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WO2024231931A1 - Protéines de fusion de type ig pour le traitement de la thrombocytopénie immunitaire - Google Patents

Protéines de fusion de type ig pour le traitement de la thrombocytopénie immunitaire Download PDF

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WO2024231931A1
WO2024231931A1 PCT/IL2024/050448 IL2024050448W WO2024231931A1 WO 2024231931 A1 WO2024231931 A1 WO 2024231931A1 IL 2024050448 W IL2024050448 W IL 2024050448W WO 2024231931 A1 WO2024231931 A1 WO 2024231931A1
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domain
fragment
dimerization
mutation
composition
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Kfir Oved
Galit Denkberg
Sharon REEF
Yelena PINZUR
Inbar ARMAN ZELMAN
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Canopy Immuno Therapeutics Ltd
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Canopy Immuno Therapeutics Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1777—Integrin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention is in the field of fusion protein generation and immune thrombocytopenia (ITP) treatment.
  • Immune thrombocytopenia is an acquired thrombocytopenia caused by autoantibodies against platelet antigens. It is one of the more common causes of thrombocytopenia in otherwise asymptomatic adults. ITP has previously been called idiopathic thrombocytopenic purpura, immune thrombocytopenic purpura, or autoimmune thrombocytopenic purpura (AITP). These terms have been replaced by "immune thrombocytopenia" to reflect the known autoantibody mechanism and the absence of purpura in some patients.
  • ITP Intra-inflammatory thrombocytopenia
  • Primary ITP is acquired immune thrombocytopenia due to autoimmune mechanisms leading to platelet destruction and platelet underproduction that is not triggered by an apparent associated condition. Secondary ITP is associated with another condition (i.e., lupus associated ITP).
  • Drug-induced immune thrombocytopenia (DITP) is thrombocytopenia due to drug-dependent platelet antibodies that cause platelet destruction. This syndrome should be distinguished from drug-induced bone marrow suppression, a non-immune phenomenon.
  • the time elapsed since diagnosis determines whether ITP is referred to as newly diagnosed, persistent, or chronic where newly diagnosed is up to three months since diagnosis, persistent is three to 12 months since diagnosis and chronic is >12 months since diagnosis.
  • ITP severity is defined based on the bleeding complications it elicits and the lab status of the patient. Severe ITP refers to ITP with bleeding symptoms sufficient to require treatment which typically occurs when platelet counts are ⁇ 20,000/uL.
  • ITP insulin-derived neuropeptide
  • IgG platelet membrane glycoproteins
  • IGB3 platelet membrane glycoproteins
  • ITP is associated with a preceding, mostly viral infection. Where antibodies against viral antigens may crossreact with normal platelet antigens (molecular mimicry).
  • alterations in immune homeostasis might induce loss of peripheral tolerance and promote the development of self- reactive antibodies. This often occurs in the setting of other autoimmune conditions including the antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE), Evans syndrome, hematopoietic cell transplantation, chronic lymphocytic leukemia (CLL) and other disorders.
  • APS antiphospholipid syndrome
  • SLE systemic lupus erythematosus
  • Evans syndrome hematopoietic cell transplantation
  • CLL chronic lymphocytic leukemia
  • Antibody production in ITP appears to be driven by CD4-positive helper T cells. Splenic macrophages appear to be the major antigen-presenting cells. Despite this likely mechanism, antiplatelet antibodies are not demonstrable in close to 50 percent of patients with ITP (i.e., sensitivity of antiplatelet antibodies is low).
  • the primary site of platelet clearance for most patients is the spleen, which removes opsonized (antibody coated) cells including platelets.
  • the prominent role of splenic clearance explains the effectiveness of splenectomy in most patients. However, clearance may occur in other tissues as well, such as the liver, bone marrow, and lymph nodes. This helps explain why ITP can persist or recur post- splenectomy .
  • ITP is a common acquired bleeding disorder.
  • a review of published reports determined an annual ITP incidence of approximately 1 to 6 per 100,000 adults. ITP is often a chronic disease in adults, thus, the prevalence exceeds significantly the incidence.
  • prevalence was ⁇ 8 per 100,000 in children and ⁇ 12 per 100,000 in adults.
  • the overall incidence was 2.9 per 100,000 person-years, with a peak in individuals >60 years, reaching 9 cases per 100,000 person-years in men >75 years of age.
  • Other studies have also documented the increasing incidence of ITP with increasing age. ITP is generally thought to be a condition that affects young women, with a female predominance in younger adults, while most studies show a similar incidence in males and females >60 years.
  • Some patients with ITP are asymptomatic, for those who do have symptoms, these are primarily related to thrombocytopenia and bleeding, but patients can also experience fatigue and a reduced quality of life. Bleeding due to thrombocytopenia may occur in up to two-thirds of patients. When present, bleeding typically occurs in the skin or mucous membranes, a pattern sometimes referred to as "platelet-type" bleeding. Although the onset of symptoms may be abrupt, it is more often insidious.
  • Petechiae - Petechiae are flat, red, discrete lesions that do not blanch under pressure; these often occur in dependent areas of the body (lower legs in ambulatory patients; sacral area in recumbent patients). Petechiae must be distinguished from vasculitic purpura.
  • Purpura - Purpura refers to a lesion caused by coalescence of petechiae. Purpura on the skin is sometimes referred to as "dry purpura.” Hemorrhagic blisters in mucous membranes, such as the oral mucosa, are sometimes called “wet purpura"; this finding may be a predictor of more severe bleeding.
  • Epistaxis - Minimal epistaxis such as only nose blowing, is common and may not be clinically important. Continuous epistaxis that requires intervention with nasal packing or cauterization may be predictive of greater risk for more serious bleeding.
  • [Oi l] Reported rates of bleeding are variable, depending on the population, bleeding definition, and the methods of reporting. In a population-based study that included 3771 patients with ITP, the risk of severe gastrointestinal or central nervous system bleeding at disease onset was ⁇ 1%. In a prospective registry that included 269 patients with ITP, 57% had bleeding occurrences, most of which were localized to the skin or mouth. Predictors of clinically important bleeding in individual studies include the degree of thrombocytopenia, previous minor bleeding, use NSAIDs, female sex, and chronic ITP.
  • ITP is defined by thrombocytopenia, and by consensus, the threshold for ITP is a platelet count ⁇ 100,000/uL.
  • the severity of thrombocytopenia in patients with ITP is variable; the greatest concern for bleeding is with platelet counts ⁇ 20,000/uL. Large platelets are often noted on the peripheral blood smear. However, the absence of large platelets cannot be used to exclude the diagnosis of ITP.
  • ITP is not characterized by abnormal platelet morphology. If present, abnormal platelet morphology should prompt consideration of a hereditary platelet disorder.
  • Fatigue is a common symptom among patients with ITP.
  • the causes of fatigue in ITP are not well understood.
  • thrombocytopenia in people with ITP is not necessarily protective against thrombosis.
  • Several studies have documented an increased risk of thrombosis in people with ITP compared with controls.
  • the pathogenesis of hypercoagulability in ITP is not well understood. It may relate to inflammation, antiphospholipid antibodies in some patients, or the effects of certain treatments such as splenectomy and thrombopoietin receptor agonists.
  • Other cell lines (white blood cells or red blood cells) are characteristically normal in ITP. Coagulation parameters are also typically normal.
  • ITP is a diagnosis of exclusion that is made in patients with isolated thrombocytopenia.
  • important components of the diagnostic evaluation include excluding other possible causes of thrombocytopenia and identifying conditions that may be responsible for secondary ITP.
  • the history should elicit recent infections, medications, and underlying conditions such as rheumatologic disorders or liver disease, which may be associated with thrombocytopenia.
  • questions about bleeding symptoms, bruising, and petechiae are informative.
  • the physical examination is focused on signs of bleeding, specifically on the skin and oral mucous membranes, which would suggest the need for more urgent evaluation and therapy; and the presence of lymphadenopathy or hepatosplenomegaly, which could suggest an underlying condition responsible for the thrombocytopenia.
  • Peripheral blood smear to confirm that thrombocytopenia is not artif actual due to platelet clumping and that there are no morphologic platelet abnormalities such as lack of platelet granules or uniformly large or small platelets, which could suggest a hereditary platelet disorder. While the presence of large platelets may be noted, there are no high- quality data to support the use of platelet size to confirm or exclude the diagnosis of ITP.
  • HIV and HCV testing is advised, because thrombocytopenia is a common presenting finding for these conditions, and treatment of the underlying infection might improve the platelet count.
  • Coagulation studies are not required in patients who have mild thrombocytopenia. However, there is a recommendation to measure prothrombin time (PT) and activated partial thromboplastin time (aPTT) in individuals with moderate or severe thrombocytopenia, those with concerns about clinically important bleeding, and/or those who have planned invasive procedures.
  • PT prothrombin time
  • aPTT activated partial thromboplastin time
  • Bone marrow examination may be indicated for patients with other unexplained cytopenias (anemia, leukopenia), dysplasia on the peripheral blood smear, other unexpected hematologic findings, or other causes of thrombocytopenia, when suspected.
  • Other testing may be indicated in patients with atypical clinical features. As an example, patients with bleeding out of proportion to the degree of thrombocytopenia may warrant evaluation for less common conditions such as type 2B von Willebrand disease, Bernard-Soulier syndrome, or other inherited or acquired platelet disorders. Genetic testing may be indicated in suspected hereditary thrombocytopenia. Details of this testing are discussed separately. Antiplatelet antibody testing suffers from low sensitivity and therefore does not effectively correlate with clinical outcomes.
  • Treatment The goal of current treatment is to treat or prevent significant bleeding, not to normalize the platelet count.
  • the risk of critical or severe bleeding is low; risk is greatest with prior bleeding, platelet count ⁇ 10,000/microL, and age >60 years.
  • Glucocorticoids typically, pulse dexamethasone
  • IVIG intravenous immune globulin
  • glucocorticoids alone rather than IVIG alone or glucocorticoids plus IVIG (Grade 2C) are indicated.
  • Some people may prefer IVIG for its faster action or side effect profile.
  • the threshold for treatment is individualized.
  • Pulse dexamethasone produces faster responses and fewer bleeding events.
  • Typical dosing is 40 mg orally or intravenously once daily for four days (no taper).
  • Pulse methylprednisolone is typically administered as 1 g intravenously once daily for three days (no taper).
  • Prednisone dosing is 1 mg/kg orally once daily for one to two weeks followed by a gradual taper (typically ⁇ 6 weeks).
  • IVIG - A typical dose is 1 g/kg daily for one or two days; one dose is often sufficient.
  • the present invention provides polypeptides comprising fragments of an extracellular domain of Integrin Subunit Alpha 2b (ITGA2B) or Integrin Beta 3 (ITGB3).
  • the invention further provides compositions comprising a fragment of a first human protein target of immune thrombocytopenia (ITP) autoantibodies and a fragment of a second human protein target of ITP autoantibodies and composition further comprising an effector moiety that is not an unmodified Fc domain.
  • ITP immune thrombocytopenia
  • Methods of treating ITP by administering a pharmaceutical composition of the invention are also provided, as are nucleic acid molecules and systems encoding the polypeptides and compositions of the invention, methods of producing those polypeptides and compositions and methods of determining suitability to be treated by a method of the invention.
  • compositions comprising: a. a first polypeptide comprising a fragment of an extracellular domain of Integrin Subunit Alpha 2b (ITGA2B) or an analog or derivative thereof, a fragment of an extracellular domain of Integrin Subunit Beta 3 (ITGB3) or an analog or derivative thereof or both and a first dimerization domain; and b. a second polypeptide comprising a fragment of ITGA2B or an analog or derivative thereof, a fragment of ITGB3 or an analog or derivative thereof or both and a second dimerization domain; wherein the first and second dimerization domains are configured to dimerize with each other.
  • ITGA2B Integrin Subunit Alpha 2b
  • ITGB3 Integrin Subunit Beta 3
  • polypeptide of the invention comprising an effector moiety.
  • the effector moiety is not an Fc domain.
  • the effector moiety is an Fc domain comprising at least one mutation that increases antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
  • ADCC antibody dependent cell cytotoxicity
  • CDC complement dependent cytotoxicity
  • the effector moiety is capable of inducing death in a cell binding the fragment.
  • 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.
  • 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.
  • the effector moiety is an Fc domain comprising SEQ ID NO: 60 or SEQ ID NO: 62 comprising a plurality of mutations selected from: L15V/F23L/R72P/Y80L/P176L, S19D/A110E/I112E, G16A/A110E/I112E, and
  • the effector moiety is conjugated to the polypeptide by a linker.
  • the first polypeptide comprises a fragment of ITGA2B or an analog or derivative thereof and the second polypeptide comprises a fragment of ITGB3 or an analog or derivative thereof.
  • the dimerizing comprises forming a covalent bond between the first dimerization domain and the second dimerization domain.
  • the protein complex comprises an immunoglobulin scaffold.
  • 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.
  • the fragment and the dimerization domain of the first, second or both polypeptide chains are separated by a linker.
  • the first polypeptide chain, the second polypeptide chain or both further comprise an Fc region of a human antibody heavy chain.
  • the Fc region is capable of inducing cytotoxicity against a cell binding the protein complex.
  • the first polypeptide chain comprises a first CH3 domain of a heavy chain of an immunoglobulin, a first CH2 domain of a heavy chain of an immunoglobulin or both and the second polypeptide chain comprises a second CH3 domain of a heavy chain of an immunoglobulin, a second CH2 domain of a heavy chain of an immunoglobulin or both.
  • the first CH3 domain comprises at least a first mutation and the second CH3 domain comprises at least a second mutation, and wherein the mutations permit heterodimerization of the first and second polypeptide chains and inhibit homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.
  • the first CH2 domain comprises at least a first mutation and the second CH2 domain comprises at least a second mutation, and wherein the mutations permit heterodimerization of the first and second polypeptide chains and inhibit homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.
  • the first mutation is selected from a mutation provided in Table 1 and the second mutation is provided in Table 1 and is a corresponding mutation to the first mutation.
  • the first mutation is a T366W mutation within a CH3 domain and the second mutation is a combination of aT366S mutation a L368A mutation and a Y407V mutation.
  • the Fc region of the first, second or both polypeptide chains is separated from the fragment or the dimerization domain by a linker.
  • the Fc region comprises at least one mutation that increases ADCC or CDC.
  • the Fc region is an Fc region comprising SEQ ID NO: 60 or SEQ ID NO: 62 comprising a plurality of mutations selected from: L15V/F23L/R72P/Y80L/P176L, S19D/A110L/I112E, G16A/A110L/I112E, and
  • the dimerization domain of the first, second or both polypeptide chains is C-terminal to the fragment and N-terminal to the Fc region.
  • the composition is devoid of an antibody variable domain.
  • the composition further comprises a third polypeptide comprising a fragment of ITGA2B or an analog or derivative thereof, a fragment of ITGB3 or an analog or derivative thereof or both and a third dimerization domain, wherein the first polypeptide further comprises a fourth dimerization domain and the third and fourth dimerization domains are capable of dimerizing to each other.
  • the third dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin and the fourth dimerization domain comprises a second hinge domain of a heavy chain and the first and the second dimerization domains dimerizes by a disulfide bond; or b. the third and fourth dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein the first and third polypeptides do not both comprise the CHI domain or the CL domain.
  • the composition further comprises a fourth polypeptide comprising a fragment of ITGA2B or an analog or derivative thereof, a fragment of ITGB3 or an analog or derivative thereof or both and a fifth dimerization domain, wherein the second polypeptide further comprises a sixth dimerization domain and the fifth and six dimerization domains are capable of dimerizing to each other.
  • the fifth dimerization domain comprises a first hinge domain of a heavy chain of an immunoglobulin and the sixth dimerization domain comprises a second hinge domain of a heavy chain and the first and the second dimerization domains dimerizes by a disulfide bond; or b.
  • the fifth and six dimerization domains each comprise a domain selected from a CHI domain of a heavy chain of an immunoglobulin and a CL domain of a light chain of an immunoglobulin and dimerize by a disulfide bond and wherein the first and third polypeptides do not both comprise the CHI domain or the CL domain.
  • the first polypeptide and the second polypeptide do not both comprise a CHI domain or both comprise a CL domain.
