Improved acetylcholine receptor molecules and use thereof

Abstract

Compositions and polypeptides comprising a fragment, analog or derivative of human acetylcholine receptor subunit alpha (ACHRA) and a fragment, analog or derivative of human acetylcholine receptor subunit gamma (ACHRG), wherein the ACHRA fragment, analog or derivative comprises mutation of phenylalanine 137 to tyrosine (F137Y), the ACHRG fragment, analog or derivative comprises a F137Y mutation, or both are provided. Pharmaceutical compositions comprising the composition, polypeptides, nucleic acid systems encoding the polypeptides of the composition and methods of treatment and determining suitability for treatment using the composition are also provided.

Description

IMPROVED ACETYLCHOLINE RECEPTOR MOLECULES AND USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 609,424, filed December 13, 2023, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (CNPY-P-007-PCT.xml; Size: 100,346 bytes; and Date of Creation: December 3, 2024) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of fusion protein generation and myasthenia gravis treatment.BACKGROUND OF THE INVENTION

[0004] 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 withhyperplasia, and 10% with thymoma. The predominant clinical feature of MG is fluctuating skeletal muscle weakness, often with true muscle fatigue.

[0005] 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.

[0006] International Patent Application WO2023 / 112028 provides molecules for treating MG that comprise a fragment of the extracellular domain from two different acetylcholine receptor subunits. Several mutations that decrease aggregation and enhance production are disclosed. Alternative mutants of the extracellular domain of acetylcholine receptor subunits are greatly needed.SUMMARY OF THE INVENTION

[0007] The present invention provides compositions and polypeptides comprising a fragment, analog or derivative of human acetylcholine receptor subunit alpha (ACHRA) and a fragment, analog or derivative of human acetylcholine receptor subunit gamma (ACHRG), wherein the ACHRA fragment, analog or derivative comprises mutation of phenylalanine 137 to tyrosine (F137Y), the ACHRG fragment, analog or derivative comprises a F137Y mutation, or both are provided. Pharmaceutical compositions comprising the composition, polypeptides, nucleic acid systems encoding the polypeptides of the composition and methods of treatment and determining suitability for treatment using the composition are also provided.

[0008] According to a first aspect, there is provided a composition, comprising a fragment, analog or derivative of human acetylcholine receptor subunit alpha (ACHRA), and a fragment, analog or derivative of human acetylcholine receptor subunit gamma (ACHRG), wherein the ACHRA fragment, analog or derivative comprises mutation of phenylalanine 137 to tyrosine (F137Y), the ACHRG fragment, analog or derivative comprises a F137Y mutation or both and wherein the phenylalanine 137 within ACHRA is with respect to SEQ ID NO: 1 and the phenylalanine 137 within ACHRG is with respect to SEQ ID NO: 2.

[0009] According to some embodiments, the fragment, analog or derivative of ACHRA comprises the F137Y mutation and the fragment, analog or derivative of ACHRG comprises the F137Y mutation.

[0010] According to some embodiments, the fragment, analog or derivative of ACHRA further comprises a W149R mutation.[Oi l] According to some embodiments, the fragment, analog or derivative of ACHRG comprises a Y 105E mutation, a Y117R mutation or both.

[0012] According to some embodiments, the fragment, analog or derivative of ACHRG comprises a Y 105E mutation and a Y117R mutation.

[0013] According to some embodiments, the fragment, analog or derivative of ACHRA comprises or consists ofSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCEIIVTHFPYDEQNCSMKEGTRTYDGSVVAINPESDQPDE SNFMESGEWVIKESRGWKHSVTYSCCPDTPYEDITYHFVMQREP (SEQ ID NO: 3).

[0014] According to some embodiments, the fragment, analog or derivative of ACHRG comprises or consists ofRNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVW IEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALECNV LVSPDGCIRWLPPAIFRSACSISVTYFPYDWQNCSLIFQSQTYSTNEIDLQLSQEDGQ TIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKP (SEQ ID NO: 4).

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

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

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

[0018] According to some embodiments, a. the first dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin and the second dimerization domain comprises a second hinge domain of a heavy chain and the first and 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.

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

[0020] According to some embodiments, the first polypeptide chain, the second polypeptide chain or both further comprise an Fc region of a human antibody heavy chain.

[0021] According to some embodiments, the Fc region is capable of inducing cytotoxicity against a cell binding the protein complex.

[0022] According to some embodiments, the Fc is from an IgG2 or IgG4 or comprises at least one mutation that reduces effector function.

[0023] According to some embodiments, the Fc region of the first, second or both polypeptide chains is separated from the fragment, analog or derivative or the dimerization domain by a linker.

[0024] According to some embodiments, the dimerization domain of the first, second or both polypeptide chains is C-terminal to the fragment.

[0025] According to some embodiments, the composition is devoid of an antibody variable domain.

[0026] According to some embodiments, the composition comprises a single polypeptide chain comprising the fragment, analog or derivative of ACHRA and the fragment, analog or derivative of ACHRG.

[0027] According to some embodiments, the fragment, analog or derivative of ACHRA is N-terminal to the fragment, analog or derivative of ACHRG.

[0028] According to some embodiments, the fragment, analog or derivative of ACHRA and the fragment, analog or derivative of ACHRG are separated by an amino acid linker.

[0029] According to some embodiments, the linker is a flexible GS linker or wherein the linker is a rigid linker.

[0030] According to some embodiments, the linker is a flexible linker comprising or consisting of (GGGS, SEQ ID NO: 5)n wherein n is an integer from 1 to 10.

[0031] According to some embodiments, the linker comprises or consists of (GGGS)7GS (SEQ ID NO: 6).

[0032] According to some embodiments, the single polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 8 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

[0033] According to some embodiments, the polypeptide chain further comprises an Fc region of a human antibody heavy chain.

[0034] According to some embodiments, the Fc region is capable of inducing cytotoxicity against a cell binding the protein complex.

[0035] According to some embodiments, the Fc region comprises at least one mutation that increases ADCC or CDC.

[0036] According to some embodiments, the Fc region is an Fc region comprising SEQ ID NO: 31 or SEQ ID NO: 49 comprising a plurality of mutations selected from: E15V / F23E / R72P / Y80E / P176E, S19D / A110E / I112E, G16A / A110E / I112E, andG16A / S47E / H48F / S104T / I112E within the SEQ ID NO: 31 or SEQ ID NO: 49.

[0037] According to some embodiments, the Fc is from an IgG2 or IgG4 or comprises at least one mutation that reduces effector function.

[0038] According to some embodiments, the FC domain comprises or consists of EPKSCDKTHTCPPCPAPEEEGGPSVFEFPPKPKDTEMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVETVEHQDWENGKEYKCKVSN KAEPAPIEKTISKAKGQPREPQVYTEPPSREEMTKNQVSETCEVKGFYPSDIAVEW ESNGQPENNYKTTPPVEDSDGSFFEYSKETVDKSRWQQGNVFSCSVMHEAEHNH YTQKSESESPGK (SEQ ID NO: 7).

[0039] According to some embodiments, the Fc region comprises at least one mutation that increases binding to FcgRIIb (FCGR2B / CD32B).

[0040] According to some embodiments, the Fc region is an Fc region comprising an S267E and E328F double mutation.

[0041] According to some embodiments, the FC region comprises or consists of EPKSCDKTHTCPPCPAPEEEGGPSVFEFPPKPKDTEMISRTPEVTCVVVDVEHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVETVEHQDWENGKEYKCKVSN KAFPAPIEKTISKAKGQPREPQVYTEPPSREEMTKNQVSETCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 87).

[0042] According to some embodiments, the Fc domain is linked to the fragment, analog or derivative of ACHRA or ACHRG by an amino acid linker.

[0043] According to some embodiments, the amino acid linker comprises or consists of GGS.

[0044] According to some embodiments, the composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

[0045] According to some embodiments, the composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, a tyrosine and at position 378.

[0046] According to some embodiments, composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

[0047] According to some embodiments, the composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, and a tyrosine at position 378.

[0048] According to some embodiments, the composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 75 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

[0049] According to some embodiments, the composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 75 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, and a tyrosine at position 378.

[0050] According to some embodiments, a polypeptide chain further comprising an N- terminal signal peptide sequence.

[0051] According to some embodiments, the signal peptide sequence consists of MGWSCIILFLVATATGVHS (SEQ ID NO: 10).

[0052] According to some embodiments, the composition further comprises an effector moiety that is not an Fc domain.

[0053] According to some embodiments, the effector moiety is selected from an amatoxin / amanitin, an anthracycline, an anthramycin-based dimer, a calicheamicin, camptothecin or an analog thereof, a duocarmycin, triptolide and a tubulin inhibitor.

[0054] 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.

[0055] According to some embodiments, the effector moiety is conjugated to an Fc domain of the composition.

[0056] According to another aspect, there is provided a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a derivative thereof comprising at least 80% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

[0057] According to some embodiments, the polypeptide comprises or consisting of the amino acid sequence of SEQ ID NO: 9 or a derivative thereof comprising at least 80% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

[0058] According to some embodiments, a derivative thereof comprises an arginine at position 149.

[0059] According to some embodiments, a derivative thereof comprises a glutamic acid at position 346, an arginine at position 358 or both.

[0060] According to some embodiments, a derivative thereof comprises a tyrosine at position 137, an arginine at position 149, a glutamic acid at position 346, an arginine at position 358 and a tyrosine at position 378.

[0061] According to some embodiments, the polypeptide further comprises an N-terminal signal peptide sequence.

[0062] According to some embodiments, the signal peptide sequence consists of SEQ ID NO: 10.

[0063] According to some embodiments, the polypeptide further comprises an effector moiety.

[0064] According to some embodiments, the effector moiety is not an Fc domain.

[0065] According to some embodiments, the effector moiety is an Fc domain comprising at least one mutation that increases antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).

[0066] According to some embodiments, the effector moiety is capable of inducing death in a cell binding the fragment.

[0067] 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.

[0068] According to some embodiments, the effector moiety is selected from: alpha- amanitin, PNU- 159682, tesirine, deruxtecan (Dxd), mertansine, MMAE, MMAF and a combination thereof.

[0069] According to some embodiments, the effector moiety is an Fc domain comprising SEQ ID NO: 31 or SEQ ID NO: 49 comprising a plurality of mutations selected from: E15V / F23E / R72P / Y80E / P176E, S19D / A110E / I112E, G16A / A110E / I112E, andG16A / S47E / H48F / S104T / I112E within the SEQ ID NO: 31 or SEQ ID NO: 49.

[0070] According to some embodiments, the effector moiety is conjugated to an Fc domain of the polypeptide.

[0071] 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.

[0072] According to some embodiments, the pharmaceutical composition is formulated for systemic administration to a subject, for administration to a human, for parenteral administration or any combination thereof.

[0073] 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, a polypeptide of the invention or a pharmaceutical composition of the invention, thereby treating myasthenia gravis.

[0074] According to some embodiments, the method further comprises reducing in the subject the levels of circulating antibodies against ACHRA, ACHRG or both prior to the administering.

[0075] According to some embodiments, the treating comprises killing B cells producing autoantibodies against ACHRA, B cells producing autoantibodies against ACHRG or both.

[0076] 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.

[0077] 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 the 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.

[0078] According to another aspect, there is provide 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 ACHRA, antibodies against ACHRG or both within the sample to the composition or the polypeptide, wherein binding of autoantibodies to the composition or polypeptide indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.

[0079] 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 preferredembodiments 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

[0080] Figures 1A-1G: (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-1D) Line graphs showing dose-dependent depletion in (IB) an exemplary highly depleted sample, (1C) an exemplary moderately depleted sample and (ID) and exemplary nondepleted 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.

[0081] Figures 2A-2B: (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.

[0082] Figures 3A-3F: Diagrams of possible embodiments of the two-chain therapeutic agent of the invention: (3A) shows a general embodiment of a molecule with two heavy chains for treating AChR positive MG, (3B) shows embodiments in which at least one of the CHI, CH2 or CH3 domains has been excluded, (3C) shows a general embodiment in which two fragments / analogs / derivatives are included in each heavy chain, (3D) shows a general embodiment in which two fragments / analogs / derivatives are included in one heavy chainwhile the other is devoid of a fragment / analog / derivative, (3E) shows a general embodiment in which two fragments / analogs / derivatives are included in one heavy chain while the other includes only one fragment / analog / derivative, and (3F) shows a general embodiment of a molecule with one heavy chain and one light chain.

[0083] Figure 4: A diagram of an embodiment of a three-chain therapeutic agent of the invention showing a general embodiment of a molecule with two heavy chains and one light chain.

[0084] Figures 5A-5C: Diagrams of generic embodiments of the two-chain therapeutic agents of the invention: (5A) shows a generic embodiment of two chains in which two contain chains contain two dimerization domain and two chains contain a single dimerization domain, (5B) shows an embodiment with optional linkers separating the various domains and fragments and (5C) shows embodiments in which two fragments / analogs / derivatives are present on each chain.

[0085] Figures 6A-6C: Diagrams of single chain therapeutic agents of the invention: (6A) shows an embodiment of a single chain molecule containing an ACHRA fragment / analog / derivative and an ACHRG fragment / analog / derivative, (6B) shows an embodiments of a single chain molecule containing an ACHRA fragment / analog / derivative and an ACHRG fragment / analog / derivative and a heavy chain constant region, and (6C) shows the single chain molecules of 6A-6B with amino acid (AA) linkers separating various domains.

[0086] Figure 7: 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.

[0087] Figure 8A-8B: Bar graphs of binding of an alpha-gamma combination molecules to various B cell hybridoma cell lines: (8A) binding of CRD-509 and CRD-600 to anti-alpha hybridoma and (8B) binding of CRD-509 and CRD-600 to anti-gamma hybridoma and to negative control hybridomas.

[0088] Figures 9A-9D: (9A-9B) Line graphs of binding of alpha and gamma mutant molecules to (9A) anti-alpha B cell hybridoma a- 192 and (9B) anti-gamma B cell hybridomag-63. (9C-D) Heat maps of binding of alpha-gamma combination molecules to (9C) various anti-alpha hybridomas and (9D) various anti-gamma hybridomas.

