically as expected.

Example 17: In-vitro analysis of complex molecule internalization [0426] The Fc domain induces cytotoxicity by being recognized by cells of the immune system. Thus, in order to act it must be displayed on the surface of the B cell to which it bound. In contrast, the various drugs tested hereinabove need to be internalized into the B cells in order to produce a toxic effect. To test the internalization of the molecules of the invention, the molecules were labeled with Zenon pHrodo iFL. This fluorescent tracker molecule is pH sensitive such that it does not fluoresce when outside the cell but does when in an acidic environment such as the lysosome. This fluorescence detected within the cell by standard flow cytometry. The labeled molecules were cultured with hybridomas expressing BCR against the relevant subunit, the B cells were washed and analyzed by FACS. Incubation with the pHrodo unconjugated was used as a negative control and the MFI was calculated as compared to this background fluorescence.

[0427] CRD-509 showed internalization to both alpha- specific and gamma- specific hybridomas but not to any hybridomas with BCRs against irrelevant proteins (Fig. 23). This confirms that the AChR ECD fusion proteins internalize into B cells and do so in a BCR specific manner. This may at least partially explain the lack of killing caused by the Fc alone which led to an in vivo booster effect (Fig. 17B).

Example 18: In-vitro analysis of cytotoxicity of non-Fc effector moieties

[0428] To test the actual cell killing produced by the non-Fc effector moieties, hybridomas were cultured with the molecules of the invention for 1-3 hours. The conjugates were then washed out by centrifugation and the cells were given fresh media and incubated for 40 hours. At the end of the culture, cell death was measured by double positive staining for annexin and PI.

[0429] CRD-213-tesirine at three different doses (2.5, 5 and 10 pg) was cultured with antialpha B cell line TIB- 175 and cell death was measured. A dose dependent induction of cell death was indeed observed demonstrating the effectiveness of the drug after internalization within the B cells (Fig. 24A). No cell death was observed in a hybridoma with BCR to an irrelevant protein (NC). CRD-213 was also conjugated to mertansine (DM-1), a tubulin inhibitor often used in antibody-drug conjugates. This drug also produced dose dependent killing in the alpha- specific hybridoma cells (Fig. 24B).

[0430] CRD-509-MMAE, CRD-509-MMAF and unconjugated CRD-509 at increasing concentrations was cultured with an anti-gamma hybridoma (g-66) and cell death was measured. An irrelevant hybridoma (204-4) was used as a control in the same co-culture. A dose dependent induction of cell death was observed for both the MMAE and MMAF conjugates (Fig. 24C). In contrast, the unconjugated CRD-509 produced only low-level baseline amounts of cell death which did not increase with dose. A near 100% cytotoxicity was achieved in the gamma hybridoma cells with the MMAE/MMAF conjugates which was significantly greater than the unconjugated molecule. Further, no killing above background was observed in the irrelevant hybridoma cells indicating that the killing is highly specific. Similar results are observed with alpha- specific hybridoma cells. CRD-509-a-amanitin also showed killing of alpha- specific hybridoma cells and gamma-specific hybridoma cells, as did CRD-509-PNU (Fig. 24F-G).

[0431] In order to further test the specificity of the killing CRD-213-tesirine was incubated in a coculture of equal numbers of beta- specific hybridoma cells (b-73) and gamma specificspecific hybridoma cells. After 1-3 hours the drug was washed away, and new media was added. Approximately 36 hours later FACS was performed to measure the number of surviving cells of each hybridoma. As expected, when CRD-213 unconjugated was added as a control, or when vehicle only was added the ratio of the two cell hybridomas was about 1:1 at the end of the culture period (Fig. 24C). When the tesirine conjugated molecule was added a dose dependent specific killing of the b-73 cells was observed. This reinforces that the molecule of the invention can distinguish between B cell populations producing autoantibodies and healthy B cell populations.

[0432] The same experiment was performed with CRD-506-tesirine. In this instance a dose dependent killing of the gamma- specific population was observed (Fig. 24D). Similar results were observed when CRD-509-a-amanitin (Fig. 24E), CRD-509-PNU (Fig. 24F) and CRD- 509-tesirine (Fig. 24G) were tested in the coculture assay.

[0433] The overall cytotoxicity of CRD-506-tesirine is summarized in Figure 24H. The overall cytotoxicity of CRD-213-tesirine is summarized in Figure 241. These experiments reinforce that the molecule of the invention can distinguish between B cell populations producing autoantibodies and healthy B cell populations.