  • the third and fourth dimerization domains or the fifth and sixth dimerization domains comprise mutations that permits dimerization of the third and fourth dimerization domains and the fifth and sixth dimerization domains and inhibit dimerization of the third dimerization domain to the fifth or sixth dimerization domain and the sixth dimerization domain to the third or fourth dimerization domain.
  • the first polypeptide comprises a fragment of ITGA2B or an analog or derivative thereof and the second polypeptide comprises a fragment of ITGB3 or an analog or derivative thereof.
  • the first polypeptide chain or the second polypeptide chain comprises both a fragment of ITGA2B or an analog or derivative thereof, and a fragment of ITGB3 or an analog or derivative thereof.
  • the fragments are separated by an amino acid linker.
  • the ITGA2B lacks a signal peptide and comprises or consists of SEQ ID NO: 1
  • the ITGB3 lacks a signal peptide and comprises or consists of SEQ ID NO: 2 or both.
  • the fragment of an extracellular domain consists of a truncation of the extracellular domain.
  • an analog or derivative thereof comprises at least 85% identity to ITGA2B or ITGB3.
  • the fragment comprises at least 20 sequential amino acids from ITGA2B or ITGB3.
  • the fragment comprises at least one B cell receptor (BCR)-specific epitope target of autoantibodies.
  • BCR B cell receptor
  • the composition comprises a first or second polypeptide comprising a sequence selected from SEQ ID NO: 5, 8 and 11-14.
  • the hinge domain, the CH2 domain or the CH3 domain comprises at least one mutation that increases or reduces antibody dependent cell cytotoxicity (ADCC).
  • ADCC antibody dependent cell cytotoxicity
  • the at least one mutation that decreases ADCC is selected from: a. a mutation of the hinge domain comprising an L19A and an L20A mutation of SEQ ID NO: 22; and b. a mutation of the CH2 domain comprising an N59A mutation of SEQ ID NO: 36.
  • the composition further comprises at least one effector moiety capable of inducing cell death in a cell binding said composition.
  • the effector moiety is not an Fc domain.
  • 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.
  • compositions of the invention comprising a composition of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.
  • ITP immune thrombocytopenia
  • the ITGA2B lacks a signal peptide and comprises or consists of SEQ ID NO: 1
  • the ITGB3 lacks a signal peptide and comprises or consists of SEQ ID NO: 2 or both.
  • the composition is a composition of the invention.
  • the composition is a pharmaceutical composition of the invention.
  • the method further comprises reducing in the subject the levels of circulating antibodies against ITGA2B, ITGB3 or both.
  • the treating comprises decreasing the concentration of circulating autoantibodies against ITGA2B, ITGB3 or both.
  • the composition comprises an Fc region and the treating comprises killing B cells producing anti-ITGA2B or anti-ITGB3 autoantibodies.
  • the B cells are autoreactive B cells producing autoantibodies against a fragment of the composition.
  • nucleic acid system comprising a nucleic acid molecule, wherein a first nucleic acid molecule encodes the first polypeptide of a composition of the invention and a second nucleic acid molecule encodes the second polypeptide of a composition of the invention.
  • the nucleic acid system of the invention further comprising, a third nucleic acid molecule encoding the third polypeptide of a composition of the invention, a fourth nucleic acid molecule encoding the fourth polypeptide of a composition of the invention, or both.
  • a method of producing a composition of the invention comprising expressing a nucleic acid system of the invention in a cell, wherein the nucleic acid system is configured to produce the encoded polypeptide in the cell, thereby producing a composition of the invention.
  • a method for producing a protein comprising: obtaining a first fragment of an extracellular domain of ITGA2B or an analog or derivative thereof or a fragment of an extracellular domain of ITGB3 or an analog or derivative thereof, and a second fragment of an extracellular domain of ITGA2B or an analog or derivative thereof or a fragment of an extracellular domain of ITGB3 or an analog or derivative thereof, linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain wherein the first and second dimerization domains are capable of dimerizing with each other, and contacting the first polypeptide and the second polypeptide under conditions sufficient to induce the dimerization; or culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding at least two polypeptide chains, wherein the two polypeptide chains are produced by: i.
  • the protein complex is a protein complex of a composition of the invention.
  • the method further comprises a. linking a third dimerization domain to the first dimerization domain or first fragment within the first polypeptide chain; obtaining a third fragment of an extracellular domain of ITGA2B or an analog or derivative thereof or a fragment of an extracellular domain of ITGB3 or an analog or derivative thereof, and linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain wherein the third dimerization domain and the fourth dimerization domain are capable of dimerizing to each other; and contacting the first, second, and third polypeptides under conditions sufficient to induce the dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a third polypeptide chain produced by: i.
  • the method further comprises a. linking a sixth dimerization domain to the second dimerization domain or second fragment within the second polypeptide chain; obtaining a fourth fragment of an extracellular domain of ITGA2B or an analog or derivative thereof or a fragment of an extracellular domain of ITGB3 or an analog or derivative thereof, and linking the fourth fragment to a fifth dimerization domain to produce a fourth polypeptide chain wherein the fifth dimerization domain and the sixth dimerization domain are capable of dimerizing to each other; and contacting the first, second, third and fourth polypeptides under conditions sufficient to induce the dimerization; or b. expressing in the host cell a nucleic acid sequence encoding a fourth polypeptide chain produced by: i.
  • the method further comprises producing at least one mutation in an extracellular domain of the protein or truncating the protein to remove a portion thereof.
  • the method further comprises linking an effector moiety to at least one of the polypeptide chains or the mutated fragment, wherein the effector moiety is capable of killing a cell that binds the at least one polypeptide.
  • the effector moiety is not an Fc domain.
  • 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.
  • the effector moiety is an Fc domain comprising at least one mutation that increases ADCC or CDC.
  • the effector moiety is an Fc domain comprising SEQ ID NO: 60 or SEQ ID NO: 62 comprising a plurality of mutations selected from: L15V/F23L/R72P/Y80L/P176L, S19D/A110E/I112E, G16A/A110E/I112E, and
  • a method of determining suitability of a subject in need thereof to be treated by a method of the invention comprising receiving a sample from the subject, contacting the sample with a protein of the invention or a composition of the invention and determining binding of autoantibodies within the sample to the protein or the composition, wherein binding of autoantibodies to the protein or the composition indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.
  • Figures 1A-1F Diagrams of five possible embodiments of the four-chain therapeutic agent of the invention: (1A) shows a general embodiment of a molecule for treating ITP, (IB) shows an embodiment in which the four chains each comprise a different protein fragment, (1C) shows an embodiment in which the four protein fragments are all the same, (ID) shows an embodiment in which the two heavy chains are identical and the two light chains are identical, (IE) shows an embodiment in which the two heavy chains are different and the two light chains are identical, and (IF) shows an embodiment in which the two heavy chains are contain the same protein fragment and the two light chains contain different protein fragments.
  • FIGS. 2A-2N Diagrams of possible embodiments of the two-chain therapeutic agent of the invention: (2A) shows general embodiments of a molecule with two heavy chains for treating ITP, (2B) shows embodiments in which at least one of the CHI, CH2 or CH3 domains has been excluded, (2C) shows an embodiment in which the two protein fragments are the same, (2D) shows an embodiment in which the two protein fragments are different, (2E) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CHI domain, (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CHI domain or a hinge domain, (2G) shows a general embodiment in which two tandem fragments are included in each heavy chain and connected via a linker, (2H) shows the tandem fragment configuration in which all the subunits are the same, (21) shows the tandem fragment configuration in which all the subunits are the same without the CHI domain, (2J) shows the tandem fragment configuration in which each heavy chain contains the
  • Figures 3A-3D Diagrams of four possible embodiments of the three-chain therapeutic agent of the invention: (3A) shows a general embodiment of a molecule with two heavy chains and one light chain, (3B) shows an embodiment in which the three protein fragments are the same, (3C) shows an embodiment in which each of the protein fragments are different, (3D) shows an embodiment in which two of the protein fragments are the same and the third is different.
  • Figure 4 Diagram of an embodiment of a four-chain therapeutic agent for treating ITP of the invention similar to those shown in Figure 1 but in which the four chains each comprise a different protein fragment and a different immunoglobulin scaffold which promotes formation of the four-chain molecule.
  • FIGS 5A-5B Diagrams of generic embodiments of the four-chain therapeutic agent of the invention: (5A) shows a generic embodiment of four chains in which two contain chains contain two dimerization domain and two chains contain a single dimerization domain, and (5B) shows an embodiment with optional linkers separating the various domains and fragments.
  • FIGS 6A-6G Diagrams of single chain therapeutic agents of the invention: (6A) shows an embodiment of a single chain molecule containing fragments from two different ITGA2B/B3 fragments, (6B) shows embodiments of fragments comprising a truncation of ITGA2B or ITGB3, (6C) shows an embodiments of a single chain molecule containing three different fragments, (6D) shows an embodiments of a single chain molecule containing four different fragments, (6E) shows an embodiments of a single chain molecule containing fragments from one or two different ITGA2B/B3 proteins/domains and a heavy chain constant region, (6F) shows an embodiments of a single chain molecule containing fragments from one or two different ITGA2B/B3 proteins/domains and a CH3-CH2 fragment of the heavy chain constant region, and (6G) shows the single chain molecules of 6A and 6C-6F with amino acid (AA) linkers separating various domains.
  • AA amino acid
  • Figure 7 Photographs of an SDS-PAGE gel, in reducing (right) and non-reducing (left) conditions showing molecules CRD-757, 758, 760 and 756.
  • FIGS 8A-8D show Figures 8A-8D.
  • Figure 9 Line graph of the increase in percent cell killing by CRD-757, and CRD- 758 by CDC. The increase is relative to CRD-760 which lacks an Fc domain.
  • compositions comprising a fragment of a first human receptor target of immune thrombocytopenia (ITP) autoantibodies or an analog or derivative thereof and a fragment of a second human protein receptor target of ITP autoantibodies or an analog or derivative thereof.
  • compositions further comprising an effector moiety that is not an unmodified Fc domain are also provided.
  • Protein complexes comprising at least two polypeptide chains wherein a first chain comprises a fragment of a first human protein target of ITP autoantibodies or an analog or derivative thereof and a first dimerization domain and a second chain comprises a fragment of a second human protein target of ITP autoantibodies or an analog or derivative thereof and a second dimerization domain capable of dimerizing with the first dimerization domain are also provided.
  • Polypeptides comprising fragments of an extracellular domain of Integrin Subunit Alpha 2b (ITGA2B) or Integrin Subunit Beta 3 (ITGB3) are also provided. Protein complexes further comprising an effector moiety that is not an unmodified Fc domain are also provided.
  • the present invention further concerns pharmaceutical composition
  • pharmaceutical composition comprising the compositions and/or protein complexes, nucleic acids encoding the polypeptides of the compositions and/or protein complexes and methods of treatment and determining suitability for treatment using the compositions and/or protein complexes; as well as methods of producing the compositions and/or protein complexes.
  • composition comprising a fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof.
  • composition comprising a fragment of a first protein target of ITP autoantibodies and a fragment of a second protein target of ITP autoantibodies or an analog or derivative thereof.
  • a protein comprising a fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof.
  • a protein comprising a fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and a fragment of a second protein target of ITP autoantibodies or an analog or derivative thereof.
  • a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and a first dimerization domain and a second polypeptide chain comprising a fragment of a second protein target of ITP autoantibodies or an analog or derivative thereof and second dimerization domain.
  • the composition comprises a protein complex comprising at least two polypeptide chains, wherein a first polypeptide chain comprises a fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and a first dimerization domain and a second polypeptide chain comprising a fragment of a second protein target of ITP autoantibodies or an analog or derivative thereof and second dimerization domain.
  • the composition comprises a protein complex of the invention.
  • the composition comprises a protein of the invention.
  • the protein is a recombinant protein.
  • the protein is a fusion protein.
  • peptide 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.
  • 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.
  • the peptide is not a cyclic peptide.
  • the fragment is not a cyclic peptide.
  • the extracellular domain is not a cyclic peptide.
  • the protein complex is an immunoglobulin (Ig)-like complex.
  • the protein complex comprises an Ig-like scaffold.
  • the protein complex comprises an Ig-like backbone.
  • the protein complex is an Ig Fc-fusion complex.
  • the composition is devoid of an antibody variable domain.
  • the protein complex is devoid of an antibody variable domain.
  • the composition is devoid of a variable domain.
  • the protein complex is devoid of a variable domain.
  • the first chain is devoid of a variable domain.
  • the second chain is devoid of a variable domain.
  • 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 ITP. In some embodiments, ITP is acquired immune thrombocytopenia. In some embodiments, ITP is secondary ITP. In some embodiments, ITP is drug-induced ITP (DITP). In some embodiments, the protein complex is for use in treating ITP.
  • ITP is acquired immune thrombocytopenia. In some embodiments, ITP is secondary ITP. In some embodiments, ITP is drug-induced ITP (DITP). In some embodiments, the protein complex is for use in treating ITP.
  • the protein complex is for use in diagnosing ITP. In some embodiments, the protein complex is for use in determining appropriate treatment in ITP. In some embodiments, the protein complex is for use in characterizing the serological response in ITP. In some embodiments, the protein complex is for use in determining the autoantibody titer in ITP.
  • 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).
  • the polypeptide chain is a recombinant polypeptide.
  • 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.
  • 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.
  • 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.
  • expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide).
  • a nucleic acid molecule of the invention is expressed in a cell to produce a polypeptide of the invention.
  • a nucleic acid complex of the invention is expressed in a cell to produce a protein complex of the invention.
  • the RNA is a vector.
  • DNA sequence or an RNA within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome.
  • the DNA sequence is in an expression vector such as plasmid or viral vector.
  • a Kozak sequence is inserted upper stream of the transcription initiating codon. In some embodiments, the Kozak sequence enhances the amount of protein expressed.
  • 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.
  • the protein is a mammalian protein. In some embodiments, the mammal is a human. In some embodiments, the protein is a transmembrane protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a subunit in a receptor. In some embodiments, the protein is a cell surface protein. In some embodiments, the cell surface protein is an integral membrane protein. In some embodiments, the cell surface protein is a plasma membrane embedded protein. In some embodiments, the cell surface protein is a membrane anchored protein. In some embodiments, the protein is an ITP-associated protein. In some embodiments, the protein is a synthetic protein.
  • the protein is a naturally occurring protein. In some embodiments, the protein is a target of ITP autoantibodies. In some embodiments, the protein is selected from Integrin Subunit Alpha 2b (ITGA2B) and Integrin Subunit Beta 3 (ITGB3). In some embodiments, the protein is ITGA2B. In some embodiments, the protein is ITGB3.
  • the term “receptor” refers to a protein expressed on the surface of a cell that is capable of binding a ligand. In some embodiments, a receptor is a protein capable of transducing a signal to the cytoplasm of the cell. In some embodiments, a receptor comprises a ligand binding domain. In some embodiments, a receptor comprises a transmembrane domain. In some embodiments, a receptor comprises an intracellular domain.
  • the fragment comprises an extracellular domain (ECD) of the protein. In some embodiments, the fragment comprises a fragment of an extracellular domain of the protein. In some embodiments, the fragment consists of the extracellular domain of a fragment thereof. In some embodiments, the fragment consists of an extracellular domain of 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.
  • the chain is devoid of an intracellular domain.
  • the fragment includes a sequence from a homologous human protein. In some embodiments, the fragment includes a sequence from a homologous nonhuman protein. In some embodiments, the fragment includes mutations in the human protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the chain comprises at least one fragment. In some embodiments, the chain comprises at least two fragments. In some embodiments, the fragments are separated by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, 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 protein stability as compared to the region of the protein that has been replaced.
  • the protein is a target of antibodies.
  • antibody includes all classes of IgA, IgD, IgE, IgG and IgM and also includes all subclasses thereof.
  • the antibody is a circulating antibody.
  • the antibody is a naturally occurring antibody.
  • the antibodies are autoantibodies.
  • autoantibodies refers to antibodies generated by a subject’s own immune system against at least one of the subject’s own proteins.
  • an autoantibody is an autoreactive antibody.
  • autoantibodies target self-antigens. Self-antigens are also known as autoantigens.
  • the autoantibodies are associated with ITP.
  • the autoantibodies characterize ITP.
