IG-like fusion protein for the treatment of pemphigus vulgaris and pemphigus foliaceus

Abstract

A composition is provided comprising a first polypeptide comprising a first fragment or analog or derivative of the extracellular domain of DSG1 or DSG3 and a dimerizing domain, and a second polypeptide comprising a second fragment or analog or derivative of the extracellular domain of DSG1 or DSG3 and a dimerizing domain. Polypeptides comprising fragments of the extracellular domain of DSG1 or DSG3 that include at least one mutation that increases solubility, decreases aggregation, or both, or that lack extracellular domains 3 and 4. Also provided are the compositions of the present invention, polypeptides, nucleotide systems and molecules encoding the polypeptides of the present invention, pharmaceutical compositions, methods for therapeutic use of the compositions or polypeptides and methods for determining suitability for therapeutic use, and methods for producing the compositions or proteins.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 451,668, titled "Ig - like Fusion Protein and Its Use," filed on March 13, 2023, and U.S. Provisional Patent Application No. 63 / 609,419, titled "Ig - like Fusion Protein for Treating Pemphigus Vulgaris and Pemphigus Foliaceus," filed on December 13, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (CNPY - P - 009 - PCT.xml; size: 257,833 bytes; creation date: February 19, 2024) are hereby incorporated by reference in their entirety.

[0003] The present invention is in the field of production of fusion proteins and treatment of pemphigus vulgaris and pemphigus foliaceus.

Background Art

[0004] Pemphigus vulgaris / pemphigus foliaceus (PV / PF) is a rare life - threatening autoimmune blistering disease that affects the skin and mucous membranes. In most cases, PV initially presents oral mucosal lesions, especially in areas exposed to frictional trauma such as the buccal and lateral tongue mucosa. The lesions begin as small blisters that easily rupture, leaving erosions and ulcers. Subsequently, many patients progress to skin lesions within the next few weeks or months. PF has a skin distribution similar to that of PV, but the mucosal surfaces are not affected.

[0005] Pemphigus vulgaris occurs worldwide, with an incidence of 0.1 to 2.7 cases per 100,000 people per year and a prevalence of 5.2 cases per 100,000 people. Pemphigus vulgaris occurs in all races and ethnic groups, but the highest incidence is seen in Ashkenazi Jews of Mediterranean origin. The average age of onset is 40 to 60 years, and the sex ratio is nearly equal.

[0006] Pathophysiology: PV and PF are caused by IgG1 and IgG4 autoantibodies against desmoglein-1 (Dsg-1) and desmoglein-3 (Dsg-3), keratinocyte adhesion proteins that constitute desmosomes. The binding of autoantibodies impairs intraepidermal adhesion, leading to acantholysis and the formation of intraepithelial blisters in the mucous membranes and skin. Pemphigus vulgaris is characterized by mucosal and skin lesions caused by autoantibodies against Dsg-3 (mucosa) or both Dsg-3 and Dsg-1 (mucocutaneous). Pemphigus foliaceus is characterized by skin lesions caused by autoantibodies against Dsg-1 alone.

[0007] Dsg-1 and Dsg-3 are transmembrane glycoproteins with values ​​of 160 and 130 kDa, respectively, and are part of the cadherin family. Structurally, they consist of an extracellular domain with five structural domains (EC1-EC5), a transmembrane domain, and a cytoplasmic domain. The distal extracellular domains of cadherin molecules (amino-terminal EC1 and EC2 domains) bind to the corresponding distal domains of adjacent cell membranes, and it is this binding that is targeted and disrupted by autoantibodies.

[0008] Four desmoglein isoforms exist, but Dsg-1 and Dsg-3 are the only isoforms expressed in squamous stratified epithelium, while Dsg-2 is expressed in monolayer epithelium and cardiomyocytes, and Dsg-4 is expressed in hair follicles. It remains unclear whether further proteins constituting the desmosome, such as desmocollin, play a role in the pathogenesis of pemphigus, and why they cannot compensate for the loss of desmoglein function.

[0009] While IgG4 is dominant during the acute phase of the disease, the remission period has been shown to be associated with IgG1. Although several hypotheses have been proposed, the triggers for autoantibody production and the mechanism by which autoantibody binding causes acantholysis remain uncertain. Such theories involve changes in intracellular signaling and cytoskeletal segregation, resulting in keratinocyte contraction; spatial impairment of desmoglein adhesion; and the formation of desmoglein-deficient desmosomes.

[0010] The pathogenic role of anti-Dsg antibodies has been well established in several studies. In vitro studies have shown that adding IgG derived from PV patient serum to cultured keratinocytes resulted in a transient increase in intracellular signaling molecules. This induced phosphorylation and dissociation of Dsg3, as well as keratin retraction from intercellular contact sites. In vivo studies have shown that passive transfer of IgG antibodies from the serum of PV patients into mice induces blister formation with features consistent with pemphigus. The N-terminal extracellular domain 1 (EC1) of DSG3 is essential for DSG3 binding, and mouse monoclonal antibodies targeting this domain are thought to have induced experimental PV in vivo. However, in humans, DSG3 targeting is more complex than in mice, and PV patients produce polyclonal antibodies targeting the extracellular domain 1 epitope, which can be overcome by p38 MAPK inhibition, suggesting that antibody effects on signaling may play a significant role beyond mere physical blockage.

[0011] Diagnosis and Treatment. Diagnosis is made through clinical findings, including biopsy of skin lesions and serology, and confirmatory laboratory tests. However, due to a lack of subjective awareness and the inability to recognize clinical features, it takes an average of 5 physicians and 10 months to confirm the diagnosis. Tissue biopsy and direct immunofluorescence remain the best diagnostic tools in combination with clinical findings, while indirect immunofluorescence and ELISA play a complementary role. More than 80% of patients with active disease produce Dsg-1 and Dsg-3 specific autoantibodies of IgG1 and IgG4, detectable by indirect immunofluorescence. FDA-approved ELISA tests are also commercially available and offer high sensitivity and specificity. Disease activity correlates with antibody titers in most cases, and the use of ELISA helps monitor disease activity and treatment response.

[0012] Before the introduction of systemic corticosteroids, the prognosis for PV was almost always fatal within the first two years after the onset of symptoms. While appropriate treatment has dramatically reduced the mortality rate from PV, it still remains at approximately 15%. Most deaths are thought to be due to complications from treatment resulting from secondary effects of first- and second-line immunosuppressants.

[0013] First-line treatment involves anti-CD20 antibodies combined with systemic corticosteroids to achieve rapid control of disease activity (reduce lesion appearance) within weeks, followed by slowly tapered steroid administration. Unfortunately, due to the chronicity of the disease, long-term systemic corticosteroid therapy is necessary to maintain the clinical response, increasing the incidence of serious adverse events, including severe infections, diabetes mellitus, osteoporosis, hypertension, and gastrointestinal ulcers, resulting from immunodeficiency.

[0014] While the majority of patients can achieve initial improvement with first-line therapy, others are unresponsive or experience frequent relapses. The main treatment options for refractory diseases include directly targeting autoantibody-mediated responses via IVIG, plasmapheresis, or rituximab. Recently, rituximab has been introduced as an adjuvant for patients unresponsive to first-line steroids and as a steroid-sparing add-on to first-line therapy. 65% of patients maintain remission (3–22 months) with rituximab in addition to immunosuppressive therapy. The overall incidence of serious adverse events to rituximab has been 1–16% in various studies, including infections that frequently lead to sepsis. Today, systemic glucocorticoids and rituximab are mainstream treatments for PV and PF and are usually very effective in controlling the disease. However, due to life-threatening adverse events associated with first-line therapy, safer and more sustained therapies are needed.

[0015] Primary endpoints in clinical studies include the proportion of patients who achieve sustained complete remission without treatment over a long period, and efficacy in reducing the Pemphigus Disease Area Index (PDAI) score within weeks compared to baseline. Generally, trials compare novel treatments to first-line steroid treatment or placebo after first-line steroid treatment.

[0016] Surrogate biomarkers and predictive biomarkers. Antibody titer is a surrogate measure that has been reported to correlate with disease activity in pemphigus. A higher risk of all-cause mortality is associated with an age of onset of 65 years or older, the presence of coronary heart disease or cardiac arrhythmias, and higher levels of anti-DSG1 and / or anti-DSG3 antibodies (≥100 U / mL). Similarly, a correlation has been reported between increased levels of anti-DSG1 and / or anti-DSG3 autoantibodies and higher disease activity. This is consistent with the fact that titers of anti-DSG1 and / or anti-DSG3 antibodies are associated with more severe mucocutaneous PV and respond less to treatment than patients with myxoid or cutaneous PV. Improved treatments for PV / PF are greatly needed. [Overview of the project]

[0017] The present invention provides polypeptides comprising a fragment of the extracellular domain of desmoglein 1 (DSG1) or desmoglein 3 (DSG3) containing at least one mutation that increases solubility, decreases aggregation, or both, or lacking extracellular domains 3 and 4, and an effector moiety that is not an unmodified Fc domain. The present invention further provides compositions comprising a fragment of a first human receptor target of pemphigus vulgaris autoantibody, a fragment of a second human receptor target of pemphigus vulgaris autoantibody, and an effector moiety that is not an unmodified Fc domain. Also provided are methods for treating pemphigus vulgaris or pemphigus foliaceus by administering the pharmaceutical compositions of the present invention, as well as nucleic acid molecules and systems encoding the polypeptides and compositions of the present invention, methods for producing such polypeptides and compositions, and methods for determining suitability for treatment by the methods of the present invention.

[0018] According to a first embodiment, a polypeptide is provided comprising a fragment of the extracellular domain of desmoglein 1 (DSG1) or desmoglein 3 (DSG3) and at least one mutation that increases the solubility of the fragment, decreases the aggregation of the fragment, or both.

[0019] In another embodiment, a polypeptide of the present invention including an effector portion is provided. In some embodiments, the effector portion is an effector portion that is not an unmodified Fc domain.

[0020] According to some embodiments, at least one mutation is located in the cadherin domain or the calcium-binding domain.

[0021] According to some embodiments, the DSG1 extracellular domain includes Sequence ID No. 2, and the mutations are selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, R97A, C9V, C9S, C28A, C28S, C78V, C78S, C204A, C204S, C206V, C206S, C426S, E69G, D103G, D136G, E179G, D215G, D247G, E231G, E291G, E332G, N61Q, and N131Q.

[0022] According to some embodiments, the DSG1 extracellular domain contains Sequence ID No. 2, and the mutations are selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, R97A, C9V, C28A, C78V, C204A, C206V, C426S, E69G, D103G, D136G, E179G, D215G, D247G, E231G, E291G, E332G, N61Q, and N131Q.

[0023] According to some embodiments, at least one mutation is a.W2A; b.D27T; c.E89S; d.W2A, D27T, and E89S; e.W2A and A80I; f.K17A and R18A; g. R10A, K17A, R18A, and R97A; h.C9V, C28A, C78V, C204A and C206V; i.C9V, C28A, C78V, C204A, C206V and C426S; j.E69G, D103G, and D136G; k.E179G, D215G, and D247G; l.E231G, E291G, and E332G; m.N61Q and N131Q; or n.C9S, C28S, C78S, C204S, and C206S is.

[0024] According to some embodiments, at least one mutation is a.W2A; b.D27T; c.E89S; d.W2A, D27T, and E89S; e.W2A and A80I; f.K17A and R18A; g.R10A, K17A, R18A, and R97A; h.C9V, C28A, C78V, C204A, and C206V; i.C9V, C28A, C78V, C204A, C206V, and C426S; j.E69G, D103G, and D136G; k.E179G, D215G, and D247G; l.E231G, E291G, and E332G; or m.N61Q and N131Q is.

[0025] According to some embodiments, the DSG3 extracellular domain comprises SEQ ID NO: 4, and the mutations are selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, K97A, C9V, C9S, C78V, C78S, C202A, C202S, C204V, C204S, E69G, D103G, D136G, E179G, D213G, D245G, E229G, E289G, E330G, N61Q, and N131Q.

[0026] According to some embodiments, the DSG3 extracellular domain includes Sequence ID No. 4, and the mutations are selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, K97A, C9V, C78V, C202A, C204V, E69G, D103G, D136G, E179G, D213G, D245G, E229G, E289G, E330G, N61Q, and N131Q.

[0027] According to some embodiments, at least one mutation is a.W2A; b.E89S; c.W2A, D27T, and E89S; d.W2A and A80I; e.K17A and R18A; f. R10A, K17A, R18A, and K97A; g. C9V, C78V, C202A, and C204V; h.E69G, D103G and D136G; i.E179G, D213G, and D245G; j.E229G, E289G, and E330G; k.N61Q and N131Q; or l. C9S, C78S, C202S, and C204S That is the case.

[0028] According to some embodiments, at least one mutation is a.W2A; b.E89S; c.W2A, D27T, and E89S; d.W2A and A80I; e.K17A and R18A; f. R10A, K17A, R18A, and K97A; g. C9V, C78V, C202A, and C204V; h.E69G, D103G and D136G; i.E179G, D213G, and D245G; j.E229G, E289G and E330G; or k.N61Q and N131Q That is the case.

[0029] According to some embodiments, the fragment consists of extracellular domains (ECs) 1 and 2 of DSG1 or DSG3, the polypeptide lacks extracellular domains (ECs) 3 and 4 of DSG1 or DSG3, EC1 of DSG1 consists of SEQ ID NO: 114, EC2 of DSG1 consists of SEQ ID NO: 115, EC3 of DSG1 consists of SEQ ID NO: 116, EC4 of DSG1 consists of SEQ ID NO: 117, EC1 of DSG3 consists of SEQ ID NO: 118, EC2 of DSG3 consists of SEQ ID NO: 119, EC3 of DSG3 consists of SEQ ID NO: 120, and EC4 of DSG3 consists of SEQ ID NO: 121.

[0030] According to some embodiments, the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs. 74-85, 90-103, and 108-113.

[0031] According to some embodiments, the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs: 81, 82, 90, 100, and 108.

[0032] In another embodiment, a polypeptide is provided comprising a fragment of the extracellular domain of desmoglein 1 (DSG1) or desmoglein 3 (DSG3), wherein the extracellular domains 3 and 4 of DSG1 or DSG3 are lacking, and the EC3 of DSG1 is sequence number 116, the EC4 of DSG1 is sequence number 117, the EC3 of DSG3 is sequence number 120, and the EC4 of DSG3 is sequence number 121.

[0033] According to some embodiments, the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs: 87, 90-91, 105, and 108-109.

[0034] According to some embodiments, the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs: 90 and 108.

[0035] According to some embodiments, the polypeptide further comprises an effector portion.

[0036] According to some embodiments, the effector portion is not in the Fc domain.

[0037] 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).

[0038] According to some embodiments, the effector portion can induce cell death in cells bound to the fragment.

[0039] 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.

[0040] According to some embodiments, the effector portion is selected from α-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0041] According to some embodiments, the effector portion is an Fc domain containing sequence number 60 or sequence number 62, and which contains multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E within sequence number 60 or sequence number 62.

[0042] According to some embodiments, the effector portion is conjugated to a polypeptide by a linker.

[0043] According to another embodiment, a. A first polypeptide comprising a fragment or analog or derivative of the extracellular domain of desmoglein 1 (DSG1), a fragment or analog or derivative of the extracellular domain of desmoglein 3 (DSG3), or both, and a first dimerization domain; and b. A second polypeptide comprising a fragment of DSG1 or an analog or derivative thereof, a fragment of DSG3 or an analog or derivative thereof, or both, and a second dimerization domain; A composition is provided which includes a first and second dimerization domain configured to dimerize with respect to each other.

[0044] According to some embodiments, dimerization involves forming a covalent bond between a first dimerization domain and a second dimerization domain.

[0045] According to some embodiments, the protein complex includes an immunoglobulin scaffold.

[0046] According to some embodiments, a. The first dimerization domain comprises the first hinge domain of the immunoglobulin heavy chain, and the second dimerization domain comprises the second hinge domain of the heavy chain, and the first and second dimerization domains dimerize 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 both a CH1 domain, nor both contains both a CL domain.

[0047] According to some embodiments, the fragments of the first, second, or both polypeptide chains and the dimerization domain are separated by a linker.

[0048] 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.

[0049] According to some embodiments, the Fc region can induce cytotoxicity against cells that bind to the protein complex.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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 and is the mutation corresponding to the first mutation.

[0054] 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.

[0055] 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.

[0056] According to some embodiments, the Fc region includes at least one mutation that increases ADCC or CDC.

[0057] According to some embodiments, the Fc region is an Fc region comprising SEQ ID NO: 60 or SEQ ID NO: 62, and containing multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E within SEQ ID NO: 60 or SEQ ID NO: 62.

[0058] According to some embodiments, the dimerization domain of the first, second, or both polypeptide chains is on the C-terminal side of the fragment and on the N-terminal side of the Fc region.

[0059] According to some embodiments, the composition lacks an antibody-variable domain.

[0060] According to some embodiments, the composition further comprises a third polypeptide comprising a fragment of DSG1 or an analog or derivative thereof, a fragment of DSG3 or an analog or derivative thereof, or both thereof, 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.

[0061] According to some embodiments, 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 dimerize 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, such that neither the first nor the third polypeptide contains both a CH1 domain, nor both contain both a CL domain.

[0062] According to some embodiments, the composition further comprises a fourth polypeptide comprising a fragment of DSG1 or an analog or derivative thereof, a fragment of DSG3 or an analog or derivative thereof, or both thereof, 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.

[0063] According to some embodiments, 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 immunoglobulin heavy chain, and the first and second dimerization domains dimerize 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, such that neither the first nor the third polypeptide contains both a CH1 domain, nor both contain both a CL domain.

[0064] According to some embodiments, the first polypeptide and the second polypeptide do not both contain a CH1 domain, nor do both contain a CL domain.

[0065] 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 that 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.

[0066] According to some embodiments, the first polypeptide comprises a fragment of DSG1 or an analog or derivative thereof, and the second polypeptide comprises a fragment of DSG3 or an analog or derivative thereof.

[0067] According to some embodiments, the first polypeptide chain or the second polypeptide chain includes both a fragment of DSG1 or an analog or derivative thereof, and a fragment of DSG3 or an analog or derivative thereof.

[0068] According to some embodiments, the fragments are separated by an amino acid linker.

[0069] According to some embodiments, DSG1 lacks a prodomain and includes or consists of SEQ ID NO: 2, DSG3 lacks a prodomain and includes or consists of SEQ ID NO: 4, or DSG1 includes a prodomain and includes or consists of SEQ ID NO: 1, DSG3 includes a prodomain and includes or consists of SEQ ID NO: 3.

[0070] According to some embodiments, the extracellular domain fragment consists of a truncation of the extracellular domain lacking an extracellular functional domain containing a sequence selected from sequence numbers 114-121.

[0071] According to some embodiments, the fragment lacks the extracellular domains 3 and 4 of DSG1 or DSG3, the EC3 of DSG1 is sequence number 116, the EC4 of DSG1 is sequence number 117, the EC3 of DSG3 is sequence number 120, and the EC4 of DSG3 is sequence number 121.

[0072] According to some embodiments, the fragment comprises or consists of the extracellular domains (ECs) 1 and 2 of DSG1 or DSG3 or analogs or derivatives thereof, wherein EC1 of DSG1 is sequence number 114, EC2 of DSG1 is sequence number 115, EC1 of DSG3 is sequence number 118, and EC2 of DSG3 is sequence number 119.

[0073] According to some embodiments, the fragment consists of an amino acid sequence selected from SEQ ID NOs: 87, 90-91, 105, and 108-109, or a derivative thereof containing at least 85% identity.

[0074] According to some embodiments, the fragment consists of an amino acid sequence selected from SEQ ID NOs: 90 and 108 or a derivative thereof having at least 85% identity.

[0075] According to some embodiments, at least one of the fragments contains a mutation in the cadherin domain or the calcium-binding domain.

[0076] According to some embodiments, the mutation increases the solubility of the composition, decreases the aggregation of the complex, or both.

[0077] According to some embodiments, the DSG1 extracellular domain includes Sequence ID No. 2, and the mutations are W2A, D27T, E89S, A80I, K17A, R18A, R10A, R97A, C9V, C9S, C28A, C28S, C78V, C78S, C204A, C204S, C206V, C206S, C426S, E69G, D103G, D136G, E179G, D215G, D247G, E231G, E291G, E332G, N61Q and The mutation is selected from N131Q, or the DSG3 extracellular domain contains Sequence ID No. 4, and the mutation is selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, K97A, C9V, C9S, C78V, C78S, C202A, C202S, C204V, C204S, E69G, D103G, D136G, E179G, D213G, D245G, E229G, E289G, E330G, N61Q and N131Q.

[0078] According to some embodiments, the extracellular domain of DSG1 includes Sequence ID No. 2, and the mutations are W2A, D27T, E89S, A80I, K17A, R18A, R10A, R97A, C9V, C28A, C78V, C204A, C206V, C426S, E69G, D103G, D136G, E179G, D215G, D247G, E231G, E291G, E332G, N61Q and N131Q The mutation is selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, K97A, C9V, C78V, C202A, C204V, E69G, D103G, D136G, E179G, D213G, D245G, E229G, E289G, E330G, N61Q and N131Q.

[0079] According to some embodiments, at least one mutation is a. W2A of sequence number 2 or 4; b. D27T of sequence number 2; c. E89S of sequence number 2 or 4; d. W2A, D27T, and E89S of sequence number 2 or 4; e. Sequence IDs 2 or 4, W2A and A80I; f. K17A and R18A of sequence number 2 or 4; g. R10A, K17A, R18A, and R97A of Sequence ID No. 2; h. R10A, K17A, R18A, and K97A of Sequence ID No. 4; i. C9V, C28A, C78V, C204A and C206V of Sequence ID No. 2; j. C9V, C78V, C202A and C204V of Sequence ID No. 4; k. C9V, C28A, C78V, C204A, C206V, and C426S of Sequence ID No. 2; i. Sequence IDs E69G, D103G, and D136G of sequence number 2 or 4; m. Sequence ID 2, E179G, D215G, and D247G; n. Sequence ID 4, E179G, D213G, and D245G; o. Sequence ID No. 2, E231G, E291G, and E332G; p. Sequence ID No. 4, E229G, E289G, and E330G; q. N61Q and N131Q of sequence number 2 or 4; r. Sequence ID No. 2 C9S, C28S, C78S, C204S and C206S; or s. Sequence ID 4 C9S, C78S, C202S and C204S That is the case.

