Ig-like fusion protein for treating immune thrombocytopenia

Abstract

A composition is provided comprising a first polypeptide comprising a first fragment or analog or derivative of the extracellular domain of ITGA2B or ITGB3 and a dimerization domain, and a second polypeptide comprising a second fragment or analog or derivative of the extracellular domain of ITGA2B or ITGB3 and a dimerization domain. Polypeptides comprising fragments of the extracellular domain of ITGA2B or ITGB3 are also provided. Pharmaceutical compositions comprising the composition, polypeptide, nucleic acid system, and molecule encoding the polypeptide of the above composition are also provided, as well as therapeutic methods using the above composition or polypeptide, methods for determining suitability for therapeutic use of the above composition or polypeptide, and methods for producing the above composition or protein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 464,654, filed on May 8, 2023, and U.S. Provisional Patent Application No. 63 / 609,416, filed on December 13, 2023, and the entire contents of each are hereby incorporated by reference herein.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (CNPY - P - 004 - PCT.xml; size: 80,793 bytes; and creation date: May 1, 2024) are hereby incorporated by reference in their entirety herein.

[0003] The present invention is in the field of fusion protein production and treatment of immune thrombocytopenia (ITP).

Background Art

[0004] Immune thrombocytopenia (ITP) is an acquired thrombocytopenia caused by autoantibodies against platelet antigens. Autoantibodies are one of the more common causes of thrombocytopenia in otherwise asymptomatic adults. ITP was previously 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 These terms have been replaced by "immune thrombocytopenia" to reflect the known autoantibody mechanism and the absence of purpura in some patients.

[0005] There are several types of ITP. Primary ITP is acquired immune thrombocytopenia caused by an autoimmune mechanism that leads to platelet destruction and insufficient platelet production, without any apparent related symptoms. Secondary ITP is associated with another condition (i.e., lupus-associated ITP). Drug-induced immune thrombocytopenia (DITP) is thrombocytopenia caused by drug-dependent platelet antibodies that lead to platelet destruction. This syndrome should be distinguished from drug-induced myelosuppression, which is a non-immune phenomenon. The time elapsed since diagnosis determines whether ITP is newly diagnosed, persistent, or chronic: newly diagnosed is up to 3 months from diagnosis, persistent is 3 to 12 months from diagnosis, and chronic is more than 12 months from diagnosis. The severity of ITP is defined based on the bleeding complications it induces and the patient's laboratory status. Severe ITP refers to ITP with bleeding symptoms sufficient to require treatment, which typically occurs when the platelet count is less than 20,000 / uL.

[0006] Pathophysiology: The pathogenesis of ITP is not fully understood. While antibody-mediated destruction leading to reduced platelet lifespan is the primary cause, other mechanisms, including autoreactive cytotoxic T cells causing impaired platelet production, as well as humoral and cellular autoimmunity against megakaryocytes, are likely important. The main mechanism is, in most cases, accompanied by specific autoantibodies (typically IgG) produced by the patient's B cells targeting platelet membrane glycoproteins such as GPIIb (ITGA2B) / GPIIIa (ITGB3). In some cases, ITP is associated with a preceding infection, mostly viral, where antibodies against viral antigens can cross-react with normal platelet antigens (molecular mimicry). In addition, alterations in immune homeostasis can induce loss of peripheral tolerance and promote the development of autoreactive antibodies. This often occurs in the context of other autoimmune conditions, including antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE), Evans syndrome, hematopoietic stem cell transplantation, chronic lymphocytic leukemia (CLL), and other disorders.

[0007] Antibody production in ITP appears to be facilitated by CD4-positive helper T cells. Splenic macrophages appear to be the primary antigen-presenting cells. Despite this likely mechanism, anti-platelet antibodies are absent in nearly 50% of ITP patients (i.e., they have low sensitivity to anti-platelet antibodies). In most patients, the primary site of platelet clearance is the spleen, which removes opsonized (antibody-coated) cells containing platelets. The prominent role of splenic clearance makes the efficacy of splenectomy evident in most patients. However, clearance can also occur in other tissues such as the liver, bone marrow, and lymph nodes. This helps explain why ITP can persist or recur after splenectomy.

[0008] Epidemiology, clinical symptoms, and detailed diagnosis. ITP is a common acquired bleeding disorder. A review of published reports has determined an annual ITP incidence of approximately 1–6 cases per 100,000 adults. ITP is often a chronic disease in adults, and therefore the prevalence significantly exceeds the incidence. A review from the United States found a prevalence of approximately 8 cases per 100,000 children and approximately 12 cases per 100,000 adults. A database review from France, limited to ITP cases requiring chronic treatment and / or hospitalization, found an overall incidence of 2.9 cases per 100,000 per year, peaking in individuals over 60 years of age, reaching 9 cases per 100,000 per year in men over 75 years of age. Other studies have also demonstrated an increased incidence of ITP with age. ITP is generally considered a condition that affects young women, and women are predominant in young adults, but most studies show similar incidences in men and women over 60 years of age.

[0009] Some ITP patients are asymptomatic, and in symptomatic patients, symptoms are primarily associated with thrombocytopenia and bleeding, but patients may also experience fatigue and a reduced quality of life. Bleeding due to thrombocytopenia can occur in up to two-thirds of patients. Bleeding, if present, typically occurs on the skin or mucous membranes, a pattern sometimes called “platelet-type” bleeding. The onset of symptoms can be sudden, but is more often insidious.

[0010] Types of bleeding symptoms: Petechiae - Petechiae are flat, red, discontinuous lesions that do not turn white under pressure. They often occur in the secondary areas of the body (lower legs in ambly patients; sacral area in supine patients). Petechiae must be distinguished from vasculitic purpura. Purpura - Purpura refers to lesions caused by the fusion of petechiae. Purpura on the skin is sometimes called "dry purpura." Hemorrhagic blisters on mucous membranes, such as the oral mucosa, are sometimes called "wet purpura." This finding may be a predictor of more severe bleeding. The emergence of new wet purpura should prompt a reassessment of platelet count and the need for ITP treatment. Epistaxis - Minimal epistaxis, such as simply blowing your nose, is common and may not be clinically significant. Persistent epistaxis requiring intervention with nasal tampon insertion or cauterization may predict a greater risk of more serious bleeding.

[0011] Reported bleeding rates vary depending on the population, definition of bleeding, and reporting method. In a population-based study including 3771 ITP patients, the risk of severe gastrointestinal or central nervous system bleeding at disease onset was less than 1%. In a prospective enrollment including 269 ITP patients, 57% experienced bleeding, most of which were localized to the skin or mouth. Predictors of clinically significant bleeding in individual studies included degree of thrombocytopenia, prior minor bleeding, NSAID use, female gender, and chronic ITP.

[0012] ITP is defined by thrombocytopenia, and according to common understanding, the threshold for ITP is a platelet count <100,000 / uL. The severity of thrombocytopenia varies in ITP patients, with bleeding being the greatest concern at platelet counts <20,000 / uL. Large platelets are often observed on peripheral blood smears. However, the absence of large platelets cannot be used to rule out ITP. Importantly, ITP does not feature abnormal platelet morphology. Abnormal platelet morphology, if present, should prompt consideration of hereditary platelet disorders.

[0013] Furthermore, patients with severe thrombocytopenia (e.g., <20,000 / uL) are more likely to experience clinically significant bleeding than those with higher platelet counts. However, the correlation between platelet count and bleeding risk is weak. Since the risk of clinically significant bleeding is low for platelet counts >20,000 / uL, it is generally not common to use platelet-increasing therapies on individuals with stable platelet counts above 30,000 / uL unless other comorbidities or drug treatments that increase bleeding risk are present. This slightly higher threshold allows for a measure of safety and accommodates small daily fluctuations in platelet count, which will be discussed separately in detail.

[0014] Fatigue is a common symptom in ITP patients. The causes of fatigue in ITP are not well understood. In addition, thrombocytopenia in people with ITP is not necessarily protective against thrombosis. Several studies have demonstrated an increased risk of thrombosis in people with ITP compared to controls. The pathogenesis of hypercoagulation in ITP is not well understood. Its pathogenesis may be related to inflammation, antiphospholipid antibodies in some patients, or the effects of certain treatments such as splenectomy and thrombopoietin receptor agonists. Other cell lineages (leukocytes or erythrocytes) are characteristically normal in ITP. Coagulation parameters are also typically normal.

[0015] ITP is a diagnostic test performed in patients with isolated thrombocytopenia. Therefore, key components of the diagnostic evaluation include excluding other possible causes of thrombocytopenia and identifying conditions that may cause secondary ITP. From the medical history, recent infections, drug treatments, and underlying conditions such as rheumatic disorders or liver disease that may be associated with thrombocytopenia should be identified. In addition, questions regarding bleeding symptoms, subcutaneous bleeding, and petechiae are helpful. Physical examination should focus on signs of bleeding, particularly on the skin and oral mucosa, as well as the presence of lymphadenopathy or hepatosplenomegaly, which may suggest an underlying condition causing thrombocytopenia, indicating a need for urgent evaluation and treatment.

[0016] Once other symptoms associated with thrombocytopenia have been ruled out, the minimum diagnostic tests that should be performed on a patient suspected of having ITP are as follows:

[0017] Peripheral blood smears are used to confirm that thrombocytopenia is not an artifact caused by platelet aggregation and that there are no morphological platelet abnormalities such as a lack of platelet granules or uniform platelet sizes that could suggest a hereditary platelet disorder. While the presence of large platelets may be observed, there is no high-quality data to support the use of platelet size to confirm or rule out the diagnosis of ITP.

[0018] Thrombocytopenia is a common finding in HIV and HCV, and since treating the underlying infection can improve platelet counts, HIV and HCV testing is recommended.

[0019] Coagulation tests are not required in patients with mild thrombocytopenia. However, measurement of prothrombin time (PT) and activated partial thromboplastin time (aPTT) is recommended in individuals with moderate or severe thrombocytopenia, individuals with clinically significant bleeding concerns, and / or individuals for whom invasive procedures are planned.

[0020] In patients with GI symptoms, a link between ITP and Helicobacter pylori infection has been reported, making H. pylori testing appropriate.

[0021] In patients with other unexplained cytopenia (anemia, leukopenia), dysplasia on peripheral blood smears, other unexpected hematological findings, or other causes of thrombocytopenia, bone marrow examination may be indicated if suspected. In patients with atypical clinical features, other tests may be indicated. For example, patients with bleeding proportional to the degree of thrombocytopenia may need evaluation for less common conditions such as von Willebrand disease type 2B, Bernard-Soulier syndrome, or other hereditary or acquired platelet disorders. If hereditary thrombocytopenia is suspected, genetic testing may be indicated. Details of this test will be described separately. Antiplatelet antibody testing has low sensitivity and does not effectively correlate with clinical outcomes.

[0022] Treatment: The goal of current treatment is not to normalize platelet counts, but to treat or prevent significant bleeding. The risk of severe or heavy bleeding is low. The risk is highest in individuals with a history of bleeding, platelet counts <10,000 / μL, and age >60 years.

[0023] In cases of severe bleeding (severe anatomical or hemodynamic impairment), platelet transfusion is indicated for all patients. Glucocorticoids (typically pulsed dexamethasone) plus intravenous immunoglobulin (IVIG) are recommended over either treatment alone (grade 2C). For severe bleeding (hemoglobin reduction ≥2 g / dL, or requiring ≥2 units of transfusion), glucocorticoids alone or glucocorticoids + IVIG (grade 2C) are indicated, rather than IVIG alone. In some individuals, IVIG may be preferred due to its rapid action or side effect profile. For mild bleeding or severe thrombocytopenia without bleeding, the treatment threshold is individualized. For most individuals with platelet counts <20,000 / uL (especially <10,000 / μL), treatment (grade 2C) is recommended over observation. Treatment with higher platelet counts may be reasonable if the risk of bleeding increases or if anticoagulation therapy is required. Observation may be reasonable in asymptomatic young individuals with low platelet counts.

[0024] Pulsed dexamethasone results in a faster response and fewer bleeding events. A typical dose is 40 mg orally or intravenously once daily for 4 days (without tapering). Pulsed methylprednisolone is typically administered intravenously at 1 g once daily for 3 days (without tapering). Prednisone is administered orally at 1 mg / kg once daily for 1-2 weeks, then tapered (typically less than 6 weeks). IVIG - a typical dose is 1 g / kg daily for 1 or 2 days. A single dose is often sufficient.

[0025] Currently, there are no treatments that target the cause of ITP, nor are there any treatments that target autoreactive antibodies or the B cells that produce these antibodies. An improved treatment approach that targets the mechanistic causes of ITP is greatly needed. [Overview of the project]

[0026] The present invention provides a polypeptide comprising a fragment of the extracellular domain of Integrin Subunit Alpha 2b (ITGA2B) or Integrin Subunit Beta 3 (ITGB3). The present invention further provides a composition comprising a fragment of the first human protein target of immune thrombocytopenia (ITP) autoantibodies and a fragment of the second human protein target of ITP autoantibodies, and a composition further comprising an effector portion other than an unmodified Fc domain. A method of treating ITP by administering the pharmaceutical composition of the present invention, as well as nucleic acid molecules and systems encoding the polypeptides and compositions of the present invention, methods of producing those polypeptides and compositions, and methods of determining suitability for treatment by the methods of the present invention are also provided.

[0027] According to a first aspect, a. A first polypeptide comprising a fragment of the extracellular domain of integrin subunit alpha 2b (ITGA2B) or an analog or derivative thereof, a fragment of the extracellular domain of integrin subunit beta 3 (ITGB3) or an analog or derivative thereof, or both, and a first dimerization domain, and b. A composition comprising 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. A composition is provided.

[0028] According to another aspect, there is provided a polypeptide of the present invention comprising an effector portion.

[0029] In some embodiments, the effector portion is not an Fc domain.

[0030] According to some embodiments, the effector portion is an Fc domain containing at least one mutation that increases antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC).

[0031] According to some embodiments, the effector portion can induce cell death in cells bound to the fragment.

[0032] According to some embodiments, the effector portion is selected from Fc domains containing at least one mutation that increases ADCC, amatoxin / amanitin, anthracyclines, anthramycin dimers, calicheamicin, camptothecin or its analogues, duocalmycin, triptolide, and tubulin inhibitors.

[0033] According to some embodiments, the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0034] According to some embodiments, the effector portion is an Fc domain containing SEQ ID NO: 60 or SEQ ID NO: 62, which contains multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 60 or SEQ ID NO: 62.

[0035] According to some embodiments, the effector portion is conjugated to a polypeptide by a linker.

[0036] According to some embodiments, 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.

[0037] According to some embodiments, dimerization involves forming a covalent bond between a first dimerization domain and a second dimerization domain.

[0038] According to some embodiments, the protein complex includes an immunoglobulin scaffold.

[0039] According to some embodiments, a. The first dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the second dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains are dimerized by disulfide bonds, or b. The first and second dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, respectively, and are dimerized by a disulfide bond, and neither the first nor the second dimerization domain contains a CH1 domain or a CL domain.

[0040] According to some embodiments, fragments and dimerization domains of the first, second, or both polypeptide chains are separated by a linker.

[0041] According to some embodiments, the first polypeptide chain, the second polypeptide chain, or both further comprise the Fc region of a human antibody heavy chain.

[0042] According to some embodiments, the Fc region can induce cytotoxicity against cells that bind to the protein complex.

[0043] According to some embodiments, the first polypeptide chain comprises a first CH3 domain of the immunoglobulin heavy chain, a first CH2 domain of the immunoglobulin heavy chain, or both, and the second polypeptide chain comprises a second CH3 domain of the immunoglobulin heavy chain, a second CH2 domain of the immunoglobulin heavy chain, or both.

[0044] According to some embodiments, the first CH3 domain comprises at least a first mutation, and the second CH3 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

[0045] According to some embodiments, the first CH2 domain comprises at least a first mutation, and the second CH2 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

[0046] According to some embodiments, the first mutation is selected from the mutations provided in Table 1, and the second mutation is a mutation provided in Table 1 that corresponds to the first mutation.

[0047] According to some embodiments, the first mutation is the T366W mutation within the CH3 domain, and the second mutation is a combination of the T366S mutation, the L368A mutation, and the Y407V mutation.

[0048] According to some embodiments, the Fc region of the first, second, or both polypeptide chains is separated from the fragment or dimerization domain by a linker.

[0049] According to some embodiments, the Fc region includes at least one mutation that increases ADCC or CDC.

[0050] According to some embodiments, the Fc region is an Fc region containing SEQ ID NO: 60 or SEQ ID NO: 62, which contains multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 60 or SEQ ID NO: 62.

[0051] According to some embodiments, the dimerization domains of the first, second, or both polypeptide chains are located on the C-terminal side of the fragment and on the N-terminal side of the Fc region.

[0052] According to some embodiments, the composition lacks an antibody-variable domain.

[0053] According to some embodiments, 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 can dimerize with each other.

[0054] According to some embodiments, a. The third dimerization domain includes the first hinge domain of the immunoglobulin heavy chain, the fourth dimerization domain includes the second hinge domain of the heavy chain, and the first and second dimerization domains are dimerized by disulfide bonds, or b. The third and fourth dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, respectively, and are dimerized by a disulfide bond, with neither the first nor the third polypeptide containing a CH1 domain or a CL domain.

[0055] According to some embodiments, 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, the second polypeptide further comprising a sixth dimerization domain, and the fifth and sixth dimerization domains can dimerize with each other.

[0056] According to some embodiments, a. The fifth dimerization domain includes the first hinge domain of the immunoglobulin heavy chain, the sixth dimerization domain includes the second hinge domain of the heavy chain, and the first and second dimerization domains are dimerized by disulfide bonds, or b. The fifth and sixth dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, respectively, and are dimerized by a disulfide bond, while the first and third polypeptides neither contain a CH1 domain nor a CL domain.

[0057] According to some embodiments, the first polypeptide and the second polypeptide either do not contain a CH1 domain, or both contain a CL domain.

[0058] According to some embodiments, the third and fourth dimerization domains, or the fifth and sixth dimerization domains, include mutations that enable dimerization of the third and fourth dimerization domains, as well as the fifth and sixth dimerization domains, and inhibit dimerization of the third dimerization domain to the fifth or sixth dimerization domain, and dimerization of the sixth dimerization domain to the third or fourth dimerization domain.

[0059] According to some embodiments, 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.

[0060] According to some embodiments, 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.

[0061] According to some embodiments, the fragments are separated by an amino acid linker.

[0062] According to some embodiments, ITGA2B lacks a signal peptide and contains or comprises SEQ ID NO: 1, and ITGB3 lacks a signal peptide and contains or comprises SEQ ID NO: 2, or both.

[0063] According to some embodiments, the extracellular domain fragment consists of truncations of the extracellular domain.

[0064] According to some embodiments, the analogues or derivatives thereof contain at least 85% identity with respect to ITGA2B or ITGB3.

[0065] According to some embodiments, the fragment comprises at least 20 consecutive amino acids derived from ITGA2B or ITGB3.

[0066] According to some embodiments, the fragment comprises at least one B cell receptor (BCR)-specific epitope target of the autoantibody.

[0067] According to some embodiments, the composition comprises a first or second polypeptide containing a sequence selected from SEQ ID NOs: 5, 8 and 11-14.

[0068] According to some embodiments, the hinge domain, CH2 domain, or CH3 domain contains at least one mutation that increases or decreases antibody-dependent cell-mediated cytotoxicity (ADCC).

[0069] According to some embodiments, at least one mutation that reduces ADCC is selected from the following: a. Hinge domain mutations including L19A and L20A mutations in Sequence ID No. 22; and b. Mutations in the CH2 domain, including the N59A mutation in Sequence ID No. 36.

[0070] According to some embodiments, the composition further comprises at least one effector portion capable of inducing cell death in cells bound to the composition.

[0071] According to some embodiments, the effects portion is not in the Fc domain.

[0072] According to some embodiments, the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0073] In another embodiment, a pharmaceutical composition is provided comprising the composition of the present invention and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0074] In another embodiment, a method is provided for treating immune thrombocytopenia (ITP) in a subject requiring such treatment, comprising administering a composition comprising a fragment of the extracellular domain of ITGA2B or ITGB3 or an analog or derivative thereof to the subject, thereby treating the ITP.

[0075] According to some embodiments, ITGA2B lacks a signal peptide and contains or comprises SEQ ID NO: 1, and ITGB3 lacks a signal peptide and contains or comprises SEQ ID NO: 2, or both.

[0076] According to some embodiments, the composition is the composition of the present invention.

[0077] According to some embodiments, the composition is the pharmaceutical composition of the present invention.

[0078] According to some embodiments, the method further includes reducing the level of circulating antibodies against ITGA2B, ITGB3, or both, in a subject.

[0079] According to some embodiments, treatment involves reducing the concentration of circulating autoantibodies against ITGA2B, ITGB3, or both.

[0080] According to some embodiments, the composition comprises an Fc region, and the treatment involves killing B cells that produce anti-ITGA2B autoantibodies or anti-ITGB3 autoantibodies.

[0081] According to some embodiments, the B cells are autoreactive B cells that produce autoantibodies against fragments of the composition.

[0082] In another embodiment, a nucleic acid system is provided comprising nucleic acid molecules, wherein a first nucleic acid molecule encodes a first polypeptide of the composition of the present invention, and a second nucleic acid molecule encodes a second polypeptide of the composition of the present invention.

[0083] According to some embodiments, the nucleic acid system of the present invention further comprises a third nucleic acid molecule encoding a third polypeptide of the composition of the present invention, a fourth nucleic acid molecule encoding a fourth polypeptide of the composition of the present invention, or both.

[0084] In another aspect, a method for producing a composition of the present invention is provided, comprising expressing a nucleic acid system of the present invention in a cell, wherein the nucleic acid system is configured to produce an encoded polypeptide in the cell, thereby producing a composition of the present invention.

[0085] In another embodiment, a method for producing a protein, To obtain a first fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof, and a second fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof. The first fragment is linked to the first dimerization domain to generate the first polypeptide chain, and the second fragment is linked to the second dimerization domain to generate the second polypeptide chain, wherein the first and second dimerization domains can dimerize with each other, and the first polypeptide and the second polypeptide are brought into contact under conditions sufficient to induce dimerization, or Culture host cells containing one or more vectors, each containing nucleic acid sequences encoding at least two polypeptide chains, where the two polypeptide chains are i. Obtaining a first fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof, and a second fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof, and ii. The first polypeptide chain is generated by linking the first fragment to the first dimerization domain, and the second polypeptide chain is generated by linking the second fragment to the second dimerization domain, wherein the first and second dimerization domains can dimerize with each other. This provides a method that includes the production of proteins.

[0086] According to some embodiments, the protein complex is the protein complex of the composition of the present invention.

[0087] According to some embodiments, the method is a. Within the first polypeptide chain, the third dimerization domain is linked to the first dimerization domain or the first fragment to obtain the third fragment of the extracellular domain of ITGA2B or its analogue or derivative, or the extracellular domain fragment of ITGB3 or its analogue or derivative, and the third fragment is linked to the fourth dimerization domain to generate the third polypeptide chain, wherein the third dimerization domain and the fourth dimerization domain can dimerize with each other, and the first, second and third polypeptides are brought into contact under conditions sufficient to induce dimerization, or To obtain a third fragment of biITGA2B or its analogues or derivatives, or a fragment of the extracellular domain of ITGB3 or its analogues or derivatives, and ii. Linking the third fragment to the fourth dimerization domain to generate a third polypeptide chain. The method further includes expressing a nucleic acid sequence encoding a third polypeptide chain generated by in a host cell, The first polypeptide chain further comprises a third dimerization domain, and the third and fourth dimerization domains can dimerize with each other.

[0088] According to some embodiments, the method is a. In the second polypeptide chain, the sixth dimerization domain is linked to the second dimerization domain or the second fragment to obtain the fourth fragment of the extracellular domain of ITGA2B or its analogue or derivative, or the extracellular domain of ITGB3 or its analogue or derivative, and the fourth fragment is linked to the fifth dimerization domain to generate the fourth polypeptide chain, wherein the fifth dimerization domain and the sixth dimerization domain can dimerize with each other, and the first, second, third and fourth polypeptides are brought into contact under conditions sufficient to induce dimerization, or To obtain a fourth fragment of the extracellular domain of biITGA2B or its analogues or derivatives, or a fragment of the extracellular domain of ITGB3 or its analogues or derivatives, and ii. Linking the fourth fragment to the fifth dimerization domain to generate the fourth polypeptide chain. The method further comprises expressing in a host cell a nucleic acid sequence encoding a fourth polypeptide chain generated by the method, The second polypeptide chain further comprises a sixth dimerization domain, and the fifth and sixth dimerization domains can dimerize with each other.

[0089] According to some embodiments, the method further includes causing at least one mutation in the extracellular domain of a protein, or truncating the protein to remove a portion thereof.

[0090] According to some embodiments, the method further comprises ligating an effector portion to at least one polypeptide chain or mutant fragment, the effector portion being able to kill cells bound to at least one polypeptide.

[0091] According to some embodiments, the effects portion is not in the Fc domain.

[0092] According to some embodiments, the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0093] According to some embodiments, the effector portion is an Fc domain containing at least one mutation that increases ADCC or CDC.

[0094] According to some embodiments, the effector portion is an Fc domain containing SEQ ID NO: 60 or SEQ ID NO: 62, which contains multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 60 or SEQ ID NO: 62.

[0095] In another aspect, a protein produced by the method of the present invention is provided.