[0434] The same experiments were performed with CRD-586-tesirine (Fig. 24J) and CRD- 586-a-amanitin (Fig. 24K). In this instance specific killing that was significantly superior to the unconjugated CRD-586 was observed for both gamma hybridoma cells (g-66) and delta hybridoma cells (delta) but not for alpha hybridoma cells (a- 192). An unrelated control hybridoma (204-4) was used as negative control and also showed no specific killing increase. The irrelevant Ig-like construct, CRD-243, did not produce specific killing of any cells.

Example 19: In-vivo analysis of cytotoxicity of non-Fc effector moieties

[0435] Next the ability of the molecules of the invention to actually treat myasthenia gravis in vivo was evaluated. EAMG was again used as the animal model of Myasthenia Gravis. EAMG was induced in female Lewis rats by active immunization with a mix of alpha, beta, gamma, and delta ECD in CFA. Titers for antibodies against all 4 subunits were measured in serum samples from the various animals. Animals with positive anti-AChR-delta/gamma subunits specific titer covering over 50% of the total AChR antibodies were treated with CRD-586-tesirine or unconjugated CRD-586 (Fig. 25). EAMG score is an evaluation system for determining MG disease severity. The 4-point scale is as follows: 0: normal strength, no symptoms; 1: normal before exercise, symptoms observed after exercise due to fatigue; 2: symptoms present without exercise; 3: severe symptoms at rest, hind limb paralysis, no grip; 4: moribund. The average score relative to day 0 was evaluated for the CRD-586-tesirine and control groups. Evaluation was done at 8 time points. The unconjugated CRD-586 had no visible effect on disease severity, however, the tesirine conjugate greatly reduced the average score by about a total of 50% at the final time point (Fig. 25). This result both demonstrates that the Fc domain is not a sufficient B cell killer and that tesirine is and can therefore treat MG when included in the molecule of the invention.

[0436] In a similar experiment, a pre-immunization treatment to eliminate autoreactive B cells was performed. It has been well established that auto-reactive B cells can be found in naive/healthy mice, especially inbred strains (see for example Ding and Yan, “Regulation of autoreactive B cells: checkpoints and activation”, Arch. Immunol. Ther. Exp., 2007, 55, 83- 89; Wang et al., “The naive B cell repertoire predisposes to antigen-induced systemic lupus erythematosus” J Immunol. 2003 May l;170(9):4826-32; and Fereidan-Esfahani et al., “IgM natural autoantibodies in physiology and the treatment of disease”, Methods Mol Biol. 2019:1904:53-81). To confirm this, blood was drawn from 67 naive 7-8-week-old C57B16 inbred female mice and a magnetic bead-based immunoassay was performed to measure anti-AChR alpha and gamma antibody titers. All mice were found to be positive for antibodies although there was a great deal of variability (Fig. 26A). The presence of these autoreactive antibodies indicates that autoreactive B cells are present even before immunization with AChR fragments.

[0437] To test the ability of the molecules of the invention to kill these autoreactive B cells, C57B16 female mice at 6 weeks of age were treated intravenously with 0.5 mg/kg CRD-509- tesirine. Three negative control groups were also tested: animals that received PBS and animals that were intravenously administered one of two irrelevant Ig-like molecules conjugated to tesirine. 18 days post treatment animals were immunized subcutaneously (S.C.) with a mix of 50pg AChR-alpha + 50pg AChR-gamma ECDs in complete Freund’s adjuvant (CFA) + 0.2mg M. Tuberculosis per mouse. Serum samples were isolated during the experiment and the anti-AChR antibodies titer was evaluated. 14 days post immunization titers were significantly lower in the CRD-509-tesirine pre-treated group as compared to irrelevant-CRD-tesirine or PBS groups (Fig. 26B). This indicates that the tesirine conjugate was able to kill the autoreactive B cells such that immunization did not produce a significant boost in autoreactive antibody production. It is thus clear that the molecules of the invention are suitable for killing MG autoreactive B cells and treating MG.

[0438] The various other molecules of the invention are also tested in vivo. Other CRD molecules and other effector moieties (alpha-amanitin, PNU, Dxd, and mertansine) are all found to effectively treat MG, kill B cells and reduce anti-AChR titer levels in vivo. All tested effector moieties are found superior to Fc.

[0439] EAMG is also induced in mice and the ability to treat MG in this organism is also evaluated and found to be similar to rats. Serum is taken and antibody titer levels are monitored. Not only to the molecules of the invention kill target B cells, but they also reduce circulating antibody levels.

