Methods and Compositions for Treating Eosinophil-Driven Diseases and Disorders - Patent application

Abstract

The present disclosure relates, in part, to compositions comprising anti-CD39 antibodies for use in methods for reducing eosinophil cells or function and / or methods for treating diseases or conditions associated with unwanted eosinophil activity.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 336,418, filed April 29, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing The specification includes a Sequence Listing that has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. The XML file, created on April 7, 2023, is named PNC-00325_SL.xml and is 86,881 bytes in size. [Background technology]

[0003] Recent studies have shown that eosinophils are also involved in several homeostatic processes, such as metabolism, tissue remodeling, and development, neuronal regulation, epithelial and microbiome regulation, and immune regulation, indicating that these cells may play an important role in metabolic regulation and organ function in healthy individuals.

[0004] Eosinophils play a homeostatic role in the body's immune response. These cells are involved in fighting some parasitic, bacterial, and viral infections, as well as certain cancers, and have a pathological role in inflammatory disorders and diseases, including asthma, sinusitis, eosinophilic gastrointestinal disorders, and eosinophilic syndrome.

[0005] Treatment of eosinophilic diseases has traditionally been achieved by nonspecifically attenuating eosinophils using glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4 / IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory diseases may not achieve adequate symptom control, highlighting the need for further investigation of the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity.

[0006] Esophageal inflammatory disorders, such as eosinophilic esophagitis (EoE), a disease characterized by high levels of eosinophils and thickened basal zones in the esophagus, are increasingly being diagnosed in children and adults. Many aspects of this disease remain unclear, including its etiology, natural history, and optimal treatment. EoE can occur in all age groups, but most frequently affects individuals aged 20 to 50. Symptoms of EoE are often similar to those of gastroesophageal reflux disease (GERD) and include vomiting, dysphagia, pain, and food impaction. This condition is painful, causes difficulty swallowing, and predisposes patients to other complications. EoE is often misdiagnosed as GERD, leading to delays in appropriate treatment for EoE patients. Currently, no locally administered anti-inflammatory drugs are approved for the treatment of conditions associated with inflammation in the upper gastrointestinal tract, particularly esophageal inflammatory conditions such as EoE. Although systemic treatment with corticosteroids such as prednisolone is effective, these agents are associated with significant side effects, including suppression of the hypothalamic-pituitary-adrenal (HPA) axis, as reflected in salivary cortisol levels, and general suppression of immune function; troublesome side effects from long-term systemic exposure, particularly in children, include growth retardation.

[0007] Furthermore, some drugs have been reported to cause eosinophilia, so-called "drug-induced eosinophilia." For example, immune checkpoint inhibitors (ICIs) are a new standard of care for some cancers. However, ICIs are also associated with frequent, potentially organ- or life-threatening immune-related adverse events (irAEs), including eosinophilic asthma and hypereosinophilic disorders, which generally mimic autoimmune or inflammatory conditions. Other drugs associated with drug-induced eosinophilia that have been reported to cause severe reactions include, but are not limited to, antimalarials (e.g., pyrimethamine and dapsone), penicillins, glycopeptides, cephalosporins, sulfonamides, tetracyclines (especially minocycline), nitrofurantoin, antituberculosis therapy, ACE inhibitors, tryptophan, anticonvulsants (e.g., phenytoin, carbamazepine, and phenobarbitone), NSAIDs, gold, H2 receptor antagonists, proton pump inhibitors, aminosalicylates, and chlorpropamide. Summary of the Invention

[0008] Eosinophils can regulate local immune and inflammatory responses, and their accumulation in blood and tissues is associated with several inflammatory and infectious diseases. Therefore, treatments targeting eosinophils may be useful for controlling a variety of diseases, including atopic disorders such as asthma and allergies, as well as diseases not primarily associated with eosinophils, such as autoimmunity and malignant tumors. The present invention relates to a therapeutic agent targeting eosinophils.

[0009] The present invention is based on the observation that eosinophils express CD39 and can be targeted by CD39-targeted eosinophil-depleting agents, such as ADCC (antibody-dependent cellular cytotoxicity)-competent and / or ADCP (antibody-dependent cellular phagocytosis)-competent anti-CD39 antibodies or CD39-targeted cytotoxic drug conjugates, to reduce the levels of eosinophils and / or eosinophil function either systemically or locally (or both). Thus, these CD39-targeted eosinophil-depleting agents have utility for use as part of the treatment of diseases in which abnormal activation or excess of eosinophils is part of the pathology, including, for example, inflammatory or autoimmune diseases and systemic or localized eosinophilia or drug-induced eosinophilia. Use as part of a therapeutic approach to prevent rejection of tissue transplants (autologous or allogeneic) is also contemplated.

[0010] With the exception of the PEO22 antibody described herein, current clinical use of anti-CD39 antibodies focuses on inhibiting the ectonucleotidase activity of CD39, i.e., aiming to reduce intratumoral levels of the enzyme activity associated with that protein, thereby lowering intratumoral levels of the immunosuppressant adenosine. These antibodies have been selected to lack any CD39-dependent cell-depleting activity, i.e., they have been deliberately selected to lack ADCC and / or ADCP function. Again, the purpose of these prior art antibodies is to inhibit the enzyme activity of CD39 so as not to kill cells expressing CD39, and the formats in which they are used do not have the eosinophil-level depletion potential of the antibodies contemplated for use in the methods and formulations of the present invention.

[0011] However, in the context of the present invention, those versions of the methods and formulations of the present invention that utilize anti-CD39 antibodies that retain ADCC and / or ADCP function, when bound to eosinophils, result in ADCC- and / or ADCP-mediated clearance of those cells. Such anti-CD39 antibodies can, by way of example, be monovalent or multivalent (including bivalent) for CD39. However, the present invention also provides bispecific antibodies that, in addition to one or more CD39-binding moieties, contain additional binding moieties that bind to one or more cell surface epitopes expressed by eosinophils.

[0012] Numerous embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one aspect, an anti-CD39 antibody or antigen-binding fragment thereof is provided that includes: (i) at least one antigen-binding domain that binds to ectonucleoside triphosphate diphosphohydrolase-1 (CD39) at a site such that the anti-CD39 antibody forms a stable immune complex; and (ii) an FcγRIIIa-binding portion that binds to the FcγRIIIa receptor and confers ADCC and / or ADSP activity against CD39+ cells to the anti-CD39 antibody.

[0013] Numerous further embodiments are provided that can be applied to aspects of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the eosinophil cells are CD39+ eosinophil cells. In some embodiments, the CD39+ eosinophil cells are characterized by the expression of (i) CD45, CD11b, Siglec-8, the alpha subunit of the IL-5 receptor (IL-5Rα or CD125), the alpha subunit of the IL-3 receptor (IL-3Rα or CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), paired immunoglobulin-like receptor A (PIRA), paired immunoglobulin-like receptor B (PIRB), L-selectin, murine eosinophils, and / or eosinophils. (ii) co-express one or more cell surface markers selected from the group consisting of chemoattractant receptor (EMR) 1, CCR3 (CD193), and a chemoattractant receptor-like molecule expressed on type 2 helper T cells (also called CRTh2, DP2, type 2 prostaglandin D2 receptor, or CD294); (ii) are CD45+CD11b+ eosinophil cells; and / or (iii) are CD45+CD11b+Siglec-8+ eosinophil cells. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof (i) upon incubation with HCC1739BL cells forms stable immune complexes characterized by loss of less than 40% of the immune complex after 24 hours, or less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or even less than 10% of the immune complex after 24 hours, and optionally, the formation of the immune complex is detected by fluorescence intensity using a fluorescently labeled secondary antibody (e.g., by way of example only, the stability of immune complexes formed with an anti-CD39 antibody can be measured using an anti-CD39 monoclonal antibody). (ii) depletion of CD39+ eosinophils; (iii) binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 30; and / or (iv) binding to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody clone A1, which binds to CD39.In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof promotes depletion of CD39+ eosinophils via ADCC-mediated killing and / or ADCP-mediated killing. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof promotes depletion of CD39+ eosinophils in the form of an antibody-drug conjugate that is taken up by and toxic to CD39+ eosinophils.

[0014] In another embodiment, the FcγRIIIa binding portion is selected from the group consisting of an Fc domain, an antibody or fragment thereof that binds to FcγRIIIa, and an FcγRIIIa binding peptide.

[0015] In yet another embodiment, the antigen-binding domain is selected from the group consisting of a Fab, Fab', F(ab')2, Fv or single-chain Fv (scFv), Fav, dsFv, sc(Fv)2, Fde, sdFv, single-domain antibody (dAb), and diabody fragment, and / or the anti-CD39 antibody or antigen-binding fragment is monoclonal. In some embodiments, the antigen-binding domain is an scFv comprising the sequence of SEQ ID NO:40.

[0016] In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises a VH domain having an amino acid sequence that can be encoded by the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid that hybridizes to the nucleic acid of SEQ ID NO: 1 under stringent conditions, and a VL domain having an amino acid sequence that can be encoded by the nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid that hybridizes to the nucleic acid of SEQ ID NO: 3 under stringent conditions (e.g., hybridization in 6x sodium chloride / sodium citrate (SSC) at 45°C, followed by washing in 0.2x SSC / 0.1% SDS at 50-65°C). In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the CDRs of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54. and a light chain having CDRs that are at least 60% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the CDRs of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54. and a variable light chain (VL) that is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises: (i) a CDR1 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 29; , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 31, and a CDR3 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 31. and (ii) a CDR1 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 32, and at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 33. and a light chain having a CDR2 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 34, and a CDR3 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 34.In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises a heavy chain having CDRs selected from the group consisting of CDRs of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54, a light chain having CDRs selected from the group consisting of CDRs of SEQ ID NOs: 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and human framework sequences, forming humanized heavy and light chains having an antigen-binding site capable of specifically binding to human CD39. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises (i) a heavy chain variable domain comprising a CDRH1 having the sequence of SEQ ID NO: 29, a CDRH2 having the amino acid sequence of SEQ ID NO: 30, and a CDRH3 having the sequence of SEQ ID NO: 31; and (ii) a light chain variable domain comprising a CDRL1 having the sequence of SEQ ID NO: 32, a CDRL2 having the sequence of SEQ ID NO: 33, and a CDRL3 having the sequence of SEQ ID NO: 34. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises a heavy chain having CDRs selected from the group consisting of CDRs of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54, a light chain having CDRs selected from the group consisting of CDRs of SEQ ID NOs: 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and human framework sequences, forming humanized heavy and light chains with antigen-binding sites capable of specifically binding to human CD39.

[0017] In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises an Fc domain of the IgG1 or IgG3 isotype, and optionally, the Fc domain is human. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is hypofucosylated or defucosylated.

[0018] In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is human or humanized.

[0019] In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is bispecific, comprising at least one additional antigen-binding site for an eosinophil antigen.

[0020] In a specific embodiment, the invention provides bispecific antibodies that bind to both CD39 and an eosinophil cell surface marker selected from Siglec-8, the alpha subunit of the IL-5 receptor (IL-5Rα or CD125), the alpha subunit of the IL-3 receptor (IL-3Rα or CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), PIRA, PIRB, L-selectin, EMR1, CCR3 (CD193), and CRTh2 (CD294), and cause eosinophil depletion, preferably by ADCC- and / or ADCP-mediated killing, or in the form of an antibody-drug conjugate that is preferentially taken up by and toxic to eosinophils.

[0021] In other embodiments, bispecifics are generated using binding domains for antigens upregulated on activated eosinophils (or eosinophils found in lesions) with bispecific antibody affinity for different antigens (CD39 and a second antigen), both of which are expressed on eosinophils, providing selectivity. Exemplary antigens for generating bispecifics using the CD39 binders of the invention include CD3, CD4, γδ TCR, CD9, CD28, CD29, CD40, CD44, CD45, CD45RO, CD48, CD58, CD63 (lysosome-associated membrane protein 3), CD66b (CEACAM8), CD66e (CEACAM5), CD67, CD69, CD80, CD86, C5αR (CD88), CD101, CD122, CD137 (tumor necrosis factor receptor superfamily member 9, induced by lymphocyte activation, 4-1BB), CD274 (programmed death ligand 1), α IIbIntegrin (CD41), α2 integrin (CD49b), α4 integrin (CD49d), αL integrin (CD11a), αM integrin (CD11b), αX integrin (CD11c), αD integrin, β2 integrin (CD18), aminopeptidase N (CD13), FcαRI (CD89), FcγRIII (CD16), FcγRII (CD32), FcεRII (CD23), granulocyte-monocyte colony-stimulating factor Rα (CD116), HLA-DR, intercellular indirect Antibody-drug conjugates that bind to eosinophils include CD54, interleukin (IL)-2Rα (CD25), IL-17RA, IL-17RB, galectin-3, neuropeptide S receptor, P-selectin glycoprotein ligand-1 (CD162), semaphorin 7A (CD108), thymic stromal lymphopoietin protein receptor (TSLPR), activated αM integrin, activated β1 integrin (CD29), activated β2 integrin, activated FcγRII, and activated CRTh2 (CD294). Such bispecifics bind to eosinophils and cause eosinophil depletion, preferably by ADCC-mediated killing and / or ADCP-mediated killing, or in the form of antibody-drug conjugates that are preferentially taken up by eosinophils and are toxic to eosinophils. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof reduces eosinophils. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof reduces eosinophils. high Decreasing induced and / or activated eosinophils, and optionally, CD39 high Induced and / or activated eosinophils are i) present in pathologies such as asthma, vasculitis, dermatitis, or sinusitis; and / or ii) located in a space selected from the group consisting of blood, bone marrow, lesions, and / or combinations thereof. In some embodiments, eosinophil-associated diseases suitable for treatment with anti-CD39 antibodies involve CD39 in a space selected from the group consisting of blood, bone marrow, lesions, and / or combinations thereof. high It is determined by the presence of induced and / or activated eosinophils.

[0022] In another aspect, a pharmaceutical formulation is provided comprising a therapeutically effective amount of at least one anti-CD39 antibody or antigen-binding fragment thereof described herein and one or more pharmaceutically acceptable excipients, buffers, or solutions. For example, the pharmaceutical formulation can comprise an effective amount of an anti-CD39 antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable excipients, buffers, or solutions to reduce eosinophil levels, activity, and / or function, and can be suitable for administration to a subject (by way of example only) with an inflammatory condition or who has undergone a tissue transplant, where administration of the anti-CD39 antibody to the subject results in a decrease in the number and / or function of CD39+ eosinophil cells.

[0023] In yet another aspect, a method for the involvement of eosinophils in a disease or condition by depleting CD39+ eosinophil cells, comprising administering to a subject having an undesired eosinophil condition an effective amount of a pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof described herein, wherein administration of the anti-CD39 antibody or antigen-binding fragment thereof reduces the number and / or function of CD39+ eosinophil cells.

[0024] In certain embodiments, CD39-targeted eosinophil-depleting agents can be used as part of a treatment for patients suffering from inflammatory or autoimmune disorders characterized by fibrosis, including pulmonary fibrosis such as cystic fibrosis, idiopathic pulmonary fibrosis, and progressive massive fibrosis; liver fibrosis such as cirrhosis and primary biliary cirrhosis; heart disease such as atrial fibrosis, endomyocardial fibrosis, and previous myocardial infarction; arthrofibrosis; Dupuytren's contracture; keloid fibrosis; mediastinal fibrosis; myelofibrosis; nephrogenic systemic fibrosis; retroperitoneal fibrosis; and scleroderma.