[0096] In another embodiment, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising: receiving a sample from the object; contacting the sample with the protein of the present invention or a composition of the present invention; and determining the binding of autoantibodies in the sample to the protein or composition, wherein the binding of autoantibodies to the protein or composition indicates that the object is suitable to be treated by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0097] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given merely as examples, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0098] [Figure 1A]Figures 1A-1F: Diagrams of five possible embodiments of the quadruple-chain therapeutic agent of the present invention: (1A) shows a general embodiment of the molecule for treating ITP; (1B) shows an embodiment in which each of the four chains contains a different protein fragment; (1C) shows an embodiment in which all four protein fragments are the same; (1D) shows an embodiment in which two heavy chains are the same and two light chains are the same; (1E) shows an embodiment in which two heavy chains are different and two light chains are the same; and (1F) shows an embodiment in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments. [Figure 1B] Figures 1A-1F: Diagrams of five possible embodiments of the quadruple-chain therapeutic agent of the present invention: (1A) shows a general embodiment of the molecule for treating ITP; (1B) shows an embodiment in which each of the four chains contains a different protein fragment; (1C) shows an embodiment in which all four protein fragments are the same; (1D) shows an embodiment in which two heavy chains are the same and two light chains are the same; (1E) shows an embodiment in which two heavy chains are different and two light chains are the same; and (1F) shows an embodiment in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments. [Figure 1C] Figures 1A-1F: Diagrams of five possible embodiments of the quadruple-chain therapeutic agent of the present invention: (1A) shows a general embodiment of the molecule for treating ITP; (1B) shows an embodiment in which each of the four chains contains a different protein fragment; (1C) shows an embodiment in which all four protein fragments are the same; (1D) shows an embodiment in which two heavy chains are the same and two light chains are the same; (1E) shows an embodiment in which two heavy chains are different and two light chains are the same; and (1F) shows an embodiment in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments. [Figure 1D]Figures 1A-1F: Diagrams of five possible embodiments of the quadruple-chain therapeutic agent of the present invention: (1A) shows a general embodiment of the molecule for treating ITP; (1B) shows an embodiment in which each of the four chains contains a different protein fragment; (1C) shows an embodiment in which all four protein fragments are the same; (1D) shows an embodiment in which two heavy chains are the same and two light chains are the same; (1E) shows an embodiment in which two heavy chains are different and two light chains are the same; and (1F) shows an embodiment in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments. [Figure 1E] Figures 1A-1F: Diagrams of five possible embodiments of the quadruple-chain therapeutic agent of the present invention: (1A) shows a general embodiment of the molecule for treating ITP; (1B) shows an embodiment in which each of the four chains contains a different protein fragment; (1C) shows an embodiment in which all four protein fragments are the same; (1D) shows an embodiment in which two heavy chains are the same and two light chains are the same; (1E) shows an embodiment in which two heavy chains are different and two light chains are the same; and (1F) shows an embodiment in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments. [Figure 1F] Figures 1A-1F: Diagrams of five possible embodiments of the quadruple-chain therapeutic agent of the present invention: (1A) shows a general embodiment of the molecule for treating ITP; (1B) shows an embodiment in which each of the four chains contains a different protein fragment; (1C) shows an embodiment in which all four protein fragments are the same; (1D) shows an embodiment in which two heavy chains are the same and two light chains are the same; (1E) shows an embodiment in which two heavy chains are different and two light chains are the same; and (1F) shows an embodiment in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments.

[0099] [Figure 2A-1]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2A-2] Same as above. [Figure 2B-1]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2B-2] Same as above. [Figure 2B-3] Same as above. [Figure 2B-4] Same as above. [Figure 2B-5] Same as above. [Figure 2B-6] Same as above. [Figure 2B-7] Same as above. [Figure 2C]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2D]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2E]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2F]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2G]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2H]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2I]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2J]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2K]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2L]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2M]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain. [Figure 2N]Figures 2A-2N: Diagrams of possible embodiments of the double-chain therapeutic agent of the present invention: (2A) shows a general embodiment of a molecule having two heavy chains for treating ITP; (2B) shows an embodiment in which at least one of the CH1 domain, CH2 domain or CH3 domain is 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 CH1 domain; (2F) shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or hinge domain; (2G) shows an embodiment in which two tandem fragments are included in each heavy chain and a linker The following are general embodiments of a molecule having one heavy chain and one light chain: (2H) shows a tandem fragment configuration in which all subunits are the same; (2I) shows a tandem fragment configuration in which all subunits are the same but do not have a CH1 domain; (2J) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same; (2K) shows a tandem fragment configuration in which each heavy chain contains two different fragments that are the same but do not have a CH1 domain; (2L) shows a tandem fragment configuration in which two heavy chains contain different fragments that are not the same; (2M) shows a tandem fragment configuration in which two chains contain different fragments that are not the same but do not have a CH1 domain; and (2N) shows a general embodiment of a molecule having one heavy chain and one light chain.

[0100] [Figure 3A] Figures 3A-3D: Diagrams of four possible embodiments of the triple-chain therapeutic agent of the present invention: (3A) shows a general embodiment of a molecule having two heavy chains and one light chain; (3B) shows an embodiment in which three protein fragments are the same; (3C) shows an embodiment in which each of the protein fragments is different; and (3D) shows an embodiment in which two of the protein fragments are the same and the third is different. [Figure 3B]Figures 3A-3D: Diagrams of four possible embodiments of the triple-chain therapeutic agent of the present invention: (3A) shows a general embodiment of a molecule having two heavy chains and one light chain; (3B) shows an embodiment in which three protein fragments are the same; (3C) shows an embodiment in which each of the protein fragments is different; and (3D) shows an embodiment in which two of the protein fragments are the same and the third is different. [Figure 3C] Figures 3A-3D: Diagrams of four possible embodiments of the triple-chain therapeutic agent of the present invention: (3A) shows a general embodiment of a molecule having two heavy chains and one light chain; (3B) shows an embodiment in which three protein fragments are the same; (3C) shows an embodiment in which each of the protein fragments is different; and (3D) shows an embodiment in which two of the protein fragments are the same and the third is different. [Figure 3D] Figures 3A-3D: Diagrams of four possible embodiments of the triple-chain therapeutic agent of the present invention: (3A) shows a general embodiment of a molecule having two heavy chains and one light chain; (3B) shows an embodiment in which three protein fragments are the same; (3C) shows an embodiment in which each of the protein fragments is different; and (3D) shows an embodiment in which two of the protein fragments are the same and the third is different.

[0101] [Figure 4] Figure 4: A diagram illustrating an embodiment of the quadruple-chain therapeutic agent for treating ITP according to the present invention, similar to that shown in Figure 1, but in which the four chains each contain a different protein fragment and a different immunoglobulin scaffold that promotes the formation of the quadruple-chain molecule.

[0102] [Figure 5A] Figures 5A-5B: (5A-5B) Figures of general embodiments of the quadruple-chain therapeutic agent of the present invention: (5A) shows a general embodiment of four chains in which two chains contain two dimerization domains and two chains contain a single dimerization domain, and (5B) shows an embodiment having an arbitrary linker for separating various domains and fragments. [Figure 5B]Figures 5A-5B: (5A-5B) Figures of general embodiments of the quadruple-chain therapeutic agent of the present invention: (5A) shows a general embodiment of four chains in which two chains contain two dimerization domains and two chains contain a single dimerization domain, and (5B) shows an embodiment having an arbitrary linker for separating various domains and fragments.

[0103] [Figure 6A] Figures 6A-6G: Figures of the single-chain therapeutic agent of the present invention: (6A) shows an embodiment of a single-chain molecule containing fragments from two different ITGA2B / B3 fragments; (6B) shows an embodiment of a fragment containing a truncation of ITGA2B or ITGB3; (6C) shows an embodiment of a single-chain molecule containing three different fragments; (6D) shows an embodiment of a single-chain molecule containing four different fragments; (6E) shows an embodiment 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 embodiment 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; (6G) shows the single-chain molecules of 6A and 6C-6F having an amino acid (AA) linker that separates various domains. [Figure 6B] Figures 6A-6G: Figures of the single-chain therapeutic agent of the present invention: (6A) shows an embodiment of a single-chain molecule containing fragments from two different ITGA2B / B3 fragments; (6B) shows an embodiment of a fragment containing a truncation of ITGA2B or ITGB3; (6C) shows an embodiment of a single-chain molecule containing three different fragments; (6D) shows an embodiment of a single-chain molecule containing four different fragments; (6E) shows an embodiment 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 embodiment 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; (6G) shows the single-chain molecules of 6A and 6C-6F having an amino acid (AA) linker that separates various domains. [Figure 6C] Figures 6A-6G: Figures of the single-chain therapeutic agent of the present invention: (6A) shows an embodiment of a single-chain molecule containing fragments from two different ITGA2B / B3 fragments; (6B) shows an embodiment of a fragment containing a truncation of ITGA2B or ITGB3; (6C) shows an embodiment of a single-chain molecule containing three different fragments; (6D) shows an embodiment of a single-chain molecule containing four different fragments; (6E) shows an embodiment 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 embodiment 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; (6G) shows the single-chain molecules of 6A and 6C-6F having an amino acid (AA) linker that separates various domains. [Figure 6D] Figures 6A-6G: Figures of the single-chain therapeutic agent of the present invention: (6A) shows an embodiment of a single-chain molecule containing fragments from two different ITGA2B / B3 fragments; (6B) shows an embodiment of a fragment containing a truncation of ITGA2B or ITGB3; (6C) shows an embodiment of a single-chain molecule containing three different fragments; (6D) shows an embodiment of a single-chain molecule containing four different fragments; (6E) shows an embodiment 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 embodiment 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; (6G) shows the single-chain molecules of 6A and 6C-6F having an amino acid (AA) linker that separates various domains. [Figure 6E]Figures 6A-6G: Figures of the single-chain therapeutic agent of the present invention: (6A) shows an embodiment of a single-chain molecule containing fragments from two different ITGA2B / B3 fragments; (6B) shows an embodiment of a fragment containing a truncation of ITGA2B or ITGB3; (6C) shows an embodiment of a single-chain molecule containing three different fragments; (6D) shows an embodiment of a single-chain molecule containing four different fragments; (6E) shows an embodiment 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 embodiment 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; (6G) shows the single-chain molecules of 6A and 6C-6F having an amino acid (AA) linker that separates various domains. [Figure 6F] Figures 6A-6G: Figures of the single-chain therapeutic agent of the present invention: (6A) shows an embodiment of a single-chain molecule containing fragments from two different ITGA2B / B3 fragments; (6B) shows an embodiment of a fragment containing a truncation of ITGA2B or ITGB3; (6C) shows an embodiment of a single-chain molecule containing three different fragments; (6D) shows an embodiment of a single-chain molecule containing four different fragments; (6E) shows an embodiment 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 embodiment 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; (6G) shows the single-chain molecules of 6A and 6C-6F having an amino acid (AA) linker that separates various domains. [Figure 6G-1]Figures 6A-6G: Figures of the single-chain therapeutic agent of the present invention: (6A) shows an embodiment of a single-chain molecule containing fragments from two different ITGA2B / B3 fragments; (6B) shows an embodiment of a fragment containing a truncation of ITGA2B or ITGB3; (6C) shows an embodiment of a single-chain molecule containing three different fragments; (6D) shows an embodiment of a single-chain molecule containing four different fragments; (6E) shows an embodiment 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 embodiment 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; (6G) shows the single-chain molecules of 6A and 6C-6F having an amino acid (AA) linker that separates various domains. [Figure 6G-2] Same as above.

[0104] [Figure 7] Figure 7: Photographs of SDS-PAGE gels showing molecules CRD-757, 758, 760, and 756 under reducing (right) and non-reducing (left) conditions.

[0105] [Figure 8A-1] Figures 8A-8D: (8A) Histograms of CRD-757 binding to 17 different hybridomas. Secondary antibody alone is used as a negative control. Binding is observed only for ITP hybridomas LK-4 and AP-3. (8B) Bar graph summarizing the increase in MFI above background for CRD-757 binding to each hybridoma observed in 8A. (8C) Histograms of CRD-758 binding to two ITP hybridomas LK-4 and AP-3. Secondary antibody alone is used as a negative control. (8D) Bar graph summarizing the increase in MFI above background for CRD-758 binding to various hybridomas. Details for each hybridoma are shown in Table 3. [Figure 8A-2] Same as above. [Figure 8A-3] Same as above. [Figure 8B]Figures 8A-8D: (8A) Histograms of CRD-757 binding to 17 different hybridomas. Secondary antibody alone is used as a negative control. Binding is observed only for ITP hybridomas LK-4 and AP-3. (8B) Bar graph summarizing the increase in MFI above background for CRD-757 binding to each hybridoma observed in 8A. (8C) Histograms of CRD-758 binding to two ITP hybridomas LK-4 and AP-3. Secondary antibody alone is used as a negative control. (8D) Bar graph summarizing the increase in MFI above background for CRD-758 binding to various hybridomas. Details for each hybridoma are shown in Table 3. [Figure 8C] Figures 8A-8D: (8A) Histograms of CRD-757 binding to 17 different hybridomas. Secondary antibody alone is used as a negative control. Binding is observed only for ITP hybridomas LK-4 and AP-3. (8B) Bar graph summarizing the increase in MFI above background for CRD-757 binding to each hybridoma observed in 8A. (8C) Histograms of CRD-758 binding to two ITP hybridomas LK-4 and AP-3. Secondary antibody alone is used as a negative control. (8D) Bar graph summarizing the increase in MFI above background for CRD-758 binding to various hybridomas. Details for each hybridoma are shown in Table 3. [Figure 8D] Figures 8A-8D: (8A) Histograms of CRD-757 binding to 17 different hybridomas. Secondary antibody alone is used as a negative control. Binding is observed only for ITP hybridomas LK-4 and AP-3. (8B) Bar graph summarizing the increase in MFI above background for CRD-757 binding to each hybridoma observed in 8A. (8C) Histograms of CRD-758 binding to two ITP hybridomas LK-4 and AP-3. Secondary antibody alone is used as a negative control. (8D) Bar graph summarizing the increase in MFI above background for CRD-758 binding to various hybridomas. Details for each hybridoma are shown in Table 3.

[0106] [Figure 9] Figure 9: Line graphs showing the increase in cell euthanasia rates by CRD-757 and CRD-758 as measured by the CDC. This increase is compared to CRD-760, which lacks the Fc domain. [Modes for carrying out the invention]

[0107] The present invention provides, in some embodiments, compositions comprising a fragment of a first human receptor target or an analog or derivative thereof of an immune thrombocytopenia (ITP) autoantibody, and a fragment of a second human protein receptor target or an analog or derivative thereof of an ITP autoantibody. Compositions further comprising an effector moiety that is not an unmodified Fc domain are also provided. Protein complexes comprising at least two polypeptide chains are also provided, wherein the first chain comprises a fragment of a first human protein target or an analog or derivative thereof of an ITP autoantibody and a first dimerization domain, and the second chain comprises a fragment of a second human protein target or an analog or derivative thereof of an ITP autoantibody and a second dimerization domain that can dimerize with the first dimerization domain. Polypeptides comprising fragments of the 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 relates to pharmaceutical compositions comprising a composition and / or protein complex, nucleic acids encoding polypeptides of the composition and / or protein complex, therapeutic methods using the composition and / or protein complex, methods for determining suitability for therapeutic use of the composition and / or protein complex, and methods for producing the composition and / or protein complex.

[0108] In a first embodiment, a composition is provided comprising a fragment of a first protein target of an ITP autoantibody, or an analog or derivative thereof.

[0109] In another embodiment, a composition is provided comprising a fragment of a first protein target of an ITP autoantibody or an analog or derivative thereof, and a fragment of a second protein target of an ITP autoantibody or an analog or derivative thereof.

[0110] In another embodiment, a protein is provided that comprises a fragment of a first protein target of an ITP autoantibody, or an analog or derivative thereof.

[0111] In another embodiment, a protein is provided comprising a fragment of a first protein target of an ITP autoantibody, or an analog or derivative thereof, and a fragment of a second protein target of an ITP autoantibody, or an analog or derivative thereof.

[0112] In another embodiment, a protein complex comprising at least two polypeptide chains is provided, the first polypeptide chain comprising a fragment of a first protein target of an ITP autoantibody or an analog or derivative thereof, and a first dimerization domain, and the second polypeptide chain comprising a fragment of a second protein target of an ITP autoantibody or an analog or derivative thereof, and a second dimerization domain.

[0113] In some embodiments, the composition comprises a protein complex comprising at least two polypeptide chains, wherein the first polypeptide chain comprises a fragment of a first protein target of an ITP autoantibody or an analog or derivative thereof, and a first dimerization domain, and the second polypeptide chain comprises a fragment of a second protein target of an ITP autoantibody or an analog or derivative thereof, and a second dimerization domain. In some embodiments, the composition comprises the protein complex of the present invention. In some embodiments, the composition comprises the protein of the present invention. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is a fusion protein.

[0114] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to polymers of amino acid residues. In other embodiments, the terms “peptide,” “polypeptide,” and “protein” as used herein encompass natural peptides, peptide mimetics (typically including non-peptide bonds or other synthetic modifications), as well as peptide analogs, peptoids, and semipeptoids, or any combination thereof. In other embodiments, the peptides, polypeptides, and proteins described have modifications that make them more stable in the body or more capable of penetrating cells. In one embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to natural amino acid polymers. In another embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids. In some embodiments, the peptide is not a cyclic peptide. In some embodiments, the fragment is not a cyclic peptide. In some embodiments, the extracellular domain is not a cyclic peptide.

[0115] In some embodiments, the protein complex is an immunoglobulin (Ig)-like complex. In some embodiments, the protein complex includes an Ig-like scaffold. In some embodiments, the protein complex includes an Ig-like backbone. In some embodiments, the protein complex is an Ig Fc fusion complex. In some embodiments, the composition lacks an antibody-variable domain. In some embodiments, the protein complex lacks an antibody-variable domain. In some embodiments, the composition lacks a variable domain. In some embodiments, the protein complex lacks a variable domain. In some embodiments, the first chain lacks a variable domain. In some embodiments, the second chain lacks a variable domain. In some embodiments, the protein complex is a multi-chain complex. In some embodiments, the composition is a therapeutic composition. In some embodiments, the protein complex is a therapeutic complex. In some embodiments, the composition is for use in therapeutic methods. In some embodiments, the protein complex is for use in therapeutic methods. In some embodiments, the composition is for use in the production of pharmaceuticals. In some embodiments, the protein complex is for use in the production of pharmaceuticals. In some embodiments, the composition is for use in the treatment of 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 the treatment of ITP. In some embodiments, the protein complex is for use in the diagnosis of ITP. In some embodiments, the protein complex is for use in determining appropriate treatment for 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 autoantibody titers in ITP.

[0116] As used herein, the term “polypeptide chain” refers to a polymer of amino acids linked by peptide bonds from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). In some embodiments, the polypeptide chain is a recombinant polypeptide. In some embodiments, the polypeptide chain contains 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 distinct embodiment of the present invention. In some embodiments, the polypeptide chain contains up to 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 distinct embodiment of the present invention.

[0117] As used herein, the term “recombinant polypeptide” refers to a protein encoded by recombinant DNA and therefore not found in nature. In some embodiments, the protein complex does not exist in nature. In some embodiments, the polypeptide chain does not exist in nature. In some embodiments, the recombinant polypeptide is a synthetic polypeptide. The term “recombinant DNA” refers to a DNA molecule formed by laboratory methods. Generally, this recombinant DNA is in the form of a vector, plasmid, or virus used to express recombinant proteins in cells. The production of recombinant proteins by cell expression is well known in the art, and the polypeptides of the present invention may be produced using any method of recombinant protein expression. Cell-free expression systems for recombinant protein production may also be used.

[0118] As used herein, the term “expression” refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. Therefore, the expression of a nucleic acid molecule may refer to the transcription of a nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA), and / or the translation of RNA into a precursor or mature protein (polypeptide). In some embodiments, the nucleic acid molecule of the present invention is expressed in a cell to produce the polypeptide of the present invention. In some embodiments, the nucleic acid complex of the present invention is expressed in a cell to produce the protein complex of the present invention. In some embodiments, RNA is a vector.

[0119] The expression of DNA sequences or RNA within cells is well known to those skilled in the art. This can be done by transfection, viral infection, or direct alteration of the cell's genome, among many other methods. In some embodiments, the DNA sequence is contained within an expression vector, such as a plasmid or viral vector. In some embodiments, a Kozak sequence is inserted upstream of the transcription start codon. In some embodiments, the Kozak sequence enhances the amount of protein being expressed.

[0120] In some embodiments, the protein complex comprises at least two polypeptide chains. In some embodiments, the protein complex comprises at least three polypeptide chains. In some embodiments, the protein complex comprises at least four polypeptide chains. In some embodiments, the protein complex comprises or consists of two polypeptide chains. In some embodiments, the protein complex comprises or consists of three polypeptide chains. In some embodiments, the protein complex comprises or consists of four chains. In some embodiments, the polypeptide chains are the same. In some embodiments, the polypeptide chains are different. In some embodiments, at least two of the polypeptide chains are the same. In some embodiments, at least two of the polypeptide chains are different.

[0121] protein In some embodiments, the protein is a mammalian protein. In some embodiments, the mammal is a human. In some embodiments, the protein is a transmembrane protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a subunit within a receptor. In some embodiments, the protein is a cell surface protein. In some embodiments, the cell surface protein is an endogenous 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-fixed protein. In some embodiments, the protein is an ITP-related protein. In some embodiments, the protein is a synthetic protein. In some embodiments, the protein is a naturally occurring protein. In some embodiments, the protein is a target of an ITP autoantibody. 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.

[0122] As used herein, the term “receptor” refers to a protein expressed on the surface of a cell that can bind to a ligand. In some embodiments, the receptor is a protein that can transmit a signal to the cytoplasm of a cell. In some embodiments, the receptor includes a ligand-binding domain. In some embodiments, the receptor includes a transmembrane domain. In some embodiments, the receptor includes an intracellular domain.

[0123] In some embodiments, the fragment includes the extracellular domain (ECD) of a protein. In some embodiments, the fragment includes a fragment of the extracellular domain of a protein. In some embodiments, the fragment consists of the extracellular domain of that fragment. In some embodiments, the fragment consists of the extracellular domain of a protein. In some embodiments, the fragment consists of a fragment of the extracellular domain of a protein. In some embodiments, the fragment includes the transmembrane domain of a protein. In some embodiments, the fragment lacks the transmembrane domain of a protein. In some embodiments, the fragment lacks the intracellular domain of a protein. In some embodiments, the chain lacks the transmembrane domain. In some embodiments, the chain lacks the intracellular domain. In some embodiments, the fragment includes a sequence derived from a homologous human protein. In some embodiments, the fragment includes a sequence derived from a homologous non-human protein. In some embodiments, the fragment includes a mutation in a human protein.

[0124] In some embodiments, the fragment contains at least five amino acids of the protein. In some embodiments, the fragment contains at least ten amino acids of the protein. In some embodiments, the fragment contains 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 distinct embodiment of the present invention. In some embodiments, the amino acids of the protein are sequential amino acids of the protein. In some embodiments, the fragment contains less than 100% of the protein. In some embodiments, the fragment contains less than 100% of the extracellular domain of the protein. In some embodiments, the fragment contains less than 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55, or less than 50% of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragment contains 100, 99, 97, 95, 90, 85, 80, 75, 70, 65, 60, 55, or less than 50% of the extracellular domain of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragment contains 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 distinct embodiment of the present invention. In some embodiments, the number of fragments is up to 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, 3 It contains 10, 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 distinct embodiment of the present invention.

[0125] In some embodiments, the chain comprises at least one fragment. In some embodiments, the chain comprises at least two fragments. In some embodiments, the fragments are separated by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, a region of protein is replaced by a region of a non-protein. In some embodiments, the substituted region contains increased protein stability compared to the substituted protein region.

[0126] In some embodiments, the protein is the target of the antibody. As used herein, the term “antibody” includes all classes of IgA, IgD, IgE, IgG, and IgM, as well as all their subclasses. In some embodiments, the antibody is a circulating antibody. In some embodiments, the antibody is a naturally occurring antibody. In some embodiments, the antibody is an autoantibody.

[0127] As used herein, the term “autoantibody” refers to an antibody produced by the subject’s own immune system against at least one of the subject’s own proteins. In some embodiments, the autoantibody is an autoreactive antibody. In some embodiments, the autoantibody targets an autoantigen. An autoantigen is also known as an autoantigen. In some embodiments, the autoantibody is associated with ITP. In some embodiments, the autoantibody characterizes ITP. In some embodiments, the autoantibody is an autoantibody of ITP. In some embodiments, the autoantibody is produced by autoreactive B cells. In some embodiments, the protein is the antigen of the antibody. In some embodiments, the fragment contains the antigen of the antibody. In some embodiments, the fragment contains at least one antigen of the antibody. In some embodiments, the fragment contains at least two antigens of the antibody. In some embodiments, the fragment contains at least one, two, three, four, five, six, seven, eight, nine, or ten antigens of the antibody. Each possibility represents a distinct embodiment of the invention. In some embodiments, the antigen of the antibody is an autoantigen. In some embodiments, the antigen is an epitope. In some embodiments, the antigen comprises at least one epitope. In some embodiments, the epitope comprises at least five amino acids. In some embodiments, the epitope comprises five to six amino acids. In some embodiments, the epitope comprises five to ten amino acids. In some embodiments, the epitope is a simple epitope. In some embodiments, the simple epitope is a linear epitope. In some embodiments, the epitope is a complex epitope. In some embodiments, the complex epitope is a 3D epitope. In some embodiments, the complex epitope is a discontinuous epitope. In some embodiments, the discontinuous epitope comprises at least two discontinuous portions of amino acids that bind to form an epitope. In some embodiments, the linker sequence is located between the two portions of the epitope.