[0440] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

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 said first and second subunits are different subunits and wherein said effector moiety is not an Fc domain or is an Fc domain comprising at least one mutation that increases antibody dependent cell cytotoxicity (ADCC).

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

3. The composition of claim 1 or 2, wherein said 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).

4. The composition of any one of claims 1 to 3, wherein said effector moiety capable of inducing death in a cell binding either of said fragments.

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

6. The composition of any one of claims 1 to 5, wherein said effector moiety is selected from: alpha-amanitin, PNU- 159682, tesirine, deruxtecan (Dxd), mertansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and a combination thereof.

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

8. The composition of 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 said fragment of a second human acetylcholine receptor subunit or an analog or derivative thereof and second dimerization domain; wherein said first and second dimerization domains are configured to dimerize with each other. The composition of claim 8, wherein said dimerizing comprises forming a covalent bond between said first dimerization domain and said second dimerization domain. The composition of claim 8 or 9, wherein said protein complex comprises an immunoglobulin scaffold. The composition of any one of claims 8 to 10, wherein a. said first dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin and said second dimerization domain comprises a second hinge domain of a heavy chain and said first and said second dimerization domains dimerizes by a disulfide bond; or b. said first and second dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein said first and second dimerization domains do not both comprise said CHI domain or said CL domain. The composition of any one of claims 8 to 10, wherein said fragment and said dimerization domain of said first, second or both polypeptide chains are separated by a linker. The composition of any one of claims 8 to 12, wherein said first polypeptide chain, said second polypeptide chain or both further comprise said effector moiety. The composition of claim 13, wherein said effector moiety is linked to said first polypeptide chain, said second polypeptide chain or both via a covalent bond. The composition of claim 13 or 14, wherein said 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 said 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 comprise an effector moiety that is not an Fc domain or comprises at least one mutation that increases ADCC within said first CH3 domain, said first CH2 domain, said second CH3 domain, said second CH2 domain or a combination thereof. The composition of claim 15, wherein said first CH3 domain, said first CH2 domain or both comprises at least a first mutation and said second CH3 domain, said second CH2 domain or both comprises at least a second mutation, and wherein said mutations permit heterodimerization of said first and second polypeptide chains and inhibit homodimerization of said first polypeptide chain and homodimerization of said second polypeptide chain. The composition of claim 16, wherein said first mutation is selected from a mutation provided in Table 1 and said second mutation is provided in Table 1 and is a corresponding mutation to said first mutation. The composition of any one of claims 13 to 17, wherein said Fc region of said first, second or both polypeptide chains is separated from said fragment or said dimerization domain by a linker. The composition of any one of claims 13 to 18, wherein said Fc is from an IgG2 or IgG4 or comprises at least one mutation that reduces effector function. The composition of any one of claims 13 to 19, wherein said dimerization domain of said first, second or both polypeptide chains is C-terminal to said fragment or N- terminal to said fragment. The composition of any one of claims 1 to 20, devoid of an antibody variable domain. 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 said first polypeptide further comprises a fourth dimerization domain and said third and fourth dimerization domains are capable of dimerizing to each other. The composition of claim 22, wherein a. said third dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin and said fourth dimerization domain comprises a second hinge domain of a heavy chain and said first and said second dimerization domains dimerizes by a disulfide bond; or b. said third and fourth dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein said first and third polypeptides do not both comprise said CHI domain or said CL domain. 