[0025] In some embodiments, the subject has a disease or condition associated with unwanted eosinophil activity. Unwanted eosinophilization activity can be caused by aberrant activation or excess of eosinophils. In some embodiments, the disease or condition is an inflammatory disorder or an autoimmune disease, e.g., a gastrointestinal inflammatory disorder, e.g., eosinophilic esophagitis and / or Crohn's disease. In some embodiments, the method further comprises administering to the subject one or more agents selected from the group consisting of glucocorticosteroids, leukotriene antagonists, mast cell stabilizers, immunomodulators, and proton pump inhibitors (PPIs). In some embodiments, the inflammatory disorder is a chronic inflammatory condition. In some embodiments, the chronic inflammatory condition is selected from the group consisting of rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, vasculitis, Sjogren's disease, diabetes, lupus erythematosus, asthma, fibrotic diseases, UV damage, and psoriasis. In some embodiments, the fibrosis is selected from the group consisting of pulmonary fibrosis, liver fibrosis, cardiac disease, arthrofibrosis, Dupuytren's contracture, keloid fibrosis, mediastinal fibrosis, myelofibrosis, nephrogenic systemic fibrosis, retroperitoneal fibrosis, and scleroderma. In some embodiments, the pulmonary fibrosis is cystic fibrosis, idiopathic pulmonary fibrosis, or progressive widespread fibrosis. In some embodiments, the liver fibrosis is hepatic fibrosis, cirrhosis, or primary biliary cirrhosis. In some embodiments, the cardiac disease is atrial fibrosis, intramyocardial fibrosis, or previous myocardial infarction. In some embodiments, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In some embodiments, the disease or condition is an inflammatory or obstructive airway disease. In some embodiments, the inflammatory or obstructive airway disease is selected from the group consisting of asthma, acute lung injury (ALI), adult / acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease, airway or lung disease (COPD, COAD, or COLD), pulmonary embolism, exacerbation of airway hyperresponsiveness due to other medications, bronchitis, and pulmonary inflammation. In some embodiments, the disease or condition is an inflammatory or allergic condition of the skin.In some embodiments, the inflammatory or allergic skin condition is selected from the group consisting of psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforme, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity vasculitis, urticaria, bullous pemphigus, lupus erythematosus, systemic lupus erythematosus, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, epidermolysis bullosa acquisita, and acne vulgaris. In some embodiments, the disease or condition is a pulmonary inflammatory disease, axial spondyloarthropathy, primary biliary cholangitis, allergic rhinitis, chronic lung disease, allergy, or eosinophilia.

[0026] In certain embodiments, CD39-targeted eosinophil depleting agents may be used as part of a treatment for drug-induced eosinophilia, such as eosinophilic asthma and hypereosinophilic disorders, secondary to ICI therapy and / or other drugs, including, but not limited to, antimalarials (e.g., pyrimethamine and dapsone), penicillins, glycopeptides, cephaloporins, sulfonamides, tetracyclines (particularly minocycline), nitrofurantoin, antituberculosis therapy, ACE inhibitors, tryptophan, anticonvulsants (e.g., phenytoin, carbamazepine, and phenobarbitone), NSAIDs, gold, H2 receptor antagonists, proton pump inhibitors, aminosalicylates, and chlorpropamide.

[0027] In certain embodiments, CD39-targeted eosinophil-depleting agents may be used as part of a treatment for inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

[0028] In certain embodiments, CD39-targeted eosinophil-depleting agents may be used as part of a treatment for atopic dermatitis, vasculitis, eosinophilic esophagitis, allergic rhinitis (including seasonal rhinitis), asthma, chronic lung disease (including chronic obstructive pulmonary disease), and allergies (e.g., peanut allergy), particularly asthma.

[0029] In some embodiments, the disease or condition is associated with the respiratory system, digestive system, cardiovascular system, endocrine system, integumentary system, musculoskeletal system, or nervous system, or is a non-neoplastic hematological disorder. In some embodiments, the disease or condition is tissue transplant rejection.

[0030] In some embodiments, the tissue graft is autologous or allogeneic. In some embodiments, the subject is a mammal, such as a human or rodent. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is administered to the subject with one or more pharmaceutically acceptable excipients, buffers, or solutions. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 0.01 to 10 mg / kg, optionally with a sustained, controlled delivery platform to avoid / limit antibody-mediated target cytosis (or antigen modulation or antigen shaving) for optimal ADCC-mediated and / or ADCP-mediated eosinophil cell depletion efficacy. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is administered to the subject one or more times daily, three times per week, twice per week, once per week, once every two weeks, once every three weeks, or once every four weeks, optionally with weekly administration. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is administered to a subject for at least 2-6 treatment cycles, or monthly for lifelong use. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is administered to a subject via parenteral administration, submucosal hydrogel administration, pulmonary administration, or topical application, where parenteral administration is by subcutaneous administration, intravenous administration, or intramuscular administration.