[0080] According to some embodiments, at least one mutation is a. W2A of sequence number 2 or 4; b. D27T of sequence number 2; c. E89S of sequence number 2 or 4; d. W2A, D27T, and E89S of sequence number 2 or 4; e. Sequence IDs 2 or 4, W2A and A80I; f. K17A and R18A of sequence number 2 or 4; g. R10A, K17A, R18A, and R97A of Sequence ID No. 2; h. R10A, K17A, R18A, and K97A of Sequence ID No. 4; i. C9V, C28A, C78V, C204A and C206V of Sequence ID No. 2; j. C9V, C78V, C202A and C204V of Sequence ID No. 4; k. C9V, C28A, C78V, C204A, C206V, and C426S of Sequence ID No. 2; i. Sequence IDs E69G, D103G, and D136G of sequence number 2 or 4; m. Sequence ID 2, E179G, D215G, and D247G; n. Sequence ID 4, E179G, D213G, and D245G; o. Sequence ID No. 2, E231G, E291G, and E332G; p. Sequence ID No. 4 E229G, E289G and E330G; or q. N61Q and N131Q with sequence numbers 2 or 4 That is the case.

[0081] According to some embodiments, the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs. 74-85, 90-103, and 108-113.

[0082] According to some embodiments, the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs: 81, 82, 90, 100, and 108.

[0083] According to some embodiments, the analog or its derivative contains at least 85% identity with DSG1 or DSG3.

[0084] According to some embodiments, the fragment comprises at least 20 consecutive amino acids derived from DSG1 or DSG3.

[0085] According to some embodiments, the fragment comprises at least one B cell receptor (BCR)-specific epitope target of the autoantibody.

[0086] According to some embodiments, the composition comprises a first or second polypeptide containing an sequence selected from Sequence IDs 5 to 73.

[0087] According to some embodiments, the hinge domain, CH2 domain, or CH3 domain contains at least one mutation that reduces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0088] According to some embodiments, at least one mutation is a. Hinge domain mutations including L19A and L20A mutations in Sequence ID No. 125; and b. Mutations in the CH2 domain, including the N59A mutation in Sequence ID No. 139 Selected from.

[0089] According to some embodiments, the composition further comprises at least one effector portion capable of inducing cell death in cells bound to the composition.

[0090] According to some embodiments, the effector portion is not in the Fc domain.

[0091] According to some embodiments, the effector portion is selected from α-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0092] In another embodiment, a pharmaceutical composition is provided comprising a polypeptide or composition of the present invention and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0093] In another embodiment, a method is provided for treating pemphigus vulgaris or pemphigus foliace in a subject requiring treatment of pemphigus vulgaris or pemphigus foliace, comprising administering a composition comprising a fragment of the extracellular domain of DSG1 or DSG3 or an analog or derivative thereof to the subject, thereby treating pemphigus vulgaris or pemphigus foliace.

[0094] According to some embodiments, DSG1 lacks a prodomain and includes or consists of SEQ ID NO: 2, DSG3 lacks a prodomain and includes or consists of SEQ ID NO: 4, or DSG1 includes a prodomain and includes or consists of SEQ ID NO: 1, DSG3 includes a prodomain and includes or consists of SEQ ID NO: 3.

[0095] According to some embodiments, the composition comprises the polypeptide of the present invention.

[0096] According to some embodiments, the composition is the composition of the present invention.

[0097] According to some embodiments, the composition is the pharmaceutical composition of the present invention.

[0098] According to some embodiments, the method further includes the step of reducing the level of circulating antibodies against DSG1, DSG3, or both, in a subject.

[0099] According to some embodiments, treatment involves reducing the concentration of circulating autoantibodies against DSG1, DSG3, or both.

[0100] According to some embodiments, the composition comprises an Fc region, and the treatment involves killing B cells that produce anti-DSG1 or anti-DSG3 autoantibodies.

[0101] According to some embodiments, the B cells are autoreactive B cells that produce autoantibodies against fragments of the composition.

[0102] In another embodiment, nucleic acid molecules encoding the polypeptide of the present invention are provided.

[0103] 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.

[0104] According to some embodiments, the nucleic acid system 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.

[0105] In another aspect, a method for producing the polypeptide or composition of the present invention is provided, comprising the step of expressing a nucleic acid system of the present invention in a cell, wherein the nucleic acid system is configured to produce a polypeptide encoded in the cell, thereby providing a method for producing the polypeptide or composition of the present invention.

[0106] In another embodiment, a method for producing a protein, A step to obtain a first fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof, and a second fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof. A step of linking a first fragment to a first dimerization domain to generate a first polypeptide chain, linking a second fragment to a second dimerization domain to generate a second polypeptide chain, so that the first and second dimerization domains can dimerize with each other, and a step of bringing the first polypeptide and the second polypeptide into contact under conditions sufficient to induce 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 i. Obtain a first fragment or analog or derivative of the extracellular domain of DSG1, or a fragment or analog or derivative of the extracellular domain of DSG3, and a second fragment or analog or derivative of the extracellular domain of DSG1, or a fragment or analog or derivative of the extracellular domain of DSG3; and ii. The first fragment can be linked to the first dimerization domain to generate a first polypeptide chain, and the second fragment can be linked to the second dimerization domain to generate a second polypeptide chain, and the first and second dimerization domains can dimerize with each other; The process generated by This includes a method for producing protein.

[0107] According to some embodiments, the protein complex is the protein complex of the composition of the present invention.

[0108] According to some embodiments, the method is a. A step of linking a third dimerization domain to a first dimerization domain or first fragment in a first polypeptide chain; obtaining a third fragment or analogue or derivative of the extracellular domain of DSG1 or a fragment or analogue or derivative of the extracellular domain of DSG3, and linking the third fragment to a fourth dimerization domain to produce a third polypeptide chain, wherein the third dimerization domain and the fourth dimerization domain can dimerize with each other; and a step of contacting the first, second and third polypeptides under conditions sufficient to induce dimerization; or b. A step of expressing a nucleic acid sequence encoding a third polypeptide chain in a host cell, wherein the third polypeptide chain is i. Obtain a third fragment of DSG1 or its analogues or derivatives, or a fragment of the extracellular domain of DSG3 or its analogues or derivatives; and ii. Linking the third fragment to the fourth dimerization domain. The process generated by The first polypeptide chain further comprises a third dimerization domain, and the third and fourth dimerization domains can dimerize with each other.

[0109] According to some embodiments, the method is a. A step of linking the sixth dimerization domain to the second dimerization domain or second fragment in the second polypeptide chain; obtaining the fourth fragment or analogue or derivative of the extracellular domain of DSG1, or the fragment or analogue or derivative of the extracellular domain of DSG3, and linking the fourth fragment to the fifth dimerization domain to produce the fourth polypeptide chain, so that the fifth dimerization domain and the sixth dimerization domain can dimerize with each other; and a step of contacting the first, second, third, and fourth polypeptides under conditions sufficient to induce dimerization; or b. A step of expressing a nucleic acid sequence encoding a fourth polypeptide chain in a host cell, wherein the fourth polypeptide chain is i. Obtain a fourth fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; and ii. Linking the fourth fragment to the fifth dimerization domain to generate the fourth polypeptide chain. The process generated by The second polypeptide chain further comprises a sixth dimerization domain, and the fifth and sixth dimerization domains can dimerize with each other.

[0110] According to some embodiments, the method further includes the steps of generating at least one mutation in the cadherin domain of a protein, or truncating the protein to remove at least one cadherin domain or a portion thereof.

[0111] According to some embodiments, the method further includes the steps of measuring the aggregation of mutant or truncated proteins and selecting mutant or truncated proteins in which aggregation has been reduced.

[0112] In another embodiment, a protein produced by the method of the present invention is provided.

[0113] In another embodiment, a method for producing a protein, a. A step to obtain a fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; b. A step of generating a mutant fragment by introducing at least one mutation into the fragment; c. A step of measuring the solubility, aggregation, or both of the mutant fragments; and d. The step of selecting at least one mutant fragment that has increased solubility, decreased aggregation, or both, compared to the obtained fragment. This includes a method for producing protein.

[0114] According to some embodiments, the method further comprises the step of 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.

[0115] According to some embodiments, the effector portion is not in the Fc domain.

[0116] According to some embodiments, the effector portion is selected from α-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

[0117] According to some embodiments, the effector portion is an Fc domain containing at least one mutation that increases ADCC or CDC.

[0118] According to some embodiments, the effector portion is an Fc domain containing sequence number 60 or sequence number 62, and which contains multiple mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E within sequence number 60 or sequence number 62.

[0119] In another embodiment, a protein produced by the method of the present invention is provided.

[0120] 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 the steps of obtaining a sample from the object, contacting the sample with the protein or 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 a method for determining the suitability of the object to be treated.

[0121] 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]

[0122] [Figure 1A] Figure 1A shows five possible embodiments of the quadruple-chain therapeutic agent of the present invention. Figure 1A shows a general embodiment of the molecule for treating DSG1 / DSG3-positive PV / PF. [Figure 1B] Figure 1B shows five possible embodiments of the four-stranded therapeutic agent of the present invention. Figure 1B shows an embodiment in which each of the four strands contains a different protein fragment. [Figure 1C] Figure 1C shows five possible embodiments of the quadruple-chain therapeutic agent of the present invention. Figure 1C shows an embodiment in which all four protein fragments are identical. [Figure 1D] Figure 1D shows five possible embodiments of the four-chain therapeutic agent of the present invention. Figure 1D shows an embodiment in which two heavy chains are identical and two light chains are identical. [Figure 1E] Figure 1E shows five possible embodiments of the four-chain therapeutic agent of the present invention. Figure 1E shows an embodiment in which two heavy chains are different and two light chains are identical. [Figure 1F] This figure shows five possible embodiments of the quadruple-chain therapeutic agent of the present invention. Figure 1F shows an embodiment in which two heavy chains contain the same protein fragment and two light chains contain different protein fragments.

[0123] [Figure 2A] Figure 2A shows possible embodiments of the double-chain therapeutic agent of the present invention. Figure 2A shows a general embodiment of a molecule having two heavy chains for treating DSG1 / DSG3-positive PV / PF. [Figure 2B] Figure 2B shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2B shows an embodiment in which at least one of the CH1, CH2, or CH3 domains is excluded. [Figure 2C] This figure shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2C shows an embodiment in which the two protein fragments are identical. [Figure 2D] This figure shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2D shows two protein fragments in different embodiments. [Figure 2E] Figure 2E shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2E shows an embodiment in which the two protein fragments are different and the molecule does not contain the CH1 domain. [Figure 2F] This figure shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2F shows an embodiment in which the two protein fragments are different and the molecule does not contain a CH1 domain or a hinge domain. [Figure 2G]This figure shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2G shows a typical embodiment in which two tandem fragments are contained in each heavy chain and linked via a linker. [Figure 2H] Figure 2H shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2H shows a tandem fragment configuration in which all subunits are identical. [Figure 2I] Figure 2I shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2I shows a tandem fragment configuration in which all subunits are identical without the CH1 domain. [Figure 2J] This figure shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2J shows a tandem fragment configuration in which each heavy chain contains two identical different fragments. [Figure 2K] Figure 2K shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2K shows a tandem fragment configuration in which each heavy chain contains two identical different fragments without the CH1 domain. [Figure 2L] Figure 2L shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2L shows a tandem fragment configuration in which the two heavy chains are different fragments that are not the same. [Figure 2M] This figure shows possible embodiments of the double-stranded therapeutic agent of the present invention. Figure 2M shows a tandem fragment configuration in which two heavy chains contain different fragments that are not identical and lack a CH3 domain. [Figure 2N] This figure shows possible embodiments of the double-chain therapeutic agent of the present invention. Figure 2N shows a typical embodiment of a molecule having one heavy chain and one light chain.

[0124] [Figure 3A] Figure 3A shows four possible embodiments of the triple-chain therapeutic agent of the present invention. Figure 3A shows a general embodiment of a molecule having two heavy chains and one light chain. [Figure 3B] Figure 3B shows four possible embodiments of the triple-stranded therapeutic agent of the present invention. Figure 3B shows an embodiment in which the three protein fragments are identical. [Figure 3C] Figure 3C shows four possible embodiments of the triple-stranded therapeutic agent of the present invention. Each protein fragment is shown in a different embodiment. [Figure 3D] Figure 3D shows four possible embodiments of the triple-stranded therapeutic agent of the present invention. Figure 3D shows an embodiment in which two of the protein fragments are the same and the third is different.

[0125] [Figure 4] This figure illustrates an embodiment of the quadruple-chain therapeutic agent for treating DSG1 / DSG3-positive PV / PF according to the present invention, similar to that shown in Figure 1, but comprising four different immunoglobulin scaffolds in which each of the four chains promotes the formation of different protein fragments and quadruple-chain molecules.

[0126] [Figure 5A] This figure shows a general embodiment of the four-stranded therapeutic agent of the present invention. Figure 5A shows a general embodiment of four strands in which two strands contain two dimerization domains and two strands contain a single dimerization domain. [Figure 5B] Figure 5B shows a general embodiment of the quadruple-stranded therapeutic agent of the present invention. Figure 5B shows an embodiment having an arbitrary linker for separating various domains and fragments.

[0127] [Figure 6A] This is a diagram of the single-stranded therapeutic agent of the present invention. Figure 6A shows an embodiment of a single-stranded molecule containing fragments derived from two different DSG1 / 3 fragments. [Figure 6B] This is a diagram of the single-strand therapeutic agent of the present invention. Figure 6B shows a fragment embodiment including truncation of DSG1 or DSG3. [Figure 6C] This is a diagram of the single-chain therapeutic agent of the present invention. Figure 6C shows an embodiment of a single-chain molecule containing three different fragments. [Figure 6D] This is a diagram of the single-chain therapeutic agent of the present invention. Figure 6D shows an embodiment of a single-chain molecule containing four different fragments. [Figure 6E] This is a diagram of the single-chain therapeutic agent of the present invention. Figure 6E shows an embodiment of a single-chain molecule containing fragments and heavy chain constant regions derived from two different DSG proteins / domains. [Figure 6F]This is a diagram of the single-chain therapeutic agent of the present invention. Figure 6F shows an embodiment of a single-chain molecule containing fragments derived from two different DSG proteins / domains and a CH3-CH2 fragment of the heavy chain constant region. [Figure 6G] This is a diagram of the single-chain therapeutic agent of the present invention. Figure 6G shows the single-chain molecule in Figures 6A and 6C-6F, which has amino acid (AA) linkers that separate various domains.

[0128] [Figure 7A] Figure 7A shows SDS-PAGE gel (left) and Western blot (right) images of molecules 290 and 292, both under reducing conditions. [Figure 7B] Figure 7B is a photograph of an SDS-PAGE gel under reducing conditions showing molecules 291, 293, 294, 295, and 296. [Figure 7C] Figure 7C is a line graph showing the depletion rate of anti-DSG3 IgG in the serum of four different human PV patients in the presence of gradually increasing concentrations of the DSG3-containing molecule of the present invention. [Figure 7D] Figure 7D is a line graph showing the mean serum values ​​of four human PV patients from Figure 7C. [Figure 7E] Figure 7E is a bar graph showing the mean depletion rate of anti-DSG3 IgG in the serum of six different human PV patients in the presence of 3.85 μM of the molecule of the present invention. [Figure 7F] Figure 7F is a bar graph showing the mean depletion rate of anti-DSG1 IgG in the serum of one human pemphigus patient in the presence of 0.77 μM of the molecule of the present invention.

[0129] [Figure 8A] Figure 8A is a photograph of an SDS-PAGE gel showing molecules CRD-291, CRD-657, and CRD-662 under non-reducing conditions. [Figure 8B] Figure 8B is a bar graph showing the monomer purity percentages determined by HPLC for molecules CRD-291, CRD-657, and CRD-700. [Figure 8C]Figure 8C is a photograph of an SDS-PAGE gel under non-reducing conditions showing molecules CRD-293, CRD-674, and CRD-679. [Figure 8D] Figure 8D is a bar graph showing the monomer purity percentages determined by HPLC for molecules CRD-293, CRD-674, and CRD-701. [Figure 8E] Figure 8E is a photograph of an SDS-PAGE gel under non-reducing conditions showing molecules CRD-294 and CRD-685.

[0130] [Figure 9A] Figure 9A is a bar graph showing the mean depletion rate of anti-DSG1 IgG in the presence of 1.54 μM of the molecule of the present invention, which contains the complete extracellular domain of DSG1. [Figure 9B] Figure 9B is a bar graph showing the mean depletion rate of anti-DSG3 IgG in the presence of 1.54 μM of the present invention molecule containing the complete extracellular domain of DSG3. [Figure 9C] Figures 9C–E are bar graphs showing the mean depletion rates of anti-DSG3 IgG in the presence of 3.85 μM of the molecule of the present invention, which contains truncation of the extracellular domain of DSG3. Figure 9C shows the mean depletion rates of all tested molecules except CRD-291 in eight different serum samples. CRD-291 was tested in only one sample, and a direct comparison of CRD-291 in that serum sample with CRD-657 in the same serum sample is provided in Figure 9D. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Figure 9F is a bar graph showing the mean depletion rate of anti-DSG1 IgG in the presence of 3.85 μM of the molecule of the present invention, which contains truncation of the extracellular domain of DSG1.

[0131] [Figure 10A]Figure 10A is a bar graph showing the MFI ratio of specific staining versus background staining of various molecules of the present invention for two different hybridomas. 5H10 is a DSG3-specific hybridoma, and g-66 is a hybridoma specific to an unrelated protein. [Figure 10B] Figure 10B is a bar graph showing the relative binding of various molecules of the present invention to the hybridoma 5H10. The binding is standardized compared to the binding of CRD-924. [Figure 10C] Figure 10C is a line graph of the binding of CRD-691 to DSG3-specific B cell hybridoma 5H10 and non-specific hybridoma a-18-C5-F6. Hybridomas were incubated in the presence of 32 nM, 10.6 nM, or 3.5 nM CRD-691. The Y-axis represents the MFI value of each sample divided by the BCR MFI value of each hybridoma. To assess BCR expression, 5H10 hybridomas were stained with PE-anti-mouse BCR, and a-18-C5-F6 hybridomas were stained with anti-rat BCR.

[0132] [Figure 11A] Figure 11A is a bar graph showing the mean depletion rate of anti-DSG3 IgG in the presence of 3.85 μM or 0.769 μM unconjugated CRD-924 or tesirin-conjugated CRD-924. [Figure 11B] Figure 11B is a bar graph showing 5H10 hybridoma cell binding with various concentrations of unconjugated or tesirin-conjugated CRD-924. The Y-axis represents the MFI value of each sample divided by the BCR MFI value. [Figure 11C] Figure 11C is a line graph showing the percentage increase in cell death induced by CRD-924-tecillin compared to the non-conjugate molecule. Death induced by CRD-924-tecillin was specific to DSG-3 hybridomas, while tecillin background death in unrelated hybridomas (co-cultured with the target hybridomas) was minimal and dose-independent. [Modes for carrying out the invention]

[0133] The present invention provides compositions comprising, in some embodiments, a fragment or analog or derivative of a first human receptor target of pemphigus vulgaris (PV) and pemphigus foliaceus (PF) autoantibodies, and a fragment or analog or derivative of a second human protein receptor target of pemphigus vulgaris and pemphigus foliaceus autoantibodies. 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 or analog or derivative of a first human protein target of pemphigus vulgaris and pemphigus foliaceus autoantibodies and a first dimerization domain, and the second chain comprises a fragment or analog or derivative of a second human protein target of pemphigus vulgaris and pemphigus foliaceus autoantibodies and a second dimerization domain that can dimerize with the first dimerization domain. Similar to protein complexes in which one of the chains contains an effector moiety that is not an unmodified Fc domain, the invention also provides polypeptides containing fragments of the extracellular domain of desmoglein 1 (DSG1) or desmoglein 3 (DSG3) that contain at least one mutation that increases solubility, decreases aggregation, or both, or that lack extracellular domains 3 and 4. Similar to polypeptides containing an effector moiety that is not an unmodified Fc domain, the invention further relates to pharmaceutical compositions comprising compositions and / or protein complexes, nucleic acids encoding polypeptides of compositions and / or protein complexes, methods for therapeutic use of compositions and / or protein complexes and methods for determining suitability for therapeutic use, and methods for producing compositions and / or protein complexes.

[0134] In a first aspect, a composition is provided comprising a fragment of a first protein target of a PV or PF autoantibody, or an analog or derivative thereof.

[0135] In another embodiment, a composition is provided comprising a fragment of a first protein target of a PV or PF autoantibody, and a fragment of a second protein target of a PV or PF autoantibody, or an analogue or derivative thereof.

[0136] In another embodiment, a protein is provided comprising a fragment or analog or derivative of a first protein target of a PV or PF autoantibody.

[0137] In another embodiment, a protein is provided comprising a fragment or analog or derivative of a first protein target of a PV or PF autoantibody, and a fragment or analog or derivative of a second protein target of a PV or PF autoantibody.

[0138] In another embodiment, a protein complex comprising at least two polypeptide chains is provided, wherein the first polypeptide chain comprises a fragment or analog or derivative of a first protein target of a PV or PF autoantibody and a first dimerization domain, and the second polypeptide chain comprises a fragment or analog or derivative of a second protein target of a PV or PF autoantibody and a second dimerization domain.

[0139] In some embodiments, the composition comprises a protein complex comprising at least two polypeptide chains, wherein the first polypeptide chain comprises a fragment or analog or derivative of a first protein target of a PV or PF autoantibody and a first dimerization domain, and the second polypeptide chain comprises a fragment or analog or derivative of a second protein target of a PV or PF autoantibody 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.