[0089] Figures 10A-10C: Line graphs showing dose-dependent depletion of MG autoantibodies from sera with (10A) predominantly anti-ACHRA autoantibodies by CRD- 991, (10B) sera with predominantly anti-ACHRG autoantibodies by CRD-997 and (10C) both sera with predominantly anti-ACHRA and sera with predominantly anti-ACHRG autoantibodies by CRD-981 and CRD-509.

[0090] Figures 11A-11B: Bar graphs of the percentage of CD138 and TACI double positive cells from (11A) spleen and (11B) bone marrow that also are bound by the CRD-991 and CRD-997 molecules.

[0091] Figure 12: Line graphs showing dose-dependent depletion of MG autoantibodies from sera contacted with CRD-981 and CRD-999.

[0092] Figures 13A-13C: Bar graphs of levels of secretion of proinflammatory cytokines (13A) IL — 6 and (13B) TNFA from peripheral white blood cells after a 24-hour culture with CRD-509. (13C) Bar graph of levels of secretion of various proinflammatory cytokines from peripheral blood cells after a 24-hour culture with CRD-999.

[0093] Figure 14A-14L: (14A-14F) Diagrams of possible generic embodiments of the four- chain therapeutic agent of the invention: (14A) shows a generic embodiment of two chains in which the chains contain a dimerization domain, and optionally a second dimerization domain, and an effector domain, (14B) shows the embodiments of 14A but with an optional linker separating the domains, (14C) shows embodiments with a chemical bond or linker between the dimerization domains, or optionally an EF, and a cytotoxic molecule (CM) such as a drug or a radio-labeled material and (14D) shows embodiments of the 14C but with an optional linker separation the domains. (14E) Diagrams of dimeric heavy chains each containing an ACHRA fragment / analog / derivative and an ACHRG fragment / analog / derivative and a cytotoxic moiety linked to various regions. (14F-14L) Diagrams of possible generic embodiments of the single-chain therapeutic agent of the invention: (14F) the molecule of 6A connected to a cytotoxic moiety, (14G) the molecule of 6A connected to an EF and CM, (14H) the molecule of 14G with the CHI 714 and hinge 713 domains it will be understood that any combination of domains from the Fc can be used, (141) the molecules of 14G with optional linkers, (14J) an ACHRAfragment / analog / derivative and an ACHRG fragment / analog / derivative linked to a CM; (14K) the molecule of 6B conjugated to a CM, (14L) the molecules of 6E with a cytotoxic moiety.

[0094] Figures 15A-15B: (15A) Line graphs of % specific cytotoxicity of CRD-981- MMAE on anti-alpha hybridoma a-18-C5-F6 and anti-gamma hybridomas g-50-Hl-E2 and G-66. A bar graph summarizing the maximum % specific cytotoxicity reached is also provided. (15B) Line graphs of % specific cytotoxicity of CRD-999-MMAE on anti-alpha hybridoma a- 122 and anti-gamma hybridomas g-50-Hl-E2 and G-66. A bar graph summarizing the maximum % specific cytotoxicity reached (with the concentration that produced that cytotoxicity) is also provided.

[0095] Figures 16A-16B: (16A) Plot of CD32B staining and CD79B staining of B cells after incubation with increasing concentrations of CRD-999 and increasing concentration of Polivy. (16B) Table of binding Kd for CRD-981 and CRD-999 to CD32B as assessed by SPR.

[0096] Figures 17A-17B: (17A) Line graph of average PTMG score and (17B) Kaplan Meier plot of % survival over time of rats that received various doses of CRD-981, or an irrelevant control molecule or vehicle only (PBS).DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention, in some embodiments, provides compositions comprising a fragment, analog or derivative of human acetylcholine receptor subunit alpha (ACHRA) and a fragment, analog or derivative of human acetylcholine receptor subunit gamma (ACHRG), wherein the ACHRA fragment, analog or derivative comprises mutation of phenylalanine 137 to tyrosine (F137Y), the ACHRG fragment, analog or derivative comprises a F137Y mutation, or both. Polypeptides comprising a fragment, analog or derivative of human ACHRA and a fragment, analog or derivative of human ACHRG, wherein the ACHRA fragment, analog or derivative comprises mutation of phenylalanine 137 to tyrosine (F137Y), the ACHRG fragment, analog or derivative comprises a F137Y mutation, or both are also provided. Polypeptides, compositions and protein complexes of the invention further comprising an effector moiety that is not an unmodified Fc domain are also provided. Thepresent invention further concerns pharmaceutical composition comprising the compositions and / or polypeptides, 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.

[0098] By a first aspect, there is provided a protein comprising a fragment, analog or derivative of acetylcholine receptor subunit alpha (ACHRA) and a fragment, analog or derivative of acetylcholine receptor subunit gamma (ACHRG).

[0099] By a first aspect, there is provided a polypeptide comprising a fragment, analog or derivative of acetylcholine receptor subunit alpha (ACHRA) and a fragment, analog or derivative of acetylcholine receptor subunit gamma (ACHRG).

[0100] By another aspect, there is provided a composition comprising a fragment, analog or derivative of acetylcholine receptor subunit alpha (ACHRA) and a fragment, analog or derivative of acetylcholine receptor subunit gamma (ACHRG).

[0101] By another aspect, there is provided a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a fragment, analog or derivative of acetylcholine receptor subunit alpha (ACHRA) and a first dimerization domain and a second polypeptide chain comprising a fragment, analog or derivative of acetylcholine receptor subunit gamma (ACHRG) and second dimerization domain.

[0102] In some embodiments, the protein is for use in treating myasthenia gravis. 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.

[0103] In some embodiments, the composition comprises a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a fragment, analog or derivative of ACHRA and a first dimerization domain and a second polypeptide chain comprising a fragment, analog or derivative of ACHRG and second dimerization domain. Insome 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. In some embodiments, the protein is a polypeptide of the invention.

[0104] 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.

[0105] 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 diagnosingmyasthenia 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. In some embodiments, ACHR is ACHRA. In some embodiments, ACHR is ACHRG. In some embodiments, ACHR is ACHRA or ACHRG. In some embodiments, ACHR is ACHRA and ACHRG.

[0106] 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.

[0107] 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.

[0108] 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 aprecursor 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.

[0109] Expression of a DNA sequence or an RNA sequence 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.

[0110] 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.

[0111] It will be understood by a skilled artisan that the extracellular domains (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

[0112] In some embodiments, the composition comprises a fragment of ACHRA. In some embodiments, the polypeptide comprises a fragment of ACHRA. In some embodiments, the composition comprises a fragment of ACHRG. In some embodiments, the polypeptide comprises a fragment of ACHRG. In some embodiments, a fragment is not the entire protein. 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 mutations in the human protein. In some embodiments, a fragment is an analog thereof. In some embodiments, a fragment is a derivative thereof.

[0113] 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.

[0114] 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 80% homology or identity. 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.

[0115] In some embodiments, the chain comprises at least one fragment, analog or derivative. In some embodiments, the chain comprises at least two fragments, analogs or derivatives. In some embodiments, the ACHRA fragment, analog or derivative is N-terminal to the ACHRG fragment, analog or derivative. In some embodiments, the ACHRG fragment, analog or derivative is N-terminal to the ACHRA fragment, analog or derivative. 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.

[0116] 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) residuesuch 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.

[0117] 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.

[0118] 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 proteinor at least one epitope of the antibodies. In some embodiments, the derivative is a derivative of a canonical sequence of the protein.

[0119] 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.

[0120] Canonical amino acid sequences of known proteins are well known in the art. They can be found in a variety of databases, including UniProt, NCBI, and the UCSC Genome Browser. Any sequence accepted as a canonical sequence may be employed. For a nonlimiting example, human 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.

[0121] 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. 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 canonicalamino acid sequence of an extracellular domain of ACHRA comprises or consists of SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDL SNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLP (SEQ ID NO: 1). In some embodiments, the extracellular domain is devoid of a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the ACHRA signal peptide comprises or consists of MEPWPLLLLFSLCSAGLVLG (SEQ ID NO: 11). In some embodiments, the ACHRA signal peptide comprises or consists of MFMCLEGGEKNLTVLVSSAVSAGLVLG (SEQ ID NO: 12).

[0122] 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: 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 ACHRG signal peptide comprises or consists of MHGGQGPLLLLLLLAVCLGAQG (SEQ ID NO: 13).

[0123] 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: 14). In some embodiments, the heavy chainsignal peptide consists of SEQ ID NO: 14. In some embodiments, the heavy chain signal peptide comprises MEFGLSWLFLVAILKGVQC (SEQ ID NO: 15). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 15. In some embodiments, the light chain signal peptide comprises MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 16). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 16. In some embodiments, the heavy chain signal peptide is the mouse heavy chain signal peptide and comprises MGWSCIILFLVATATGVHS (SEQ ID NO: 10). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 10.

[0124] In some embodiments, the ACHRA fragment, analog or derivative comprises mutation of phenylalanine 137 (F137). In some embodiments, the ACHRG fragment, analog or derivative comprises mutation of phenylalanine 137 (F137). In some embodiments, numbering within ACHRA is with respect to SEQ ID NO: 1. In some embodiments, numbering within ACHRG is with respect to SEQ ID NO: 2. In some embodiments, F137 is within SEQ ID NO: 1. In some embodiments, F137 is within SEQ ID NO: 2. It will be understood by a skilled artisan that if amino acids are added or removed from the ACHRA or ACHRG extracellular domain (e.g., a fragment with an N-terminal truncation) then the numbering will need to be shifted accordingly. For example, if a 5-amino acid N-terminal truncation of ACHRA / ACHRG were present then the phenylalanine in questions would not be the 137 amino acid but rather the 132. Similarly, if a 20 amino acid long N-terminal signal peptide were included in the polypeptide chain, then the phenylalanine is question would be shifted back 20 amino acid sand would be Fl 57. A skilled artisan would be able to identify this phenylalanine within the sequence regardless of deletions, additions or substitutions within the sequence of the ACHRA / ACHRG ECD. In some embodiments, F137 is mutated to tyrosine (F137Y). In some embodiments, the ACHRA fragment, analog or derivative comprises an F137Y mutation. In some embodiments, the ACHRG fragment, analog or derivative comprises an F137Y mutation. In some embodiments, the ACHRA fragment, analog or derivative comprises an F137Y mutation and the ACHRG fragment, analog or derivative comprises an F137Y mutation. In some embodiments, F137 mutation is a F137Y mutation. In some embodiments, F137 is mutated to alanine (F137A). In some embodiments, the ACHRA fragment, analog or derivative comprises an F137A mutation. In some embodiments, the ACHRG fragment, analog or derivative comprises an F137A mutation. In some embodiments, the ACHRA fragment, analog or derivative comprises an F137Amutation and the ACHRG fragment, analog or derivative comprises an F137A mutation. In some embodiments, F137 mutation is a F137A mutation. In some embodiments, F137 is mutated to serine (F137S). In some embodiments, the ACHRA fragment, analog or derivative comprises an F137S mutation. In some embodiments, the ACHRG fragment, analog or derivative comprises an F137S mutation. In some embodiments, the ACHRA fragment, analog or derivative comprises an F137S mutation and the ACHRG fragment, analog or derivative comprises an F137S mutation. In some embodiments, F137 mutation is a F137S mutation. The phenylalanine within the human cys loop is problematic due to the aggregation it produces. Mutating this amino acid to tyrosine produces the desired result of reducing aggregation. Serine and alanine both lack side chains that would produce aggregation and it is well known in protein engineering that tyrosine, alanine and serine can often be interchanged.

[0125] In some embodiments, the ACHRA fragment, analog or derivative further comprises a mutation of tryptophan 149 (W149). In some embodiments, W149 is with respect to SEQ ID NO: 1. In some embodiments, W149 is within SEQ ID NO: 1. Once again a skilled artisan will be able to determine the position of this tryptophan even if additions, deletions or substitutions have been made within SEQ ID NO: 1. In some embodiments, the tryptophan is mutated to arginine (W149R). In some embodiments, the ACHRA fragment analog or derivative comprises a W149R mutation. In some embodiments, the W149 mutation is a W 149R mutation.

[0126] In some embodiments, a fragment of ACHRA comprises the F137 mutation. In some embodiments, an analog of ACHRA comprises the Fl 37 mutation. In some embodiments, a derivative of ACHRA comprises the F137 mutation. It will be understood by a skilled artisan that in embodiments where ACHRA comprises the F137 mutation any modification to ACHRA must still retain the Fl 37 mutation. In some embodiments, a fragment of ACHRA comprises the W149 mutation. In some embodiments, an analog of ACHRA comprises the W149 mutation. In some embodiments, a derivative of ACHRA comprises the W149 mutation. It will be understood by a skilled artisan that in embodiments, where ACHRA comprises the W149 mutation any modification to ACHRA must still retain the W149 mutation.

[0127] In some embodiments, the ACHRG fragment, analog or derivative further comprises a mutation of threonine 105 (Y 105). In some embodiments, Y 105 is with respect to SEQ ID NO: 2. In some embodiments, Y105 is within SEQ ID NO: 2. Once again a skilled artisan will be able to determine the position of this threonine even if additions, deletions or substitutions have been made within SEQ ID NO: 2. In some embodiments, the threonine is mutated to glutamic acid (Y105E). In some embodiments, the ACHRG fragment analog or derivative comprises a Y105E mutation. In some embodiments, the Y105 mutation is a Y105E mutation.

[0128] In some embodiments, the ACHRG fragment, analog or derivative further comprises a mutation of threonine 117 (Y117). In some embodiments, Y117 is with respect to SEQ ID NO: 2. In some embodiments, Y117 is within SEQ ID NO: 2. Once again a skilled artisan will be able to determine the position of this threonine even if additions, deletions or substitutions have been made within SEQ ID NO: 2. In some embodiments, the threonine is mutated to arginine (Y117R). In some embodiments, the ACHRG fragment analog or derivative comprises a Y117R mutation. In some embodiments, the Y117 mutation is a Y117R mutation.