[0031] The patent on file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0032] [Figure 1]We demonstrate that the fucosylated counterparts PEONP22 and PEOAF22 exhibit higher ADCC activity (NK cytotoxicity) against CD39high HCC1739BL cells than their parent fully fucosylated clone, PEOWT22. Using the fully human anti-CD39 monoclonal antibody PEOWT22, we generated its ADCC-enhanced counterparts PEOAF22 and PEONP22 using two different chemical defucosylation methods without genetic modification. To assess their ADCC profiles, target cells (CFSE-labeled HCC1739BL, an Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cell line) were incubated with serially diluted human IgG1 isotype control antibody (KLH-hIgG1) or PEOWT22, PEOAF22, or PEONP22 for 30 minutes at 37°C in 5% CO2. The cells were then co-cultured with NK-92-CD16 V / V effector cells (E:T = 1:8) for 6 h at 37 °C. Target cell death was analyzed by flow cytometry, and cytotoxicity was determined by the % of CFSE+P / I+ cells. [Figure 2] This shows that the IgG1 Fc fraction confers ADCC activity to the antibody. The hCD39 Ref antibody shares its antigen-binding site with antibodies in the art. However, the Ref antibody used in this example was generated with an Fc portion specifically designed to have ADCC function. Therefore, both the hCD39 Ref antibody and PEOWT22 of the present invention contain the same ADCC-competent human IgG1 Fc fraction. NK cytotoxicity against HCC1739BL cells was performed as described in Figure 1 to determine the ADCC cell killing profiles of hCD39 Ref and PEOWT22. Note that hCD39 Ref and PEOWT22 exhibit similar ADCC activity profiles. [Figure 3]We show that the further optimized defucosylated counterpart PEO22 exerts higher ADCC activity than PEONP22. PEO22 is a further ADCC-enhanced version of the parent clone PEOWT22 by optimizing the defucosylation process. NK cytotoxicity against HCC1739BL cells was performed as described in Figure 1 to determine the ADCC cell killing profiles of PEO22 and PEONP22. [Figure 4] This shows that PEOWT22-induced ADCC activity is selective for cells highly expressing human CD39. Various Raji cell lines expressing different levels of human CD39, such as Raji cells (Raji-hCD39neg), hCD39-transfected Raji cells expressing high levels of human CD39 (Raji-hCD39hi), or hCD39-transfected Raji cells expressing low levels of human CD39 (Raji-hCD39lo), were used as target cells and preincubated with serially diluted PEOWT22 for 30 minutes at 37°C in 5% CO2. Effector cells (Jurkat cells stably expressing luciferase and hCD16a-158V) were then added to the culture (E:T = 6:1) and incubated for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background (RLU). RLU: relative light units. [Figure 5] This study demonstrates that PEOWT22 does not exert ADCC activity against CD39-low normal endothelial cells (HUVECs), suggesting a low potential for systemic side effects. Both human melanoma cells (SK-MEL-28; endogenously expressing moderate levels of CD39) and human umbilical vein endothelial cells (HUVECs; endogenously expressing low levels of CD39) were used as target cells and preincubated with serially diluted PEOWT22 for 30 minutes at 37°C in 5% CO2. Effector cells (i.e., Jurkat cells stably expressing luciferase and hCD16a-158V) were then added to the culture (E:T = 6:1) and incubated for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background. RLU: relative light units. [Figure 6-1] Most of the human / rabbit chimeric anti-human CD39 monoclonal antibodies target the same epitope as the reference anti-human CD39 monoclonal antibody clone A1 (epitope competition assay using HCC1739BL cells). HCC1739BL cells were incubated with a panel of 18 anti-human CD39 monoclonal antibodies (human / rabbit chimeric clones; unconjugated; 2 μg / mL) for 30 minutes at 4°C, washed, and stained with PE-conjugated mouse anti-human CD39 monoclonal antibody (clone A1) for 30 minutes at 4°C. The cells were then analyzed by flow cytometry to detect PE median fluorescence intensity (MFI). Cells incubated with medium instead of chimeric antibodies were used as a control. [Figure 6-2] Most of the human / rabbit chimeric anti-human CD39 monoclonal antibodies target the same epitope as the reference anti-human CD39 monoclonal antibody clone A1 (epitope competition assay using HCC1739BL cells). HCC1739BL cells were incubated with a panel of 18 anti-human CD39 monoclonal antibodies (human / rabbit chimeric clones; unconjugated; 2 μg / mL) for 30 minutes at 4°C, washed, and stained with PE-conjugated mouse anti-human CD39 monoclonal antibody (clone A1) for 30 minutes at 4°C. The cells were then analyzed by flow cytometry to detect PE median fluorescence intensity (MFI). Cells incubated with medium instead of chimeric antibodies were used as a control. [Figure 7]This shows that human / rabbit chimeric antibodies that do not compete for the same epitope as clone A1 have high ADCC activity. HCC1739BL cells were used as target cells and preincubated with serially diluted chimeric antibodies for 30 minutes at 37°C in 5% CO2. Effector cells (i.e., Jurkat cells stably expressing luciferase and hCD16a-158V) were then added to the culture (E:T = 6:1) and incubated for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background. RLU: relative light units. Note that among these high ADCC clones, only the PEO23 antibody competes for the same epitope as clone A1 (see Figure 6). [Figure 8-1] This shows that the human / rabbit chimeric antibody that fully competes for the same epitope as clone A1 has low or no ADCC activity. Luc-reporter ADCC assays were performed as shown in Figure 7 above. PEO18 served as a positive control. [Figure 8-2] This shows that the human / rabbit chimeric antibody that fully competes for the same epitope as clone A1 has low or no ADCC activity. Luc-reporter ADCC assays were performed as shown in Figure 7 above. PEO18 served as a positive control. [Figure 9]This shows that antibodies with high ADCC activity form stable immune complexes on the cell membrane, while antibodies with low or no ADCC activity do not. Exemplary anti-human CD39 antibodies (2 μg / ml) with high, low, or no ADCC activity were incubated with HCC1739BL cells at 37°C for 24 hours in 5% CO2 or at 4°C for 20 minutes, followed by staining with a secondary antibody (anti-human IgG (Fc-specific), AlexaFluor® 488) for 30 minutes at 4°C. The cells were then washed and analyzed by flow cytometry. The difference in AF488 MFI between the 20-minute and 24-hour treatments represents the loss of human CD39 on the cell membrane, calculated as described in Materials and Methods. Note that clone PEO26 (low ADCC activity) and clones PEO27, PEO28, and PEO29 (no ADCC activity) do not form stable immune complexes on the cell membrane after 24 hours of incubation (e.g., loss of CD39 is greater than 40%). On the other hand, clones PEONP22, PEO19, PEO20, and PEO21 (high ADCC activity) form stable antibody-antigen immune complexes (e.g., loss of CD39 is less than 30%). Hu / Ra: human / rabbit chimeric antibody; hIgG1: humanized rabbit antibody, IgG1 isotype. [Figure 10-1] Percentages of granulocyte subpopulations in the blood and bone marrow of healthy mice are shown. Blood and bone marrow (BM) samples were collected from healthy human (h)CD39KI mice and analyzed by flow cytometry. Granulocytes were gated on eosinophils (EO) (CD45+CD11b+SSChighSiglec-F+), neutrophils (Neu) (CD45+CD11b+Ly-6G+), and basophils (Baso) (CD45+SSClowCD200R3+FcεR1α+). The percentages of each cell subtype were calculated and expressed as % of CD45+CD11b+ cells. n = 8 mice per sex. [Figure 10-2]Percentages of granulocyte subpopulations in the blood and bone marrow of healthy mice are shown. Blood and bone marrow (BM) samples were collected from healthy human (h)CD39KI mice and analyzed by flow cytometry. Granulocytes were gated on eosinophils (EO) (CD45+CD11b+SSChighSiglec-F+), neutrophils (Neu) (CD45+CD11b+Ly-6G+), and basophils (Baso) (CD45+SSClowCD200R3+FcεR1α+). The percentages of each cell subtype were calculated and expressed as % of CD45+CD11b+ cells. n = 8 mice per sex. [Figure 10-3] Percentages of granulocyte subpopulations in the blood and bone marrow of healthy mice are shown. Blood and bone marrow (BM) samples were collected from healthy human (h)CD39KI mice and analyzed by flow cytometry. Granulocytes were gated on eosinophils (EO) (CD45+CD11b+SSChighSiglec-F+), neutrophils (Neu) (CD45+CD11b+Ly-6G+), and basophils (Baso) (CD45+SSClowCD200R3+FcεR1α+). The percentages of each cell subtype were calculated and expressed as % of CD45+CD11b+ cells. n = 8 mice per sex. [Figure 10-4] Percentages of granulocyte subpopulations in the blood and bone marrow of healthy mice are shown. Blood and bone marrow (BM) samples were collected from healthy human (h)CD39KI mice and analyzed by flow cytometry. Granulocytes were gated on eosinophils (EO) (CD45+CD11b+SSChighSiglec-F+), neutrophils (Neu) (CD45+CD11b+Ly-6G+), and basophils (Baso) (CD45+SSClowCD200R3+FcεR1α+). The percentages of each cell subtype were calculated and expressed as % of CD45+CD11b+ cells. n = 8 mice per sex. [Figure 11-1]These results demonstrate that eosinophils are the granulocyte subpopulation expressing the highest levels of CD39 on their membranes in mice. CD39 is a highly selective phenotypic biomarker for eosinophils in mouse granulocytes. Blood and BM from healthy hCD39KI mice were analyzed for human CD39 (hCD39) expression on the granulocyte cell surface by flow cytometry. The hCD39 expression levels of each subpopulation are shown relative to the isotype control. Note that CD39 expression on eosinophils is much higher than that on neutrophils, while basophils express very little CD39 protein (ranking of CD39 expression levels: eosinophils > neutrophils > basophils). n = 8 mice. [Figure 11-2] These results demonstrate that eosinophils are the granulocyte subpopulation expressing the highest levels of CD39 on their membranes in mice. CD39 is a highly selective phenotypic biomarker for eosinophils in mouse granulocytes. Blood and BM from healthy hCD39KI mice were analyzed for human CD39 (hCD39) expression on the granulocyte cell surface by flow cytometry. The hCD39 expression levels of each subpopulation are shown relative to the isotype control. Note that CD39 expression on eosinophils is much higher than that on neutrophils, while basophils express very little CD39 protein (ranking of CD39 expression levels: eosinophils > neutrophils > basophils). n = 8 mice. [Figure 12] These results demonstrate that the ADCC-enhancing PEO22 results in a higher blood eosinophil depletion effect compared to its low-ADCC counterpart, PEOWT22. Healthy hCD39KI mice were treated twice (days 0 and 2) with saline or 1 mg / kg of PEO22 or PEOWT22 (ranking of ADCC activity: PEO22 > PEOWT22) i.p. Blood samples were collected and analyzed by flow cytometry on days 0 (before treatment) and 3 at the time of final granulocyte collection. Note that both the PEO22 and PEOWT22 antibodies can selectively deplete EO without reducing the amount of Neu. Such in vivo EO depletion effects positively correlate with their in vitro ADCC activity (PEO22 > PEOWT22). n = 1 mouse per group. [Figure 13-1] We demonstrate that PEO22 selectively depletes blood and bone marrow eosinophils in healthy hCD39KI mice. Healthy hCD39KI mice were treated with saline or PEO22 (1 mg / kg) i.p. every other day for a total of four doses. Blood and BM samples were collected one day after the last dose and analyzed by flow cytometry. Note that PEO22 depletes eosinophils but not neutrophils, despite reducing CD39 membrane expression on both neutrophils and eosinophils. n = 4–8 mice per group. ns: non-significant; *p<0.05; ***p<0.001; ****p<0.0001 vs. control saline group (paired t-test, one-tailed). [Figure 13-2] We demonstrate that PEO22 selectively depletes blood and bone marrow eosinophils in healthy hCD39KI mice. Healthy hCD39KI mice were treated with saline or PEO22 (1 mg / kg) i.p. every other day for a total of four doses. Blood and BM samples were collected one day after the last dose and analyzed by flow cytometry. Note that PEO22 depletes eosinophils but not neutrophils, despite reducing CD39 membrane expression on both neutrophils and eosinophils. n = 4–8 mice per group. ns: non-significant; *p<0.05; ***p<0.001; ****p<0.0001 vs. control saline group (paired t-test, one-tailed). [Figure 14] This shows that eosinophils are significantly increased in asthmatic mice. Blood and BM samples were collected from eosinophilic asthmatic hCD39KI mice one day after the final OVA (in) challenge and analyzed by flow cytometry. Data obtained from healthy animals were used as a reference for comparison. n = 8 mice per group. **p < 0.01 vs. healthy animals (unpaired t-test, one-tailed). [Figure 15]We demonstrate that PEO22 selectively depletes eosinophils in asthmatic hCD39KI mice. Asthmatic hCD39KI mice were treated with saline or PEO22 (1 mg / kg) i.p. every other day for a total of four doses. Blood and BM samples were collected one day after the last dose and analyzed by flow cytometry. Note that eosinophil CD39 expression was also dramatically reduced after PEO22 treatment in both compartments. n = 8 mice per group. *p < 0.05, **p < 0.01, ****p < 0.0001 vs. saline control (paired t-test, one-tailed). [Figure 16-1] These results demonstrate that PEO22 selectively depletes the CD39high eosinophil subpopulation in asthmatic hCD39KI mice. Asthmatic hCD39KI mice were treated with saline or PEO22 (1 mg / kg) i.p. every other day for a total of four doses. One day after the last dose, blood and BM samples were collected and analyzed by flow cytometry. Data from healthy mice were used as a reference for comparison. Note that in healthy mice, most eosinophils are highly CD39high—"inducible" in response to daily environmental challenges. PEO22 treatment restores the absolute number of EOs in asthmatic mice to levels comparable to those present in healthy mice and shifts the EO subpopulation from CD39high to CD39low. n = 8 mice per group. ****p < 0.0001 vs. saline control (unpaired t-test, one-tailed). [Figure 16-2]These results demonstrate that PEO22 selectively depletes the CD39high eosinophil subpopulation in asthmatic hCD39KI mice. Asthmatic hCD39KI mice were treated with saline or PEO22 (1 mg / kg) i.p. every other day for a total of four doses. One day after the last dose, blood and BM samples were collected and analyzed by flow cytometry. Data from healthy mice were used as a reference for comparison. Note that in healthy mice, most eosinophils are highly CD39high—"inducible" in response to daily environmental challenges. PEO22 treatment restores the absolute number of EOs in asthmatic mice to levels comparable to those present in healthy mice and shifts the EO subpopulation from CD39high to CD39low. n = 8 mice per group. ****p < 0.0001 vs. saline control (unpaired t-test, one-tailed). [Figure 17] This figure shows that PEO22 significantly depletes eosinophils in the lungs of asthmatic hCD39KI mice and improves disease severity. Asthmatic hCD39KI mice were treated with saline or PEO22 (1 mg / kg) i.p. every other day for a total of four doses. Lung samples were collected one day after the last dose and analyzed by a pathologist. Note that the PEO22-treated group showed a significant improvement in the focal eosinophilic inflammation, primarily in peribronchial and perivascular areas, a characteristic sign of OVA-induced asthma seen in the saline control group. n = 8 mice per group. Images were taken at 100x magnification (bar 100 μm) and 400x magnification (bar 10 μm). Representative images from one animal per group are shown. Solid asterisks highlight perivascular areas infiltrated with eosinophils. White asterisks indicate areas of the vessel wall infiltrated with eosinophils. There is a significant reduction in lung-infiltrating eosinophils in both regions of the PEO22-treated group. [Figure 18]This figure shows that PEO22 ameliorates pulmonary eosinophilic vasculitis in asthmatic hCD39KI mice. Asthmatic hCD39KI mice were treated with saline or PEO22 (1 mg / kg) i.p. every other day for a total of four doses. Lung samples were collected one day after the last dose and analyzed by a pathologist. Note that the PEO22-treated group showed signs of reduced eosinophil numbers and activity within the vascular wall when compared to the saline control group. n = 8 mice per group. Images were taken at 100x magnification (bar 100 μm) and 400x magnification (bar 10 μm). Representative images from one animal per group are shown. Solid asterisks highlight perivascular areas infiltrated with eosinophils. White asterisks indicate eosinophil-infiltrated vascular walls. [Figure 19-1] We demonstrate that CD39high phenotypically and functionally defines an activated eosinophil subpopulation in asthmatic mice. Blood and BM samples from asthmatic hCD39KI mice were collected 1 day after the final OVA challenge and analyzed by flow cytometry for eosinophil biomarkers, including previously reported activation markers. Note that EO cells highly expressing CD39 (CD39high subtype) comprise the majority of the EO subpopulation in both the blood and bone marrow of asthmatic mice. n = 10 mice per group. [Figure 19-2] We demonstrate that CD39high phenotypically and functionally defines an activated eosinophil subpopulation in asthmatic mice. Blood and BM samples from asthmatic hCD39KI mice were collected 1 day after the final OVA challenge and analyzed by flow cytometry for eosinophil biomarkers, including previously reported activation markers. Note that EO cells highly expressing CD39 (CD39high subtype) comprise the majority of the EO subpopulation in both the blood and bone marrow of asthmatic mice. n = 10 mice per group. [Figure 20-1]This study demonstrates that IL-5Rα is not a selective biomarker for eosinophils in mouse granulocytes, as demonstrated in both healthy and asthmatic mice. Blood and BM samples from asthmatic hCD39KI mice collected 1 day after the final OVA challenge were analyzed for IL-5Rα expression on granulocytes by flow cytometry. Data from healthy mice were used in parallel for comparison. Note that neutrophils, but not eosinophils, are the granulocyte subpopulation that highly express IL-5Rα in the blood and BM of both healthy and asthmatic mice. n = 8 mice per group. [Figure 20-2] This study demonstrates that IL-5Rα is not a selective biomarker for eosinophils in mouse granulocytes, as demonstrated in both healthy and asthmatic mice. Blood and BM samples from asthmatic hCD39KI mice collected 1 day after the final OVA challenge were analyzed for IL-5Rα expression on granulocytes by flow cytometry. Data from healthy mice were used in parallel for comparison. Note that neutrophils, but not eosinophils, are the granulocyte subpopulation that highly express IL-5Rα in the blood and BM of both healthy and asthmatic mice. n = 8 mice per group. [Figure 21] This shows that blood eosinophils are significantly increased in mice with dermatitis. hCD39KI mice were subjected to atopic dermatitis model induction as detailed in Materials and Methods. Blood samples were collected two days after the final OVA challenge and analyzed by flow cytometry. Data from healthy animals were used as a reference for comparison. n = 5-8 mice per group. **p < 0.01 vs. healthy animals (unpaired t-test, one-tailed). [Figure 22]This figure shows that PEO22 reduces skin tissue infiltration eosinophils and ameliorates characteristic signs of dermatitis—acanthosis, hyperkeratosis, necrotic keratinocytes, and inflammatory infiltrates. hCD39KI mice subjected to dermatitis model induction were treated with saline or PEO22 (1 mg / kg) i.p. for a total of three doses. Skin samples were collected two days after the final dose and analyzed by a pathologist. n = 8 mice per group in the dermatitis model. Images were taken at 400x magnification (bar 10 μm). Representative images from one animal per group are shown. Skin lesions from healthy mice are shown in parallel for comparison. Solid asterisks indicate localized inflammatory areas infiltrated with eosinophils, demonstrating a significant reduction in skin tissue inflammation in the PEO22-treated group. [Figure 23] These results demonstrate that PEO22 selectively depletes blood eosinophils in murine eosinophilic sinusitis. As detailed in the Materials and Methods section, hCD39KI mice subjected to eosinophilic sinusitis model induction were treated with saline or PEO22 (3 mg / kg) i.p. every other day for a total of four doses. One day after the last dose, blood samples were collected and analyzed by flow cytometry. Data from healthy animals were used as a reference for comparison. Note that blood EO cells were significantly increased in the sinusitis model, in contrast to healthy mice, and decreased in the PEO22-treated group. n = 5 mice per group in the sinusitis model. **p < 0.01 for each group (one-way ANOVA followed by Tukey's multiple comparison test). [Figure 24]These results demonstrate that PEO22 reduces the subepithelial infiltrating inflammatory cells dominated by eosinophils, which are the etiology of eosinophilic sinusitis, and improves local lesions. hCD39KI mice subjected to eosinophilic sinusitis model induction were treated with saline or PEO22 (3 mg / kg) i.p. every other day for a total of four doses. Nasal samples were collected one day after the final administration and analyzed by a pathologist. n = 5 mice per group. The nasal cavity was consistently sampled at level II (from the incised papilla to the first palatine eminence), and pathology images were focused on the nasal septum. Images were taken at 100x magnification (bar 100 μm) and 400x magnification (bar 10 μm). Representative images from one animal per group are shown. Solid asterisks indicate subepithelial infiltrating inflammatory cells dominated by eosinophils. [Figure 25-1] Figure 1 shows the percentages of granulocyte subpopulations in human blood. Blood samples from healthy humans were analyzed by flow cytometry. Granulocytes were gated on eosinophils (EO) (CD45+SSChighSiglec-8+), neutrophils (Neu) (CD45+SSChighSiglec-8-), and basophils (Baso) (CD45+SSClowHLA-DR-IL-3Rα+). The percentages of each cell subtype were calculated and expressed as % of CD45+SSChigh (EO and Neu) and % of CD45+SSClow (Baso). [Figure 25-2] Figure 1 shows the percentages of granulocyte subpopulations in human blood. Blood samples from healthy humans were analyzed by flow cytometry. Granulocytes were gated on eosinophils (EO) (CD45+SSChighSiglec-8+), neutrophils (Neu) (CD45+SSChighSiglec-8-), and basophils (Baso) (CD45+SSClowHLA-DR-IL-3Rα+). The percentages of each cell subtype were calculated and expressed as % of CD45+SSChigh (EO and Neu) and % of CD45+SSClow (Baso). [Figure 26-1]These results demonstrate that eosinophils are the granulocyte subpopulation expressing the highest levels of CD39 on their membranes in humans. CD39 is a highly selective phenotypic biomarker for eosinophils in human granulocytes. Blood samples from healthy individuals were analyzed for hCD39 expression on the granulocyte cell surface by flow cytometry. The hCD39 expression levels of each subtype are shown relative to isotype controls. Note that CD39 expression on eosinophils is much higher than that of neutrophils, while basophils express very little CD39 protein (ranking of CD39 expression levels: eosinophils > neutrophils > basophils). [Figure 26-2] These results demonstrate that eosinophils are the granulocyte subpopulation expressing the highest levels of CD39 on their membranes in humans. CD39 is a highly selective phenotypic biomarker for eosinophils in human granulocytes. Blood samples from healthy individuals were analyzed for hCD39 expression on the granulocyte cell surface by flow cytometry. The hCD39 expression levels of each subtype are shown relative to isotype controls. Note that CD39 expression on eosinophils is much higher than that of neutrophils, while basophils express very little CD39 protein (ranking of CD39 expression levels: eosinophils > neutrophils > basophils). [Figure 27] Similar to healthy mice, most eosinophils in healthy humans are CD39high. Blood eosinophils in healthy humans were further subdivided based on hCD39 expression analyzed by flow cytometry. Note that the majority of eosinophils in healthy humans are of the CD39high-inducible subtype. [Figure 28] We demonstrate that ADCC-enhanced PEO22 exhibits increased ADCC activity against human eosinophils compared to its low-ADCC counterpart, PEOWT22. Isolated human EO target cells were incubated with serially diluted PEOWT22 or PEO22 for 30 minutes at 37°C in 5% CO2. The cells were then co-cultured with isolated and activated human NK effector cells (E:T = 1:5) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and cytotoxicity was determined by the % of 7-AAD+EO cells. [Figure 29] We demonstrate that PEO22 exhibits ADCP activity against human eosinophils. Purple-labeled human EO target cells were incubated with isotype control (10 μg / mL) or PEO22 (1 or 10 μg / mL) for 30 minutes at 37°C in 5% CO2. The cells were then co-cultured with human macrophage effector cells (E:T = 3:1) for 6 hours at 37°C. Bound macrophages were collected and analyzed by flow cytometry, and ADCP was determined by the percentage of Pacific Blue+ (EO-engulfing) macrophage cells. Macrophages incubated with ADCP medium alone were used as a negative control for the phagocytosis assay. [Figure 30-1] Conformational epitope mapping and a list of major putative epitope candidates are shown. [Figure 30-2] Conformational epitope mapping and a list of major putative epitope candidates are shown. DETAILED DESCRIPTION OF THE INVENTION

[0033] I. Overview Effective treatment of inflammatory diseases is often challenging due to the heterogeneity of their pathophysiology. With improved understanding of the underlying disease mechanisms, it is now clear that eosinophils play a complex pathophysiological role in a wide range of inflammatory diseases, including a central role in type 2 inflammatory diseases.

[0034] Eosinophils are a specialized subset of granulocytes that circulate in peripheral blood and home to several body tissues. In addition to their traditional association with helminth immunity and allergy, it has become increasingly clear that eosinophils play important roles in several homeostatic and pathological conditions. For example, in allergies, eosinophils are involved in the pathogenesis of atopic dermatitis, allergic rhinitis, and asthma. They are also fundamentally involved in autoimmune disorders such as eosinophilic esophagitis, eosinophilic gastroenteritis, acute and chronic eosinophilic pneumonia, and eosinophilic granulomatosis with polyangiitis (a rare form of vasculitis). Furthermore, secondary (or reactive) eosinophilia can also be induced / caused by drugs such as ICIs.