[0140] 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 peptoids and semipeptoids or any combination thereof, which are natural peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications), and peptide analogs. In other embodiments, the peptides, polypeptides, and proteins described have modifications that make them more stable in the body or more permeable to cells. In one embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0141] 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 manufacture of pharmaceuticals. In some embodiments, the protein complex is for use in the manufacture of pharmaceuticals. In some embodiments, the composition is for use in the treatment of PV. In some embodiments, the composition is for use in the treatment of PF. In some embodiments, the protein complex is for use in the treatment of PV. In some embodiments, the protein complex is intended for use in the treatment of PF. In some embodiments, the protein complex is intended for use in the diagnosis of PV. In some embodiments, the protein complex is intended for use in the diagnosis of PF. In some embodiments, the protein complex is intended for use in determining appropriate treatment for PV. In some embodiments, the protein complex is intended for use in determining appropriate treatment for PF. In some embodiments, the protein complex is intended for use in characterizing the serological response in PV.In some embodiments, the protein complex is used to characterize the serological response in PF. In some embodiments, the protein complex is used to determine the autoantibody titer in PV. In some embodiments, the protein complex is used to determine the autoantibody titer in PF.

[0142] 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.

[0143] 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. Production of recombinant proteins by cell expression is well known in the art, and the polypeptides of the present invention can be produced using any method of recombinant protein expression. Cell-free expression systems for recombinant protein production may also be used.

[0144] 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.

[0145] Expressing DNA sequences or RNA within cells is well known to those skilled in the art. This can be done by many methods, including transfection, viral infection, or direct modification of the cell's genome. In some embodiments, the DNA sequence is contained in 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 expression.

[0146] 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 two polypeptide chains or consists of two polypeptide chains. In some embodiments, the protein complex comprises three polypeptide chains or consists of three polypeptide chains. In some embodiments, the protein complex comprises four polypeptide chains or consists of four polypeptide 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.

[0147] 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 in 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 a PV or PF-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 a PV or PF autoantibody. In some embodiments, the protein is selected from desmoglein 1 (DSG1) and desmoglein 3 (DSG3). In some embodiments, the protein is DSG1. In some embodiments, the protein is DSG3.

[0148] 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 signals 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.

[0149] 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.

[0150] In some embodiments, the fragment contains at least 5 amino acids of the protein. In some embodiments, the fragment contains at least 10 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 consecutive 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.

[0151] The extracellular regions of DSG1 and DSG3 each contain four extracellular domains (ECs). These are referred to as EC1, EC2, EC3, and EC4, from the N-terminus to the C-terminus of the extracellular region. In some embodiments, EC1 of DSG1 contains or consists of EWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFS (SEQ ID NO: 114). In some embodiments, EC2 of DSG1 contains or consists of MATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYME (SEQ ID NO: 115). In some embodiments, EC3 of DSG1 includes or consists of QSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVF (Sequence ID 116). In some embodiments, EC4 of DSG1 includes or consists of RPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPN (Sequence ID 117). In some embodiments, EC1 of DSG3 includes or consists of EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFS (Sequence ID 118).In some embodiments, EC2 of DSG3 includes or consists of QQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFR (SEQ ID NO: 119). In some embodiments, EC3 of DSG3 includes or consists of DSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAF (SEQ ID NO: 120). In some embodiments, EC4 of DSG3 includes or consists of RPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEK (SEQ ID NO: 121).

[0152] In some embodiments, the fragment includes EC1 of DSG1. In some embodiments, the fragment includes EC2 of DSG1. In some embodiments, the fragment includes EC3 of DSG1. In some embodiments, the fragment includes EC4 of DSG1. In some embodiments, the fragment includes EC1 of DSG3. In some embodiments, the fragment includes EC2 of DSG3. In some embodiments, the fragment includes EC3 of DSG3. In some embodiments, the fragment includes EC4 of DSG3. In some embodiments, the fragment includes or consists of EC1 and EC2 of DSG1. In some embodiments, the fragment includes or consists of EC1 and EC2 of DSG3. In some embodiments, the fragment lacks EC3 of DSG1. In some embodiments, the fragment lacks EC3 of DSG3. In some embodiments, the fragment lacks EC4 of DSG1. In some embodiments, the fragment lacks EC4 of DSG3. In some embodiments, the fragment lacks both EC3 and EC4 of DSG1. In some embodiments, the fragment lacks EC3 and EC4 of DSG3. In some embodiments, the fragment includes an amino acid sequence selected from SEQ ID NOs. 87, 90-91, 105, and 108-109. In some embodiments, the fragment consists of an amino acid sequence selected from SEQ ID NOs. 87, 90-91, 105, and 108-109. In some embodiments, the fragment is derived from DSG1 and includes or consists of a sequence selected from SEQ ID NOs. 87 and 90-91. In some embodiments, the fragment is derived from DSG3 and includes or consists of a sequence selected from SEQ ID NOs. 105 and 108-109. It will be understood that each sequence represents a distinct embodiment of the present invention.

[0153] In some embodiments, the chain contains at least one fragment. In some embodiments, the chain contains 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, the linker contains increased solubility compared to the protein region excluded from the chain. In some embodiments, the protein region is replaced by a non-protein protein region. In some embodiments, the substituted region contains increased solubility compared to the substituted protein region. In some embodiments, the substituted region contains increased protein stability compared to the substituted protein region.

[0154] In some embodiments, a protein is the target of the antibody. As used herein, the term “antibody” includes all classes of IgA, IgD, IgE, IgG, and IgM, and 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.

[0155] 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 its own antigen. The antigen is also known as an autoantigen. In some embodiments, the autoantibody is associated with PV or PF. In some embodiments, the autoantibody characterizes PV or PF. In some embodiments, the autoantibody is an autoantibody of PV or PF. 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 contains at least one epitope. In some embodiments, the epitope contains at least 5 amino acids. In some embodiments, the epitope contains 5 to 6 amino acids. In some embodiments, the epitope contains 5 to 10 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 contains 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.

[0156] 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 with 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, e.g., isoleucine, valine, leucine, or methionine; substitution of a polar (hydrophilic) residue, e.g., between arginine and lysine, glutamine and asparagine, or glycine and serine; substitution of a basic residue, e.g., lysine, arginine, or histidine; or substitution of an acidic residue, e.g., between aspartic acid or glutamic acid. Each possibility represents a distinct embodiment of the 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 be similar to 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 of the canonical sequence of the protein.

[0157] 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 PV or PF autoantibodies. In some embodiments, the analog can still capture PV or PF autoantibodies. In some embodiments, the analog can still treat PV or PF. 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 mutations of the canonical sequence.

[0158] 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 may have no substituted or removed amino acids (analogs), and rather may have further 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.

[0159] 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 PV or PF autoantibodies. In some embodiments, the derivative can still capture PV or PF autoantibodies. In some embodiments, the derivative can still treat PV or PF. In some embodiments, the derivative is a mutant protein or fragment.

[0160] 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 recognized as a canonical sequence can be used. As a non-limiting example, human DSG1 is encoded by the DSG1 gene, its canonical nucleic acid sequence can be found in Entrez gene 1828, its canonical protein-coding mRNA sequence can be found in NM_001942, and its canonical amino acid sequence can be found in NP_001933 and UniProt number Q02413. Similarly, human DSG3 is encoded by the DSG3 gene, its canonical nucleic acid sequence can be found in Entrez gene 1830, its canonical protein-coding mRNA sequence can be found in NM_001944, and its canonical amino acid sequence can be found in NP_001935 and UniProt number P32926. 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.

[0161] In some embodiments, the canonical amino acid sequence of the extracellular domain of DSG1 is EFRIQVRDYNTKNGTIKWHSIRRQKREWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFSMATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMNNFLDREQYGQYALAVRGSDRDGGADGMSA It comprises or consists of ECECNIKILDVNDNIPYMEQSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVFRPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPN (SEQ ID NO: 1). In some embodiments, DSG1 lacks its prodomain. In some embodiments, the prodomain is the N-terminal prodomain. In some embodiments, the prodomain consists of amino acids 1-26 of SEQ ID NO: 1.In some embodiments, the canonical amino acid sequence of DSG1 lacks a prodomain, and EWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFSMATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVND NIPYMEQSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVFRPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPN (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 DSG1 signal peptide comprises or consists of MDWSFFRVVAMLFIFLVVVEVNS (SEQ ID NO: 158).

[0162] In some embodiments, the canonical amino acid sequence of the extracellular domain of DSG3 is ELRIETKGQYDEEEMTMQQAKRRQKREWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCE CNIKVKDVNDNFPMFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEK (SEQ ID NO: 3) is included or consists of the above. In some embodiments, DSG3 lacks its prodomain. In some embodiments, the prodomain is the N-terminal prodomain. In some embodiments, the prodomain consists of amino acids 1-26 of SEQ ID NO: 3.In some embodiments, the canonical amino acid sequence of DSG3 lacks a prodomain, and EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFP MFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEK (SEQ ID NO: 4) is included or consists of the DSG3 signal peptide. In some embodiments, the extracellular domain lacks a signal peptide. In some embodiments, the extracellular domain further includes a signal peptide. In some embodiments, the DSG3 signal peptide includes or consists of MMGLFPRTTGALAIFVVVILVHG (SEQ ID NO: 159). In some embodiments, DSG1 or DSG3 signal peptides are used.

[0163] 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 κ light chain. In some embodiments, the signal peptide is of the λ light chain. In some embodiments, the heavy chain signal peptide includes MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 122). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 122. In some embodiments, the light chain signal peptide includes MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 123). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 123. In some embodiments, the signal peptide includes or consists of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 160). In some embodiments, the signal peptide includes or consists of MGWSCIILFLVATATGVHS (SEQ ID NO: 161).

[0164] 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.

[0165] In some embodiments, the protein or fragment contains a mutation that reduces aggregation. In some embodiments, aggregation is the aggregation of a protein. In some embodiments, aggregation is the aggregation of a fragment. In some embodiments, aggregation is the aggregation of an extracellular domain. In some embodiments, aggregation is the aggregation of a complex. In some embodiments, aggregation is the aggregation of a polypeptide. In some embodiments, reducing aggregation includes increasing solubility. In some embodiments, reducing aggregation includes increasing stability. In some embodiments, the protein or fragment contains a mutation that increases solubility. In some embodiments, the protein or fragment contains a mutation that increases the stability of the protein or fragment. In some embodiments, the protein is a polypeptide. In some embodiments, the mutation is an insertion. In some embodiments, the protein is a surface protein and contains a mutation that increases solubility. In some embodiments, the fragment is the extracellular domain of a surface protein and contains an insertion that increases solubility.

[0166] In some embodiments, the mutation is a point mutation. In some embodiments, the mutation is selected from mutations in at least one amino acid selected from W28, D53, E115, A106, K43, R44, R36, R123, C35, C54, C104, C230, C232, C452, E95, D129, D162, E205, D241, D273, E257, E317, E358, N87 and N157 in SEQ ID NO: 1. In some embodiments, the mutation is selected from mutations in at least one amino acid selected from W2, D27, E89, A80, K17, R18, R10, R97, C9, C28, C78, ​​C204, C206, C426, E69, D103, D136, E179, D215, D247, E231, E291, E332, N61, and N131 in SEQ ID NO: 2. In some embodiments, the mutation is selected from at least one amino acid mutation selected from W28, D53, E115, A106, K43, R44, R36, K123, C35, C104, C228, C230, E95, D129, D162, E205, D239, D271, E255, E315, E356, N87, and N157 in SEQ ID NO: 3. In some embodiments, the mutation is selected from at least one amino acid mutation selected from W2, D27, E89, A80, K17, R18, R10, K97, C9, C78, ​​C202, C204, E69, D103, D136, E179, D213, D245, E229, E289, E330, N61, and N131 in SEQ ID NO: 4. In some embodiments, the mutation is W28A or W2A. In some embodiments, the mutation is D53T or D27T. In some embodiments, the mutation is E115S or E89S. In some embodiments, the mutation is A106I or A80I. In some embodiments, the mutation is K43A or K17A. In some embodiments, the mutation is R44A or R18A. In some embodiments, the mutation is K123A or K97A. In some embodiments, the mutation is R123A or R97A. In some embodiments, the mutation is C35V or C9V. In some embodiments, the mutation is C54A or C28A.In some embodiments, the mutation is C104V or C78V. In some embodiments, the mutation is C228A, C230A, C202A or C204A. In some embodiments, the mutation is C230V, C232V, C204V or C206V. In some embodiments, the mutation is E95G or E69G. In some embodiments, the mutation is D129G or D103G. In some embodiments, the mutation is D162G or D136G. In some embodiments, the mutation is E205G or E179G. In some embodiments, the mutation is D239G, D241G, D213G or D215G. In some embodiments, the mutation is D271G, D273G, D245G or D247G. In some embodiments, the mutation is E255G, E257G, E229G, or E231G. In some embodiments, the mutation is E315G, E317G, E289G, or E291G. In some embodiments, the mutation is E356G, E358G, E330G, or E332G. In some embodiments, the mutation is a mutation to alanine. In some embodiments, the mutation is a mutation to glycine.

[0167] In some embodiments, the DSG1 extracellular domain contains Sequence ID No. 2, and the mutation is selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, R97A, C9V, C9S, C28A, C28S, C78V, C78S, C204A, C204S, C206V, C206S, C426S, E69G, D103G, D136G, E179G, D215G, D247G, E231G, E291G, E332G, N61Q, and N131Q. In some embodiments, at least one mutation is W2A. In some embodiments, at least one mutation is D27T. In some embodiments, at least one mutation is E89S. In some embodiments, at least one mutation is W2A, D27T, and E89S. In some embodiments, at least one mutation is W2A and A80I. In some embodiments, at least one mutation is K17A and R18A. In some embodiments, at least one mutation is R10A, K17A, R18A, and R97A. In some embodiments, at least one mutation is C9V, C28A, C78V, C204A, C206V, and C426S. In some embodiments, at least one mutation is C9V, C28A, C78V, C204A, and C206V. In some embodiments, at least one mutation is E69G, D103G, and D136G. In some embodiments, at least one mutation is E179G, D215G, and D247G. In some embodiments, at least one mutation is E231G, E291G, and E332G. In some embodiments, at least one mutation is N61Q and N131Q. In some embodiments, at least one mutation is C9S, C28S, C78S, C204S, and C206S. If Sequence ID No. 1 containing the prodomain is used, it will be understood that each of these amino acid positions is increased by 26.

[0168] In some embodiments, the DSG3 extracellular domain includes SEQ ID NO: 4, and the mutations are selected from W2A, D27T, E89S, A80I, K17A, R18A, R10A, K97A, C9V, C9S, C78V, C78S, C202A, C202S, C204V, C204S, E69G, D103G, D136G, E179G, D213G, D245G, E229G, E289G, E330G, N61Q, and N131Q. In some embodiments, at least one mutation is W2A. In some embodiments, at least one mutation is D27T. In some embodiments, at least one mutation is E89S. In some embodiments, at least one mutation is W2A, D27T, and E89S. In some embodiments, at least one mutation is W2A and A80I. In some embodiments, at least one mutation is K17A and R18A. In some embodiments, at least one mutation is R10A, K17A, R18A and K97A. In some embodiments, at least one mutation is C9V, C78V, C202A and C204V. In some embodiments, at least one mutation is E69G, D103G and D136G. In some embodiments, at least one mutation is E179G, D213G and D245G. In some embodiments, at least one mutation is E229G, E289G and E330G. In some embodiments, at least one mutation is N61Q and N131Q. In some embodiments, at least one mutation is C9S, C78S, C202S and C204S. If Sequence ID No. 3, which contains the prodomain, is used, it will be understood that each of these amino acid positions increases by 26.

[0169] In some embodiments, the fragment comprises an amino acid sequence selected from SEQ ID NOs. 74-85, 90-103, and 108-113. In some embodiments, the fragment consists of an amino acid sequence selected from SEQ ID NOs. 74-85, 90-103, and 108-113. In some embodiments, the fragment comprises SEQ ID NOs. 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 102, 103, 108, 109, 110, 111, 112, or 113. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragments consist of sequence numbers 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 102, 103, 108, 109, 110, 111, 112, or 113. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the fragments include sequence number 81. In some embodiments, the fragments consist of sequence number 81. In some embodiments, the fragments include sequence number 82. In some embodiments, the fragments consist of sequence number 82. In some embodiments, the fragments include sequence number 90. In some embodiments, the fragments consist of sequence number 90. In some embodiments, the fragments include sequence number 100. In some embodiments, the fragments consist of sequence number 100. In some embodiments, the fragments include sequence number 108. In some embodiments, the fragments consist of sequence number 108. In some embodiments, the extracellular domain is derived from DSG1, and the fragment includes or consists of one of SEQ ID NOs. 74-85 and 90-93. In some embodiments, the extracellular domain is derived from DSG3, and the fragment includes or consists of one of SEQ ID NOs. 94-103 and 108-113. In some embodiments, the extracellular domain is derived from DSG1, and the fragment includes or consists of SEQ ID NOs. 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 91, 92, or 93. Each possibility represents a distinct embodiment of the present invention.In some embodiments, the extracellular domain is derived from DSG3, and the fragment includes or consists of SEQ ID NOs: 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 108, 109, 110, 111, 112, or 113. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the extracellular domain is derived from DSG1, and the fragment includes or consists of SEQ ID NOs: 81. In some embodiments, the extracellular domain is derived from DSG1, and the fragment includes or consists of SEQ ID NOs: 82. In some embodiments, the extracellular domain is derived from DSG1, and the fragment includes or consists of SEQ ID NOs: 90. In some embodiments, the extracellular domain is derived from DSG3, and the fragment includes or consists of SEQ ID NOs: 100. In some embodiments, the extracellular domain is derived from DSG3, and the fragment includes or consists of SEQ ID NOs: 108.

[0170] In some embodiments, the mutation is multiple mutations. In some embodiments, the multiple mutations consist of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the multiple is 2. In some embodiments, the multiple is 4. In some embodiments, the multiple is 5. In some embodiments, the multiple mutations are selected from the set of mutations shown in Table 3. In some embodiments, the multiple mutations are selected from the set of mutations shown in Table 4. In some embodiments, the multiple mutations are selected from the set of mutations shown in Table 5. In some embodiments, the multiple mutations include the mutations C35, C104, C228, and C230. In some embodiments, the mutation is in SEQ ID NO: 3. In some embodiments, the mutation is in SEQ ID NO: 1. In some embodiments, the multiple mutations include the mutations C9, C78, ​​C202, and C204. In some embodiments, the mutation is in SEQ ID NO: 4. In some embodiments, the mutation is in SEQ ID NO: 2. In some embodiments, the multiple mutations include mutations at C35, C54, C104, C230, and C232. In some embodiments, the mutation is in SEQ ID NO: 1. In some embodiments, the mutation is in SEQ ID NO: 3. In some embodiments, the multiple mutations include mutations at C9, C28, C78, ​​C204, and C206 in SEQ ID NO: 2. In some embodiments, the multiple mutations include mutations at C9, C28, C78, ​​C204, and C206 in SEQ ID NO: 4. In some embodiments, C is mutated to V. In some embodiments, C is mutated to A. In some embodiments, the derivative is a derivative of the mutant fragment. In some embodiments, the derivative contains mutations and has at least 85% sequence identity with the mutant fragment.

[0171] In some embodiments, the fragment includes an extracellular functional domain. In some embodiments, the functional domain is a cadherin domain. In some embodiments, the functional domain is a calcium-binding domain. In some embodiments, the mutation is in the extracellular functional domain. In some embodiments, the mutation is in the cadherin domain. In some embodiments, the mutation alters binding to cadherin. In some embodiments, the mutation decreases binding to cadherin. In some embodiments, altering means decreasing. In some embodiments, the mutation is in the calcium-binding domain. In some embodiments, the mutation decreases binding to calcium. In some embodiments, decreasing means removing. In some embodiments, the decrease is at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 92, 95, 97, 99, or 100%. Each possibility represents a distinct embodiment of the invention. In some embodiments, the extracellular functional domain includes four cadherin domains. In some embodiments, the cadherin domains are EC1 to EC4.

[0172] Since the protein complex of the present invention is intended to bind to antibodies and B cells, it will be understood by those skilled in the art that it is advantageous that it does not bind to itself or to other copies of the therapeutic molecule. Therefore, mutations and truncations that reduce aggregation but do not interfere with autoantibody binding are advantageous. In some embodiments, the fragment comprises 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. In some embodiments, the truncation lacks EC1. In some embodiments, the truncation lacks EC2. In some embodiments, the truncation lacks EC3. In some embodiments, the truncation lacks EC4. In some embodiments, the truncation lacks EC1 and EC2. In some embodiments, the truncation lacks EC2 and EC3. In some embodiments, the truncation lacks EC3 and EC4. In some embodiments, the functional domain is selected from SEQ ID NOs: 114-117. In some embodiments, the functional domain is selected from SEQ ID NOs: 118-121.

[0173] In some embodiments, the derivative is a derivative of the truncation. In some embodiments, the derivative has at least 85% identity with the truncation and does not further contain a stretch of homologous / identical amino acids to the sequence derived from DSG1 or DSG3. Thus, it will be understood that a sequence having sequence identity with the truncation is not an untruncated sequence. In some embodiments, the truncation contains at least one mutation. In some embodiments, the derivative has at least 85% identity with one of sequence numbers 74-85, 90-103 and 108-113 and contains one or more mutations. In some embodiments, the derivative has at least 85% identity with one of sequence numbers 87, 90-91, 105 and 108-109 and lacks EC3 and EC4.

[0174] Dimerization domain In some embodiments, dimerizing domains can dimerize with each other. In some embodiments, a first dimerizing domain can dimerize with a second dimerizing domain. In some embodiments, the first and second dimerizing domains can dimerize with each other. In some embodiments, being able to dimerize means being configured to dimerize. In some embodiments, dimerization occurs under physiological conditions. In some embodiments, dimerization occurs in body fluids. In some embodiments, body fluids are blood. In some embodiments, body fluids are plasma. In some embodiments, body fluids are serum. In some embodiments, dimerization occurs within a subject. In some embodiments, dimerization occurs in vivo. In some embodiments, dimerization occurs in vitro.

[0175] 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 each other. 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 just 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 α / β. Furthermore, the upper hinge domain can be manipulated by cysteine ​​substitution / mutation to serine to prevent dimerization. In some embodiments, the dimerizing domain contains or consists of the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 124).

[0176] 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.