[0128] As used herein, the term “analog” includes any peptide having a substantially identical amino acid sequence to that of a protein, but in which one or more residues are conservedly substituted by functionally similar residues. In some embodiments, the analog exhibits similar functionality to the original protein. Examples of conservative substitutions include substitution of a nonpolar (hydrophobic) residue to another residue, e.g., isoleucine, valine, leucine, or methionine; substitution of a polar (hydrophilic) residue to another residue, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine; substitution of a basic residue to another residue, e.g., lysine, arginine, or histidine; or substitution of an acidic residue to another residue, e.g., aspartic acid or glutamic acid. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the substitution lies outside the antigenic region of the protein. In some embodiments, the substitution lies outside the epitope of the antibody. In some embodiments, the analog is still a target of the antibody. In some embodiments, the analog retains the binding of an autoantibody. The analog may have deletions or mutations that result in an amino acid sequence different from the canonical amino acid sequence of the protein. Furthermore, the analog may resemble a fragment of the protein, in which case the fragment must contain at least 50 consecutive amino acids of the protein or at least one epitope of the antibody. In some embodiments, the analog is an analog to the canonical sequence of the protein.

[0129] In some embodiments, the protein analog comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the protein analog comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the protein analog comprises an amino acid sequence having at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the analog can still bind to ITP autoantibodies. In some embodiments, the analog can still capture ITP autoantibodies. In some embodiments, the analog can still treat ITP. In some embodiments, the analog comprises at least one substitution. In some embodiments, the analogues include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. Each possibility represents a distinct embodiment of the invention. In some embodiments, the substitutions are variations of the canonical sequence.

[0130] As used herein, the term “derivative” refers to any polypeptide based on a protein that still retains antibody binding. A derivative is not merely a fragment of a protein, but does not have substituted or removed amino acids (analogs), and rather may have additional modifications made to the protein, such as post-translational modifications. Furthermore, a derivative may be a derivative of a protein fragment, in which case the fragment must contain at least 50 consecutive amino acids of the protein or at least one epitope of the antibody. In some embodiments, the derivative is a derivative of the canonical sequence of a protein.

[0131] In some embodiments, the protein derivative includes an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% homology to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the protein derivative includes an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity to the canonical amino acid sequence of the protein. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the protein derivative includes an amino acid sequence having at least 85% identity to the canonical amino acid sequence of the protein. In some embodiments, the derivative can still bind to ITP autoantibodies. In some embodiments, the derivative can still capture ITP autoantibodies. In some embodiments, the derivative can still treat ITP. In some embodiments, the derivative is a mutant protein or fragment.

[0132] The canonical amino acid sequences of known proteins are well known in the art. They can be found in various databases, including UniProt, NCBI, and UCSC Genome Browser. Any sequence accepted as a canonical sequence may be used. In a non-limiting example, human ITGA2B (also known as GPIIb) is encoded by the ITGA2B gene, its canonical nucleic acid sequence can be found in Entrez gene 3674, its canonical protein-coding mRNA sequence can be found in NM_000419, and its canonical amino acid sequence can be found in NP_000410 and UniProt number P08514. Similarly, human ITGB3 (also known as GPIIIa) is encoded by the ITGB3 gene, whose canonical nucleic acid sequence can be found at Entrez gene 3690, whose canonical protein-coding mRNA sequence can be found at NM_000212, and whose canonical amino acid sequence can be found at NP_000203 and UniProt number P05106. In some embodiments, the canonical sequence is identical to a sequence present in at least 50, 60, 70, 75, 80, 90, 95, 97, or 99% of the population. Each possibility represents a distinct embodiment of the invention. In some embodiments, the canonical sequence is identical to the most common sequence present in the population. In some embodiments, the population is a disease population. In some embodiments, the population is a population with an autoimmune disease.

[0133] In some embodiments, the canonical amino acid sequence of the extracellular domain of ITGA2B is:(Sequence ID 1) is included or consists of. In some embodiments,The extracellular domain lacks 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).

[0134] In some embodiments, the canonical amino acid sequence of the extracellular domain of ITGB3 includes or consists of (SEQ ID NO: 2).In some embodiments, the extracellular domain lacks a signal peptide. In some embodiments, the extracellular domain further comprises a signal peptide. In some embodiments, the ITGB3 signal peptide comprises or consists of MRARPRPRPLWATVLALGALAGVGVG (SEQ ID NO: 16). In some embodiments, the ITGA2B signal peptide or the ITGB3 signal peptide is used.

[0135] In some embodiments, the signal peptide is the signal peptide of the antibody chain. In some embodiments, the single peptide is of the antibody heavy chain. In some embodiments, the signal peptide is of the antibody light chain. In some embodiments, the signal peptide is of the kappa light chain. In some embodiments, the signal peptide is of the lambda light chain. In some embodiments, the heavy chain signal peptide includes MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 17). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 17. In some embodiments, the light chain signal peptide includes MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 18). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 18. In some embodiments, the signal peptide includes or 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 includes or consists of MGWSCIILFLVATATGVHS (SEQ ID NO: 20). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 20.

[0136] In some embodiments, the first protein and the second protein are the same protein. In some embodiments, the first and second proteins are the same protein, but the fragments are different fragments. In some embodiments, the fragments are different fragments. In some embodiments, the fragments contain or consist of different sequences. In some embodiments, the first and second proteins are different proteins.

[0137] In some embodiments, the fragment includes an extracellular functional domain. In some embodiments, the functional domain is a ligand-binding domain. In some embodiments, the ligand is selected from laminin, collagen, and fibronectin.

[0138] In some embodiments, the fragment includes a truncation of an extracellular domain. In some embodiments, the fragment consists of a truncation of an 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, the derivative is a derivative of the truncation. In some embodiments, the derivative contains at least 85% identity with respect to the truncation and does not further contain a stretch of homologous / identical amino acids to the sequence derived from ITGA2B or ITGB3. Thus, it is understood that a sequence having sequence identity with respect to the truncation is not an untruncated sequence. In some embodiments, the truncation contains at least one mutation. In some embodiments, the derivative contains at least 85% identity with respect to any one of SEQ ID NOs: 1. In some embodiments, the derivative contains at least 85% identity with respect to any one of SEQ ID NOs: 2.

[0140] Dimerization domain In some embodiments, the dimerizing domains can dimerize with each other. In some embodiments, the first dimerizing domain can dimerize with the second dimerizing domain. In some embodiments, the first and second dimerizing domains can dimerize with each other. In some embodiments, "can dimerize" means "is configured to dimerize". In some embodiments, dimerization occurs under physiological conditions. In some embodiments, dimerization occurs in body fluids. In some embodiments, the body fluid is blood. In some embodiments, the body fluid is plasma. In some embodiments, the body fluid is serum. In some embodiments, dimerization occurs within a subject. In some embodiments, dimerization occurs in vivo. In some embodiments, dimerization occurs in vitro.

[0141] As used herein, the term “dimerizing domain” refers to an amino acid sequence that, upon contact with another amino acid sequence (another dimerizing domain), binds to it and forms a dimer. Dimerizing domains are well known in the art, as many protein sequences are known to bind to one another. In some embodiments, dimerization involves the formation of a covalent bond between dimerizing domains. In some embodiments, dimerization involves an electrostatic bond. In some embodiments, dimerization does not involve an electrostatic bond. In some embodiments, dimerization is reversible. In some embodiments, dimerization is irreversible. In some embodiments, dimerization involves a bond formed between dimerizing domains. In some embodiments, the bond is a chemical bond. In some embodiments, the bond is a disulfide bond. In some embodiments, the bond is a peptide bond. Examples of dimerizing domains include, to name a few, the hinge domain of an antibody heavy chain, the CH1 / CL domain of an antibody heavy / light chain, and the ECD domain of TCR alpha / beta. In addition, the upper hinge domain may be manipulated by cysteine ​​substitution / mutation to serine to prevent dimerization. In some embodiments, the dimerization domain contains or consists of the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 21).

[0142] In some embodiments, the dimerization domain includes or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, the Ig hinge domain is a heavy chain hinge domain. In some embodiments, Ig is human Ig. In some embodiments, the immunoglobulin is selected from IgA, IgD, IgE, IgG, and IgM. In some embodiments, the immunoglobulin is IgG. In some embodiments, IgG is IgG1. In some embodiments, IgG is IgG2. In some embodiments, IgG is IgG3. In some embodiments, IgG is selected from IgG1 and IgG3. In some embodiments, IgG is IgG4. In some embodiments, both the first and second dimerization domains are 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 distinct embodiment of the present invention.

[0143] In some embodiments, the hinge domain includes 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 IgG1 hinge includes or consists of SEQ ID NO: 22. In some embodiments, the hinge domain includes 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 includes or consists of SEQ ID NO: 23. In some embodiments, the hinge domain includes 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 includes or consists of SEQ ID NO: 24. In some embodiments, the hinge domain includes the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGP (SEQ ID NO: 25). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IgG3 hinge includes or consists of SEQ ID NO: 25. In some embodiments, the hinge domain includes the CPXCP (SEQ ID NO: 26) motif. In some embodiments, X in SEQ ID NO: 26 is selected from P and R. In some embodiments, SEQ ID NO: 26 is CPPCP (SEQ ID NO: 27). In some embodiments, SEQ ID NO: 26 is CPRCP (SEQ ID NO: 28). In some embodiments, the hinge domain includes EPKSCDKTHTCPPCP (SEQ ID NO: 29). Thus, it is understood that the hinge region may be considered to terminate after the CPXCP motif.

[0144] In some embodiments, the dimerization domain includes or consists of an Ig CH1 domain. In some embodiments, the dimerization domain includes or consists of an Ig heavy chain CH1 domain. In some embodiments, the dimerization domain includes or consists of an Ig light chain. In some embodiments, the dimerization domain includes 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 CH1 domain of the Ig heavy chain dimerizes with the light chain CL domain. In some embodiments, the first dimerization domain includes or consists of a CH1 domain, and the second dimerization domain includes or consists of a CL domain. In some embodiments, both the first and second dimerization domains include a hinge domain. In some embodiments, neither the first nor the second dimerization domains include a CH1 domain. In some embodiments, neither the first nor the second dimerization domains include a CL domain. In some embodiments, neither the first nor the second polypeptide chain contains a CH1 domain. In some embodiments, neither the first nor the second polypeptide chain contains a CL domain. In some embodiments, neither the first nor the second polypeptide chain contains a CH1 domain.

[0145] In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEITPTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 30). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 30. In some embodiments, SEQ ID NO: 30 is the IgG1 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEITPTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 31). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 31. In some embodiments, SEQ ID NO: 31 is the IgG2 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEITPTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 32). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 32. In some embodiments, SEQ ID NO: 32 is the IgG3 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEITPTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 33). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 33. In some embodiments, SEQ ID NO: 33 is the IgG4 CH1 domain.

[0146] In some embodiments, the Ig CL kappa domain comprises the amino acid sequence AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSITPTKSFNRGEC (SEQ ID NO: 34). In some embodiments, the Ig CL kappa domain consists of SEQ ID NO: 34. In some embodiments, the Ig CL lambda domain comprises the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSITPKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 35). In some embodiments, the Ig CL lambda domain consists of SEQ ID NO: 35.

[0147] Effects pedal section In some embodiments, the composition includes an effector moiety. In some embodiments, the first polypeptide chain includes an effector moiety. In some embodiments, the second polypeptide chain includes an effector moiety. In some embodiments, both the first and second polypeptide chains include an effector moiety. As used herein, the term “moiety” refers to a portion of a molecule that may include an entire functional group or a portion of a functional group as a substructure. The term “moiety” may also refer to a portion of a molecule that exhibits a particular set of chemical and / or pharmacological features similar to the corresponding molecule. As used herein, the term “effector moiety” refers to a molecule or fragment of a molecule that performs a cytotoxic effect. In some embodiments, the effector moiety is an effector molecule.

[0148] In some embodiments, the effector portion can induce a cytotoxic effect. In some embodiments, the effector portion is configured to induce a cytotoxic effect. In some embodiments, the effector portion can induce death. In some embodiments, the effector portion 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 toward target cells. In some embodiments, death is within the target cells. In some embodiments, the cytotoxic effect occurs at the time of binding. In some embodiments, death occurs at the time of binding. In some embodiments, the cytotoxic effect is toward target cells bound to the composition. In some embodiments, death is the death of target cells bound to the composition. In some embodiments, the cytotoxic effect is toward cells bound by a protein complex. In some embodiments, the cytotoxic effect is toward cells bound to a protein complex. In some embodiments, death is the death of cells bound by a protein complex. In some embodiments, death is the death of cells bound to a 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 to the composition means binding to the fragment. In some embodiments, binding to the protein complex means binding to the fragment. In some embodiments, the fragment is at least one of the fragments. In some embodiments, the fragment is one of the fragments. In some embodiments, the fragment is both fragments.

[0149] In some embodiments, the effector portion is a cytotoxic portion. In some embodiments, the effector portion is a toxin. In some embodiments, the effector portion is a poison. In some embodiments, the effector portion is a chemotherapeutic agent. In some embodiments, the effector portion is an anticancer agent. In some embodiments, the effector portion is an engager. In some embodiments, the engager binds to cytotoxic cells. In some embodiments, binding to cytotoxic cells means recruiting cytotoxic cells. In some embodiments, binding means being bound by.

[0150] In some embodiments, the effector portion recruits a cytotoxic agent. In some embodiments, the cytotoxic agent is a cytotoxic cell. In some embodiments, the cytotoxic cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is a CD8-positive T cell. In some embodiments, the effector portion induces antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector portion induces complement-dependent cell-mediated cytotoxicity (CDC).

[0151] In some embodiments, the effector portion binds to a receptor on the cell surface of cytotoxic cells. Examples of receptors include, but are not limited to, CD3, CD8, CD56, CD14, and CD16. In some embodiments, the receptor is a marker of cytotoxic cells. In some embodiments, the receptor is specific to cytotoxic cells. In some embodiments, the receptor is CD3. In some embodiments, the effector portion is an activator that binds to CD3. In some embodiments, the engager is an activator that binds to CD3. In some embodiments, CD3 is human CD3. In some embodiments, the activator that binds to CD3 is an anti-CD3 antibody or its antigen-binding fragment. In some embodiments, the receptor is CD16. In some embodiments, the effector portion is an activator that binds to CD16. In some embodiments, the engager is an activator that binds to CD16. In some embodiments, CD16 is human CD16. In some embodiments, the activator that binds to CD16 is an anti-CD16 antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is a single-chain antibody. In some embodiments, the antibody or its antigen-binding fragment is a single-domain antibody. In some embodiments, the antibody or its antigen-binding fragment is a single-strand variable fragment (scFv). Anti-CD3 activators are well known in the art, and any such conjugate activator may be used. For example, an anti-human CD3 scFv known as OKT3 may be used as the activator. In some embodiments, the cytotoxic moiety is selected from alpha-amanitin, a radioactive moiety, and an anti-CD3 conjugate activator.Other examples of human anti-CD3 antibodies include muromonab (trade name Orthoclone OKT3), a mouse monoclonal anti-human CD3 antibody (DrugBank accession number DB00075); teplizumab, a humanized version of the mouse OKT3 anti-CD3 monoclonal antibody (DrugBank accession number DB06606); UCHT1, a mouse monoclonal anti-human CD3 antibody; UCHT1 mutant-9, a humanized version of the UCHT1 clone; and bispecific CD19-CD3 blinatumomab (DrugBank accession number DB09052). Examples of human anti-CD16 include AFM13, a bispecific tetravalent innate cell engager (ICE®) that targets CD30 on tumor cells and CD16A on NK cells and macrophages, and GTB-3550 (CD16 / IL-15 / CD33), a triplicate killer cell engager.

[0152] In some embodiments, the composition includes an Fc region. In some embodiments, the effector portion is not an Fc region. In some embodiments, not being an Fc region means not being an unmodified Fc region. In some embodiments, the composition includes an effector portion that is not an Fc region. In some embodiments, the composition includes an effector portion other than an Fc region. In some embodiments, the composition lacks an Fc region. In some embodiments, the protein includes an effector portion that is not an Fc region. In some embodiments, the protein includes an effector portion other than an Fc region. In some embodiments, the protein lacks an Fc region. In some embodiments, the engager is an Fc region. In some embodiments, the engager is not an Fc region. In some embodiments, the composition includes an effector portion that is superior in killing compared to Fc. In some embodiments, superior in killing means superior in killing B cells. In some embodiments, Fc is unmodified Fc. In some embodiments, Fc is unmutated Fc. In some embodiments, Fc is naturally occurring Fc. In some embodiments, Fc is not naturally occurring Fc. In some embodiments, Fc is human Fc. In some embodiments, a superior Fc is an Fc containing at least one mutation that increases ADCC. In some embodiments, the Fc region is an Fc domain. In some embodiments, the Fc region is an Fc fragment. In some embodiments, the first polypeptide chain contains an Fc region. In some embodiments, the second polypeptide chain contains an Fc region. In some embodiments, both the first and second polypeptide chains contain an Fc region. In some embodiments, the Fc region is the Fc region of an antibody heavy chain. In some embodiments, the antibody heavy chain is a human antibody heavy chain. In some embodiments, the heavy chain is an IgG heavy chain. In some embodiments, IgG is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, IgG is selected from IgG1 and IgG3. In some embodiments, IgG is IgG1. In some embodiments, IgG is IgG2. In some embodiments, IgG is IgG3.In some embodiments, IgG is IgG4.

[0153] In some embodiments, the Fc region can induce a cytotoxic effect. In some embodiments, the Fc domain includes DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 57). In some embodiments, the Fc domain contains EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 58). It is understood that SEQ ID NO: 58 contains five additional N-terminal amino acids compared to SEQ ID NO: 57. Thus, the numbering in this specification is given with respect to SEQ ID NO: 57, but the numbering for SEQ ID NO: 58 can be found by adding 5. In some embodiments, the Fc region can induce a cytotoxic effect. In some embodiments, the Fc domain includes DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 59).In some embodiments, the Fc domain contains EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 60). It is understood that SEQ ID NO: 60 contains five additional N-terminal amino acids compared to SEQ ID NO: 59. Thus, although the numbering herein is given with respect to SEQ ID NO: 59 (or the equivalent SEQ ID NO: 57), 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. These two sequences are interchangeable, and it is understood that if a mutation is given to sequence number 57, it will also apply to sequence number 59, and vice versa. Similarly, sequence numbers 58 and 60 also differ by only two amino acids, and these two sequences are interchangeable.

[0154] In some embodiments, the Fc domain consists of SEQ ID NO: 57. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 57. In some embodiments, the Fc domain includes or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 57. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 58. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 58. In some embodiments, the Fc domain includes or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 58. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 59. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 59. In some embodiments, the Fc domain contains or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 59. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 60. In some embodiments, the Fc domain of IgG1 contains or consists of SEQ ID NO: 60. In some embodiments, the Fc domain contains or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 60. Each possibility represents a distinct embodiment of the present invention.

[0155] In some embodiments, the Fc region can induce a cytotoxic effect. In some embodiments, the Fc region is configured to induce a cytotoxic effect. In some embodiments, the cytotoxic effect is directed towards target cells. In some embodiments, the cytotoxic effect occurs upon binding. In some embodiments, the cytotoxic effect is directed towards cells bound by a protein complex. In some embodiments, the cytotoxic effect is directed towards cells that bind to a protein complex. In some embodiments, the cytotoxic effect is mediated by the binding of immune cells to the Fc region. In some embodiments, the cytotoxic effect is mediated by the activation of immune cells by the Fc region. In some embodiments, the cytotoxic effect is mediated by the recruitment of immune cells by the Fc region. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are macrophages. In some embodiments, the T cells are cytotoxic T cells. In some embodiments, the T cells are CD8-positive T cells. In some embodiments, the Fc region induces antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the Fc region induces complement-dependent cell injury (CDC).

[0156] In some embodiments, the Fc region includes an Ig hinge. In some embodiments, the Fc region includes an Ig CH2 domain. In some embodiments, the Fc region includes an Ig heavy chain CH2 domain. In some embodiments, the Fc region includes an Ig CH3 domain. In some embodiments, the Fc region includes an Ig heavy chain CH3 domain. In some embodiments, the Fc region includes or consists of both an Ig CH2 domain and an Ig CH3 domain. In some embodiments, the Fc region includes or consists of both an Ig heavy chain CH2 domain and an Ig heavy chain CH3 domain. In some embodiments, the first chain includes a first portion of the Fc region, and the second chain includes a second portion of the Fc region. In some embodiments, the first portion includes a CH2 domain, a CH3 domain, or both. In some embodiments, the second portion includes a CH2 domain, a CH3 domain, or both. In some embodiments, the interface between the first portion of the Fc region and the second portion of the Fc region results in a functional Fc region. In some embodiments, the interface includes contact. In some embodiments, the interface includes adjacent positioning. In some embodiments, the interface includes the formation of the protein complex of the present invention. In some embodiments, the interface includes the dimerization of the first and second dimerization domains. In some embodiments, the CH2 domain is an Ig CH2 domain. In some embodiments, the CH2 domain is a heavy chain CH2 domain. In some embodiments, the CH3 domain is an Ig CH3 domain. In some embodiments, the CH3 domain is a heavy chain CH3 domain.

[0157] In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 36). In some embodiments, the CH2 domain consists of SEQ ID NO: 36. In some embodiments, SEQ ID NO: 36 is the IgG1 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO: 37). In some embodiments, the CH2 domain consists of SEQ ID NO: 37. In some embodiments, SEQ ID NO: 37 is the IgG2 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 38). In some embodiments, the CH2 domain consists of SEQ ID NO: 38. In some embodiments, SEQ ID NO: 38 is the IgG4 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO: 39). In some embodiments, the CH2 domain consists of SEQ ID NO: 39. In some embodiments, SEQ ID NO: 39 is the IgG3 CH2 domain.

[0158] In some embodiments, the CH3 domain includes the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40). In some embodiments, the CH3 domain includes the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41). In some embodiments, the CH3 domain consists of SEQ ID NO: 40. In some embodiments, the CH3 domain consists of SEQ ID NO: 41. In some embodiments, SEQ ID NO: 40 is the IgG1 CH3 domain. In some embodiments, SEQ ID NO: 41 is the IgG1 CH3 domain. In some embodiments, the sequence of SEQ ID NO: 40 is a sequence primarily found in humans of European and American descent. In some embodiments, SEQ ID NO: 41 is a sequence primarily found in humans of Asian descent. In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42). In some embodiments, the CH3 domain consists of SEQ ID NO: 42. In some embodiments, SEQ ID NO: 42 is the IgG2 CH3 domain. In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 43). In some embodiments, the CH3 domain consists of SEQ ID NO: 43. In some embodiments, SEQ ID NO: 43 is the IgG4 CH3 domain.In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 44). In some embodiments, the CH3 domain consists of SEQ ID NO: 44. In some embodiments, SEQ ID NO: 44 is the IgG3 CH3 domain.

[0159] In some embodiments, Fc contains a mutation. In some embodiments, the CH3 domain contains a mutation. In some embodiments, the first CH3 domain contains a first mutation. In some embodiments, the second CH3 domain contains a second mutation. In some embodiments, the CH2 domain contains a mutation. In some embodiments, the first CH2 domain contains a first mutation. In some embodiments, the second CH2 domain contains a second mutation. In some embodiments, both the CH2 and CH3 domains contain mutations. In some embodiments, the first CH2 domain and the first CH3 domain each contain a first mutation. In some embodiments, the second CH2 domain and the second CH3 domain each contain a second mutation. In some embodiments, the mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the second mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the mutation enables heterodimerization. In some embodiments, the mutation enables heterodimerization of the first and second chains. In some embodiments, enabling means promoting. In some embodiments, enabling means enhancing.

[0160] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutations or modifications may be used to construct the polypeptide of the present invention. In some embodiments, an IgG-derived region is replaced by an IgA-derived region. In some embodiments, a TCR-derived region is inserted into the first CH3 domain, and a TCRb-derived region is inserted into the second CH3 domain. In some embodiments, the mutation is an insertion of a TCR-derived region. In some embodiments, the TCR is selected from TCR and TCRb. In some embodiments, the mutation is an insertion of a different Ig-derived region. Examples of these mutations can be found in Table 1. In some embodiments, the mutation is selected from the mutations in Table 1. In some embodiments, the first mutation is selected from the group of mutations shown in the row and second column of Table 1, and the second mutation is the group of mutations shown in the same row of Table 1 in the third column. The mutations in Table 1 are shown with the Kabat numbering of IgG1 unless otherwise indicated. Corresponding mutations can be produced in other IGs, specifically other IgGs. In some embodiments, the first mutation is T366Y and the second mutation is Y407T. In some embodiments, the first mutations are S354C and T366W and the second mutations are Y349C, T366S, L368A and Y407V. In some embodiments, the first mutations are S364H and F405A and the second mutations are Y349T and T392F. In some embodiments, the first mutations are T350V, L351Y, F405A and Y407V and the second mutations are T350V, T366L, K392L and T394W. In some embodiments, the first mutations are K392D and K409D and the second mutations are E356K and D399K. In some embodiments, the first mutations are D221E, P228E, and L368E, and the second mutations are D221R, P228R, and K409R. In some embodiments, the first mutations are K360E and K409W, and the second mutations are Q347R, D399V, and F405T.In some embodiments, the first mutations are K360E, K409W, and Y349C, and the second mutations are Q347R, D399V, F405T, and S354C. In some embodiments, the first mutation is F405L, and the second mutation is K409R. In some embodiments, the first mutations are K360D, D399M, and Y407A, and the second mutations are E345R, Q347R, T366V, and K409V. In some embodiments, the first mutations are Y349S, K370Y, T366M, and K409V, and the second mutations are E356G, E357D, S364Q, and Y407A. In some embodiments, the first mutation is T366K, and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, as well as Y349D and R355D. In some embodiments, the first mutations are T366K and C351K, and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, as well as Y349D and R355D. In some embodiments, the first mutations are L351D and L368E, and the second mutations are L351K and T366K. In some embodiments, the first mutations are L368D and K370S, and the second mutations are E357Q and S364K. In some embodiments, the first mutation is T366W, and the second mutations are T366S, L368A, and Y407V. In some embodiments, Ig is IgG2, and the first mutations are C223E, P228E, and L368E, and the second mutations are C223R, E225R, P228R, and K409R. In some embodiments, the first mutation is S354C or T366W, and the second mutations are Y349C, T366S, L368A, or Y407V. In some embodiments, the first mutation is S364H or F405A, and the second mutation is Y349T or T392F.In some embodiments, the first mutation is T350V, L351Y, F405A, or Y407V, and the second mutation is T350V, T366L, K392L, or T394W. In some embodiments, the first mutation is K392D or K409D, and the second mutation is E356K or D399K. In some embodiments, the first mutation is D221E, P228E, or L368E, and the second mutation is D221R, P228R, or K409R. In some embodiments, the first mutation is K360E or K409W, and the second mutation is Q347R, D399V, or F405T. In some embodiments, the first mutation is K360E, K409W, or Y349C, and the second mutation is Q347R, D399V, F405T, or S354C. In some embodiments, the first mutation is K360D, D399M, or Y407A, and the second mutation is E345R, Q347R, T366V, or K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, or K409V, and the second mutation is E356G, E357D, S364Q, or Y407A. In some embodiments, the first mutation is L351D or L368E, and the second mutation is L351K or T366K. In some embodiments, the first mutation is L368D or K370S, and the second mutation is E357Q or S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A, or Y407V. In some embodiments, Ig is IgG2, and the first mutation is C223E, P228E, or L368E, and the second mutation is C223R, E225R, P228R, or K409R. In some embodiments, the CH3 domain contains or consists of GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45).In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 46). In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47). In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 48).