[0035] The present invention provides that certain CD39-targeting agents, such as ADCC-competent and / or ADCP-competent anti-CD39 antibodies, can selectively target and ablate CD39-expressing eosinophils, resulting in a reduction in the number of eosinophils located throughout the body and / or in target tissues (such as sites of inflammation or lesions, or bone marrow, which are sites of eosinophil production). The resulting reduction in the number and / or function of CD39+ eosinophil cells can result in a change in the inflammatory phenotype of the tissue.

[0036] Thus, in some aspects, the present invention relates to a CD39-targeted eosinophil-depleting agent. The term "CD39-targeted eosinophil-depleting agent" refers to any agent (e.g., antibody, small molecule, aptamer, etc.) that specifically binds to CD39 on the surface of eosinophils and induces eosinophil cell death. In some embodiments, the CD39-targeted eosinophil-depleting agent is an anti-CD39 antibody, such as an ADCC-competent and / or ADCP-competent anti-CD39 antibody.

[0037] Using anti-CD39 antibodies as an example of a CD39-targeted eosinophil depleting agent, the antibodies that can be selected for use in the methods of the present invention can form more stable immune complexes with CD39, resulting in a more potent ADCC killing effect. The inventors have observed that antibodies that cannot form stable immune complexes with CD39 result in a reduction of CD39 from the surface, but this is through a mechanism of increased CD39 shedding or cytosis (internalization), and do not have the same effect in terms of being able to remove CD39-expressing cells by antibody-dependent cellular cytotoxicity. Without being bound by theory, using anti-CD39 antibodies as an example of a CD39-targeted eosinophil depleting agent further suggests that the antibodies that can be selected for use in the methods of the present invention can form more stable immune complexes with CD39, resulting in a more potent ADCC killing effect. ADCP is another major Fc effector function, by which antibody-opsonized target cells activate FcγRs on the surface of macrophages (such as the FcγRIIIa receptor described herein, which is expressed on NK cells and confers ADCC killing function on NK cells) to induce phagocytosis, resulting in internalization, degradation, and ultimately target cell killing. Hypofucosylation or defucosylation of therapeutic monoclonal antibodies has been shown to result in enhanced FcγRIIIa receptor binding and subsequent ADCC-mediated target cell depletion by NK cells, as well as ADCP-mediated target cell depletion by macrophages (see Figures 1, 3, 12, 13, 15-18, 22-24, 28, and 29 herein; see also Dagher et al., 2022, Eur. Respir. J. 59:2004306, where the target cells in both experimental settings are eosinophils).

[0038] Summarized below are exemplary characteristics of certain preferred anti-CD39 monoclonal antibodies that are differently taught for use in therapeutic anti-CD39 antibodies described in the literature.

[0039] The antibodies of the invention deplete the CD39+ eosinophil cell population through FcγRIIIa receptor-dependent activity (eg, ADCC and / or ADCP).

[0040] For example, Figures 1 and 3 show that reducing the fucosylation (also known as hypofucosylation or defucosylation) of the fully human anti-CD39 monoclonal antibody clone PEOWT22, either by using a fucosylation inhibitor (PEOAF22) or by optimizing the production process (PEONP22 and PEO22), dramatically improved its ADCC activity against CD39+ cells in vitro. The ranking of ADCC activity was: PEO22 > PEONP22 > PEOWT22. This phenomenon was also demonstrated when using human NK cells (as effector cells) and human eosinophils (as target cells) in an in vitro ADCC assay: PEO22 exhibited significantly enhanced NK cytotoxicity against eosinophils (EC50 = 2.94E-04 μg / mL), in contrast to its fully fucosylated counterpart, PEOWT22 (EC50 = 3.03E-03 μg / mL) (Figure 28). Furthermore, PEO22 also exhibits ADCP activity against eosinophils in an in vitro phagocytosis assay using human macrophages (as effector cells) and human eosinophils (as target cells) (Figure 29). As expected, all of these in vitro CD39-targeted eosinophil-depleting activities are accompanied by enhanced anti-eosinophil activity of the defucosylated antibody PEO22 in vivo (Figures 12, 13, 15-18, and 22-24).

[0041] CD39 high defines phenotypically and / or functionally inducible and / or activated eosinophil subpopulations.

[0042] Eosinophils are heterogeneous, with different eosinophil subpopulations—resident and inducible eosinophils—existing in various tissues, which may impact the efficacy of eosinophil-targeted therapies. Characterization of eosinophil subpopulations is an important goal of eosinophil research. Therefore, for the development of safer, more selective, and more effective therapies for treating diseases or conditions associated with unwanted eosinophil activity, it is valuable to determine the phenotype of different eosinophil subpopulations using membrane surface markers to distinguish between homeostatic and inflammatory eosinophils. That is, therapeutic intervention with biological agents that completely deplete tissue and circulating eosinophils, or conversely, those that maintain a minimal proportion of eosinophils, especially resident or homeostatic ones, in tissues, may have very different effects, especially considering the administration of these treatments over long periods of time. Similarly, the use of authentic biomarkers of inducible eosinophils (e.g., circulating eosinophils, organ-specific eosinophils) may also help select the best eosinophil-targeting approach among biologic agents with increasing therapeutic efficacy. However, eosinophil subtypes have not been fully characterized in humans (Messnil et al., 2016, J. Clin. Invest. 126:3279-3295; Kanda et al., 2021, Allergol. Int. 70(1):9-18; Lombardi et al., 2022, Curr. Res. Immunol. 3:42-53).

[0043] Herein, we consider the following: 1) almost all eosinophils express CD39 (eosinophil cells are CD39+); and 2) CD39 high Phenotypically and / or functionally, we show that induced and / or activated eosinophils represent the majority of eosinophil cells in both mice and humans.

[0044] As an example, Figure 16 shows that eosinophils are divided into two distinct subpopulations, namely CD39, in both healthy and asthmatic mice based on cell surface CD39 expression levels analyzed by flow cytometry. high and CD39 low Furthermore, we show that these CD39high Eosinophils exhibit "inducible" functional properties and expand in response to the induction of inflammation: in asthmatic mice, CD39 expression in peripheral blood and bone marrow is significantly increased. high An increase in the number of eosinophil subpopulations is observed (Figure 16). This is accompanied by an increase in the number of infiltrating eosinophils in the lungs (e.g., localized eosinophilic inflammation, vasculitis, mainly in peribronchial and perivascular areas) (Figures 17 and 18). All are positively correlated with increased disease severity (Figures 17 and 18). This phenomenon is also seen in mouse models of atopic dermatitis and eosinophilic sinusitis (Figures 22 and 24). More importantly, a similar CD39 expression profile of eosinophils is also observed in human blood (Figures 25-27), suggesting favorable clinical translation of CD39-targeted eosinophil-depleting agents, such as the subject anti-CD39 antibodies of the present invention.

[0045] Notably, the majority of eosinophils express CD39 under hemostatic conditions (e.g., in healthy mice and healthy humans). high Inducible subtype, i.e., 80-90% CD39 high and 10–20% CD39 low (Figures 16 and 27). In eosinophil-associated diseases (EADs) such as asthma, these CD39 high Eosinophils were enlarged and increased (Fig. 16), and CD39 high This suggests that eosinophils are "inducible" and responsive to inflammatory inducers. high are selective phenotypic and / or functional biomarkers of "activated" eosinophils, which are the etiologic agent of EAD.

[0046] Unlike CD39, other previously reported phenotypic biomarkers of "activated" eosinophils in EAD, such as CD49b and CD101, which are termed "upregulated" on eosinophils, are unable to distinguish activated eosinophil subpopulations from their homeostatic counterparts (or at least to a much lesser extent than CD39) (Figure 19). As another example, FASENRA® (benralizumab), a humanized IgG1 anti-IL-5Rα monoclonal antibody that is defucosylated for enhanced ADCC to deplete eosinophils and basophils, binds directly to the surface of eosinophils and is the first and only anti-eosinophil biologic approved for eosinophilic asthma (available on the World Wide Web at fasenrahcp.com). However, in eosinophilic asthma, IL-5Rα is not upregulated. high The eosinophil subpopulation accounts for only a small fraction of eosinophils, i.e., approximately 20% in the blood and less than 50% in the bone marrow (Figure 19). Furthermore, neutrophils express much higher levels of IL-5Rα than eosinophils (Kda et al., 2021, Allergol. Int. 70(1):9-18 - Figure 3; and Figure 20 herein). Therefore, the depletion activity of IL-5Rα-targeting eosinophil-depleting agents, such as benralizumab, lacks eosinophil cell selectivity.

[0047] These data suggest that CD39 high These data demonstrate that CD39 functions as a bona fide phenotypic and / or functional biomarker for inducible and / or activated eosinophil subtypes in EAD, providing further evidence for the high selectivity and potentially high efficacy of CD39-targeted eosinophil-depleting agents for treating diseases or conditions associated with unwanted eosinophil activity. In other words, our data herein also support the use of CD39 as a biomarker to aid in the stratification of EAD patients. high This suggests the potential clinical utility of eosinophils.

[0048] The ADCC activity of the anti-CD39 antibody of the present invention is high cells, e.g., CD39 highSelective for induced and / or activated eosinophils.

[0049] For example, Figures 4 and 5 show that the ADCC activity of PEOWT22 inhibits CD39 in vitro. high These results demonstrate that the antibody is selective for the Raji-hCD39hi cells in FIG. 4 and the SK-MEL-28 cells in FIG. 5.

[0050] As a further example shown in Figure 16, PEO22 inhibits CD39 in vivo. high Induced and activated eosinophils can be selectively targeted and eliminated, resulting in the systemic elimination of these CD39 eosinophils in EADs such as eosinophilic asthma. high PEO22 treatment also significantly reduces the number of tissue-infiltrating eosinophils in pathological lesions in mouse models of asthma, vasculitis, atopic dermatitis, and eosinophilic sinusitis (Figs. 17, 18, 22, and 24). high Reduction in the number and / or function of eosinophil subpopulations results in an improvement in the inflammatory phenotype of the tissue and a decrease in disease severity (Figures 17, 18, 22, and 24).

[0051] In contrast, as additional examples shown in Figures 12 and 13, neutrophils, which express much lower CD39 levels than eosinophils (Figure 11), are not depleted by in vivo PEO22 treatment.

[0052] In summary, this functional trait should confer specificity to the antibody of interest and avoid systemic side effects, resulting in safer, more selective, and more effective anti-CD39 antibodies for treating EADs (diseases or conditions associated with unwanted eosinophil activity).

[0053] The formation of a stable immune complex between the anti-CD39 antibody and an antigen on the target cell membrane confers high ADCC activity to the antibody.

[0054] As an example shown in Figure 9, the stability of antibody-antigen immune complexes on the surface of target cells was examined using antibodies selected from three groups: ADCC-high (i.e., PEONP22, hCD39 Ref, and human / rabbit chimeric clones PEO19, PEO20, PEO21, and PEO25), ADCC-low (human / rabbit chimeric clone PEO26), or ADCC-negative (human / rabbit chimeric clones PEO27, PEO28, and PEO29). A strong positive correlation was clearly observed between the stability of such immune complexes and the ADCC activity of the antibodies. That is, the higher the stability of the antibody-antigen immune complex, the higher the ADCC activity of the antibody.

[0055] The different epitopes of an anti-CD39 antibody are directly related to the ADCC activity of the antibody.

[0056] As an example, by comparing the epitopes of the human / rabbit chimeric anti-hCD39 antibodies of the present invention with those of the commercially available anti-hCD39 monoclonal antibody clone A1, Figures 6-8 show that the anti-CD39 antibodies bind to CD39 in a manner that is non-competitive or only partially competitive with clone A1, and thus have a high probability of exhibiting high ADCC activity. Of the six ADCC-high antibodies, five (PEO18, PEO19, PEO20, PEO21, and PEO24) except for PEO23 exhibit such properties (Figures 6 and 7). In contrast, all ADCC-low (PEO26, PEO30, PEO31, and PEO32) and ADCC-negative (PEO27, PEO28, PEO29, PEO33, PEO34, PEO35, PEO36, and PEO37) antibodies exhibit epitopes that completely overlap with clone A1 (Figures 6 and 8).

[0057] In certain embodiments, rather than focusing on direct inhibition of CD39 NTPase activity, as has been the focus for prior art anti-CD39 therapeutic antibodies in oncological use (Perrot et al., 2019, Cell Reports 27:2411-2425; Li et al., 2019, Cancer Discovery 9(12):1754-1773; and WO / 2017 / 089334), anti-CD39 antibodies useful in the present invention (whether or not they inhibit NTPase activity) can be engineered to have a human constant region with an lgG1 Fc domain. This design confers FcγRIIIa receptor-dependent cellular activities, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and (optionally) complement-dependent cytotoxicity (CDC) against CD39+ cells. Consequently, such cellular activities result in the elimination and depletion of CD39+ cells.

[0058] In certain embodiments, certain antibodies encompassed by the present invention have been shown to bind to epitopes on CD39 that are non-competitive or only partially competitive with the binding of monoclonal antibody clone A1 to CD39.

[0059] II. Definition To facilitate the understanding of this invention, several terms and phrases are defined below.

[0060] "CD39," also known as "cluster of differentiation 39," "ectonucleoside triphosphate diphosphohydrolase-1," or (gene) "ENTPD1" and (protein) "NTPDase1," is a cell-surface-located ectonucleotidase with an extracellular-facing catalytic site that catalyzes the hydrolysis of γ- and β-phosphate residues of triphosphonucleosides and diphosphonucleosides to monophosphonucleoside derivatives (e.g., hydrolyzing P2 receptor ligands such as ATP, ADP, UTP, and UDP) (ENZYME entry: EC 3.6.1.5) (Junger et al., 2011, Nat. Rev. Immunol. 11:201-212). A representative human NTPDase1 protein sequence is provided in UniProtKB entry "P49961 (ENTP1_HUMAN)," and a representative human coding sequence for the enzyme is provided in GenBank accession S73813.

[0061] Representative human CD39 cDNA and protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). For example, at least seven human CD39 transcript variants are known to encode six different human CD39 isoforms. Human CD39 isoform 1 is available under accession numbers NM_001776.5 and NP_001767.3. The transcript variants represent the longest transcript and encode isoform 1. Human CD39 isoform 2, available under accession numbers NM_001098175.1 and NP_001091645.1, differs from transcript variant 1 by using an alternative 5' exon, which results in a distinct 5' untranslated region (UTR) and translation initiation at an alternative start codon, resulting in a longer, distinct N-terminus. Human CD39 isoform 3, available under accession numbers NM_001164178.1 and NP_001157650.1, uses an alternative 5' exon that differs from transcript variant 1, resulting in a distinct 5' UTR and translation initiation at an alternative start codon, resulting in a longer, distinct N-terminus. Human CD39 isoform 4, available under accession numbers NM_001164179.1 and NP_001157651.1, uses an alternative in-frame splice site compared to transcript variant 1, resulting in a shorter isoform. Human CD39 isoform 5, available under accession numbers NM_001164181.1 and NP_001157653.1, uses an alternative exon in the 5' region, resulting in a distinct 5' UTR and translation initiation at a downstream start codon compared to transcript variant 1, resulting in a shorter isoform. Human CD39 isoform 6, available under accession numbers NM_001164182.1 and NP_001157654.1, lacks an alternative exon, resulting in a distinct 5'UTR and translation initiation at a downstream start codon compared to transcript variant 1, resulting in a shorter isoform.Human CD39 isoform 6 is also encoded by another transcript variant available under accession numbers NM_001164183.1 and NP_001157655.1, which lacks two alternative internal exons, resulting in a distinct 5'UTR and translation initiation at a downstream start codon compared to transcript variant 1, resulting in a shorter isoform.