[0177] In some embodiments, the hinge domain includes the amino acid sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 125). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 125. In some embodiments, the IgG1 hinge includes or consists of SEQ ID NO: 125. In some embodiments, the hinge domain includes the amino acid sequence EPKCCVECPPCPAPPAAAP (SEQ ID NO: 126). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 126. In some embodiments, the IgG2 hinge includes or consists of SEQ ID NO: 126. In some embodiments, the hinge domain includes the amino acid sequence ESKYGPPCPPCPAPEFLGGP (SEQ ID NO: 127). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 127. In some embodiments, the IgG4 hinge includes or consists of SEQ ID NO: 127. In some embodiments, the hinge domain contains the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGP (SEQ ID NO: 128). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 128. In some embodiments, the IgG3 hinge contains or consists of SEQ ID NO: 128. In some embodiments, the hinge domain contains the CPXCP (SEQ ID NO: 129) motif. In some embodiments, X in SEQ ID NO: 129 is selected from P and R. In some embodiments, SEQ ID NO: 129 is CPPCP (SEQ ID NO: 130). In some embodiments, SEQ ID NO: 129 is CPRCP (SEQ ID NO: 131). In some embodiments, the hinge domain contains EPKSCDKTHTCPPCP (SEQ ID NO: 132). Thus, it will be understood that the hinge region can be considered to terminate after the CPXCP motif.

[0178] 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 CLκ domain. In some embodiments, the CL domain is a CLλ 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 contain a CH1 domain. In some embodiments, neither the first nor the second dimerization domains contain a CL domain. In some cases, neither the first nor the second polypeptide chain contains a CH1 domain. In other cases, neither the first nor the second polypeptide chain contains a CL domain.

[0179] In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 133). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 133. In some embodiments, SEQ ID NO: 133 is the IgG1 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 134). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 134. In some embodiments, SEQ ID NO: 134 is the IgG2 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 135). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 135. In some embodiments, SEQ ID NO: 135 is the IgG3 CH1 domain. In some embodiments, the Ig CH1 domain includes the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 136). In some embodiments, the Ig CH1 domain consists of SEQ ID NO: 136. In some embodiments, SEQ ID NO: 136 is the IgG4 CH1 domain.

[0180] In some embodiments, the Ig CLκ domain contains the amino acid sequence AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 137). In some embodiments, the Ig CLκ domain consists of SEQ ID NO: 137. In some embodiments, the Ig CLλ domain contains the amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 138). In some embodiments, the Ig CLλ domain consists of SEQ ID NO: 138.

[0181] Effects 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 either 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 properties similar to the corresponding molecule. As used herein, the term “effector moiety” refers to a molecule or fragment of a molecule that exerts a cytotoxic effect. In some embodiments, the effector moiety is an effector molecule.

[0182] 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 occurs in target cells. In some embodiments, the cytotoxic effect occurs upon binding. In some embodiments, death occurs upon 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 of the fragments.

[0183] 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 organizer. In some embodiments, the organizer binds to cytotoxic cells. In some embodiments, binding to cytotoxic cells means recruiting cytotoxic cells. In some embodiments, binding means being bound by ~.

[0184] 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).

[0185] In some embodiments, the effector portion binds to a receptor on the cell surface of a cytotoxic cell. Examples of receptors include, but are not limited to, CD3, CD8, CD56, CD14, and CD16. In some embodiments, the receptor is a marker for 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 active agent that binds to CD3. In some embodiments, the engager is an active agent that binds to CD3. In some embodiments, CD3 is human CD3. In some embodiments, the active agent 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 active agent that binds to CD16. In some embodiments, the engager is an active agent that binds to CD16. In some embodiments, CD16 is human CD16. In some embodiments, the active agent 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 agents are well known in the art, and any such conjugate may be used. For example, an anti-human CD3 scFv known as OKT3 may be used as the agent. In some embodiments, the cytotoxic moiety is selected from α-amanitin, a radioactive moiety, and an anti-CD3 conjugate.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®) targeting CD30 on tumor cells and CD16A on NK cells and macrophages; and GTB-3550 (CD16 / IL-15 / CD33), a triplicate killer cell engager.

[0186] 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 cells compared to Fc. In some embodiments, superior in killing cells means superior in killing B cells. In some embodiments, Fc is unmodified Fc. In some embodiments, Fc is non-mutant Fc. In some embodiments, Fc is 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.

[0187] In some embodiments, the Fc region can induce a cytotoxic effect. In some embodiments, the Fc domain includes DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 164). In some embodiments, the Fc domain contains EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 171). It will be understood that SEQ ID NO: 171 contains five additional N-terminal amino acids compared to SEQ ID NO: 164. Thus, while the numbering in this specification is given with respect to SEQ ID NO: 164, the numbering of SEQ ID NO: 171 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: 165).In some embodiments, the Fc domain contains EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 155). It will be understood that SEQ ID NO: 155 contains five additional N-terminal amino acids compared to SEQ ID NO: 165. Thus, although the numbering in this specification is given with respect to SEQ ID NO: 165 (or the equivalent SEQ ID NO: 164), the numbering of SEQ ID NO: 155 can be found by adding 5. SEQ ID NO: 164 and SEQ ID NO: 165 differ by two amino acids. These two sequences may be interchangeable, and it will be understood that if mutations are given to sequence number 164, they will also apply to sequence number 165, and vice versa. Similarly, sequence numbers 171 and 155 also differ by only two amino acids, and these two sequences may also be interchangeable.

[0188] In some embodiments, the Fc domain consists of SEQ ID NO: 164. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 164. 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 with SEQ ID NO: 164. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 171. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 171. 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 with SEQ ID NO: 171. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain consists of SEQ ID NO: 165. In some embodiments, the Fc domain of IgG1 includes or consists of SEQ ID NO: 165. In some embodiments, the Fc domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology with SEQ ID NO: 165. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the Fc domain comprises SEQ ID NO: 155. In some embodiments, the Fc domain of IgG1 comprises or consists of SEQ ID NO: 155. In some embodiments, the Fc domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology with SEQ ID NO: 155. Each possibility represents a distinct embodiment of the present invention.

[0189] 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 the protein complex. In some embodiments, the cytotoxic effect is directed towards cells that bind to the 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).

[0190] 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 generates 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 dimerization of 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.

[0191] In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 139). In some embodiments, the CH2 domain consists of SEQ ID NO: 139. In some embodiments, SEQ ID NO: 139 is the IgG1 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO: 140). In some embodiments, the CH2 domain consists of SEQ ID NO: 140. In some embodiments, SEQ ID NO: 40 is the IgG2 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 141). In some embodiments, the CH2 domain consists of SEQ ID NO: 141. In some embodiments, SEQ ID NO: 141 is the IgG4 CH2 domain. In some embodiments, the CH2 domain contains the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO: 142). In some embodiments, the CH2 domain consists of SEQ ID NO: 142. In some embodiments, SEQ ID NO: 142 is the IgG3 CH2 domain.

[0192] In some embodiments, the CH3 domain includes the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 143). In some embodiments, the CH3 domain includes the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 144). In some embodiments, the CH3 domain consists of SEQ ID NO: 143. In some embodiments, the CH3 domain consists of SEQ ID NO: 144. In some embodiments, SEQ ID NO: 143 is the IgG1 CH3 domain. In some embodiments, SEQ ID NO: 144 is the IgG1 CH3 domain. In some embodiments, the sequence of SEQ ID NO: 143 is a sequence found primarily in European and American humans. In some embodiments, SEQ ID NO: 144 is a sequence found primarily in Asian humans. In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 145). In some embodiments, the CH3 domain consists of SEQ ID NO: 145. In some embodiments, SEQ ID NO: 145 is the IgG2 CH3 domain. In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 146). In some embodiments, the CH3 domain consists of SEQ ID NO: 146. In some embodiments, SEQ ID NO: 146 is the IgG4 CH3 domain.In some embodiments, the CH3 domain contains the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 147). In some embodiments, the CH3 domain consists of SEQ ID NO: 147. In some embodiments, SEQ ID NO: 147 is the IgG3 CH3 domain.

[0193] 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 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 strands. In some embodiments, enabling is facilitating. In some embodiments, enabling is enhancing.

[0194] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutation or modification may be used to construct the polypeptide of the present invention. In some embodiments, an IgG-derived region is replaced with 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 third column of the same row in Table 1. The mutations in Table 1 are shown using IgG1 Kabat numbering unless otherwise specified; corresponding mutations can be generated in other IGs, particularly in 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 GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 148).In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 149). In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 150). In some embodiments, the CH3 domain includes or consists of GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 151).

[0195] [Table 1]

[0196] 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.

[0197] In some embodiments, the mutation reduces effector function. In some embodiments, the effector function includes ADCC, CDC, or both. In some embodiments, the reduction in effector function includes a reduction in cytotoxicity. In some embodiments, reduction means disabling. In some embodiments, Fc is derived from IgG1 or IgG3, and the mutation reduces effector function. In some embodiments, Fc is derived from IgG1 and includes at least one mutation that reduces effector function. Mutations that reduce effector function are well known in the art, and any such mutation can be used. Examples of such mutations can be found in Saunders, 2019, “Conceptual approaches to modulating antibody effector functions and circulation half-life,” Front Immunol., Jun 7;10:1296, which is incorporated herein by reference in its entirety.

[0198] 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. Furthermore, mutations such as S228P and L235E in IgG4 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, 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 mutation of L19 and L20 in SEQ ID NO: 125 to A19 and A20. In some embodiments, the LALA mutation hinge includes the L19A and L20A mutations in SEQ ID NO: 125. In some embodiments, the Fc domain includes a hinge domain that includes SEQ ID NO: 162. In some embodiments, the Fc domain containing the LALA mutation includes SEQ ID NO: 162. In some embodiments, the Fc domain includes a hinge domain that consists of SEQ ID NO: 162. In some embodiments, the LALA mutation hinge domain consists of SEQ ID NO: 162. In some embodiments, the LALA mutation is the L234A and L235A mutations in Fc. In some embodiments, at least one mutation that reduces ADCC is the N297A mutation. In some embodiments, the N297A mutation is located within the CH2 domain. In some embodiments, the N297A mutation is the mutation of asparagine 59 in SEQ ID NO: 139 to alanine.In some embodiments, the N297A mutant CH2 domain includes the N59A mutation of SEQ ID NO: 139. In some embodiments, the Fc domain includes the CH2 domain including SEQ ID NO: 163. In some embodiments, the Fc domain includes the CH2 domain consisting of SEQ ID NO: 163. In some embodiments, the N297A mutant CH2 domain consists of SEQ ID NO: 163.

[0199] In some embodiments, the mutations that reduce cytotoxicity include LALA mutations. In some embodiments, the mutations that reduce cytotoxicity include PG-LALA mutations. In some embodiments, the mutation is a mutation of proline 329 to glycine in the IgG1 human heavy chain (P329G). In some embodiments, the P-to-G mutation is a mutation of P109 to G in SEQ ID NO: 164. In some embodiments, the mutation is a mutation of leucine 234 to alanine in the IgG1 human heavy chain (L234A). In some embodiments, the L-to-A mutation is a mutation of L14 to A in SEQ ID NO: 164. In some embodiments, the mutation is a mutation of leucine 235 to alanine in the IgG1 human heavy chain (L235A). In some embodiments, the L-to-A mutation is a mutation of L15 to A in SEQ ID NO: 164. In some embodiments, the mutations include P109G, L14A, and L15A in SEQ ID NO: 164. In some embodiments, the multiple mutations include L14A and L15A of SEQ ID NO: 164. In some embodiments, the multiple mutations include P329G, L234A and L235A of the IgG1 human heavy chain. In some embodiments, the multiple mutations include L234A and L235A of the IgG1 human heavy chain. It will be understood by those skilled in the art that parallel mutations can also be performed on the IgG3 heavy chain or the heavy chain of non-human IgG1. In some embodiments, the multiple mutations that reduce cytotoxicity include YTE mutations. In some embodiments, the mutation is a mutation of methionine 252 to tyrosine (M252Y) of the IgG1 human heavy chain. In some embodiments, the M to Y mutation is a mutation of M32 to Y of SEQ ID NO: 164. In some embodiments, the mutation is a mutation of serine 254 to threonine (S254T) of the IgG1 human heavy chain. In some embodiments, the S to T mutation is a mutation of S34 to T of SEQ ID NO: 164. In some embodiments, the mutation is a mutation of threonine 256 to glutamate in the IgG1 human heavy chain (T256E). In some embodiments, the T-to-E mutation is a mutation of T36 to E in SEQ ID NO: 164. In some embodiments, multiple mutations include M32Y, S34T, and T36E in SEQ ID NO: 164.In some embodiments, the multiple mutations include M252Y, S254T, and T256E of the IgG1 human heavy chain. In some embodiments, the mutation is the mutation (N297) at asparagine 297 of 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 of SEQ ID NO: 164 (N77A or N77Q).

[0200] In some embodiments, the mutation increases the half-life of a molecule, peptide, polypeptide, or protein complex. In some embodiments, the mutation that increases half-life is 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 7. In some embodiments, the mutation is a mutation of asparagine 434 to histidine (N434H). In some embodiments, the N434H mutant Fc domain contains the N214H mutation of SEQ ID NO: 164 or 165. In some embodiments, the mutation is a mutation of valine 308 to proline (V308P). In some embodiments, the H435A mutant Fc domain contains the H215A mutation of SEQ ID NO: 164 or 165. In some embodiments, the mutation weakens binding to FcRN. In some embodiments, the mutation that weakens binding is a mutation of histidine 435 to alanine (H435A). In some embodiments, the H435A mutant Fc domain includes the H215A mutation of SEQ ID NO: 164 or 165. In some embodiments, the mutation that increases binding to FcRn is multiple mutations. In some embodiments, the multiple mutations include or consist of a mutation of methionine 252 to tyrosine (M252Y), a mutation of serine 234 to threonine, and a mutation of threonine 256 to glutamate (T256E) (also known as YTE). In some embodiments, the M252Y / S254T / T256E mutant Fc domain includes the M32Y, S34T, and T35E mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of methionine 428 to leucine (M428L) and a mutation of 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: 164 or 165. In some embodiments, the multiple mutations include or consist of M428L and a mutation to alanine of asparagine 434 (N434A) (also known as LA).In some embodiments, the M428L / N434A mutant Fc domain includes the M208L and N214A mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of threonine 250 to glutamine (T250Q) and a mutation of 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: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of histidine 433 to lysine (H433K) and a mutation of asparagine 434 to phenylalanine (N434F). In some embodiments, the H433K / N434F mutant Fc domain includes the H213K and N214F mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist 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: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of threonine 307 to alanine (T307A), a mutation of glutamate 380 to alanine (E380A), and a mutation of asparagine 434 to alanine (N434A). In some embodiments, the T307A / E380A / N434A mutant Fc domain includes the T87A, E160A, and N214A mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of mutations of methionine 252 to tyrosine (M252Y), valine 308 to proline (V308P), and asparagine 343 to tyrosine (N343Y). In some embodiments, the M252Y / V308P / N343Y mutant Fc domain includes the M32Y, V88P, and N123Y mutations of SEQ ID NO: 164 or 165.In some embodiments, the multiple mutations include or consist of M252Y, a mutation of valine 308 to proline (V308P), and a mutation of asparagine 434 to tyrosine (N434Y). In some embodiments, the M252Y / V308P / N434Y mutant Fc domain includes the M32Y, V88P, and N214Y mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of histidine 258 to aspartic acid (H258D), threonine 307 to glutamine (T307Q), and alanine 378 to valine (A378V). In some embodiments, the H258D / T307Q / A378V mutant Fc domain includes the H38D, T87Q, and A158V mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of leucine 309 to aspartic acid (L309D), a mutation of glutamine 311 to histidine (Q311H), and a mutation of 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: 164 or 165. In some embodiments, the multiple mutations that weaken binding include or consist of a mutation of isoleucine 253 to alanine (I253A), H435A, and a mutation of 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: 164 or 165. In some embodiments, the multiple mutations that weaken binding include or consist of I253A, a mutation of histidine 310 to alanine (H310A), and H435A. In some embodiments, the I253A / H310A / H435A mutant Fc domain includes the I33A, H90A, and H215A mutations of SEQ ID NO: 164 or 165.

[0201] [Table 2]

[0202] 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 N-glycan is bound to N297, the mutation disables 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: 139. In some embodiments, the N297A mutant Fc domain contains the N77A mutation of SEQ ID NO: 164 or 165. In some embodiments, the N297Q mutant CH2 domain includes the N59Q mutation of SEQ ID NO: 139. In some embodiments, the N297Q mutant Fc domain includes the N77Q mutation of SEQ ID NO: 164 or 165. In some embodiments, the N297G mutant CH2 domain includes the N59G mutation of SEQ ID NO: 139. In some embodiments, the N297G mutant Fc domain includes the N77G mutation of SEQ ID NO: 164 or 165. In some embodiments, the mutations are multiple mutations that reduce binding to the Fc receptor. In some embodiments, the multiple mutations include or consist of mutations of glycine 236 to arginine (G236R) and mutations of 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: 125 and a CH2 domain containing the L90R mutation of SEQ ID NO: 139. In some embodiments, the G236R / L328R mutant Fc domain includes the G16R and L108R mutations of sequence number 164 or 165.In some embodiments, the multiple mutations include or consist of a mutation of serine 298 to glycine (S298G) and a mutation of threonine 299 to alanine (T299A). In some embodiments, the S298G / T299A mutant CH2 domain includes the S60G and T61A mutations of SEQ ID NO: 139. In some embodiments, the S298G / T299A mutant Fc domain includes the S78G and T79A mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of leucine 234 to phenylalanine (L234F), a mutation of leucine 235 to glutamic acid (L235E), and a mutation of aspartic acid 265 to arginine (D265A). In some embodiments, the L234F / L235E / D265A mutation Fc includes a hinge domain containing the L19F and L20E mutations of SEQ ID NO: 125 and a CH2 domain containing the D27A mutation of SEQ ID NO: 139. In some embodiments, the L234F / L235E / D265A mutation Fc domain includes the L14F, L15E and D45A mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of leucine 234 to alanine (L234A), a mutation of leucine 235 to alanine (L235A), and a mutation of proline 329 to glycine (P329G). In some embodiments, the L234A / L235A / P329G mutation Fc includes a hinge domain containing the L19A and L20A mutations of SEQ ID NO: 125 and a CH2 domain containing the P91G mutation of SEQ ID NO: 139. In some embodiments, the L234A / L235A / P329G mutant Fc domain includes the L14A, L15A, and P109G mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of L234F, L235E, and a mutation from proline 331 to serine (P331S). In some embodiments, the L234F / L235E / P331S mutant Fc includes a hinge domain containing the L19F and L20E mutations of SEQ ID NO: 125 and a CH2 domain containing the P93S mutation of SEQ ID NO: 139.In some embodiments, the L234F / L235E / P331S mutant Fc domain includes the L14F, L15E, and P111S mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of a mutation of leucine 235 to alanine (L235A), a mutation of glycine 237 to alanine (G237A), and a mutation of glutamic acid 318 to alanine (E318A). In some embodiments, the L235A / G237A / E318A mutant Fc domain includes a hinge domain containing the L20A and G22A mutations of SEQ ID NO: 125 and a CH2 domain containing the E80A mutation of SEQ ID NO: 139. In some embodiments, the L235A / G237A / E318A mutant Fc domain includes the L15A, G17A, and E98A mutations of SEQ ID NO: 164 or 165.

[0203] 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 cleavase 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.

[0204] In some embodiments, the mutation is one 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 of serine 267 to glutamate (S267E). In some embodiments, the S267E mutant CH2 domain contains the S29E mutation of SEQ ID NO: 139. In some embodiments, the S267E mutant Fc domain contains the S47E mutation of SEQ ID NO: 164 or 165. In some embodiments, the mutation is a mutation of proline 238 to aspartate (P238D). In some embodiments, the P238D mutant hinge domain contains the P23D mutation of SEQ ID NO: 125. In some embodiments, the P238D mutant Fc domain contains the P18D mutation of SEQ ID NO: 164 or 165. In some embodiments, the mutations are multiple mutations that increase binding to the Fc receptor. In some embodiments, the multiple mutations include or consist of S267E and a mutation to phenylalanine at leucine 328 (L328F) (also known as SELF). In some embodiments, the S267E / L328F mutant CH2 domain includes the S29E and L90F mutations of SEQ ID NO: 139. In some embodiments, the S267E / L328F mutant Fc domain includes the S47E and L108F mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of S267E and a mutation to phenylalanine at histidine 268 (H268F) and a mutation to threonine at serine 324 (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: 139. In some embodiments, the S267E / H268F / S324T mutant Fc domain includes the S47E, H48F, and S104T mutations of SEQ ID NO: 164 or 165.In some embodiments, the multiple mutations include or consist of mutations of glycine 237 to aspartic acid (G237D), P238D, proline 271 to glycine (P271G), and 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: 125, and a mutant CH2 domain containing the P33G and A92R mutations of SEQ ID NO: 139. In some embodiments, the G237D / P238D / P271G / A330R mutant Fc domain includes the G17D, P18D, P51G, and A110R mutations of SEQ ID NO: 164 or 165. In some embodiments, the multiple mutations include or consist of the G237D, P238D, the mutation of histidine 268 to aspartic acid (H268D), P271G, and A330R (also known as V11) mutations. 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: 125 and a mutant CH2 domain containing the H30D, P33G, and A92R mutations of SEQ ID NO: 139. 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: 164 or 165. In some embodiments, the multiple mutations include or consist of the mutation of 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: 125, and a mutant CH2 domain containing the H30D, P33G, and A92R mutations of SEQ ID NO: 139.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 164 or 165.

[0205] The S267E mutation was found to increase affinity for inhibitory FcγRIIB and also for 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. The EFT mutation was found to increase FcγRIIB binding 18-fold compared to human WT IgG1. EFT also increased CDC, ADCC, and antibody-dependent phagocytic (ADCP) activity through enhanced binding to C1q and activated FcG receptors. In some embodiments, the mutations that increased ADCC were multiple mutations in EFT. P238D showed enhanced binding to FcγRIIB with an affinity approximately 4.3-fold increase compared to WT human IgG1. P238D also significantly decreased binding to all other activated Fcg receptors. V9 significantly increased antibody affinity to hFcγRIIB by approximately 32-fold compared to WT IgG1. V9 was also found to decrease affinity to the hFcγRIIA R131 allele by approximately 3-fold compared to WT IgG1. V11 significantly increased antibody affinity to hFcγRIIB by approximately 96-fold compared to human WT IgG1, while decreasing affinity to hFcγRIIA R131 by approximately 3-fold. 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 exhibited decreased FcγRI binding (0.061-fold change compared to wt IgG1) and decreased FcγRIIA-H131 binding (0.068-fold change compared to wt IgG1). It should be noted that V12 slightly improves binding to FcγRIIA-R131, resulting in a twofold increase in binding compared to WT hIgG1.