[0161] [Table 1]

[0162] In some embodiments, the Fc domain includes at least one mutation that increases effector function. In some embodiments, the Fc domain includes at least one mutation that increases CDC, ADCC, or both. In some embodiments, the Fc domain includes at least one mutation that increases CDC. In some embodiments, the Fc domain includes at least one mutation that increases ADCC. In some embodiments, the Fc domain includes at least one mutation that increases antibody effector function. In some embodiments, the Fc domain includes at least one mutation that increases antibody stability. In some embodiments, stability is half-life. In some embodiments, half-life is circulating half-life. In some embodiments, half-life is blood half-life. In some embodiments, blood is serum.

[0163] In some embodiments, the Fc domain contains at least one mutation that reduces antibody effector function. In some embodiments, the Fc domain contains at least one mutation that reduces ADCC. In some embodiments, the at least one mutation that reduces ADCC is an LALA mutation. As used herein, an LALA mutation refers to a mutation of two consecutive leucine residues to an alanine residue. In some embodiments, the LALA mutation is located within a hinge domain. In some embodiments, the hinge domain is the hinge domain of IgG1. In some embodiments, the LALA mutation is the L19 and L20 mutations of SEQ ID NO: 22 to A19 and A20. In some embodiments, the LALA mutation hinge contains the L19A and L20A mutations of SEQ ID NO: 22. In some embodiments, the Fc domain contains a hinge domain containing EPKSCDKTHTCPPCPAPEAA (SEQ ID NO: 49). In some embodiments, the Fc domain containing the LALA mutation contains SEQ ID NO: 49. In some embodiments, the Fc domain contains a hinge domain consisting of SEQ ID NO: 49. In some embodiments, the LALA variant hinge domain consists of SEQ ID NO: 49. In some embodiments, the LALA variants are L234A and L235A variants of Fc. In some embodiments, at least one variant that reduces ADCC is the N297A variant. In some embodiments, the N297A variant is located within the CH2 domain. In some embodiments, the N297A variant is the asparagine variant of SEQ ID NO: 36 to alanine. In some embodiments, the N297A variant CH2 domain contains the N59A variant of SEQ ID NO: 36. In some embodiments, the Fc domain contains a CH2 domain containing SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 50). In some embodiments, the Fc domain contains a CH2 domain consisting of SEQ ID NO: 50. In some embodiments, the N297A mutant CH2 domain consists of Sequence ID No. 50.Mutations that produce the above functions are well known in the art, and any such mutation can be used. Examples of such mutations can be found in at least KOSAunders, 2019, "Conceptual approaches to modulating antibody effector functions and circulation half-life", Front, Immunol., 2019 Jun 7;10:1296, which is incorporated herein by reference in its entirety.

[0164] It is known to those skilled in the art that IgG2 and IgG4 have significantly reduced effector function and are generally not cytotoxic in nature. In addition, mutations in IgG4, such as S228P and L235E, are known to further reduce effector function. Furthermore, mutations that reduce the cytotoxicity / effector function of IgG1 and IgG3 are well known in the art. In some embodiments, IgG contains at least one mutation. In some embodiments, the mutation is multiple mutations. In some embodiments, the mutation reduces cytotoxicity. In some embodiments, the mutation increases stability. In some embodiments, the mutation reduces aggregation. In some embodiments, multiple mutations that reduce cytotoxicity include LALA mutations. In some embodiments, multiple mutations that reduce cytotoxicity include PG-LALA mutations. In some embodiments, the mutation is a mutation from proline 329 to glycine (P329G) in the IgG1 human heavy chain. In some embodiments, the P to G mutation is the P109 to G mutation of SEQ ID NO: 57 or 59. In some embodiments, the mutation is a mutation from leucine 234 to alanine (L234A) in the IgG1 human heavy chain. In some embodiments, the L-to-A mutation is a mutation from L14 to A in SEQ ID NO: 57 or 59. In some embodiments, the mutation is a mutation from leucine 235 to alanine (L235A) in the IgG1 human heavy chain. In some embodiments, the L-to-A mutation is a mutation from L15 to A in SEQ ID NO: 57 or 59. In some embodiments, the multiple mutations include P109G, L14A, and L15A in SEQ ID NO: 57 or 59. In some embodiments, the multiple mutations include L14A and L15A in SEQ ID NO: 57 or 59. In some embodiments, the multiple mutations include P329G, L234A, and L235A in the IgG1 human heavy chain. In some embodiments, the multiple mutations include L234A and L235A in the IgG1 human heavy chain. Those skilled in the art will understand that parallel mutations can also be performed on IgG3 heavy chains or non-human IgG1 heavy chains. In some embodiments, the multiple mutations that reduce cytotoxicity include YTE mutations.In some embodiments, the mutation is a mutation from methionine 252 to tyrosine (M252Y) in the IgG1 human heavy chain. In some embodiments, the M to Y mutation is the M32 to Y mutation in SEQ ID NO: 57 or 59. In some embodiments, the mutation is a mutation from serine 254 to threonine (S254T) in the IgG1 human heavy chain. In some embodiments, the S to T mutation is the S34 to T mutation in SEQ ID NO: 57 or 59. In some embodiments, the mutation is a mutation from threonine 256 to glutamate (T256E) in the IgG1 human heavy chain. In some embodiments, the T to E mutation is the T36 to E mutation in SEQ ID NO: 57 or 59. In some embodiments, multiple mutations include M32Y, S34T, and T36E in SEQ ID NO: 57 or 59. In some embodiments, multiple mutations include M252Y, S254T, and T256E in the IgG1 human heavy chain. In some embodiments, the mutation is a mutation at asparagine 297 (N297) in the IgG1 human heavy chain. In some embodiments, asparagine is mutated to alanine (N297A). In some embodiments, asparagine is mutated to glutamine (N297Q). In some embodiments, asparagine is N77 (N77A or N77Q) of SEQ ID NO: 57 or 59.

[0165] In some embodiments, the mutation increases the half-life of a molecule, peptide, polypeptide, or protein complex. In some embodiments, the mutation that increases the half-life is the mutation that increases binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutation that increases binding to FcRn is selected from the mutations shown in Table 4. In some embodiments, the mutation is a mutation from asparagine 434 to histidine (N434H). In some embodiments, the N434H mutant Fc domain contains the N214H mutation of SEQ ID NO: 57 or 59. In some embodiments, the mutation is a mutation from valine 308 to proline (V308P). In some embodiments, the H435A mutant Fc domain contains the 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 a mutation from histidine 435 to alanine (H435A). In some embodiments, the H435A mutant Fc domain contains the H215A mutation of SEQ ID NO: 57 or 59. In some embodiments, the mutations that increase binding to FcRn are multiple mutations. In some embodiments, the multiple mutations include or consist of a mutation from methionine 252 to tyrosine (M252Y), a mutation from serine 234 to threonine, and a mutation from threonine 256 to glutamate (T256E) (also known as YTE). In some embodiments, the M252Y / S254T / T256E mutant Fc domain contains the M32Y, S34T, and T35E mutations of SEQ ID NO: 57 or 59. In some embodiments, the multiple mutations include or consist of a mutation from methionine 428 to leucine (M428L) and a mutation from asparagine 434 to serine (N434S) (also known as LS). In some embodiments, the M428L / N434S mutant Fc domain includes the M208L and N214S mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of M428L and the asparagine 434 to alanine mutation (N434A) (also known as LA).In some embodiments, the M428L / N434A mutant Fc domain includes the M208L and N214A mutations of SEQ ID NO: 57 or 59. In some embodiments, a plurality of mutations include or consist of a mutation from threonine 250 to glutamine (T250Q) and a mutation from methionine 428 to leucine (M428L) (also known as QL). In some embodiments, the T250Q / M428L mutant Fc domain includes the T30Q and M208L mutations of SEQ ID NO: 57 or 59. In some embodiments, a plurality of mutations include a mutation from histidine 433 to lysine (H433K) and a mutation from asparagine 434 to phenylalanine (N434F). In some embodiments, the H433K / N434F mutant Fc domain includes the H213K and N214F mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the M252Y / S254T / T256E / H433K / N434F mutant Fc domain includes the M32Y, S34T, T35E, H213K, and N214F mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of the threonine 307 to alanine mutation (T307A), the glutamate 380 to alanine mutation (E380A), and the asparagine 434 to alanine mutation (N434A). In some embodiments, the T307A / E380A / N434A mutant Fc domain includes the T87A, E160A, and N214A mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of a methionine 252 to tyrosine mutation (M252Y), a valine 308 to proline mutation (V308P), and an asparagine 343 to tyrosine mutation (N343Y). In some embodiments, the M252Y / V308P / N343Y mutant Fc domain includes the M32Y, V88P, and N123Y mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of M252Y, a valine 308 to proline mutation (V308P), and an asparagine 434 to tyrosine mutation (N434Y).In some embodiments, the M252Y / V308P / N434Y mutant Fc domain includes the M32Y, V88P, and N214Y mutations of SEQ ID NO: 57 or 59. In some embodiments, a plurality of mutations include or consist of a histidine 258 to aspartic acid mutation (H258D), a threonine 307 to glutamine mutation (T307Q), and an alanine 378 to valine mutation (A378V). In some embodiments, the H258D / T307Q / A378V mutant Fc domain includes the H38D, T87Q, and A158V mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of a mutation from leucine 309 to aspartic acid (L309D), a mutation from glutamine 311 to histidine (Q311H), and a mutation from asparagine 434 to serine (N434S). In some embodiments, the L309D / Q311H / N434S mutant Fc domain includes the L89D, Q91H, and N214A mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality that attenuate binding includes or consists of a mutation from isoleucine 253 to alanine (I253A), H435A, and a mutation from histidine 436 to alanine (H436A). In some embodiments, the I253A / H435A / H436A mutant Fc domain includes the I33A, H215A, and H216A mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality of attenuating molecules include or consist of I253A, a histidine 310-to-alanine mutation (H310A), and H435A. In some embodiments, the I253A / H310A / H435A mutant Fc domain includes the I33A, H90A, and H215A mutations of SEQ ID NO: 57 or 59. Any position in SEQ ID NO: 58 and SEQ ID NO: 60 that is equivalent to those enumerated in SEQ ID NO: 57 and SEQ ID NO: 59 (by adding five amino acids to the numbering) is also expressly enumerated and intended herein.

[0166] [Table 2]

[0167] In some embodiments, the mutation is one that reduces binding to the Fc receptor. In some embodiments, the Fc receptor is FcγR. In some embodiments, FcγR is FcγRI. In some embodiments, the mutation is one that reduces binding to C1q. In some embodiments, the mutation that reduces binding to the Fc receptor reduces ADCC. In some embodiments, the mutation is a mutation at N297. Since the N-glycan is linked to N297, the mutation in this case invalidates glycosylation of this residue. In some embodiments, the mutation at N297 is a mutation to alanine (N297A). In some embodiments, the mutation at N297 is a mutation to glutamine (N297Q). In some embodiments, the mutation at N297 is a mutation to glycine (N297G). In some embodiments, the N297A mutant CH2 domain contains the N59A mutation of SEQ ID NO: 36. In some embodiments, the N297A mutant Fc domain contains the N77A mutation of SEQ ID NO: 57 or 59. In some embodiments, the N297Q mutant CH2 domain includes the N59Q mutation of SEQ ID NO: 36. In some embodiments, the N297Q mutant Fc domain includes the N77Q mutation of SEQ ID NO: 57 or 59. In some embodiments, the N297G mutant CH2 domain includes the N59G mutation of SEQ ID NO: 36. In some embodiments, the N297G mutant Fc domain includes the N77G mutation of SEQ ID NO: 57 or 59. In some embodiments, the mutation is a plurality of mutations that reduce binding to the Fc receptor. In some embodiments, the plurality includes or consists of a mutation from glycine 236 to arginine (G236R) and a mutation from leucine 328 to arginine (L328R). In some embodiments, the G236R / L328R mutant Fc includes a hinge domain containing the G21R mutation of SEQ ID NO: 22 and a CH2 domain containing the L90R mutation of SEQ ID NO: 36. In some embodiments, the G236R / L328R mutant Fc domain includes the G16R and L108R mutations of SEQ ID NO: 57 or 59. In some embodiments, a plurality of mutations include, or consist of, a serine 298 to glycine mutation (S298G) and a threonine 299 to alanine mutation (T299A).In some embodiments, the S298G / T299A mutant CH2 domain includes the S60G and T61A mutations of SEQ ID NO: 36. In some embodiments, the S298G / T299A mutant Fc domain includes the S78G and T79A mutations of SEQ ID NO: 57 or 59. In some embodiments, multiple mutations include or consist of leucine 234 to phenylalanine (L234F), leucine 235 to glutamic acid (L235E), and aspartic acid 265 to arginine (D265A). In some embodiments, the L234F / L235E / D265A mutant Fc includes a hinge domain containing the L19F and L20E mutations of SEQ ID NO: 22 and a CH2 domain containing the D27A mutation of SEQ ID NO: 36. In some embodiments, the L234F / L235E / D265A mutant Fc domain includes the L14F, L15E, and D45A mutations of SEQ ID NO: 57 or 59. In some embodiments, multiple mutations include or consist of a leucine 234 to alanine (L234A), a leucine 235 to alanine (L235A), and a proline 329 to glycine (P329G). In some embodiments, the L234A / L235A / P329G mutant Fc domain includes a hinge domain containing the L19A and L20A mutations of SEQ ID NO: 22 and a CH2 domain containing the P91G mutation of SEQ ID NO: 36. In some embodiments, the L234A / L235A / P329G mutant Fc domain includes the L14A, L15A, and P109G mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of L234F, L235E, and a proline 331 to serine mutation (P331S). In some embodiments, the L234F / L235E / P331S mutation Fc includes a hinge domain containing the L19F and L20E mutations of SEQ ID NO: 22 and a CH2 domain containing the P93S mutation of SEQ ID NO: 36. In some embodiments, the L234F / L235E / P331S mutation Fc domain includes the L14F, L15E, and P111S mutations of SEQ ID NO: 57 or 59.In some embodiments, the plurality includes or consists of mutations from leucine 235 to alanine (L235A), from glycine 237 to alanine (G237A), and from glutamic acid 318 to alanine (E318A). In some embodiments, the L235A / G237A / E318A mutation Fc includes a hinge domain containing the L20A and G22A mutations of SEQ ID NO: 22 and a CH2 domain containing the E80A mutation of SEQ ID NO: 36. In some embodiments, the L235A / G237A / E318A mutation Fc domain includes the L15A, G17A, and E98A mutations of SEQ ID NO: 57 or 59.

[0168] In some embodiments, Fc is modified to reduce its binding to the Fc receptor. In some embodiments, the modification is the removal of glycosylation. In some embodiments, Fc glycosylation is removed enzymatically. In some embodiments, enzymatic deglycosylation is carried out using a deglycosylase. In some embodiments, enzymatic deglycosylation is carried out using a cleavage that cleaves the sugar. Examples of enzymes for deglycosylation include, but are not limited to, peptide-N-glycosidase F (PNGase) and endoglycosidase H (Endo H). Kits for deglycosylation are also commercially available.

[0169] In some embodiments, the mutation is a mutation that increases binding to the Fc receptor. In some embodiments, the Fc receptor is selected from FcγRI, FcγRIIA, FcγRIIIA, and FcγRIIIB. In some embodiments, the Fc receptor is FcγRI. In some embodiments, the mutation is a mutation from serine 267 to glutamate (S267E). In some embodiments, the S267E mutant CH2 domain contains the S29E mutation of SEQ ID NO: 36. In some embodiments, the S267E mutant Fc domain contains the S47E mutation of SEQ ID NO: 57 or 59. In some embodiments, the mutation is a mutation from proline 238 to aspartate (P238D). In some embodiments, the P238D mutant hinge domain contains the P23D mutation of SEQ ID NO: 22. In some embodiments, the P238D mutant Fc domain contains the P18D mutation of SEQ ID NO: 57 or 59. In some embodiments, the mutation is multiple mutations that increase binding to the Fc receptor. In some embodiments, the multiple includes or consists of S267E and a mutation from leucine 328 to phenylalanine (L328F) (also known as SELF). In some embodiments, the S267E / L328F mutant CH2 domain includes the S29E and L90F mutations of SEQ ID NO: 36. In some embodiments, the S267E / L328F mutant Fc domain includes the S47E and L108F mutations of SEQ ID NO: 57 or 59. In some embodiments, the multiple includes or consists of S267E and a mutation from histidine 268 to phenylalanine (H268F) and a mutation from serine 324 to threonine (S324T) (also known as EFT). In some embodiments, the S267E / H268F / S324T mutant CH2 domain includes the S29E, H30F and S86T mutations of SEQ ID NO: 36. In some embodiments, the S267E / H268F / S324T mutant Fc domain includes the S47E, H48F, and S104T mutations of sequence number 57 or 59.In some embodiments, the plurality includes or consists of mutations from glycine 237 to aspartic acid (G237D), P238D, from proline 271 to glycine (P271G), and from alanine 330 to arginine (A330R) (also known as V9). In some embodiments, the G237D / P238D / P271G / A330R mutant polypeptide includes a mutant hinge domain containing the G22D and P23D mutations of SEQ ID NO: 22, and a mutant CH2 domain containing the P33G and A92R mutations of SEQ ID NO: 36. In some embodiments, the G237D / P238D / P271G / A330R mutant Fc domain includes the G17D, P18D, P51G, and A110R mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of mutations from G237D, P238D, histidine 268 to aspartic acid (H268D), P271G, and A330R (also known as V11). In some embodiments, the G237D / P238D / H268D / P271G / A330R mutant polypeptide includes a mutant hinge domain containing the G22D and P23D mutations of SEQ ID NO: 22, and a mutant CH2 domain containing the H30D, P33G, and A92R mutations of SEQ ID NO: 36. In some embodiments, the G237D / P238D / H268D / P271G / A330R mutant Fc domain includes the G17D, P18D, H48D, P51G, and A110R mutations of SEQ ID NO: 57 or 59. In some embodiments, the plurality includes or consists of mutations from glutamic acid 233 to aspartic acid (E233D), G237D, P238D, H268D, P271G, and A330R (also known as V12). In some embodiments, the E233D / G237D / P238D / H268D / P271G / A330R mutant polypeptide includes a mutant hinge domain containing the E18D, G22D, and P23D mutations of SEQ ID NO: 22, and a mutant CH2 domain containing the H30D, P33G, and A92R mutations of SEQ ID NO: 36. In some embodiments, the E233D / G237D / P238D / H268D / P271G / A330R mutant Fc domain includes the E13D, G17D, P18D, H48D, P51G, and A110R mutants of sequence number 57 or 59.

[0170] The S267E mutation was found to enhance affinity for inhibitory FcγRIIB and activated FcγRIIa. The SELF mutation in hIgG1 resulted in a substantial 430-fold increase in binding to FcγRIIB, with minimal changes in binding to FcγRI and FcγRIIA-H131 compared to human WT IgG1. EFT mutations were found to increase FcγRIIB binding by 18-fold compared to human WT IgG1. EFTs also increased CDC, ADCC, and antibody-dependent phagocytic (ADCP) activity through enhanced binding to C1q and activator FcG receptors. In some embodiments, mutations that increase ADCC are EFT plurality of mutations. P238D showed enhanced binding to FcγRIIB with an affinity increase of approximately 4.3-fold compared to WT human IgG1. P238D also significantly reduced binding to any other activated Fcg receptor. V9 significantly enhanced antibody affinity to hFcγRIIB by approximately 32-fold compared to WT IgG1. V9 was also found to reduce affinity to the hFcγRIIA R131 allele to approximately one-third compared to WT IgG1. V11 significantly enhanced antibody affinity to hFcγRIIB by approximately 96-fold compared to human WT IgG1, while reducing affinity to hFcγRIIA R131 to approximately one-third. V12 showed a significant enhancement of binding to FcγRIIB, with a 217-fold change compared to human WT IgG1. The V12 mutation also showed no detectable binding to the FcγRIIIA allotype and reduced FcγRI binding (0.061-fold change compared to WT IgG1) and FcγRIIA-H131 (0.068-fold change compared to wt IgG1). It should be noted that V12 slightly improves binding to FcγRIIA-R131, showing a twofold increase in binding compared to WT hIgG1.

[0171] Mutations that produce the above functions are well known in the art, and any such mutation can be used. Examples of such mutations can be found in at least KOSaunders, 2019, "Conceptual approaches to modulating antibody effector functions and circulation half-life", Front, Immunol., 2019 Jun 7;10:1296, which is incorporated herein by reference in its entirety. Table 1 of Saunders provides Fc modifications that enhance antibody effector function. Table 2 of Saunders provides Fc modifications that improve antibody circulation half-life. Table 3 of Saunders provides Fc modifications that inhibit antibody effector function. Table 1 of Saunders provides Fc modifications that enhance antibody effector function. Table 2 of Saunders provides Fc modifications that improve antibody circulation half-life. Table 3 of Saunders provides Fc modifications that inhibit antibody effector function. It will be understood by those skilled in the art that parallel mutations can also be performed on IgG3 heavy chains or non-human IgG1 heavy chains. It will be understood that the numbers shown herein relate to full-length IgG including variable domains. The number can be modified to correspond to the positions of these amino acids only within the Fc portion of IgG.

[0172] In some embodiments, the mutation increases effector function. In some embodiments, the mutation increases ADCC. In some embodiments, the mutation is not a mutation that increases CDC. In some embodiments, the mutation increases ADCC but not CDC. It will be understood by those skilled in the art that unmodified Fc is not cytotoxic enough to overcome the booster effect brought about by the molecules of the present invention, but Fc containing a mutation that increases ADCC is cytotoxic. In some embodiments, the effector function includes ADCC. In some embodiments, the effector function includes ADCC but not CDC. In some embodiments, the increase in effector function includes an increase in cytotoxicity. In some embodiments, Fc is derived from IgG1 or IgG3, and the mutation increases effector function. In some embodiments, Fc is derived from IgG1 and includes 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, which is incorporated in its entirety herein by reference.

[0173] In some embodiments, the mutations that increase ADCC are multiple mutations that increase ADCC. In some embodiments, the multiple mutations include mutations in human IgG1 from 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). In some embodiments, the multiple mutations include mutations in SEQ ID NO: 57 from 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). In some embodiments, the multiple mutations include mutations from serine 239 to aspartic acid (S239D) and from isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include mutations from serine 19 to aspartic acid (S19D) and from isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 57. In some embodiments, the S239D / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include mutations from serine 239 to aspartic acid (S239D), from alanine 330 to leucine (A330L), and from isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include mutations from serine 19 to aspartic acid (S19D), alanine 110 to leucine (A110L), and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 57. In some embodiments, the S239D / A330L / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include mutations from glycine 236 to alanine (G236A), alanine 330 to leucine (A330L), and isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include mutations from glycine 16 to alanine (G16A), alanine 110 to leucine (A110L), and isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 57.In some embodiments, the multiple mutations include mutations in human IgG1 from glycine 236 to alanine (G236A), serine 267 to glutamic acid (S267E), histidine 268 to phenylamine (H268F), serine 324 to threonine (S324T), and isoleucine 332 to glutamic acid (I332E). In some embodiments, the multiple mutations include mutations in SEQ ID NO: 57 from glycine 16 to alanine (G16A), serine 47 to glutamic acid (S47E), histidine 48 to phenylamine (H48F), serine 104 to threonine (S104T), and isoleucine 112 to glutamic acid (I112E). In some embodiments, the multiple mutations include mutations from serine 298 to alanine (S298A), glutamic acid 333 to alanine (E333A), and lysine 334 to alanine (K334A) within human IgG1. In some embodiments, the multiple mutations include mutations from serine 78 to alanine (S78A), glutamic acid 113 to alanine (E113A), and lysine 114 to alanine (K114A) within SEQ ID NO: 57. In some embodiments, the multiple mutations include mutations from proline 247 to isoleucine (P247I) and alanine 339 to glutamine (A339Q) within human IgG1. In some embodiments, the multiple mutations include mutations from proline 27 to isoleucine (P27I) and alanine 119 to glutamine (A119Q) within SEQ ID NO: 57. In some embodiments, the multiple mutations include mutations from glycine 236 to alanine (G236A), serine 239 to aspartic acid (S239D), and isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include mutations from glycine 16 to alanine (G16A), serine 19 to aspartic acid (S19D), and isoleucine 112 to glutamic acid (I112E) within Sequence ID No. 57. In some embodiments, the G236A / S239D / I332E mutations also increase ADCP.In some embodiments, the multiple mutations include mutations in the first heavy chain of human IgG1 from 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), as well as mutations in the second heavy chain of IgG1 from aspartic acid 270 to glutamic acid (D270E), lysine 326 to aspartic acid (K26D), alanine 330 to methionine (A330M), and lysine 334 to glutamic acid (K334E). In some embodiments, the multiple mutations include mutations in the first chain of SEQ ID NO: 57 from 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), as well as mutations in the second chain of SEQ ID NO: 57 from 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). It is understood that any of the mutations listed above given for SEQ ID NO: 57 also apply to SEQ ID NO: 59. In fact, these also apply to sequence numbers 58 and 60, but in these sequences, any numbering given above in this specification must be increased by 5.