[0062] The nucleic acid and polypeptide sequences of CD39 orthologs in organisms other than humans are well known, including, for example, mouse CD39 (NM_009848.3 and NP_033978.1), rat CD39 (NM_022587.1 and NP_072109.1), bovine CD39 (NM_174536.2 and NP_776961.1), frog CD39 (NM_001006795.1 and NP_001006796.1), and zebrafish CD39 (NM_001003545.1 and NP_001003545.1).

[0063] Extensive glycosylation of CD39 is associated with its cell surface expression and activity, such that deletion of glycosylated residues or mutation of glycosylated residues to non-glycosylated residues significantly reduces CD39 activity (e.g., deletion or mutation of glycosylated residues 73 at the N-terminus, 333 in the middle, and / or 429 and / or 458 at the C-terminus of rat CD39, or corresponding residues in its orthologs; see Wu et al. (2005) Mol. Biol. Cell. 16:1661-1672). Similarly, mutation of conserved residues in any one or more of the apyrase conserved regions (ACRs) 1 to 5 results in reduced CD39 activity (Schulte am Esch et al. (1999) Biochem. 38:2248-2258; Yang et al. (2001) Biochem. 40:3943-4940; Wang and Guidotti (1998) J. Biol. Chem. 273:11392-11399).

[0064] Modulation (e.g., reduction) of CD39 activity can be measured in any number of ways (e.g., by measurements described herein, such as using a control, ratio, comparison to baseline, etc.). For example, a CD39 activity modulator can reduce the catalytic activity of an ectonucleotidase or overall CD39 activity compared to the level of such ectonucleotidase in the presence of a test agent. In one embodiment, CD39 activity is determined by analyzing the concentration of adenosine in a sample. The concentration can be assessed over time. In another embodiment, ATP is added to the sample being tested, and the remaining concentrations of ATP, AMP, or adenosine are determined or assessed. Modulation (e.g., decrease) in this context can mean a decrease of 1%, 5%, 10%, >20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 200%, 500%, 1000%, or more. In one embodiment, the increase is detected over time.

[0065] In certain embodiments, cells such as eosinophils exhibit high levels of expression of a gene (e.g., CD39) or other biomarker of interest (e.g., flow cytometry side scatter, also known as SSC). In one embodiment, high levels can be determined using any of the methods described herein for determining the expression level of a gene or other biomarker. For example, CD39 high Exemplary, non-limiting methods for defining eosinophils include analysis of blood (or blood derivatives) or bone marrow, e.g., blood or bone marrow obtained from healthy or asthmatic hCD39KI mice (obtained from Purinomia Animal Facology), or blood obtained from healthy human donors, using the detection antibodies listed in Tables 1-3, such as using a Cytek® Aurora flow cytometer, under the antibody-based flow cytometry assays described herein in Exemplary Materials and Methods.

[0066] In certain embodiments, CD39 highEosinophils, in addition to expressing markers characteristic of eosinophils and / or having functional characteristics of eosinophils, can be detected using an antibody-based flow cytometry assay described herein in exemplary materials and methods, e.g., using a Cytek® Aurora flow cytometer, e.g., using the detection antibodies listed in Tables 1-3, and can be present in at least 10 3 Eosinophils can be identified as those expressing CD39 at a sufficiently high level that the fluorescent intensity of the CD39 detection antibody is 0.05.

[0067] In some embodiments, CD39 of cells such as eosinophils high The population may be at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more, or any range (inclusive), e.g., 60-99%, 65-95%, 70-90%, 70-80%, etc., but not exceeding a high level (e.g., by way of example only, the fluorescence intensity of the CD39 detection antibody is at least 10 under this particular flow cytometry assay). 3 , e.g. at least 10 4 , 10 5 , 10 6 , 10 7 , or 10 3 ~10 7 , 10 4 ~10 6 , 10 3 ~10 6 In some embodiments, the CD39 expression of cells such as eosinophils is expressed in any range (inclusive) of CD39. low The population may be less than 41, 40, 39, 38, 37, 36, 35, 30, 25, 20, 15, 10, 5, or any range (inclusive) of, e.g., 4-41%, 10-35%, 15-25%, etc., but not exceeding a low level (e.g., by way of example only, the fluorescence intensity of a CD39 detection antibody is less than 10 under this particular flow cytometry assay). 3 Less than, e.g., 10 3 , 10 2 , 10 1 , 10 0 , or 10 0~10 3 , 10 1 ~10 2 , 10 1 ~10 3 In some embodiments, the population includes a population of cells expressing CD39 in any range (inclusive), such as less than or equal to CD45+CD11b+CD39 high Siglec-8+ eosinophil cells (CD45+CD11b+CD39 in mice) high CD39 (equivalent to Siglec-F+ eosinophil cells) high The cells may represent 60-99% of the total population, such as the CD45+CD11b+Siglec-8+CD39+ eosinophil population, or any range (inclusive) such as 60-95%, 60-80%, 70-80%, or 75-80%.

[0068] In certain embodiments, cells such as eosinophils are "induced and / or activated" eosinophils. In one embodiment, any of the methods described herein for determining the expression levels of genes or other biomarkers can be used to determine the cell subtype. For example, a representative, non-limiting method for defining "induced and / or activated" eosinophils involves analyzing blood (or blood derivatives) or bone marrow, e.g., blood or bone marrow obtained from healthy or asthmatic hCD39KI mice (obtained from Purinomia Animal Facology) or blood obtained from healthy human donors, using the detection antibodies listed in Tables 1-3, or an antibody-based flow cytometry assay described herein in the exemplary materials and methods, such as using a Cytek® Aurora flow cytometer. In some embodiments, "induced and / or activated" eosinophils are: i) present in pathologies such as asthma, vasculitis, dermatitis, or sinusitis; and / or ii) located within a space selected from the group consisting of blood, bone marrow, lesions, and / or combinations thereof. In some embodiments, the "induced and / or activated" eosinophils are CD39 high In some embodiments, "induced and / or activated" eosinophils are CD45+CD11b+CD39 highSiglec-8+ eosinophil population (which is CD45+CD11b+CD39 high In some embodiments, "induced and / or activated" eosinophils are CD45+CD11b+CD39 high Siglec-8 high It is an eosinophil population.

[0069] A "CD39 antibody" (alternatively, "anti-CD39 antibody") refers to an antibody that selectively binds to one or more epitopes of the NTPDase1 protein, and includes monoparatopic antibodies, as well as biparatopic and other multiparatopic antibodies.

[0070] An "immune complex" (also known as an antigen-antibody complex or antigen-bound antibody) may, in some embodiments, refer to a composition formed by binding of an antigen (e.g., expressed in a medium such as a cell, or alone) to an antibody. A "stable immune complex" is one in which the loss of interaction between the antigen and antibody is less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the immune complex after 24 hours, or any range (inclusive) of 40%-35%, 35%-30%, 30%-25%, 25%-20%, 20%-15%, or 15%-10% (e.g., when the antibody is incubated with cells expressing the antigen, such as HCC1739BL cells). In some embodiments, immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody (e.g., by way of example only, the stability of immune complexes formed with an anti-CD39 antibody can be determined by incubating an anti-CD39 monoclonal antibody (mAb) (e.g., 2 μg / mL or higher) with HCC1739BL cells for different periods of time, followed by detecting the presence of immune complexes with a fluorescently conjugated secondary antibody).

[0071] a. Antibodies and other polypeptides As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site, typically located within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen-binding site of an antibody (formatted to include an Fc or other FcγRIII-binding domain), and any other modified immunoglobulin molecule containing an antigen-binding site, so long as the antibody exhibits the desired biological activity.

[0072] As used herein, the term "antigen-binding portion" or "antibody-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human CD39). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, e.g., an anti-CD39 antibody described herein, include (i) an Fab fragment, i.e., a V L , V H (ii) a monovalent fragment consisting of the V, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V HExamples of antibody fragments include (vi) dAb fragments consisting of a domain (Ward et al., 1989, Nature 341:544-546), and (vi) isolated complementarity-determining regions (CDRs), or (vii) combinations of two or more isolated CDRs, optionally joined by a synthetic linker. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These and other potential configurations are described in Chan and Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0073] The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. Generally, heavy and light chain variable regions each consist of four framework regions (FRs) and three complementarity-determining regions (CDRs), also known as "hypervariable regions." The CDRs within each chain are held together in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variation (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda, Md.), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol. 273:927-948). Furthermore, those skilled in the art sometimes use a combination of these two methods to determine CDRs.

[0074] Antibodies can be of any of the five major immunoglobulin classes, namely, IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the distinctiveness of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively; preferred CD39 antibodies are of the IgG1 and IgG3 isotypes for most effective engagement with FcγRIII (i.e., Kd of 10 or greater). -7 (See below).

[0075] In certain embodiments, antibodies are "hypofucosylated" and may be "defucosylated." A "hypofucosylated" antibody preparation refers to an antibody preparation in which less than 50% of the oligosaccharide chains contain α-1,6-fucose. Typically, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5% or less than 1% of the oligosaccharide chains in a "hypofucosylated" antibody preparation contain α-1,6 fucose. A "defucosylated" antibody lacks α-1,6-fucosyl in the carbohydrate attached to the CH2 domain of the IgG heavy chain.

[0076] As used herein, the term "monoclonal antibody" refers to an antibody displaying a single binding specificity and affinity for a particular epitope, or a composition of antibodies in which all of the antibodies display a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies are derived from a single cell or antibody-encoding nucleic acid and propagated without the intentional introduction of any sequence modifications. Thus, the term "human monoclonal antibody" refers to a monoclonal antibody having variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas obtained, for example, by fusing B cells obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome containing human heavy chain and light chain transgenes) to immortalized cells.

[0077] As used herein, the term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequence. Typically, humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and / or binding capacity. In some cases, residues of the Fv framework regions of the human immunoglobulin are replaced with corresponding residues in an antibody from the non-human species. Humanized antibodies can be further modified by substitution of additional residues either in the Fv framework regions and / or within the replaced non-human residues to improve and optimize the specificity, affinity, and / or binding capacity of the antibody. A humanized antibody may comprise variable domains containing all or substantially all of the CDRs corresponding to the non-human immunoglobulin, while all or substantially all of the framework regions are those of human immunoglobulin sequences. In some embodiments, the variable domain comprises framework regions of human immunoglobulin sequences. In some embodiments, the variable domains comprise framework regions of human immunoglobulin consensus sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Humanized antibodies are generally considered to be distinct from chimeric antibodies.

[0078] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art.

[0079] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from more than one species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and / or binding capacity, and the constant regions are homologous to the sequences of antibodies from another species (usually human) to avoid eliciting an immune response in that species.

[0080] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcgRI, FcγRIII, and FcgRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcgRIIB) receptor.

[0081] An "FcγRIII binding portion" is a peptide, protein, nucleic acid, or other moiety that, when associated with the antigen-binding site of an anti-CD39 antibody, is capable of binding to FcγRIII (CD16) and, optionally, mediating antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). Heavy chain Fc fragments containing the CH2 and CH3 domains of IgG1 and IgG3 isotypes are FcγRIII binding portions.

[0082] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, an epitope can be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids (also called linear epitopes) are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding (also called conformational epitopes) are typically lost upon protein denaturation. An epitope typically comprises at least 3, more usually at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

[0083] As used herein, the terms "specifically binds to" or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody moiety that binds to this target with higher affinity, stronger avidity, more readily, and / or for longer than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or even 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0084] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), and other modifications known in the art. Because polypeptides encompassed by the present invention can be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0085] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to align amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides encompassed by the present invention are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the coding regions of target proteins or antibodies. In some embodiments, the identity exists over a region of nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequence being compared, such as a nucleotide sequence encoding a protein of interest.

[0086] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains is generally defined in the art and includes amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, replacing tyrosine with phenylalanine is a conservative substitution. In general, conservative substitutions in the sequences of polypeptides, soluble proteins, and / or antibodies encompassed by the present invention do not abolish binding of the polypeptide, soluble protein, or antibody containing that amino acid sequence to its target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0087] An "isolated" polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0088] As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0089] As used herein, the term "fusion protein" or "fusion polypeptide" refers to a hybrid protein expressed by a nucleic acid molecule comprising the nucleotide sequences of at least two genes.

[0090] As used herein, the term "linker" or "linker region" refers to a linker inserted between a first polypeptide (e.g., an anti-CD39 antibody) and a second polypeptide (e.g., an Fc or other FcγRIII binding moiety; an scFV, a Vhh domain, etc. that binds to different proteins to create a bispecific antibody format that maintains the bivalency of CD39). In some embodiments, the linker is a peptide linker. The linker should not adversely affect the expression, secretion, or biological activity of the polypeptide. Preferably, the linker is not antigenic and does not elicit an immune response.

[0091] b. Treatment As used herein, the term "effective amount" refers to an amount to provide a therapeutic or prophylactic benefit.

[0092] As used herein, the term "treatment" refers to an action by an individual to alter the course of clinical disease, and may be prophylactic or interventional to alter the course of clinical pathology. This term includes, but is not limited to, treatment to prevent the onset or recurrence of disease, alleviate symptoms, reduce any direct or indirect pathological consequences of disease, prevent metastasis, reduce the rate of disease progression, reduce or ameliorate disease, or improve prognosis.

[0093] The term "subject" refers to any animal (e.g., mammal) that is to receive a particular treatment, including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein, e.g., with reference to a human subject.

[0094] As used herein, the terms "agonist" and "agonistic" refer to or describe a therapeutic moiety that can directly or indirectly substantially induce, activate, promote, increase, or enhance the biological activity of a target and / or pathway. The term "agonist" is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.

[0095] As used herein, the terms "antagonist" and "antagonistic" refer to or describe a therapeutic moiety that can directly or indirectly, partially or completely, block, inhibit, reduce, or neutralize the biological activity of a target and / or pathway. The term "antagonist" is used herein to include any agent that partially or completely blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest.

[0096] As used herein, the terms "modulation" and "modulating" refer to a change or alteration in biological activity. Modulation includes, but is not limited to, stimulating activity or inhibiting activity. Modulation can be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, pathway, system, or other biological target of interest.

[0097] As used herein, the term "immune response" includes responses from both the innate and adaptive immune systems. It includes both cell-mediated and / or humoral immune responses. It includes both T cell and B cell responses, as well as responses from other cells of the immune system, such as natural killer (NK) cells, monocytes, macrophages, etc.

[0098] The term "pharmaceutically acceptable" refers to a substance that is approved or approvable by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, including humans.

[0099] The term "pharmaceutically acceptable excipient, carrier, or adjuvant" or "acceptable pharmaceutical carrier" refers to an excipient, carrier, or adjuvant that can be administered to a subject together with at least one agent of the present disclosure and that does not impair its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic effect. Generally, those skilled in the art and the US FAD consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.