[0129] In some embodiments, a fragment of ACHRG comprises the Y 105 mutation. In some embodiments, an analog of ACHRG comprises the Y105 mutation. In some embodiments, a derivative of ACHRG comprises the Y105 mutation. It will be understood by a skilled artisan that in embodiments where ACHRG comprises the Y 105 mutation any modification to ACHRG must still retain the Y105 mutation. In some embodiments, a fragment of ACHRG comprises the Y117 mutation. In some embodiments, an analog of ACHRG comprises the Y117 mutation. In some embodiments, a derivative of ACHRG comprises the Y117 mutation. It will be understood by a skilled artisan that in embodiments where ACHRG comprises the Y117 mutation any modification to ACHRG must still retain the Y117 mutation.

[0130] In some embodiments, the ACHRA fragment, analog or derivative comprise a mutation of Fl 37 and a mutation of W 149. In some embodiments, the ACHRA fragment analog or derivative comprises a F137Y mutation and a W149R mutation. In some embodiments, the ACHRG fragment, analog or derivative comprise a mutation of Y 105 and a mutation of Y117. In some embodiments, the ACHRG fragment analog or derivativecomprises a Y105E mutation and a Y117R mutation. In some embodiments, the ACHRG fragment, analog or derivative comprise a mutation of Y105 and a mutation of F137. In some embodiments, the ACHRG fragment analog or derivative comprises a Y105E mutation and a F137Y mutation. In some embodiments, the ACHRG fragment, analog or derivative comprise a mutation of F137 and a mutation of Y117. In some embodiments, the ACHRG fragment analog or derivative comprises a F137Y mutation and a Y117R mutation. In some embodiments, the ACHRG fragment, analog or derivative comprise a mutation of F137, Y105 and a mutation of Y117. In some embodiments, the ACHRG fragment analog or derivative comprises a F137Y mutation, a Y 105E mutation and a Y117R mutation.

[0131] In some embodiments, the fragment, analog or derivative of ACHRA comprises the amino acid sequenceSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCEIIVTHFPYDEQNCSMKLGTRTYDGSVVAINPESDQPDL SNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLP (SEQ ID NO: 3). In some embodiments, the ACHRA fragment, analog or derivative consists of SEQ ID NO: 3. In some embodiments, the composition comprises SEQ ID NO: 3. In some embodiments, the polypeptide comprises SEQ ID NO: 3. In some embodiments, a polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, a derivative of an ACHRA ECD fragment comprises or consists of SEQ ID NO: 3.

[0132] In some embodiments, an analog or derivative comprises at least 70, 75, 80, 85, 90, 92, 95, 97, 99 or 100% homology or identity to SEQ ID NO: 3. Each possibility represents a separate embodiment of the invention. In some embodiments, an analog or derivative comprises at least 70% homology or identity to SEQ ID NO: 3. In some embodiments, an analog or derivative comprises at least 80% homology or identity to SEQ ID NO: 3. In some embodiments, an analog or derivative comprises at least 85% homology or identity to SEQ ID NO: 3. In some embodiments, an analog or derivative comprises at least 95% homology or identity to SEQ ID NO: 3. In some embodiments, the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 3 while retaining a tyrosine at position 137. In some embodiments, the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 3 while retaining an arginine at position 149. In some embodiments,the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 3 while retaining a tyrosine at position 137 and an arginine at position 149.

[0133] In some embodiments, the fragment, analog or derivative of ACHRG comprises the amino acid sequenceRNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVW IEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALECNV LVSPDGCIRWLPPAIFRSACSISVTYFPYDWQNCSLIFQSQTYSTNEIDLQLSQEDGQ TIEWIFIDPEAFTENGEWAIQHRPAKMEEDPAAPAQEAGHQKVVFYEEIQRKP (SEQ ID NO: 4). In some embodiments, the ACHRG fragment, analog or derivative consists of SEQ ID NO: 4. In some embodiments, the composition comprises SEQ ID NO: 4. In some embodiments, the polypeptide comprises SEQ ID NO: 4. In some embodiments, a polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, a derivative of an ACHRG ECD fragment comprises or consists of SEQ ID NO: 4.

[0134] In some embodiments, an analog or derivative comprises at least 70, 75, 80, 85, 90, 92, 95, 97, 99 or 100% homology or identity to SEQ ID NO: 4. Each possibility represents a separate embodiment of the invention. In some embodiments, an analog or derivative comprises at least 70% homology or identity to SEQ ID NO: 4. In some embodiments, an analog or derivative comprises at least 80% homology or identity to SEQ ID NO: 4. In some embodiments, an analog or derivative comprises at least 85% homology or identity to SEQ ID NO: 4. In some embodiments, an analog or derivative comprises at least 95% homology or identity to SEQ ID NO: 4. In some embodiments, the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 4 while retaining a tyrosine at position 137. In some embodiments, the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 4 while retaining a glutamic acid at position 105. In some embodiments, the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 4 while retaining an arginine at position 117. In some embodiments, the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 4 while retaining a tyrosine at position 137 and a glutamic acid at position 105. In some embodiments, the analog or derivative comprises a given level or homology or identity to SEQ ID NO: 4 while retaining a tyrosine at position 137 and an arginine at position 117. In some embodiments, the analog or derivative comprises a given level or homology or identityto SEQ ID NO: 4 while retaining a tyrosine at position 137, a glutamic acid at position 105 and an arginine at position 117.

[0135] 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 fragment is an extracellular domain of a surface protein and comprises an insertion that increases solubility. In some embodiments, ACHRA or ACHRG 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, W149R 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, ACHRG comprises parallel mutations. In some embodiments, parallel mutation are mutations to amino acids with homology.

[0136] In some embodiments, the alpha subunit extracellular domain into which mutations are made comprises or consists of SEQ ID NO: 1. In some embodiments, the gamma subunit extracellular domain into which mutations are made comprises or consists of SEQ ID NO: 2.

[0137] By another aspect, there is provided a protein comprises SEQ ID NO: 3. By another aspect, there is provided a protein comprising SEQ ID NO: 4.

[0138] 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 to phenylalanine.

[0139] In some embodiments, the fragment, analog or derivative comprises a mutation that decreases aggregation. In some embodiments, aggregation comprises auto-dimerization. In some embodiments, aggregation comprises multimerization. In some embodiments, a mutation is a plurality of mutations. In some embodiments, a plurality is two. In some embodiments, a plurality is three.

[0140] In some embodiments, the protein is ACHRA and the mutation is deletion of N 141. In some embodiments, N141 is within SEQ ID NO: 1. In some embodiments, N141 is within SEQ ID NO: 3. 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: 3. 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, 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: 3. 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: 3.

[0141] 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. Themutation 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: 2. In some embodiments, M84 is within SEQ ID NO: 4. In some embodiments, the protein is ACHRG and the mutation is mutation of tyrosine 105. In some embodiments, mutation of tyrosine 105 reduces alphagamma 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 (Y 105D). In some embodiments, tyrosine 105 is mutated to a positively charged amino acid. In some embodiments, tyrosine 105 is mutated to arginine (Y 105R). In some embodiments, tyrosine 105 is mutated to lysine (Y105K). In some embodiments, tyrosine 105 is mutated to histidine (Y105H). In some embodiments, Y 105 is within SEQ ID NO: 2. 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: 2. In some embodiments, the mutation is a plurality of mutations and comprises at least two mutations selected from mutation of M84, Y105 and Y117. 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, Y105E and Y117R. In some embodiments, the mutation isa 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 Y 117R. 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 Y117R and Y105E.Dimerization domains

[0142] 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.

[0143] 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: 17).

[0144] In some embodiments, the dimerization domain comprises or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, an Ig hinge domain is a heavy chain hinge domain. In some embodiments, the Ig is a human Ig. In some embodiments, the immunoglobulin is elected from IgA, IgD, IgE, IgG and IgM. In some embodiments, the immunoglobulin is IgG. In some embodiments, the IgG is IgGl. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is selected from IgGl and IgG3. In some embodiments, the IgG is IgG4. In some embodiments, the first and second dimerization domains are both Ig hinge domains. In some embodiments, the first and second dimerization domains are identical. In some embodiments, the first and second dimerization domains are at least 95% identical. In some embodiments, the first and second dimerization domains are at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% identical. Each possibility represents a separate embodiment of the invention.

[0145] In some embodiments, the hinge domain comprises the amino acid sequence EPKSCDKTHTCPPCPAPELLGGP (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 IgGl hinge comprises or consists of SEQ ID NO: 18. In some embodiments, the hinge domain comprises the amino acid sequence EPKCCVECPPCPAPPAAAP (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 IgG2 hinge comprises or consists of SEQ ID NO: 19. In some embodiments, the hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFLGGP (SEQ ID NO: 20). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IgG4 hinge comprises or consists of SEQ ID NO: 20. In some embodiments, the hinge domain comprises the amino acid sequenceELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC PRCPAPELLGGP (SEQ ID NO: 21). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IgG3 hinge comprises or consists of SEQ ID NO: 21. In some embodiments, the hinge domain comprises a CPXCP (SEQ ID NO: 22) motif. In some embodiments, the X in SEQ ID NO: 22 is selected from P and R. In some embodiments, SEQ ID NO: 22 is CPPCP (SEQ ID NO: 23). In someembodiments, SEQ ID NO: 22 is CPRCP (SEQ ID NO: 24). In some embodiments, the hinge domain comprises EPKSCDKTHTCPPCP (SEQ ID NO: 25). It will thus be understood that the hinge region can be considered to end after the CPXCP (SEQ ID NO: 22) motif.

[0146] 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.

[0147] In some embodiments, an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (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 IgGl CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequenceASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 27). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 27. In some embodiments, SEQ ID NO: 27 is the IgG2 CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequenceASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 28). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 28. In some embodiments,SEQ ID NO: 28 is the IgG3 CHI domain. In some embodiments, an Ig CHI domain comprises of the amino acid sequenceASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV EQSSGEYSESSVVTVPSSSEGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 29). In some embodiments, an Ig CHI domain consists of SEQ ID NO: 29. In some embodiments, SEQ ID NO: 29 is the IgG4 CHI domain.

[0148] In some embodiments, an Ig CL Kappa domain comprises of the amino acid sequenceAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 55). In some embodiments, an Ig CL Kappa domain consists of SEQ ID NO: 30. In some embodiments, an Ig CL Lambda domain comprises of the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVET TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 30). In some embodiments, an Ig CL Lambda domain consists of SEQ ID NO: 30.Effector moiety

[0149] 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.

[0150] 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 someembodiments, 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.

[0151] 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.

[0152] 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).

[0153] 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 CD16. 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.

[0154] 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 Fcregion. 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, the Fc is not a naturally occurring Fc. In some embodiments, an Fc is a human Fc. In some embodiments, a superior Fc is an Fc comprising at least one mutation that increases ADCC. In some embodiments, an Fc region is an Fc domain. In some embodiments, an Fc region is an Fc fragment. In some embodiments, the first polypeptide chain comprises an Fc region. In some embodiments, the second polypeptide chain comprises an Fc region. In some embodiments, both the first and second polypeptide chains comprise an Fc region. In some embodiments, the Fc region is an Fc region of an antibody heavy chain. In some embodiments, the antibody heavy chain is a human antibody heavy chain. In some embodiments, the heavy chain is an IgG heavy chain. In some embodiments, the IgG is selected from IgGl, IgG2, IgG3 and IgG4. In some embodiments, the IgG is selected from IgGl and IgG3. In some embodiments, the IgG is IgGl. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is IgG4.

[0155] In some embodiments, the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 31). In some embodiments, the Fc domain consists of SEQ ID NO: 31. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 31. 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: 31. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 48). It will be understood that SEQ ID NO: 48 contains 5 additional N-terminal amino acids as compared to SEQ ID NO: 31. As such, while numbering herein is given with respect to SEQ ID NO: 31 the numbering for SEQ ID NO: 48 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: 49). In some embodiments, the Fc region is capable of inducing a cytotoxic effect. In some embodiments, the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 7). In some embodiments, the Fc domain consists of SEQ ID NO: 7. 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: 7. Each possibility represents a separate embodiment of the invention. It will be understood that SEQ ID NO: 7 contains 5 additional N-terminal amino acids as compared to SEQ ID NO: 49. As such, while numbering herein is given with respect to SEQ ID NO: 49 (or SEQ ID NO: 31 which is equivalent) the numbering for SEQ ID NO: 7 can be found by adding 5. SEQ ID NO: 31 and SEQ ID NO: 49 differ by two amino acids. The two sequences can be interchanged and when mutations are given with respect to SEQ ID NO: 31 it will be understood that they apply also to SEQ ID NO: 49 and vice-versa. So too SEQ ID NO: 48 and SEQ ID NO: 7 also differ by only two amino acids and these two sequences can be interchanged.

[0156] In some embodiments, the Fc domain consists of SEQ ID NO: 31. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 31. 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: 31. Each possibility represents aseparate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 48. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 48. 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: 48. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 49. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 49. 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: 49. Each possibility represents a separate embodiment of the invention. In some embodiments, the Fc domain consists of SEQ ID NO: 7. In some embodiments, the Fc domain of IgGl comprises or consists of SEQ ID NO: 7. 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: 7. Each possibility represents a separate embodiment of the invention.

[0157] 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).

[0158] In some embodiments, the Fc region comprises an Ig hinge. 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 heavychain 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.

[0159] In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 32). In some embodiments, a CH2 domain comprises the amino acid sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAK (SEQ ID NO: 33). In some embodiments, the CH2 domain consists of SEQ ID NO: 32. In some embodiments, SEQ ID NO: 32 is the IgGl CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO:34). In some embodiments, the CH2 domain consists of SEQ ID NO: 34. In some embodiments, SEQ ID NO: 34 is the IgG2 CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO:35). In some embodiments, the CH2 domain consists of SEQ ID NO: 35. In some embodiments, SEQ ID NO: 35 is the IgG4 CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPRE EQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO: 36). In some embodiments, the CH2 domain consists of SEQ ID NO: 36. In some embodiments, SEQ ID NO: 36 is the IgG3 CH2 domain.