  • the autoantibodies are autoantibodies of ITP.
  • autoantibodies are generated by auto-reactive B cells.
  • the protein is an antigen of the antibodies.
  • the fragment comprises an antigen of the antibodies.
  • the fragment comprises at least one antigen of the antibodies. In some embodiments, the fragment comprises at least two antigens of the antibodies. In some embodiments, the fragment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigens of the antibodies. Each possibility represents a separate embodiment of the invention.
  • an antigen of the antibodies is an autoantigen.
  • the antigen is an epitope. In some embodiments, the antigen includes at least one epitope. In some embodiments, an epitope comprises at least 5 amino acids. In some embodiments, an epitope comprises 5-6 amino acids. In some embodiments, an epitope comprises 5-10 amino acids. In some embodiments, an epitope is a simple epitope.
  • a simple epitope is a linear epitope.
  • an epitope is a complex epitope.
  • a complex epitope is a 3D epitope.
  • a complex epitope is a discontinuous epitope.
  • a discontinuous epitope comprises at least two discontinuous sections of amino acids that combine to form an epitope.
  • a linker sequence is between the two sections of the epitope.
  • 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.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • the substitution is outside of an antigenic region of the protein. In some embodiments, the substitution is outside an epitope of the antibodies.
  • 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.
  • 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.
  • 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.
  • an analog to the protein comprises an amino acid sequence with at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the analog is still able to bind ITP autoantibodies. In some embodiments, the analog is still able to sequester ITP autoantibodies. In some embodiments, the analog is still able to treat ITP. 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.
  • derivative 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.
  • a derivative may be a derivative of a fragment of the protein, however, in such a case the fragment must comprise at least 50 consecutive amino acids of the protein or at least one epitope of the antibodies.
  • the derivative is a derivative of a canonical sequence of the protein.
  • 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.
  • 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.
  • a derivative to the protein comprises an amino acid sequence with at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the derivative is still able to bind ITP autoantibodies. In some embodiments, the derivative is still able to sequester ITP autoantibodies. In some embodiments, the derivative is still able to treat ITP. In some embodiments, a derivative is the protein or fragment with a mutation.
  • 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.
  • human ITGA2B also known as GPIIb
  • GPIIb canonical nucleic acid sequence
  • its canonical nucleic acid sequence can be found in Entrez gene 3674
  • its canonical protein coding mRNA sequence can be found in NM_000419
  • its canonical amino acid sequence can be found in NP_000410 and UniProt number P08514.
  • 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.
  • a canonical sequence is a sequence identical to the most prevalent sequence present in a population.
  • the population is a disease population.
  • the population is a population with the autoimmune disease.
  • a canonical amino acid sequence of an extracellular domain of ITGA2B comprises or consists of
  • the extracellular domain is devoid of a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the ITGA2B signal peptide comprises or consists of MARALCPLQALWLLEWVLLLLGPCAAPPAWA (SEQ ID NO: 15).
  • a canonical amino acid sequence of an extracellular domain of ITGB3 comprises or consists of
  • the extracellular domain is devoid of a signal peptide.
  • the extracellular domain further comprises a signal peptide.
  • the ITGB3 signal peptide comprises or consists of MRARPRPRPLWATVLALGALAGVGVG (SEQ ID NO: 16).
  • the ITGA2B or the ITGB3 signal peptide is used.
  • the signal peptide is a signal peptide of an antibody chain.
  • the single peptide is of an antibody heavy chain.
  • the signal peptide is of an antibody light chain.
  • the signal peptide is of the Kappa light chain.
  • the signal peptide is of the Lambda light chain.
  • the heavy chain signal peptide comprises MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 17).
  • the heavy ch in signal peptide consists of SEQ ID NO: 17.
  • the light chain signal peptide comprises MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 18).
  • the light chain signal peptide consists of SEQ ID NO: 18. In some embodiments, the signal peptide comprises of consists of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 19). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 19. In some embodiments, the signal peptide comprises or consists of MGWSCIILFLVATATGVHS (SEQ ID NO: 20). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 20.
  • the first protein and the second protein are the same protein. In some embodiments, the first and second proteins are the same proteins, and the fragments are different fragments. In some embodiments, the fragments are different fragments. In some embodiments, the fragments comprise or consist of different sequences. In some embodiments, the first and second proteins are different proteins.
  • the fragment comprises an extracellular functional domain.
  • the functional domain is a ligand binding domain.
  • the ligand is selected from laminins, collagens and fibronectin.
  • the fragment comprises a truncation of the extracellular domain. In some embodiments, the fragment consists of a truncation of the extracellular domain. In some embodiments, the truncation lacks at least one extracellular functional domain. In some embodiments, the truncation lacks at least two extracellular functional domains. [0139] In some embodiments, a derivative is a derivative of the truncation. In some embodiments, the derivative comprises at least 85% identity to the truncation and does not further comprise a stretch of amino acids homologous/identical to a sequence from ITGA2B or ITGB3.
  • a sequence with sequence identity to a truncation is not a sequence which is not truncated.
  • the truncation comprises at least one mutation.
  • the derivative comprises at least 85% identity to any one of SEQ ID NO: 1.
  • the derivative comprises at least 85% identity to any one of SEQ ID NO: 2.
  • dimerization domains are capable of dimerizing with each other.
  • the first dimerization domain is capable of dimerization with the second dimerization domain.
  • the first and second dimerization domains are capable of dimerizing with each other.
  • capable of dimerizing is configured to dimerize.
  • dimerization is under physiological conditions.
  • dimerization is within a bodily fluid.
  • the bodily fluid is blood.
  • the bodily fluid is plasma.
  • the bodily fluid is serum.
  • dimerization is within a subject.
  • dimerization is in vivo. In some embodiments, dimerization is in vitro.
  • 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.
  • the bond is a chemical bond. In some embodiments, the bond is a disulfide bond. In some embodiments, the bond is a peptide bond.
  • dimerization domain include the hinge domain of antibody heavy chains, the CH1/CL domains of antibody heavy/light chains, and the ECD domains of TCR alpha/beta to name but a few. Additionally, the upper hinge domain can be engineered with cysteine substitutions/mutations to serine in order to prevent dimerization. In some embodiments, the dimerization domain comprises or consists of the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 21).
  • the dimerization domain comprises or consists of an immunoglobulin (Ig) hinge domain.
  • an Ig hinge domain is a heavy chain hinge domain.
  • the Ig is a human Ig.
  • the immunoglobulin is elected from IgA, IgD, IgE, IgG and IgM.
  • the immunoglobulin is IgG.
  • the IgG is IgGl.
  • the IgG is IgG2.
  • the IgG is IgG3.
  • the IgG is selected from IgGl and IgG3.
  • the IgG is IgG4.
  • 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.
  • the hinge domain comprises the amino acid sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 22). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the IgGl hinge comprises or consists of SEQ ID NO: 22. In some embodiments, the hinge domain comprises the amino acid sequence EPKCCVECPPCPAPPAAAP (SEQ ID NO: 23). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgG2 hinge comprises or consists of SEQ ID NO: 23. In some embodiments, the hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFLGGP (SEQ ID NO: 24). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the IgG4 hinge comprises or consists of SEQ ID NO: 24. In some embodiments, the hinge domain comprises the amino acid sequence
  • the hinge domain consists of the amino acid sequence of SEQ ID NO: 25.
  • the IgG3 hinge comprises or consists of SEQ ID NO: 25.
  • the hinge domain comprises a CPXCP (SEQ ID NO: 26) motif.
  • the X in SEQ ID NO: 26 is selected from P and R.
  • SEQ ID NO: 26 is CPPCP (SEQ ID NO: 27).
  • SEQ ID NO: 26 is CPRCP (SEQ ID NO: 28).
  • the hinge domain comprises EPKSCDKTHTCPPCP (SEQ ID NO: 29). It will thus be understood that the hinge region can be considered to end after the CPXCP motif.
  • 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.
  • the first dimerization domain comprises or consists of a CHI domain
  • the second dimerization domain comprises or consists of a CL domain.
  • the first and second dimerization domains both comprise a hinge domain.
  • the first and second dimerization domains do not both comprise a CHI domain.
  • the first and second dimerization domains do not both comprise a CL domain.
  • the first and second polypeptide chains do not both comprise a CHI domain.
  • the first and second polypeptide chains do not both comprise a CL domain.
  • the first and second polypeptide chains are both devoid of a CHI domain.
  • an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEITPTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 30).
  • an Ig CHI domain consists of SEQ ID NO: 30.
  • SEQ ID NO: 30 is the IgGl CHI domain.
  • an Ig CHI domain comprises of the amino acid sequence
  • an Ig CHI domain consists of SEQ ID NO: 31.
  • SEQ ID NO: 31 is the IgG2 CHI domain.
  • an Ig CHI domain comprises of the amino acid sequence ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEITPTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 32).
  • an Ig CHI domain consists of SEQ ID NO: 32.
  • SEQ ID NO: 32 is the IgG3 CHI domain.
  • an Ig CHI domain comprises of the amino acid sequence
  • an Ig CHI domain consists of SEQ ID NO: 33.
  • SEQ ID NO: 33 is the IgG4 CHI domain.
  • an Ig CL Kappa domain comprises of the amino acid sequence AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSITPTKSFNRGEC (SEQ ID NO: 34).
  • an Ig CL Kappa domain consists of SEQ ID NO: 34.
  • an Ig CL Lambda domain comprises of the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSITPKAGVET TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 35).
  • an Ig CL Lambda domain consists of SEQ ID NO: 35.
  • the composition comprises an effector moiety.
  • the first polypeptide chain comprises an effector moiety.
  • the second polypeptide chain comprises an effector moiety.
  • 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.
  • the term “effector moiety” refers to a molecule or fragment of a molecule that carriers out a cytotoxic effect.
  • an effector moiety is an effector molecule.
  • the effector moiety is capable of inducing a cytotoxic effect. In some embodiments, the effector moiety is configured to induce a cytotoxic effect. In some embodiments, the effector moiety is capable of inducing death. In some embodiments, the effector moiety is configured to induce death. In some embodiments, death is cell death. In some embodiments, death is apoptosis. In some embodiments, death is necrosis. In some embodiments, death is cell mediated death. In some embodiments, death is phagocytosis. In some embodiments, the cytotoxic effect is against a target cell. In some embodiments, death is in a target cell. In some embodiments, the cytotoxic effect is upon binding.
  • 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.
  • 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.
  • the effector moiety recruits a cytotoxic agent.
  • the cytotoxic agent is a cytotoxic cell.
  • the cytotoxic cell is an immune cell.
  • the immune cell is a T cell.
  • the immune cell is a natural killer (NK) cell.
  • the immune cell is a macrophage.
  • the T cell is a cytotoxic T cell.
  • the T cell is a CD8 positive T cell.
  • the effector moiety induces antibody-dependent cell cytotoxicity (ADCC).
  • the effector moiety induces complement-dependent cytotoxicity (CDC).
  • the effector moiety binds a receptor on a cell surface of the cytotoxic cell.
  • receptors include, but are not limited to CD3, CD8, CD56, CD14 and CD16.
  • the receptor is a marker of the cytotoxic cell.
  • the receptor is unique to the cytotoxic cell.
  • the receptor is CD3.
  • the effector moiety is an agent that binds CD3.
  • the engager is an agent that binds CD3.
  • CD3 is human CD3.
  • the agent that binds CD3 is an anti-CD3 antibody or antigen binding fragment thereof.
  • the receptor is CD16.
  • the effector moiety is an agent that binds CD16.
  • the engager is an agent that binds CD16.
  • CD16 is human CD16.
  • the agent that binds CD16 is an anti-CD16 antibody or antigen binding fragment thereof.
  • the antibody of antigen binding fragment thereof is a single chain antibody.
  • the antibody of antigen binding fragment thereof is a single domain antibody.
  • 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.
  • the anti-human CD3 scFv known as OKT3 may be used as the agent.
  • the cytotoxic moiety is selected from alpha-amanitin, a radioactive moiety and an anti-CD3 binding agent.
  • 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).
  • human anti-CD16 examples 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.
  • AFM13 a bispecific tetravalent Innate Cell Engager (ICE®) targeting CD30 on tumor cells and CD16A on NK cells and macrophages
  • GTB-3550 CD16/IL-15/CD33
  • the composition comprises an Fc region. In some embodiments, the effector moiety is not an Fc region. In some embodiments, not an Fc region is not an unmodified Fc region. In some embodiments, the composition comprises an effector moiety that is not an Fc region. In some embodiments, the composition comprises an effector moiety other than an Fc region. In some embodiments, the composition is devoid of an Fc region. In some embodiments, the protein comprises an effector moiety that is not an Fc region. In some embodiments, the protein comprises an effector moiety other than an Fc region. In some embodiments, the protein is devoid of an Fc region. In some embodiments, the engager is an Fc region.
  • the engager is not an Fc region.
  • 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.
  • an Fc is an unmodified Fc. In some embodiments, an Fc is an unmutated Fc. In some embodiments, an Fc is a naturally occurring Fc. In some embodiments, an Fc is 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.
  • the first polypeptide chain comprises an Fc region.
  • the second polypeptide chain comprises an Fc region.
  • both the first and second polypeptide chains comprise an Fc region.
  • the Fc region is an Fc region of an antibody heavy chain.
  • the antibody heavy chain is a human antibody heavy chain.
  • the heavy chain is an IgG heavy chain.
  • the IgG is selected from IgGl, IgG2, IgG3 and IgG4.
  • the IgG is selected from IgGl and IgG3.
  • the IgG is IgGl.
  • the IgG is IgG2.
  • the IgG is IgG3.
  • the IgG is IgG4.
  • the Fc region is capable of inducing a cytotoxic effect.
  • the Fc domain comprises
  • the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 58). It will be understood that SEQ ID NO: 58 contains 5 additional N-terminal amino acids as compared to SEQ ID NO: 57.
  • the Fc region is capable of inducing a cytotoxic effect.
  • the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 59).
  • the Fc domain comprises EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 60). It will be understood that SEQ ID NO: 60 contains 5 additional N-terminal amino acids as compared to SEQ ID NO: 59.
  • SEQ ID NO: 59 (or SEQ ID NO: 57 which is equivalent) the numbering for SEQ ID NO: 60 can be found by adding 5.
  • SEQ ID NO: 57 and SEQ ID NO: 59 differ by two amino acids. The two sequences can be interchanged and when mutations are given with respect to SEQ ID NO: 57 it will be understood that they apply also to SEQ ID NO: 59 and vice-versa. So too SEQ ID NO: 58 and SEQ ID NO: 60 also differ by only two amino acids and these two sequences can be interchanged.
  • the Fc domain consists of SEQ ID NO: 57.
  • the Fc domain of IgGl comprises or consists of SEQ ID NO: 57.
  • 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: 57.
  • the Fc domain consists of SEQ ID NO: 58.
  • the Fc domain of IgGl comprises or consists of SEQ ID NO: 58.
  • 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: 58. Each possibility represents a separate embodiment of the invention.
  • the Fc domain consists of SEQ ID NO: 59.
  • the Fc domain of IgGl comprises or consists of SEQ ID NO: 59.
  • 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: 59.
  • the Fc domain consists of SEQ ID NO: 60.
  • the Fc domain of IgGl comprises or consists of SEQ ID NO: 60. 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: 60. Each possibility represents a separate embodiment of the invention.
  • the Fc region is capable of inducing a cytotoxic effect. In some embodiments, the Fc region is configured to induce a cytotoxic effect. In some embodiments, the cytotoxic effect is against a target cell. In some embodiments, the cytotoxic effect is upon binding. In some embodiments, the cytotoxic effect is against a cell bound by the protein complex. In some embodiments, the cytotoxic effect is against a cell binding the protein complex. In some embodiments, the cytotoxic effect is mediated by immune cell binding to the Fc region. In some embodiments, the cytotoxic effect is mediated by immune cell activation by the Fc region. In some embodiments, the cytotoxic effect is mediated by immune cell recruitment by the Fc region.
  • 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).
  • ADCC antibody-dependent cell cytotoxicity
  • CDC complement-dependent cytotoxicity
  • 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 heavy chain CH2 and an Ig heavy chain CH3 domain. In some embodiments, the first chain comprises a first portion of an Fc region and the second chain comprises a second portion of the Fc region.