[0100] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of an anti-CD39 antibody effective to "treat" a disease or disorder in a subject, such as a mammal.

[0101] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow, reduce symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder as well as those prone to have the disorder and those in whom the disorder is to be prevented.

[0102] c. Other Whenever an embodiment is described herein using the phrase "comprising," it is understood that analogous embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein using the phrase "consisting essentially of," it is also understood that analogous embodiments otherwise described using the term "consisting of" are also provided.

[0103] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. For example, a reference to "about X" includes a description of "X."

[0104] Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "both A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0105] III. Anti-CD39 antibody a. Monoclonal antibodies Anti-CD39 antibodies useful in the methods and pharmaceutical preparations of the present invention may be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, 1975, Nature 256:495. In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. In some embodiments, monoclonal antibodies, e.g., rabbit monoclonal antibodies, can be produced using single B cell cloning techniques, such as those described in Rashidian and Lloyd, 2020, Methods Mol. Biol. 2070:423-441, the contents of which are incorporated herein by reference in their entirety.

[0106] The immunizing agent typically comprises a CD39 polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are usually used. Hybridoma cells can be seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), substances that prevent the growth of HGPRT-deficient cells.

[0107] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level antibody expression by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which are available, for example, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., 1984, J. Immunol. 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0108] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are readily known to those skilled in the art. The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, 1980, Anal. Biochem. 107:220.

[0109] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0110] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0111] Monoclonal antibodies may be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding monoclonal antibodies encompassed by the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells encompassed by the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector and then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, resulting in the synthesis of monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., supra), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide may be substituted for the constant domains of an antibody encompassed by the invention, or may be substituted for the variable domains of one antigen-binding site of an antibody encompassed by the invention to create a chimeric bivalent antibody.

[0112] b. Human and humanized antibodies Anti-CD39 antibodies encompassed by the present invention may further include humanized or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions corresponding to those of a human immunoglobulin consensus sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0113] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and colleagues (Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536) by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (U.S. Patent No. 4,816,567). In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0114] Human antibodies can also be produced using various techniques known in the art, such as phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581), and yeast display (Chao et al., 2006, Nat. Protoc. 1(2):755-68). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77 (1985) and Boerner et al., 1991, J. Immunol. 147(1):86-95). Similarly, human antibodies can be produced by, for example, introducing human immunoglobulin loci into transgenic animals, e.g., mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; and in the following scientific publications: Marks et al., 1992, Bio / Technology 10:779-783; Lonberg et al., 1994, Nature 368:856-859; Morrison, 1994, Nature 368:812-13; Fishwild et al., 1996, Nature Biotechnology 14:845-51; Neuberger, 1996, Nature Biotechnology 14:826; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93.

[0115] Antibodies can also be affinity matured using known selection and / or mutagenesis methods, as described above. Preferred affinity-matured antibodies have 5-fold, more preferably 10-fold, and even more preferably 20 or 30-fold better affinity than the starting antibody (generally murine, humanized, or human) from which the mature antibody is prepared.

[0116] c. Bispecific antibodies The anti-CD39 antibodies described herein include bispecific molecules. Anti-CD39 antibodies, or antigen-binding portions thereof, may be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibodies described herein may, in fact, be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds to two or more different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create the bispecific molecules described herein, the antibodies described herein can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, resulting in a bispecific molecule.

[0117] Thus, provided herein are bispecific molecules comprising at least one first binding specificity for CD39 and a second binding specificity for a second target epitope. In embodiments described herein in which the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0118] In a specific embodiment, the invention provides bispecific antibodies that bind to both CD39 and an eosinophil cell surface antigen selected from Siglec-8, IL-5Rα (CD125), IL-3Rα (CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), PIRA, PIRB, L-selectin, EMR1, CCR3 (CD193), and CRTh2 (CD294), and cause eosinophil depletion, preferably by ADCC- and / or ADCP-mediated killing, or in the form of an antibody-drug conjugate that is preferentially taken up by and toxic to eosinophils.

[0119] In other embodiments, bispecifics are generated using binding domains for antigens upregulated on activated eosinophils (or eosinophils found in lesions) with bispecific antibody affinity for different antigens (CD39 and a second antigen), both of which are expressed on eosinophils, providing selectivity. Exemplary antigens for generating bispecifics using the CD39 binders of the invention include CD3, CD4, γδ TCR, CD9, CD28, CD29, CD40, CD44, CD45, CD45RO, CD48, CD58, CD63 (lysosome-associated membrane protein 3), CD66b (CEACAM8), CD66e (CEACAM5), CD67, CD69, CD80, CD86, C5αR (CD88), CD101, CD122, CD137 (tumor necrosis factor receptor superfamily member 9, induced by lymphocyte activation, 4-1BB), CD274 (programmed death ligand 1), α IIbIntegrin (CD41), α2 integrin (CD49b), α4 integrin (CD49d), αL integrin (CD11a), αM integrin (CD11b), αX integrin (CD11c), αD integrin, β2 integrin (CD18), aminopeptidase N (CD13), FcαRI (CD89), FcγRIII (CD16), FcγRII (CD32), FcεRII (CD23), granulocyte-monocyte colony-stimulating factor Rα (CD116), HLA-DR, intercellular indirect These include antibody-drug conjugates for eosinophils, such as eosinophil-1 (CD54), interleukin (IL)-2Rα (CD25), IL-17RA, IL-17RB, galectin-3, neuropeptide S receptor, P-selectin glycoprotein ligand-1 (CD162), semaphorin 7A (CD108), thymic stromal lymphopoietin protein receptor (TSLPR), activated αM integrin, activated β1 integrin (CD29), activated β2 integrin, activated FcγRII, and activated CRTh2 (CD294). Such bispecifics bind to eosinophils and cause eosinophil depletion, preferably by ADCC-mediated killing and / or ADCP-mediated killing, or in the form of antibody-drug conjugates that are preferentially taken up by eosinophils and are toxic to eosinophils.

[0120] In one embodiment, the bispecific molecules described herein comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv. The antibody can also be a dimer of a light or heavy chain, or any minimal fragment thereof, such as an Fv or single-chain (scFv) construct.

[0121] Binding of bispecific molecules to their specific targets can be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by employing a labeled reagent (e.g., an antibody) that is specific for the complex of interest.

[0122] Methods for producing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, 1983, Nature 305:537-539). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) generate a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. Similar techniques are disclosed in WO 93 / 08829, published May 13, 1993, and in Traunecker et al., 1991, EMBO J. 10:3655-3659.

[0123] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies, see, e.g., Suresh et al., 1986, Methods in Enzymology 121:210.

[0124] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created at the interface of a second antibody molecule by replacing the large amino acid side chain(s) with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers.

[0125] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., 1985, Science 229:81, describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0126] Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., 1992, J. Exp. Med. 175:217-225, describe the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0127] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., 1992, J. Immunol. 148(5):1547-1553). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448 provides an alternative mechanism for making bispecific antibody fragments. The fragments comprise a light-chain variable domain (V) connected by a linker that is too short to allow pairing between the two domains on the same chain. L ) connected to the heavy chain variable domain (V H ) is included. Therefore, the V of one fragment H and V L The domain is complementary to the V L and V H The Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., 1994, J. Immunol. 152:5368.

[0128] Antibodies with more than two valencies are contemplated. As one non-limiting example, trispecific antibodies can be prepared. See, e.g., Tutt et al., 1991, J. Immunol. 147:60.

[0129] d. Heteroconjugate antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO91 / 00360; WO92 / 200373; EP03089). Antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, as well as those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0130] e. Effector function operation For example, it may be desirable to modify antibodies encompassed by the present invention with respect to effector function to further enhance the effectiveness of the anti-CD39 antibody in depleting eosinophils. For example, cysteine ​​residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability, complement-mediated cell killing, antibody-dependent cellular cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP). See Caron et al., 1992, J. Exp Med. 176:1191-1195 and Shopes, 1992, J. Immunol., 148:2918-2922.

[0131] f. Representative anti-CD39 antibody sequences [Table 1] [Table 2] [Table 3] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable that is at least 60% identical to SEQ ID NO:2, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID NO:2, and is capable of specifically binding to human CD39.

[0132] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one light chain variable that is at least 60% identical to SEQ ID NO: 4, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID NO: 4, and is capable of specifically binding to human CD39.

[0133] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having human framework sequences associated with the CDRs of the VH domain set forth in SEQ ID NOs: 29, 30, and 31, and the CDRs of the corresponding VL domain set forth in SEQ ID NOs: 32, 33, and 34. The CDRs are preferably identical, but may differ by one, two, or three amino acids in each CDR, so long as the resulting antibody specifically binds to human CD39.

[0134] In certain embodiments, the heavy and light chains of the anti-CD39 antibody have variable domains that can be encoded by nucleic acids that are identical to the coding sequences set forth in SEQ ID NO: 1 (VH) and SEQ ID NO: 3 (VL) or that hybridize thereto under stringent conditions (e.g., 6x sodium chloride / sodium citrate (SSC) at 45°C, washed in 0.2x SSC / 0.1% SDS at 50-65°C).

[0135] [Table 4-1] [Table 4-2] [Table 4-3] [Table 5] In some embodiments, the anti-CD39 antibodies provided herein promote: (i) stable immune complex formation when incubated with HCC1739BL cells, characterized by less than 30% loss of immune complexes after 24 hours (optionally, immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody); (ii) antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) against CD39+ cells; (iii) depletion of CD39+ eosinophils; (iv) binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 30 (e.g., 1) IYLTDCMERAR, 2) LRMESEELADR, 3) RVKGPGISKFV, 4) DCMERARE. and / or (v) binds to one or more linear or conformational CD39 epitopes such as those selected from the group consisting of RVKGPGISKFV and DCMERAREVIPR, LTDCMERAREVIPR and SLSNYPFDFQGAR, or any combination of CRVKGPGISKF, GAYGWITINYLLGKFSQK, and ILRDPCFHPGYKK; and / or (v) binds to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody clone A1, which binds to CD39.