[0160] In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 37). In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38). In some embodiments, the CH3 domain consists of SEQ ID NO: 37. In some embodiments, the CH3 domain consists of SEQ ID NO: 38. In some embodiments, SEQ ID NO: 37 is the IgGl CH3 domain. In some embodiments, SEQ ID NO: 38 is the IgGl CH3 domain. In some embodiments, the SEQ ID NO: 37 sequence is the sequence found predominantly is humans of European and American descent. In some embodiments, SEQ ID NO: 38 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: 39). In some embodiments, the CH3 domain consists of SEQ ID NO: 39. In some embodiments, SEQ ID NO: 39 is the IgG2 CH3 domain. In some embodiments, a CH3 domain comprises the amino acid sequenceGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 40). In some embodiments, the CH3 domain consists of SEQ ID NO: 40. In some embodiments, SEQ ID NO: 40 is the IgG4 CH3 domain. In some embodiments, a CH3 domain comprises the amino acid sequenceGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPP MLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 41 In some embodiments, the CH3 domain consists of SEQ ID NO: 41. In some embodiments, SEQ ID NO: 41 is the IgG3 CH3 domain.

[0161] 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.

[0162] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutations or alterations may be used for constructing the polypeptides of the invention. In some embodiments, a region from an IgG is replaced with a region from an IgA. In some embodiments, a region from a TCRa is inserted into the first CH3 domain and a region from TCRb is inserted in to the second CH3 domain. In some embodiments, the mutation is insertion of a region from a TCR. In some embodiments, the TCR is selected from TCRa and TCRb. In some embodiments, the mutation is insertion of a region from a different Ig. Examples of these mutations can be found in Table 1. In some embodiments, the mutation is selected from a mutation in Table 1. In some embodiments, the first mutation is selected from a group of mutations provided in a row and the second column of Table 1 and the second mutation is the group of mutations provided in that same row of Table 1 in the third column. The mutations in Table 1 are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in other IgGs. In some embodiments, the first mutation is T366Y, and the second mutation is Y407T. In some embodiments, thefirst mutation is S354C and T366W and the second mutation is Y349C, T366S, L368A, and Y407V. In some embodiments, the first mutation is S364H and F405A and the second mutation is Y349T and T392F. In some embodiments, the first mutation is T350V, L351Y, F405A, and Y407V and the second mutation is T350V, T366L, K392L, and T394W. In some embodiments, the first mutation is K392D, and K409D and the second mutation is E356K, and D399K. In some embodiments, the first mutation is D221E, P228E, and L368E and the second mutation is D221R, P228R, and K409R. In some embodiments, the first mutation is K360E, and K409W and the second mutation is Q347R, D399V, and F405T. In some embodiments, the first mutation is K360E, K409W, and Y349C and the second mutation is Q347R, D399V, F405T, and S354C. In some embodiments, the first mutation is F405L and the second mutation is K409R. In some embodiments, the first mutation is K360D, D399M, and Y407A and the second mutation is E345R, Q347R, T366V, and K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, and K409V and the second mutation is E356G, E357D, S364Q, and Y407A. In some embodiments, the first mutation is T366K, and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, and Y349D and R355D. In some embodiments, the first mutation is T366K and C351K and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, and Y349D and R355D. In some embodiments, the first mutation is L351D and L368E and the second mutation is L351K and T366K. In some embodiments, the first mutation is L368D and K370S and the second mutation is E357Q and S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A and Y407V. In some embodiments, the Ig is IgG2, and the first mutation is C223E, P228E, and L368E and the second mutation is C223R, E225R, P228R, and K409R. In some embodiments, the first mutation is S354C or T366W and the second mutation is Y349C, T366S, L368A, or Y407V. In some embodiments, the first mutation is S364H or F405A and the second mutation is Y349T or T392F. In some embodiments, the first mutation is T350V, L351Y, F405A, or Y407V and the second mutation is T350V, T366L, K392L, or T394W. In some embodiments, the first mutation is K392D, or K409D and the second mutation is E356K, or D399K. In some embodiments, the first mutation is D221E, P228E, or L368E and the second mutation is D221R, P228R, or K409R. In some embodiments, the first mutation is K360E, or K409W and the second mutation is Q347R, D399V, or F405T.In some embodiments, the first mutation is K360E, K409W, or Y349C and the second mutation is Q347R, D399V, F405T, or S354C. In some embodiments, the first mutation is K360D, D399M, or Y407A and the second mutation is E345R, Q347R, T366V, or K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, or K409V and the second mutation is E356G, E357D, S364Q, or Y407A. In some embodiments, the first mutation is L351D or L368E and the second mutation is L351K or T366K. In some embodiments, the first mutation is L368D or K370S and the second mutation is E357Q or S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A or Y407V. In some embodiments, the Ig is IgG2, and the first mutation is C223E, P228E, or L368E and the second mutation is C223R, E225R, P228R, or K409R. In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 43). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45).

[0163] Table 1: Mutations for enhancing heterodimerization and inhibiting homodimerization of CH3 domains.

[0164] In some embodiments, the Fc domain comprises at least one mutation that increases effector function. In some embodiments, the Fc domain comprises at least one mutation that increases CDC, ADCC or both. In some embodiments, the Fc domain comprises at least one mutation that increases CDC. In some embodiments, the Fc domain comprises at least one mutation that increases ADCC. In some embodiments, the Fc domain comprises at least one mutation that increases antibody effector function. In some embodiments, the Fc domain comprises at least one mutation that increases antibody stability. In some embodiments,stability is half-life. In some embodiments, half-life is circulation half-life. In some embodiments, half-life is half-life in blood. In some embodiments, blood is serum.

[0165] 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.

[0166] 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 Pl 14 of SEQ ID NO: 7 to G. In some embodiments, the P to G mutation is mutation of P 109 of SEQ ID NO: 31 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 L19 of SEQ ID NO: 7 to A. In some embodiments, the L to A mutation is mutation of L14 of SEQ ID NO: 31 to A. In some embodiments, the mutation is mutation of leucine 235 of the IgGl human heavy chain to alanine (L235A). In some embodiments, the L to A mutation is mutation of LI 20 of SEQ ID NO: 7 to A. In some embodiments, the L to A mutation is mutation of L15 of SEQ ID NO: 31 to A. In some embodiments, the plurality of mutation comprises P114G, L19A and L20A of SEQ ID NO: 7. In some embodiments,the plurality of mutation comprises P109G, L14A and L15A of SEQ ID NO: 31. In some embodiments, the plurality of mutation comprises L19A and L20A of SEQ ID NO: 7. In some embodiments, the plurality of mutation comprises L14A and L15A of SEQ ID NO: 31. In some embodiments, the plurality of mutation comprises P329G, L234A and L235A of the IgGl human heavy chain. In some embodiments, the plurality of mutation comprises L234A and L235A of the IgGl human heavy chain. It will be understood by a skilled artisan that parallel mutation can also be performed in the IgG3 heavy chain or the heavy chains of non-human IgGls. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the YTE mutations. In some embodiments, the mutation is mutation of methionine 252 of the IgGl human heavy chain to tyrosine (M252Y). In some embodiments, the M to Y mutation is mutation of M37 of SEQ ID NO: 7 to Y. In some embodiments, the M to Y mutation is mutation of M32 of SEQ ID NO: 31 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 S39 of SEQ ID NO: 36 to T. In some embodiments, the S to T mutation is mutation of S34 of SEQ ID NO: 31 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 T41 of SEQ ID NO: 7 to E. In some embodiments, the T to E mutation is mutation of T36 of SEQ ID NO: 31 to E. In some embodiments, the plurality of mutation comprises M37Y, S39T and T41E of SEQ ID NO: 7. In some embodiments, the plurality of mutation comprises M32Y, S34T and T36E of SEQ ID NO: 31. 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 N82 of SEQ ID NO: 7 (N82A or N82Q). In some embodiments, the asparagine is N77 of SEQ ID NO: 31 (N77A or N77Q). 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.

[0167] In some embodiments, the at least one mutation that decreases ADCC is a N297A mutation. In some embodiments, the N297A mutation is within the CH2 domain. In some embodiments, the N297A mutation is mutation of asparagine 59 of SEQ ID NO: 32 to alanine. In some embodiments, an N297A mutated CH2 domain comprises an N59A mutation of SEQ ID NO: 32. In some embodiments, the Fc domain comprises a CH2 domain comprising SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYASTYRVVSVETVEHQDWENGKEYKCKVSNKAEPAPIEKTISKAK (SEQ ID NO: 50). In some embodiments, the Fc domain comprises a CH2 domain consisting of SEQ ID NO: 50. In some embodiments, the N297A mutated CH2 domain consists of SEQ ID NO: 50.

[0168] 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: 31 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: 31 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: 31 to A. In some embodiments, the plurality of mutation comprises P109G, L14A and L15A of SEQ ID NO: 31. In some embodiments, the plurality of mutation comprises L14A and L15A of SEQ ID NO: 31. In some embodiments, the plurality of mutation comprises P329G, L234A and L235A of the IgGl human heavy chain. In some embodiments, the plurality of mutation comprises L234A and L235A of the IgGl human heavy chain. It will be understood by a skilled artisan that parallel mutation can also be performed in the IgG3 heavy chain or the heavy chains of non-human IgGls. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the YTE mutations. In some embodiments, the mutation is mutation of methionine 252 of the IgGl human heavy chain to tyrosine (M252Y). In some embodiments, the M to Y mutation is mutation of M32 of SEQ ID NO: 31 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: 31 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: 31 to E. In some embodiments, the plurality of mutation comprises M32Y, S34T and T36E of SEQ ID NO: 31. 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: 31 (N77A or N77Q).

[0169] 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 5. 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: 31 or 49. 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: 31 or 49. 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: 31 or 49. 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: 31 or 49. In some embodiments, the plurality comprises or consists of mutation of methionine 428 to leucine (M428L) and mutation of asparagine 434 to serine (N434S) (also termed LS). In some embodiments, an M428L / N434S mutated Fc domain comprises an M208L and N214S mutation of SEQ ID NO: 31 or 49. In some embodiments, the plurality comprises or consists of M428L and mutation of asparagine 434 to alanine (N434A) (also termed LA). In some embodiments, anM428L / N434A mutated Fc domain comprises an M208L and N214A mutation of SEQ ID NO: 31 or 49. 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: 31 or 49. 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: 31 or 49. 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: 31 or 49. 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: 31 or 49. 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: 31 or 49. 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: 31 or 49. 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 A 158V mutation of SEQ ID NO: 31 or 49. 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: 31 or 49. In some embodiments, the plurality that attenuates binding comprises or consists of mutation of isoleucine 253 to alanine (I253A), H435A and mutationof 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: 31 or 49. In some embodiments, the plurality that attenuates binding comprises or consists of 1253 A, mutation of histidine 310 to alanine (H310A) and H435A. In some embodiments, an I253A / H310A / H435A mutated Fc domain comprises an I33A, H90A and H215A mutation of SEQ ID NO: 31 or 49.

[0170] Table 5: Mutations influencing FcRn binding

[0171] 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: 32. In some embodiments, an N297A mutated Fc domain comprises an N77A mutation of SEQ ID NO: 31 or 49. In some embodiments, an N297Q mutated CH2 domain comprises an N59Q mutation of SEQ ID NO: 32. In some embodiments, an N297Q mutated Fc domain comprises an N77Q mutation of SEQ ID NO: 31 or 49. In some embodiments, an N297G mutated CH2 domain comprises an N59G mutation of SEQ ID NO: 32. In some embodiments, an N297G mutated Fc domain comprises an N77G mutation of SEQ ID NO: 31 or 49. In some embodiments, the mutationis 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: 18 and a CH2 domain comprising a L90R mutation of SEQ ID NO: 32. In some embodiments, a G236R / L328R mutated Fc domain comprises an G16R and L108R mutation of SEQ ID NO: 31 or 49. 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: 32. In some embodiments, a S298G / T299A mutated Fc domain comprises an S78G and T79A mutation of SEQ ID NO: 31 or 49. 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 L19F and L20E mutation of SEQ ID NO: 18 and a CH2 domain comprising a D27A mutation of SEQ ID NO: 32. In some embodiments, a L234F / L235E / D265A mutated Fc domain comprises an L14F, L15E and D45A mutation of SEQ ID NO: 31 or 49. 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: 18 and a CH2 domain comprising a P91G mutation of SEQ ID NO: 32. In some embodiments, a L234A / L235A / P329G mutated Fc domain comprises an L14A, L15A and P109G mutation of SEQ ID NO: 31 or 49. 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: 18 and a CH2 domain comprising a P93S mutation of SEQ ID NO: 32. In some embodiments, a L234F / L235E / P331S mutated Fc domain comprises an L14F, L15E and PH IS mutation of SEQ ID NO: 31 or 49. 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: 18 and a CH2 domain comprising a E80A mutation of SEQ IDNO: 32. In some embodiments, a L235A / G237A / E318A mutated Fc domain comprises an L15A, G17A and E98A mutation of SEQ ID NO: 31 or 49.

[0172] 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.

[0173] 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 Fc receptor is FcgRIIb (FCGR2B / CD32B). In some embodiments, the at least one mutation is the double mutation S267E and E328F. 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: 32. In some embodiments, an S267E mutated Fc domain comprises an S47E mutation of SEQ ID NO: 31 or 49. 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: 18. In some embodiments, a P238D mutated Fc domain comprises an P18D mutation of SEQ ID NO: 31 or 49. 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 (E328F) (also termed SEEF). In some embodiments, an S267E / E328F mutated CH2 domain comprises an S29E and L90F mutation of SEQ ID NO: 32. In some embodiments, an S267E / L328F mutated Fc domain comprises an S47E and L108F mutation of SEQ ID NO: 31 or 49. 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: 32. In some embodiments, an S267E / H268F / S324T mutated Fc domain comprises an S47E, H48F and S 104T mutation of SEQ ID NO: 31 or 49. 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: 18 and a mutated CH2 domain comprising a P33G and A92R mutation of SEQ ID NO: 32. In some embodiments, a G237D / P238D / P271G / A330R mutated Fc domain comprises a G17D, P18D, P51G and A110R mutation of SEQ ID NO: 31 or 49. 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: 18 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 32. 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: 31 or 49. 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 E28D, G22D and P23D mutation of SEQ ID NO: 18 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 32. 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: 31 or 49.