  • the first portion comprises a CH2 domain, a CH3 domain or both.
  • the second portion comprises a CH2 domain, a CH3 domain or both.
  • interface of the first portion of an Fc region and the second portion of an Fc region produces a functional Fc region.
  • interface comprises contact.
  • interface comprises adjacent positioning.
  • interface comprises formation of the protein complex of the invention.
  • interface comprises dimerization of the first and second dimerization domains.
  • the CH2 domain is an Ig CH2 domain.
  • the CH2 domain is a heavy chain CH2 domain.
  • the CH3 domain is an Ig CH3 domain.
  • the CH3 domain is a heavy chain CH3 domain.
  • a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 36).
  • the CH2 domain consists of SEQ ID NO: 36.
  • SEQ ID NO: 36 is the IgGl CH2 domain.
  • a CH2 domain comprises the amino acid sequence
  • the CH2 domain consists of SEQ ID NO: 37.
  • SEQ ID NO: 37 is the IgG2 CH2 domain.
  • a CH2 domain comprises the amino acid sequence
  • the CH2 domain consists of SEQ ID NO: 38.
  • SEQ ID NO: 38 is the IgG4 CH2 domain.
  • a CH2 domain comprises the amino acid sequence
  • the CH2 domain consists of SEQ ID NO: 39.
  • SEQ ID NO: 39 is the IgG3 CH2 domain.
  • a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPI TPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40).
  • a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPI TPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41).
  • the CH3 domain consists of SEQ ID NO: 40. In some embodiments, the CH3 domain consists of SEQ ID NO: 41. In some embodiments, SEQ ID NO: 40 is the IgGl CH3 domain. In some embodiments, SEQ ID NO: 41 is the IgGl CH3 domain. In some embodiments, the SEQ ID NO: 40 sequence is the sequence found predominantly is humans of European and American descent. In some embodiments, SEQ ID NO: 41 is the sequence found predominantly in humans of Asian descent.
  • a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42).
  • the CH3 domain consists of SEQ ID NO: 42.
  • SEQ ID NO: 42 is the IgG2 CH3 domain.
  • a CH3 domain comprises the amino acid sequence
  • the CH3 domain consists of SEQ ID NO: 43.
  • SEQ ID NO: 43 is the IgG4 CH3 domain.
  • a CH3 domain comprises the amino acid sequence
  • the CH3 domain consists of SEQ ID NO: 44.
  • SEQ ID NO: 44 is the IgG3 CH3 domain.
  • the Fc comprises a mutation.
  • a CH3 domain comprises a mutation.
  • the first CH3 domain comprises a first mutation.
  • the second CH3 domain comprises a second mutation.
  • a CH2 domain comprises a mutation.
  • the first CH2 domain comprises a first mutation.
  • the second CH2 domain comprises a second mutation.
  • the CH2 and CH3 domains both comprise mutations.
  • the first CH2 domain and first CH3 domains each comprise a first mutation.
  • the second CH2 domain and the second CH3 domain each comprise a second mutation.
  • 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.
  • a region from an IgG is replaced with a region from an IgA.
  • 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.
  • the mutation is insertion of a region from a TCR.
  • the TCR is selected from TCRa and TCRb.
  • the mutation is insertion of a region from a different Ig. Examples of these mutations can be found in Table 1.
  • the mutation is selected from a mutation in Table 1.
  • the first mutation is selected from a group of mutation provided in a row and the second column of Table 1 and the second mutation is the group of mutations provided in that same row of Table 1 in the third column.
  • the mutations in Table 1 are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in other IgGs.
  • the first mutation is T366Y
  • the second mutation is Y407T.
  • the first mutation is S354C and T366W and the second mutation is Y349C, T366S, L368A, and Y407V.
  • the first mutation is S364H and F405A and the second mutation is Y349T and T392F.
  • the first mutation is T350V, E351Y, F405A, and Y407V and the second mutation is T350V, T366E, K392E, and T394W.
  • the first mutation is K392D, and K409D and the second mutation is E356K, and D399K.
  • the first mutation is D221E, P228E, and E368E and the second mutation is D221R, P228R, and K409R.
  • 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.
  • the first mutation is Y349S, K370Y, T366M, and K409V and the second mutation is E356G, E357D, S364Q, and Y407A.
  • the first mutation is T366K
  • the second mutation is selected from C351D, Y349E, Y349D, E368E, E368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, and Y349D and R355D.
  • the first mutation is T366K and C351K and the second mutation is selected from C351D, Y349E, Y349D, E368E, E368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, and Y349D and R355D.
  • the first mutation is E351D and E368E and the second mutation is E351K and T366K.
  • the first mutation is E368D and K370S and the second mutation is E357Q and S364K.
  • the first mutation is T366W, and the second mutation is T366S, E368A and Y407V.
  • the Ig is IgG2
  • the first mutation is C223E, P228E, and E368E and the second mutation is C223R, E225R, P228R, and K409R.
  • the first mutation is S354C or T366W and the second mutation is Y349C, T366S, E368A, or Y407V.
  • the first mutation is S364H or F405A and the second mutation is Y349T or T392F.
  • the first mutation is T350V, E351Y, F405A, or Y407V and the second mutation is T350V, T366E, K392E, or T394W.
  • the first mutation is K392D, or K409D and the second mutation is E356K, or D399K.
  • the first mutation is D221E, P228E, or E368E and the second mutation is D221R, P228R, or K409R.
  • the first mutation is K360E, or K409W and the second mutation is Q347R, D399V, or F405T.
  • the first mutation is K360E, K409W, or Y349C and the second mutation is Q347R, D399V, F405T, or S354C.
  • the first mutation is K360D, D399M, or Y407A and the second mutation is E345R, Q347R, T366V, or K409V.
  • the first mutation is Y349S, K370Y, T366M, or K409V and the second mutation is E356G, E357D, S364Q, or Y407A.
  • the first mutation is E351D or E368E and the second mutation is E351K or T366K.
  • the first mutation is E368D or K370S and the second mutation is E357Q or S364K.
  • the first mutation is T366W
  • the second mutation is T366S, L368A or Y407V.
  • the Ig is IgG2
  • the first mutation is C223E, P228E, or L368E and the second mutation is C223R, E225R, P228R, or K409R.
  • the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP ITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTEPPSREEMTKNQVSESCAVKGFYPSDIAVEWESNGQPENNYKTTPI TPEDSDGSFFEVSKETVDKSRWQQGNVFSCSVMHEAEHNHYTQKSESESPGK (SEQ ID NO: 46).
  • the CH3 domain comprises or consists of GQPREPQVYTEPPSREEMTKNQVSEYCEVKGFYPSDIAVEWESNGQPENNYKTTPI TPEDSDGSFFEYSKETVDKSRWQQGNVFSCSVMHEAEHNHYTQKSESESPGK (SEQ ID NO: 47). In some embodiments, the CH3 domain comprises or consists of GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPI TPLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 48).
  • Table 1 Mutations for enhancing heterodimerization and inhibiting homodimerization of CH3 domains.
  • 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.
  • the Fc domain comprises at least one mutation that decreases antibody effector function. In some embodiments, the Fc domain comprises at least one mutation that decreases ADCC. In some embodiments, the at least one mutation that decreases ADCC is a LALA mutation.
  • the LALA mutation refers to mutation of two successive leucine residues to alanine residues. In some embodiments, the LALA mutation is within the hinge domain. In some embodiments, the hinge domain is the hinge domain of IgGl. In some embodiments, the LALA mutation is mutation of L19 and L20 of SEQ ID NO: 22 to A19 and A20. In some embodiments, a LALA mutation hinge comprises an L19A and an L20A mutation of SEQ ID NO: 22.
  • the Fc domain comprises a hinge domain comprising EPKSCDKTHTCPPCPAPEAA (SEQ ID NO: 49.
  • an Fc domain comprising a LALA mutation comprises SEQ ID NO: 49.
  • the Fc domain comprises a hinge domain consisting of SEQ ID NO:
  • a LALA mutated hinge domain consists of SEQ ID NO: 49.
  • the LALA mutation is a L234A and L235A mutation of the Fc.
  • the at least one mutation that decreases ADCC is a N297A mutation.
  • the N297A mutation is within the CH2 domain.
  • the N297A mutation is mutation of asparagine 59 of SEQ ID NO: 36 to alanine.
  • an N297A mutated CH2 domain comprises an N59A mutation of SEQ ID NO: 36.
  • the Fc domain comprises a CH2 domain comprising SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKSEQ ID NO:
  • the Fc domain comprises a CH2 domain consisting of SEQ ID NO: 50.
  • the N297A mutated CH2 domain consists of SEQ ID NO: 50. 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.
  • the IgG comprises at least one mutation.
  • the mutation is a plurality of mutations.
  • the mutation decreases cytotoxicity.
  • the mutation increases stability.
  • the mutation decreases aggregation.
  • 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.
  • 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: 57 OR 59 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: 57 OR 59 to A.
  • the mutation is mutation of leucine 235 of the IgGl human heavy chain to alanine (L235A).
  • the L to A mutation is mutation of L15 of SEQ ID NO: 57 OR 59 to A.
  • the plurality of mutation comprises P109G, L14A and L15A of SEQ ID NO: 57 OR 59.
  • the plurality of mutation comprises L14A and L15A of SEQ ID NO: 57 OR 59.
  • the plurality of mutation comprises P329G, L234A and L235A of the IgGl human heavy chain.
  • the plurality of mutation comprises L234A and L235A of the IgGl human heavy chain.
  • the plurality of mutations that decreases cytotoxicity comprise the YTE mutations.
  • the mutation is mutation of methionine 252 of the IgGl human heavy chain to tyrosine (M252Y).
  • the M to Y mutation is mutation of M32 of SEQ ID NO: 57 OR 59 to Y.
  • the mutation is mutation of serine 254 of the IgGl human heavy chain to threonine (S254T).
  • the S to T mutation is mutation of S34 of SEQ ID NO: 57 OR 59 to T.
  • the mutation is mutation of threonine 256 of the IgGl human heavy chain to glutamic acid (T256E).
  • the T to E mutation is mutation of T36 of SEQ ID NO: 57 OR 59 to E.
  • the plurality of mutation comprises M32Y, S34T and T36E of SEQ ID NO: 57 OR 59.
  • the plurality of mutation comprises M252Y, S254T and T256E of the IgGl human heavy chain.
  • the mutation is mutation of asparagine 297 of the IgGl human heavy chain (N297).
  • 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: 57 OR 59 (N77A or N77Q).
  • the mutation increases the half-life of the molecule, peptide, polypeptide or protein complex.
  • a mutation that increases half-life is a mutation that increases binding to the neonatal Fc receptor (FcRn).
  • a mutation that increases binding to FcRn is selected from the mutations provided in Table 4.
  • the mutation is mutation of asparagine 434 to histidine (N434H).
  • N434H mutated Fc domain comprises an N214H mutation of SEQ ID NO: 57 or 59.
  • the mutation is mutation of valine 308 to proline (V3O8P).
  • an H435A mutated Fc domain comprises an H215A mutation of SEQ ID NO: 57 or 59. 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: 57 or 59. In some embodiments, the mutation that increases binding to FcRn is a plurality of mutations.
  • 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).
  • M252Y/S254T/T256E mutated Fc domain comprises an M32Y, S34T and T35E mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of mutation of methionine 428 to leucine (M428E) and mutation of asparagine 434 to serine (N434S) (also termed ES).
  • an M428E/N434S mutated Fc domain comprises an M208E and N214S mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of M428E and mutation of asparagine 434 to alanine (N434A) (also termed LA).
  • N434A asparagine 434 to alanine
  • an M428L/N434A mutated Fc domain comprises an M208L and N214A mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of mutation of threonine 250 to glutamine (T250Q) and mutation of methionine 428 to leucine (M428L) (also termed QL).
  • an T250Q/M428L mutated Fc domain comprises an T30Q and M208L mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of mutation of histidine 433 to lysine (H433K) and mutation of asparagine 434 to phenylalanine (N434F).
  • an H433K/N434F mutated Fc domain comprises an H213K and N214F mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of M252Y, S254T, T256E, H433K and N434F.
  • an M252Y/S254T/T256E/H433K/N434F mutated Fc domain comprises an M32Y, S34T, T35E, H213K and N214F mutation of SEQ ID NO: 57 or 59.
  • 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).
  • an T307A/E380A/N434A mutated Fc domain comprises an T87A, E160A and N214A mutation of SEQ ID NO: 57 or 59.
  • 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).
  • M252Y/V308P/N343Y mutated Fc domain comprises an M32Y, V88P and N123Y mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of M252Y, mutation of valine 308 to proline (V3O8P) and mutation of asparagine 434 to tyrosine (N434Y).
  • an M252Y/V308P/N434Y mutated Fc domain comprises an M32Y, V88P and N214Y mutation of SEQ ID NO: 57 or 59.
  • 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).
  • H258D/T307Q/A378V mutated Fc domain comprises an H38D, T87Q and A 158V mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of mutation of leucine 309 to aspartic acid (L309D), mutation of glutamine 311 to histidine (Q311H) and mutation of asparagine 434 to serine (N434S).
  • L309D/Q311H/N434S mutated Fc domain comprises an L89D, Q91H and N214A mutation of SEQ ID NO: 57 or 59.
  • the plurality that attenuates binding comprises or consists of mutation of isoleucine 253 to alanine (I253A), H435A and mutation of histidine 436 to alanine (H436A).
  • an I253A/H435A/H436A mutated Fc domain comprises an I33A, H215A and H216A mutation of SEQ ID NO: 57 or 59.
  • the plurality that attenuates binding comprises or consists of 1253 A, mutation of histidine 310 to alanine (H310A) and H435A.
  • an I253A/H310A/H435A mutated Fc domain comprises an I33A, H90A and H215A mutation of SEQ ID NO: 57 or 59.
  • the mutation is a mutation that decreases binding to an Fc receptor.
  • the Fc receptor is FcyR.
  • FcyR is FcyRI.
  • the mutation is a mutation that decreases binding to Clq.
  • a mutation that decreases binding to Fc receptor decreases ADCC.
  • the mutation is mutation of N297. As N-glycans are linked to N297 its mutation abrogates the glycosylation of this residue.
  • mutation of N297 is mutation to alanine (N297A).
  • mutation of N297 is mutation to glutamine (N297Q).
  • mutation of N297 is mutation to glycine (N297G).
  • an N297A mutated CH2 domain comprises an N59A mutation of SEQ ID NO: 36.
  • an N297A mutated Fc domain comprises an N77A mutation of SEQ ID NO: 57 or 59.
  • an N297Q mutated CH2 domain comprises an N59Q mutation of SEQ ID NO: 36.
  • an N297Q mutated Fc domain comprises an N77Q mutation of SEQ ID NO: 57 or 59.
  • an N297G mutated CH2 domain comprises an N59G mutation of SEQ ID NO: 36.
  • an N297G mutated Fc domain comprises an N77G mutation of SEQ ID NO: 57 or 59.
  • the mutation is a plurality of mutations that decrease binding to an Fc receptor.
  • the plurality comprises or consists of glycine 236 to arginine (G236R) and mutation of leucine 328 to arginine (L328R).
  • G236R/L328R mutated Fc comprises a hinge domain comprising a G21R mutation of SEQ ID NO: 22 and a CH2 domain comprising a L90R mutation of SEQ ID NO: 36.
  • a G236R/L328R mutated Fc domain comprises an G16R and L108R mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of serine 298 to glycine (S298G) and mutation of threonine 299 to alanine (T299A).
  • S298G/T299A mutated CH2 domain comprises a S60G and T61A mutation of SEQ ID NO: 36.
  • a S298G/T299A mutated Fc domain comprises an S78G and T79A mutation of SEQ ID NO: 57 or 59.
  • 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).
  • L234F/L235E/D265A mutated Fc comprises a hinge domain comprising a L19F and L20E mutation of SEQ ID NO: 22 and a CH2 domain comprising a D27A mutation of SEQ ID NO: 36.
  • a L234F/L235E/D265A mutated Fc domain comprises an L14F, L15E and D45A mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of leucine 234 to alanine (L234A), leucine 235 to alanine (L235A) and mutation of proline 329 to glycine (P329G).