[0174] In some embodiments, the ADCC-enhanced Fc domain contains the L15V / F23L / R72P / Y80L / P176L mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 57 and 59. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 61). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs. 61. In some embodiments, the Fc containing the L235V / F243L / R292P / Y300L / P396L mutation is SEQ ID NO: 61. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97, or 99% identical to SEQ ID NO: 61 and contains the L15V / F23L / R72P / Y80L / P176L mutation.

[0175] In some embodiments, the ADCC-enhanced Fc domain contains the S19D / A110L / I112E mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 57 and 59. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 62). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs. 62. In some embodiments, the Fc containing the S19D / A110L / I112E mutation is sequence number 62. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97, or 99% identical to sequence number 62 and contains the S19D / A110L / I112E mutation.

[0176] In some embodiments, the mutation increases CDC. In some embodiments, the CDC-increasing mutation is multiple mutations that increase CDC. In some embodiments, the CDC-increasing Fc domain contains the G16A / S47E / H48F / S104T / I112E mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 57 and 59. In some embodiments, the CDC-increasing Fc domain contains EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVTNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 63). In some embodiments, the Fc domain with increased CDC is comprised of SEQ ID NO: 63. In some embodiments, the Fc containing the G16A / S47E / H48F / S104T / I112E mutation is SEQ ID NO: 63. In some embodiments, the Fc domain with increased CDC is at least 75, 80, 85, 90, 92, 95, 97 or 99% identical to SEQ ID NO: 63 and contains the G16A / S47E / H48F / S104T / I112E mutation.

[0177] In some embodiments, the ADCC-enhanced Fc domain contains the G16A / A110L / I112E mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NOs. 57 and 59. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 64). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs. 64. In some embodiments, the Fc containing the G16A / A110L / I112E mutation is sequence number 64. In some embodiments, the Fc domain with increased ADCC is at least 75, 80, 85, 90, 92, 95, 97, or 99% identical to sequence number 64 and contains the G16A / A110L / I112E mutation.

[0178] In some embodiments, the effector domain is selected from SEQ ID NOs: 61-64. In some embodiments, the effector domain includes any one of SEQ ID NOs: 61-64. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 61-64. In some embodiments, the effector domain is selected from SEQ ID NOs: 61, 62, and 64. In some embodiments, the effector domain includes any one of SEQ ID NOs: 61, 62, and 64. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 61, 62, and 64. In some embodiments, the effector domain contains at least 75, 80, 85, 90, 92, 95, 97, or 99% identity with any one of SEQ ID NOs: 61, 62, and 64 and retains increased ADCC compared to the control Fc domain. In some embodiments, the control Fc domain is an unmodified Fc domain. In some embodiments, the unmodified Fc is a naturally occurring Fc. In some embodiments, the unmodified Fc is a naturally occurring human Fc.

[0179] In some embodiments, Fc is modified to increase ADCC. In some embodiments, the modification is the removal of fucosylation. In some embodiments, Fc fucosylation is removed enzymatically. In some embodiments, Fc is defucosylated. In some embodiments, the method involves defucosylation of the molecule. In some embodiments, the molecule of the present invention is produced in a cell line engineered to produce the defucosylated molecule.

[0180] In some embodiments, the mutation increases CDC. In some embodiments, multiple mutations increase CDC. In some embodiments, multiple mutations include mutations in human IgG1 from glycine 236 to alanine (G236A), serine 267 to glutamic acid (S267E), histidine 268 to phenylamine (H268F), serine 324 to threonine (S324T), and isoleucine 332 to glutamic acid (I332E). In some embodiments, multiple mutations include mutations in SEQ ID NO: 57 from glycine 16 to alanine (G16A), serine 47 to glutamic acid (S47E), histidine 48 to phenylamine (H48F), serine 104 to threonine (S104T), and isoleucine 112 to glutamic acid (I112E). In some embodiments, the multiple mutations include mutations from lysine 326 to tryptophan (K326W) and from glutamate 333 to serine (E333S) within human IgG1. In some embodiments, the multiple mutations include mutations from lysine 106 to tryptophan (K106W) and from glutamate 113 to serine (E113S) within SEQ ID NO: 57. In some embodiments, the multiple mutations include mutations from glutamate 345 to arginine (E345R), from glutamate 430 to glycine (E430G), and from serine 440 to tyrosine (S440Y) within human IgG1. In some embodiments, the multiple mutations include mutations from glutamate 125 to arginine (E125R), from glutamate 210 to glycine (E210G), and from serine 220 to tyrosine (S220Y) within SEQ ID NO: 57. It is understood that any of the mutations given for sequence number 57 and listed above also apply to sequence number 59. In fact, they also apply to sequence numbers 58 and 60, but in these sequences, any numbering given above herein must be increased by 5.

[0181] In some embodiments, the effector portion is a drug. In some embodiments, the protein is an ITGA2B / ITGB3 ECD drug conjugate. In some embodiments, the protein is an ITGA2B / ITGB3-Fc drug conjugate. In some embodiments, the complex is an ITGA2B / ITGB3 ECD drug conjugate. In some embodiments, the complex is an ITGA2B / ITGB3 ECD fragment drug conjugate. In some embodiments, the complex is an ITGA2B / ITGB3-Fc drug conjugate. In some embodiments, the effector portion is cytotoxic. In some embodiments, the effector portion is radioactive. In some embodiments, the effector portion is a radioactive moiety. In some embodiments, the effector portion is radiolabeled. In some embodiments, the effector portion is a chemotherapeutic agent. In some embodiments, the effector portion is not a chemotherapeutic agent. In some embodiments, the effector portion is toxic to non-replicating cells. In some embodiments, the toxicity is lethal. In some embodiments, the effector portion is sufficient to kill cells. Drug conjugation, particularly drug conjugation to antibody scaffolds, is well known in the art, and any conjugation method can be used.

[0182] In some embodiments, the effector portion is amatoxin. In some embodiments, the effector portion is amanitin. Amatoxins are a group of toxic compounds found in poisonous mushrooms. They consist of eight amino acid residues arranged in a large bicyclic motif and inhibit RNA polymerase. Amatoxins are also known as amanitin. In some embodiments, amanitin is selected from alpha-amanitin, beta-amanitin, gamma-amanitin, epsilon-amanitin, amanulin, amanulinic acid, amaninamide, amanin, and proamanulin. In some embodiments, amanitin is alpha-amanitin. In some embodiments, the effector portion is alpha-amanitin.

[0183] In some embodiments, the chemotherapeutic agent is an anthracycline. In some embodiments, the effector portion is an anthracycline. Anthracyclines are a class of drugs extracted from Streptomyces bacteria that cause cytotoxicity by intercalating into DNA and primarily inhibiting topoisomerase. Examples of anthracyclines include, but are not limited to, doxorubicin, daunorubicin, epirubicin, nemorubicin, PNU-159682, radirubicin, and idarubicin. In some embodiments, the anthracycline is PNU-159682.

[0184] In some embodiments, the chemotherapeutic agent is an anthramycin dimer. In some embodiments, the anthramycin dimer is pyrrolobenzodiazepine (PBD). In some embodiments, the chemotherapeutic agent is PBD. In some embodiments, the anthramycin dimer is indolinobenzodiazepine dimer (IGN). In some embodiments, the chemotherapeutic agent is pyridinobenzodiazepine (PDD). In some embodiments, the anthramycin dimer is PDD. In some embodiments, the effector portion is PBD. In some embodiments, the effector portion is PDD. PBD and PDD are families of DNA supraclution binders that inhibit DNA and RNA synthesis. In some embodiments, PBD is a PBD dimer. Examples of PBD and PDD include, but are not limited to, anthramycin, SJG-136, NS 694501, and FGX2-62. In some embodiments, PBD is anthramycin. In some embodiments, the effector portion is anthramycin. In some embodiments, anthramycin is anthramycin-methyl ether (AME). In some embodiments, anthramycin is an anthramycin dimer. In some embodiments, PBD is tesirin (SG3249). In some embodiments, tesirin is SG3199. In some embodiments, the chemotherapeutic agent is SG3249. In some embodiments, the chemotherapeutic agent is SG3199.

[0185] In some embodiments, the chemotherapeutic agent is calicheamicin. In some embodiments, the effector portion is calicheamicin. Calicheamicin is a class of antibiotics derived from the bacterium Micromonospora echinospora that bind to the DNA minor groove and cause strand breaks. Examples of calicheamicin include, but are not limited to, calicheamicin gamma 1, esperamicin, and ozogamicin.

[0186] In some embodiments, the chemotherapeutic agent is camptothecin or an analogue. In some embodiments, the effector portion is camptothecin or an analogue. In some embodiments, the effector portion is camptothecin. Examples of camptothecin analogues include, but are not limited to, exatecan, SN-38, and deruxtecan (Dxd). In some embodiments, the camptothecin analogue is Dxd. In some embodiments, the chemotherapeutic agent is Dxd. In some embodiments, the effector portion is Dxd.

[0187] In some embodiments, the chemotherapeutic agent is duocalmycin. In some embodiments, the effector portion is duocalmycin. Duocalmycin is a small molecule isolated from Streptomyces bacteria that binds to the DNA minor groove and alkylates adenine bases. Examples of duocalmycin include, but are not limited to, duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, duocalmycin TM, duocalmycin MA, and CC-1065.

[0188] In some embodiments, the chemotherapeutic agent is triptolide. In some embodiments, the effector portion is triptolide.

[0189] In some embodiments, the effector portion is a tubulin inhibitor. In some embodiments, the effector portion is a meitansinoid. In some embodiments, the meitansinoid is a thiol-containing meitansinoid. Meitansinoids or meitansin are known to be tubulin inhibitors that inhibit microtubule aggregation by binding tubulin to the ryzoxin binding site. In some embodiments, the meitansinoid is meltansine (DM-1). In some embodiments, meltansine is emtansine. In some embodiments, the tubulin inhibitor is auristatin. In some embodiments, auristatin is selected from monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). In some embodiments, the tubulin inhibitor is tubulicine. In some embodiments, tubulicine is tubulicine A. In some embodiments, auristatin is MMAE. In some embodiments, auristatin is MMAF. In some embodiments, the effector portion is MMAE. In some embodiments, the effector portion is MMAF.

[0190] In some embodiments, the effector portion is a combination of parts. In some embodiments, the effector portion is a plurality of effector portions. In some embodiments, the effector portion is a combination of cytotoxic portions. In some embodiments, the effector portion includes at least two cytotoxic portions selected from the group consisting of amatoxin, anthracycline, pyrrolobenzodiazepine, calicheamycin, camptothecin, duochamycin, triptolide, and tubulin inhibitors. In some embodiments, the effector portion includes at least two cytotoxic portions selected from the group consisting of amatoxin, anthracycline, pyrrolobenzodiazepine, calicheamycin, camptothecin, duochamycin, triptolide, and meitansinoids.

[0191] Third and fourth chains In some embodiments, the protein complex further comprises a third polypeptide chain. In some embodiments, the third polypeptide chain comprises a third fragment of the protein target of the ITP autoantibody. In some embodiments, the third fragment is different from the first fragment. In some embodiments, the third fragment is different from the second fragment. In some embodiments, the third fragment is the same as the first fragment. In some embodiments, the first fragment is the same as the second fragment. In some embodiments, the third fragment is the same as the first and second fragments. In some embodiments, "same as" means the same sequence. In some embodiments, "different" means a different sequence.

[0192] In some embodiments, the third polypeptide further comprises a third dimerization domain. In some embodiments, the first polypeptide further comprises a fourth dimerization domain. In some embodiments, the third and fourth dimerization domains can dimerize with each other. In some embodiments, the third and fourth dimerization domains are configured to dimerize with each other. In some embodiments, the third dimerization domain is not configured to dimerize with the first dimerization domain. In some embodiments, the third dimerization domain is not configured to dimerize with the second dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize with the first dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize with the second dimerization domain. In some embodiments, "configured to dimerize" means "can dimerize". In some embodiments, the third and fourth dimerization domains are different from the first and second dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, and the third and fourth dimerization domains are CH1 / CL domains. In some embodiments, the first and second dimerization domains are CH1 / CL domains, and the third and fourth dimerization domains are hinge domains.

[0193] In some embodiments, the protein complex further comprises a fourth polypeptide chain. In some embodiments, the fourth polypeptide chain comprises a fourth fragment of the protein target of the ITP autoantibody. In some embodiments, the fourth fragment is different from the first fragment. In some embodiments, the fourth fragment is different from the second fragment. In some embodiments, the fourth fragment is different from the third fragment. In some embodiments, the fourth fragment is the same as the first fragment. In some embodiments, the fourth fragment is the same as the second fragment. In some embodiments, the fourth fragment is the same as the third fragment. In some embodiments, the fourth fragment is the same as the first, second, and third fragments. In some embodiments, the first, second, and third fragments are all the same. In some embodiments, the first, second, third, and fourth fragments are all different. In some embodiments, "same as" means the same sequence. In some embodiments, "different" means different sequences. In some embodiments, "different" means derived from different proteins. In some embodiments, "different" means derived from the same protein but containing different sequences. In some embodiments, different means originating from the same protein but from different regions of the protein. In some embodiments, at least two of the first, second, third, and fourth proteins are part of a single protein complex. In some embodiments, the protein complex is a mammalian complex. In some embodiments, the protein complex is a human complex.

[0194] In some embodiments, the fourth polypeptide further comprises a fifth dimerization domain. In some embodiments, the second polypeptide further comprises a sixth dimerization domain. In some embodiments, the fifth and sixth dimerization domains can dimerize with each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerize with each other. In some embodiments, the fifth dimerization domain is not configured to dimerize into the first dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize into the third dimerization domain. In some embodiments, the fifth dimerization domain is not configured to dimerize into the fourth dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the first dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the third dimerization domain. In some embodiments, the sixth dimerization domain is not configured to dimerize into the fourth dimerization domain. In some embodiments, the fifth and sixth dimerization domains are different from the first and second dimerization domains. In some embodiments, the fifth and sixth dimerization domains are different from the third and fourth dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, the third and fourth dimerization domains are CH1 / CL domains, and the fifth and sixth dimerization domains are CH1 / CL domains. In some embodiments, the first and second dimerization domains are CH1 / CL domains, the third and fourth dimerization domains are hinge domains, and the fifth and sixth dimerization domains are hinge domains. In some embodiments, neither the first polypeptide nor the second polypeptide contains a CH1 domain.In some embodiments, both the first and second polypeptides contain a CH1 domain. Both the first and second polypeptides contain a CL domain. In some embodiments, neither the first nor the second polypeptide contains a CL domain. In some embodiments, the first polypeptide contains a CH1 domain and the second polypeptide contains a CL domain. In some embodiments, the third polypeptide contains a CL domain and the fourth polypeptide contains a CH1 domain. In some embodiments, the first polypeptide contains a CL domain and the second polypeptide contains a CH1 domain. In some embodiments, the third polypeptide contains a CH1 domain and the fourth polypeptide contains a CL domain.

[0195] In some embodiments, the third and fourth dimerization domains include mutations that enable 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 include mutations that enable 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 include mutations that enable 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 include mutations that enable dimerization of the fifth and sixth dimerization domains and inhibit dimerization of the sixth dimerization domain to the third, sixth, or both dimerization domains.

[0196] Alternate configuration In some embodiments, the composition comprises a polypeptide chain comprising a fragment or analog or derivative of a first protein target of the ITP autoantibody and a fragment or analog or derivative of a second protein target of the ITP autoantibody. In some embodiments, the polypeptide chain is a single polypeptide chain. In some embodiments, the single chain comprises a fragment of the first protein and a fragment of the second protein. In some embodiments, the polypeptide chain further comprises a fragment or analog or derivative of a third protein target of the ITP autoantibody. In some embodiments, the polypeptide chain further comprises a fragment or analog or derivative of a fourth protein target of the ITP autoantibody. In some embodiments, the polypeptide chain further comprises an Fc region. In some embodiments, the polypeptide chain further comprises an effector portion.

[0197] In some embodiments, a fragment or analogue or derivative of the first protein target of the ITP autoantibody is separated by a linker from a fragment or analogue or derivative of the second protein target of the ITP autoantibody. In some embodiments, a fragment or analogue or derivative of the third protein target of the ITP autoantibody is separated by a linker from a fragment or analogue or derivative of the first or second protein target of the ITP autoantibody. In some embodiments, a fragment or analogue or derivative of the fourth protein target of the ITP autoantibody is separated by a linker from a fragment or analogue or derivative of the first, second or third protein target of the ITP autoantibody. In some embodiments, the fragment is separated from the Fc region by a linker. In some embodiments, the effector portion is separated by a linker. In some embodiments, the effector portion is separated from the fragment by a linker. In some embodiments, the fragment and the dimerization domain are separated by a linker. In some embodiments, the dimerization domain and the Fc region are separated by a linker. In some embodiments, the fragment and the Fc region are separated by a linker. In some embodiments, the effector portion is separated by a linker. In some embodiments, the effector portion is separated by a fragment via a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a bond. In some embodiments, the bond is a peptide bond. In some embodiments, the bond is an amino acid bond. In some embodiments, the linker is a flexible linker. Linkers are well known in the art, and any linker can be used.

[0198] In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is a polyethylene glycol (PEG) linker. In some embodiments, the PEG linker is a Gly3-PEG-azide linker. In some embodiments, the linker is a dibenzocyclooctin group (DBCO) linker. In some embodiments, the DBCO linker is a DBCO-C6 linker. In some embodiments, the DBCO linker is a DBCO-Gly5-EDA linker. In some embodiments, the linker is a dimethylethylenediamine (DMEDA) linker. In some embodiments, the linker is an N-dimethylethylenediamine (DMAE) linker. In some embodiments, the linker is a glutathione linker. In some embodiments, the linker is a CLICK linker. In some embodiments, the CLICK linker is a CLICK-DBCO linker. In some embodiments, the CLICK linker is a CLICK-azide linker. In some embodiments, the linker is a disulfide linker. In some embodiments, the linker is a thiol linker. In some embodiments, the linker is an azide linker. In some embodiments, the linker is a maleimide (Mal) linker. In some embodiments, the Mal linker is a maleimide caproyl linker. In some embodiments, the Mal linker is a Mal-C6 linker. In some embodiments, the Mal linker is a Mal-Gly5-EDA linker. In some embodiments, the linker is a lysine linker. In some embodiments, the linker is an asparagine linker. In some embodiments, the linker is an acid-unstable linker. In some embodiments, the linker is a cleavable linker. In some embodiments, cleavable means protease-cleavable. In some embodiments, the cleavable linker is a glutathione-cleavable linker. In some embodiments, the linker is an incleavable linker. Other examples of linkers include, for example, SPDB linkers, SMCC linkers, MCC linkers, and butanoic acid linkers.In some embodiments, the linker is a p-aminobenzyl (PAB) linker. In some embodiments, the linker is a p-aminocarbamate (PABC) linker. In some embodiments, the linker is a maleimidocaproyl (mc) linker. In some embodiments, the linker contains mc. In some embodiments, the linker is a Val-Cit-PAB linker. In some embodiments, the linker is a Val-Cit-PABC linker. In some embodiments, the linker is a Val-Cit-PAB-MMAE linker. In some embodiments, the linker is an mc-VC-PABC-MMAE linker. In some embodiments, the linker is an mc-MMAF linker. In some embodiments, the linker is a monomethyl auristatin E (MMAE) linker. Examples of peptide linkers include, but are not limited to, Val-Cit-PAB linkers, Phe-Lys(Trt)-PAB linkers, and Ala-Ala-Asn-PAB linkers. In some embodiments, the linker is a mixture of linkers. In some embodiments, the linker is a DBCO-PEG linker. In some embodiments, the linker is a PBCO-PEG-DMEDA linker. In some embodiments, the linker is a DBCO-PEG-VC-PAB-DMEDA linker. In some embodiments, VC in the linker is replaced by EVC. In some embodiments, VC in the linker is replaced by EVA. In some embodiments, the fragments and dimerization domains are linked by non-cleavable linkers. In some embodiments, the fragments and dimerization domains are linked by cleavable linkers. In some embodiments, the effector portions are linked by cleavable linkers. In some embodiments, the effector portions are linked by non-cleavable linkers.

[0199] In some embodiments, conjugation means linking. In some embodiments, conjugation is via coupling. In some embodiments, conjugates are directly conjugated. In some embodiments, conjugates are conjugated via a linker. In some embodiments, the effector portion is conjugated by a linker.

[0200] In some embodiments, conjugation involves conjugating an amino acid linker, a portion, or both, and includes elongation of the amino acid sequence of the active substance chain of the present invention. The nucleic acid molecule encoding the active substance of the present invention may be modified to include a coding sequence of a linker, a portion, or both, so that a complete conjugate is produced at translation. In some embodiments, the conjugate is a fusion protein. Methods for linking and conjugating portions 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 for linking and conjugating to an IgG scaffold are also well known. Methods of linking / conjugating include, but are not limited to, natural cysteine ​​reduction (including natural hinge reduction, also referred to herein as natural cysteine ​​conjugation), manipulated cysteine ​​reduction, disulfide crosslinking, lysine conjugation, and enzymatic conjugation. Examples of enzymatic conjugations include, but are not limited to, click chemistry, saltase-assisted SMAC technology, transglutaminase addition of amine azides, and glycan remodeling.

[0201] Natural cysteine ​​conjugation was performed as follows: CRD protein was reduced using TCEP and incubated at 37°C for 90 minutes. Subsequently, DMA and linker payload were added, followed by incubation at room temperature for 2 hours. Finally, the conjugated material was purified by size exclusion chromatography.

[0202] In some embodiments, the conjugation is site-specific. In some embodiments, the conjugation is not random. In some embodiments, the conjugation or linkage is directed to the IgG backbone. In some embodiments, the conjugation or linkage is not directed to the ITGA2B or ITGB3 fragment. In some embodiments, the conjugation or linkage does not interfere with antibody binding to the ITGA2B or ITGB3 fragment. In some embodiments, the antibody is an autoantibody. In some embodiments, the conjugation or linkage is directed to the dimerization domain. In some embodiments, the conjugation or linkage is directed to the hinge region. In some embodiments, the conjugation or linkage is directed to the CH2 region. In some embodiments, the conjugation or linkage is directed to the CH3 region. In some embodiments, the conjugation or linkage is directed to the CH1 region. In some embodiments, the conjugation or linkage is directed to the CL region. In some embodiments, the linkage or conjugate is directed to a native amino acid residue. In some embodiments, the linkage or conjugate is directed to an engineered amino acid residue. In some embodiments, the residue is cysteine. Examples of manipulated cysteine ​​include, but are not limited to, A231C, S239C, N325C, L328C, D265C, and S442C of the IgG heavy chain. In some embodiments, the residue is lysine. In some embodiments, the residue is asparagine. In some embodiments, glycan remodeling is used to link to asparagine. In some embodiments, asparagine is N297 of the IgG heavy chain. In some embodiments, the residue is glutamine. In some embodiments, N297 is converted, manipulated, or mutated to glutamine (N297Q). In some embodiments, glutamine is Q295 of the IgG heavy chain. Examples of manipulated glutamine include, but are not limited to, Q297.Unless otherwise indicated, the references provide Kabat numbering for IgG1. Corresponding mutations can be produced in other IGs, specifically in other IgGs. In some embodiments, the conjugation or linkage is to the C-terminus or N-terminus of the active ingredient chain of the present invention. In some embodiments, the conjugation or linkage is to the C-terminus. In some embodiments, the conjugation or linkage is to the N-terminus. In some embodiments, the terminus is the terminus of the heavy chain. In some embodiments, the terminus is the terminus of the light chain. In some embodiments, the conjugation or linkage is to multiple sites.

[0203] In some embodiments, the linker is long enough to inhibit steric hindrance between different parts of the chain. In some embodiments, the linker is long enough to inhibit steric hindrance between different parts of the conjugate. In some embodiments, the linker is long enough to allow the antibody to bind to the fragment without steric hindrance from another part of the chain. In some embodiments, the linker is long enough to allow the antibody to bind to the fragment without steric hindrance from another part of the conjugate. In some embodiments, the linker is long enough to allow cells to bind to the fragment without steric hindrance from another part of the chain. In some embodiments, the linker is long enough to allow cells to bind to the fragment without steric hindrance from another part 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 acid lengths. Each possibility represents a distinct embodiment of the invention. In some embodiments, the linker is at least 1 amino acid length. In some embodiments, the linker is at least 5 amino acid length. In some embodiments, the linker is at least 10 amino acids long. In some embodiments, the linker is at least 15 amino acids long. In some embodiments, the linker is up to 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids long. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the linker is up to 10 amino acids long. In some embodiments, the linker is up to 20 amino acids long. In some embodiments, the linker is up to 50 amino acids long. In some embodiments, the linker is up to 100 amino acids long.

[0204] In some embodiments, the linker includes GGGGS (sequence number 6). In some embodiments, the linker consists of sequence number 6. In some embodiments, the linker includes (GGGGS)n, where n is an integer. In some embodiments, the linker consists of (GGGGS)n, where n is an integer. In some embodiments, the linker includes GSAGSAAGSGEF (sequence number 51). In some embodiments, the linker includes or consists of (GGGS)nGS, where n is an integer. In some embodiments, n is selected from 1, 2, 3, 4, and 5. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, the linker includes or consists of sequence number 7. In some embodiments, the linker includes or consists of GGGGSGGGGSGGGGSGGGGSGGGGS (sequence number 52). In some embodiments, the linker consists of (GGGS)n, where n is an integer.