[0174] 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, byapproximately 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. In some embodiments, the Fc region comprises an S267E and L328F (SELF) double mutation. In some embodiments, the hinge, CH2 and CH3 domains comprising the SELF double mutation comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 87). In some embodiments, the Fc region comprising the SELF double mutation comprises SEQ ID NO: 87. In some embodiments, the Fc region comprising the SELF double mutation consists of SEQ ID NO: 87.

[0175] 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.

[0176] 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.

[0177] 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: 31. 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: 31. 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: 31. In some embodiments, the S239D / A330L / I332E mutations alsoincrease 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: 31. 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: 31. 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: 31. 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 (Al 19Q) within SEQ ID NO: 31. 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: 31. 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 comprisesmutation 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: 31 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: 31. It will be understood that all of the above recited mutations given with respect to SEQ ID NO: 31 also apply to SEQ ID NO: 49. Indeed, they also apply to SEQ ID NO: 48 and SEQ ID NO: 7, but all numbering given hereinabove must be increased by 5 for these sequences.

[0178] 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: 31 and SEQ ID NO: 49. In some embodiments, the Fc domain with increased ADCC comprisesEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 51). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 51. In some embodiments, the Fc comprising the L15V / F23L / R72P / Y80L / P176L mutations is SEQ ID NO: 51. 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: 51 and comprises L15V / F23L / R72P / Y80L / P176L mutations.

[0179] 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: 31 and SEQ ID NO: 49. In some embodiments, the Fc domain with increased ADCC comprisesEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 52). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 52. In some embodiments, the Fc comprising theS19D / A110L / I112E mutations is SEQ ID NO: 52. 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: 52 and comprises S19D / A110L / I112E mutations.

[0180] 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: 31 and SEQ ID NO: 49. In some embodiments, the Fc domain with increased CDC comprisesEPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVT NKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO: 53). In some embodiments, the Fc domain with increased CDC consists of SEQ ID NO: 53. In some embodiments, the Fc comprising the G16A / S47E / H48F / S104T / I112E mutations is SEQ ID NO: 53. 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: 53 and comprises G16A / S47E / H48F / S104T / I112E mutations.

[0181] 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: 31 and SEQ ID NO: 49. In some embodiments, the Fc domain with increased ADCC comprisesEPKSCDKTHTCPPCPAPEEEAGPSVFEFPPKPKDTEMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVETVEHQDWENGKEYKCKVSN KAEPEPEEKTISKAKGQPREPQVYTEPPSREEMTKNQVSETCEVKGFYPSDIAVEW ESNGQPENNYKTTPPVEDSDGSFFEYSKETVDKSRWQQGNVFSCSVMHEAEHNH YTQKSESESPGK (SEQ ID NO: 54). In some embodiments, the Fc domain with increased ADCC consists of SEQ ID NO: 54. In some embodiments, the Fc comprising the G16A / A110E / I112E mutations is SEQ ID NO: 54. 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: 54 and comprises G16A / A110E / I112E mutations.

[0182] In some embodiments, the effector domain is selected from SEQ ID NO: 51-54. In some embodiments, the effector domain comprises any one of SEQ ID NO: 51-54. In someembodiments, the effector domain consists of any one of SEQ ID NO: 51-54. In some embodiments, the effector domain is selected from SEQ ID NO: 51, 52 and 54. In some embodiments, the effector domain comprises any one of SEQ ID NO: 51, 52 and 54. In some embodiments, the effector domain consists of any one of SEQ ID NO: 51, 52 and 54. 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: 51, 52 and 54 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.

[0183] 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.

[0184] 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: 31. 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 (E113S) within SEQ ID NO: 31. 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: 31. It will be understood that all of the above recited mutations given with respect to SEQ ID NO: 31also apply to SEQ ID NO: 49. Indeed, they also apply to SEQ ID NO: 48 and SEQ ID NO: 7, but all numbering given hereinabove must be increased by 5 for these sequences.

[0185] In some embodiments, the effector moiety is a drug. In some embodiments, the protein is a PLA2R1 ECD drug conjugate. In some embodiments, the protein is a PLA2R1- Fc drug conjugate. In some embodiments, the complex is a PLA2R1 ECD drug conjugate. In some embodiments, the complex is a PLA2R1-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.

[0186] 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.

[0187] 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.

[0188] 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-baseddimer 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 inhibit DNA and RNA synthesis. In some embodiments, the PBD is a PBD dimer. Examples of PBDs and PDDs include, but are not limited to anthramycin, SJG-136, NS 694501 and FGX2-62. In some embodiments, the PBD is anthramycin. In some embodiments, the effector moiety is anthramycin. In some embodiments, anthramycin is anthramycin-methyl-ether (AME). In some embodiments, anthramycin is an anthramycin based dimer. In some embodiments, the PBD is tesirine (SG3249). In some embodiments, tesirine is SG3199. In some embodiments, the chemotherapeutic SG3249. In some embodiments, the chemotherapeutic is SG3199.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] In some embodiments, the chemotherapeutic is triptolide. In some embodiments, the effector moiety is triptolide.

[0193] 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.

[0194] 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

[0195] 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.

[0196] 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.

[0197] 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 arepart 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.

[0198] 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 firstpolypeptide 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.

[0199] 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

[0200] In some embodiments, the composition comprises a polypeptide chain comprising the fragment, analog or derivative of ACHRA and the fragment, analog or derivative of ACHRG. In some embodiments, the polypeptide chain is a single polypeptide chain. In some embodiments, the single chain comprises the fragment of ACHRA and the fragment of ACHRG. In some embodiments, the polypeptide chain further comprises an Fc region.

[0201] In some embodiments, the fragment, analog or derivative of ACHRA is separated from the fragment, analog or derivative of ACHRG by a linker. In some embodiments, a fragment, analog or derivative is separated from the Fc region by a linker. In some embodiments, an ACHRA fragment, analog or derivative is separated from the Fc region by a linker. In some embodiments, an ACHRG fragment, analog or derivative is separated from the Fc region by a linker. In some embodiments, the effector moiety is separated attached by a linker. In some embodiments, the effector moiety is separated by the fragment by a linker.

[0202] In some embodiments, the fragment, analog or derivative and the dimerization domain are separated by a linker. In some embodiments, the dimerization domain and the Fcregion are separated by a linker. In some embodiments, the fragment and the Fc region are separated by a linker. In some embodiments, the effector moiety is attached by a linker. In some embodiments, the effector moiety and fragment are separated by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a bond. In some embodiments, the bond is a peptide bond. In some embodiments, the bond is an amino acid bond. In some embodiments, the linker is a flexible linker. Linkers are well known in the art and any linker may be used.

[0203] 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-MMAE 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-DMED A 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.

[0204] In some embodiments, conjugated is linked. In some embodiments, conjugation is via a bond. In some embodiments, the conjugate is directly conjugated. In some embodiments, the conjugate is conjugated via a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the effector moiety is conjugated by a linker.

[0205] 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.

[0206] 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.

[0207] 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 sites.

[0208] 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.

[0209] 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: 5). In some embodiments, the linker comprises GGGGS (SEQ ID NO: 46). In some embodiments, the linker is GGS. In some embodiments, linker comprises GGS. In some embodiments, the linker consists of GGS. In some embodiments, the linker comprises (GGGS)n wherein n is an integer. In some embodiments, the linker comprises (GGGS, SEQ ID NO: 5)n wherein n is an integer. In some embodiments, the linker consists of (GGGS, SEQ ID NO: 5)n wherein n is an integer. In some embodiments, the linker comprises (GGGS)nGS wherein n is an integer. In some embodiments, the linker consists of (GGGS)nGS wherein n is an integer. In someembodiments, 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 7. In some embodiments, the linker comprises GGGSGGGSGGGSGGGSGGGSGGGSGGGSGS (SEQ ID NO: 6). In some embodiments, the linker is a rigid linker. In some embodiments, the rigid linker comprises EAAAK (SEQ ID NO: 47). In some embodiments, the rigid linker consists of SEQ ID NO: 47. 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. In some embodiments, n is 7.

[0210] In some embodiments, the dimerization domain is C-terminal to the fragments, analogs or derivates. In some embodiments, a fragment, analog or derivative is C-terminal to the dimerization domain. In some embodiments, the Fc region is C-terminal to the fragments, analogs or derivatives. In some embodiments, a fragment, analog or derivative 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 a fragment, analog or derivative. In some embodiments, the fragment, analogs or derivatives are N-terminal to the dimerization domain. In some embodiments, the Fc region is N-terminal to a fragment, analog or derivative. In some embodiments, the fragments, analogs or derivatives are 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.

[0211] In some embodiments, the single polypeptide chain comprises SEHETREVAKEFKDYSSVVRPVEDHRQVVEVTVGEQEIQEINVDEVNQIVTTNVRE KQQWVDYNEKWNPDDYGGVKKIHIPSEKIWRPDEVEYNNADGDFAIVKFTKVEE QYTGHITWTPPAIFKSYCEIIVTHFPYDEQNCSMKEGTRTYDGSVVAINPESDQPDE SNFMESGEWVIKESRGWKHSVTYSCCPDTPYEDITYHFVMQREPGGGSGGGSGGGSGGGSGGGSGGGSGGGSGSRNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKL TLTNLISLNEREEALTTNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPD IVLENNVDGVFEVALECNVLVSPDGCIRWLPPAIFRSACSISVTYFPYDWQNCSLIF QSQTYSTNEIDEQESQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMEEDPAAPAQE AGHQKVVFYEEIQRKP (SEQ ID NO: 8). In some embodiments, the single polypeptide chain comprises a sequence with at least 70, 75, 80, 85, 90, 92, 95, 97, or 99% homology or identity to SEQ ID NO: 8. Each possibility represents a separate embodiment of the invention. In some embodiments, a sequence comprises at least 80% homology or identity. In some embodiments, a sequence comprises at least 85% homology or identity. In some embodiments, a sequence comprises at least 95% homology or identity. In some embodiments, the sequence with homology or identity to SEQ ID NO: 8 comprises tyrosine at position 137. In some embodiments, the sequence with homology or identity to SEQ ID NO: 8 comprises tyrosine at position 378. In some embodiments, the sequence with homology or identity to SEQ ID NO: 8 comprises tyrosine at position 137 and tyrosine at position 378. In some embodiments, the sequence with homology or identity to SEQ ID NO: 8 comprises arginine at position 149. In some embodiments, the sequence with homology or identity to SEQ ID NO: 8 comprises glutamic acid at position 346. In some embodiments, the sequence with homology or identity to SEQ ID NO: 8 comprises arginine at position 358. In some embodiments, the sequence with homology or identity to SEQ ID NO: 8 comprises tyrosine at position 137, arginine at position 149, glutamic acid at position 346, arginine at position 358 and tyrosine at position 378. In some embodiments, SEQ ID NO: 8 is linked to a dimerization domain. In some embodiments, a sequence with homology or identity to SEQ ID NO: 8 is linked to a dimerization domain. In some embodiments, SEQ ID NO: 8 is linked to an Fc domain. In some embodiments, a sequence with homology or identity to SEQ ID NO: 8 is linked to an Fc domain. In some embodiments, the single polypeptide chain consists of SEQ ID NO: 8.

[0212] In some embodiments, the single polypeptide chain comprises SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCEIIVTHFPYDEQNCSMKLGTRTYDGSVVAINPESDQPDL SNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPGGGSGGGSGGG SGGGSGGGSGGGSGGGSGSRNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPD IVLENNVDGVFEVALECNVLVSPDGCIRWLPPAIFRSACSISVTYFPYDWQNCSLIF QSQTYSTNEIDLQLSQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQE AGHQKVVFYLLIQRKPGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9). In some embodiments, the single polypeptide chain comprises a sequence with at least 70, 75, 80, 85, 90, 92, 95, 97, or 99% homology or identity to SEQ ID NO: 9. Each possibility represents a separate embodiment of the invention. In some embodiments, a sequence comprises at least 80% homology or identity. In some embodiments, a sequence comprises at least 85% homology or identity. In some embodiments, a sequence comprises at least 95% homology or identity. In some embodiments, the sequence with homology or identity to SEQ ID NO: 9 comprises tyrosine at position 137. In some embodiments, the sequence with homology or identity to SEQ ID NO: 9 comprises tyrosine at position 378. In some embodiments, the sequence with homology or identity to SEQ ID NO: 9 comprises tyrosine at position 137 and tyrosine at position 378. In some embodiments, the sequence with homology or identity to SEQ ID NO: 9 comprises arginine at position 149. In some embodiments, the sequence with homology or identity to SEQ ID NO: 9 comprises glutamic acid at position 346. In some embodiments, the sequence with homology or identity to SEQ ID NO: 9 comprises arginine at position 358. In some embodiments, the sequence with homology or identity to SEQ ID NO: 9 comprises tyrosine at position 137, arginine at position 149, glutamic acid at position 346, arginine at position 358 and tyrosine at position 378.