[0206] Mutations resulting in the functions listed above are well known in the art, and any such mutation can be used. Examples of such mutations can be found, at least, in KOSAunders, 2019, “Conceptual approaches to modulating antibody effector function and circulation half-life”, Front, Immunol., 2019 Jun 7;10:1296, which is incorporated herein in its entirety by reference. 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 in IgG3 heavy chains or non-human IgG1 heavy chains. It will be understood that the numbers shown herein are with respect to full-length IgG including the variable domain. The numbers may be shifted to correspond to the positions of these amino acids only in the Fc portion of IgG.

[0207] In some embodiments, the mutation increases effector function. In some embodiments, the mutation increases ADCC. In some embodiments, the mutation is not one 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 sufficiently cytotoxic to overcome the booster effect brought about by the molecules of the present invention, but Fc containing a mutation that increases ADCC is sufficiently 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 herein by reference in its entirety.

[0208] In some embodiments, the mutations that increase ADCC are multiple mutations that increase ADCC. In some embodiments, the multiple mutations include a mutation of leucine 235 to valine (L235V), a mutation of phenylalanine 243 to leucine (F243L), a mutation of arginine 292 to proline (R292P), a mutation of tyrosine 300 to leucine (Y300L), and a mutation of proline 296 to leucine (P396L) within human IgG1. In some embodiments, the multiple mutations include a mutation of leucine 15 to valine (L15V), a mutation of phenylalanine 23 to leucine (F23L), a mutation of arginine 72 to proline (R72P), a mutation of tyrosine 80 to leucine (Y80L), and a mutation of proline 176 to leucine (P176L) within SEQ ID NO: 164. In some embodiments, the multiple mutations include a mutation of serine 239 to aspartic acid (S239D) and a mutation of isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include a mutation of serine 19 to aspartic acid (S19D) and a mutation of isoleucine 112 to glutamic acid (I112E) within SEQ ID NO: 164. In some embodiments, the S239D / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include a mutation of serine 239 to aspartic acid (S239D), a mutation of alanine 330 to leucine (A330L), and a mutation of isoleucine 332 to glutamic acid (I332E) within human IgG1. In some embodiments, the multiple mutations include a mutation of serine 19 to aspartic acid (S19D), alanine 110 to leucine (A110L), and isoleucine 112 to glutamic acid (I112E) in SEQ ID NO: 164. In some embodiments, the S239D / A330L / I332E mutation also increases ADCP. In some embodiments, the multiple mutations include a mutation of glycine 236 to alanine (G236A), alanine 330 to leucine (A330L), and isoleucine 332 to glutamic acid (I332E) in human IgG1.In some embodiments, the multiple mutations include a mutation of glycine 16 to alanine (G16A), a mutation of alanine 110 to leucine (A110L), and a mutation of isoleucine 112 to glutamate (I112E) in SEQ ID NO: 164. In some embodiments, the multiple mutations include a mutation of glycine 236 to alanine (G236A), a mutation of serine 267 to glutamate (S267E), a mutation of histidine 268 to phenylalanine (H268F), a mutation of serine 324 to threonine (S324T), and a mutation of isoleucine 332 to glutamate (I332E) in human IgG1. In some embodiments, the multiple mutations include a mutation of glycine 16 to alanine (G16A), serine 47 to glutamate (S47E), histidine 48 to phenylalanine (H48F), serine 104 to threonine (S104T), and isoleucine 112 to glutamate (I112E) in SEQ ID NO: 164. In some embodiments, the multiple mutations include a mutation of serine 298 to alanine (S298A), glutamate 333 to alanine (E333A), and lysine 334 to alanine (K334A) in human IgG1. In some embodiments, the multiple mutations include a mutation of serine 78 to alanine (S78A), glutamate 113 to alanine (E113A), and lysine 114 to alanine (K114A) in SEQ ID NO: 164. In some embodiments, the multiple mutations include a mutation of proline 247 to isoleucine (P247I) and a mutation of alanine 339 to glutamine (A339Q) within human IgG1. In some embodiments, the multiple mutations include a mutation of proline 27 to isoleucine (P27I) and a mutation of alanine 119 to glutamine (A119Q) within SEQ ID NO: 164. In some embodiments, the multiple mutations include a mutation of 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 a mutation of glycine 16 to alanine (G16A), a mutation of serine 19 to aspartic acid (S19D), and a mutation of isoleucine 112 to glutamic acid (I112E) in SEQ ID NO: 164. In some embodiments, the G236A / S239D / I332E mutation also increases ADCP. In some embodiments, the mutations include mutations in the first heavy chain of human IgG1, such as a mutation of lysine 234 to tyrosine (L234Y), a mutation of lysine 235 to glutamine (L235Q), a mutation of glycine 236 to tryptophan (G236W), a mutation of serine 239 to methionine (S239M), a mutation of histidine 268 to aspartic acid (H268D), a mutation of aspartic acid 270 to glutamic acid (D270E), and a mutation of serine 298 to alanine (S298A), as well as mutations in the second heavy chain of IgG1, such as a mutation of aspartic acid 270 to glutamic acid (D270E), a mutation of lysine 326 to aspartic acid (K26D), a mutation of alanine 330 to methionine (A330M), and a mutation of lysine 334 to glutamic acid (K334E). In some embodiments, the mutations include mutations in the first chain of SEQ ID NO: 164, such as a mutation of lysine 14 to tyrosine (L14Y), lysine 15 to glutamine (L15Q), glycine 16 to tryptophan (G16W), serine 19 to methionine (S19M), histidine 48 to aspartic acid (H48D), aspartic acid 50 to glutamic acid (D50E), and serine 78 to alanine (S78A), as well as mutations in the second chain of SEQ ID NO: 164, such as a mutation of aspartic acid 50 to glutamic acid (D50E), lysine 326 to aspartic acid (K106D), alanine 110 to methionine (A110M), and lysine 114 to glutamic acid (K114E). It will be understood that all the mutations listed above given for sequence number 164 also apply to sequence number 165. In fact, they also apply to sequence numbers 171 and 155, but all the numbering given above in this specification must be increased by 5 for these sequences.

[0209] 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: 164 and 165. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs: 166). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs: 166. In some embodiments, the Fc containing the L15V / F23L / R72P / Y80L / P176L mutation is sequence number 166. 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 166 and contains the L15V / F23L / R72P / Y80L / P176L mutation.

[0210] 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. 164 and 165. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs. 167). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs. 167. In some embodiments, the Fc containing the S19D / A110L / I112E mutation is sequence number 167. 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 167 and contains the S19D / A110L / I112E mutation.

[0211] In some embodiments, the Fc domain with increased CDC contains the G16A / S47E / H48F / S104T / I112E mutation within the Fc domain. In some embodiments, the Fc domain is selected from SEQ ID NO: 164 and SEQ ID NO: 165. In some embodiments, the Fc domain with increased CDC contains EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEFEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVTNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 168). In some embodiments, the Fc domain with increased CDC consists of SEQ ID NO: 168. In some embodiments, the Fc containing the G16A / S47E / H48F / S104T / I112E mutation is SEQ ID NO: 168. 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: 168 and contains the G16A / S47E / H48F / S104T / I112E mutation.

[0212] 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: 164 and 165. In some embodiments, the ADCC-enhanced Fc domain contains EPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NOs: 169). In some embodiments, the ADCC-enhanced Fc domain consists of SEQ ID NOs: 169. In some embodiments, the Fc containing the G16A / A110L / I112E mutation is sequence number 169. 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 169 and contains the G16A / A110L / I112E mutation.

[0213] In some embodiments, the effector domain is selected from SEQ ID NOs: 166 - 169. In some embodiments, the effector domain comprises any one of SEQ ID NOs: 166 - 169. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 166 - 169. In some embodiments, the effector domain is selected from SEQ ID NOs: 166, 167, and 169. In some embodiments, the effector domain comprises any one of SEQ ID NOs: 166, 167, and 169. In some embodiments, the effector domain consists of any one of SEQ ID NOs: 166, 167, and 169. In some embodiments, the effector domain comprises at least 75, 80, 85, 90, 92, 95, 97, or 99% identity with any one of SEQ ID NOs: 166, 167, and 169 and retains increased ADCC compared to a control Fc domain. In some embodiments, the control Fc domain is an unmodified Fc domain. In some embodiments, the unmodified Fc is a naturally found Fc. In some embodiments, the unmodified Fc is a naturally found human Fc.

[0214] In some embodiments, the Fc is modified to increase ADCC. In some embodiments, the modification is removal of fucosylation. In some embodiments, the Fc fucosylation is enzymatically removed. In some embodiments, the Fc is defucosylated. In some embodiments, the method comprises a step of defucosylating the molecule. In some embodiments, the molecule of the invention is produced in a cell line engineered to produce a defucosylated molecule.

[0215] In some embodiments, the mutation increases CDC. In some embodiments, multiple mutations increase CDC. In some embodiments, multiple mutations include a mutation in human IgG1 of glycine 236 to alanine (G236A), serine 267 to glutamate (S267E), histidine 268 to phenylalanine (H268F), serine 324 to threonine (S324T), and isoleucine 332 to glutamate (I332E). In some embodiments, multiple mutations include a mutation in SEQ ID NO: 164 of glycine 16 to alanine (G16A), serine 47 to glutamate (S47E), histidine 48 to phenylalanine (H48F), serine 104 to threonine (S104T), and isoleucine 112 to glutamate (I112E). In some embodiments, the multiple mutations include a mutation of lysine 326 to tryptophan (K326W) and a mutation of glutamate 333 to serine (E333S) within human IgG1. In some embodiments, the multiple mutations include a mutation of lysine 106 to tryptophan (K106W) and a mutation of glutamate 113 to serine (E113S) within SEQ ID NO: 164. In some embodiments, the multiple mutations include a mutation of glutamate 345 to arginine (E345R), glutamate 430 to glycine (E430G), and serine 440 to tyrosine (S440Y) within human IgG1. In some embodiments, the multiple mutations include a mutation of glutamate 125 to arginine (E125R), glutamate 210 to glycine (E210G), and serine 220 to tyrosine (S220Y) within SEQ ID NO: 164. It will be understood that all the mutations listed above given for sequence number 164 also apply to sequence number 165. In fact, they also apply to sequence numbers 171 and 155, but all the numbering given above in this specification must be increased by 5 for these sequences.

[0216] In some embodiments, the effector portion is a drug. In some embodiments, the protein is a DSG-ECD drug conjugate. In some embodiments, the protein is a DSG-ECD fragment drug conjugate. In some embodiments, the protein is a DSG-Fc drug conjugate. In some embodiments, the complex is a DSG-ECD drug conjugate. In some embodiments, the complex is a DSG-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, toxicity means lethal. In some embodiments, the effector portion is sufficient to kill cells. Drug conjugation, particularly drug conjugation to antibody backbones, is well known in the art and any conjugation method can be used.

[0217] In some embodiments, the effector moiety is amatoxin. In some embodiments, the effector moiety 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 α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanulin, amanulinic acid, amaninamide, amanin, and proamanulin. In some embodiments, amanitin is α-amanitin. In some embodiments, the effector moiety is α-amanitin.

[0218] 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 bacterium that cause cytotoxicity by intercalating into DNA and inhibiting topoisomerase, among other things. 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.

[0219] 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 moiety is PBD. In some embodiments, the effector moiety 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 moiety 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.

[0220] 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, which binds to the DNA minor groove and causes strand breaks. Examples of calicheamicin include, but are not limited to, calicheamicin γ1, esperamycin, and ozogamicin.

[0221] 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.

[0222] 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, which binds to the DNA subgroove 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.

[0223] In some embodiments, the chemotherapeutic agent is a triptolide. In some embodiments, the effector portion is a triptolide.

[0224] 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 assembly by binding to tubulin at a 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.

[0225] In some embodiments, the effector portion is a combination of portions. 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.

[0226] 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 a protein target of a PV or PF 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" means having the same sequence. In some embodiments, "different" means having different sequences.

[0227] 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 into the first dimerization domain. In some embodiments, the third dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize into the first dimerization domain. In some embodiments, the fourth dimerization domain is not configured to dimerize into the second dimerization domain. In some embodiments, "configured to dimerize" means "capable of dimerizing". In some embodiments, the third and fourth dimerization domains are different from the first and second dimerization domains. In some embodiments, the first and second dimerization domains are hinge domains, and the third and fourth dimerization domains are CH1 / CL domains. In some embodiments, the first and second dimerization domains are CH1 / CL domains, and the third and fourth dimerization domains are hinge domains.

[0228] 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 PV or PF 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" means having the same sequence. In some embodiments, "different" means having different sequences. In some embodiments, "different" means originating from different proteins. In some embodiments, "different" means originating from the same protein but containing different sequences. In some embodiments, "different" means that they originate 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.

[0229] 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.

[0230] 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.

[0231] Alternative configuration In some embodiments, the composition comprises a polypeptide chain comprising a fragment or analog or derivative of a first protein target of a PV or PF autoantibody and a fragment or analog or derivative of a second protein target of a PV or PF autoantibody. In some embodiments, the polypeptide chain is a single-strand polypeptide. In some embodiments, the single strand 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 a PV or PF autoantibody. In some embodiments, the polypeptide chain further comprises a fragment or analog or derivative of a fourth protein target of a PV or PF autoantibody. In some embodiments, the polypeptide chain further comprises an Fc region. In some embodiments, the polypeptide chain further comprises an effector moiety.

[0232] In some embodiments, a fragment or analogue or derivative of the first protein target of the PV or PF autoantibody is separated from a fragment or analogue or derivative of the second protein target of the PV or PF autoantibody by a linker. In some embodiments, a fragment or analogue or derivative of the third protein target of the PV or PF autoantibody is separated from a fragment or analogue or derivative of the first or second protein target of the PV or PF autoantibody by a linker. In some embodiments, a fragment or analogue or derivative of the fourth protein target of the PV or PF autoantibody is separated from a fragment or analogue or derivative of the first, second or third protein target of the PV or PF autoantibody by a linker. In some embodiments, the fragment is separated from the Fc region by a linker. In some embodiments, the effector portion is separated or linked by a linker. In some embodiments, the effector portion is separated from the fragment by a linker.

[0233] 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 dimerization domain and the effector moiety are separated by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a bond. In some embodiments, the bond is a peptide bond. In some embodiments, the bond is an amino acid bond. In some embodiments, the linker is a flexible linker. Linkers are well known in the art, and any linker can be used.

[0234] 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 a mc-VC-PABC-MMAE linker. In some embodiments, the linker is a mc-MMAF linker. In some embodiments, the linker is a monomethyl auristatin E (MMAE) linker. Examples of peptide linkers include, but are not limited to, Val-Cit-PAB linker, Phe-Lys(Trt)-PAB linker, and Ala-Ala-Asn-PAB linker. 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, the VC in the linker is replaced by EVC. In some embodiments, the VC in the linker is replaced by EVA. In some embodiments, the fragment and the dimerization domain are linked by a non-cleavable linker. In some embodiments, the fragment and the dimerization domain are linked by a cleavable linker. In some embodiments, the effector moiety is linked by a cleavable linker. In some embodiments, the effector moiety is linked by a non-cleavable linker.

[0235] In some embodiments, conjugation means being linked together. In some embodiments, conjugation is via linking. 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.

[0236] In some embodiments, conjugation involves conjugating an amino acid linker, a portion, or both, and includes elongation of the amino acid sequence of the drug chain of the present invention. The nucleic acid molecule encoding the drug of the present invention can be modified to include a linker, a portion, or both of the coding sequences, 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 for 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 conjugation include, but are not limited to, click chemistry, saltase-assisted SMAC technology, transglutaminase addition of amine azides, and glycan remodeling.

[0237] 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.

[0238] 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 DSG1 or DSG3 fragment. In some embodiments, the conjugation or linkage does not interfere with antibody binding to the DSG1 or DSG3 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, linking or conjugating is directed to a native amino acid residue. In some embodiments, linking or conjugating 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 in 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 in the IgG heavy chain. In some embodiments, the residue is glutamine. In some embodiments, N297 is converted to glutamine, manipulated, or mutated (N297Q). In some embodiments, glutamine is Q295 in the IgG heavy chain. An example of a manipulated glutamine is, but is not limited to, Q297.Unless otherwise specified, sites are indicated using IgG1 Kabat numbering; corresponding mutations can be produced in other IGs, particularly other IgGs. In some embodiments, conjugation or linking is to the C-terminus or N-terminus of the drug chain of the present invention. In some embodiments, conjugation or linking is to the C-terminus. In some embodiments, conjugation or linking 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, conjugation or linking is to multiple sites.

[0239] In some embodiments, the linker is long enough to suppress steric hindrance between different parts of the chain. In some embodiments, the linker is long enough to suppress steric hindrance between different parts of the conjugate. In some embodiments, the linker is long enough to allow antibody binding to the fragment without steric hindrance from another part of the chain. In some embodiments, the linker is long enough to allow antibody binding to the fragment without steric hindrance from another part of the conjugate. In some embodiments, the linker is long enough to allow cell binding to the fragment without steric hindrance from another part of the chain. In some embodiments, the linker is long enough to allow cell binding 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 present 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 acid length. 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.

[0240] In some embodiments, the linker includes GGGGS (sequence number 152). In some embodiments, the linker consists of sequence number 99. 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 154). 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 GGGGSGGGGSGGGGSGGGGSGGGGS (sequence number 153). In some embodiments, the linker consists of (GGGS)n, where n is an integer.

[0241] In some embodiments, the linker is a rigid linker. In some embodiments, the rigid linker includes EAAAK (Sequence ID 170). In some embodiments, the rigid linker consists of Sequence ID 170. 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.

[0242] 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.

[0243] In some embodiments, the epitope spans at least two fragments. In some embodiments, the epitope spans a first and a second fragment. In some embodiments, the epitope spans a first and a third fragment. In some embodiments, the epitope spans a first and a fourth fragment. In some embodiments, the epitope spans a second and a third fragment. In some embodiments, the epitope spans a second and a fourth fragment. In some embodiments, the epitope spans a third and a fourth fragment. In some embodiments, the epitope spans two proteins. In some embodiments, the epitope spans two proteins in a protein complex. In some embodiments, the epitope spans three fragments. In some embodiments, the epitope spans three proteins. In some embodiments, the epitope spans four fragments. In some embodiments, the epitope spans four proteins. In some embodiments, the epitope is a complex epitope. In some embodiments, the epitope is a B cell receptor (BCR) specific epitope.

[0244] In some embodiments, all fragments originate from DSG1. In some embodiments, all fragments originate from DSG3. In some embodiments, the complex contains fragments derived from DSG1 and fragments derived from DSG3. In some embodiments, the DSG1-derived fragment is mutated. In some embodiments, the DSG3-derived fragment is mutated. In some embodiments, both the DSG1-derived and DSG3-derived fragments are mutated. In some embodiments, the DSG1 and DSG3 fragments have the same or equivalent mutations. In some embodiments, the DSG1 and DSG3 fragments have different non-equivalent mutations. Equivalent mutations are mutations of the same amino acid but have slightly different numbering positions due to differences in the DSG1 / 3 sequences. In some embodiments, equivalent mutations are homologous mutations. In some embodiments, the complex contains a DSG1 truncation. In some embodiments, the complex contains a DSG3 truncation. In some embodiments, the complex contains both DSG1 and DSG3 mutations. In some embodiments, the DSG1 and DSG3 truncations are truncations of the same length. In some embodiments, the truncations of DSG1 and DSG3 are truncations of different lengths. In some embodiments, the truncations of DSG1 and DSG3 are truncations of the same domain.

[0245] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 155). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 155. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 155.

[0246] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 156). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 156. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 156.

[0247] In some embodiments, the first polypeptide includes a fragment linked to EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 157). In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 157. In some embodiments, both the first and second polypeptides include a fragment linked to SEQ ID NO: 157.

[0248] In some embodiments, the first polypeptide includes a fragment linked to SEQ ID NO: 137. In some embodiments, the second polypeptide includes a fragment linked to SEQ ID NO: 137. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 137. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 137.

[0249] In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 155. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 156. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 157. In some embodiments, the third polypeptide includes a fragment linked to SEQ ID NO: 137. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 155. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 156. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 157. In some embodiments, the fourth polypeptide includes a fragment linked to SEQ ID NO: 137.

[0250] In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence selected from SEQ ID NOs: 5-73. 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: 5-73, 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 polypeptide chain comprises or consists of an amino acid sequence selected from Table 3. In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence selected from Table 4. In some embodiments, the polypeptide chain comprises or consists of an amino acid sequence selected from Table 5. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NOs: 5-73. Each sequence represents a distinct embodiment of the present invention. In some embodiments, the complex comprises or consists of two polypeptide chains selected from SEQ ID NOs: 5-73, 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 two chains are the same chain. In some embodiments, the two chains are different chains.