  • an L234A/L235A/P329G mutated Fc comprises a hinge domain comprising a L19A and L20A mutation of SEQ ID NO: 22 and a CH2 domain comprising a P91G mutation of SEQ ID NO: 36.
  • a L234A/L235A/P329G mutated Fc domain comprises an L14A, L15A and P109G mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of L234F, L235E and mutation of proline 331 to serine (P331S).
  • an L234F/L235E/P331S mutated Fc comprises a hinge domain comprising a L19F and L20E mutation of SEQ ID NO: 22 and a CH2 domain comprising a P93S mutation of SEQ ID NO: 36.
  • a L234F/L235E/P331S mutated Fc domain comprises an L14F, L15E and PH IS mutation of SEQ ID NO: 57 or 59.
  • 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).
  • L235A/G237A/E318A mutated Fc comprises a hinge domain comprising a L20A and G22A mutation of SEQ ID NO: 22 and a CH2 domain comprising a E80A mutation of SEQ ID NO: 36.
  • a L235A/G237A/E318A mutated Fc domain comprises an L15A, G17A and E98A mutation of SEQ ID NO: 57 or 59.
  • the Fc is modified to decrease binding to Fc receptor.
  • the modification is removal of glycosylation.
  • Fc glycosylation is removed enzymatically.
  • enzymatic de-glycosylation is performed with a deglycosylase.
  • 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.
  • the mutation is a mutation that increases binding to an Fc receptor.
  • the Fc receptor is selected from FcyRI, FcyRIIA, FcyRIIIA, and FcyRIIIB.
  • the Fc receptor is FcyRI.
  • the mutation is mutation of serine 267 to glutamic acid (S267E).
  • S267E mutated CH2 domain comprises an S29E mutation of SEQ ID NO: 36.
  • an S267E mutated Fc domain comprises an S47E mutation of SEQ ID NO: 57 or 59.
  • the mutation is mutations of proline 238 to aspartic acid (P238D).
  • a P238D mutated hinge domain comprises an P23D mutation of SEQ ID NO: 22.
  • a P238D mutated Fc domain comprises an P18D mutation of SEQ ID NO: 57 or 59.
  • the mutation is a plurality of mutations that increase binding to an Fc receptor.
  • the plurality comprises or consists of S267E and mutation of leucine 328 to phenylalanine (L328F) (also termed SELF).
  • an S267E/L328F mutated CH2 domain comprises an S29E and L90F mutation of SEQ ID NO: 36.
  • an S267E/L328F mutated Fc domain comprises an S47E and L108F mutation of SEQ ID NO: 57 or 59.
  • 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).
  • an S267E/H268F/S324T mutated CH2 domain comprises an S29E, H30F and S86T mutation of SEQ ID NO: 36.
  • an S267E/H268F/S324T mutated Fc domain comprises an S47E, H48F and S104T mutation of SEQ ID NO: 57 or 59.
  • 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).
  • a G237D/P238D/P271G/A330R mutated polypeptide comprises a mutated hinge domain comprising a G22D and P23D mutation of SEQ ID NO: 22 and a mutated CH2 domain comprising a P33G and A92R mutation of SEQ ID NO: 36.
  • a G237D/P238D/P271G/A330R mutated Fc domain comprises a G17D, P18D, P51G and Al 10R mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of mutation of G237D, P238D, histidine 268 to aspartic acid (H268D), P271G and A33OR (also termed VI 1).
  • a G237D/P238D/H268D/P271G/A330R mutated polypeptide comprises a mutated hinge domain comprising a G22D and P23D mutation of SEQ ID NO: 22 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 36.
  • a G237D/P238D/H268D/P271G/A330R mutated Fc domain comprises a G17D, P18D, H48D, P51G and A110R mutation of SEQ ID NO: 57 or 59.
  • the plurality comprises or consists of mutation of glutamic acid 233 to aspartic acid (E233D), G237D, P238D, H268D, P271G and A33OR (also termed V12).
  • a E233D/G237D/P238D/H268D/P271G/A330R mutated polypeptide comprises a mutated hinge domain comprising a E18D, G22D and P23D mutation of SEQ ID NO: 22 and a mutated CH2 domain comprising a H30D, P33G and A92R mutation of SEQ ID NO: 36.
  • a E233D/G237D/P238D/H268D/P271G/A330R mutated Fc domain comprises a E13D, G17D, P18D, H48D, P51G and A110R mutation of SEQ ID NO: 57 or 59.
  • 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.
  • a mutation that increases ADCC is the EFT plurality of mutations.
  • P238D demonstrated enhanced binding to FcyRIIB with about 4.3-fold increased affinity in comparison to WT human IgGl.
  • P238D also significantly reduces the binding toward all other activating Fcg receptors.
  • V9 significantly enhanced the affinity of antibodies toward hFcyRIIB, by approximately a 32-fold change in comparison to WT IgGl.
  • V9 also was found to reduce the affinity toward hFcyRIIA R131 allele by about 3-fold in comparison to WT IgGl.
  • Vl l was found to significantly enhance the affinity of antibodies for hFcyRIIB by approximately 96-fold, while reducing the affinity toward hFcyRIIA R131 by about 3-fold in comparison to human WT IgGl .
  • V12 demonstrated significant enhancement of binding toward FcyRIIB, with 217-fold change in comparison to human WT IgGl.
  • V12 mutations also show no detectable binding toward FcyRIIIA allotypes, reduced FcyRI binding (0.061-fold change relative to WT IgGl) and FcyRIIA-H131 (0.068-fold change relative to wt IgGl). It should be noted that VI 2 slightly improves the binding toward FcyRIIA-R131, with a 2-fold binding increase in compared to WT hlgGl.
  • 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.
  • 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.
  • 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.
  • a mutation that increases ADCC is a plurality of mutations that increase ADCC.
  • 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.
  • 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: 57.
  • the plurality of mutations comprises mutation of serine 239 to aspartic acid (S239D) and isoleucine 332 to glutamic acid (I332E) within human IgGl.
  • the plurality of mutations comprises mutation of serine 19 to aspartic acid (S19D) and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 57.
  • the S239D/I332E mutations also increase ADCP.
  • 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.
  • 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: 57.
  • the S239D/A330L/I332E mutations also increase ADCP.
  • 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.
  • 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: 57.
  • 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.
  • 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: 57.
  • 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.
  • 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: 57.
  • the plurality of mutations comprises mutation of proline 247 to isoleucine (P247I), and alanine 339 to glutamine (A339Q) within human IgGl.
  • 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: 57.
  • 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.
  • 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: 57.
  • the G236A/S239D/I332E mutations also increase ADCP.
  • 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.
  • the plurality of mutations comprises mutation of mutation of lysine 14 to tyrosine (L14Y), lysine 15 to glutamine (L15Q), glycine 16 to tryptophan (G16W), serine 19 to methionine (S19M), histidine 48 to aspartic acid (H48D), aspartic acid 50 to glutamic acid (D50E) and serine 78 to alanine (S78A) within a first chain of SEQ ID NO: 57 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: 57.
  • the Fc domain with increased ADCC comprises L15V/F23L/R72P/Y80L/P176L mutations within the Fc domain.
  • the Fc domain is selected from SEQ ID NO: 57 and SEQ ID NO: 59.
  • the Fc domain with increased ADCC comprises
  • the Fc domain with increased ADCC consists of SEQ ID NO: 61.
  • the Fc comprising the L235V/F243L/R292P/Y300L/P396L mutations is SEQ ID NO: 61.
  • the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 61 and comprises L15V/F23L/R72P/Y80L/P176L mutations.
  • the Fc domain with increased ADCC comprises S19D/A110E/I112E mutations within the Fc domain.
  • the Fc domain is selected from SEQ ID NO: 57 and SEQ ID NO: 59.
  • the Fc domain with increased ADCC comprises
  • the Fc domain with increased ADCC consists of SEQ ID NO: 62.
  • the Fc comprising the S19D/A110E/I112E mutations is SEQ ID NO: 62.
  • the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 62 and comprises S19D/A110E/I112E mutations.
  • the mutation increases CDC.
  • a mutation that increases CDC is a plurality of mutations that increase CDC.
  • the Fc domain with increased CDC comprises G16A/S47E/H48F/S 104T/I112E mutations within the Fc domain.
  • the Fc domain is selected from SEQ ID NO: 57 and SEQ ID NO: 59.
  • the Fc domain with increased CDC comprises
  • the Fc domain with increased CDC consists of SEQ ID NO: 63.
  • the Fc comprising the G16A/S47E/H48F/S104T/I112E mutations is SEQ ID NO: 63.
  • the Fc domain with increased CDC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 63 and comprises G16A/S47E/H48F/S104T/I112E mutations.
  • the Fc domain with increased ADCC comprises G16A/A110L/I112E mutations within the Fc domain.
  • the Fc domain is selected from SEQ ID NO: 57 and SEQ ID NO: 59.
  • the Fc domain with increased ADCC comprises
  • the Fc domain with increased ADCC consists of SEQ ID NO: 64.
  • the Fc comprising the G16A/A110L/I112E mutations is SEQ ID NO: 64.
  • the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 64 and comprises G16A/A110L/I112E mutations.
  • the effector domain is selected from SEQ ID NO: 61-64. In some embodiments, the effector domain comprises any one of SEQ ID NO: 61-64. In some embodiments, the effector domain consists of any one of SEQ ID NO: 61-64. In some embodiments, the effector domain is selected from SEQ ID NO: 61, 62 and 64. In some embodiments, the effector domain comprises any one of SEQ ID NO: 61, 62 and 64. In some embodiments, the effector domain consists of any one of SEQ ID NO: 61, 62 and 64.
  • the effector domain comprises at least 75, 80, 85, 90, 92, 95, 97 or 99% identity to any one of SEQ ID NO: 61, 62 and 64 and retains increased ADCC as compared to a control Fc domain.
  • the control Fc domain is an unmodified Fc domain.
  • unmodified Fc is an Fc found in nature.
  • unmodified Fc is a human Fc found in nature.
  • the Fc is modified to increase ADCC.
  • the modification is removal of fucosylation.
  • Fc fucosylation is removed enzymatically.
  • the Fc is afucosylated.
  • the method comprises performing afucosylation of the molecule.
  • the molecules of the invention are produced in a cell line engineered to produce afucosylated molecules.
  • the mutation increases CDC. In some embodiments, a plurality of mutations increases CDC.
  • 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.
  • G236A glycine 236 to alanine
  • S267E serine 267 to glutamic acid
  • H268F histidine 268 for phenylamine
  • S324T serine 324 to threonine
  • I332E isoleucine 332 to glutamic acid
  • 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: 57.
  • the plurality of mutation comprises mutation of lysine 326 to tryptophan (K326W) and glutamic acid 333 to serine (E333S) within human IgGl.
  • the plurality of mutations comprises mutation of lysine 106 to tryptophan (K106W) and glutamic acid 113 to serine (El 13S) within SEQ ID NO: 57.
  • 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.
  • 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: 57. It will be understood that all of the above recited mutations given with respect to SEQ ID NO: 57 also apply to SEQ ID NO: 59. Indeed, they also apply to SEQ ID NO: 58 and SEQ ID NO: 60, but all numbering given hereinabove must be increased by 5 for these sequences.
  • the effector moiety is a drug.
  • the protein is an ITGA2B/ITGB3 ECD drug conjugate.
  • the protein is an ITGA2B/ITGB3-Fc drug conjugate.
  • the complex is an ITGA2B/ITGB3 ECD drug conjugate.
  • the complex is an ITGA2B/ITGB3 ECD fragment drug conjugate.
  • the complex is an ITGA2B/ITGB3 -Fc drug conjugate.
  • the effector moiety is cytotoxic.
  • the effector moiety is radioactive.
  • the effector moiety is a radioactive moiety.
  • 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.
  • 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.
  • the chemotherapeutic is an anthracy cline.
  • 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.
  • the chemotherapeutic is an anthramycin -based dimer.
  • the anthramycin-based dimer is a pyrrolobenzodiazepine (PBD).
  • the chemotherapeutic is PBD.
  • the anthramycin-based dimer is an indolinobenzodiazepine dimers (IGN).
  • the chemotherapeutic is a pyrridinobenzodiazepine (PDD).
  • the anthramycin-based dimer is PDD.
  • 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.
  • the PBD is a PBD dimer.
  • PBDs and PDDs include, but are not limited to anthramycin, SJG-136, NS 694501 and FGX2-62.
  • the PBD is anthramycin.
  • the effector moiety is anthramycin.
  • anthramycin is anthramycin-methyl-ether (AME).
  • anthramycin is an anthramycin based dimer.
  • the PBD is tesirine (SG3249). In some embodiments, tesirine is SG3199.
  • the chemotherapeutic SG3249. In some embodiments, the chemotherapeutic is SG3199. [0185] 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.
  • the chemotherapeutic is camptothecin or an analog thereof.
  • the effector moiety is camptothecin or an analog thereof.
  • the effector moiety is camptothecin.
  • Examples of analogs of camptothecin include, but are not limited to exatecan, SN-38, and deruxtecan (Dxd).
  • the camptothecin analog is Dxd.
  • the chemotherapeutic is Dxd.
  • the effector moiety is Dxd.
  • the chemotherapeutic is a duocarmycin.
  • 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.
  • the chemotherapeutic is triptolide.
  • the effector moiety is triptolide.
  • the effector moiety is a tubulin inhibitor.
  • the effector moiety is a maytansinoid.
  • 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.
  • the maytansinoid is mertansine (DM-1).
  • mertansine is emtansine.
  • the tubulin inhibitor is an auristatin.
  • the auristatin is selected from Monomethyl auristatin E (MMAE) and Monomethyl auristatin F (MMAF).
  • the tubulin inhibitor is a tubulysin.
  • the tubulysin is tubulysin A.
  • the auristatin is MMAE.
  • the auristatin is MMAF.
  • the effector moiety is MMAE.
  • the effector moiety is MMAF. [0190]
  • the effector moiety is a combination of moieties. In some embodiments, the effector moiety is a plurality of effector moieties.
  • the effector moiety is a combination of cytotoxic moieties.
  • 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.
  • 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.
  • the protein complex further comprises a third polypeptide chain.
  • the third polypeptide chain comprises a third fragment of a protein target of ITP autoantibodies.
  • the third fragment is different than the first fragment.
  • the third fragment is different than the second fragment.
  • the third fragment is the same as the first fragment.
  • the first fragment is the same as the second fragment.
  • the third fragment is the same as the first and second fragments.
  • the same as is the same sequence. In some embodiments, different is a different sequence.
  • the third polypeptide further comprises a third dimerization domain.
  • the first polypeptide further comprises a fourth dimerization domain.
  • the third and fourth dimerization domains are capable of dimerizing to each other.
  • the third and fourth dimerization domains are configured to dimerizing to each other.
  • the third dimerization domain is not configured to dimerize to the first dimerization domain.
  • the third dimerization domain is not configured to dimerize to the second dimerization domain.
  • the fourth dimerization domain is not configured to dimerize to the first dimerization domain.
  • 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.
  • the protein complex further comprises a fourth polypeptide chain.
  • the fourth polypeptide chain comprises a fourth fragment of a protein target of ITP autoantibodies.
  • the fourth fragment is different than the first fragment.
  • the fourth fragment is different than the second fragment.
  • the fourth fragment is different than the third fragment.
  • the fourth fragment is the same as the first fragment.
  • the fourth fragment is the same as the second fragment.
  • the fourth fragment is the same as the third fragment.
  • 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.
  • the first, second, third and fourth fragments are all different. In some embodiments, the same as is the same sequence. In some embodiments, different is a different sequence. In some embodiments, different is from a different protein. In some embodiments, different is from the same protein but comprising a different sequence. In some embodiments, different is from the same protein but from a different region of the protein. In some embodiments, at least two of the first, second, third and fourth proteins are part of a single protein complex. In some embodiments, the protein complex is a complex in mammals. In some embodiments, the protein complex is a complex in humans.
  • the fourth polypeptide further comprises a fifth dimerization domain.
  • the second polypeptide further comprises a sixth dimerization domain.
  • the fifth and sixth dimerization domains are capable of dimerizing to each other.
  • the fifth and sixth dimerization domains are configured to dimerizing to each other.
  • the fifth dimerization domain is not configured to dimerize to the first dimerization domain.
  • the fifth dimerization domain is not configured to dimerize to the second dimerization domain.