[0213] In some embodiments, the single polypeptide chain comprises SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRL KQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLL QYTGHITWTPPAIFKSYCEIIVTHFPYDEQNCSMKLGTRTYDGSVVAINPESDQPDL SNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPGGGSGGGSGGG SGGGSGGGSGGGSGGGSGSRNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKL TLTNLISLNEREEALTTNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPD IVLENNVDGVFEVALECNVLVSPDGCIRWLPPAIFRSACSISVTYFPYDWQNCSLIFQSQTYSTNEIDLQLSQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQE AGHQKVVFYLLIQRKPGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 75). In some embodiments, the single polypeptide chain comprises a sequence with at least 70, 75, 80, 85, 90, 92, 95, 97, or 99% homology or identity to SEQ ID NO: 75. Each possibility represents a separate embodiment of the invention. In some embodiments, a sequence comprises at least 80% homology or identity. In some embodiments, a sequence comprises at least 85% homology or identity. In some embodiments, a sequence comprises at least 95% homology or identity. In some embodiments, the sequence with homology or identity to SEQ ID NO: 75 comprises tyrosine at position 137. In some embodiments, the sequence with homology or identity to SEQ ID NO: 75 comprises tyrosine at position 378. In some embodiments, the sequence with homology or identity to SEQ ID NO: 75 comprises tyrosine at position 137 and tyrosine at position 378. In some embodiments, the sequence with homology or identity to SEQ ID NO: 75 comprises arginine at position 149. In some embodiments, the sequence with homology or identity to SEQ ID NO: 75 comprises glutamic acid at position 346. In some embodiments, the sequence with homology or identity to SEQ ID NO: 75 comprises arginine at position 358. In some embodiments, the sequence with homology or identity to SEQ ID NO: 75 comprises tyrosine at position 137, arginine at position 149, glutamic acid at position 346, arginine at position 358 and tyrosine at position 378.

[0214] 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. Further, it will be understood that a signal peptide can be present at the N-terminus. Any signal peptide that results in the secretion of the polypeptide can be used. In some embodiments, the signal peptide comprises SEQ ID NO: 10.

[0215] In some embodiments, the protein complex comprises a first polypeptide comprising or consisting of SEQ ID NO: 8 and a second polypeptide comprising or consisting of SEQ ID NO: 8. In some embodiments, the protein complex comprises a first polypeptide comprising or consisting of SEQ ID NO: 9 and a second polypeptide comprising or consisting of SEQ ID NO: 9. In some embodiments, the protein complex comprises a firstpolypeptide comprising or consisting of SEQ ID NO: 75 and a second polypeptide comprising or consisting of SEQ ID NO: 75.

[0216] 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.Effector moiety

[0217] In some embodiments, the composition comprises an effector moiety. In some embodiments, the polypeptide comprises an effector moiety. In some embodiments, the effector moiety is an Fc domain. In some embodiments, the effector moiety is not an Fc domain. In some embodiments, the effector moiety is a non-Fc moiety. In some embodiments, the composition comprises an effector moiety other than an Fc region. In some embodiments, the polypeptide comprises an effector moiety other than an Fc region. In some embodiments, the effector moiety comprises an Fc domain and a non-Fc moiety. In some embodiments, the non-Fc moiety is conjugated to the Fc domain.

[0218] In some embodiments, the effector moiety is a drug. In some embodiments, the protein is an ACHRA ECD drug conjugate. In some embodiments, the protein is an ACHRG ECD drug conjugate. In some embodiments, the protein is an ACHRA ECD and ACHRG ECD 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 effectormoiety 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.

[0219] 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. They 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.

[0220] 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.

[0221] 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 inhibit DNA and RNA synthesis. In some embodiments, the PBD is a PBD dimer. Examples of PBDs and PDDs include, but are not limited to anthramycin, SJG-136, NS 694501 and FGX2-62. In some embodiments, the PBD is anthramycin. In some embodiments, the effector moiety is anthramycin. In some embodiments, anthramycin is anthramycin-methyl-ether (AME). In some embodiments, anthramycin is an anthramycin based dimer. In some embodiments, the PBD is tesirine(SG3249). In some embodiments, tesirine is SG3199. In some embodiments, the chemotherapeutic SG3249. In some embodiments, the chemotherapeutic is SG3199.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] In some embodiments, the chemotherapeutic is triptolide. In some embodiments, the effector moiety is triptolide.

[0226] 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 MMAE and MMAF. In some embodiments, the tubulin inhibitor is a tubulysin. In some embodiments, the tubulysin is tubulysin A.

[0227] 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.Pharmaceutical compositions

[0228] By another aspect, there is provided a pharmaceutical composition comprising a protein of the invention.

[0229] By another aspect, there is provided a pharmaceutical composition comprising a polypeptide chain of the invention.

[0230] By another aspect, there is provided a pharmaceutical composition comprising a protein complex of the invention.

[0231] By another aspect, there is provided a pharmaceutical composition comprises a composition of the invention.

[0232] 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 asethyl 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.

[0233] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0234] 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.

[0235] 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, improvedbreathing, reduced autoantibody titer, improved synapsis function or any other measure of improvement.

[0236] 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.

[0237] 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.

[0238] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.Methods of treatment

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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

[0247] By another aspect, there is provided a nucleic acid molecule encoding a protein of the invention.

[0248] 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.

[0249] 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.

[0250] By another aspect, there is provided a nucleic acid molecule encoding a polypeptide chain of a composition of the invention.

[0251] By another aspect, there is provided a nucleic acid molecule encoding a composition of the invention.

[0252] 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.

[0253] 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.

[0254] 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 someembodiments, 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.

[0255] 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 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.

[0256] 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.

[0257] 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.

[0258] 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).

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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 isthe 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] In some embodiments, the nucleic acid molecule is a single nucleic acid molecule. In some embodiments, the first and second nucleic acid molecules are different molecules. In some embodiments, the first and second nucleic acid molecule are the same molecule. In some embodiments, any two of the first, second, third and fourth nucleic acid molecules are different molecules. In some embodiments, any two of the first, second, third and fourth nucleic acid molecules are the same molecule. In some embodiments, any three of the first, second, third and fourth nucleic acid molecules are different molecules. In some embodiments, the first, second, and third nucleic acid molecules are different molecules. In some embodiments, any three of the first, second, third and fourth nucleic acid molecules are the same molecule. In some embodiments, all of the first, second, third and fourth nucleic acid molecules are different molecules. In some embodiments, all of the first, second, third and fourth nucleic acid molecules are the same molecule.Patient selection

[0268] 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.

[0269] 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.

[0270] By another aspect, there is provided a method of determining suitability of a subject to be treated by a method of the invention, the method comprising receiving a sample fromthe subject, contacting the sample with a protein 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.

[0271] 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 known to be positive for anti-ACHRA autoantibodies. In some embodiments, the subject is known to be positive for anti-ACHRG autoantibodies. In some embodiments, the subject is known to be positive for both anti-ACHRA and ACHRG autoantibodies. 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.

[0272] 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.

[0273] In some embodiments, the autoantibodies are myasthenia gravis autoantibodies. In some embodiments, the autoantibodies are against ACHRA. In some embodiments, the autoantibodies are antibodies against ACHRA. In some embodiments, the autoantibodies are against ACHRG. In some embodiments, the autoantibodies are antibodies against ACHRG. In some embodiments, autoantibodies are pathologic autoantibodies. In some embodiments, the autoantibodies are disease causing autoantibodies.

[0274] In some embodiments, contacting is incubating. In some embodiments, contacting is under conditions sufficient for binding of antibodies to the protein complex. In someembodiments, 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.

[0275] 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%.

[0276] 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 suchmethod may be used. Examples of such methods 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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."

[0281] 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.

[0282] 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.

[0283] 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

[0284] 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:

[0285] 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.

[0286] 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:

[0287] 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.

[0288] 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 ofalpha / 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:

[0289] 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.

[0290] Figures 3A-3E show some embodiments of the invention in which only two chains are combined. In Figures 3A-3B, protein complex 201 comprises 2 polypeptide chains which specifically are two heavy chains. Heavy chains 215 and 216 can optionally include a CH2212, 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. Chain 215 contains the fragment, analog or derivative of ACHRA 230 and chain 216 contains the fragment, analog or derivative of ACHRG 231. Figure 3B 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.3B). In place of the variable region one chain has a fragment, analog or derivative of ACHRA230 while the other chain has a fragment, analog or derivative of ACHRG 231. It is advantageous to design the molecule such that predominantly heterodimers of 215 and 216 are formed and not homodimers. 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.

[0291] In Figure 3C alternative configurations comprising two heavy chains are shown. Instead of containing a single fragment, analog or derivative in place of the variable region, two tandem fragments, one that is an ACHRA fragment, analog or derivative 230 and one that is an ACHRG fragment, analog or derivative 231, 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. 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. Of course, the heavy chains need not be identical as various technologies may be used to favor heterodimerization over homodimerization.

[0292] Of course, if the two heavy chains are not identical the molecule can be designed with only one of the heavy chains containing two fragments, analogs or derivatives 230 and231 and with the other heavy chain containing no fragment 230 (Fig. 3D) or a single fragment (Fig. 3E). The same fragment could be repeated on both chains or a different one could be used.

[0293] In Figure 3F, 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 beintroduced 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 one chain has an ACHRA fragment, analog or derivative 230 and the other has an ACHRG fragment, analog or derivative 231.

[0294] 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. 4). Figure 4 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 one chain has a fragment, analog or derivative of ACHRA 330 and the other has a fragment, analog or derivative of ACHRG 331. It will be understood by a skilled artisan that either the ACHRA fragment, analog or derivative or the ACHRG fragment, analog or derivative could be on the light chain or either or both of the heavy chains.

[0295] 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 5A-5C show a generic protein complex 501. In Figure 5A 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 can optionally further comprise a third dimerization domain (DD3) 514 which can dimerize specifically with a fourth dimerization domain (DD4) 524 of second chain 516 or alternatively with a DD4 524 on another chain. Chain 516 can further optionally comprise DD4 524. Each of the chains also comprises a fragment, analog or derivative. One chain will contain an ACHRA fragment, analog or derivative 530 and the other will contain an ACHRG fragment, analog or derivative 531.

[0296] Figure 5B shows an alternative embodiment to Figure 5A in which each distinct domain is separated by a linker 590. 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 5B also could employ linkers between any orall of the various domains / fragments. Figure 5C shows embodiments similar to 5A and 5B but in which each chain contains both an ACHRA fragment, analog or derivative 530 and an ACHRG fragment, analog or derivative 531.

[0297] In Figure 6A-6C single chain embodiments of the invention are depicted. Figure 6A shows a single chain fusion protein 601 containing a fragment, analog or derivative of ACHRA 630 and a fragment, analog or derivative of ACHRG 631. It will of course be understood that while fragment, analog or derivative 630 is depicted as positioned N- terminal to fragment, analog or derivative 631 it could also be the reverse with fragment, analog or derivative 631 N-terminal to fragment, analog or derivative 630. As shown in Figure 6B, 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 6C depicts embodiments, of 6A and 6B with linkers separating the fragments, analogs or derivatives and also the Fc region. 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 without the CHI domain are also envisioned.

[0298] Although not depicted in Figures 3-6, 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 3-6 may include such mutations in one or many of the fragments.Example 4: Mutations that decrease subunit aggregation

[0299] 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. Bi-interaction surfaces on the subunits were disrupted and hydrophobicsurfaces were made more hydrophilic. The mutants were designed to reduce aggregation while not significantly disrupting autoantibody epitopes and antibody binding.

[0300] Tables 2-3 provide the mutations made in the alpha, and gamma subunits. The molecules were expressed by transient expression in CHO cells and the proteins were purified by affinity chromatography on a nickel-column. 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.

[0301] Table 2: Alpha subunit mutants

[0302] Table 3: Gamma subunit mutants

[0303] 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.

[0304] 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. The mutants were generally as effective as the WT ACHRA or WT ACHRG fragments.Example 5: Ability of the ECDs to bind B cells

[0305] 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. A- 192 hybridoma binds alpha subunit, b-73 hybridoma binds beta subunit, g-66 binds gamma subunit and 23-F3-H1 binds delta subunit .204-4 hybridoma targeting Coxsackie virus B4(CB4) was used as a negative control hybridoma. 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 7, each subunit molecule specific bound to thehybridoma cells expressing antibodies against that subunit. Non-specific binding to other hybridomas was similar to the binding to the negative control. This indicates that not only can these molecules 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.Example 6: Molecules comprising combinations of ECDs

[0306] 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 3), 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.

[0307] Table 4: Combination molecules

[0308] 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 toproduce close to an additive effect, indicating that all the subunits present are still binding their target autoantibodies when included in the molecule.

[0309] 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-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 to anti-alpha hybridoma cells and not a negative control hybridoma (Fig. 8A). Similarly, both molecules bound to anti-gamma hybridomas (Fig. 8B). 5 different negative control hybridoma lines were tested and both molecules showed very little non-specific binding. 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.Example 7: Superior molecule comprising ACHRA ECD and ACHRG ECD

[0310] Next, superior alpha and gamma mutant subunit polypeptides were generated. The human ACHRA ECD was mutated with the already tested W 149R mutation and additionally a mutation of phenylalanine 137 to tyrosine (ACHRA F137Y, W149R, SEQ ID NO: 73, CRD-991). The human ACHRG ECD was mutated with the already tested Y 105E mutation and the Y117R mutation and additionally a mutation of phenylalanine 137 to tyrosine (ACHRG Y105E, Y117R, F137Y, SEQ ID NO: 74, CRD-997).

[0311] The ability of CRD-991 to bind B cell hybridomas expression anti-alpha MG autoantibodies was tested. CRD-642 (bearing the W149R mutation) was used as a control. The a- 192 hybridoma was incubated in the presence of increasing concentrations of the CRD molecules (Fig. 9A). Following incubation of the hybridoma cells with the molecules, cells were washed (DPBS+1% FBS) three times and incubated with PE-conjugated anti-human polyclonal Antibody (pAb). Incubation with PE-conjugated anti-human pAb only, was used as a negative control. Cells were washed three times with buffer and were subsequently analyzed by flow cytometry (CytoFlex, Beckman Coulter). The hybridoma was also stained with PE-aRAT BCR. Surprisingly, CRD-991 displayed superior binding to the B cells as compared to CRD-642, even though it included an additional mutation.

[0312] Binding of CRD-997 to anti-gamma B cell hybridomas was tested in the same way. CRD-510 (bearing the Y 105E / Y 117R mutations) was used as a control. The g-63 hybridomawas incubated in the presence of increasing concentrations of the CRD molecules (Fig. 9B). Surprisingly, CRD-997 displayed superior binding to the B cells as compared to CRD-510, even though it included an additional mutation.