[0136] The representative anti-CD39 antibody sequences listed above by sequence identification number correspond to the following: Picture 1 (Introduction to PEOWT22 vH Pictures) gag gtg caa ctg gtg gag tct ggg gga ggt gtg gta agg cct ggg ggg 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr 20 25 30 gct atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca gtt ata tca tat gat gta agc aat aaa tac gca gac tcc gtg 192 Ala Val Ile Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240 Lys Gly Arg Phe Thr Ile Arg Asp Asn Serves Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga tct tac tac tac tac tac ggt atg gac gtc tgg ggc caa ggg 336 Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110 acc acg gtc acc gtc tcc tca 357 Thr Thr Val Thr Val Ser Ser 115 Sequence number 2 (Clone PEOWT22 vH amino acid sequence) Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly<\ 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Arg Asp Asn Serves Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Served Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Only Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 Picture 3 (PeOWT22 vL Picture Picture) gat gtt gtg atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48 Asp Val Val Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgc cgg gca agt cag agc att agc agc tac 96 Asp Arg Val Thr With Thr Cys Arg Only Serves Gln Ser With Arg Tyr 20 25 30 tta gcc tgg tac cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Wing Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gtc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag cca 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tca cct cgg 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg 85 90 95 acg ttc ggc caa ggg aca cga ctg gag att aaa 321 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 Figure 4(PeOWT22 vL Picture Kit) Asp Val Val Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 Sequence number 5 (clone PEO18 vH domain nucleic acid sequence) cag tca gtg aag gag gcc ggg ggt cgc ctg gta acg cct gga gga tcc 48 Gln Ser Val Lys Glu Ala Gly Gly Arg Leu Val Thr Pro Gly Gly Ser 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt gcg tat gga 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Gly 20 25 30 ata agt tgg gtc cgc cag gct cca ggg aag gga ctg gaa tgg atc gga 144 Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 atc att tat agt agt ggt agg act tac tac gcg aac tgg gcg aaa ggc 192 Ile Ile Tyr Ser Ser Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg tcg acc acg gtg gat ctg aaa atg 240 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 acc agt ctg aca acc gag gac acg gcc gcc tat ttc tgt gcc aga tca 288 Thr Ser Leu Thr Thr Glu Asp Thr Ala Ala Tyr Phe Cys Ala Arg Ser 85 90 95 cgg gct ggt att agt agt ggt gat ggt ttt gat tcc tgg ggc cca ggc 336 Arg Ala Gly Ile Ser Ser Gly Asp Gly Phe Asp Ser Trp Gly Pro Gly 100 105 110 acc ctg gtc acc gtc tcc tca 357 Thr Leu Val Thr Val Ser Ser 115 SEQ ID NO: 6 (Clone PEO18 vH domain amino acid sequence) Gln Ser Val Lys Glu Ala Gly Gly Arg Leu Val Thr Pro Gly Gly Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Gly 20 25 30 Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Ser Ser Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu Thr Thr Glu Asp Thr Ala Ala Tyr Phe Cys Ala Arg Ser 85 90 95 Arg Ala Gly Ile Ser Ser Gly Asp Gly Phe Asp Ser Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 SEQ ID NO:7(クローンPEO18 vL domain nucleic acid SEQ ID NO: 7) gcc ctt gtg atg acc cag act cca tcc tcc gtg tct gca gct gtg gga 48 Ala Leu Val Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt cag aac att tac agc aat 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Asn Ile Tyr Ser Asn 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc cag ctc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Gln Leu Leu Ile 35 40 45 tac agg gca tcc act ctg gca tct ggg gtc cca tcg cgg ttc aaa ggc 192 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 agt gca tct ggg aca gaa tac act ctc acc atc agc ggt gtg cag tgt 240 Ser Ala Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Gln Cys 65 70 75 80 gac gat gct gcc act tac tat tgt caa cag ggt ttt gat agt agt aac 288 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Asp Ser Ser Asn[[ID=十七]] 85 90 95 att gat aat act ttc ggc gga ggg acc gag gtg gtg gtc aca 330 Ile Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Thr 100 105 110 Sequence number 8 (Clone PEO18 vL domain amino acid sequence) Ala Leu Val Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Gln Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Ala Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Gln Cys 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Asp Ser Ser Asn 85 90 95 Ile Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Thr 100 105 110 sequence number 9(クローンPEO19 vH domain nucleic acid sequence) cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca cac 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr His 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt aag agt ata 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Ser Ile 20 25 30 ata agt tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tac atc gga 144 Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 atc att ggt agt agt ggt agc aca tac tac gcg aac tgg gcg aaa ggc 192 Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg tcg acc acg gtg gat ctg aga atg 240 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Arg Met 65 70 75 80 acc agt ctg aca ccc gag gac acg gcc acc tat ttc tgt gcc aga gga 288 Thr Ser Leu Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 ctt ctt tat tct ggt aat aaa tcg tgg ggc ccg ggc acc ctg gtc acc 336 Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr Leu Val Thr 100 105 110 gtc tcc tca 345 Val Ser Ser 115 SEQ ID NO: 10 (Clone PEO19 vH domain amino acid sequence) Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr His 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Ser Ile 20 25 30 Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Arg Met 65 70 75 80 Thr Ser Leu Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 Movie 11(PEO19 Photograph vL) gcc att gat atg acc cag act cca tcc tcc gtg tct gca gct gtg gga 48 Ala Ile Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aac tgc cag tcc agt cag agt gtt tta ctg aac 96 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Leu Asn 20 25 30 aac caa tta tcc tgg tgg cag cag aaa cca ggg cag cct ccc aag ctc 144 Leu Gln Asn Served as Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 ctg atc tat gat gca tcc act ctg gaa tct ggg gtc cca tct cgg ttc 192 Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe 50 55 60 aca ggc agt gga tct ggg aca cag ttc act ctc acc atc agc gac ctg 240 Thr Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 gag tgt gac gat gct gcc act tac tat tgt tta ggc ggt tat agt ggg 288 Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Ser Gly 85 90 95 aac ctt tat gct ttc ggc gga ggg acc gag gtg cta gtc aaa 330 Asn Leu Tyr Ala Phe Gly Gly Gly Thr Glu Val Leu Val Lys 100 105 110 クローンPEO19 vL domain amino acid sequence 12 Ala Ile Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Leu Asn 20 25 30 Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe 50 55 60 Thr Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Ser Gly 85 90 95 Asn Leu Tyr Ala Phe Gly Gly Gly Thr Glu Val Leu Val Lys 100 105 110 SEQ ID NO: 13 (Clone PEO20 vH domain nucleic acid sequence) cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt agc tat gca 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25 30 ata agt tgg gtc cgc cag gct cca ggg aag ggg ctc gaa tat atc gcg 144 Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Ala 35 40 45 atc att aat agt tat ggt acc aca tac tac gcg agc tgg gcg aaa ggc 192 Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 cga gtc acc atc tcc aaa acc tcg agc acg gtg gat ctg aaa atc tcc 240 Arg Val Thr Ile Ser Lys Thr Ser Ser Thr Val Asp Leu Lys Ile Ser 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga ggc gat 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp 85 90 95 agt tat ggt agt ggt gtt ggt ttg ggc ttg tgg ggc cca ggc acc ctg 336 Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly Pro Gly Thr Leu 100 105 110 gtc acc gtc tcc tca 351 Val Thr Val Ser Ser 115 Accession No. 14 (Clone PEO20 vH Domain Amino Acid Sequence) Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25 30 Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Ala 35 40 45 Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Val Thr Ile Ser Lys Thr Ser Ser Thr Val Asp Leu Lys Ile Ser 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp 85 90 95 Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 Picture 15(PEO20 vL picture picture) gcc tat gat atg acc cag act cca gcc tct gtg gag gta gct gtg gga 48 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aag tgc cag gcc agt cag aac att tac agc aat 96 Gly Thr Val Thr With Lys Cys Gln Only Serves Gln Asn With Tyr Ser Asn 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aag ctc ctc atc 144 Lion Wing Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Lion Ile 35 40 45 tac agg gca tcc agt ctg gca tct ggg gtc ccg tcg cgg ttc agt ggc 192 Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct ggg aca gag ttc act ctc acc atc agc ggt gtg cag tgt 240 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Val Gln Cys 65 70 75 80 gac gat gct gcc act tac tac tgt caa cag ggt ttt agt agt aat aat 288 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser Ser Asn Asn 85 90 95 gtt gat aat act ttc ggc gga ggg acc gag gtg gtg gtc aaa 330 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 SEQ ID NO: 16 (Clone PEO20 vL domain amino acid sequence) Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Val Gln Cys 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser Ser Asn Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 Picture 17(PEO21 vH Picture Kit) cag tcg gtg gag gag tcc ggg ggt cgc ctc gtc acg cct ggg aca ccc 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 ctg aca ctc acc tgc acc gtc tcc gga ttc tcc ctc agt agc tat gca 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Tyr Ala 20 25 30 atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tac atc gga 144 Met Ser Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 atc att agt agt agt ggt agc aca tac tac gcg agc tgg gcg aaa ggc 192 Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg aaa atc tcc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Ser 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga gat cgt 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg 85 90 95 gtt att tat agt att ggt ccg tat tat ttt aat ttg tgg ggc cca ggc 336 Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu Trp Gly Pro Gly 100 105 110 acc ctg gtc acc gtc tcc tca 357 Thr Leu Val Thr Val Ser Ser 115 Sequence No. 18 (Amino Acid Sequence of Clone PEO21 vH Domain) Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Ser 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg [[ID=2�]] 85 90 95 Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 SEQ ID NO: 19 (Clone PEO21 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca tcc tcc gtg tct gca act gtg gga 48 Ala Tyr Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala Thr Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt gag atc att tat agc aat 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ile Ile Tyr Ser Asn 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cct ccc aag ctc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 tat ggc gca tcc act ctg gca tct ggg gtc cca tcg cgg ttc aaa ggc 192 Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 agt gga tct ggg aca gag tac act ctc acc atc agc gac ctg cag tgt 240 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Gln Cys 65 70 75 80 gac gat gct gcc act tac tac tgt caa cag agt ttt agt agt aat aat 288 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Ser Asn Asn 85 90 95 gtt ggg aat att ttc ggc gga ggg acc gag gtg gtg gtc aaa 330 Val Gly Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 Sequence number 20 (clone PEO21 vL domain amino acid sequence) Ala Tyr Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala Thr Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ile Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Gln Cys 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Ser Asn Asn 85 90 95 Val Gly Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 sequence number 21(クローンPEO23 vH domain nucleic acid sequence) cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 ctg aca ctc acc tgc aca gcc tct gga ttc tcc ctc agt acc cat gca 96 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr His Ala 20 25 30 ata aac tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tgg atc ggg 144 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 atc act tat gct agt ggt agg aca tat tac gcg agc tgg gcg aaa ggc 192 Ile Thr Tyr Ala Ser Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg aaa atc ​​acc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga aat ggg 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Gly 85 90 95 gct gat gaa aca ttt tac tac ttt gac ttg tgg ggc cca ggc acc ctg 336 Ala Asp Glu Thr Phe Tyr Tyr Phe Asp Leu Trp Gly Pro Gly Thr Leu 100 105 110 gtc acc gtc tcc tca 351 Val Thr Val Ser Ser 115 クローンPEO23 vH domain amino acid sequence 22 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr His Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Thr Tyr Ala Ser Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Gly 85 90 95 Ala Asp Glu Thr Phe Tyr Tyr Phe Asp Leu Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 Sequence No. 23 (Clone PEO23 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca gcc tcc gtg gag gca gct gtg gga 48 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aag tgc cag gcc agt cag aat att aat act tgg 96 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Asn Thr Trp 20 25 30 tta tcc tgg tat cag cag aag gca ggg cag cct ccc aag ctc ctg atc 144 Leu Ser Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 tac agg gca tcc act ctg gca tct ggg gtc tca tcg cgg ttc aaa ggc 192 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 agt gga tct ggg aca cag ttc act ctc acc atc agc ggc gtg gag tgt 240 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 gcc gat gct gcc act tac tac tgt caa caa tat gat gct agt att aat 288 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ala Ser Ile Asn 85 90 95 att gat aat gct ttc ggc gga ggg acc gag gtg gtg gtc aaa 330 Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 Sequence number 24 (clone PEO23 vL domain amino acid sequence) Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Asn Thr Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ala Ser Ile Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 Accession No. 25 (Clone PEO24 vH domain nucleic acid sequence) cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt agc aat gca 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asn Ala [[ID=2跟]]20 25 30 atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tat atc gga 144 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 att att agg aat aat gat atc aca tac tac gcg agc tgg gcg aaa ggc 192 Ile Ile Arg Asn Asn Asp Ile Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg ata atc ​​acc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga ggg ggt 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly 85 90 95 ggt tct tac agt att gtc ttc tgg aac tta tgg ggc cca ggc acc ctg 336 Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu 100 105 110 gtc acc gtc tcc tca 351 Val Thr Val Ser Ser 115 クローンPEO24 vH domain amino acid sequence 26 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asn Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Arg Asn Asn Asp Ile Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly 85 90 95 Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 Sequence No. 27 (Clone PEO24 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca gcc tct gtg gag gta gct gtg gga 48 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt gag agg att tat agc aat 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Arg Ile Tyr Ser Asn 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aaa ctc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 tat tat gca tcc act ctg gca tct ggg gtc tca tcg cgg ttc aaa ggc 192 Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 agt gga tct ggg aca cag ttc act ctc acc atc agc ggc gtg cag tgt 240 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln Cys 65 70 75 80 gcc gat gct gcc act tac tac tgt cag cag ggt tat agt aat aat aat 288 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Asn Asn Asn 85 90 95 gtt gac aat act ttc ggc gga ggg acc gag gtg gtg gtc aga 330 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Arg 100 105 110 Sequence No. 28 (Clone PEO24 vL domain amino acid sequence)​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Asn Asn Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Arg 100 105 110 SEQ ID NO: 29 (clone PEOWT22 vH domain CDR1 amino acid sequence) Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 SEQ ID NO: 30 (clone PEOWT22 vH domain CDR2 amino acid sequence) Ile Ser Tyr Asp Val Ser Asn Lys 1 5 SEQ ID NO: 31 (clone PEOWT22 vH domain CDR3 amino acid sequence) Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 SEQ ID NO: 32 (clone PEOWT22 vL domain CDR1 amino acid sequence) Gln Ser Ile Ser Arg Tyr 1 5 SEQ ID NO: 33 (clone PEOWT22 vL domain CDR2 amino acid sequence) Asp Ala Ser 1 Figure 34(PEOWT22 vL CDR3 file) Gln Gln Phe Gly Arg Ser Pro Arg Thr 1 5 35 (in the log of PEOWT22) gag gtg caa ctg gtg gag tct ggg gga ggt gtg gta agg cct ggg ggg 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr 20 25 30 gct atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca gtt ata tca tat gat gta agc aat aaa tac gca gac tcc gtg 192 Ala Val Ile Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga tct tac tac tac tac tac ggt atg gac gtc tgg ggc caa ggg 336 Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110 acc acg gtc acc gtc tcc tca gcc tcc act aag ggc cca tcc gtg ttc 384 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 cca ctg gca ccc tct agt aag agc aca tct ggg ggt act gcc gct ctg 432 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 gga tgt ctg gtg aag gat tac ttc cca gag cca gtc acc gtg tcc tgg 480 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 aac agc ggg gcc ctg act tcc ggt gtc cat acc ttt cca gct gtg ctg 528 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 cag tca tcc ggc ctg tac agc ctg agc tct gtg gtc acc gtc ccc agt 576 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 tca tcc ctg gga aca cag act tat atc tgc aac gtg aat cac aag cca 624 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 tcc aat aca aaa gtc gac aag aaa gtg gaa ccc aag agc tgt gat aaa 672 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 acc cat aca tgc ccc cct tgt cct gct cca gag ctg ctg gga gga cca 720 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 tcc gtg ttc ctg ttt cca ccc aag cct aaa gac act ctg atg att tct 768 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 cga acc ccc gaa gtc aca tgc gtg gtc gtg gac gtg tcc cac gag gat 816 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 cct gaa gtc aag ttc aac tgg tac gtg gat ggc gtc gag gtg cat aat 864 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 gcc aag aca aaa cca cga gag gaa cag tac aac agt acc tat cgt gtc 912 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 gtg tca gtc ctg aca gtg ctg cac cag gac tgg ctg aac ggg aag gaa 960 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 tat aag tgc aaa gtg agc aat aag gca ctg ccc gcc cct atc gag aaa Tyr Lys Cys Lys Will Be Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 aca att tct aag gct aaa gga cag cct agg gaa cca cag gtg tac act Thr Is Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 ctg cct cca tca cgg gac gag ctg aca aag aac cag gtc agt ctg act 1104 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 tgt ctg gtg aaa ggg ttc tat cct tct gat atc gcc gtg gag tgg gaa 1152 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 agt aat ggt cag cca gag aac aat tac aag acc aca ccc cct gtc ctg 1200 Serving Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 gac tct gat ggg agt ttc ttt ctg tat tcc aag ctg acc gtg gat aaa 1248 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 agc cgg tgg cag cag ggt aat gtc ttt agt tgt tca gtg atg cac gag 1296 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 gca ctg cac aat cac tac acc cag aaa tca ctg tca ctg tca cca ggt 1344 Only Leu His Asn His Tyr Thr Gln Lys Served Leu Ser Leu Served Pro Gly 435 440 445 yes it is 1350 Lys Photo 36 (See PEOWT22 on Facebook page) Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385,390,395,400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys PEOWT22 37-inch (2000) gat gtt gtg atg acc cag tct ca tcc tcc ctg tct gca tct gta gga 48 Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgc cgg gca agt cag agc att agc agg tac 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gtc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag cca 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tca cct cgg 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg 85 90 95 acg ttc ggc caa ggg aca cga ctg gag att aaa cga act gtg gct gca 336 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttc aac agg gga gag tgt tag 645 Phe Asn Arg Gly Glu Cys 210 SEQ ID NO: 38 (Full-length vL chain amino acid sequence of clone PEOWT22) Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 39 (in the profile of PEOWT22 scFv gat gtt gtg atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48 Asp Val Val Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgc cgg gca agt cag agc att agc agc tac 96 Asp Arg Val Thr With Thr Cys Arg Only Serves Gln Ser With Arg Tyr 20 25 30 tta gcc tgg tac cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Wing Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gtc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag cca 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tca cct cgg 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg 85 90 95 acg ttc ggc caa ggg aca cga ctg gag att aaa ggc gga tcc tct agg 336 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Ser Ser Arg 100 105 110 tca agt tcc agc ggc ggc ggt ggc agc gga ggc ggc ggt gag gtg caa 384 Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln 115 120 125 ctg gtg gag tct ggg gga ggt gtg gta agg cct ggg ggg tcc ctg aga 432 Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg 130 135 140 ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat gct atg cac 480 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met His 145 150 155 160 tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg gca gtt ata 528 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile 165 170 175 tca tat gat gta agc aat aaa tac tac gca gac tc gtg aag ggc cga 576 Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 ttc acc atc tcc aga gac aat tcc aac aac acg ctg tat ctg caa atg 624 Phe Thr Ile Serves Arg Asp Asn Serves Lys Asn Thr Leu Tyr Leu Gln Met 195 200 205 aac agc ctg aga gct gag gac acg gct gtg tat tac tgt gcg aga tct 672 Asn Ser Leu Arg Ala Glu Asp Thr Val Tyr Tyr Cys Ala Arg Ser 210 215 220 tac tac tac tac tac ggt atg gac gtc tgg gc caa ggg acc acg gtc 720 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 225 230 235 240 acc gtc tcc tca 732 Thr Val Ser Ser sequence number 40(クローンPEOWT22 scFv amino acid sequence) Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Ser Ser Arg 100 105 110 Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met His 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile 165 170 175 Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser 210 215 220 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 225 230 235 240 Thr Val Ser Ser Sequence number 41 (Clone PEO25 vH domain nucleic acid sequence) cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt aac aat gca 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Asn Asn Ala 20 25 30 atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tat atc gga 144 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 atc att agg agt agt ggt agt aca tat tac gcg aac tgg gca aaa ggc 192 Ile Ile Arg Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 cgg ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg ata atc acc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga ggg ggt 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly 85 90 95 ggt tct tac agt att gtc ttc tgg aac ttg tgg ggc cca ggc acc ctg 336 Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu 100 105 110 gtc acc gtc tcc tca 351 Val Thr Val Ser Ser 115 Sequence number 42 (Clone PEO25 vH domain amino acid sequence) Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Asn Asn Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Arg Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly 85 90 95 Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 SEQ ID NO: 43 (Clone PEO25 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca gcc tct gtg gag gta gct gtg gga 48 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt gag agg att tat agc aat 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Arg Ile Tyr Ser Asn 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aag ctc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 tat tat aca tcc act ctg gca tct ggg gtc tca tcg cgg ttc aaa ggc 192 Tyr Tyr Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 agt gga tct ggg aca cag ttc act ctc acc atc agc ggc gtg gag tgt 240 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 gcc gat gct gcc act tac tac tgt caa cag ggt tat agt agt agt aat 288 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ser Ser Asn 85 90 95 gtt gac aat act ttc ggc gga ggg acc gag gtg gtg gtc aaa ggt 333 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly 100 105 110 SEQ ID NO: 44 (clone PEO25 vL domain amino acid sequence) Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Arg Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ser Ser Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly 100 105 110 SEQ ID NO: 45 (Humanized PEO20 vH domain nucleic acid sequence) ggc gag cag cag ctg gtg gag agc ggc gga ggc ctg gtg cag cct gga 48 Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 gga agc ctg agg ctg agc tgc gcc gtg tcc ggc ttc agc ctg agc agc 96 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser 20 25 30 tac gcc atc agc tgg gtg agg cag gcc ccc gga aag ggc ctg gag tac 144 Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 atc gcc atc atc aac agc tac ggc acc acc tac tac gcc agc tgg gcc 192 Ile Ala Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 aag ggc aga gtg acc atc tcc aag gat tcc tcc aag aac acc gtg tac 240 Lys Gly Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 ctg cag atg ggc tcc ctg aga gcc gag gat atg gcc gtg tac ttt tgc 288 Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys 85 90 95 gcc aga ggc gat tcc tac ggc tcc ggc gtg ggc ctg ggc ctg tgg gga 336 Ala Arg Gly Asp Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly 100 105 110 cct gga acc ctg gtg aca gtg tcc tcc 363 Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 SEQ ID NO: 46 (Amino acid sequence of humanized PEO20 vH domain) Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser 20 25 30 Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 Ile Ala Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Asp Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly 100 105 110 Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 SEQ ID NO: 47 (Humanized PEO20 vL domain nucleic acid sequence) gga gac tac cag atg aca cag tcc cct agc acc ctg tcc gcc tcc gtg 48 Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val 1 5 10 15 ggc gac aga gtg aca atc acc tgt cag gcc tcc cag aat atc tac agc 96 Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Ser 20 25 30 aat ctg gcc tgg tac cag cag aag cct ggc aag agg ccc aag ctg ctg 144 Lion Asn Trp Tyr Gln Gln Lys Pro Gly Lys Arg Pro Lys Leu Lion 35 40 45 atc tac aga gcc agc tcc ctg gcc tcc ggc gtg cca tct aga ttt tcc 192 Ile Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 ggc tcc ggc agc ggc aca gag ttt acc ctg aca atc ​​agc agc ctg cag 240 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 ccc gat gat ttc gcc acc tac tac tgt cag cag ggc ttc agc agc aat 288 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser Ser Ser Asn 85 90 95 aat gtg gac aat aca ttt ggc ggc ggc aca aag gtg gag atc aag 333 Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 48 (Photo of PEO20 vL Photograph) Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Ser 20 25 30 Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Arg Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser Ser Asn 85 90 95 Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Sequence number 49 (humanized PEO19 vH domain nucleic acid sequence) ggc gag cag cag ctg gtg gag agc ggc gga ggc ctg gtg cag cct gga 48 Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 gga agc ctg agg ctg agc tgc gcc gtg tcc ggc ttt tcc ctg agc aag 96 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Lys 20 25 30 agc atc atc agc tgg gtg agg cag gcc cct ggc aag ggc ctg gag tac 144 Ser Ile Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 atc ggc atc atc ggc agc agc ggc tcc acc tac tac gcc aac tgg gcc 192 Ile Gly Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala 50 55 60 aag ggc aga ttc aca atc tcc aag gac tcc tcc aag aat acc gtg tac 240 Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 ctg cag atg ggc tcc ctg agg gcc gag gat atg gcc gtg tac ttt tgt 288 Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys 85 90 95 gcc aga ggc ctg ctg tac tcc ggc aat aag tcc tgg ggc ccc ggc aca 336 Ala Arg Gly Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr 100 105 110 ctg gtg acc gtg agc tcc 354 Leu Val Thr Val Ser Ser 115 SEQ ID NO: 50 (Amino acid sequence of humanized PEO19 vH domain) Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Lys 20 25 30 Ser Ile Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 Ile Gly Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 Movie 51 (Picture of PEO19 vL Picture Photo) ggc gac atc gtg atg acc cag tcc ccc gat tcc ctg gcc gtg tcc ctg 48 Gly Asp Is With Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu 1 5 10 15 ggc gag aga gcc aca atc ​​aat tgt cag tcc tcc cag agc gtg ctg ctg 96 Gly Glu Arg With Three Ile Asn Cys Gln Ser Gln Ser Val Leu Leu 20 25 30 aac aat cag ctg tcc tgg ttc cag cag aag cct ggc cag cct ccc aag 144 Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys 35 40 45 ctg ctg atc tac gac gcc tcc aca ctg gag tcc ggc gtg ccc gat agg 192 Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Asp Arg 50 55 60 ttc agc ggc tcc ggc agc ggc acc gac ttt acc ctg acc atc tcc agc 240 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 ctg cag gcc gag gat gtg gcc gtg tac tac tgc ctg ggc ggc tac agc 288 Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gly Gly Tyr Ser 85 90 95 ggc aac ctg tac gcc ttt ggc ggc ggc acc aag gtg gag atc aag 333 Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Sequence No. 52 (Amino Acid Sequence of Humanized PEO19 vL Domain) Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu 1 5 10 15 Gly Glu Arg Ala Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Leu 20 25 30 Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys 35 40 45 Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gly Gly Tyr Ser 85 90 95 Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 SEQ ID NO: 53 (Humanized PEO21 vH domain nucleic acid sequence) ggc gag cag cag ctg gtg gag tcc ggc gga ggc ctg gtg cag cca gga 48 Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 gga agc ctg agg ctg tcc tgt gcc gtg agc ggc ttc tcc ctg agc tcc 96 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser 20 25 30 tac gcc atg agc tgg gtg agg cag gcc ccc gga aag ggc ctg gag tac 144 Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 atc ggc atc atc agc agc agc ggc agc aca tac tac gcc agc tgg gcc 192 Ile Gly Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 aag ggc agg ttc aca atc agc aag gat tcc tcc aag aat aca gtg tac 240 Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 ctg cag atg ggc tcc ctg agg gcc gag gac atg gcc gtg tac ttc tgt 288 Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys 85 90 95 gcc aga gac agg gtc atc tat tcc atc ggc cct tac tac ttc aac ctg 336 Ala Arg Asp Arg Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu 100 105 110 tgg ggc ccc ggc aca ctg gtg aca gtg tcc agc 369 Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 SEQ ID NO: 54 (Amino Acid Sequence of Humanized PEO21 vH Domain) Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser 20 25 30 Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 Ile Gly Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys 85 90 95 Only Arg Asp Arg Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu 100 105 110 Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 55 (Photo of PEO21 vL Photo Photo) ggc gat tac cag atg aca cag tcc ccc tcc tcc ctg agc gcc tcc gtg 48 Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Leu Ser Ala Ser Val 1 5 10 15 gga gat agg gtg acc atc aca tgc cag gcc agc gag atc atc tac agc 96 Gly Asp Arg Val Thr With Thr Cys Gln Only Ser Glu With Tyr Ser 20 25 30 aat ctg gcc tgg tac cag cag aag ccc ggc aag ccc ccc aag ctg ctg 144 Liver Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Lion 35 40 45 atc tac ggc gcc tcc aca ctg gcc agc ggc gtg cct agc aga ttc agc 192 Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 ggc agc ggc tcc ggc acc gat tac acc ctg aca atc tcc agc ctg cag 240 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 cct gag gat ttt gcc aca tac tac tgt cag cag tcc ttc agc tcc aat 288 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Ser Asn 85 90 95 aac gtg ggc aac atc ttc ggc ggc ggc aca aag gtg gag atc aag 333 Asn Val Gly Asn Ile Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Sequence number 56 (humanized PEO21 vL domain amino acid sequence) Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ile Ile Tyr Ser 20 25 30 Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Ser Asn 85 90 95 Asn Val Gly Asn Ile Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110