  • the fifth dimerization domain is not configured to dimerize to the third dimerization domain.
  • the fifth dimerization domain is not configured to dimerize to the fourth dimerization domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • the first polypeptide and second polypeptide do not both comprise a CHI domain.
  • first polypeptide and second polypeptide both both comprise a CHI domain
  • first polypeptide and second polypeptide both both comprise a CL domain.
  • first polypeptide and second polypeptide do not both comprise a CL domain.
  • the first polypeptide comprises a CHI domain
  • the second polypeptide comprises a CL domain
  • the third polypeptide comprises a CL domain and the fourth polypeptide comprise a CHI domain.
  • the first polypeptide comprises a CL domain and the second polypeptide comprises a CHI domain.
  • the third polypeptide comprises a CHI domain
  • the fourth polypeptide comprise a CL domain.
  • 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.
  • the composition comprises a polypeptide chain comprising the fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and the fragment of a second protein target of ITP autoantibodies or an analog or derivative thereof.
  • the polypeptide chain is a single polypeptide chain.
  • the single chain comprises the fragment of the first protein and the fragment of the second protein.
  • the polypeptide chain further comprises a fragment of a third protein target of ITP autoantibodies or an analog or derivative thereof.
  • the polypeptide chain further comprises a fragment of a fourth protein target of ITP autoantibodies or an analog or derivative thereof.
  • the polypeptide chain further comprises an Fc region.
  • the polypeptide chain further comprises an effector moiety.
  • the fragment of the first protein target of ITP autoantibodies or an analog or derivative thereof is separated from the fragment of the second protein target of ITP autoantibodies or an analog or derivative thereof by a linker.
  • the fragment of the third protein target of ITP autoantibodies or an analog or derivative thereof is separated from the fragment of the first or the second protein target of ITP autoantibodies or an analog or derivative thereof by a linker.
  • the fragment of the fourth protein target of ITP autoantibodies or an analog or derivative thereof is separated from the fragment of the first, the second or the third protein target of ITP autoantibodies or an analog or derivative thereof by a linker.
  • a fragment is separated from the Fc region by a linker.
  • the effector moiety is separated attached by a linker.
  • the effector moiety is separated by the fragment by a linker.
  • the fragment and the dimerization domain are separated by a linker.
  • the dimerization domain and the Fc region are separated by a linker.
  • the fragment and the Fc region are separated by a linker.
  • the effector moiety is separated attached by a linker.
  • the effector moiety is separated by the fragment by a linker.
  • the linker is an amino acid linker.
  • 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.
  • a linker is a chemical linker.
  • chemical linker is a polyethylene glycol (PEG) linker.
  • the PEG linker is a Gly3- PEG-azide linker.
  • the linker is a dibenzocyclooctyne group (DBCO) linker.
  • the DBCO linker is a DBCO-C6 linker.
  • the DBCO linker is a DBCO-Gly5-EDA linker.
  • the linker is dimethylethylenediamine (DMEDA) linker.
  • 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.
  • DMAE N- dimethylethylenediamine
  • 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.
  • linkers include for example SPDB linkers, SMCC linkers, MCC linkers, and butanoic acid linkers.
  • the linker is a p-aminobenzyl (PAB) linker.
  • the linker is a p- aminocarbamate (PABC) linker.
  • the linker is a Maleimidocaproyl (me) linker.
  • the linker comprises me.
  • the linker is a Val-Cit-PAB linker.
  • the linker is a Val-Cit-PABC linker.
  • the linker is a Val-Cit-PAB -MM AE linker.
  • 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.
  • MMAE monomethyl auristatin E
  • 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.
  • the linker is a DB CO-PEG- VC-PAB-DMED A linker.
  • VC in the linker is replaced with EVC.
  • VC in the linker is replaced with EVA.
  • the fragment and the dimerization domains are linked by a non-cleavable linker.
  • the fragment and the dimerization domains are linked by a cleavable linker.
  • the effector moiety is linked by a cleavable linker.
  • the effector moiety is linked by a non-cleavable linker.
  • 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 effector moiety is conjugated by a linker.
  • 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.
  • 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.
  • enzymatic conjugation include, but are not limited to: Click chemistry, sortase assisted-SMAC technology, transglutaminase addition of amine azide, and glycan remodeling.
  • 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 ITGA2B or ITGB3 fragment. In some embodiments, the conjugation or linking does not interfere with antibody binding to an ITGA2B or ITGB3 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.
  • 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.
  • the asparagine is N297 of the heavy chain of IgG.
  • the residue is a glutamine.
  • N297 is converted, engineered, or mutated to glutamine (N297Q).
  • 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.
  • the linking or conjugating is to a C- or N- terminus of a chain of the agent of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • the linker comprises GGGGS (SEQ ID NO: 6). In some embodiments, the linker consists of SEQ ID NO: 6. In some embodiments, the linker comprises (GGGGS)n wherein n is an integer. In some embodiments, the linker consists of (GGGGS)n wherein n is an integer. In some embodiments, the linker comprises GSAGSAAGSGEF (SEQ ID NO: 51). In some embodiments, the linker comprises or consists of (GGGS)nGS wherein n is an integer. In some embodiments, n is selected from 1, 2 ,3, 4 and 5. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.
  • n is 5. In some embodiments, the linker comprises or consists of SEQ ID NO: 7. In some embodiments, the linker comprises or consists of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 52). In some embodiments, the linker consists of (GGGS)n wherein n is an integer.
  • the linker is a rigid linker.
  • the rigid linker comprises EAAAK (SEQ ID NO: 65).
  • the rigid linker consists of SEQ ID NO: 65.
  • the rigid linker comprises (EAAAK)n where n is an integer.
  • the rigid linker consists of (EAAAK)n where n is an integer.
  • the rigid linker comprises (EAAAK)nGS where n is an integer.
  • the rigid linker consists of (EAAAK)nGS where n is an integer.
  • the rigid linker comprises (EAAAK)nGGS where n is an integer.
  • 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.
  • the dimerization domain is C-terminal to the fragment. In some embodiments, the fragment is C-terminal to the dimerization domain. In some embodiments, the Fc region is C-terminal to the fragment. In some embodiments, the fragment is C-terminal to the Fc region. In some embodiments, the dimerization domain is C-terminal to the Fc region. In some embodiments, the Fc region is C-terminal to the dimerization domain. In some embodiments, the dimerization domain is N-terminal to the fragment. In some embodiments, the fragment is N-terminal to the dimerization domain. In some embodiments, the Fc region is N-terminal to the fragment. In some embodiments, the fragment is N-terminal to the Fc region. In some embodiments, the dimerization domain is N-terminal to the Fc region. In some embodiments, the dimerization domain is N-terminal to the Fc region. In some embodiments, the dimerization domain is N-terminal to the Fc region. In some embodiments, the
  • the epitope spans at least two fragments. In some embodiments, the epitope spans the first and second fragments. In some embodiments, the epitope spans the first and third fragments. In some embodiments, the epitope spans the first and fourth fragments. In some embodiments, the epitope spans the second and third fragments. In some embodiments, the epitope spans the second and fourth fragments. In some embodiments, the epitope spans the third and fourth fragments. In some embodiments, the epitope spans two proteins. In some embodiments, the epitope spans two proteins in a protein complex. In some embodiments, the epitope spans three fragments. In some embodiments, the epitope spans three proteins.
  • the epitope spans four fragments. In some embodiments, the epitope spans four proteins. In some embodiments, the epitope is a complex epitope. In some embodiments, the epitope is a B cell receptor (BCR)-specific epitope. [0208] In some embodiments, all fragments are from ITGA2B. In some embodiments, all fragments are from ITGB3. In some embodiments, the complex comprises a fragment from ITGA2B and a fragment from ITGB3. In some embodiments, a fragment from ITGA2B is mutated. In some embodiments, a fragment from ITGB3 is mutated. In some embodiments, both a fragment from ITGA2B and a fragment from ITGB3 are mutated.
  • the ITGA2B fragment and the ITGB3 fragment bear the same or equivalent mutations. In some embodiments, the ITGA2B and ITGB3 fragment bear different and nonequivalent mutations. Equivalent mutations are mutations of the same amino acid, but with a slightly different numbered position due to differences in ITGA2B/B3 sequence. In some embodiments, an equivalent mutation is a homologous mutation.
  • the complex comprises an ITGA2B truncation. In some embodiments, the complex comprises an ITGB3 truncation. In some embodiments, the complex comprises both a ITGA2B and ITGB3 mutation. In some embodiments, the ITGA2B and ITGB3 truncations are truncations of the same length. In some embodiments, the ITGA2B and ITGB3 truncations are truncations of different length. In some embodiments, the ITGA2B and ITGB3 truncations are truncations of the same domains.
  • the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVETVEHQDWENGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 53).
  • the second polypeptide comprises a fragment linked to SEQ ID NO: 53.
  • the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 53.
  • the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPITPLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 54).
  • the second polypeptide comprises a fragment linked to SEQ ID NO: 54. In some embodiments, the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 54. [0211] In some embodiments, the first polypeptide comprises a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 55). In some embodiments, the second polypeptide comprises a fragment linked to SEQ ID NO: 55. In some embodiments, the first and second polypeptide
  • the first polypeptide comprises a fragment linked to SEQ ID NO: 9.
  • the second polypeptide comprises a fragment linked to SEQ ID NO: 9.
  • the first and second polypeptides both comprises a fragment linked to SEQ ID NO: 9.
  • the first polypeptide comprises a fragment linked to SEQ ID NO: 10.
  • the second polypeptide comprises a fragment linked to SEQ ID NO: 10.
  • the first and second polypeptides both comprise a fragment linked to SEQ ID NO: 910.
  • the first polypeptide comprises a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56).
  • the second polypeptide comprises a fragment linked to SEQ ID NO: 56.
  • the third polypeptide comprises a fragment linked to SEQ ID NO: 56.
  • the fourth polypeptide comprises a fragment linked to SEQ ID NO: 56.
  • the third polypeptide comprises a fragment linked to SEQ ID NO: 53. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 54. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 55. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 56. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 9. In some embodiments, the third polypeptide comprises a fragment linked to SEQ ID NO: 10. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 53. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 54.
  • the fourth polypeptide comprises a fragment linked to SEQ ID NO: 55. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 56. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 9. In some embodiments, the fourth polypeptide comprises a fragment linked to SEQ ID NO: 10.
  • a polypeptide chain comprises or consists of an amino acid sequence selected from SEQ ID NO: 5, 8 and 11-14. Each sequence represents a separate embodiment of the invention. In some embodiments, a polypeptide chain comprises or consists of an amino acid sequence selected from SEQ ID NO: 5, 8 and 11- Mor an analog or derivative thereof with at least 85% identity. Each sequence represents a separate embodiment of the invention. In some embodiments, a polypeptide chain comprises or consists of an amino acid sequence selected from Table 2. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 5, 8 and 11-14. Each sequence represents a separate embodiment of the invention.
  • the complex comprises or consists of two polypeptide chains selected from SEQ ID NO: 5, 8 and l l-14or an analog or derivative thereof comprising at least 85% identity.
  • Each sequence represents a separate embodiment of the invention.
  • the two chains are the same chain. In some embodiments, the two chains are different chains.
  • the complex comprises or consists of CRD-755. In some embodiments, the complex comprises or consists of CRD-756. In some embodiments, the complex comprises or consists of CRD-757. In some embodiments, the complex comprises or consists of CRD-758.
  • 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.
  • 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.
  • composition comprising a protein or polypeptide of the invention.
  • composition comprising a protein complex of the invention.
  • a pharmaceutical composition comprises a composition of the invention.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant.
  • carrier refers to any component of a pharmaceutical composition that is not the active agent.
  • 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.
  • sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethy
  • 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 nontoxic, 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.
  • compositions 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.
  • the carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.
  • the pharmaceutical composition comprises a therapeutically effective amount of the protein complex of the invention.
  • the pharmaceutical composition comprises a therapeutically effective amount of the conjugate of the invention.
  • therapeutically effective amount refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • 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.
  • an effective amount is an amount sufficient to treat at least one symptom of a disease.
  • the disease is ITP.
  • the disease is PF.
  • the disease is ITP.
  • ITP is characterized by autoantibodies against the protein.
  • ITP is characterized by autoantibody against ITGA2B and/or ITGB3.
  • treatment 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.
  • 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 ITP is well known in the art and may include any acceptable measure for assessing improvement of a ITP symptom.
  • This may include, Rituximab, steroids, steroid- sparing immunosuppressants (such as azathioprine, mycophenolate and cyclophosphamide) dapsone, intravenous immunoglobulin (IVIG) and the like. Treatment may include improved quality of life, suppression of blister formation, reduction of autoantibodies and killing of autoreactive B cells.
  • immunosuppressants such as azathioprine, mycophenolate and cyclophosphamide
  • IVIG intravenous immunoglobulin
  • 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. [0225] 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.
  • suitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal.
  • the administering is intravenous administering.
  • the administering is topical administration.
  • the administering is selected from oral, intravenous, intramuscular, intraperitoneal, intertumoral, topical, or subdermal administration.
  • administering is administering to a site of disease.
  • 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.
  • a method of treating ITP in a subject in need thereof comprising administering to the subject a protein or polypeptide of the invention, thereby treating ITP in a subject.
  • a method of treating ITP in a subject in need thereof comprising administering to the subject a protein complex of the invention, thereby treating ITP in a subject.
  • a method of treating ITP in a subject in need thereof comprising administering to the subject a composition of the invention, thereby treating ITP in a subject.
  • the administering is administering a pharmaceutical composition of the invention.
  • ITP is characterized by antibodies against the protein.
  • the protein is a target of ITP antibodies. It will be understood by the skilled artisan that a protein complex will be designed with fragments of proteins which are targeted by ITP antibodies in the subject.
  • antibodies are autoantibodies.
  • the disease is ITP and the autoantibodies are against ITGA2B, ITGB3 or both.
  • treating comprises lowering antibody concentration.
  • treating comprises lower antibody number.
  • antibody concentration is circulating antibody concentration.
  • treating comprises depleting antibodies.
  • treating comprises killing B cells.
  • the B cell are autoreactive B cells.
  • killing B cells is specific B cell killing.
  • treating comprises killing B cells that produce the antibodies.
  • treating comprises killing B cells that produce the antibodies and the not substantially killing other B cells.
  • treating comprises killing B cell that produce antibodies against the protein complex.
  • treating comprises killing B cell that produce antibodies against the fragment.
  • treating comprises killing B cell that produce antibodies against a fragment of the protein complex.
  • 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.
  • 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.
  • 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.
  • 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. [0233] 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.
  • 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.
  • the method comprises plasmapheresis of the antibodies before administering.
  • the method comprises administering a B cell targeting therapy before administering the therapeutic of the invention.
  • a B cell targeting therapy is an anti-B cell therapy.
  • the B cell targeting therapy is B cell lethal therapy.
  • the B cell targeting therapy is a pan B cell therapy.
  • the B cell targeting therapy is not a targeted therapy.
  • a “targeted B cell therapy” is a therapy that targets only specific B cell clones that produce specific antibodies.
  • 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.
  • the B cell targeting therapy is rituximab.
  • 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.
  • 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 ITP 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 ITP autoantibodies or an analog or derivative thereof and second dimerization domain.
  • nucleic acid molecule encoding a protein of the invention.
  • nucleic acid molecule encoding a polypeptide chain of a composition of the invention.
  • nucleic acid molecule encoding a composition of the invention.
  • nucleic acid molecule encoding a fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and fragment of a second human protein target of ITP autoantibodies or an analog or derivative thereof.
  • the nucleic acid system further comprises a third nucleic acid molecule that encodes a third polypeptide of the protein complex of the invention. In some embodiments, the nucleic acid system further comprises a fourth nucleic acid molecule that encodes a fourth polypeptide of the protein complex of the invention. In some embodiments, a first nucleic acid molecule encodes the first polypeptide of the invention. In some embodiments, a second nucleic acid molecule encodes the second polypeptide of the invention. In some embodiments, a third nucleic acid molecule encodes the third polypeptide of the invention. In some embodiments, a fourth nucleic acid molecule encodes the fourth polypeptide.
  • the nucleic acid molecule is a vector.
  • the vector is an expression vector.