[0313] The ACHRA F137Y / W149R fragment was linked to the ACHRG Y105E / Y117R / F137Y fragment with a (GGGS)7GS, resulting in SEQ ID NO: 8. SEQ ID NO: 8 was then linked to an Fc domain with a GGS linker to produce SEQ ID NO: 9. This molecule was termed CRD-981.

[0314] The ability of CRD-981 to bind various B cell hybridomas expressing MG autoantibodies was tested. CRD-509 (alpha and gamma ECD, with gamma bearing the Y105E / Y117R mutations) was used as a control. Four hybridomas expressing BCR against AChR alpha (a-18-C5-F6, a-122-Hl-G6, a-198 and a-192) were incubated in the presence of 5nM CRD molecules (Fig. 9C). The background staining level of each tested hybridoma was determined as the MFI value of the hybridoma cell line incubated in the presence of secondary antibody only. Fold change from background levels was calculated as the MFI value of CRD molecule on specific hybridoma divided by the MFI value of the same hybridoma with secondary antibody only. CRD-981 was found to be at least comparable to CRD-509 and for several of the hybridomas was actually a superior binder.

[0315] Next, the test was repeated with four hybridomas expressing BCR against AChR gamma (g-63, g-50-Hl-E2, g-22-F5-Cl and g-66) (Fig. 9D). Binding to hybridomas by CRD-981 was at least comparable to binding by CRD-509 and for two of the hybridomas was actually superior.

[0316] Depletion of MG antibodies from human sera was also tested with the new mutant alpha and new mutant gamma molecules, plus the combination molecule. MG patient serum samples with predominantly anti-ACHRA IgGs (n=ll), were incubated with increasing concentrations (0, 0.02 nM, 0.91 nM, 4.6 nM, 22.8 nM, 113.8 nM and 569nM) of CRD-991 coated beads for 1 hour with shaking (400 RPM). Following incubation, the CRD coated beads were separated from the depleted sera by centrifugation (3200 g) and the sera were assessed for their anti- ACHR titer using EUROIMMUN ELISA. The depletion rate for each serum was calculated as 1 -(depleted serum / non-depleted serum). CRD-991 successfully depleted alpha autoreactive antibodies with a calculated Kd of 10.19 nM (Fig. 10A).

[0317] MG patient serum samples with predominantly anti-ACHRG IgGs (n=l l), were incubated with increasing concentrations (0, 0.02 nM, 0.91 nM, 4.6 nM, 22.8 nM, 113.8 nM and 569nM) of CRD-997 coated beads and analyzed as before. CRD-997 successfully depleted gamma autoreactive antibodies with a calculated Kd of 14.05 nM (Fig. 10B).

[0318] MG patient serum samples with predominantly anti-ACHRA IgGs (n=8) or predominantly anti-ACHRG IgGs (n=8) were incubated with increasing concentrations of CRD-509 or CRD-981 coated beads as before. The results of the depletion are provided in Figure 10C. Similar to the individual subunits, CRD-981 was significantly better at sequestering anti-ACHRA autoantibodies than CRD-509 (Kd: 12.8 vs 165.2, Kd paired t- testpval = 0.0034), and also at sequestering anti-ACHRG autoantibodies (Kd: 15.7 vs. 29.7). Thus, CRD-981 is a surprisingly superior molecule for treating subjects with predominantly anti-ACHRA autoantibodies, predominantly anti-ACHRG autoantibodies or subjects with both.

[0319] Next the ability to bind primary B cells was tested. Female C57BL / 6 mice, 8 weeks of age, 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. At 7 weeks post immunization, the mice were immunized for the second time (boost) subcutaneously (S.C.) with a mix of 50pg AChR-alpha + 50pg AChR-gamma ECDs in complete Freund’s adjuvant (CFA) per mouse. At 5 weeks after the boosting, mice were sacrificed and cells were isolated from the spleen and bone-marrow.

[0320] Samples were treated with red blood cell lysis buffer, washed and blocked. Anti- CD138 BV605 and anti-TACI BV421 antibodies were used to identify plasmablast and plasma cell subpopulations. Cells were also contacted with CRD-991 and CRD-997 conjugated to PE (biotinylated molecules were conjugated to streptavidin PE tetramers) for 1 hour at 4°C. The percentage of CRD positive cells (the two CRD molecules could not be distinguished) in each subpopulation was determined. A population of CD138 and TACI double positive B cells that were recognized by the CRD molecules were clearly present in both the spleen (Fig. 11A) and the bone marrow (Fig. 11B) in the mice that were immunized. A control irrelevant CRD did not recognize a population in the immunized mice. In naive mice the population recognized by CRD-991 / 997 was absent as staining with thesemolecules was comparable to the control CRD. This indicates that primary B cells can be recognized by the new mutant molecules.

[0321] SEQ ID NO: 8 was also linked to CH2CH3 bearing the SELF (S267E+L328F) double mutation to produce SEQ ID NO: 75. This molecule was termed CRD-999. The SELF mutation increases binding of IgGs to FcyRIIB and does the same to the CRD molecules of the invention. This enhances B cell targeting. As the purpose of the various mutations inserted into the alpha and gamma subunits was to decrease aggregation, the amount of monomeric CRD-999 produced was measured. Expressing unmutated alpha domain fused to the FC resulted in production of only about 2% monomers. In contrast expression of CRD- 999 produced about 28% monomers, indicating that the alterations introduced are highly effective at decreasing aggregation.

[0322] Further, the introduction of the SELF mutation did not impair serum autoantibody depletion rates; indeed, rather unexpectedly CRD-999 was slightly superior to CRD-981 in its ability to deplete autoantibodies from patient sera (n=l 1) (Fig. 12).Example 8: CRDs of the invention do not induce an inflammatory response

[0323] Next, it was tested if the CRD molecules of the invention induce an inflammatory response, e.g., inflammatory cytokine secretion from peripheral white blood cells. This would indicate a general activation of the immune system. Secretion of TNFA and IL-6 from peripheral white blood cells (PBMCs) was measured after 24-hour exposure to CRD-509 or its’ controls (no molecule as a negative control and Alemtuzumab as a positive control). CRD-509 produced no increase in secretion of IL-6 (Fig. 13A) and did not produce a significant increase in TNFA (Fig. 13B). For CRD-999, non-specific normal human IgGl was used as a negative control and alemtuzumab was again used as a positive control. Secretion of TNFA, IFNg, IL-2, II- lb and IL-6 from PBMCs was measured after 24-hour exposure to CRD-999. CRD-999 induced a low pro-inflammatory response that was similar to non-specific IgGl treatment (Fig. 13C). Indeed, CRD-999 consistently and surprisingly for all tested cytokines, produced slightly lower levels than the IgG control. Similar results are observed for CRD-998.Example 9: Effector moiety compositions

[0324] 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 dimerizationand effector domains parallel molecules can be generated. Figures 14A-14D show a generic protein complex 701. In Figure 14A 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 optionally comprises a third dimerization domain (DD3) 714 which can dimerize specifically with an optional fourth dimerization domain (DD4) 724 of second chain 716. The first chain also comprises a fragment / analog / derivative of ACHRA 730, while the second chain also comprises a fragment / analog / derivative of ACHRG 731. Chains 715 and 716 can also have pairs of fragments / analogs / derivatives such as an N-terminal fragment / analog / derivative of ACHRA 730 followed a fragment / analog / derivative of ACHRG 731 and then the dimerization domains 714 / 714 and 763 / 773 and finally the EF domain 711. Figure 14B shows the inclusion of optional linkers between the various domains of the molecules from 14A. It will be understood by a skilled artisan that only some of these linkers may be present or that they all may be, and that all linkers may be the same or they may be different.

[0325] Figure 14C shows an alternative embodiment, in which a cytotoxic molecule (CM, such as a toxin, radiolabeled molecule or poison) 791 is connected by a chemical bond or linker 795 to chain 715. Figure 14D shows a cytotoxic moiety conjugated to molecules with linkers. It will be understood that embodiments in which CM 791 is connected to chain 716 or to both chains are also contemplated. Further, though CM 791 is depicted as being conjugated to DD1, it could also be conjugated to DD2, DD3, DD4 or even one of the fragments / analogs / derivatives. Further, while CM 791 is depicted as being conjugated to the C-terminus of the chain CM 791 can in fact be conjugated anywhere within molecule 701 by any conjugation method known in the art. It will be understood by a skilled artisan that when a cytotoxic molecule is employed the additional inclusion of an EF is not essential, although an EF can be included as well. Further, more than one CM 791 can be employed. 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 795 is depicted, but it will be understood that direct conjugation of CM 791 is also envisioned.

[0326] Figure 14E depicts embodiments in which two heavy chains each comprising both an ACHRA fragment / analog / derivative and an ACHRG fragment / analog / derivative are linked to a cytotoxic moiety. It will be understood that any of the agents 201, 301, 501 and 601 shown in Figure 3-6 can be conjugated to CM 791 either directly or through a linker 795 to produce agent 701.

[0327] As shown in Figure 14D, CM 791 can be linked to any of CHI 714, hinge 713, CH2 712 and CH3 711. Similarly, attachment of CM 791 can be to both chains or only to a single chain. The attachment 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. It will be understood that CM attachment as shown in Figure 14D can be done as well to all the molecules of Figures 3- 4. 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.

[0328] Figure 14F shows embodiments of a conjugate of the invention in which the cytotoxic molecule 791 is conjugated directly to a fragment / analog / derivative of ACHRA 730 or ACHRG 731, optionally by a linker 795. It will be understood that fragments comprising mutations that increase solubility and / or decrease aggregation can be used.

[0329] Figures 14G-14I show embodiments of the single chain agents of the invention shown in Figures 6A-6C 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, to a domain within the Fc, to one of the fragments / analogs / derivatives or any part of the single chain, can be linked via linker 795 (Fig. 14J-14L).

[0330] CRD-981 and CRD-999 were both conjugated to the tubulin inhibitor monomethyl auristatin E (MMAE) by a cleavable linker via a site-specific conjugation. The two drug conjugated molecules were then incubated with anti-alpha and anti-gamma B cell hybridoma lines for 72 hours and then live cells were counted by flow cytometry. %Cytotoxicity was calculated by the following formula: %Cytotoxicity = 100*(l-(Number of cells in treated well (x nM)) / (Number of cells in untreated well (0 nM))). % Specific cytotoxicity was calculated as the delta between CRD-MMAE % cytotoxicity minus Control non AChRrelated % cytotoxicity in each tested concentration. CRD-981-MMAE (Fig. 15A) and CRD- 999-MMAE (Fig. 15B) both killed significant amounts of both alpha and gamma hybridomas with cell killing reaching essentially 100 percent for some of the hybridomas.Example 10: Enhanced B cell binding via FcgRIIb

[0331] The SELF double mutation is reported to increase IgG binding to FcgRIIb (CD32B). This receptor is highly expressed on B cells and so it was hypothesized that incorporation of this double mutation in the CH2CH3 backbone of the CRD molecules would enhance B cell binding as well. To test this PBMCs derived from healthy human donor were incubated in the presence of 0-250 nM of CRD-999 or a control anti-CD79b molecule for 1 hour at 4° C. Cells were than washed and stained with an antibody cocktail containing the following antibodies - anti-human CD3 BV510, anti-human CD56 BV786, anti-human CD14 BV786, anti-human CD20 AF700, anti-human CD79b PE and anti-human CD32b APC. B cells were identified as based on staining CD3 negative, CD56 negative, CD 14 negative and CD20 positive. MFI (median) for the anti-CD32B and anti-CD79b was measured in the gated B cells for all tested concentrations. CD79b staining was unchanged regardless of CRD-999 concentration, but CD32B staining decreased as the concentration of CRD-999 went up (Fig. 16A). This indicates that CRD-999 is blocking CD32B detection by the antibody (by binding the receptor), resulting in a lower APC MFI in a dose dependent manner. Anti-CD79b MMAE (commercial Polivy) was tested as a negative control that targets B cells, but not via CD32b. It is thus confirmed that the SELF mutation in the molecules of the invention increases B cell binding.

[0332] The ability of CRD-981 and CRD-999 to bind CD32B was also tested by surface plasmon resonance (SPR). Test samples were injected into a flow cell to which a CaptureSelect reagent had been coupled via streptavidin. Once the test samples were coupled to the surface, binding to CD32B was measured in the mobile phase. Though both molecules bound to CD32B, CRD-999 bound with a significantly lower Kd than CRD-981 (Fig. 16B). Taken together, this data all indicates that the SELF mutation is useful in enhancing the binding of the molecules of the invention to B cells.Example 11: Unconjugated CRD-981 reduces MG score and mortality in a rat model

[0333] The EAMG (Experimental Autoimmune Myasthenia Gravis) model involves inducing myasthenia gravis symptoms by triggering the autoimmune cellular processes,leading to the production of autoantibodies. The passive transfer MG (PTMG) model, on the other hand, allows for studying the consistent and reproducible effects of these autoantibodies by bypassing the variability associated with the autoimmune cellular processes. The use of the PTMG model for pre-clinical evaluation of a therapeutic is justified when the effect includes inhibiting the autoantibody binding or preserving the function and structure of the neuromuscular junction (NMJ) during antibody attack. The PTMG rat model is induced by intraperitoneal (I.P.) injection of an anti-Alpha-AChR antibody (Mab35) which induces a disease state within 12 hours to 2-3 days. The antibodies cause a NMJ disruption in a relatively short 24-72-hour window. Seven hours after Mab35 antibody administration, CRD-981 was intravenously administered at different doses (7.5, 3 and 1.5 mg / kg).

[0334] Female Lewis rats, 5-6 weeks of age (-100 gr), were intraperitoneally injected with anti-Alpha-AChR antibody (Mab35) and assessed for changes in clinical score. Animals were scored according to their disease symptoms: 0, no disease; 1, reduced grip strength in front paws; 2, loss of grip in front paws; 3, loss of grip and hind limb weakness and wasting; 4, Moribund. Half scores were given for intermediate symptoms. Animals were also weighed at frequent intervals. Rats were sacrificed when weight loss exceeded 20% of the original body weight, or when clinical score reached 3.5 (loss of grip and hind limb paralysis and respiratory distress). After the onset of clinical symptoms, rats were assessed at frequent intervals. Seven hours after Mab35 antibody administration, CRD-981 was intravenously administered at the selected doses. An irrelevant Antigen-Fc-Fusion or vehicle-only were intravenously administered to the control groups.