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 said second polypeptide further comprises a sixth dimerization domain and said fifth and six dimerization domains are capable of dimerizing to each other. The composition of claim 24, wherein a. said fifth dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin and said sixth dimerization domain comprises a second hinge domain of a heavy chain and said first and said second dimerization domains dimerizes by a disulfide bond; or b. said fifth and six dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein said first and third polypeptides do not both comprise said CHI domain or said CL domain. The composition of any one of claims 1 to 25, comprising a single polypeptide chain comprising said fragment of a 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. The composition of claim 26, wherein said 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. The composition of claim 26 or 27, wherein said fragments are separate by an amino acid linker, optionally wherein said linker is a flexible GS linker or wherein said linker is a rigid linker. The composition of any one of claims 26 to 28, wherein said polypeptide chain further comprises an Fc region of a human antibody heavy chain and a 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 said second polypeptide chain further comprises a fourth human acetylcholine receptor subunit. The composition of any one of claims 26 to 29, further comprising a second polypeptide chain comprises a third human acetylcholine receptor subunit or an analog or derivative thereof, optionally wherein said second polypeptide chain further comprises a fourth human acetylcholine receptor subunit. The composition of any one of claims 5 to 30, wherein said effector moiety is linked to said fragments by a linker. The composition of any one of claims 1 to 31, wherein said complex comprises at least one amino acid sequence selected from SEQ ID NO: 64 to 69 or a derivative thereof comprising at least 80% identity thereto. The composition of any one of claims 1 to 32, wherein at least one of said fragments comprise a mutation that increases stability or solubility of said fragment. The composition of claim 33, wherein said mutation comprises replacement of a cys loop within an acetylcholine receptor subunit with CDVSGVDTESGATNC (SEQ ID NO: 44). The composition of claim 33 or 34, wherein said acetylcholine receptor subunit is selected from: 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. The composition of any one of claims 1 to 35, wherein an analog or derivative thereof comprises at least 85% identity to said human protein. The composition of any one of claims 1 to 36, wherein said fragment comprises at least 20 sequential amino acids from said protein. The composition of any one of claims 1 to 37, wherein said fragment comprises at least one B cell receptor (BCR) -specific epitope target of said autoantibodies. The composition of any one of claims 1 to 38, wherein said fragment comprises at least one mutation that decreases aggregation of the fragment. The composition of claim 39, wherein said fragment is selected from: a. a fragment of ACHRA and comprises a mutation selected from: deletion of N141, F100G, W149R, V155A, Y93F, Y93H, Y93R and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 1 or a AChRa with increased solubility comprising SEQ ID NO: 131; b. a fragment of ACHRG and comprises a mutation selected from: M84S, Y 105E, Y117E, Y117R, and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 6 or a AChRa with increased solubility comprising SEQ ID NO: 133; and c. a fragment of ACHRD and comprises a mutation selected from: C108A, C108I, Y119R, deletion of N141, L151E and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 8 or a AChRa with increased solubility comprising SEQ ID NO: 134. The composition of any one of claims 8 to 40, wherein said first polypeptide chain and said 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. The composition of any one of claims 26 to 40, wherein said single polypeptide chain is selected from: SEQ ID NO: 94, SEQ ID NO: 104, and SEQ ID NO: 108-129. A polypeptide comprising a fragment of a first human acetylcholine receptor subunit comprising at least one mutation that decreases aggregation of the fragment, and an effector moiety that is not an Fc domain or is an Fc domain comprising at least one mutation that increases ADCC, wherein said fragment is selected from: a. a fragment of ACHRA and comprises a mutation selected from: deletion of N141, F100G, W149R, V155A, Y93F, Y93H, Y93R and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 1 or a AChRa with increased solubility comprising SEQ ID NO: 131; b. a fragment of ACHRG and comprises a mutation selected from: M84S, Y 105E, Y117E, Y117R, and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 6 or a AChRa with increased solubility comprising SEQ ID NO: 133; and c. a fragment of ACHRD and comprises a mutation selected from: C108A, C108I, Y119R, deletion of N141, L151E and a combination thereof within a wild-type AChRa comprising SEQ ID NO: 8 or a AChRa with increased solubility comprising SEQ ID NO: 134. The polypeptide of claim 43, further comprising replacement of a cys loop within an acetylcholine receptor subunit with CDVSGVDTESGATNC (SEQ ID NO: 44) and wherein said subunit is ACHRA and said cys loop consists of CEIIVTHFPFDEQNC (SEQ ID NO: 39), said subunit is ACHRG and said cys loop consists of CSISVTYFPFDWQNC (SEQ ID NO: 41), or said subunit is ACHRD and said cys loop consists of CPISVTYFPFDWQNC (SEQ ID NO: 42). The polypeptide of claim 43 or 44, further comprising a second fragment of a second acetylcholine receptor subunit linked to said first fragment by an amino acid linker; and optionally further comprising a fragment from a third, fourth, or fifth acetylcholine receptor subunit. The polypeptide of any one of claims 43 to 45, further comprising an Fc region of a human antibody heavy chain, optionally wherein said Fc region is separated from said fragment by an amino acid linker. The polypeptide of any one of claims 43 to 46, comprising a sequence selected from SEQ ID NO: 72-91. The polypeptide of any one of claims 43 to 47, wherein said