[0205] In some embodiments, the linker is a rigid linker. In some embodiments, the rigid linker includes EAAAK (SEQ ID NO: 65). In some embodiments, the rigid linker consists of SEQ ID NO: 65. In some embodiments, the rigid linker includes (EAAAK)n, where n is an integer. In some embodiments, the rigid linker consists of (EAAAK)n, where n is an integer. In some embodiments, the rigid linker includes (EAAAK)nGS, where n is an integer. In some embodiments, the rigid linker consists of (EAAAK)nGS, where n is an integer. In some embodiments, the rigid linker includes (EAAAK)nGGS, where n is an integer. In some embodiments, the rigid linker consists of (EAAAK)nGGS, where n is an integer. In some embodiments, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each possibility represents a distinct embodiment of the present invention. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0206] In some embodiments, the dimerization domain is located on the C-terminal side of the fragment. In some embodiments, the fragment is located on the C-terminal side of the dimerization domain. In some embodiments, the Fc region is located on the C-terminal side of the fragment. In some embodiments, the fragment is located on the C-terminal side of the Fc region. In some embodiments, the dimerization domain is located on the C-terminal side of the Fc region. In some embodiments, the Fc region is located on the C-terminal side of the dimerization domain. In some embodiments, the dimerization domain is located on the N-terminal side of the fragment. In some embodiments, the fragment is located on the N-terminal side of the dimerization domain. In some embodiments, the Fc region is located on the N-terminal side of the fragment. In some embodiments, the fragment is located on the N-terminal side of the Fc region. In some embodiments, the dimerization domain is located on the N-terminal side of the Fc region. In some embodiments, the Fc region is located on the N-terminal side of the dimerization domain.

[0207] In some embodiments, the epitope extends to at least two fragments. In some embodiments, the epitope extends to first and second fragments. In some embodiments, the epitope extends to first and third fragments. In some embodiments, the epitope extends to first and fourth fragments. In some embodiments, the epitope extends to second and third fragments. In some embodiments, the epitope extends to second and fourth fragments. In some embodiments, the epitope extends to third and fourth fragments. In some embodiments, the epitope extends to two proteins. In some embodiments, the epitope extends to two proteins within a protein complex. In some embodiments, the epitope extends to three fragments. In some embodiments, the epitope extends to three proteins. In some embodiments, the epitope extends to four fragments. In some embodiments, the epitope extends to 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 derived from ITGA2B. In some embodiments, all fragments are derived from ITGB3. In some embodiments, the complex includes a fragment derived from ITGA2B and a fragment derived from ITGB3. In some embodiments, the fragment derived from ITGA2B is mutated. In some embodiments, the fragment derived from ITGB3 is mutated. In some embodiments, both the fragment derived from ITGA2B and the fragment derived from ITGB3 are mutated. In some embodiments, the ITGA2B fragment and the ITGB3 fragment have the same or equivalent mutations. In some embodiments, the ITGA2B fragment and the ITGB3 fragment have different, non-equivalent mutations. Equivalent mutations are mutations at the same amino acid but have slightly different numbering positions due to differences in the ITGA2B / B3 sequences. In some embodiments, equivalent mutations are homologous mutations. In some embodiments, the complex includes an ITGA2B truncation. In some embodiments, the complex includes an ITGB3 truncation. In some embodiments, the complex includes both ITGA2B mutations and ITGB3 mutations. In some embodiments, the ITGA2B truncation and the ITGB3 truncation are truncations of the same length. In some embodiments, the ITGA2B truncation and the ITGB3 truncation are truncations of different lengths. In some embodiments, the ITGA2B truncation and the ITGB3 truncation are truncations of the same domain.

[0209] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 53. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 53.

[0210] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 54. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 54.

[0211] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPITPLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 55. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 55.

[0212] In some embodiments, the first polypeptide includes a fragment linked to SEQ ID NO: 9. In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 9. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 9. In some embodiments, the first polypeptide includes a fragment linked to SEQ ID NO: 10. In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 10. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 910.

[0213] In some embodiments, the first polypeptide includes a fragment linked to AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 56.

[0214] In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 53. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 54. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 55. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 9. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 10. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 53. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 54. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 55. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 56. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 9. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 10.

[0215] In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence selected from SEQ ID NOs: 5, 8, and 11-14. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence selected from SEQ ID NOs: 5, 8, and 11-14, or an analog or derivative thereof having at least 85% identity. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the 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 NOs: 5, 8, and 11-14. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the two chains are the same chain. In some embodiments, the two chains are different chains.

[0216] In some embodiments, the complex includes or consists of CRD-755. In some embodiments, the complex includes or consists of CRD-756. In some embodiments, the complex includes or consists of CRD-757. In some embodiments, the complex includes or consists of CRD-758. In some embodiments, the polypeptide chain includes or consists of a sequence having at least 70% identity with the sequence provided herein. In some embodiments, the polypeptide chain includes or consists of a sequence having at least 75% identity with the sequence provided herein. In some embodiments, the polypeptide chain includes or consists of a sequence having at least 80% identity with the sequence provided herein. In some embodiments, the polypeptide chain includes or consists of a sequence having at least 85% identity with the sequence provided herein. In some embodiments, the polypeptide chain includes or consists of a sequence having at least 90% identity with the sequence provided herein. In some embodiments, the polypeptide chain includes or consists of a sequence having at least 95% identity with the sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 97% identity with the sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 99% identity with the sequence provided herein.

[0217] Pharmaceutical composition In another embodiment, a pharmaceutical composition comprising the protein or polypeptide of the present invention is provided.

[0218] In another embodiment, a pharmaceutical composition comprising the protein complex of the present invention is provided.

[0219] In another embodiment, a pharmaceutical composition comprising the composition of the present invention is provided.

[0220] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or adjuvant. As used herein, the terms “carrier,” “adjuvant,” or “excipient” refer to any component of the pharmaceutical composition that is not an activator. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as physiological saline. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt, gelatin, and talc; excipients, e.g., cocoa butter and suppository waxes; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate, and agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer; and other non-toxic, suitable substances used in pharmaceutical formulations. Some non-limiting examples of substances that may function as carriers in this specification include sugars, starches, cellulose and their derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers, and other non-toxic, pharmaceutically acceptable substances used in other pharmaceutical formulations. Wetting and lubricating agents such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions intended herein.Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those skilled in the art, for example, those listed in the following: The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, which are incorporated herein by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextrose solution, Hanks' 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, for example, Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005), each of which is incorporated herein in its entirety by reference. The compositions described herein may also be contained in artificially constructed structures, such as liposomes, ISCOMS, sustained-release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum.Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, and lamellar layers. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids, as well as sterols, such as cholesterol. The choice of lipids is generally determined by considerations such as liposome size and stability in blood. Various methods for preparing liposomes are available, as outlined in, for example, Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York; see also U.S. Patents 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0221] The carrier may contain the pharmaceutical compositions presented herein in total in an amount of about 0.1% to about 99.99999% by weight.

[0222] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the protein conjugate of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the conjugate of the present invention. The term “therapeutic effective amount” refers to the amount of drug effective in treating a disease or disorder in a mammal. In some embodiments, the therapeutic effective amount is the amount that is effective in the required dose and for the required duration to achieve the desired therapeutic or prophylactic outcome. The exact dosage form and regimen are determined by the physician depending on the patient’s condition. In some embodiments, the effective amount is an amount sufficient to treat at least one symptom of the disease. In some embodiments, the disease is ITP. In some embodiments, the disease is PF. In some embodiments, the disease is ITP. In some embodiments, ITP is characterized by autoantibodies against the protein. In some embodiments, ITP is characterized by autoantibodies against ITGA2B and / or ITGB3.

[0223] As used herein, the terms “treatment” or “to treat” a disease, disorder, or condition encompass alleviation of at least one symptom, reduction of its severity, or inhibition of its progression. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For a treatment to be effective, a useful composition or method herein may only reduce the severity of the disease, disorder, or condition, reduce the severity of its associated symptoms, or improve the quality of life of the patient or subject. Treatments of ITP are well known in the art and may include any acceptable means for evaluating improvement of ITP symptoms. These may include rituximab, steroids, steroid-sparing immunosuppressants (azathioprine, mycophenolic acid, and cyclophosphamide), dapsone, intravenous immunoglobulin (IVIG), and the like. Treatments may include improvement of quality of life, suppression of blister formation, reduction of autoantibodies, and killing of autoreactive B cells.

[0224] In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the pharmaceutical composition is formulated for administration to a human. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0225] As used herein, terms such as “administer,” “dosage,” etc., refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice. One embodiment of this subject provides intravenous administration of a therapeutically effective dose of the composition to a patient in need. Other preferred routes of administration may include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal. In some embodiments, administration is intravenous. In some embodiments, administration is topical. In some embodiments, administration is selected from oral, intravenous, intramuscular, intraperitoneal, intratumoral, topical, or subdermal administration. In some embodiments, administration is administration to the site of disease.

[0226] The dosage administered depends on the recipient's age, health condition, and weight, the type of concomitant therapy if any, the frequency of treatment, and the nature of the desired effect.

[0227] Treatment method In another aspect, a method is provided for treating ITP in a subject requiring the present invention, comprising administering the protein or polypeptide of the present invention to a subject to thereby treat the ITP in the subject.

[0228] In another aspect, a method is provided for treating ITP in a subject requiring the same, comprising administering the protein complex of the present invention to a subject, thereby treating the ITP in the subject.

[0229] In another aspect, a method is provided for treating ITP in a subject requiring the present invention, comprising administering the composition of the present invention to the subject, thereby treating the ITP in the subject.

[0230] In some embodiments, administration is the administration of the pharmaceutical composition of the present invention. In some embodiments, ITP is characterized by an antibody against a protein. In some embodiments, the protein is the target of the ITP antibody. It will be understood by those skilled in the art that the protein complex is designed using a fragment of the protein targeted by the ITP antibody in the subject. In some embodiments, the antibody is an autoantibody. In some embodiments, the disease is ITP, and the autoantibody is against ITGA2B, ITGB3, or both.

[0231] In some embodiments, treatment includes reducing antibody concentration. In some embodiments, treatment includes reducing the number of antibodies. In some embodiments, antibody concentration is circulating antibody concentration. In some embodiments, treatment includes depleting antibodies. In some embodiments, treatment includes killing B cells. In some embodiments, B cells are autoreactive B cells. In some embodiments, B cell killing is specific B cell killing. In some embodiments, treatment includes killing antibody-producing B cells. In some embodiments, treatment includes killing antibody-producing B cells but substantially not killing other B cells. In some embodiments, treatment includes killing B cells that produce antibodies against a protein complex. In some embodiments, treatment includes killing B cells that produce antibodies against a fragment. In some embodiments, treatment includes killing B cells that produce antibodies against a fragment of a protein complex.

[0232] In some embodiments, reducing an antibody involves binding an antibody. In some embodiments, reducing an antibody involves removing at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% of an antibody. Each possibility represents a distinct embodiment of the invention. In some embodiments, the antibody is an autoantibody. In some embodiments, the antibody is an antibody in a subject. In some embodiments, the antibody is a circulating antibody. In some embodiments, the autoantibody is an autoantibody against a protein or fragment. In some embodiments, the autoantibody is a cytotoxic autoantibody. In some embodiments, the autoantibody includes an IgG1 autoantibody. In some embodiments, the autoantibody includes IgG3. In some embodiments, the autoantibody includes both an IgG1 autoantibody and an IgG3 autoantibody. In some embodiments, the autoantibody includes an IgG1 autoantibody, an IgG2 autoantibody, and an IgG3 autoantibody. In some embodiments, the autoantibody includes an IgG1 autoantibody, an IgG3 autoantibody, and an IgG4 autoantibody. In some embodiments, the autoantibodies include IgG1 autoantibodies, IgG2 autoantibodies, IgG3 autoantibodies, and IgG4 autoantibodies. In some embodiments, reduction means removing at least 25% of the antibodies. In some embodiments, reduction means removing at least 50% of the antibodies. In some embodiments, reduction means removing at least 70% of the antibodies. In some embodiments, reduction means removing at least 75% of the antibodies. In some embodiments, the percentage of antibodies is the percentage of autoantibodies. In some embodiments, the percentage of antibodies is the percentage of antibodies against a protein or fragment. In some embodiments, the percentage of antibodies is the percentage of antibodies associated with the disease.

[0233] In some embodiments, the method further includes reducing antibodies in a subject. In some embodiments, the reduction is pre-administration. In some embodiments, reducing antibodies means reducing circulating antibodies. In some embodiments, the antibodies are autoantibodies. In some embodiments, the antibodies are against proteins. In some embodiments, the antibodies are against proteins from which the fragments originate. In some embodiments, the antibodies are against proteins from which at least one of the fragments originates. In some embodiments, the reduction means reducing antibodies against any protein from which at least one of the fragments originates. In some embodiments, the antibodies are against protein complexes. Methods for reducing antibodies are well known in the art and include, for example, plasmapheresis, intravenous Ig (IVIg), antibody filtering, and B-cell targeted therapy, any of which may be used. In some embodiments, the method includes plasmapheresis of antibodies before administration. In some embodiments, the method includes administering B-cell targeted therapy before administering the therapeutic agent of the present invention. In some embodiments, the B-cell targeted therapy is anti-B-cell therapy. In some embodiments, the B-cell targeted therapy is B-cell lethal therapy. In some embodiments, B-cell targeted therapy is pan-B-cell therapy. In some embodiments, B-cell targeted therapy is not targeted therapy. As used herein, “targeted B-cell therapy” is a therapy that targets only specific B-cell clones that produce specific antibodies. In some embodiments, anti-B-cell therapy is anti-B-cell antibody. B-cell targeted antibodies are well known in the art and include, in a non-limiting example, anti-CD20 antibodies. Antibodies for the treatment of anti-CD20 are well known in the art and include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tiuxetan, tocitumomab, and ubrituximab. In some embodiments, B-cell targeted therapy is rituximab.

[0234] nucleic acid In another embodiment, a nucleic acid system is provided comprising at least two nucleic acid molecules, wherein the first nucleic acid molecule encodes a first polypeptide chain of the protein complex of the present invention, and the second nucleic acid molecule encodes a second polypeptide chain of the protein complex of the present invention.

[0235] In another embodiment, a nucleic acid system is provided comprising at least two nucleic acid molecules, wherein the first nucleic acid molecule encodes a first polypeptide chain comprising a fragment of a first human protein target of an ITP autoantibody or an analog or derivative thereof and a first dimerization domain, and the second nucleic acid molecule encodes a second polypeptide chain comprising a fragment of a second human protein target of an ITP autoantibody or an analog or derivative thereof and a second dimerization domain.

[0236] In another embodiment, nucleic acid molecules encoding the protein of the present invention are provided.

[0237] In another embodiment, nucleic acid molecules encoding the polypeptide chain of the composition of the present invention are provided.

[0238] In another embodiment, nucleic acid molecules encoding the composition of the present invention are provided.

[0239] In another embodiment, nucleic acid molecules are provided that encode a fragment or analog or derivative of a first protein target of an ITP autoantibody and a fragment or analog or derivative of a second human protein target of an ITP autoantibody.

[0240] In some embodiments, the nucleic acid system further comprises a third nucleic acid molecule encoding a third polypeptide of the protein complex of the present invention. In some embodiments, the nucleic acid system further comprises a fourth nucleic acid molecule encoding a fourth polypeptide of the protein complex of the present invention. In some embodiments, the first nucleic acid molecule encodes the first polypeptide of the present invention. In some embodiments, the second nucleic acid molecule encodes the second polypeptide of the present invention. In some embodiments, the third nucleic acid molecule encodes the third polypeptide of the present invention. In some embodiments, the fourth nucleic acid molecule encodes the fourth polypeptide.

[0241] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the nucleic acid molecule includes an open reading frame encoding a polypeptide chain. The expression of open reading frames in cells is well known to those skilled in the art. This can be done by transfection, viral infection, or direct alteration of the cell's genome, among many other methods. Expression vectors are well known in the art, and any vector suitable for the target cell expressing the protein complex of the present invention may be used.

[0242] The vector nucleic acid sequence generally contains at least one origin of replication for intracellular proliferation and optionally additional elements, such as heterologous polynucleotide sequences, expression regulatory elements (e.g., promoters, enhancers), selection markers (e.g., antibiotic resistance), and polyadenine sequences. In some embodiments, the vector includes a promoter. In some embodiments, the promoter is configured for expression in target cells in which the protein complex of the present invention is expressed.

[0243] The vector may be a DNA plasmid delivered via a non-viral or viral method. Viral vectors may be retroviral vectors, herpesvirus vectors, adenovirus vectors, adeno-associated virus vectors, or poxvirus vectors. Promoters may be active in mammalian cells. Promoters may be viral promoters. Promoters may be active in bacterial cells. Promoters may be active in human cells. Promoters may be active in fibroblasts. As used herein, the term “promoter” refers to a group of transcriptional regulatory modules concentrated around the start site of RNA polymerase, i.e., RNA polymerase II. Promoters consist of separate functional modules, each consisting of approximately 7–20 bp of DNA, containing one or more recognition sites for transcriptional activators or repressor proteins.

[0244] In some embodiments, the open reading frame is operably ligated to the promoter. The term “operably ligated” is intended to mean that the nucleotide sequence of interest is ligated to one or more regulatory elements in a manner that enables the expression of the nucleotide sequence (for example, in an in vitro transcription / translation system if the vector is introduced into a host cell, or in the host cell).

[0245] In some embodiments, the vector is introduced into cells by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with a viral vector, and high-velocity ballistic penetration by small particles containing nucleic acids within or on the surface of a matrix of small beads or particles (Klein et al., Nature 327. 70-73 (1987)).

[0246] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. RNAP II catalyzes the transcription of DNA to synthesize mRNA and precursors of most snRNA and microRNA.

[0247] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, pCI available from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0248] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses such as retroviruses are used according to the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that enables the expression of proteins under the guidance of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any other promoter shown to be effective for expression in eukaryotic cells.

[0249] In some embodiments, recombinant viral vectors that offer advantages such as horizontal transmission and target specificity are used for in vivo expression. In one embodiment, horizontal transmission is a process inherent in the life cycle of retroviruses, for example, in which a single infected cell produces many progeny virions that germinate and infect neighboring cells. In one embodiment, the result is rapid infection of a large area that was not initially infected by the original viral particle. In one embodiment, a viral vector that cannot spread horizontally is produced. In one embodiment, this feature may be useful when the desired objective is to introduce a specific gene into only a local number of target cells.

[0250] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are commonly described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988), and Gilboa et al. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, see U.S. Patent Nos. 5,464,764 and 5,487,992 for information on positive-negative selection methods.

[0251] In one embodiment, a plant expression vector is used. In one embodiment, the expression of the polypeptide coding sequence is promoted by several promoters. In some embodiments, viral promoters such as the CaMV 35S RNA promoter and 19S RNA promoter [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter for TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters such as the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)], or heat shock promoters such as soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)] etc. are used. In one embodiment, constructs are introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to those skilled in the art. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insect and mammalian host cell lines well known in the art can also be used in the present invention.

[0252] It is understood that in addition to containing elements necessary for transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the present invention can also include sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0253] In some embodiments, the nucleic acid molecule is a single nucleic acid molecule. In some embodiments, the first and second nucleic acid molecules are different molecules. In some embodiments, the first and second nucleic acid molecules are the same molecule. In some embodiments, any two of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, any two of the first, second, third, and fourth nucleic acid molecules are the same molecule. In some embodiments, any three of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, the first, second, 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, the first, second, third, and fourth nucleic acid molecules are all different molecules. In some embodiments, the first, second, third, and fourth nucleic acid molecules are all the same molecule.

[0254] Generation method A method for producing a protein by another embodiment, To obtain a first fragment of the extracellular domain of a first human receptor or its analog or derivative, and a second fragment of the extracellular domain of a second human receptor or its analog or derivative, wherein the first and second human receptors are targets of an ITP autoantibody, and the first fragment is linked to the second fragment to produce a single polypeptide chain. This provides a method that includes the production of proteins.

[0255] A method for producing a protein by another embodiment, To obtain a first fragment or analogue or derivative of the extracellular domain of a first human receptor and a second fragment or analogue or derivative of the extracellular domain of a second human receptor, wherein the first and second human receptors are targets of an ITP autoantibody, the first fragment is linked to the second fragment to produce a single polypeptide chain, and the single polypeptide chain is linked to an effector portion that is not an unmodified Fc domain. This provides a method that includes the production of proteins.

[0256] A method for generating a protein complex, in another embodiment, To obtain a first fragment or analog or derivative of the first protein target of the ITP autoantibody, and a second fragment or analog or derivative of the second protein target of the ITP autoantibody; to ligate the first fragment to the first dimerization domain to generate a first polypeptide chain; and to ligate the second fragment to the second dimerization domain to generate a second polypeptide chain. This provides a method that includes generating a protein complex.

[0257] A method for generating a protein complex, in another embodiment, Obtaining a first fragment or analog or derivative of the first protein target of the ITP autoantibody, and a second fragment or analog or derivative of the second protein target of the ITP autoantibody, linking the first fragment to the first dimerization domain to generate a first polypeptide, linking the second fragment to the second dimerization domain to generate a second polypeptide chain, and linking the first polypeptide, the second polypeptide chain, or both to an effector portion that is not an unmodified Fc domain. This provides a method that includes generating a protein complex.

[0258] A method for producing a protein by another embodiment, Culture of host cells containing one or more vectors containing nucleic acid sequences encoding a single polypeptide chain, where the single polypeptide chain is i. Obtain a first fragment or analog or derivative of the extracellular domain of a first human receptor and a second fragment or analog or derivative of the extracellular domain of a second human receptor, where the first and second human receptors are targets of ITP autoantibodies and are different proteins, and ii. Produced by linking the first fragment to the second fragment to produce a single polypeptide chain, This provides a method that includes the production of proteins.

[0259] A method for producing a protein by another embodiment, Culture of host cells containing one or more vectors containing nucleic acid sequences encoding a single polypeptide chain, where the single polypeptide chain is i. Obtain a first fragment or analog or derivative of the extracellular domain of a first human receptor and a second fragment or analog or derivative of the extracellular domain of a second human receptor, where 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. Generated by ligating a single polypeptide chain to an effector portion that is not an unmodified Fc domain, This provides a method that includes the production of proteins.

[0260] A method for generating a protein complex, in another embodiment, Culture host cells containing one or more vectors, each containing nucleic acid sequences encoding at least two polypeptide chains, where the two polypeptide chains are i. To obtain a first fragment or analog or derivative of the first protein target of the ITP autoantibody, and a second fragment or analog or derivative of the second protein target of the ITP autoantibody, and ii. The first polypeptide chain is produced by ligating the first fragment to the first dimerization domain, and the second polypeptide chain is produced by ligating the second fragment to the second dimerization domain. This provides a method that includes generating a protein complex.

[0261] A method for generating a protein complex, in another embodiment, Culture host cells containing one or more vectors, each containing nucleic acid sequences encoding at least two polypeptide chains, where the two polypeptide chains are i. Obtain a first fragment or analog or derivative of the first protein target of the ITP autoantibody, and a second fragment or analog or derivative of the second protein target of the ITP autoantibody. ii. Linking the first fragment to the first dimerization domain to generate a first polypeptide chain, and linking the second fragment to the second dimerization domain to generate a second polypeptide chain, and iii. Generated by ligating the first polypeptide chain, the second polypeptide chain, or both, to an effector portion that is not an unmodified Fc domain. This provides a method that includes generating a protein complex.

[0262] In some embodiments, the protein is the protein of the present invention. In some embodiments, the protein is a polypeptide. In some embodiments, the truncation is the truncation of the present invention. In some embodiments, the protein is the polypeptide of the present invention. In some embodiments, the mutation is the mutation of the present invention. In some embodiments, the protein complex is the protein complex of the present invention. In some embodiments, the protein composition is the composition of the present invention. In some embodiments, the protein is the protein of the present invention. In some embodiments, the protein is the polypeptide chain of the present invention. In some embodiments, the fragment is the fragment of the present invention. In some embodiments, the derivative is the derivative of the present invention. In some embodiments, the analog is the analog of the present invention. In some embodiments, the dimerization domain is the dimerization domain of the present invention. In some embodiments, the composition, protein complex, protein, fragment, analog, derivative or dimerization domain is as described above herein. In some embodiments, the method further comprises linking a protein, polypeptide or protein complex to an effector moiety. In some embodiments, the effector moiety is not an Fc domain. In some embodiments, the effector moiety does not contain an Fc portion. In some embodiments, the effector moiety is not an unmodified Fc domain. In some embodiments, the effector moiety is an Fc domain comprising at least one mutation that increases ADCC.

[0263] In some embodiments, the protein is a human protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the first and second proteins are the same protein. In some embodiments, the first and second proteins are different proteins. In some embodiments, the first and second proteins are targets of 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 contains a fragment of the extracellular domain. In some embodiments, the fragment consists of an extracellular domain.

[0264] In some embodiments, the first and second dimerization domains can dimerize with each other. In some embodiments, the first and second dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first and second polypeptides. In some embodiments, contacting comprises incubating the polypeptides together. In some embodiments, contacting is intracellular. In some embodiments, contacting is in vitro. In some embodiments, contacting is under conditions sufficient to allow dimerization. In some embodiments, enabling is induction. In some embodiments, the conditions are sufficient to allow dimerization of the polypeptides. In some embodiments, the conditions are physiological conditions.

[0265] In some embodiments, the method further includes inserting a third dimerization domain into the first polypeptide. In some embodiments, insertion is ligation. In some embodiments, insertion is inserting a nucleic acid sequence encoding the third dimerization domain into a nucleic acid molecule or vector encoding the first polypeptide. In some embodiments, ligation is ligating the third dimerization domain to the first dimerization domain. In some embodiments, ligation is ligating the third dimerization domain to the first fragment.