[0251] In some embodiments, the complex includes or consists of CRD-290. In some embodiments, the complex includes or consists of CRD-291. In some embodiments, the complex includes or consists of CRD-292. In some embodiments, the complex includes or consists of CRD-293. In some embodiments, the complex includes or consists of CRD-294. In some embodiments, the complex includes or consists of CRD-295. In some embodiments, the complex includes or consists of CRD-296. In some embodiments, the complex includes or consists of CRD-650. In some embodiments, the complex includes or consists of CRD-652. In some embodiments, the complex includes or consists of CRD-653. In some embodiments, the complex includes or consists of CRD-654. In some embodiments, the complex includes or consists of CRD-655. In some embodiments, the complex includes or consists of CRD-656. In some embodiments, the complex includes or consists of CRD-657. In some embodiments, the complex includes or consists of CRD-658. In some embodiments, the complex includes or consists of CRD-659. In some embodiments, the complex includes or consists of CRD-660. In some embodiments, the complex includes or consists of CRD-661. In some embodiments, the complex includes or consists of CRD-662. In some embodiments, the complex includes or consists of CRD-663. In some embodiments, the complex includes or consists of CRD-664. In some embodiments, the complex includes or consists of CRD-666. In some embodiments, the complex includes or consists of CRD-667. In some embodiments, the complex includes or consists of CRD-668. In some embodiments, the complex includes or consists of CRD-669. In some embodiments, the complex includes or consists of CRD-670.In some embodiments, the complex includes or consists of CRD-671. In some embodiments, the complex includes or consists of CRD-672. In some embodiments, the complex includes or consists of CRD-673. In some embodiments, the complex includes or consists of CRD-674. In some embodiments, the complex includes or consists of CRD-675. In some embodiments, the complex includes or consists of CRD-676. In some embodiments, the complex includes or consists of CRD-677. In some embodiments, the complex includes or consists of CRD-678. In some embodiments, the complex includes or consists of CRD-679. In some embodiments, the complex includes or consists of CRD-680. In some embodiments, the complex includes or consists of CRD-681. In some embodiments, the complex includes or consists of CRD-683. In some embodiments, the complex includes or consists of CRD-684. In some embodiments, the complex includes or consists of CRD-685. In some embodiments, the complex includes or consists of CRD-686. In some embodiments, the complex includes or consists of CRD-688. In some embodiments, the complex includes or consists of CRD-689. In some embodiments, the complex includes or consists of CRD-690. In some embodiments, the complex includes or consists of CRD-691. In some embodiments, the complex includes or consists of CRD-692. In some embodiments, the complex includes or consists of CRD-693. In some embodiments, the complex includes or consists of CRD-695. In some embodiments, the complex includes or consists of CRD-696. In some embodiments, the complex includes or consists of CRD-697. In some embodiments, the complex includes or consists of CRD-698. In some embodiments, the complex includes or consists of CRD-699.In some embodiments, the complex comprises or consists of CRD-700. In some embodiments, the complex comprises or consists of CRD-701. In some embodiments, the complex comprises or consists of CRD-702. In some embodiments, the complex comprises or consists of CRD-703. In some embodiments, the complex comprises or consists of CRD-923. In some embodiments, the complex comprises or consists of CRD-924. In some embodiments, the complex comprises or consists of CRD-925. In some embodiments, the complex comprises or consists of CRD-926. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 70% identity with the sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 75% identity with the sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 80% identity with the sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 85% identity with the sequence provided herein. In some embodiments, the polypeptide chain comprises or consists of a sequence having at least 90% identity with the sequence provided herein. In some embodiments, the polypeptide chain comprises 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.

[0252] Pharmaceutical composition In another embodiment, a pharmaceutical composition comprising the protein or polypeptide of the present invention is provided.

[0253] In another embodiment, a pharmaceutical composition comprising the protein complex of the present invention is provided.

[0254] In another embodiment, a pharmaceutical composition comprising the composition of the present invention is provided.

[0255] 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 the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline solution. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline and 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 can function as carriers in this specification include sugars, starches, cellulose and their derivatives, tragacanth powder, 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 compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions intended herein.Appropriate pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those skilled in the art, for example, as described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the 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, all of 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 further inactive ingredients, as well as effective formulations and administration procedures, are well known in the art and are described in standard texts, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed. Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005), each of which is incorporated herein by reference in its entirety.The compositions described herein may also be contained in artificially 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, lamellar layers, and the like. Liposomes for use with the peptides described herein are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is generally determined considering factors such as the size of the liposome and its stability in blood. Various methods for preparing liposomes are available, as reviewed, for example, in Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and also see U.S. Patents 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0256] The carriers may, in total, constitute about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0257] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the protein complex 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 amount” refers to the amount of drug effective in treating a disease or disorder in a mammal. In some embodiments, the therapeutic amount is the amount effective in the required dose and 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 the amount sufficient to treat at least one symptom of the disease. In some embodiments, the disease is PV. In some embodiments, the disease is PF. In some embodiments, the disease is PV or PF. In some embodiments, PV or PF is characterized by autoantibodies against the protein. In some embodiments, PV is characterized by autoantibodies against DSG1 and / or DSG3. In some embodiments, PF is characterized by autoantibodies against DSG1.

[0258] 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 is only required to reduce the severity of the disease, disorder, or condition, reduce the severity of its associated symptoms, or provide an improvement in the quality of life of the patient or subject. Treatments of PV or PF are well known in the art and may include any acceptable measure for evaluating improvement in PV or PF symptoms. These may include rituximab, steroids, steroid-sparing immunosuppressants (e.g., azathioprine, mycophenolic acid, and cyclophosphamide), dapsone, intravenous immunoglobulin (IVIG), etc. Treatments may include improvement in quality of life, suppression of blister formation, reduction of autoantibodies, and death of autoreactive B cells.

[0259] 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.

[0260] As used herein, terms such as “administering” and “dosing” 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 aspect of the subject matter of the present invention provides intravenous administration of a therapeutically effective amount of the subject matter of the present invention to a patient in need. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal. In some embodiments, administering is intravenous administration. In some embodiments, administration is topical administration. In some embodiments, administering is selected from oral, intravenous, intramuscular, intraperitoneal, intertumorial, topical, or subcutaneous administration. In some embodiments, administering is administration to the site of disease.

[0261] 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.

[0262] Treatment method In another aspect, a method for treating PV or PF in a subject requiring treatment of PV or PF is provided, comprising administering the protein or polypeptide of the present invention to a subject to thereby treat PV or PF in the subject.

[0263] In another aspect, a method is provided for treating PV or PF in a subject requiring treatment of PV or PF, comprising administering the protein complex of the present invention to the subject, thereby treating PV or PF in the subject.

[0264] In another aspect, a method for treating PV or PF in a subject requiring treatment of PV or PF is provided, comprising administering a composition of the present invention to the subject, thereby treating PV or PF in the subject.

[0265] In some embodiments, administration refers to administering the pharmaceutical composition of the present invention. In some embodiments, PV or PF is characterized by an antibody against a protein. In some embodiments, the protein is the target of the PV or PF 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 PV or PF antibody in the subject. In some embodiments, the antibody is an autoantibody. In some embodiments, the disease is PV and the autoantibody is against DSG1, DSG3, or both. In some embodiments, the disease is PF and the autoantibody is against DSG1.

[0266] 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, killing B cells is specific B cell death. In some embodiments, treatment includes killing antibody-producing B cells. In some embodiments, treatment includes killing antibody-producing B cells but not substantially killing other B cells. In some embodiments, treatment includes killing B cells that produce antibodies against protein complexes. In some embodiments, treatment includes killing B cells that produce antibodies against fragments. In some embodiments, treatment includes killing B cells that produce antibodies against fragments of protein complexes.

[0267] In some embodiments, reducing an antibody involves binding to the antibody. In some embodiments, reducing means removing at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% of the 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 IgG1 and IgG3 autoantibodies. In some embodiments, the autoantibody includes IgG1, IgG2, and IgG3 autoantibodies. In some embodiments, the autoantibody includes IgG1, IgG3, and IgG4 autoantibodies. In some embodiments, the autoantibody includes IgG1, IgG2, IgG3, 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 proteins or fragments. In some embodiments, the percentage of antibodies is the percentage of disease-related antibodies.

[0268] In some embodiments, the method further includes a step of reducing antibodies in a subject. In some embodiments, the reduction occurs before administration. In some embodiments, the reduction is to reduce 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 is to reduce antibodies against all proteins 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, plasma separation and exchange, intravenous Ig (IVIg), antibody filtering, and B-cell targeted therapy, any of which may be used. In some embodiments, the method includes a step of plasma separation and exchange of antibodies before administration. In some embodiments, the method includes a step of 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, the 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 non-limiting examples include 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.

[0269] 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.

[0270] 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 or analog or derivative of a first human protein target of a PV or PF autoantibody and a first dimerization domain, and the second nucleic acid molecule encodes a second polypeptide chain comprising a fragment or analog or derivative of a second human protein target of a PV or PF autoantibody and a second dimerization domain.

[0271] In another embodiment, nucleic acid molecules encoding the protein of the present invention are provided.

[0272] In another embodiment, nucleic acid molecules encoding the polypeptide chain of the composition of the present invention are provided.

[0273] In another embodiment, nucleic acid molecules encoding the composition of the present invention are provided.

[0274] In another embodiment, nucleic acid molecules are provided that encode a fragment or analog or derivative of a first protein target of a PV or PF autoantibody, and a fragment or analog or derivative of a second human protein target of a PV or PF autoantibody.

[0275] 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.

[0276] 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 modification 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.

[0277] A vector nucleic acid sequence generally includes at least one origin of replication for intracellular proliferation, and optionally further 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 expressing the protein complex of the present invention.

[0278] The vector may be a DNA plasmid delivered by 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 that assemble around the start site of RNA polymerase, i.e., RNA polymerase II. Promoters consist of individual functional modules, each consisting of approximately 7–20 bp of DNA and containing one or more recognition sites for transcriptional activators or repressor proteins.

[0279] In some embodiments, the open reading frame is operably linked to a promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that enables the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in a host cell if the vector is introduced into a host cell).

[0280] 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-speed ballistic penetration by small particles having nucleic acids either within or on the surface of a matrix of small beads or particles (Klein et al., Nature 327. 70-73 (1987)).

[0281] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme present in eukaryotic cells. It catalyzes the transcription of DNA to synthesize mRNA and the precursors of most snRNAs and microRNAs.

[0282] 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, pCI, pMbac, pPbac, pBK-RSV, and pBK-CMV, pTRES, and derivatives thereof, all available from Invitrogen.

[0283] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine 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 vectors that enable protein expression under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary cancer virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any other promoters that have been shown to be effective for expression in eukaryotic cells.

[0284] In some embodiments, recombinant viral vectors that offer advantages such as lateral infection and target specificity are used for in vivo expression. In one embodiment, lateral infection is a process inherent in the life cycle of retroviruses, for example, in which a single infected cell produces many progeny virions that budding infect neighboring cells. In one embodiment, the result is that a wide area is rapidly infected, with the majority not initially infected by the original viral particle. In one embodiment, a viral vector that cannot spread laterally 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.

[0285] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), 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. Furthermore, for information on the positive-negative selection method, see U.S. Patent Nos. 5,464,764 and 5,487,992.

[0286] In one embodiment, a plant expression vector is used. In one embodiment, the expression of a polypeptide coding sequence is driven by several promoters. In some embodiments, viral promoters are used, such as the 35S RNA and 19S RNA promoters for CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter for TMV [Takamatsu et al., EMBO J.6:307-311 (1987)]. 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)], 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 systems, which are well known in the art, can also be used in accordance with the present invention.

[0287] In addition to containing elements necessary for the transcription and translation of the inserted coding sequence (encoding the polypeptide), it will be understood that the expression construct of the present invention may also include sequences manipulated to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0288] 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, all of the first, second, third, and fourth nucleic acid molecules are different molecules. In some embodiments, all of the first, second, third, and fourth nucleic acid molecules are the same molecule.

[0289] Production method A method for producing a protein, in another embodiment, A step of obtaining 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 PV or PF autoantibodies; and a step of ligating the first fragment to the second fragment to produce a single-chain polypeptide. This includes a method for producing protein.

[0290] In another embodiment, a method for producing a protein is provided, comprising the steps of: obtaining a first fragment or an analog or derivative of the extracellular domain of a first human protein target of a pemphigus vulgaris / pemphigus foliaceus autoantibody; and ligating the first fragment to an effector portion that is not an unmodified Fc domain.

[0291] A method for producing a protein, in another embodiment, A step of obtaining a first fragment of the extracellular domain of DSG1 or DSG3 or an analog or derivative thereof, and a step of truncating DSG1 or DSG3 or an analog or derivative thereof, wherein truncation reduces aggregation of the first fragment. This includes a method for producing protein.

[0292] A method for producing a protein, in another embodiment, The steps include: obtaining a first fragment of the extracellular domain of DSG1 or DSG3, or an analog or derivative thereof; truncating DSG1 or DSG3, or an analog or derivative thereof, such that truncation reduces aggregation of the first fragment; and ligating the truncated fragment to an effector portion that is not an unmodified Fc domain. This includes a method for producing protein.

[0293] A method for producing a protein, in another embodiment, A step of obtaining 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 pemphigus vulgaris / pemphigus foliaceus autoantibodies and are different proteins; and a step of ligating the first fragment to the second fragment to produce a single-chain polypeptide, and further ligating the polypeptide chain to an effector portion that is not an unmodified Fc domain. This includes a method for producing protein.

[0294] A method for producing a protein, in another embodiment, A step of obtaining a first fragment of the extracellular domain of DSG1 or DSG3, or an analog or derivative thereof; and a step of inducing a mutation in DSG1 or DSG3, or an analog or derivative thereof, wherein the mutation reduces the aggregation of the first fragment. This includes a method for producing protein.

[0295] A method for producing a protein, in another embodiment, A step of obtaining a first fragment of the extracellular domain of DSG1 or DSG3, or an analog or derivative thereof; a step of generating a mutant fragment by introducing at least one mutation into the first fragment that reduces the aggregation of the first fragment; and a step of ligating the mutant fragment to an effector portion that is not an unmodified Fc domain. This includes a method for producing protein.

[0296] A method for producing a protein, in another embodiment, a. A step to obtain a fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; b. A step of generating a mutant fragment by introducing at least one mutation into the fragment; c. A step of measuring the solubility, aggregation, or both of the mutant fragments; and d. The step of selecting at least one mutant fragment that has increased solubility, decreased aggregation, or both, compared to the obtained fragment. This includes a method for producing protein.

[0297] A method for producing a protein, in another embodiment, a. A step to obtain a fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; b. A step of generating a mutant fragment by introducing at least one mutation into the fragment; c. A step of measuring the solubility, aggregation, or both of the mutant fragments; and d. A step of selecting at least one mutant fragment that has increased solubility, decreased aggregation, or both, compared to the obtained fragment; and e. The process of ligating the selected mutant fragment to an effector region that is not an unmodified Fc domain. This includes a method for producing protein.

[0298] A method for producing a protein complex, in another embodiment, Steps to obtain a first fragment or analog or derivative of a first protein target of a PV or PF autoantibody, and a second fragment or analog or derivative of a second protein target of a PV or PF autoantibody; steps to link the first fragment to a first dimerization domain to generate a first polypeptide, and link the second fragment to a second dimerization domain to generate a second polypeptide chain. A method is provided that includes and thereby produces a protein complex.

[0299] A method for producing a protein complex, in another embodiment, The steps include: obtaining a first fragment or analogue or derivative of a first protein target of a PV or PF autoantibody, and a second fragment or analogue or derivative of a second protein target of a PV or PF autoantibody; ligating the first fragment to a first dimerization domain to generate a first polypeptide; ligating the second fragment to a second dimerization domain to generate a second polypeptide chain; and ligating the first polypeptide chain, the second polypeptide chain, or both to an effector portion that is not an unmodified Fc domain. A method is provided that includes and thereby produces a protein complex.

[0300] A method for producing a protein, in another embodiment, A step of culturing host cells containing one or more vectors comprising nucleic acid sequences encoding single-stranded polypeptides, wherein the single-stranded polypeptide is i. 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, such that the first and second human receptors are targets of PV or PF autoantibodies and are different proteins; and ii. To generate a single-stranded polypeptide by linking the first fragment to the second fragment. The process generated by This includes a method for producing protein.

[0301] A method for producing a protein, in another embodiment, A step of culturing host cells containing one or more vectors comprising nucleic acid sequences encoding single-stranded polypeptides, wherein the single-stranded polypeptide is i. 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 PV or PF autoantibodies and are different proteins; ii. Linking the first fragment to the second fragment to produce a single-stranded polypeptide; and iii. Concatenating a single-stranded polypeptide to an effector region other than the unmodified Fc domain. The process generated by This includes a method for producing protein.

[0302] A method for producing a protein complex, in another embodiment, 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 i. Obtain a first fragment or analog or derivative of a first protein target of a PV or PF autoantibody, and a second fragment or analog or derivative of a second protein target of a PV or PF autoantibody; and ii. To generate a first polypeptide chain by linking the first fragment to the first dimerization domain, and to generate a second polypeptide chain by linking the second fragment to the second dimerization domain. The process generated by A method is provided that includes and thereby produces a protein complex.

[0303] A method for producing a protein complex, in another embodiment, 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 i. Obtain a first fragment or analog or derivative of a first protein target of a PV or PF autoantibody, and a second fragment or analog or derivative of a second protein target of a PV or PF autoantibody; ii. Linking the first fragment to the first dimerization domain to generate the first polypeptide chain, and linking the second fragment to the second dimerization domain to generate the second polypeptide chain; and iii. Linking the first polypeptide chain, the second polypeptide chain, or both to an effector portion that is not an Fc domain. The process generated by A method is provided that includes and thereby produces a protein complex.

[0304] 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 a truncation of the present invention. In some embodiments, the protein is a polypeptide of the present invention. In some embodiments, the mutation is a 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 a polypeptide chain of the present invention. In some embodiments, the fragment is the fragment of the present invention. In some embodiments, the derivative is a derivative of the present invention. In some embodiments, the analog is an analog of the present invention. In some embodiments, the dimerizing domain is the dimerizing domain of the present invention. In some embodiments, the composition, protein complex, protein, fragment, analog, derivative, or dimerizing domain is as described above. In some embodiments, the method further includes the step of linking the protein, polypeptide, or protein complex to an effector moiety. In some embodiments, the effector moiety is not an Fc domain. In some embodiments, the effector moiety does not contain an Fc moiety. In some embodiments, the effector moiety is not an unmodified Fc domain. In some embodiments, the effector portion is an Fc domain containing at least one mutation that increases ADCC.

[0305] 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 PV or PF autoantibodies. In some embodiments, the first and second proteins are targets of autoantibodies associated with PV or PF. In some embodiments, PV or PF 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.

[0306] 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 includes the step of contacting the first and second polypeptides. In some embodiments, contacting includes incubating the polypeptides together. In some embodiments, contacting is performed intracellularly. In some embodiments, contacting is performed in vitro. In some embodiments, contacting is performed 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.

[0307] In some embodiments, the method further includes the step of 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.

[0308] In some embodiments, the method further includes the steps of obtaining a third fragment or analog or derivative of a third protein target of a PV or PF autoantibody, 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 includes the step of contacting the first, second and third polypeptide chains. In some embodiments, the method further includes the step of 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 includes the step of expressing the first, second and third polypeptide chains in a cell.

[0309] In some embodiments, the method further includes the step of inserting a fifth dimerization domain into a second polypeptide. In some embodiments, insertion is ligation. In some embodiments, insertion is inserting a nucleic acid sequence encoding the fifth dimerization domain into a nucleic acid molecule or vector encoding the second polypeptide. In some embodiments, 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.

[0310] In some embodiments, the method further includes the steps of obtaining a fourth fragment or analog or derivative of a fourth protein target of a PV or PF autoantibody, 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 includes the step of contacting the first, second, third and fourth polypeptide chains. In some embodiments, the method further includes the step of 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 includes the step of expressing the first, second, third and fourth polypeptide chains in a cell.

[0311] In some embodiments, the method further includes the step of inserting the Fc region into a first strand. In some embodiments, the method further includes the step of inserting the Fc region into a second strand. In some embodiments, the method further includes the step of inserting the Fc region into a third strand. In some embodiments, the method further includes the step of inserting the Fc region into a fourth strand. In some embodiments, the method further includes the step of 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.

[0312] In some embodiments, the Fc region is inserted into the C-terminal side of the dimerization domain. In some embodiments, the Fc region is inserted into the C-terminal side of the fragment. In some embodiments, the Fc region is inserted into the N-terminal side of the dimerization domain. In some embodiments, the Fc region is inserted into the N-terminal side of the fragment. In some embodiments, the fragment is inserted into or ligated to the C-terminal side of the dimerization domain. In some embodiments, the fragment is inserted into or ligated to the N-terminal side of the dimerization domain.

[0313] In some embodiments, the method further includes the step of 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.

[0314] In some embodiments, the method further includes the step of generating at least one mutation in a protein. In some embodiments, the mutation is generated in a fragment. In some embodiments, the mutation is generated in an extracellular domain. In some embodiments, the mutation is generated in a cadherin domain of a fragment. In some embodiments, the method further includes the step of truncating a protein. In some embodiments, the method further includes the step of truncating a fragment. In some embodiments, the method further includes the step of 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.

[0315] In some embodiments, the method further includes the step of measuring protein aggregation. In some embodiments, the method further includes the step of measuring the aggregation of mutant proteins. In some embodiments, the method further includes the step of measuring the aggregation of truncated proteins. In some embodiments, the method further includes the step of selecting proteins with low aggregation. In some embodiments, low aggregation is aggregation below a predetermined threshold. In some embodiments, low aggregation is substantial aggregation. In some embodiments, the method further includes the step of selecting mutant proteins with reduced aggregation. In some embodiments, the method further includes the step of selecting truncated proteins with reduced aggregation. In some embodiments, reduced means compared to a non-mutant or non-truncated protein. In some embodiments, reduced means compared to a control protein. In some embodiments, reduced means compared to a WT extracellular domain. In some embodiments, reduced means a reduction of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100%. Each possibility represents a distinct embodiment of the present invention. In some embodiments, a reduction includes a reduction of at least 50%. In some embodiments, a reduction includes a reduction of at least 25%. Methods for measuring aggregation are well known in the art, and any such method, including those provided herein, may be used. In some embodiments, aggregation is measured under non-reducing conditions.

[0316] In some embodiments, the method further includes the step of measuring the solubility, aggregation, or both of the obtained fragments. In some embodiments, an increase means a significant increase. In some embodiments, a decrease means a reduction. In some embodiments, a decrease means a significant decrease. In some embodiments, significant means statistically significant.

[0317] 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.

[0318] In another embodiment, a protein complex produced by the method of the present invention is provided.

[0319] In another aspect, a protein produced by the method of the present invention is provided.

[0320] In another embodiment, a composition produced by the method of the present invention is provided.

[0321] Patient selection In another embodiment, a method is provided for determining the suitability of a subject to be treated by the method of the present invention, comprising the steps of obtaining a sample from the subject, 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 subject is suitable to be treated by the method of the present invention, and thereby a method for determining the suitability of the subject to be treated.

[0322] In another embodiment, a method is provided for determining the suitability of a subject to be treated by the method of the present invention, comprising the steps of obtaining a sample from the subject, 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 subject is suitable to be treated by the method of the present invention, and thereby a method for determining the suitability of the subject to be treated.

[0323] In another embodiment, a method is provided for determining the suitability of a subject to be treated by the method of the present invention, comprising the steps of obtaining a sample from the subject, 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 subject is suitable to be treated by the method of the present invention, and thereby determining the suitability of the subject to be treated.