effector moiety is selected from an Fc domain comprising at least one mutation that increases ADCC, an amatoxin/amanitin, an anthracycline, an anthramycin-based dimer, a calicheamicin, camptothecin or an analog thereof, a duocarmycin, triptolide and a tubulin inhibitor. The polypeptide of any one of claims 43 to 48, wherein said effector moiety is selected from alpha-amanitin, PNU- 159682, tesirine, Dxd, mertansine, MMAE, MMAF and a combination thereof. The polypeptide of any one of claims 43 to 48, wherein said effector moiety is an Fc domain comprising SEQ ID NO: 12 or SEQ ID NO: 141 comprising a plurality of mutations selected from: L15V/F23L/R72P/Y80L/P176L, and S19D/A110L/I112E, G16A/A110L/I112E within said SEQ ID NO: 12 or SEQ ID NO: 141. A pharmaceutical composition comprising a composition of any one of claims 1 to 42 or a polypeptide of any one of claims 43 to 50 and a pharmaceutically acceptable carrier, excipient or adjuvant; optionally wherein said pharmaceutical composition is formulated for systemic administration to a subject. A method of treating myasthenia gravis in a subject in need thereof, the method comprising administering to said subject a composition of any one of claims 1 to 42 or a polypeptide of any one of claims 43 to 50 or a pharmaceutical composition of claims 51, thereby treating myasthenia gravis. The method of claim 52, further comprising reducing in said subject the levels of circulating antibodies against at least said first human acetylcholine receptor subunit prior to said administering. The method of claim 52 or 53, further comprising reducing in said subject the levels of circulating antibodies against a human acetylcholine receptor subunit within a protein complex comprising said first human acetylcholine receptor subunit or said second human acetylcholine receptor subunit. The method of any one of claims 52 to 54, wherein said treating comprises decreasing the concentration of circulating autoantibodies against said human acetylcholine receptor subunits. The method of any one of claims 52 to 55, wherein said treating comprises killing B cells producing said autoantibodies. The method of claim 55, wherein said B cells are autoreactive B cells producing autoantibodies against a fragment of said composition or polypeptide. A nucleic acid system comprising a nucleic acid molecule, wherein a first nucleic acid molecule encodes said first polypeptide chain of a composition of any one of claims 8 to 25 and 31 to 42 and a second nucleic acid molecule encodes said second polypeptide chain of a composition of any one of claims 8 to 25 and 4 to 42 or said nucleic acid molecule encodes a single polypeptide of chain of a composition of any one of claims 26 to 42 or a polypeptide of any one of claims 43 to 50. The nucleic acid system of claim 58, further comprising, a third nucleic acid molecule encoding said third polypeptide chain of a composition of any one of claims 22 to 25 and 31 to 42, a fourth nucleic acid molecule encoding said fourth polypeptide chain of a composition of any one of claims 24 or 25 and 31 to 42, or both. A method of producing a composition of any one of claims 1 to 42 or the polypeptide of any one of claims 43 to 50, the method comprising expressing the nucleic acid system of claim 58 or 59 in a cell, wherein said nucleic acid system is configured to produce said encoded polypeptide in said cell, thereby producing a composition of any one of claims 1 to 42 or the polypeptide of any one of claims 43 to 50. A method for producing a protein 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 analog or derivative thereof, wherein said first and second human acetylcholine receptor subunits are different subunits, linking said first fragment to said second fragment to produce a single polypeptide chain and linking said single polypeptide chain to an effector moiety that is not an Fc domain or is an Fc domain comprising at least one mutation that increases ADCC; or culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single polypeptide chain, and linking said single polypeptide chain to an effector moiety that is not an Fc domain or is an Fc domain comprising at least one mutation that increases ADCC, wherein the single polypeptide chain is produced by: i. 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 analog or derivative thereof, wherein said first and second human acetylcholine receptor subunit are different subunits; and ii. linking said first fragment to said second fragment to produce a single polypeptide chain; thereby producing a protein. ethod for 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 analog or derivative thereof, wherein said first and second human acetylcholine receptor subunit are different proteins, linking said first fragment to a first dimerization domain to produce a first polypeptide chain and linking said second fragment to a second dimerization domain to produce a second polypeptide chain wherein said first and second dimerization domains are capable of dimerizing with each other and contacting said first polypeptide and said second polypeptide under conditions sufficient to induce said dimerization and linking said first polypeptide chain, said second polypeptide chain or both to an effector moiety that is not an Fc domain or is an Fc domain comprising at least one mutation that increases ADCC; or culturing 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 is an Fc domain comprising at least one mutation that increases ADCC, wherein the two polypeptide chains are produced by: i. 