[0266] In some embodiments, the method further comprises obtaining a third fragment of a third protein target of an ITP autoantibody or an analog or derivative thereof, and ligating it to a fourth dimerization domain to generate a third polypeptide chain. In some embodiments, the third and fourth dimerization domains can dimerize with each other. In some embodiments, the third and fourth dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first, second, and third polypeptide chains. In some embodiments, the method further comprises expressing a nucleic acid sequence encoding the third polypeptide chain in a host cell. In some embodiments, the third polypeptide chain is generated by obtaining a third fragment of a third protein and ligating it to a fourth dimerization domain to generate a third polypeptide chain. In some embodiments, the method comprises expressing the first, second, and third polypeptide chains in a cell.

[0267] In some embodiments, the method further includes inserting the fifth dimerization domain into the second polypeptide. In some embodiments, insertion is ligation. In some embodiments, insertion is inserting the nucleic acid sequence encoding the fifth dimerization domain into a nucleic acid molecule or vector encoding the second polypeptide. In some embodiments, ligation is ligating the fifth dimerization domain to the second dimerization domain. In some embodiments, ligation is ligating the fifth dimerization domain to the second fragment.

[0268] In some embodiments, the method further comprises obtaining a fourth fragment of a fourth protein target of an ITP autoantibody or an analog or derivative thereof, and ligating it to a sixth dimerization domain to generate a fourth polypeptide chain. In some embodiments, the fifth and sixth dimerization domains can dimerize with each other. In some embodiments, the fifth and sixth dimerization domains are configured to dimerize with each other. In some embodiments, the method further comprises contacting the first, second, third, and fourth polypeptide chains. In some embodiments, the method further comprises expressing a nucleic acid sequence encoding the fourth polypeptide chain in a host cell. In some embodiments, the fourth polypeptide chain is generated by obtaining a fourth fragment of a fourth protein and ligating it to a sixth dimerization domain to generate a fourth polypeptide chain. In some embodiments, the method comprises expressing the first, second, third, and fourth polypeptide chains in a cell.

[0269] In some embodiments, the method further includes inserting the Fc region into a first strand. In some embodiments, the method further includes inserting the Fc region into a second strand. In some embodiments, the method further includes inserting the Fc region into a third strand. In some embodiments, the method further includes inserting the Fc region into a fourth strand. In some embodiments, the method further includes inserting a portion of the Fc region into the first strand and a portion of the Fc region into the second strand, where the interface of the two portions generates a complete Fc region. In some embodiments, the Fc region is not an unmodified Fc region. In some embodiments, the Fc region includes at least one mutation that increases ADCC.

[0270] In some embodiments, the Fc region is inserted into the dimerization domain at the C-terminal end. In some embodiments, the Fc region is inserted into the fragment at the C-terminal end. In some embodiments, the Fc region is inserted into the dimerization domain at the N-terminal end. In some embodiments, the Fc region is inserted into the fragment at the N-terminal end. In some embodiments, the fragment is inserted or ligated into the dimerization domain at the C-terminal end. In some embodiments, the fragment is inserted or ligated into the dimerization domain at the N-terminal end.

[0271] In some embodiments, the method further includes inserting a linker between at least two portions of a polypeptide chain. In some embodiments, the linker is inserted between a fragment and a dimerization domain. In some embodiments, the linker is inserted between a fragment and an Fc region. In some embodiments, the linker is inserted between an Fc region and a dimerization domain. In some embodiments, the linker is inserted between a dimerization domain and another dimerization domain. In some embodiments, the linker is inserted between a fragment and another fragment. In some embodiments, the linker is inserted between a fragment of a first protein and a fragment of a second protein.

[0272] In some embodiments, the method further comprises introducing at least one mutation into a protein. In some embodiments, the mutation is introduced into a fragment. In some embodiments, the mutation is introduced into an extracellular domain. In some embodiments, the mutation is introduced into a cadherin domain of the fragment. In some embodiments, the method further comprises truncating a protein. In some embodiments, the method further comprises truncating a fragment. In some embodiments, the method further comprises truncating an extracellular domain. In some embodiments, the truncation removes at least one extracellular functional domain. In some embodiments, the truncation removes at least one cadherin domain.

[0273] In some embodiments, the method further includes measuring the binding of autoantibodies to a protein. In some embodiments, the method further includes measuring the binding of autoantibodies to a fragment. In some embodiments, the method further includes measuring the binding of autoantibodies to a protein. In some embodiments, the method further includes measuring the binding of autoantibodies to a fragment. In some embodiments, the autoantibody is an autoantibody against ITGB3. In some embodiments, the autoantibody is an autoantibody against ITGA2B. In some embodiments, the autoantibody is in serum. In some embodiments, the autoantibody is in blood. In some embodiments, the measurement is to measure the binding in serum. In some embodiments, the measurement is to measure the binding in blood. In some embodiments, the serum or blood is derived from a subject suffering from ITP. In some embodiments, the binding is depletion. In some embodiments, the measurement is to measure the depletion of autoantibodies from serum / blood by the protein. In some embodiments, the protein is conjugated to an artificial scaffold. In some embodiments, conjugated means immobilized. In some embodiments, the artificial scaffold is beads. In some embodiments, the beads are paramagnetic beads. In some embodiments, the beads are Sepharose beads. In some embodiments, the beads are avidin beads. In some embodiments, the avidin is streptavidin.

[0274] In some embodiments, a fragment is selected that binds to at least a predetermined threshold of the autoantibody. In some embodiments, the method further includes selecting a fragment that binds to at least a predetermined threshold of the autoantibody. In some embodiments, the threshold is a threshold percentage of the autoantibody. 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 distinct embodiment of the invention. In some embodiments, the threshold is at least 20%. In some embodiments, the threshold is at least 40%. In some embodiments, the threshold is at least 50%. In some embodiments, the threshold is at least 70%. In some embodiments, the threshold is at least 75%. In some embodiments, the threshold is at least 80%.

[0275] In some embodiments, the protein is for use in the method of the present invention. In some embodiments, the polypeptide is for use in the method of the present invention. In some embodiments, the protein complex is for use in the method of the present invention. In some embodiments, the method is a therapeutic method. In some embodiments, the method is a diagnostic method. In some embodiments, the method is a therapeutic method. In some embodiments, the method is a method for determining suitability for treatment.

[0276] In another aspect, a protein complex produced by the method of the present invention is provided.

[0277] In another aspect, a protein produced by the method of the present invention is provided.

[0278] In another aspect, a composition produced by the method of the present invention is provided.

[0279] Patient Selection In another aspect, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising receiving a sample from the object, contacting the sample with a composition of the present invention, and determining the binding of antibodies in the sample to the composition, wherein the binding of antibodies to the composition indicates that the object is suitable to be treated by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0280] In another aspect, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising receiving a sample from the object, contacting the sample with a protein complex of the present invention, and determining the binding of antibodies in the sample to the protein complex, wherein the binding of antibodies to the protein complex indicates that the object is suitable to be treated by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0281] In another aspect, a method is provided for determining the suitability of an object to be treated by the method of the present invention, comprising receiving a sample from the object, contacting the sample with the protein or polypeptide of the present invention, and determining the binding of antibodies in the sample to the protein, wherein the binding of antibodies to the protein or polypeptide indicates that the object is suitable to be treated by the method of the present invention, and thereby determining the suitability of the object to be treated.

[0282] In some embodiments, the subject is one that requires it. In some embodiments, the subject is one of those described above in this specification. In some embodiments, the subject has ITP. In some embodiments, the subject is known to be positive for autoantibodies associated with ITP. In some embodiments, the subject is serologically positive. In some embodiments, the subject is serologically negative. In some embodiments, the subject is naive to treatment. In some embodiments, the treatment is a treatment for ITP. In some embodiments, the subject has been treated before and has relapsed.

[0283] In some embodiments, the method includes obtaining a sample from a subject. In some embodiments, the sample includes tissue. In some embodiments, the sample is biopsy material. In some embodiments, the sample is body fluid. In some embodiments, the body fluid is blood. In some embodiments, the body fluid is serum. In some embodiments, the body fluid is plasma. In some embodiments, the body fluid is a fluid containing antibodies. In some embodiments, the body fluid is selected from at least one of blood, serum, plasma, intestinal fluid, saliva, tumor fluid, urine, interstitial fluid, cerebrospinal fluid, and feces.

[0284] In some embodiments, contact is incubation. In some embodiments, contact is conditions sufficient for the antibody to bind to the protein complex. In some embodiments, the conditions include a sufficient amount of time for the antibody to bind to the protein complex. In some embodiments, the conditions include physiological conditions. In some embodiments, the protein complex is added to the sample. In some embodiments, the protein complex is dissolved in body fluids. In some embodiments, the antibody is an autoantibody. In some embodiments, the antibody is an antibody against a protein.

[0285] In some embodiments, the composition further comprises a detectable portion. In some embodiments, the protein complex further comprises a detectable portion. In some embodiments, the protein further comprises a detectable portion. In some embodiments, the method further comprises contacting the composition, complex and / or protein with a peptide containing the detectable portion. In some embodiments, the peptide is configured to bind to the composition, protein and / or complex. In some embodiments, the peptide is specific to the composition, protein and / or complex. As used herein, the term “specific binding” means binding to a specific molecule while excluding other molecules. In some embodiments, the peptide is specific to the composition, protein and / or complex by excluding other proteins in the sample. In some embodiments, the peptide is specific to the composition, protein and / or complex by excluding naturally occurring antibodies in the sample. In some embodiments, the peptide is specific to the composition, protein and / or complex by excluding antibodies in the sample. In some embodiments, determining binding includes detecting the portion. In some embodiments, determining includes isolating the protein complex. In some embodiments, determining includes eluting antibodies from the complex. Methods for protein identification are well known in the art, and any such method may be used. Examples of such methods include protein sequencing by Western blotting, ELISA, FACS analysis, and mass spectrometry. In some embodiments, sequencing involves ELISA. In some embodiments, the ELISA is a competitive ELISA. In some embodiments, the competitive ELISA involves competition with an antibody. In some embodiments, the antibody is an antibody associated with the disease.

[0286] In some embodiments, binding is positive binding. In some embodiments, binding is binding exceeding a predetermined threshold. In some embodiments, binding is specific binding. In some embodiments, binding is binding to at least one of the fragments of the protein complex. In some embodiments, binding is binding to at least two of the fragments of the protein complex. In some embodiments, binding is binding to at least three of the fragments of the protein complex. In some embodiments, binding is binding to at least four of the fragments of the protein complex. In some embodiments, binding is at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% of the antibody in the sample. Each possibility represents a distinct embodiment of the invention. In some embodiments, binding is at least 50% of the antibody in the sample. In some embodiments, binding is at least 70% of the antibody in the sample. In some embodiments, binding is at least 75% of the antibody in the sample. In some embodiments, the percentage of antibody is the percentage of autoantibodies. In some embodiments, the percentage of antibody is the percentage of antibodies against the protein. In some embodiments, the percentage of antibodies is the percentage of antibodies associated with the disease.

[0287] As used herein, the term "approximately," when combined with a value, refers to a range of ±10% of the reference value. For example, a length of approximately 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0288] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. For example, a reference to “a polynucleotide” includes multiple such polynucleotides, and a reference to “the polypeptide” includes one or more polypeptides and their equivalents known to those skilled in the art, and so on. It should be further noted that the claims may be drafted to exclude any element. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as “alone,” “only,” etc., in connection with the enumeration of elements of the claims or the use of “negative” limitations.

[0289] Where conventions similar to “at least one of A, B, and C, etc.” are used, such constructions are generally intended to mean that a person skilled in the art would understand the convention (for example, “a system having at least one of A, B, and C” is not limiting, but includes systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or systems having A, B and C together). It will further be understood by a person skilled in the art that substantially any disjunctive word and / or phrase representing two or more alternative terms should be understood, whether in the specification, claims, or drawings, to contemplate the possibility of including one of those terms, either of those terms, or both of those terms. For example, the phrase “A or B” is understood to include the possibility of “A” or “B,” or “A and B.”

[0290] It is understood that certain features of the Invention, described in the context of separate embodiments for clarity, may be provided in combination in a single embodiment. Conversely, various features of the Invention, described in the context of a single embodiment for brevity, may be provided individually or in any preferred partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if each and all combinations were individually and expressly disclosed. In addition, all partial combinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if each and all such partial combinations were individually and expressly disclosed herein.

[0291] Further objectives, advantages, and novel features of the present invention will become apparent to those skilled in the art by considering the following embodiments, which are not intended to be limiting. In addition, experimental support is found in each of the various embodiments and aspects of the present invention described above and claimed in the following claims.

[0292] Experimental support for each of the various embodiments and aspects of the present invention described above and claimed in the following claims is found in the following examples. [Examples]

[0293] In general, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular techniques, biochemical techniques, immunological techniques, microbiological techniques, and recombinant DNA techniques. Such techniques are well described in the literature. For example, the following are all incorporated by reference: "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook," Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology," Volumes I-III, Coligan, JE, ed. (1994); Stites et al.See also (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); and Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996). Other general references are provided throughout this document.

[0294] Example 1 Autoantibodies against ITGA2B and ITGB3 are the primary cause of ITP PF. These two integrins bind to fibrinogen, forming a dimer that is the primary target of ITP autoantibodies. Therefore, we developed therapeutic agents targeting autoantibodies against ITGA2B alone, ITGB3 alone, and both proteins simultaneously. We also tested the complete extracellular domains of these integrins.

[0295] Long-term remission in ITP patients requires the removal of a significant proportion of autoreactive B cells that produce an autoantibody pool. While simply removing autoantibodies from circulation is potentially effective in treating ITP / PF symptoms, long-lived B cells continuously produce new autoantibodies, requiring repeated treatments throughout the subject's remaining lifespan. Importantly, B cells that produce autoantibodies express B cell receptors (BCRs), which are the same membrane-bound forms of these autoantibodies, on their surface. This makes it possible to target the B cells themselves with therapeutic agents containing autoantibody BCR-specific epitopes. By linking the target epitope to the Fc region of the antibody heavy chain, the therapeutic agent can lead to the specific killing of autoantibody-producing B cells. This method is robust against the potential evasion of specific subpopulations that can occur when using agents that target specific differentiation markers on the cell surface (e.g., CD19, CD38, BCMA), as any cell possessing autoreactive BCRs is targeted regardless of its differentiation state. This approach is also beneficial for protecting and preserving non-autoreactivity, including protective (e.g., antiviral, antibacterial) subpopulations that are damaged by therapies targeting nonspecific differentiation markers (e.g., CD20, CD38, BCMA), regardless of whether they possess an autoreactive BCR.

[0296] Figure 1A shows one embodiment of the therapeutic agent of the present invention. The immunoglobulin (Ig)-like protein complex 101 comprises four polypeptide chains: two heavy-chain-like polypeptides 110 and two light-chain-like polypeptides 120. Chain 110 can be dimerized via disulfide bonds between them. Furthermore, chain 110 may contain any or all of the CH3 domain 111, CH2 domain 112, hinge region 113, and CH1 domain 114. In this embodiment, the CH3 domain 111, CH2 domain 112, and hinge region 113 all contain disulfide bonds and act as dimerizing domains, but other dimerizing domains can also be used. These domains are well known in the art and can be selected from, for example, any of the human (or non-human) IgG1 domain, IgG2 domain, IgG3 domain, IgG4 domain, IgA domain, IgM domain, IgE domain, and IgD domain. Those skilled in the art will understand that the Fc portions of IgG1 and IgG3 incorporated into chain 110 enable the molecule to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). Chain 120 can dimerize with chain 110 via disulfide bonds found in the CH1 domain 114 and CL domain 124.

[0297] Unlike naturally occurring or synthetic antibodies, chains 110 and 120 lack a variable region. Instead of a variable region, each chain has a fragment 130 derived from the extracellular portion of human integrin subunit alpha 2b or integrin subunit beta 3. Each chain can be generated to have the same or a derivative of ITGA2B or ITGB3, or a different fragment / derivative. In fact, as shown in Figure 1B, the two heavy chains 115 and 116 can be manipulated 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 same applies to light chains 125 and 126, which can contain, for example, a fragment of the extracellular domain of ITGB3 133 and another fragment (the same or a different) 134, respectively. Therefore, therapeutic molecules can be designed to have four copies of a protein or domain (Figure 1C), two copies of each of two different proteins or domains (Figure 1D), one copy of each of four different protein fragments or domains (Figure 1B), or any other combination thereof. In fact, molecules are modular enough that they can be manipulated to have three copies of one protein / domain and one copy of another, or two copies of one protein / domain and one copy of two other. Figure 1E shows an embodiment in which two light chains are identical but two heavy chains are different. Figure 1F also shows an embodiment in which two heavy chains are identical but two light chains are different. Importantly, therapeutic molecules can be manipulated to contain any combination of different domains of two proteins that can bind to autoantibodies from a wide variety of patients, not just some patients. Any chain can contain any protein, fragment, domain, or variant, and it will be understood by those skilled in the art that the combinations of chains and subunits shown in Figures 1A–1F are illustrative and not limiting.

[0298] Figures 2A–2N illustrate several embodiments of the present invention in which only two chains are combined. In Figures 2A–F, the protein complex 201 specifically comprises two polypeptide chains, which are two heavy chains. The heavy chains 215 and 216 may optionally contain hinge domains 213, CH2 212, CH3 211, and / or CH1 214 domains. In this embodiment, the heavy chain hinges 213, CH2 212, and CH3 211 all dimerize via disulfide bonds. Only one of these three domains is required for dimerization, although other dimerizing domains are also conceivable. The extracellular domain is either ITGA2B or ITGB3 230. Protein complex 201 having only a fragment of the ITGA2B extracellular domain or the ITGB3 extracellular domain, for example, fragment 231 of ITGA2B, or fragment 232 of ITGA2B, is also conceivable. Protein complex 201 is also conceivable, having two different fragments derived from the same protein, or from each of two different proteins (e.g., fragment 231 on one chain and fragment 233 on the other chain). These fragments represent any fragment of the extracellular domain of either protein, which can be used in any combination.

[0299] Figure 2B shows molecules lacking the CH2 domain 212, the CH3 domain 211, or the CH1 domain 214. Molecules lacking both the hinge 213 and the CH1 domain 214 are also shown. Combinations lacking two of these domains, with or without the hinge 213, are also conceivable (Figure 2B). Instead of a variable region, each chain has a fragment 230 derived from the extracellular portion of ITGA2B and / or ITGB3. Although not shown, it will be understood by those skilled in the art that any fragment derived 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 (Figure 2C) or a different protein, fragment, or variant (Figure 2D). For ease of use, the extracellular domains of ITGA2B 235 and ITGB3 236 are shown, but it will be understood that fragments or variants of these domains can also be used. When two different subunits are used, it is advantageous to design the molecule so that primarily heterodimers of 215 and 216 are formed, and homodimers are not formed. The same applies to the heterodimerization of chains 115 and 116 in Figure 1. The same applies to Figure 2A. There are numerous techniques known in the art for designing mutations in the CH3 / CH2 domain that inhibit homodimerization and promote heterodimerization, e.g., knob-in-holes, DuoBodies, etc. Any such technique may be used. Removal of CH1 by direct conjugation to the hinge region (Figure 2E), or additional removal of the hinge by direct conjugation to CH2 (Figure 2F) is also possible. In any figure, if an extracellular domain is shown, it is understood that it is intended to include any fragments, variants, and mutants of that extracellular domain.

[0300] Figure 2G shows an alternative configuration containing two heavy chains. Instead of containing a single fragment 230 instead of the variable region, two tandem fragments 230 are used. These fragments may be separated by an optional linker 290. This configuration is structurally similar to a single-chain antibody in which the heavy chain variable domain and light chain variable domain reside on a single peptide, and is essentially equivalent to the molecule shown in Figure 1D. Heavy chains 215 and 216 may optionally contain the hinge 213, CH2 212, CH3 211, and / or CH1 214 domains. Dimerization is as described above. For simplicity, an example containing all three CH domains is shown as an example lacking the CH1 domain. Molecules lacking the hinge, CH2 domain, or CH3 domain, or any two / three of these domains, are also conceivable (as long as at least one dimerization region is retained). Fragment 230 may originate from either ITGA2B or ITGB3, and it is understood that it may contain the entire extracellular domain, only a part of it, or a variant. Therefore, two repeats from the same protein / fragment can be inserted into a single strand (Figure 2H-I, two ITGA2B fragments 231), or two different proteins / fragments can be combined on one strand (Figure 2J-K, ITGA2B fragment 231 and ITGB3 fragment 234). Naturally, the heavy chains do not need to be identical, as various techniques can be used to promote heterodimerization rather than homodimerization (Figure 2L-M, ITGA2B fragment 231 and ITGB3 fragment 234 on one strand, and ITGB3 fragment 233 and ITGA2B fragment 232 on the other strand). As mentioned above, the CH1 domain 214 may be included (Figures 2H, 2J, 2L) or excluded (Figures 2I, 2K, 2M), and the same applies to hinge 213, CH2 212 and / or CH3 211, as long as one dimerization domain (e.g., hinge, CH2, CH3) remains.

[0301] In Figure 2N, the protein complex 201 comprises two polypeptide chains, specifically a heavy chain 215 and a light chain 220. In such embodiments, the dimerization domains are the CH1 domain 214 and the CL domain 224. The heavy chain 215 may optionally contain a CH3 domain 211, a CH2 domain 212, and / or a hinge region 213. The absence of the hinge / CH2 / CH3 domain is one option to eliminate homodimerization of the two heavy chains 215. Alternatively, a cysteine ​​substitution / mutation (e.g., to serine or glutamine) may be introduced into the hinge, or one of the CH2 / CH3 region mutations that promote heterodimerization and inhibit homodimerization may be used. Instead of a variable region, each chain has a fragment 230 derived from the extracellular component of ITGA2B or ITGB3, or any fragment or variant thereof.

[0302] The construction of a protein complex 301 having three chains, heavy chain 315, heavy chain 316, and light chain 320, is also envisioned (Figures 3A-3D). Figure 3A shows one possible embodiment in which heavy chain 316 contains a CL domain 364 instead of a CH1 domain. The method described herein can be used to ensure the 315 / 316 heterodimer. Heavy chains 315 and 316 may optionally contain a CH3 domain 311, a CH2 domain 312, and / or a hinge region 313, or different dimerization domains may be used. The CL domain 324 in light chain 320 can only dimerize with the CH1 domain 314 in heavy chain 315. Instead of a variable region, each chain has a fragment 330 derived from the extracellular portion of ITGA2B / B3. All three strands may contain the same fragment (e.g., ITGA2B extracellular domain 335, Figure 3B), all three strands may contain different fragments (e.g., ITGB3 336, ITGA2B fragment 331, and ITGA2B fragment 332, Figure 3C), or the three strands may contain two different fragments, one of which is repeated (e.g., ITGA2B 335 and ITGB3 fragment 333, Figure 3D). Figure 3D may also have two identical fragments, either as a light chain and one as a heavy chain, and thus it will be understood by those skilled in the art that this embodiment has three different configurations.

[0303] This configuration, using one of the heavy chains containing a CL domain instead of a CH1 domain, can also enable the formation of protein complexes with four different fragments. Similar to the protein complex in Figure 1B, the protein complex 401 shown in Figure 4 has four different fragments on each chain. In this embodiment, fragments ITGA2B Frag.431, ITGB3 Frag.434, ITGA2B Frag.432, and ITGB3 Frag.433 are used, but those skilled in the art will understand that any four fragments could be used. Embodiments can also be envisioned in which different fragments from the same protein may be on different chains. In this embodiment, the second light chain 426 contains a CH1 domain 474 so that it can dimerize with the CL domain 464 of the heavy chain 416. The heavy chain 415 contains a CH1 domain 414, and the light chain 425 contains a CL domain 424. This ensures that chain 425 can dimerize only with chain 415, and chain 426 can dimerize only with chain 416. As described above in this specification, any mutations in the CH2 domain 412 and CH3 domain 413 can be used to promote heterodimerization of chains 415 and 416. The hinge region 413, CH2 domain 412, and CH3 domain 411 are all used here as dimerization domains between the two heavy chains, but any dimerization domain (other than CH1 / CL) can be used.

[0304] In the embodiments described above, an immunoglobulin skeleton is shown and described, but it will be understood by those skilled in the art that similar molecules can be generated by selecting other dimerization domains. Figures 5A and 5B show a typical protein complex 501. In Figure 5A, the first chain 515 contains a first dimerization domain (DD1) 563 which can specifically dimerize with a second dimerization domain (DD2) 573 of the second chain 516. Chain 515 further contains a third dimerization domain (DD3) 514 which can specifically dimerize with a fourth dimerization domain (DD4) 524 of the third chain 525. Chain 516 further contains a fifth dimerization domain (DD5) 564 which can specifically dimerize with a sixth dimerization domain (DD6) 574 of the fourth chain 526. Each of the four chains also contains a fragment 530 of a human protein target of an ITP / PF autoantibody. These could all be the same fragment having the same amino acid sequence, or they could be different sequences (either from the same protein or from different proteins), or variants / derivatives, or proteins or fragments.

[0305] Figure 5B shows an alternative embodiment of Figure 5A, where each distinct domain is separated by a linker. Those skilled in the art will understand that any of these linkers are arbitrary and that combinations of linkers are conceivable. It will be further understood that the configuration of Figure 5B also allows for the use of linkers between any or all of the various domains / fragments. Similarly, linkers can be inserted between the CH1 domain, hinge region, CH2 domain, CH3 domain and / or ITGA2B / B3 fragments. Thus, the linkers shown in Figure 5B can be extrapolated to the same positions within the immunoglobulin backbone molecule (Figures 1A-4).