  • nucleic acid molecule comprises an open reading frame encoding the polypeptide chain. Expressing of an open reading frame within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. Expression vectors are well known in the art and any vector compatible with a target cell in which the protein complex of the invention is being expressed may be used.
  • 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.
  • expression control element e.g., a promoter, enhancer
  • selectable marker e.g., antibiotic resistance
  • poly-Adenine sequence e.g., poly-Adenine sequence.
  • the vector comprises a promoter.
  • the promoter is configured for expression in a target cell in which the protein complex of the invention is being expressed.
  • 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.
  • the open reading frame is operably linked to a promoter.
  • 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).
  • 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.
  • electroporation e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • Heat shock e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • infection by viral vectors e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • Heat shock
  • 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.
  • 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.
  • expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention.
  • SV40 vectors include pSVT7 and pMT2.
  • vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • 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.
  • recombinant viral vectors which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression.
  • lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
  • the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles.
  • 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.
  • plant expression vectors are used.
  • the expression of a polypeptide coding sequence is driven by a number of promoters.
  • 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. 3:17-311 (1987)] are used.
  • plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.
  • 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.
  • the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
  • 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.
  • a method for producing a protein comprising: obtaining a first fragment of an extracellular domain of a first human receptor or an analog or derivative thereof, and a second fragment of an extracellular domain of a second human receptor or analog or derivative thereof, wherein the first and second human receptors are targets of ITP autoantibodies and linking the first fragment to the second fragment to produce a single polypeptide chain; thereby producing a protein.
  • a method for producing a protein comprising: obtaining a first fragment of an extracellular domain of a first human receptor or an analog or derivative thereof, and a second fragment of an extracellular domain of a second human receptor or analog or derivative thereof, wherein the first and second human receptors are targets of ITP autoantibodies, linking the first fragment to the second fragment to produce a single polypeptide chain and linking the single polypeptide chain to an effector moiety that is not an unmodified Fc domain; thereby producing a protein.
  • a method for producing a protein complex comprising: obtaining a first fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and a second fragment of a second protein target of ITP autoantibodies or an analog or derivative thereof, linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain; thereby producing a protein complex.
  • a method for producing a protein complex comprising: obtaining a first fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and a second fragment of a second protein target of ITP autoantibodies or an analog or derivative thereof, linking the first fragment to a first dimerization domain to produce a first polypeptide and linking the second fragment to a second dimerization domain to produce a second polypeptide chain, and linking the first polypeptide, the second polypeptide chain or both to an effector moiety that is not an unmodified Fc domain; thereby producing a protein complex.
  • a method for producing a protein comprising: culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single polypeptide chain, wherein the single polypeptide chain is produced by: i. obtaining a first fragment of an extracellular domain of a first human receptor or an analog or derivative thereof and a second fragment of an extracellular domain of a second human receptor or analog or derivative thereof, wherein the first and second human receptors are targets of ITP autoantibodies and are different proteins; and ii. linking the first fragment to the second fragment to produce a single polypeptide chain; thereby producing a protein.
  • a method for producing a protein comprising: culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single polypeptide chain, wherein the single polypeptide chain is produced by: i. obtaining a first fragment of an extracellular domain of a first human receptor or an analog or derivative thereof and a second fragment of an extracellular domain of a second human receptor or analog or derivative thereof, wherein the first and second human receptors are targets of ITP autoantibodies and are different proteins; ii. linking the first fragment to the second fragment to produce a single polypeptide chain; and iii. linking the single polypeptide chain to an effector moiety that is not an unmodified Fc domain; thereby producing a protein.
  • a method for producing a protein complex comprising: culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding at least two polypeptide chains, wherein the two polypeptide chains are produced by: i. obtaining a first fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and a second fragment of second protein target of ITP autoantibodies or analog or derivative thereof; and ii. linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain; thereby producing a protein complex.
  • a method for producing a protein complex comprising: culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding at least two polypeptide chains, wherein the two polypeptide chains are produced by: i. obtaining a first fragment of a first protein target of ITP autoantibodies or an analog or derivative thereof and a second fragment of second protein target of ITP autoantibodies or analog or derivative thereof; ii. linking the first fragment to a first dimerization domain to produce a first polypeptide chain and linking the second fragment to a second dimerization domain to produce a second polypeptide chain; and iii. linking the first polypeptide chain, the second polypeptide chain or both to an effector moiety that is not an unmodified Fc domain; thereby producing a protein complex.
  • the protein is a protein of the invention. In some embodiments, the protein is a polypeptide. In some embodiments, the truncation is a truncation of the invention. In some embodiments, the protein is a polypeptide of the invention. In some embodiments, the mutation is a mutation of the invention. In some embodiments, the protein complex is a protein complex of the invention. In some embodiments, the protein composition is a composition of the invention. In some embodiments, the protein is a protein of the invention. In some embodiments, the protein is a polypeptide chain of the invention. In some embodiments, the fragment is a fragment of the invention. In some embodiments, the derivative is a derivative of the invention.
  • the analog is an analog of the invention.
  • the dimerization domain is a dimerization domain of the invention.
  • the composition, protein complex, protein, fragment, analog, derivative or dimerization domain is such as is described hereinabove.
  • the method further comprises linking the protein, polypeptide or protein complex to an effector moiety.
  • the effector moiety is not an Fc domain.
  • the effector moiety does not comprise an Fc moiety.
  • the effector moiety is not an unmodified Fc domain.
  • the effector moiety is an Fc domain comprising at least one mutation that increases ADCC.
  • the protein is a human protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the first and second protein are the same protein. In some embodiments, the first and second protein are different proteins. In some embodiments, the first and second proteins are targets of ITP autoantibodies. In some embodiments, the first and second proteins are targets of autoantibodies associated with ITP . In some embodiments, ITP is characterized by autoantibodies against the first and second proteins. In some embodiments, the protein is a receptor, and the fragment is a fragment of the extracellular domain. In some embodiments, the fragment comprises a fragment of the extracellular domain. In some embodiments, the fragment consists of the extracellular domain.
  • the first and second dimerization domains are capable of dimerizing to each other. In some embodiments, the first and second dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first and second polypeptides. In some embodiments, the contacting comprises incubating the polypeptides together. In some embodiments, the contacting is in a cell. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is under conditions sufficient to allow dimerization. In some embodiments, allowing is inducing. In some embodiments, the conditions are sufficient to allow dimerization of the polypeptides. In some embodiments, the conditions are physiological conditions.
  • the method further comprises inserting a third dimerization domain into the first polypeptide.
  • inserting is linking.
  • inserting is inserting a nucleic acid sequence encoding the third dimerization domain into a nucleic acid molecule or vector encoding the first polypeptide.
  • the linking is linking the third dimerization domain to the first dimerization domain.
  • the linking is linking the third dimerization domain to the first fragment.
  • the method further comprises obtaining a third fragment of a third protein target of ITP autoantibodies or an analog or derivative thereof and linking it to a fourth dimerization domain to produce a third polypeptide chain.
  • the third and fourth dimerization domains are capable of dimerization to each other.
  • the third and fourth dimerization domains are configured to dimerize to each other.
  • the method further comprises contacting the first, second and third polypeptide chains.
  • the method further comprises expressing in the host cell a nucleic acid sequence encoding a third polypeptide chain.
  • the third polypeptide chain is produced by obtaining a third fragment of a third protein and linking it to a fourth dimerization domain to produce a third polypeptide chain.
  • the method comprises expression the first, second and third polypeptide chains in a cell. [0267]
  • the method further comprises inserting a fifth dimerization domain into the second polypeptide.
  • inserting is linking.
  • inserting is inserting a nucleic acid sequence encoding the fifth dimerization domain into a nucleic acid molecule or vector encoding the second polypeptide.
  • the linking is linking the fifth dimerization domain to the second dimerization domain.
  • the linking is linking the fifth dimerization domain to the second fragment.
  • the method further comprises obtaining a fourth fragment of a fourth protein target of ITP autoantibodies or an analog or derivative thereof and linking it to a sixth dimerization domain to produce a fourth polypeptide chain.
  • the fifth and sixth dimerization domains are capable of dimerization to each other.
  • the fifth and sixth dimerization domains are configured to dimerize to each other.
  • the method further comprises contacting the first, second, third and fourth polypeptide chains.
  • the method further comprises expressing in the host cell a nucleic acid sequence encoding a fourth polypeptide chain.
  • the fourth polypeptide chain is produced by obtaining a fourth fragment of a fourth protein and linking it to a sixth dimerization domain to produce a fourth polypeptide chain.
  • the method comprises expression the first, second, third and fourth polypeptide chains in a cell.
  • the method further comprises inserting an Fc region into the first chain. In some embodiments, the method further comprises inserting an Fc region into the second chain. In some embodiments, the method further comprises inserting an Fc region into the third chain. In some embodiments, the method further comprises inserting an Fc region into the fourth chain. In some embodiments, the method further comprises inserting a portion of an Fc region into the first chain and a portion of the Fc region into the second chain wherein and interface of the two portions produces a complete Fc region. In some embodiments, the Fc region is not an unmodified Fc region. In some embodiments, the Fc region comprises at least one mutation that increases ADCC.
  • an Fc region is inserted C-terminally to a dimerization domain. In some embodiments, an Fc region is inserted C-terminally to a fragment. In some embodiments, an Fc region is inserted N-terminally to a dimerization domain. In some embodiments, an Fc region is inserted N-terminally to a fragment. In some embodiments, a fragment is inserted or linked C-terminally to a dimerization domain. In some embodiments, a fragment is inserted or linked N-terminally to a dimerization domain.
  • the method further comprises inserting a linker between at least two sections of a polypeptide chain.
  • the linker is inserted between a fragment and a dimerization domain.
  • the linker is inserted between a fragment and an Fc region.
  • the linker is inserted between an Fc region and a dimerization domain.
  • the linker is inserted between a dimerization domain and another dimerization domain.
  • the linker is inserted between a fragment and another fragment.
  • the linker is inserted between a fragment of a first protein and a fragment of a second protein.
  • the method further comprises producing at least one mutation in the protein.
  • the mutation is made in fragment.
  • the mutation is made in extracellular domain.
  • the mutation is made in a cadherin domain of the fragment.
  • the method further comprises truncating the protein.
  • the method further comprises truncating the fragment.
  • the method further comprises truncating the extracellular domain.
  • the truncation removes at least one extracellular functional domain.
  • the truncation removes at least one cadherin domain.
  • the method further comprises measuring binding of autoantibodies to the protein. In some embodiments, the method further comprises measuring binding of autoantibodies to the fragment. In some embodiments, the method further comprises measuring binding of autoantibodies to the protein. In some embodiments, the method further comprises measuring binding of autoantibodies to the fragment. In some embodiments, the autoantibodies are autoantibodies to ITGB3. In some embodiments, the autoantibodies are autoantibodies to ITGA2B. In some embodiments, the autoantibodies are in serum. In some embodiments, the autoantibodies are in blood. In some embodiments, the measuring is measuring binding in serum. In some embodiments, the measuring is measuring binding in blood.
  • the serum or blood is from a subject suffering from ITP.
  • binding is depletion.
  • the measuring is measuring the depletion of the autoantibodies from the serum/blood by the protein.
  • the protein is conjugated to an artificial scaffold.
  • conjugated to is immobilized on.
  • the artificial scaffold is a bead.
  • the bead is a paramagnetic bead.
  • the bead is a Sepharose bead.
  • the bead is an avidin bead. In some embodiments, avidin is streptavidin.
  • a fragment is selected that binds at least a predetermined threshold of autoantibodies.
  • the method further comprises selecting a fragment that binds at least a predetermined threshold of autoantibodies.
  • the threshold is a threshold percent of autoantibodies. In some embodiments, the threshold is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97 or 99%. Each possibility represents a separate embodiment of the invention.
  • the threshold is at least 20%.
  • the threshold is at least 40%.
  • the threshold is at least 50%.
  • the threshold is at least 70%.
  • the threshold is at least 75%. In some embodiments, the threshold is at least 80%.
  • the protein is for use in a method of the invention.
  • the polypeptide is for use in a method of the invention.
  • the protein complex is for use in a method of the invention.
  • the method is a therapeutic method.
  • the method is a diagnostic method.
  • the method is a method of treatment.
  • the method is a method of determining suitability for treatment.
  • composition produced by a method of the invention.
  • a method of determining suitability of a subject to be treated by a method of the invention 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.
  • a method of determining suitability of a subject to be treated by a method of the invention 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.
  • a method of determining suitability of a subject to be treated by a method of the invention comprising receiving a sample from the subject, contacting the sample with a protein or polypeptide of the invention and determining binding of antibodies within the sample to the protein, wherein binding of the antibodies to the protein or polypeptide indicates the subject is suitable to be treated by a method of the invention, thereby determining suitability of the subject to be treated.
  • 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 ITP. In some embodiments, the subject is known to be positive for autoantibodies associated with ITP. 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 ITP. In some embodiments, the subject has received treatment and has relapsed.
  • the method comprises obtaining the sample from the subject.
  • the sample comprises tissue.
  • the sample is a biopsy.
  • the sample is a bodily fluid.
  • the bodily fluid is blood.
  • the bodily fluid is serum.
  • the bodily fluid is plasma.
  • the bodily fluid is a fluid that comprises antibodies.
  • 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.
  • contacting is incubating. In some embodiments, contacting is under conditions sufficient for binding of antibodies to the protein complex.
  • 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.
  • the composition further comprises a detectable moiety.
  • the protein complex further comprises a detectable moiety.
  • the protein further comprises a detectable moiety.
  • the method further comprises contacting the composition, complex and/or protein with a peptide comprising a detectable moiety.
  • the peptide is configured to bind the composition, protein and/or complex.
  • the peptide is specific to the composition, protein and/or complex.
  • the term “specific binding” refers to binding to a specific molecule to the exclusion of other molecules.
  • the peptide is specific to the composition, protein and/or complex to the exclusion of other proteins in the sample.
  • 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.
  • the determining binding comprises detecting the moiety. In some embodiments, the determining comprises isolating the protein complex. In some embodiments, the determining comprises eluting antibodies from the complex. Methods of protein identification are well known in the art and any such method may be used. Examples of such method include western blotting, ELISA, FACS analysis and protein sequencing, such as by mass spectrometry. In some embodiments, the determining comprises ELISA. In some embodiments, the ELISA is a competitive ELISA. In some embodiments, the competitive ELISA comprises competition with antibodies. In some embodiments, the antibodies are antibodies associated with the disease.
  • 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.
  • 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.
  • a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
  • BCR B-cell receptor
  • 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 differentiation state.
  • This approach is also beneficial in protecting and preserving non-autoreactive, including protective (e.g., anti-viral, anti-bacterial) subpopulations, which are damaged by treatments that are targeting nonspecific differentiation markers (e.g., CD20, CD38, BCMA) regardless of whether or not they are carrying an autoreactive BCR.
  • FIG. 1A shows one embodiment of the therapeutic agent of the invention.
  • Immunoglobulin (Ig)-like protein complex 101 comprises four polypeptide chains: two heavy-chain-like polypeptides 110 and two light-chain-like polypeptides 120. Chains 110 are able to dimerize via disulfide bonds between them. Further, chains 110 may comprise any or all of CH3 domain 111, CH2 domain 112, hinge region 113 and CHI domain 114. In this embodiment, the CH3 domain 111, CH2 domain 112, and hinge region 113 all comprise disulfide bonds and act as dimerization domains, though use of other dimerization domains is also possible.
  • IgGl human (or non-human) IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD domains for example.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • Chains 120 are able to dimerize with chains 110 via disulfide bonds found in CHI domain 114 and CL domain 124.
  • Chains 110 and 120 are devoid of variable regions, unlike naturally occurring or manmade antibodies. In place of the variable region each chain has a fragment 130 from the extracellular portion of the human Integrin Subunit Alpha 2b or Integrin Subunit Beta 3. Each chain can be generated to have the same fragment or derivative of ITGA2B or ITGB3 or different fragments/derivatives. Indeed, as shown in Figure IB, the two heavy chains 115 and 116 can be engineered separately such that chain 115 contains, for example a fragment of the extracellular domain of ITGA2B 131 and chain 116 contains a fragment of the extracellular domain of ITGA2B 132.