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 analog or derivative thereof, wherein said first and second human acetylcholine receptor subunit are different proteins; and ii. linking said first fragment to a first dimerization domain to produce a first polypeptide chain and linking said second fragment to a second dimerization domain to produce a second polypeptide chain wherein said first and second dimerization domains are capable of dimerizing with each other; thereby producing a protein complex. The method of claim 61, wherein said protein is a single polypeptide chain of a composition of any one of claims 26 to 42. The method of claim 62, wherein said protein complex is a protein complex of a composition of any one of claims 8 to 25 and 21 to 42. The method of claim 61 or 64, further comprising a. linking a third dimerization domain to said first dimerization domain or first fragment within said first polypeptide chain; obtaining a third fragment of an extracellular domain of a third human acetylcholine receptor subunit or an analog or derivative thereof, and linking said third fragment to a fourth dimerization domain to produce a third polypeptide chain wherein said third dimerization domain and said fourth dimerization domain are capable of dimerizing to each other; and contacting said first, second, and third polypeptides under conditions sufficient to induce said dimerization; or b. expressing in said host cell a nucleic acid sequence encoding a third polypeptide chain produced by: i. obtaining a third fragment of an extracellular domain of a third human acetylcholine receptor subunit or an analog or derivative thereof; and ii. linking said third fragment to a fourth dimerization domain to produce a third polypeptide chain; wherein said first polypeptide chain further comprises a third dimerization domain and wherein said third dimerization domain and said fourth dimerization domain or capable of dimerizing to each other. The method of claim 65, further comprising a. linking a sixth dimerization domain to said second dimerization domain or second fragment within said second polypeptide chain; obtaining a fourth fragment of an extracellular domain of a fourth human acetylcholine receptor subunit or an analog or derivative thereof, and linking said fourth fragment to a fifth dimerization domain to produce a fourth polypeptide chain wherein said fifth dimerization domain and said sixth dimerization domain are capable of dimerizing to each other; and contacting said first, second, third and fourth polypeptides under conditions sufficient to induce said dimerization; or b. expressing in said host cell a nucleic acid sequence encoding a fourth polypeptide chain produced by: i. obtaining a fourth fragment of an extracellular domain of a fourth human acetylcholine receptor subunit or an analog or derivative thereof; and ii. linking said fourth fragment to a fifth dimerization domain to produce a fourth polypeptide chain; wherein said second polypeptide chain further comprises a sixth dimerization domain and wherein said fifth dimerization domain and said sixth dimerization domain or capable of dimerizing to each other. A method of producing a polypeptide, the method comprising: a. obtaining a first fragment of an extracellular domain of a first human acetylcholine receptor subunit or an analog or derivative thereof; b. generating in said first fragment at least one mutation that decreases aggregation of the first fragment to produce a mutated first fragment; and c. linking said mutated first fragment to an effector moiety that is not an Fc domain or is an Fc domain comprising at least one mutation that increases ADCC; thereby producing a polypeptide. The method of any one of claims 61 to 67, wherein an analog or derivative thereof comprises at least 85% identity to said human protein. The method of any one of claims 61 to 68, wherein said effector moiety is selected from an Fc domain comprising at least one mutation that increases ADCC, an amatoxin/amanitin, an anthracycline, an anthramycin-based dimer, an auristatin, a calicheamicin, camptothecin or an analog thereof, a duocarmycin, triptolide and a tubulin inhibitor. The method of claim 69, wherein said effector moiety is selected from: alpha- amanitin, PNU- 159682, tesirine, deruxtecan (Dxd), mertansine, MMAE, MMAF and a combination thereof. The method of claim 69, wherein said effector moiety is an Fc domain comprising

SEQ ID NO: 12 or SEQ ID NO: 141 comprising a plurality of mutations selected from: L15V/F23L/R72P/Y80L/P176L, S19D/A110L/I112E, and

G16A/A110L/I112E within said SEQ ID NO: 12 or SEQ ID NO: 141. A protein complex or protein produced by a method of any one of claims 59 to 68. A method of determining suitability of a subject in need thereof to be treated by a method of any one of claims 50 to 55, the method comprising receiving a sample from the subject, contacting said 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 against an acetylcholine receptor subunit within said sample to said composition or said polypeptide, wherein binding of autoantibodies to said composition indicates said subject is suitable to be treated by a method of any one of claims 50 to 55, thereby determining suitability of the subject to be treated. The method of claim 70, wherein binding of at least 20% of autoantibodies against AChR in the sample to said composition or polypeptide indicates said subject is suitable to be treated by a method of any one of claims 50 to 55.