[0306] Figures 6A to 6F illustrate single-stranded embodiments of the present invention. Figure 6A shows a single-stranded fusion protein 601 containing the ITGA2B extracellular domain 635 and the ITGB3 extracellular domain 636. Figure 6B shows a single strand containing only a fragment of ITGA2B or only a fragment of ITGB3. For simplicity, a common fragment of ITGA2B 631 and a common fragment of ITGB3 633 are shown, but it is understood that any fragment of the extracellular domain may be used. It is further understood that any permutation of Figures 6A and 6B may be generated such that two fragments from different proteins are in the same single strand. Two different fragments may be used. Specifically, fragments from two different proteins may be used. Figures 6C and 6D show similar embodiments, but containing three and four fragments from different proteins, respectively. It is understood that any fragment or the entire extracellular domain may be used. As shown in Figure 6E, the single-stranded molecule may also contain a heavy-chain constant region having at least a CH3 domain 611 or a CH2 domain 612, and optionally a CH1 domain 614, a hinge region 613, and / or a CH2 domain 612 or a CH3 domain 611. Figure 6F shows an embodiment in which the CH1 domain has been removed. Finally, any of the domains of the single-stranded molecule can be separated using an amino acid linker. Figure 6G shows embodiments in which two, three, or four fragments are separated by linker 690, and embodiments in which linker 690 also separates the C-terminal fragment 635 from the CH1 domain 614 or a CH2 domain 612. A linker that replaces the hinge region is also shown, but this is not necessary, and it is understood that the hinge may be held by a linker that connects the C-terminal fragment 635 to the hinge. Although linkers for separating the CH1 domain 614, hinge region 613, CH2 domain 612, and CH3 domain 611 are not shown, it will be understood by those skilled in the art that any or all of these domains can be separated by linkers. Furthermore, it will be understood that these various linkers may all contain the same sequence or may be constructed from different amino acid sequences.

[0307] Example 2 In the first batch, the complete extracellular domain of ITGB3 (SEQ ID NO: 2, lacking the signal peptide) was transiently expressed in 20 ml of CHO cells (Figure 7). To further facilitate protein purification, molecules were generated using 8X His tags and AVI tags, and the tags were isolated from the C-terminus of the extracellular domain using a positively charged linker EAAAKEAAAK (SEQ ID NO: 4) (SEQ ID NO: 3, CRD-760). It is known in the art that the complete extracellular domain of ITGA2B (SEQ ID NO: 1, lacking the signal peptide) cannot be readily expressed on its own because it is retained in the ER during secretion. Therefore, the extracellular domain of ITGA2B alone was not tested.

[0308] Additional molecules combining the extracellular domain of ITGB3 with an IgG hinge, as well as the CH2 and CH3 domains (containing the S293C mutation), were also generated in CHO cells (SEQ ID NO: 5, CRD-756) (Figure 7). The C-terminus of the extracellular domain was ligated to the hinge using a (GGGGS)3 linker (SEQ ID NO: 6, SEQ ID NO: 7, with three repeats). A similar construct containing the extracellular domain of ITGA2B (SEQ ID NO: 8, CRD-755) was also generated, but when introduced into CHO cells, no expression was observed, suggesting that this protein is still retained in the Golgi.

[0309] Molecules with mutations in the Fc region to promote heterodimerization rather than homodimerization were also generated (nob-in-hole method: T366W mutation in the CH3 domain, SEQ ID NO: 9; and T366S / L368A / Y407V triple mutation in the CH3 domain, SEQ ID NO: 10). ITGB3 was fused to the knob molecule containing the T366W mutation (SEQ ID NO: 13, CRD-758 chain 1) and the hole molecule containing the triple mutation (SEQ ID NO: 12, CRD-757 chain 2). ITGA2B was also fused to the knob molecule (SEQ ID NO: 11, CRD-757 chain 1) and the hole molecule (SEQ ID NO: 14, CRD-758 chain 2). Surprisingly, both heterodimer molecules (CRD-757 and CDR-758) were expressed in CHO cells and were not captured or degraded like the ITGA2B extracellular domain alone (Figure 7). Therefore, fusion of the ITGA2B extracellular domain to Fc appears to be a viable strategy for generating this target for autoantibodies.

[0310] The generated molecules are summarized in Table 2, which shows the identifiers used throughout this specification, the predicted molecular weight (MW), predicted isoelectric point (pI, (M-1*cm-1)), predicted extinction coefficient (EC), and actual yield for each molecule. Each molecule was expressed at its predicted molecular weight (CRD-756 at 208 kda, CRD-757 and CRD-758 at 236 kda, and CRD-760 at 80.5 kda) as observed by SDS-PAGE. The reduced forms were also expressed at their predicted molecular weights (ITGA2B GPIIb-CH2-CH3 at approximately 132 kda, and ITGB3 GPIIIa-CH2-CH3 at approximately 104 kda).

[0311] [Table 3]

[0312] Example 3 Next, the binding ability of various molecules to actual pathogenic antibodies is determined using serum samples from ITP patients. Molecular binding is investigated using human serum samples positive for anti-ITGB3 and / or ITGA2B IgG antibodies. Autoantibody titers are determined using Monoclonal Antibody-Specific Immobilization of Platelet Antigens (MAIPA) assays and / or ELISAs. Molecules are biotinylated and attached to avidin-coated Sepharose beads, and serum samples are incubated separately with various ITGB3 or ITGA2B / B3-containing molecules at gradually increasing concentrations. Any molecule containing the ITGB3 extracellular domain can bind to anti-ITGB3 antibodies, regardless of the presence of the ITGA2B extracellular domain. Human serum samples positive for anti-ITGB3 IgG are depleted using the ITGB3 extracellular domain molecule. Human serum samples positive for anti-ITGA2B IgG are depleted using the ITGA2B / B3 complex molecule. The anti-ITGA2B titer is determined by subtracting the depletion rate of anti-ITGB3 alone from the depletion rate of anti-ITGA2B / B3. Both ITGA2B / B3-containing molecules undergo robust depletion of approximately 40% or more.

[0313] In addition to testing for ITGB3-containing molecules, a molecule containing only ITGA2B is also included as a negative control. As expected, all ITGB3-containing molecules can deplete anti-ITGB3 antibodies, but none of the ITGA2B-only molecules can. In the reverse experiment, ITGA2B-containing molecules can bind to anti-ITGA2B antibodies in serum. When ITGB3-containing molecules are used as a negative control, they do not bind to anti-ITGA2B antibodies.

[0314] Example 4 To test the molecular ability to target autoreactive B cells themselves and not merely capture autoantibodies, CRD-757 and CRD-758 were incubated with at least 17 different mouse / rat hybridomas (listed in Table 3) at 37°C for 40 minutes. Two of the hybridomas, LK-4 and AP-3, are ITP hybridomas and produce antibodies against ITGB3. The other hybridomas all produce antibodies against other antigens. After incubation, the cells were washed twice with FACS buffer (DPBS containing 1% FBS) and then incubated with anti-human IgG Fc region PE conjugate polyclonal antibody. After this second incubation, the cells were washed twice again and analyzed using flow cytometry (CytoFlex by Beckman Coulter) (Figure 8A, results for CRD-757 are shown, but the results for CRD-758 were comparable). Cells incubated with only the secondary antibody were used as a negative control (Pe-MFI). The mean fluorescence intensity (MFI) factor change from the background value was calculated by dividing the PE-MFI of CRD-757 / CRD-758 stained hybridoma cells by their negative control background MFI (Figure 8B). CRD-757 strongly bound to both hybridomas expressing BCR to ITGB3, but no binding to the control hybridoma was observed. Similar results were obtained using CDR-758, which also specifically bound to the two ITP hybridomas (Figures 8C-8D). When molecules with unrelated extracellular domains were used, no binding to hybridomas expressing BCR to ITGB3 was observed.

[0315] [Table 4]

[0316] This data demonstrates that the molecules of the present invention can be used to target B cells and provide a permanent cure for ITP, and that the molecules of the present invention do not merely temporarily reduce autoantibody levels. Furthermore, importantly, in the development of a therapeutic agent that can be used for both ITGA2B autoantibody patients and ITGB3 autoantibody patients, heterodimers containing both the ITGA2B extracellular domain and the ITGB3 extracellular domain were still able to bind to the BCR for ITGB3 only.

[0317] Example 5 Since we investigated protein complexes and single-strand proteins containing dimerization domains, we will also investigate soluble molecules consisting only of the extracellular domain of ITGA2B / B3, or its fragments or derivatives.

[0318] Example 6: Specific B-cell killing by the molecule of the present invention CRD-757 and CRD-758 were tested for their ability to kill anti-ITGB3 B cell hybridoma cells. These two molecules were compared to CRD-760, which contains the ITGB3 ECD but lacks the Fc domain. AP3 hybridoma cells expressing the BCR against ITGB3 were cultured using gradually increasing concentrations of the molecules (0.16–20 μg / ml). 33.3% guinea pig serum was added to RPMI medium to induce CDC. After 3 hours of incubation, dead cells were labeled with propidium iodide (PI) and quantified by flow cytometry. The percentage increase in cytotoxicity was calculated compared to CRD-760, which was expected to cause no cytotoxicity. Both molecules resulted in a significant increase in cell killing, but surprisingly, CRD-758 was significantly superior to CRD-757 (Figure 9). This result is highly unexpected, given that both ECD fragments are identical, and the only way to distinguish the molecules is by switching a knob-in-hole mutation between the two strands. Furthermore, when these molecules were tested for their ability to kill anti-ITGA2B B cell hybridomas, both were also superior to molecules lacking Fc.

[0319] The active ingredient of the present invention, comprising an ITGA2B / B3 molecule fused to a cytotoxic Fc domain, is generally tested for its ability to kill B cells. The cytotoxic activity of the ITGA2B / B3 fusion molecule is determined using ITP hybridomas LK-4 and AP-3, which produce antibodies against ITGB3. The ITGA2B / B3 molecule of the present invention results in a high level of specific killing, killing nearly 100% of anti-ITGB3 expressing hybridoma cells. This indicates that the ITGA2B / B3 complex structure is functional for cell binding and cell killing.

[0320] Other effector molecules are tested. These include the ITGA2B / B3 molecules of the present invention conjugated to alpha-amanitin, tecilin, Dxd, PNU-159682, MMAE, MMAF, and triptolide. All of these exhibit superior killing compared to that produced by the unmodified Fc domain. Fc domains with mutations that increase ADCC are also tested. Fc mutations as described above are introduced into the Fc domain, and killing is tested in anti-ITGA2B hybridomas and anti-ITGB3 hybridomas. The killing is specific to these hybridomas but not to hybridomas against other targets, and the killing is superior to that produced by the unmodified Fc domain.

[0321] It is well established that potentially autoreactive B cells can be found in naive / healthy mice, especially in inbred lines (see, for example, Ding and Yan, "Regulation of autoreactive B cells: checkpoints and activation," Arch.Immunol.Ther.Exp., 2007, 55, 83-89; Wang et al., "The naive B cell repertoire predisposes to antigen-induced systemic lupus erythematosus," J Immunol. 2003 May 1; 170(9): 4826-32; and Fereidan-Esfahani et al., "IgM natural autoantibodies in physiology and the treatment of disease," Methods Mol Biol. 2019: 1904: 53-81). To confirm this, blood was collected from 6-8 week old naive C57Bl6 inbred female mice and an immunoassay based on magnetic beads was performed to measure the titer of anti-ITGA2B or anti-ITGB3 antibodies. All mice were found to be positive for the antibodies. The presence of these autoreactive antibodies indicates the presence of autoreactive B cells even before immunization with ITGA2B / ITGB3 fragments.

[0322] To test the ability of the molecules of the present invention to kill these autoreactive B cells, 6-8 week old female C57BL6 mice are intravenously immunized with the naked ITGA2B-Fc molecule of the present invention or the drug-conjugated ITGB3-Fc molecule of the present invention (at doses of 0.5 mg / kg or higher) over a total of 4-8 injections, twice weekly. Subcutaneous injection is also tested. Negative control groups include mice administered with PBS and mice administered with an unrelated Ig-like molecule conjugated to a drug. Serum samples are isolated during the experiment and autoantibody titers are assessed. At the end of the immunization period, relative antibody titers against ITGA2B are compared using an ITGA2B-specific immunoassay (ELISA), and / or ITGB3 titers are compared using an ITGB3 ELISA. After immunization, animals immunized with the naked molecule of the present invention show an increase in autoantibody titers, while no increase in titer is observed in animals immunized with the drug-conjugated molecule. This demonstrates that the drug conjugate molecule of the present invention can kill ITP autoreactive B cells and treat this disease. The same test will be performed with the ITGA2B-ITGB3 molecule combination of the present invention.

[0323] Various molecules of the present invention are also tested in vivo. All of these molecules have been found to effectively treat ITP, kill autoreactive B cells, and reduce autoantibody titer levels in vivo. All of the tested effector molecules have been found to be superior to Fc.

[0324] ITP is also induced in mice by subcutaneous injection of the ITGA2B / B3 ECD fragment. The ability of the molecule of the present invention to treat ITP in this organism will be confirmed. Serum will be collected and antibody titer levels will be monitored. The molecule of the present invention not only kills target B cells but also reduces circulating antibody levels.

[0325] While the present invention has been described in conjunction with its specific embodiments, it is evident that many alternative, modified, and variant forms are apparent to those skilled in the art. Therefore, it is intended to encompass all such alternative, modified, and variant forms that fall within the spirit and broad scope of the appended claims.

Claims

1. a. A first polypeptide comprising a fragment of the extracellular domain of integrin subunit alpha 2b (ITGA2B) or an analog or derivative thereof, a fragment of the 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. A composition comprising, The first and second dimerization domains are configured to dimerize with each other. composition.

2. The composition according to claim 1, wherein 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.

3. The composition according to claim 1 or 2, wherein the dimerization comprises the formation of a covalent bond between the first dimerized domain and the second dimerized domain.

4. The composition according to any one of claims 1 to 3, wherein the protein complex comprises an immunoglobulin scaffold.

5. a. The first dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the second dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains are dimerized by a disulfide bond, or b. The first and second dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, wherein neither the first nor the second dimerization domain contains the CH1 domain or the CL domain. The composition according to any one of claims 1 to 4.

6. The composition according to any one of claims 1 to 5, wherein the fragments and dimerization domains of the first, second, or both polypeptide chains are separated by a linker.

7. The composition according to any one of claims 1 to 6, wherein the first polypeptide chain, the second polypeptide chain, or both further comprises the Fc region of a human antibody heavy chain.

8. The composition according to claim 7, wherein the Fc region can induce cytotoxicity against cells bound to the protein complex.

9. The composition according to claim 7 or 8, wherein the first polypeptide chain comprises a first CH3 domain of an immunoglobulin heavy chain, a first CH2 domain of an immunoglobulin heavy chain, or both, and the second polypeptide chain comprises a second CH3 domain of an immunoglobulin heavy chain, a second CH2 domain of an immunoglobulin heavy chain, or both.

10. The composition according to claim 9, wherein the first CH3 domain comprises at least a first mutation, and the second CH3 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

11. The composition according to claim 9 or 10, wherein the first CH2 domain comprises at least a first mutation, and the second CH2 domain comprises at least a second mutation, the mutations enabling heterodimerization of the first and second polypeptide chains and inhibiting homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

12. The composition according to claim 10 or 11, wherein the first mutation is selected from the mutations provided in Table 1, and the second mutation is a mutation provided in Table 1 that corresponds to the first mutation.

13. The composition according to claim 12, wherein the first mutation is a T366W mutation in the CH3 domain, and the second mutation is a combination of a T366S mutation, an L368A mutation, and a Y407V mutation.

14. The composition according to any one of claims 7 to 13, wherein the Fc region of the first, second, or both polypeptide chains is separated from the fragment or the dimerization domain by a linker.

15. The composition according to any one of claims 7 to 14, wherein the Fc region comprises at least one mutation that increases ADCC.

16. The composition according to claim 15, wherein the Fc region is an Fc region containing SEQ ID NO: 57 or SEQ ID NO: 59, which contains a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 57 or SEQ ID NO:

59.

17. The composition according to any one of claims 7 to 16, wherein the dimerization domain of the first, second, or both of the polypeptide chains is located on the C-terminal side of the fragment and on the N-terminal side of the Fc region.

18. A composition according to any one of claims 1 to 17, lacking an antibody variable domain.

19. The composition according to any one of claims 1 to 18, further comprising 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 dimerizing domain, wherein the first polypeptide further comprises a fourth dimerizing domain, and the third and fourth dimerizing domains can dimerize with each other.

20. a. The third dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the fourth dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains are dimerized by disulfide bonds, or b. The third and fourth dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, and the first and third polypeptides both do not contain the CH1 domain or the CL domain. The composition according to claim 19.

21. The composition according to claim 19 or 20, further comprising 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 sixth dimerization domains are dimerizable with each other.

22. a. The fifth dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, the sixth dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains are dimerized by disulfide bonds, or b. The fifth and sixth dimerization domains each contain a domain selected from the CH1 domain of the immunoglobulin heavy chain and the CL domain of the immunoglobulin light chain, and are dimerized by a disulfide bond, and the first and third polypeptides both do not contain the CH1 domain or the CL domain. The composition according to claim 21.

23. The composition according to any one of claims 5 to 22, wherein both the first polypeptide and the second polypeptide either do not contain a CH1 domain or both contain a CL domain.

24. The composition according to any one of claims 19 to 23, wherein the third and fourth dimerizing domains, or the fifth and sixth dimerizing domains, include mutations that enable dimerization of the third and fourth dimerizing domains, as well as the fifth and sixth dimerizing domains, and inhibit dimerization of the third dimerizing domain to the fifth or sixth dimerizing domain, and dimerization of the sixth dimerizing domain to the third or fourth dimerizing domain.

25. The composition according to any one of claims 1 to 24, wherein 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.

26. The composition according to any one of claims 1 to 25, wherein 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.

27. The composition according to claim 26, wherein the fragments are separated by an amino acid linker.

28. The composition according to any one of claims 1 to 27, wherein ITGA2B lacks a signal peptide and contains or comprises SEQ ID NO: 1, and ITGB3 lacks a signal peptide and contains or comprises SEQ ID NO: 2, or both.

29. The composition according to any one of claims 1 to 28, wherein the extracellular domain fragment consists of a truncation of the extracellular domain.

30. The composition according to any one of claims 1 to 29, wherein the analog or derivative thereof contains at least 85% identity with respect to ITGA2B or ITGB3.

31. The composition according to any one of claims 1 to 30, wherein the fragment comprises at least 20 consecutive amino acids derived from ITGA2B or ITGB3.

32. The composition according to any one of claims 1 to 31, wherein the fragment comprises at least one B cell receptor (BCR) specific epitope target of an autoantibody.

33. The composition according to any one of claims 1 to 32, comprising a first or second polypeptide containing a sequence selected from sequence numbers 5, 8 and 11 to 14.

34. The composition according to any one of claims 5 to 33, wherein the hinge domain, the CH2 domain, or the CH3 domain contains at least one mutation that reduces ADCC.

35. The at least one mutation that reduces ADCC is a. Mutations of the hinge domain, including the L19A and L20A mutations of Sequence ID No. 22, and b. The composition according to claim 34, selected from the mutations of the CH2 domain, including the N59A mutation of Sequence ID No.

36.

36. The composition according to any one of claims 1 to 35, further comprising at least one effector portion capable of inducing cell death in cells bound to the composition.

37. The composition according to claim 36, wherein the effector portion is not an Fc domain.

38. The composition according to claim 37, wherein the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

39. A pharmaceutical composition comprising the composition according to any one of claims 1 to 38 and a pharmaceutically acceptable carrier, excipient, or adjuvant.

40. A method for treating immune thrombocytopenia (ITP) in a subject requiring such treatment, comprising administering a composition comprising a fragment of the extracellular domain of ITGA2B or ITGB3 or an analog or derivative thereof to the subject, thereby treating the ITP.

41. The method according to claim 40, wherein the fragment is linked to an effector domain capable of inducing cell death in cells to which the fragment is bound.

42. The method according to claim 40 or 41, wherein ITGA2B lacks a signal peptide and contains or comprises SEQ ID NO: 1, and ITGB3 lacks a signal peptide and contains or comprises SEQ ID NO: 2, or both.

43. The method according to claim 40 or 42, wherein the composition is the composition described in any one of claims 1 to 38.

44. The method according to any one of claims 40 to 43, wherein the composition is the pharmaceutical composition described in claim 39.

45. The method according to any one of claims 40 to 44, further comprising reducing the level of circulating antibodies against ITGA2B, ITGB3, or both, in the subject.

46. The method according to any one of claims 40 to 45, wherein the treatment comprises reducing the concentration of circulating autoantibodies against ITGA2B, ITGB3, or both.

47. The method according to any one of claims 40 to 46, wherein the composition comprises an Fc region, and the treatment comprises killing B cells that produce anti-ITGA2B autoantibodies or anti-ITGB3 autoantibodies.

48. The method according to claim 47, wherein the B cells are autoreactive B cells that produce autoantibodies against the fragments of the composition.

49. A nucleic acid system comprising a nucleic acid molecule, wherein the first nucleic acid molecule encodes the first polypeptide of the composition according to any one of claims 1 to 38, and the second nucleic acid molecule encodes the second polypeptide of the composition according to any one of claims 1 to 38.

50. The nucleic acid system according to claim 49, further comprising a third nucleic acid molecule encoding the third polypeptide of the composition according to any one of claims 19 to 38, a fourth nucleic acid molecule encoding the fourth polypeptide of the composition according to any one of claims 19 to 38, or both.

51. A method for producing the composition according to any one of claims 1 to 38, comprising expressing a nucleic acid system according to claim 49 or 50 in a cell, wherein the nucleic acid system is configured to produce the encoded polypeptide in the cell, thereby producing the composition according to any one of claims 1 to 38.

52. A method for producing proteins, A step of obtaining a first fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof, and a second fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof. A step of generating a first polypeptide chain by linking the first fragment to a first dimerization domain, and generating a second polypeptide chain by linking the second fragment to a second dimerization domain, wherein the first and second dimerization domains are capable of dimerizing with each other; and a step of bringing the first polypeptide and the second polypeptide into contact under conditions sufficient to induce the dimerization, or A step of culturing host cells comprising one or more vectors comprising nucleic acid sequences encoding at least two polypeptide chains, wherein the two polypeptide chains are i. A step of obtaining a first fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof, and a second fragment of the extracellular domain of ITGA2B or an analog or derivative thereof, or a fragment of the extracellular domain of ITGB3 or an analog or derivative thereof, and ii. A step of generating a first polypeptide chain by linking the first fragment to a first dimerization domain, and generating a second polypeptide chain by linking the second fragment to a second dimerization domain, wherein the first and second dimerization domains are capable of dimerizing with each other. Steps generated by A method that includes and thereby produces protein.

53. The method according to claim 52, wherein the protein complex is a protein complex of the composition according to any one of claims 1 to 38.

54. a. In the first polypeptide chain, the third dimerization domain is linked to the first dimerization domain or the first fragment to obtain the third fragment of the extracellular domain of ITGA2B or its analog or derivative, or the extracellular domain of ITGB3 or its analog or derivative, and the third fragment is linked to the fourth dimerization domain to generate the third polypeptide chain, wherein the third dimerization domain and the fourth dimerization domain can dimerize with each other, and the first, second and third polypeptides are brought into contact under conditions sufficient to induce the dimerization, or b. i. Obtaining a third fragment of ITGA2B or its analogue or derivative, or a fragment of the extracellular domain of ITGB3 or its analogue or derivative, and ii. Linking the third fragment to the fourth dimerization domain to generate a third polypeptide chain. The method further includes expressing a nucleic acid sequence encoding a third polypeptide chain generated by the method within the host cell, The first polypeptide chain further comprises a third dimerization domain, and the third dimerization domain and the fourth dimerization domain can dimerize with each other. The method according to claim 52 or 53.

55. a. In the second polypeptide chain, the sixth dimerization domain is linked to the second dimerization domain or the second fragment to obtain a fourth fragment of the extracellular domain of ITGA2B or its analog or derivative, or a fragment of the extracellular domain of ITGB3 or its analog or derivative, and the fourth fragment is linked to the fifth dimerization domain to generate a fourth polypeptide chain, wherein the fifth dimerization domain and the sixth dimerization domain can dimerize with each other, and the first, second, third and fourth polypeptides are brought into contact under conditions sufficient to induce the dimerization, or b. i. Obtain a fourth fragment of the extracellular domain of ITGA2B or its analogue or derivative, or a fragment of the extracellular domain of ITGB3 or its analogue or derivative, and ii. Linking the fourth fragment to the fifth dimerization domain to generate a fourth polypeptide chain. The method further includes expressing a nucleic acid sequence encoding a fourth polypeptide chain generated by the method within the host cell, The second polypeptide chain further comprises a sixth dimerization domain, and the fifth dimerization domain and the sixth dimerization domain can dimerize with each other. The method according to claim 54.

56. The method according to any one of claims 52 to 55, further comprising linking an effector portion to at least one of the polypeptide chains, wherein the effector portion can kill cells bound to the at least one polypeptide.

57. The method according to claim 56, wherein the effector portion is not in the Fc domain.

58. The method according to claim 57, wherein the effector portion is selected from alpha-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

59. The method according to claim 56, wherein the effector portion is an Fc domain containing at least one mutation that increases ADCC.

60. The method according to claim 59, wherein the effector portion is an Fc domain containing SEQ ID NO: 57 or SEQ ID NO: 59, which contains a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E in SEQ ID NO: 57 or SEQ ID NO:

59.

61. A protein produced by the method described in any one of claims 52 to 60.

62. A method for determining the suitability of an object to be treated by the method of any one of claims 40 to 48, comprising: receiving a sample from the object; contacting the sample with the protein described in claim 61 or the composition described in any one of claims 1 to 38; and determining the binding of autoantibodies in the sample to the protein or the composition, wherein the binding of autoantibodies to the protein or the composition indicates that the object is suitable to be treated by the method of any one of claims 40 to 48, thereby determining the suitability of the object to be treated.

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