[0324] In some embodiments, the subject is one who needs it. In some embodiments, the subject is one of the above-described subjects. In some embodiments, the subject has PV or PF. In some embodiments, the subject is known to be positive for autoantibodies associated with PV or PF. 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 the treatment for PV or PF. In some embodiments, the subject has been treated before and has relapsed.

[0325] In some embodiments, the method includes the step of obtaining a sample from a subject. In some embodiments, the sample includes tissue. In some embodiments, the sample is a biopsy. In some embodiments, the sample is a body fluid. In some embodiments, the body fluid is blood. In some embodiments, the body fluid is serum. In some embodiments, the body fluid is plasma. In some embodiments, the body fluid is a liquid 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.

[0326] In some embodiments, contact is incubation. In some embodiments, contact is carried out under conditions sufficient for the antibody to bind to the protein complex. In some embodiments, the conditions include sufficient 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.

[0327] 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 the step of 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, the 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 a disease-related antibody.

[0328] 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 fragment of the protein complex. In some embodiments, binding is binding to at least two fragments of the protein complex. In some embodiments, binding is binding to at least three fragments of the protein complex. In some embodiments, binding is binding to at least four 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.

[0329] 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.

[0330] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless specifically indicated in the context. 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. It should be further noted that the claims may be constructed to exclude any element whatsoever. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as “solely,” “only,” etc., in connection with the enumeration of elements of the claims or the use of “negative” limitations.

[0331] Where conventions similar to “at least one of A, B, and C, etc.” are used, such structures are generally intended to be understood by those skilled in the art (for example, “a system having at least one of A, B, and C” includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or systems having A, B and C together). It will further be understood by those skilled in the art that substantially any disjunct word and / or disjunct phrase presenting two or more alternative terms should be understood, whether in the specification, claims, or drawings, to be intended to include 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 possibilities of “A” or “B” or “A and B”.

[0332] For clarity, certain features of the Invention described in the context of separate embodiments may also 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 also be provided separately or in any suitable partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if every possible combination were disclosed individually and expressly. Furthermore, all partial combinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if every possible such partial combination were disclosed herein individually and expressly.

[0333] 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. Furthermore, each of the various embodiments and aspects of the present invention described above and claimed in the following claims is experimentally supported in the following embodiments.

[0334] Various embodiments and aspects of the present invention described above and claimed in the following claims are experimentally supported in the following examples. [Examples]

[0335] In general, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular, biochemical, immunological, microbiological, and recombinant DNA techniques. Such techniques are well described in the literature. For example, all are 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); Methods described in 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 (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), “Strategies for Protein Purification and Characterization—A Laboratory Course Manual,” CSHL Press (1996). Other general references are provided throughout this document.

[0336] Example 1 Autoantibodies against DSG1 and DSG3 are the main cause of PV, and DSG1 autoantibodies are the main cause of PF. While DSG3 autoantibodies were previously thought to be the main driving factor of PV, it has become increasingly clear in recent years that both proteins play important roles. Indeed, many patients have autoantibodies against both proteins. Desmogleins are involved in tight junction formation, and therefore their extracellular domains contain several interaction domains (cadherin-like domains) that enable homocohesion (cohesion between one type of desmoglein, e.g., DSG1 / DSG1 cohesion or DSG3 / DSG3 cohesion) as well as heterocohesion between different desmogleins. DSG1 and DSG3 each contain an N-terminal prodomain that must be cleaved for normal function. After this, both proteins contain four distinct binding domains in their extracellular regions. Therefore, therapeutic agents targeting DSG1 alone, DSG3 alone, and both proteins simultaneously have been developed. Various combinations of extracellular domains have been tested, along with various mutations to reduce homocohesion and heterocohesion.

[0337] Long-term remission in PV patients requires the removal of a significant proportion of autoreactive B cells that produce an autoantibody pool. While potentially effective in treating PV / PF symptoms, simply removing autoantibodies from circulation necessitates repeated treatment for the remainder of the patient's lifespan, as long-lived B cells continuously produce new autoantibodies. 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 allows the B cells themselves to be targeted by therapeutic agents containing one or more autoantibody BCR-specific epitopes. By linking the target epitope to the Fc region of the antibody heavy chain, the therapeutic agent can direct the specific death of autoantibody-producing B cells. This approach is also robust against the potential evasion of specific subpopulations that can occur when using drugs that target specific differentiation markers on the cell surface (e.g., CD19, CD38, BCMA), as all cells carrying autoreactive BCRs are targeted regardless of their differentiation state. This approach is also beneficial for protecting and preserving non-autoreactive subpopulations, including defensive (e.g., antiviral, antibacterial) subpopulations that are damaged by therapies targeting nonspecific differentiation markers (e.g., CD20, CD38, BCMA), whether or not they carry autoreactive BCRs.

[0338] 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, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD domains. 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 be dimerized with chain 110 via disulfide bonds found in the CH1 domain 114 and CL domain 124.

[0339] 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 component of human desmoglein 1 or desmoglein 3. Each chain can be generated to have the same or a different fragment / derivative of DSG1 or DSG3. In fact, as shown in Figure 1B, the two heavy chains 115 and 116 can be manipulated separately so that chain 115 contains, for example, the extracellular domains (ECs) 1 and 2 of DSG1 (131) and chain 116 contains ECs 3 and 4 of DSG1 (132). The same applies to light chains 125 and 126, which can contain, for example, ECs 1 and 2 (133) and ECs 2 and 4 (134) of DSG3, respectively. Therefore, therapeutic molecules can be designed with four copies of a protein or domain (Figure 1C), two copies each of two different proteins or domains (Figure 1D), or one copy each of four different protein fragments or domains (Figure 1B), or any other combination thereof. In fact, molecules are modular enough to be manipulated with three copies of one protein / domain and one copy of another protein / domain, or two copies of one protein / domain and one copy each of two other proteins / domains. 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 include any combination of various domains of two proteins that can bind to autoantibodies from a wide variety of patients, not just some patients. It will be understood by those skilled in the art that any chain can contain any protein, fragment, domain, or variant, and the combinations of chains and subunits shown in Figures 1A-1F are illustrative and not limiting.

[0340] Figures 2A–2N show several embodiments of the present invention in which only two chains are combined. In Figures 2A–2F, the protein complex 201 comprises two polypeptide chains, specifically 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 hinges 213, CH2 212, and CH3 211 of the heavy chains all dimerize via disulfide bonds. Only one of these three domains is required for dimerization, although other dimerizing domains are also conceivable. An extracellular domain 230 of either DSG1 or DSG3 is included. Protein complex 201 is also envisioned to consist of only fragments of the extracellular domains of DSG1 or DSG3, for example, fragment 231 containing only EC1 and EC2 of DSG1, fragment 232 containing only EC3 and EC4 of DSG1, fragment 233 containing only EC1 and EC2 of DSG1, and fragment 234 containing only EC3 and EC4 of DSG3. Protein complex 201 is also envisioned to consist of two different fragments from the same protein or from each of two proteins (e.g., fragment 231 on one chain and fragment 233 on the other chain). While these specific fragments are shown herein, any fragment of the extracellular domain of either protein may be used in any combination.

[0341] Figure 2B shows molecules that do not contain 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 the variable region, each chain has a fragment 230 derived from the extracellular portion of DSG1 and / or DSG3. It will be understood by those skilled in the art that any fragment derived from the extracellular portion of DSG1 and / or DSG3, although not shown, can also be used. Each chain can be generated to have the same DSG protein, fragment or variant (Figure 2C) or different subunits (Figure 2D). For simplicity, the extracellular domains 235 of DSG1 and 236 of DSG3 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, such as knob-in-holes and DuoBody, that inhibit homodimerization and promote heterodimerization. Any such technique may be used. Removal of CH1 by direct conjugation to the hinge region (Figure 2E) or further removal of the hinge region by direct conjugation to CH2 (Figure 2F) is also possible. For all figures, if an extracellular domain is shown, it will be understood that all fragments, variants, and mutants of that extracellular domain are also intended to be included.

[0342] 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 can optionally contain 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, along with an example lacking the CH1 domain. Molecules lacking the hinge, CH2, or CH3 domain, or any two or three of these domains, are also conceivable (as long as at least one dimerization region is retained). It will be understood that fragment 230 can originate from either DSG1 or DSG3 and may contain the entire extracellular domain, only a part of it, or a variant. Therefore, repeats of two identical proteins / fragments can be inserted into a single strand (Figure 2H-I, two DSG1 EC 1 / 2 fragments 231), or two different proteins / fragments can be combined on a single strand (Figure 2J-K, DSG1 EC 1 / 2 fragment 231 and DSG3 EC 3 / 4 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-2M, DSG1 EC 1 / 2 fragment 231 and DSG3 EC 3 / 4 fragment 234 on one strand, and DSG3 EC 1 / 2 fragment 233 and DSG1 EC 3 / 4 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 dimerized domain (e.g., hinge, CH2, CH3) remains.

[0343] 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., a substitution / mutation to serine or glutamine) may be introduced into the hinge, or one of the mutations in the CH2 / CH3 region that promotes heterodimerization and inhibits homodimerization may be used. Instead of a variable region, each chain has a fragment 230 derived from the extracellular component of DSG1 or DSG3, or any fragment or variant thereof.

[0344] 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 above method of this specification can be used to secure 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 use different dimerizing domains. 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 human desmoglein 1 / 3. All three strands may contain the same fragment (e.g., DSG1 extracellular domain 335, Figure 3B), all three strands may contain different fragments (e.g., DSG3 336, DSG1 EC 1 / 2 fragment 331, and DSG1 3 / 4 fragment 332, Figure 3C), or the three strands may contain two different fragments, one of which is repeated (e.g., DSG1 335 and DSG3 EC 1 / 2 fragment 333, Figure 3D). Figure 3D may also have two identical fragments as either a light chain and a heavy chain, and thus it will be understood by those skilled in the art that this embodiment has three different configurations.

[0345] This configuration, in which one of the heavy chains contains 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 DSG1 EC 1 / 2 431, DSG3 EC 3 / 4 434, DSG1 EC 3 / 4 432, and DSG3 EC 1 / 2 433 are used, but those skilled in the art will understand that any four fragments can be used. Embodiments can also be envisioned in which different fragments from the same protein may be present 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 herein above, mutations in any CH2 domain 412 and CH3 domain 413 can be used to promote heterodimerization of chains 415 and 416. Hinge region 413, CH2 domain 412, and CH3 domain 411 are all used here as dimerization domains between the two heavy chains, but any dimerization domain (other than CH1 / CL) can be used.

[0346] 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–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 a PV / PF autoantibody. These could all be the same fragment with the same amino acid sequence, or they could be different sequences (derived from either the same or different proteins), or variants / derivatives, or proteins or fragments.

[0347] Figure 5B shows an alternative embodiment of Figure 5A, where each distinct domain is separated by a linker. All of these linkers are arbitrary, and it will be understood by those skilled in the art that combinations of linkers are possible. 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 DSG1 / 3 fragments. Thus, the linkers shown in Figure 5B can be extrapolated to the same positions in the immunoglobulin backbone molecule (Figures 1A-4).

[0348] Figures 6A–6F illustrate single-stranded embodiments of the present invention. Figure 6A shows a single-stranded fusion protein 601 containing the DSG1 extracellular domain 635 and the DSG3 extracellular domain 636. Figure 6B shows a single strand containing only a fragment of DSG1 or only a fragment of DSG3. For simplicity, fragment 631 containing only EC1 and EC2 of DSG1 and fragment 633 containing only EC1 and EC2 of DSG3 are shown, but it will be understood that any fragment of the extracellular domain may be used. It will be further understood that any permutation of Figures 6A and 6B may be generated such that two fragments from different proteins are present 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 will be understood that any fragment or the entire extracellular domain may be used. As shown in Figure 6E, the single-stranded molecule may also include 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, an amino acid linker can be used to separate any of the domains of the single-stranded molecule. Figure 6G shows embodiments in which two, three, or four fragments are all separated by linker 690, as well as embodiments in which linker 690 also separates the C-terminal fragment 635 from the CH1 domain 614 or a CH2 domain 612. It is also shown that the linker replaces the hinge region, but this is not necessary, and it will be understood that the hinge can be held by a linker that connects the C-terminal fragment 635 to the hinge region. 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 all these various linkers may contain the same sequence or may be constructed from different amino acid sequences.

[0349] Example 2 In the first batch, the complete extracellular domain of DSG1 (SEQ ID NO: 1, lacking the signal peptide but containing the prodomain) or the complete extracellular domain of DSG3 (SEQ ID NO: 3, lacking the signal peptide but containing the prodomain) was transiently expressed in 250 ml of CHO cells (Figure 7A). For easier protein purification, molecules with 8X His tags and AVI tags were generated (SEQ ID NOs: 5-6 for DSG1 and DSG3, respectively). The tags were isolated from the C-terminus of the extracellular domain using the positively charged linker EAAAKEAAAK (SEQ ID NO: 65).

[0350] Further molecules were generated by combining the extracellular domain with the IgG hinge and the CH2 and CH3 domains (SEQ ID NOs. 5 and 6 for DSG1 and DSG3, respectively). The C-terminus of the extracellular domain was linked to the hinge using the (GGGGS)3 linker (SEQ ID NOs. 66, 3 repeats). Similarly, molecules were generated by combining the extracellular domain with the CLκ domain (SEQ ID NOs. 7 and 8 for DSG1 and DSG3, respectively). Molecules were also generated with mutations in the Fc region to promote heterodimerization rather than homodimerization (knob-in-hole approach: DSG3-containing molecule, T366W mutation in the CH3 domain of SEQ ID NOs. 9; DSG1-containing molecule, T366S / L368A / Y407V triple mutation in the CH3 domain of SEQ ID NOs. 10). Two heavy chains intended to heterodimerize were expressed together, as with the DSG3 light chain and DSG1 heavy chain and the DSG1 light chain and DSG3 heavy chain (Figure 7B). The molecules produced are summarized in Table 2, which provides the identifier used throughout this specification for various molecules, the expected molecular weight (MW), the expected isoelectric point (pI, (M-1*cm-1)), the expected extinction coefficient (EC), and the actual yield.

[0351] [Table 3]

[0352] Next, the binding ability of various molecules to actual pathogenic antibodies was determined in serum samples from PV patients. Molecular binding was examined using four human serum samples (titer >20 RU / mL) positive for anti-DSG3 IgG antibody. Autoantibody titers were determined using Euroimmun ELISA. Molecules were biotinylated, bound to avidin-coated Sepharose beads, and serum samples were incubated separately with various DSG-3-containing molecules at gradually increasing concentrations. As shown in Figure 7C, all molecules containing the DSG-3 extracellular domain were able to bind to the anti-DSG3 antibody, regardless of whether the DSG-3 extracellular domain was present in the heavy or light chain, or whether the DSG-1 extracellular domain was present. However, while over 80% antibody depletion was possible in three of the samples, only about 40% depletion was achieved in one subject's sample. CRD-239, an Fc fusion molecule containing the extracellular domain of an unrelated protein, was used as a negative control for both antigen-side and Fc-nonspecific binding, showing no effect on autoantibody levels and demonstrating that binding is antigen-specific and Fc-independent. All serum mean depletion for anti-DSG-3 was nearly equivalent (Figure 7D).

[0353] A concentration of 0.77 μM was found to result in over 90% binding for all molecules tested and was therefore selected for further analysis. In addition to testing DSG3-containing molecules, DSG1-only molecules were also included as negative controls. As expected, all DSG3-containing molecules were able to deplete anti-DSG3 antibodies in all patient serum, while DSG1-only molecules were not (Figure 7E). Molecules with DSG3 fused to the heavy chain yielded better depletion than molecules with DSG3 fused to the light chain.

[0354] A single human serum sample positive for anti-DSG1 IgG antibody (titer > 20 RU / mL) was also used to test the ability of the DSG1 molecule to deplete autoantibodies. Using a reference concentration of 0.77 μM again, the DSG1-containing molecule was able to deplete autoantibody levels (Figure 7F). The assay was repeated multiple times with single samples. Interestingly, the heavy-chain-containing molecule performed better than the molecule without the dimerization domain. Furthermore, the DSG3-containing molecule was comparable to the negative control.

[0355] Example 3 In particular, molecules containing dimerizing domains showed a slight decrease in yield, while heavy-chain heterodimers yielded surprisingly high yields (see Table 2). However, when these transient expressions were repeated on a smaller scale (20 ml scale), both Fc-fusion molecules of DSG1 (see Table 3) and DSG3 (see Table 4) yielded significantly lower yields. These analyses were performed on denatured gels, while on non-reducing gels, smears of very heavy aggregates were observed. Similarly, visual observation of the molecules showed high levels of intermolecular aggregation. This is not surprising, as the purpose of desmogleins is essentially to generate tight junctions. Molecular adhesion led to aggregation, poor isolation, and low yields. To improve this problem, various mutants (mutants) of DSG1 and DSG3 were generated. The mutations were designed to reduce homo- and hetero-adhesion. Truncations containing only a portion of the four EC domains were also investigated (DSG1 EC 1 / 2 / 3, DSG1 EC 2 / 3 / 4, DSG1 EC 1 / 2, DSG1 EC 3 / 4, DSG3 EC 1 / 2 / 3, DSG3 EC 2 / 3 / 4, DSG3 EC 1 / 2, DSG3 EC 3 / 4, SEQ ID NOs. 57-64). A summary of DSG1-containing molecules and their characteristics is shown in Table 3. A summary of DSG3-containing molecules and their characteristics is shown in Table 4.

[0356] [Table 4] TIFF2026516401000006.tif92159

[0357] [Table 5] TIFF2026516401000008.tif65159

[0358] The molecule CRD-291, containing the WT DSG3 extracellular domain, was electrophoresed on a non-reducing gel. A band was clearly present at approximately 160 kDa, the expected weight of the molecule's dimer (Figure 8A). However, smearing was observed even at higher molecular weights, and large amounts of protein were so highly aggregated that they could barely enter the gel. In contrast, the molecule CRD-657, containing mutations that eliminate four cysteines (two cysteines in EC1 and two in EC2), showed a significant reduction in aggregation, and a primary band was observed at the expected molecular weight. Surprisingly, this advantage was only observed when the cysteines were mutated to valine or alanine. However, when the cysteines were converted to serine (CRD-700), no improvement in expression was observed. Calculations of the purity of the resulting monomers showed that CRD-291 and CRD-657 were comparable, each producing approximately 20% monomer (Figure 8B). In contrast, CRD-700 essentially produced no monomer. The molecule CRD-662, a truncation containing only extracellular domains 1 and 2, also showed a significant decrease in aggregates and had a primary band at the expected weight (110 kDa for dimers). Similar results were observed for the DSG1-containing molecules CRD-293 (WT), CRD-674 (with five cysteine ​​mutations, three in EC1 and two in EC2), and CRD-679 (truncation) (Figure 8C). In this case as well, the mutation of cysteine ​​to serine (CRD-701) did not result in improved expression and reduced monomer yield (Figure 8D). These results were also confirmed by SEC-HPLC (data not shown). Cysteine ​​mutations within the truncated molecules resulted in even better yields (CRD-923 and CRD-924 in Tables 3 and 4).

[0359] Indeed, as seen in Tables 3 and 4, some, though not all, mutations / truncations resulted in more robust yields. In the case of DSG1, the W28A mutation nearly doubled the yield produced, while the D53T and E117S mutations did not increase the yield. Combining all three mutations negated the effect of the W28A mutation. However, the combination of the W28A mutation and the A106I mutation yielded an even better effect on yield, which was consistent only with the K43A, R44A double mutation. Several other mutation combinations also yielded more modest improvements. Surprisingly, the quintuple cysteine ​​mutation within the EC1-EC2 fragment resulted in one of the best yields.

[0360] In the case of DSG3, the W28A mutation did not affect the yield, but the E117S mutation increased the yield by more than four times. Triple mutations of W28A, D53T, and E117D, and double mutations of W28A and A106I both resulted in increases of more than three times, but the double mutation of K43A, R44A was by far the best, resulting in a seven-fold increase in yield. Truncation of DSG3 also resulted in yield improvements. Removal of the fourth extracellular domain (EC4) resulted in moderate improvement, and expression of EC1+EC2 alone or EC3+EC4 alone resulted in even better yields. Truncation removing only EC1 resulted in even better yields. Interestingly, truncation of DSG1 expressed alone did not improve the yield and actually resulted in a lower yield than the entire unmodified extracellular domain. In the case of DSG3, quadruple cysteine ​​mutations within the EC1-EC2 fragment also resulted in surprisingly high yields; in fact, in this batch, all the DSG3 proteins tested yielded the highest yields.

[0361] We investigated combinations incorporating both DSG1 and DSG3 with several variants and truncations. Molecules containing both WT DSG1 and WT DSG3 extracellular domains (CRD-294) generated high molecular weight smears on non-reducing gels (Figure 8E). The use of truncated forms of both DSG1 and DSG3 (only the EC1 and EC2 domains, respectively; CRD-685) helped reduce aggregate formation and generated primary bands at the expected molecular weights. These results were also confirmed by SEC-HPLC (data not shown). The complete molecular generation results are summarized in Table 5. Both inclusion of the W28A mutation and removal of the EC4 domain resulted in moderate improvements in yield, but inclusion of only EC1 and EC2 was exceptionally good, and when combined with the W28A mutation, a more than fourfold increase in yield was observed. Here again, truncation of EC1-EC2 with all cysteine ​​mutations yielded by far the best yield. This was true for both molecules combining DSG1 EC1-EC2 truncation and DSG3 EC1-EC2 truncation (CRD-925, CRD-926). Several other mutations tested did not yield improved results.

[0362] [Table 6]

[0363] Autoantibody depletion using novel molecules was tested as before. Five human serum samples positive for anti-DSG1 IgG were depleted using a DSG1 / DSG3 knob-in-hole combination molecule (CRD-685) and a DSG1-only molecule with lower aggregation and higher yield (CRD-673). Both molecules resulted in greater depletion than an Fc fusion molecule with an unrelated extracellular domain (CRD-753), with the DSG1-only molecule resulting in over 40% depletion (Figure 9A). Similarly, six human serum samples positive for anti-DSG3 IgG were depleted using DSG1 EC1 / 2 and DSG3 EC1 / 2 fragments combined via a knob-in-hole approach (CRD-685), a DSG3 EC1 / 2 fragment Fc fusion molecule (CRD-662), and DSG-3 ECD EC1~EC4 molecules (CRD-290). Both CRD-685 and 662 were able to cause moderate depletion, but CRD-290 depleted approximately 50% of the antibody (Figure 9B).