[0335] CRD-981 showed a dose-dependent, statistically significant reduction of both MG score (Fig. 17A) and mortality rate (Fig. 17B) as was compared with control treated animals. The peak score decreased significantly after 32 hours from 3.7 to 1.1 (p< 10A-l 1) with the administration of 7.5 mg / kg of CRD-981. Subsequent to 55 hours, mortality decreased from 85% to 0% at 7.5 mg / kg of CRD-981. Doses of 3 and 1.5 mg / kg of CRD-981 reduced mortality to 10%. Thus, the unconjugated molecule without the SELF mutation is already an effective therapeutic molecule based on its ability to sequester autoantibodies. However, to produce a more permanent treatment by killing autoreactive B cells, drug conjugation and SELF mutations are needed.I l l

[0336] In order to test the ability of the molecules to target autoreactive B cells themselves and not merely sequester autoantibodies, the various molecules of the invention are incubated with various hybridomas some of which produce anti-alpha / gamma antibodies and some which produce antibodies to other irrelevant proteins. The incubation is for 30 min at 4 degrees Celsius. Following incubation cells are washed twice with FACS buffer (DPBS with 1% FBS) and then incubated with an anti-human-IgG Fc region fluorophore-conjugated antibody. Following this second incubation, cells are again washed twice and analyzed on a flow cytometry (CytoFlex by Beckman Coulter). Cells incubated with secondary antibody alone are used as a negative control. Mean fluorescent intensity (MFI) fold change from background values is computed. The molecules of the invention strongly bind to hybridomas expressing BCR against ACHRA / ACHRG, whereas no binding to the control hybridomas is observed. When a molecule with an irrelevant extracellular domain is used, no binding to the hybridoma expressing BCR against ACHRA / ACHRG is observed. This data indicates that the molecules of the invention can be used to target B cells and provide a lasting cure to MG and not just transiently reduce autoantibody levels.Example 12: In vivo MG treatment

[0337] CRD-981 and CRD-999 are also tested for their ability to kill autoreactive B cell and treat MG. CRD-981-MMAE and CRD-999-MMAE are tested. A pre-immunization treatment to eliminate autoreactive B cells is performed to test the in vivo efficacy. 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 is drawn from naive 7-8-week-old C57B16 inbred female mice and an ELISA is performed to measure anti-ACHRA and anti-ACHRG antibody titers. All mice are found to be positive for antibodies although there is a great deal of variability. The presence of these autoreactive antibodies indicates that autoreactive B cells are present even before immunization with ACHR fragments.

[0338] To test the ability of the molecules of the invention to kill these autoreactive B cells, C57B16 female mice at 6 weeks of age are treated intravenously with 0.5 mg / kg CRD-981- MMAE or CRD-999-MMAE. Two negative control groups are also tested: animals that received PBS and animals that were intravenously administered an irrelevant Ig-like molecules conjugated to MMAE. 18 days post treatment animals were immunized subcutaneously (S.C.) with 50 pg ACHRA and 50 pg ACHRG in complete Freund’s adjuvant (CFA) + 0.2 mg M. Tuberculosis per mouse. Serum samples are isolated during the experiment and the anti-ACHR antibodies’ titer evaluated. 14 days post immunization titers are significantly lower in the MMAE conjugate pre-treated groups as compared to irrelevant- CRD-MMAE or PBS groups. This indicates that the MMAE conjugate is able to kill the autoreactive B cells such that immunization does not produce a significant boost in autoreactive antibody production. CRD-999-MMAE is found to be a superior killer as compared to CRD-981-MMAE, likely due to its increased binding to B cells via CD32B.

[0339] Other effector moieties (alpha-amanitin, PNU, Dxd, MMAE, MMAF and mertansine) are all found to effectively treat MG, kill B cells and reduce anti- ACHRA / ACHRG titer levels in vivo. All tested effector moieties are found superior to Fc. Similarly, incorporation of the SELF mutation is found to produce a superior effect for all tested conjugates.

[0340] Additional molecules comprising a F137S or a F137A mutation in the alpha ECD, the gamma ECD and both are also tested. The F137S and F137A mutations are found to be comparable to the F137Y mutation at reducing aggregation and retaining functionality.

[0341] 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, analog or derivative of human acetylcholine receptor subunit alpha (ACHRA), and a fragment, analog or derivative of human acetylcholine receptor subunit gamma (ACHRG), wherein said ACHRA fragment, analog or derivative comprises mutation of phenylalanine 137 to tyrosine (F137Y), said ACHRG fragment, analog or derivative comprises a F137Y mutation or both and wherein said phenylalanine 137 within ACHRA is with respect to SEQ ID NO: 1 and said phenylalanine 137 within ACHRG is with respect to SEQ ID NO: 2.

2. The composition of claim 1, wherein said fragment, analog or derivative of ACHRA comprises said F137Y mutation and said fragment, analog or derivative of ACHRG comprises said F137Y mutation.

3. The composition of claim 1 or 2, wherein said fragment, analog or derivative of ACHRA further comprises a W149R mutation.

4. The composition of any one of claims 1 to 3, wherein said fragment, analog or derivative of ACHRG comprises a Y 105E mutation, a Y117R mutation or both.

5. The composition of claim 4, wherein said fragment, analog or derivative of ACHRG comprises a Y 105E mutation and a Y117R mutation.

6. The composition of any one of claims 1 to 5, wherein said fragment, analog or derivative of ACHRA comprises or consists of SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTT NVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAI VKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPYDEQNCSMKLGTRTYDGS VVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFV MQRLP (SEQ ID NO: 3).

7. The composition of any one of claims 1 to 6, wherein said fragment, analog or derivative of ACHRG comprises or consists of RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALT TNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVF EVALECNVLVSPDGCIRWLPPAIFRSACSISVTYFPYDWQNCSLIFQSQTYST NEIDLQLSQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAG HQKVVFYLLIQRKP (SEQ ID NO: 4).

8. The composition of any one of claims 1 to 7, comprising a protein complex comprising a. a first polypeptide chain comprising said fragment, analog or derivative of ACHRA and a first dimerization domain; and b. a second polypeptide chain comprising said fragment, analog or derivative of ACHRG and second dimerization domain; wherein said first and second dimerization domains are configured to dimerize with each other.

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

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

11. 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.

12. The composition of any one of claims 8 to 11, wherein said fragment, analog or derivative and said dimerization domain of said first, second or both polypeptide chains are separated by a linker.

13. The composition of any one of claims 8 to 12, wherein said first polypeptide chain, said second polypeptide chain or both further comprise an Fc region of a human antibody heavy chain.

14. The composition of claim 13, wherein said Fc region is capable of inducing cytotoxicity against a cell binding said protein complex.

15. The composition of any one of claims 9 to 13, wherein said Fc is from an IgG2 or IgG4 or comprises at least one mutation that reduces effector function.

16. The composition of any one of claims 13 to 15, wherein said Fc region of said first, second or both polypeptide chains is separated from said fragment, analog or derivative or said dimerization domain by a linker.

17. The composition of any one of claims 8 to 15, wherein said dimerization domain of said first, second or both polypeptide chains is C-terminal to said fragment.

18. The composition of any one of claims 1 to 17, devoid of an antibody variable domain.

19. The composition of any one of claims 1 to 18, comprising a single polypeptide chain comprising said fragment, analog or derivative of ACHRA and said fragment, analog or derivative of ACHRG.

20. The composition of claim 19, wherein said fragment, analog or derivative of ACHRA is N-terminal to said fragment, analog or derivative of ACHRG.

21. The composition of claim 19 or 20, wherein said fragment, analog or derivative of ACHRA and said fragment, analog or derivative of ACHRG are separated by an amino acid linker.

22. The composition of claim 21, wherein said linker is a flexible GS linker or wherein said linker is a rigid linker.

23. The composition of claim 22, wherein said linker is a flexible linker comprising or consisting of (GGGS, SEQ ID NO: 5)nwherein n is an integer from 1 to 10.

24. The composition of claim 23, wherein said linker comprises or consists of (GGGS)7GS (SEQ ID NO: 6).

25. The composition of any one of claims 19 to 24, wherein said single polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 8 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

26. The composition of any one of claims 19 to 25, wherein said polypeptide chain further comprises an Fc region of a human antibody heavy chain.

27. The composition of claim 26, wherein said Fc region is capable of inducing cytotoxicity against a cell binding said protein complex.

28. The composition of claims 26 or 27, wherein said Fc region comprises at least one mutation that increases binding to FcgRIIb (FCGR2B / CD32B).

29. The composition of claim 28, wherein said Fc region is an Fc region comprising an S267E and L328F double mutation.

30. The composition of claim 29, wherein said FC region comprises or consists of EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 87).

31. The composition of any one of claims 26 to 30, wherein said Fc domain is linked to said fragment, analog or derivative of ACHRA or ACHRG by an amino acid linker.

32. The composition of claim 31, wherein said amino acid linker comprises or consists of GGS.

33. The composition of any one of claims 19 to 32, wherein said composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, and a tyrosine at position 378.

34. The composition of any one of claims 28 to 32, wherein composition comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 75 or a derivative thereof comprising at least 85% identity thereto and comprising a tyrosine at position 137, and a tyrosine at position 378.

35. The composition of any one of claims 1 to 34, wherein a polypeptide chain further comprising an N-terminal signal peptide sequence.

36. The composition of claim 35, wherein said signal peptide sequence consists of MGWSCIILFLVATATGVHS (SEQ ID NO: 10).

37. The composition of any one of claims 1 to 36, further comprising an effector moiety that is not an Fc domain.

38. The composition of claim 37, wherein said effector moiety is selected from an amatoxin / amanitin, an anthracycline, an anthramycin-based dimer, a calicheamicin, camptothecin or an analog thereof, a duocarmycin, triptolide and a tubulin inhibitor.

39. The composition of claim 37 or 38, 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.

40. The composition of any one of claims 37 to 39, wherein said effector moiety is conjugated to an Fc domain of said composition.

41. A polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a derivative thereof comprising at least 80% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

42. The polypeptide of claim 41, comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or a derivative thereof comprising at least 80% identity thereto and comprising a tyrosine at position 137, a tyrosine at position 378 or both.

43. The polypeptide of claim 41 or 42, wherein a derivative thereof comprises an arginine at position 149.

44. The polypeptide of any one of claims 41 to 43, wherein a derivative thereof comprises a glutamic acid at position 346, an arginine at position 358 or both.

45. The polypeptide of any one of claims 39 to 42, wherein a derivative thereof comprises a tyrosine at position 137, an arginine at position 149, a glutamic acid at position 346, an arginine at position 358 and a tyrosine at position 378.

46. The polypeptide of any one of claims 41 to 44, further comprising an N-terminal signal peptide sequence.

47. The polypeptide of claim 45, wherein said signal peptide sequence consists of SEQ ID NO: 10.

48. The polypeptide of any one of claims 41 to 46, further comprising an effector moiety.

49. The polypeptide of claim 48, wherein said effector moiety is not an Fc domain.

50. The polypeptide of claim 48, wherein said effector moiety is an Fc domain comprising at least one mutation that increases antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).

51. The polypeptide of any one of claims 48 to 50, wherein said effector moiety is capable of inducing death in a cell binding said fragment.

52. The polypeptide of claim 51, 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.

53. The polypeptide of claim 46 or 47, wherein said effector moiety is selected from: alpha-amanitin, PNU- 159682, tesirine, deruxtecan (Dxd), mertansine, MMAE, MMAF and a combination thereof.

54. The polypeptide of any one of claims 50 to 53, wherein said effector moiety is an Fc domain comprising SEQ ID NO: 31 or SEQ ID NO: 49 comprising a plurality of mutations selected from: L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E within said SEQ ID NO: 31 or SEQ ID NO: 49.

55. The polypeptide of any one of claims 48 to 54, wherein said effector moiety is conjugated to an Fc domain of said polypeptide.

56. A pharmaceutical composition comprising a composition of any one of claims 1 to 40 or a polypeptide of any one of claims 41 to 55 and a pharmaceutically acceptable carrier, excipient or adjuvant.

57. The pharmaceutical composition of claim 56, wherein said pharmaceutical composition is formulated for systemic administration to a subject, for administration to a human, for parenteral administration or any combination thereof.

58. 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 40, a polypeptide of any one of claims 41 to 55 or a pharmaceutical composition of claim 56 or 57, thereby treating myasthenia gravis.

59. The method of claim 58, further comprising reducing in said subject the levels of circulating antibodies against ACHRA, ACHRG or both prior to said administering.

60. The method of claim 58 or 59, wherein said treating comprises killing B cells producing autoantibodies against ACHRA, B cells producing autoantibodies against ACHRG or both.

61. 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 18 and 33 to 40 and a second nucleic acid molecule encodes said second polypeptide chain of a composition of any one of claims 8 to 18 and 33 to 40 or said nucleic acid molecule encodes a single polypeptide of chain of a composition of any one of claims 19 to 40 or a polypeptide of any one of claims 41 to 55.

62. A method of producing a composition of any one of claims 1 to 40 or the polypeptide of any one of claims 41 to 55, the method comprising expressing the nucleic acid system of claim 61 in a cell, wherein said nucleic acid system is configured to producesaid encoded polypeptide in said cell, thereby producing a composition of any one of claims 1 to 40 or the polypeptide of any one of claims 41 to 55.

63. A method of determining suitability of a subject in need thereof to be treated by a method of any one of claims 58 to 60, the method comprising receiving a sample from the subject, contacting said sample with a composition of any one of claims 1 to 40 or a polypeptide of any one of claims 41 to 55 and determining binding of autoantibodies against ACHRA, antibodies against ACHRG or both within said sample to said composition or said polypeptide, wherein binding of autoantibodies to said composition or polypeptide indicates said subject is suitable to be treated by a method of any one of claims 58 to 60, thereby determining suitability of the subject to be treated.

Images