  • the therapeutic molecule can be designed with four copies of a protein or domain (Fig. 1C), two copies each of two different proteins or domains (Fig. ID), or one copy each of four different protein fragments or domains (Fig. IB) or any other combination thereof.
  • the molecule is sufficiently modular that it could be engineered with three copies of one protein/domain and one copy of another, or two copies of one protein/domain and one copy of two others.
  • Figure IE shows embodiments where the two light chains are identical, but the two heavy chains are different.
  • Figure IF shows embodiments where the two heavy chains are identical, and the two light chains are different.
  • the therapeutic molecule can be engineered to comprise any combination of the various domains of the two proteins which will be able to bind autoantibodies from a wide variety of patients and not just some patients.
  • any chain can include any protein, fragment, domain or variant, and the combinations of chains and subunit depicted in Figures 1A-1F are meant only to be illustrative and not limiting.
  • Figures 2A-2N show some embodiments of the invention in which only two chains are combined.
  • protein complex 201 comprises 2 polypeptide chains which specifically are two heavy chains.
  • Heavy chains 215 and 216 can optionally include a hinge domain 213, CH2 212, CH3 211 and/or CHI 214 domain.
  • the heavy chain hinge 213, CH2212, and CH3 211 all dimerizes via disulfide bonds. Only one of these 3 domains is needed for dimerization and other dimerization domains are also envisioned.
  • the extracellular domain of either ITGA2B or ITGB3 230 is included.
  • Protein complex 201 is also envisioned with only a fragment of the ITGA2B or ITGB3 extracellular domain, such as fragment 231 of ITGA2B, or fragment 232 of ITGA2B.
  • a protein complex 201 with two different fragments either from the same protein or from each of the two proteins (such as fragment 231 on one chain and fragment 233 on the other) is also envisioned. These fragments represent any fragment of the extracellular domain of either protein which may be used in any combination.
  • FIG. 2B shows the molecule without CH2 domain 212, CH3 domain 211 or CHI domain 214. Molecules lacking both hinge 213 and CHI domain 214 are also depicted. Combinations lacking two of these domains are also envisioned, with or without hinge 213 (Fig. 2B).
  • each chain has a fragment 230 from the extracellular portion of ITGA2B and/or ITGB3. Although it is not depicted, it will be understood by a skilled artisan that any fragment from the extracellular portion of ITGA2B and/or ITGB3 can also be used.
  • Each chain can be generated to have the same protein, fragment or variant (Fig. 2C) or different (Fig. 2D).
  • FIG. 2G alternative configurations comprising two heavy chains are shown. Instead of containing a single fragment 230 in place of the variable region, two tandem fragments 230 are used. These fragments may be separated by optional linker 290.
  • This configuration is similar in structure to a single chain antibody in which the heavy and light chain variable domains are on a single peptide and is essentially equivalent to the molecule shown in Figure ID.
  • Heavy chains 215 and 216 can optionally include a hinge 213, CH2 212, CH3 211 and/or CHI 214 domain. Dimerization is as described above. For simplicity an example containing all three CH domains is shown as is an example lacking the CHI domain.
  • fragment 230 can be from either ITGA2B or ITGB3 and can include the whole extracellular domain or only a portion or a variant thereof.
  • a repeat of two of the same protein/fragment can be inserted on a single chain (Fig. 2H-I, two ITGA2B fragments 231) or two different proteins/fragments can be combined on one chain (Fig. 2J- K, a ITGA2B fragment 231 and a ITGB3 fragment 234).
  • CHI domain 214 can be included (Fig. 2H, 2J, 2L) or excluded (Fig. 21, 2K, 2M) and the same is true for hinge 213, CH2 212 and/or CH3 211 so long as one dimerization domain (e.g., hinge, CH2, CH3) remains.
  • protein complex 201 comprises 2 polypeptide chains which specifically are a heavy chain 215 and a light chain 220.
  • 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/CH2/CH3 domains is one option for eliminating homodimerization of two heavy chains 215.
  • cysteine substitutions/mutations may be introduced into the hinge or one of the mutations in the CH2/CH3 regions that promote heterodimerization and inhibit homodimerization may be employed.
  • each chain has a fragment 230 from the extracellular portion, or any fragment or variant thereof, of ITGA2B or ITGB3.
  • FIG. 3A-3D 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. 3A-3D).
  • Figure 3A shows one possible embodiment in which heavy chain 316 comprises a CL domain 364 in place of a CHI domain.
  • 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.
  • each chain has a fragment 330 from the extracellular portion of ITGA2B/B3.
  • the three chains can all contain the same fragment (for example the ITGA2B extracellular domain 335, Fig. 3B), all three chains can contain different fragments (for example ITGB3 336, ITGA2B fragment 331 and ITGA2B fragment 332, Fig. 3C), or the three chains can contain two different fragments in which one is repeated (for example, ITGA2B 335 and ITGB3 fragment 333, Fig. 3D).
  • Figure 3D could also have the two identical fragments as the light chain and either of the heavy chains, thus there are 3 different configurations to this embodiment.
  • This configuration with one of the heavy chains comprising a CL domain in place of a CHI domain can also allow for the formation of the protein complex with four different fragments.
  • protein complex 401 depicted in Figure 4 has four different fragments on each chain.
  • the fragments ITGA2B Frag. 431, ITGB3 Frag. 434, ITGA2B Frag. 432 and ITGB3 Frag. 433 are employed but a skilled artisan will appreciate that any four fragments can be used.
  • Embodiments are also envisioned in which different fragments from the same protein can be on different chains.
  • the second light chain 426 contains a CHI domain 474 so that it can dimerize with the CL domain 464 in heavy chain 416.
  • Heavy chain 415 will contain a CHI domain 414 and light chain 425 will contain a CL domain 424. This ensures that chain 425 can dimerize only with chain 415 and chain 426 can dimerize only with chain 416.
  • mutations in the optional CH2 domains 412 and the CH3 domains 413 can be employed to promote heterodimerization of chains 415 and 416.
  • Hinge region 413, CH2 domain 412 and CH3 domain 411 are all used here as dimerization domains between the two heavy chains, though any dimerization domain (other than CH1/CL) can be employed.
  • FIGS 5A-5B show a generic protein complex 501.
  • the first chain 515 contains a first dimerization domain (DD1) 563 which can dimerize specifically with a second dimerization domain (DD2) 573 of second chain 516.
  • Chain 515 further comprises a third dimerization domain (DD3) 514 which can dimerize specifically with a fourth dimerization domain (DD4) 524 of third chain 525.
  • DD1 first dimerization domain
  • DD3 dimerization domain
  • DD4 dimerization domain
  • Chain 516 further comprises a fifth dimerization domain (DD5) 564 which can dimerize specifically with a sixth dimerization domain (DD6) 574 of fourth chain 526.
  • Each of the four chains also comprises a fragment 530 of a human protein target of ITP/PF autoantibodies. These can all be the same fragment with the same amino acid sequence, or they can be different sequences (either from the same protein or from different proteins) or variants/derivatives or the proteins or fragments.
  • Figure 5B shows an alternative embodiment to Figure 5A in which each distinct domain is separated by a linker. It will be understood by a skilled artisan that all of these linkers are optional, and that combination of linkers is envisioned. It will be further understood that the configurations of Figures 5B also could employ linkers between any or all of the various domains/fragments. Similarly, linkers can be inserted between the CHI domain, hinge region, CH2 domain, CH3 domain and/or the ITGA2B/B3 fragments. Thus, the linkers shown in Figure 5B can be extrapolated to the same positions in the immunoglobulin backbone molecules (Fig. 1A-4).
  • Figure 6A-6F single chain embodiments of the invention are depicted.
  • Figure 6A shows a single chain fusion protein 601 containing the ITGA2B extracellular domain 635 and the ITGB3 extracellular domain 636.
  • Figure 6B shows a single chain comprising only a fragment of ITGA2B or a fragment of ITGB3.
  • a generic fragment of ITGA2B 631 and a generic fragment of ITGB3 633 are shown, although it will be understood that any fragment of the extracellular domain may be used.
  • any permutation of Figures 6A and 6B may be generated such that two fragments from different proteins are in the same single chain, two different fragments can be used. Specifically fragments from two different proteins can be used.
  • Figures 6C and 6D show a similar embodiment but containing 3 and 4 fragments from different proteins respectively. It will be understood that any fragment or the complete extracellular domain can be used.
  • the single chain can also contain a heavy chain constant region with at least a CH3 domain 611 or CH2 domain 612 and optionally CHI domain 614, hinge region 613 and/or CH2 domain 612 or CH3 domain 611.
  • Figure 6F shows the embodiment in which the CHI domain has been removed.
  • amino acid linkers can be used to separate any of the domains of the single chain.
  • Figure 6G depicts embodiments, with 2, 3, or 4 fragments all separated by linkers 690 as well as embodiments in which linker 690 also separates the C-terminal fragment 635 from CHI domain 614 or CH2 domain 612. It will be understood that while the linker is shown also replacing the hinge region this is not required and the hinge could be retained with the linker linking C-terminal fragment 635 to it. 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.
  • ITGA2B was also fused both to the knob molecule (SEQ ID NO: 11, CRD-757 chain 1) and the hole molecule (SEQ ID NO: 14, CRD-758 chain 2).
  • both heterodimer molecules CRD-757 and CDR-758 were expressed in CHO cells and did not get trapped and degraded as the ITGA2B extracellular domain alone did (Fig. 7). It thus appears that fusion of the ITGA2B extracellular domain to Fc is a viable strategy for producing this target of autoantibodies.
  • the produced molecules are summarized in Table 2, which provides the identifier used herein throughout, expected molecular weight (MW), expected isoelectric point (pl, (M-l*cm-l)), expected extinction coefficient (EC) and actual yield of the various molecules. All molecules were expressed at their expected molecular weights (208 Kda for CRD-756, 236Kda for CRD-757 and CRD-758, and 80.5Kda for CRD-760) as observed by the SDS- PAGE. The reduced forms were expressed at their expected molecular weights as well (ITGA2B GPIIb-CH2-CH3 ⁇ 132Kda and ITGB3 GPIIIa-CH2-CH3 ⁇ 104Kda).
  • the binding capacity of the various molecules to actual pathogenic antibodies is determined in ITP patients’ serum samples.
  • Human sera samples positive for anti-ITGB3 and/or ITGA2B IgG antibodies are used to examine molecule binding.
  • Autoantibody titer is determined using Monoclonal Antibody-Specific Immobilization of Platelet Antigens (MAIPA) assay and/or ELISA.
  • MAIPA Monoclonal Antibody-Specific Immobilization of Platelet Antigens
  • the molecules are biotinylated and attached to avidin coated Sepharose beads, and sera samples are separately incubated with increasing concentrations of the various ITGB3 or ITGA2B/B3 containing molecules. All the molecules that include the ITGB3 extracellular domain are able to bind the anti-ITGB3 antibodies, regardless of whether the ITGA2B extracellular domain was present or not.
  • Human sera samples positive for anti-ITGB3 IgG are depleted using ITGB3 extracellular domain molecules.
  • Human sera samples positive for anti-ITGA2B IgG are depleted using ITGA2B/B3 combined molecules.
  • the anti-ITGA2B titers are determined by calculating the percentage of depletion for anti- ITGA2B/B3 minus the percentage of depletion for anti- ITGB3 along. All ITGA2B/B3 containing molecules produce robust depletion of about 40% or greater.
  • ITGA2B only molecules are also included as negative controls. As expected, all the ITGB3 containing molecules, but none of the ITGA2B only molecules, are able to deplete anti-ITGB3 antibodies. The inverse experiments are performed and ITGA2B containing molecules can bind anti-ITGA2B antibodies in serum. ITGB3 containing molecules are used as negative control and do not bind anti-ITGA2B antibodies.
  • CRD-757 and CRD-758 were incubated with at least 17 different murine/rat hybridomas (listed in Table 3) for 40 min at 37 degrees Celsius.
  • Two of the hybridomas, LK-4 and AP-3, are ITP hybridomas and produce antibodies against ITGB3. All the other hybridomas produce antibodies against other antigens.
  • FACS buffer DPBS with 1% FBS
  • CRD-757 and CRD-758 were tested for their ability to kill anti-ITGB3 B cell hybridoma cells.
  • the two molecules were compared to CRD-760 which contains the ITGB3 ECD but lacks an Fc domain.
  • AP3 hybridoma cells, expressing BCR against ITGB3, were cultured with increasing concentrations of the molecules (0.16-20 pg/ml).
  • RPMI medium was supplemented with 33.3% guinea pig serum to induce CDC. After a 3 -hour incubation dead cells were labeled with propidium iodide (PI) and quantified by flow cytometry. The percent increase in killing was calculated as compared to CRD-760 which would not be expected to cause any cytotoxicity.
  • PI propidium iodide
  • Agents of the invention comprising ITGA2B/B3 molecules fused to a cytotoxic Fc domain are tested generally for their ability to kill B cells.
  • the cytotoxic activity of the ITGA2B/B3 fusion molecules is determined on ITP hybridomas, EK-4 and AP-3, that produce antibodies against ITGB3.
  • the ITGA2B/B3 molecules of the invention produce a high level of specific killing with nearly 100% of anti-ITGB3 expressing hybridoma cells killed. This indicates that ITGA2B/B3 combined structure is functional for cell binding and cell killing.
  • effector molecules are tested. These include ITGA2B/B3 molecules of the invention conjugated to alpha-amanitin, Tesirine, Dxd, PNU- 159682, MMAE, MMAF, and triptolide. All show superior killing to that produced by an unmodified Fc domain. Fc domains with mutations that increase ADCC are also tested. Fc mutations such as are described hereinabove are generated in the Fc and killing is tested in anti- ITGA2B and anti- ITGB3 hybridomas. Killing is specific to these hybridomas and not hybridomas against other targets and the killing is superior to that produced by an unmodified Fc.
  • the various molecules of the invention are also tested in vivo. These molecules are all found to effectively treat ITP, kill autoreactive B cells and reduce autoantibody titer levels in vivo. All tested effector moieties are found superior to Fc.
  • ITP is also induced in mice by the subcutaneous injection of ITGA2B/B3 ECD fragments. The ability to treat ITP in this organism with the molecules of the invention is confirmed. Serum is taken and antibody titer levels are monitored. Not only do the molecules of the invention kill target B cells, but they also reduce circulating antibody levels.

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Abstract

L'invention concerne des compositions comprenant un premier polypeptide comprenant un premier fragment d'un domaine extracellulaire d'ITGA2B ou ITGB3 ou un analogue ou un dérivé de celui-ci et un domaine de dimérisation et un second polypeptide comprenant un second fragment d'un domaine extracellulaire d'ITGA2B ou ITGB3 ou un analogue ou un dérivé de celui-ci et un domaine de dimérisation. L'invention concerne également des polypeptides comprenant des fragments d'un domaine extracellulaire d'ITGA2B ou d'ITGB3. L'invention concerne en outre des compositions pharmaceutiques comprenant la composition, le polypeptide, des systèmes d'acide nucléique et des molécules codant pour les polypeptides de la composition et des méthodes de traitement et de détermination de l'aptitude au traitement à l'aide des compositions ou des polypeptides ; ainsi que des procédés de production des compositions ou des protéines.
PCT/IL2024/050448 2023-05-08 2024-05-08 Protéines de fusion de type ig pour le traitement de la thrombocytopénie immunitaire Pending WO2024231931A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20060263774A1 (en) * 2002-11-01 2006-11-23 Genentech, Inc. Compositions and methods for the treatment of immune related diseases

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060263774A1 (en) * 2002-11-01 2006-11-23 Genentech, Inc. Compositions and methods for the treatment of immune related diseases

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Title
AKUTA K., KASHIWAGI H., YUJIRI T., NISHIURA N., MORIKAWA Y., KATO H., HONDA S., KANAKURA Y., TOMIYAMA Y.: "A unique phenotype of acquired Glanzmann thrombasthenia due to non‐function‐blocking anti‐αIIbβ3 autoantibodies", JOURNAL OF THROMBOSIS AND HAEMOSTASIS, JOHN WILEY & SONS, vol. 17, no. 1, 1 January 2019 (2019-01-01), pages 206 - 219, XP093235902, ISSN: 1538-7836, DOI: 10.1111/jth.14323 *

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