[0364] Next, eight human serum samples positive for anti-DSG3 IgG were depleted using the following DSG3 molecules: a DSG3 molecule with a complete extracellular domain and no dimerization domain (CRD-290); a DSG3 molecule with a complete extracellular domain and CH2 and CH3 (CRD-291); a DSG3 molecule with a complete extracellular domain, all cysteines in EC1 and EC2 mutated, and CH2-CH3 (CRD-657); a DSG3 molecule containing only EC3 and EC4 and CH2-CH3 (CRD-664); a DSG3 molecule containing only EC1 and EC2, all cysteines in EC1 and EC2 mutated, and CH2-CH3 (CRD-924); and two DSG3 / DSG1 knob-...

Claims

1. A polypeptide comprising a fragment of the extracellular domain of desmoglein 1 (DSG1) or desmoglein 3 (DSG3), and at least one mutation that increases the solubility of the fragment, decreases the aggregation of the fragment, or both.

2. The polypeptide according to claim 1, wherein at least one of the mutations is located in the cadherin domain or the calcium-binding domain.

3. The polypeptide according to claim 1 or 2, wherein the extracellular domain of DSG1 comprises SEQ ID NO: 2, and the mutation is selected from C9V, C28A, C78V, C204A, C206V, W2A, D27T, E89S, A80I, K17A, R18A, R10A, R97A, C9S, C28S, C78S, C204S, C206S, C426S, E69G, D103G, D136G, E179G, D215G, D247G, E231G, E291G, E332G, N61Q, and N131Q.

4. The aforementioned at least one mutation is a. C9V, C28A, C78V, C204A, and C206V; b. W2A; c. D27T; d. E89S; e. W2A, D27T, and E89S; f. W2A and A80I; g. K17A and R18A; h. R10A, K17A, R18A, and R97A; i. C9V, C28A, C78V, C204A, C206V and C426S; j. E69G, D103G, and D136G; k. E179G, D215G and D247G; l. E231G, E291G, and E332G; m. N61Q and N131Q; or n. C9S, C28S, C78S, C204S and C206S The polypeptide according to claim 3.

5. The polypeptide according to claim 1 or 2, wherein the extracellular domain of the DSG3 comprises SEQ ID NO: 4, and the mutation is selected from C9V, C78V, C202A, C204V, W2A, D27T, E89S, A80I, K17A, R18A, R10A, K97A, C9S, C78S, C202S, C204S, E69G, D103G, D136G, E179G, D213G, D245G, E229G, E289G, E330G, N61Q, and N131Q.

6. The aforementioned at least one mutation is a. C9V, C78V, C202A, and C204V; b. W2A; c. E89S; d. W2A, D27T, and E89S; e. W2A and A80I; f. K17A and R18A; g. R10A, K17A, R18A, and K97A; h. E69G, D103G and D136G; i. E179G, D213G, and D245G; j. E229G, E289G, and E330G; k. N61Q and N131Q; or l. C9S, C78S, C202S, and C204S The polypeptide according to claim 3.

7. The polypeptide according to any one of claims 1 to 6, wherein the fragment consists of extracellular domains (ECs) 1 and 2 of DSG1 or DSG3, the polypeptide lacks extracellular domains (ECs) 3 and 4 of DSG1 or DSG3, and EC1 of DSG1 consists of SEQ ID NO: 114, EC2 of DSG1 consists of SEQ ID NO: 115, EC3 of DSG1 consists of SEQ ID NO: 116, EC4 of DSG1 consists of SEQ ID NO: 117, EC1 of DSG3 consists of SEQ ID NO: 118, EC2 of DSG3 consists of SEQ ID NO: 119, EC3 of DSG3 consists of SEQ ID NO: 120, and EC4 of DSG3 consists of SEQ ID NO:

121.

8. The polypeptide according to any one of claims 1 to 7, wherein the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs. 74-85, 90-103 and 108-113, and optionally the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs. 81, 82, 90, 100 and 108.

9. A polypeptide comprising a fragment of the extracellular domain of desmoglein 1 (DSG1) or desmoglein 3 (DSG3), wherein the extracellular domains 3 and 4 of DSG1 or DSG3 are lacking, and the EC3 of DSG1 is sequence number 116, the EC4 of DSG1 is sequence number 117, the EC3 of DSG3 is sequence number 120, and the EC4 of DSG3 is sequence number 121.

10. The polypeptide according to claim 9, wherein the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs. 87, 90-91, 105, and 108-109, and optionally comprises or consists of an amino acid sequence selected from SEQ ID NOs. 90 and 108.

11. The polypeptide according to any one of claims 1 to 10, further comprising an effector portion.

12. The polypeptide according to claim 11, wherein the effector portion is not an Fc domain.

13. The polypeptide according to claim 11, wherein 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).

14. The polypeptide according to any one of claims 11 to 13, wherein the effector portion can induce cell death in cells bound to the fragment.

15. The polypeptide according to claim 14, wherein the effector portion is selected from an Fc domain containing at least one mutation that increases ADCC or CDC, amatoxin / amanitin, anthracycline, anthramycin dimer, calicheamycin, camptothecin or its analogues, duocalmycin, triptolide, and a tubulin inhibitor.

16. The polypeptide according to any one of claims 11 to 15, wherein the effector portion is selected from α-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

17. The polypeptide according to any one of claims 11 to 15, wherein the effector portion is an Fc domain containing sequence number 164 or sequence number 165, and the sequence number 164 or sequence number 165 contains a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E.

18. The polypeptide according to any one of claims 11 to 17, wherein the effector portion is conjugated to the polypeptide by a linker.

19. a. A first polypeptide comprising a fragment of the extracellular domain of desmoglein 1 (DSG1) or an analog or derivative thereof, a fragment of the extracellular domain of desmoglein 3 (DSG3) or an analog or derivative thereof, or both, and a first dimerization domain; and b. A second polypeptide comprising a fragment of DSG1 or an analog or derivative thereof, a fragment of DSG3 or an analog or derivative thereof, or both, and a second dimerization domain; A composition comprising the first and second dimerizing domains configured to dimerize with each other.

20. The composition according to claim 19, wherein the dimerization comprises the formation of a covalent bond between the first dimerized domain and the second dimerized domain.

21. The composition according to claim 19 or 20, wherein the protein complex comprises an immunoglobulin scaffold.

22. a. The first dimerization domain comprises a first hinge domain of the immunoglobulin heavy chain, the second dimerization domain comprises a second hinge domain of the heavy chain, and the first and second dimerization domains dimerize via 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 a CH1 domain, nor both contain a CL domain. The composition according to any one of claims 19 to 21.

23. The composition according to any one of claims 19 to 22, wherein the fragments of the first, second, or both polypeptide chains and the dimerizing domains are separated by a linker.

24. The composition according to any one of claims 19 to 23, wherein the first polypeptide chain, the second polypeptide chain, or both further comprise the Fc region of a human antibody heavy chain.

25. The composition according to claim 24, wherein the Fc region can induce cytotoxicity against cells bound to the protein complex.

26. The composition according to claim 24 or 25, 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.

27. The composition according to claim 26, wherein the first CH3 domain comprises at least a first mutation, and the second CH3 domain comprises at least a second mutation, wherein the mutations enable heterodimerization of the first and second polypeptide chains and inhibit homodimerization of the first polypeptide chain and homodimerization of the second polypeptide chain.

28. The composition according to claim 26 or 27, 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 the second polypeptide chain.

29. The composition according to claim 27 or 28, wherein the first mutation is selected from the mutations provided in Table 1, and the second mutation is a mutation provided in Table 1 and is a mutation corresponding to the first mutation.

30. The composition according to claim 29, 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.

31. The composition according to any one of claims 24 to 30, wherein the Fc region of the first, second, or both of the polypeptide chains is separated from the fragment or the dimerization domain by a linker.

32. The composition according to any one of claims 24 to 31, wherein the Fc region comprises at least one mutation that increases ADCC or CDC.

33. The composition according to claim 32, wherein the Fc region is an Fc region containing sequence number 164 or sequence number 165, and the sequence number 164 or sequence number 165 contains a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E.

34. The composition according to any one of claims 24 to 33, wherein the dimerization domains of the first, second, or both of the polypeptide chains are located on the C-terminal side of the fragment and on the N-terminal side of the Fc region.

35. A composition according to any one of claims 19 to 34, lacking an antibody variable domain.

36. The composition according to any one of claims 19 to 35, further comprising a third polypeptide comprising a fragment of DSG1 or an analog or derivative thereof, a fragment of DSG3 or an analog or derivative thereof, or both thereof, 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.

37. 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 dimerize via disulfide bonds; or b. The composition according to claim 36, wherein the third and fourth dimerizing domains each comprise a domain selected from the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and are dimerized by a disulfide bond, and neither the first nor the third polypeptide contains the CH1 domain or the CL domain.

38. The composition according to claim 36 or 37, further comprising a fourth polypeptide comprising a fragment of DSG1 or an analog or derivative thereof, a fragment of DSG3 or an analog or derivative thereof, or both thereof, and a fifth dimerization domain, wherein the second polypeptide further comprises a sixth dimerization domain, and the fifth and sixth dimerization domains can dimerize with each other.

39. 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 dimerize via disulfide bonds; or b. The composition according to claim 38, wherein the fifth and sixth dimerizing domains each comprise a domain selected from the CH1 domain of the heavy chain of an immunoglobulin and the CL domain of the light chain of an immunoglobulin, and are dimerized by a disulfide bond, and neither the first nor the third polypeptide contains the CH1 domain or the CL domain.

40. The composition according to any one of claims 19 to 39, wherein neither the first polypeptide nor the second polypeptide contains a CH1 domain, nor both contain a CL domain.

41. The composition according to any one of claims 36 to 40, 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 and the fifth and sixth dimerizing domains, and that 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.

42. The composition according to any one of claims 19 to 41, wherein the first polypeptide comprises a fragment of DSG1 or an analog or derivative thereof, and the second polypeptide comprises a fragment of DSG3 or an analog or derivative thereof.

43. The composition according to any one of claims 19 to 42, wherein the first polypeptide chain or the second polypeptide chain comprises both a fragment of DSG1 or an analog or derivative thereof and a fragment of DSG3 or an analog or derivative thereof.

44. The composition according to claim 43, wherein the aforementioned fragments are separated by an amino acid linker.

45. The composition according to any one of claims 19 to 44, wherein DSG1 lacks a prodomain and contains or consists of SEQ ID NO: 2, DSG3 lacks a prodomain and contains or consists of SEQ ID NO: 4, or DSG1 contains a prodomain and contains or consists of SEQ ID NO: 1, and DSG3 contains a prodomain and contains or consists of SEQ ID NO:

3.

46. The composition according to any one of claims 19 to 45, wherein the extracellular domain fragment consists of a truncation of the extracellular domain lacking an extracellular functional domain containing a sequence selected from sequence numbers 114 to 121.

47. The composition according to claim 46, wherein the fragment lacks the extracellular domains 3 and 4 of DSG1 or DSG3, the EC3 of DSG1 is sequence number 116, the EC4 of DSG1 is sequence number 117, the EC3 of DSG3 is sequence number 120, and the EC4 of DSG3 is sequence number 121.

48. The composition according to claim 46 or 47, wherein the fragment comprises or consists of the extracellular domains (ECs) 1 and 2 of DSG1 or DSG3 or analogs or derivatives thereof, wherein EC1 of DSG1 is sequence number 114, EC2 of DSG1 is sequence number 115, EC1 of DSG3 is sequence number 118, and EC2 of DSG3 is sequence number 119.

49. The composition according to claim 48, wherein the fragment comprises an amino acid sequence selected from SEQ ID NOs: 87, 90-91, 105, and 108-109, or a derivative thereof having at least 85% identity.

50. The composition according to any one of claims 19 to 49, wherein at least one of the fragments comprises a mutation in the cadherin domain or the calcium-binding domain.

51. The composition according to claim 50, wherein the mutation increases the solubility of the composition, reduces the aggregation of the complex, or both.

52. The DSG1 extracellular domain contains Sequence ID No. 2, and the mutation is selected from C9V, C28A, C78V, C204A, C206V, W2A, D27T, E89S, A80I, K17A, R18A, R10A, R97A, C9S, C28S, C78S, C204S, C206S, C426S, E69G, D103G, D136G, E179G, D215G, D247G, E231G, E291G, E332G, N61Q and N131Q, or The composition according to claim 50 or 51, wherein the DSG3 extracellular domain comprises SEQ ID NO: 4, and the mutation is selected from C9V, C78V, C202A, C204V, W2A, D27T, E89S, A80I, K17A, R18A, R10A, K97A, C9S, C78S, C202S, C204S, E69G, D103G, D136G, E179G, D213G, D245G, E229G, E289G, E330G, N61Q, and N131Q.

53. The aforementioned at least one mutation is a. C9V, C28A, C78V, C204A and C206V of Sequence ID No. 2; b. C9V, C78V, C202A, and C204V of Sequence ID No. 4; c. W2A of sequence number 2 or 4; d. D27T of Sequence ID 2; e. E89S of sequence number 2 or 4; f. W2A, D27T, and E89S of sequence number 2 or 4; g. W2A and A80I of Sequence ID No. 2 or 4; h. K17A and R18A of Sequence ID No. 2 or 4; i. Sequence ID No. 2 R10A, K17A, R18A and R97A; j. R10A, K17A, R18A and K97A of Sequence ID No. 4; k. C9V, C28A, C78V, C204A, C206V and C426S of Sequence ID No. 2; l. E69G, D103G, and D136G of sequence numbers 2 or 4; m. Sequence ID No. 2, E179G, D215G, and D247G; n. Sequence ID No. 4, E179G, D213G, and D245G; o. Sequence ID No. 2, E231G, E291G, and E332G; p. Sequence ID No. 2, E229G, E289G, and E330G; q. Sequence IDs N61Q and N131Q of sequence numbers 2 or 4; r. C9S, C28S, C78S, C204S and C206S of Sequence ID No. 2; or s. Sequence ID No. 4 C9S, C78S, C202S and C204S The composition according to claim 52.

54. The composition according to any one of claims 50 to 53, wherein the fragment comprises or consists of an amino acid sequence selected from SEQ ID NOs. 74 to 85, 90 to 103 and 108 to 113.

55. The composition according to any one of claims 19 to 54, wherein the analog or derivative comprises at least 85% identity with DSG1 or DSG3.

56. The composition according to any one of claims 19 to 55, wherein the fragment comprises at least 20 consecutive amino acids derived from DSG1 or DSG3.

57. The composition according to any one of claims 19 to 56, wherein the fragment comprises at least one B cell receptor (BCR) specific epitope target of an autoantibody.

58. The composition according to any one of claims 19 to 57, comprising a first or second polypeptide containing a sequence selected from sequence numbers 5 to 73.

59. The composition according to any one of claims 14 to 48, wherein the hinge domain, the CH2 domain, or the CH3 domain contains at least one mutation that reduces ADCC.

60. The at least one mutation that reduces ADCC, a. Mutations of the hinge domain, including the L19A and L20A mutations of Sequence ID No. 125; and b. Mutations in the CH2 domain, including the N59A mutation of Sequence ID No. 139 A composition according to claim 59, selected from the following.

61. The composition according to any one of claims 19 to 60, further comprising at least one effector portion capable of inducing cell death in cells bound to the composition.

62. The composition according to claim 61, wherein the effector portion is not an Fc domain.

63. The composition according to claim 62, wherein the effector portion is selected from α-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

64. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 18 or a composition according to any one of claims 19 to 63, and a pharmaceutically acceptable carrier, excipient, or adjuvant.

65. A method for treating pemphigus vulgaris or pemphigus foliace in a subject requiring treatment for pemphigus vulgaris or pemphigus foliace, comprising administering to the subject a composition comprising a fragment of the extracellular domain of DSG1 or DSG3 or an analog or derivative thereof, thereby treating pemphigus vulgaris or pemphigus foliace.

66. The method according to claim 65, wherein DSG1 lacks a prodomain and includes or consists of SEQ ID NO: 2, DSG3 lacks a prodomain and includes or consists of SEQ ID NO: 4, or DSG1 includes a prodomain and includes or consists of SEQ ID NO: 1, and DSG3 includes a prodomain and includes or consists of SEQ ID NO:

3.

67. The method according to claim 65 or 66, wherein the composition comprises the polypeptide described in any one of claims 1 to 18.

68. The method according to any one of claims 65 to 67, wherein the composition is the composition according to any one of claims 19 to 63.

69. The method according to any one of claims 65 to 68, wherein the composition is the pharmaceutical composition described in claim 64.

70. The method according to any one of claims 65 to 69, further comprising reducing the level of circulating antibodies against DSG1, DSG3, or both, in the subject.

71. The method according to any one of claims 65 to 70, wherein the treatment comprises reducing the concentration of circulating autoantibodies against DSG1, DSG3, or both.

72. The method according to any one of claims 65 to 71, wherein the composition comprises an Fc region, and the treatment comprises killing B cells that produce anti-DSG1 or anti-DSG3 autoantibodies.

73. The method according to claim 72, wherein the B cells are autoreactive B cells that produce autoantibodies against the fragments of the composition.

74. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 18.

75. 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 19 to 63, and the second nucleic acid molecule encodes the second polypeptide of the composition according to any one of claims 19 to 63.

76. The nucleic acid system according to claim 75, further comprising a third nucleic acid molecule encoding the third polypeptide of the composition according to any one of claims 37 to 63, a fourth nucleic acid molecule encoding the fourth polypeptide of the composition according to any one of claims 39 to 63, or both.

77. A method for producing a polypeptide according to any one of claims 1 to 18 or a composition according to any one of claims 19 to 63, comprising expressing a nucleic acid system according to claim 75 or 76 in a cell, wherein the nucleic acid system is configured to produce the encoded polypeptide in the cell, thereby producing a polypeptide according to any one of claims 1 to 18 or a composition according to any one of claims 19 to 63.

78. A method for producing protein, A step to obtain a first fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof, and a second fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof. A step of linking the first fragment to a first dimerization domain to generate a first polypeptide chain, linking the second fragment to a second dimerization domain to generate a second polypeptide chain, so that the first and second dimerization domains can dimerize 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 i. To obtain a first fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof, and a second fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; and ii. The first fragment can be linked to a first dimerization domain to generate a first polypeptide chain, the second fragment can be linked to a second dimerization domain to generate a second polypeptide chain, and the first and second dimerization domains can dimerize with each other; The process generated by A method that includes and thereby produces protein.

79. The method according to claim 78, wherein the protein complex is a protein complex of the composition according to any one of claims 19 to 61.

80. a. A step of linking a third dimerization domain to the first dimerization domain or the first fragment in the first polypeptide chain; a step of obtaining a third fragment or analogue or derivative of the extracellular domain of DSG1 or a fragment or analogue or derivative of the extracellular domain of DSG3, and linking the third fragment to a fourth dimerization domain to generate a third polypeptide chain, wherein the third dimerization domain and the fourth dimerization domain can dimerize with each other; and a step of contacting the first, second and third polypeptides under conditions sufficient to induce the dimerization; or b. A step of expressing a nucleic acid sequence encoding a third polypeptide chain in the host cell, wherein the third polypeptide chain is i. To obtain a third fragment of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; and ii. Linking the third fragment to the fourth dimerization domain to generate a third polypeptide chain. The process generated by The method according to claim 78 or 79, further comprising, wherein 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.

81. a. A step of linking the sixth dimerization domain to the second dimerization domain or the second fragment in the second polypeptide chain; a step of obtaining a fourth fragment or analogue or derivative of the extracellular domain of DSG1, or a fragment or analogue or derivative of the extracellular domain of DSG3, and linking the fourth fragment to the fifth dimerization domain to produce a fourth polypeptide chain, such that the fifth dimerization domain and the sixth dimerization domain can dimerize with each other; and a step of contacting the first, second, third and fourth polypeptides under conditions sufficient to induce the dimerization; or b. A step of expressing a nucleic acid sequence encoding a fourth polypeptide chain in the host cell, wherein the fourth polypeptide chain is i. To obtain a fourth fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; and ii. Linking the fourth fragment to the fifth dimerization domain to generate a fourth polypeptide chain. The process generated by 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 80.

82. The method according to any one of claims 78 to 81, further comprising the steps of generating at least one mutation in the cadherin domain of the protein, or truncating the protein to remove at least one cadherin domain or a portion thereof.

83. The method according to claim 82, further comprising the steps of measuring the aggregation of the mutant or truncated protein, and selecting the mutant or truncated protein in which aggregation has been reduced.

84. A method for producing protein, a. A step of obtaining a fragment of the extracellular domain of DSG1 or an analog or derivative thereof, or a fragment of the extracellular domain of DSG3 or an analog or derivative thereof; b. A step of generating a mutant fragment by causing at least one mutation in the aforementioned fragment; c. A step of measuring the solubility, aggregation, or both of the mutant fragments; and d. A step of selecting at least one mutant fragment that has increased solubility, decreased aggregation, or both, compared to the obtained fragments. A method that includes and thereby produces protein.

85. The method according to any one of claims 78 to 83, further comprising the step of linking an effector portion to at least one of the polypeptide chain or the mutant fragment, wherein the effector portion can kill cells bound to the at least one polypeptide.

86. The method according to claim 85, wherein the effector portion is not an Fc domain.

87. The method according to claim 86, wherein the effector portion is selected from α-amanitin, PNU-159682, tesirin, deruxtecan (Dxd), meltansine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and combinations thereof.

88. The method according to claim 85, wherein the effector portion is an Fc domain containing at least one mutation that increases ADCC or CDC.

89. The method according to claim 88, wherein the effector portion is an Fc domain containing sequence number 164 or sequence number 165, and the sequence number 164 or sequence number 165 contains a plurality of mutations selected from L15V / F23L / R72P / Y80L / P176L, S19D / A110L / I112E, G16A / A110L / I112E, and G16A / S47E / H48F / S104T / I112E.

90. A protein produced by the method described in any one of claims 78 to 89.

91. A method for determining the suitability of an object to be treated by the method of any one of claims 65 to 73, comprising the steps of: obtaining a sample from the object; contacting the sample with a protein according to any one of claims 1 to 18, 90 or a composition according to any one of claims 19 to 63; 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 65 to 73, and thereby determining the suitability of the object to be treated.

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