[0137] For use in human patients, it may be desirable to humanize these antibodies, replacing both the heavy and light chain constant regions with human constant regions, as well as replacing the variable region framework regions with human antibody framework regions. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is a humanized version of a rabbit antibody.

[0138] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable domain that is at least 60% identical to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and is capable of specifically binding to human CD39.

[0139] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one light chain variable that is at least 60% identical to SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and is capable of specifically binding to human CD39.

[0140] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0141] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having human framework sequences combining with the CDRs of the VH domain selected from SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and the CDRs of the corresponding VL domain selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. The CDRs are preferably identical, but may differ by one, two, or three amino acids in each CDR, so long as the resulting antibody specifically binds to human CD39.

[0142] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, 2008, Front. Biosci. 13:1619-1633, and are described, e.g., in Riechmann et al., 1988, Nature 332:323-329; Queen et al., 1989, Proc. Natl. Acad. Sci. USA 86:10029-10033; U.S. Patent Nos. 5,821,337; 7,527,791; 6,982,321; and 7,087,409; Kashmiri et al., 2005, Methods 36:25-34 (describing specificity-determining region (SDR) grafting); Padlan, 1991, Mol. Immunol. 28:489-498 (describing "resurfacing"); Dall'Acqua et al., 2005, Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al., 2005, Methods 36:61-68 and Klimka et al., 2000, Br. J. Cancer 83:252-260 (describing the "guided selection" approach to FR shuffling).

[0143] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. 1993, J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of particular subpopulations of light chain variable regions or heavy chain variable regions (see, e.g., Carter et al. 1992, Proc. Natl. Acad. Sci. USA 89:4285; and Presta et al. 1993, J. Immunol. 151:2623), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, 2008, Front. Biosci. 13:1619-1633); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., 1997, J. Biol. Chem. 272:10678-10684, and Rosok et al., 1996, J. Biol. Chem. 271:22611-22618).

[0144] In certain embodiments, the anti-CD39 antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-7, and Lonberg, 2008, Curr. Opin. Immunol. 20:450-459.

[0145] For example, human antibodies may be prepared by administering an immunogen to a transgenic animal that has been engineered to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, 2005, Nat. Biotech. 23:1117-1125 (see also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE; U.S. Pat. No. 5,770,429, which describes HuMAB technology; U.S. Pat. No. 7,041,870, which describes the KM MOUSE; and U.S. Patent Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE technology). The human variable regions from intact antibodies produced by such animals can be further modified by combining them with different human constant regions.

[0146] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, 1984, J. Immunol. 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., 1991, J. Immunol. 147:86.) Human antibodies produced by human B cell hybridoma technology are also described in Li et al., 2006, Proc. Natl. Acad. Sci USA 103:3557-3562. Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, 2005, Histology and Histopathology 20(3):927-937, and Vollmers and Brandlein, 2005, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91.

[0147] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage, yeast, or bacterial display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0148] By way of example, anti-CD39 antibodies encompassed by the present invention can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). A variety of methods are known in the art for generating, for example, phage or yeast display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), or described, for example, in McCafferty et al., 1990, Nature 348:552-554; Clackson et al., 1991, Nature 352:624-628; Marks et al., 1992, J. Mol. Biol. 222:581-597; Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., 2004, J. Mol. Biol. 338(2):299-310; Lee et al. al., 2004, J. Mol. Biol. 340(5):1073-1093; Fellouse, 2004, Proc. Natl. Acad. Sci. USA 101(34):12467-12472; and Lee et al., 2004, J. Immunol. Methods 284(1-2):119-132.

[0149] As an example of phage display technology, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage, as described in Winter et al., 1994, Ann. Rev. Immunol., 12:433-455. Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of antigens, including non-self and even self-antigens, without any immunization, as described in Griffiths et al., 1993, EMBO J. 12:725-734. Finally, as described in Hoogenboom and Winter, 1992, J. Mol. Biol. 227:381-388, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0150] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0151] FcyRIII binding can also be increased by state-of-the-art methods, such as altering the amino acid sequence of the Fc portion of the antibody or modifying the glycosylation of the Fc portion (see, e.g., EP 2235061). In certain embodiments, the antibodies of the invention are produced by cells such that, when glycosylated, less than 50% of the oligosaccharide chains on the antibody contain α-1,6-fucose. Typically, in "hypofucosylated" antibody preparations, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5% or less than 1% of the oligosaccharide chains contain α-1,6 fucose. "Defucosylated" antibodies lack α-1,6-fucosyl in the carbohydrate attached to the CH2 domain of the IgG heavy chain. Mori et al., 2007, Cytotechnology 55(2-3):109-114, and Satoh et al., 2006, Expert Opin Biol Ther. 6:1161-1173 relate to a FUT8 (α-1,6-fucosyltransferase) gene knockout CHO line to produce defucosylated antibodies.

[0152] IV. Expression Vectors In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding the anti-CD39 antibodies described herein. For example, a recombinant expression vector can be a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding the polypeptide chain of an anti-CD39 antibody operably linked to suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises an assembly of (1) genetic element(s) that play a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can also be incorporated. DNA regions are "operably linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence that enables extracellular secretion of the translated protein by the host cell. In other embodiments in which the recombinant protein is expressed without a leader or transport sequence, an N-terminal methionine residue can be included. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0153] The choice of expression control sequences and expression vectors will depend on the choice of host. A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli-derived plasmids, including pCR1, pBR322, pMB9, and their derivatives, as well as broad-host-range plasmids such as M13 and other filamentous single-stranded DNA phages.

[0154] Suitable host cells for expressing the polypeptide chains of anti-CD39 antibodies (or proteins used as targets) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin, as described below. Cell-free translation systems may also be used. Appropriate cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are well known to those skilled in the art.

[0155] Various mammalian cell culture systems are used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells can be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblast), C127 (derived from mouse mammary tumor), 3T3 (derived from mouse fibroblast), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical carcinoma), BHK (derived from hamster kidney fibroblast), and HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other non-transcribed sequences flanking the 5' or 3' ends, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences.

[0156] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a powerful method for producing correctly folded, biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

[0157] In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody light chain that comprises a variable region that is at least 60% identical to SEQ ID NO:1, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID NO:1, and that is capable of specifically binding to human CD39.

[0158] In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody heavy chain that comprises a variable region that is at least 60% identical to SEQ ID NO:2, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID NO:2, and that is capable of specifically binding to human CD39.

[0159] V. Encoded Anti-CD39 Antibodies for In Vivo Delivery Therapeutic vectors for delivering the coding sequence for the anti-CD39 antibody to be expressed in the patient can be viral, non-viral, or physical. See, e.g., Rosenberg et al., 1988, Science 242:1575-1578, and Wolff et al., 1989, Proc. Natl. Acad. Sci. USA 86:9011-9014. For a discussion of methods and compositions for use in gene therapy, see Eck et al., 1996, in Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Ninth Edition, Hardman et al., eds., McGraw-Hill, New York, Chapter 5, pp. 77-101; Wilson et al., 1997, *Clin. Exp. Immunol.* 107(Suppl. 1):31-32; Wivel et al., 1998, *Hematology / Oncology Clinics of North America*, *Gene Therapy*, SLEck, ed., 12(3):483-501; Romano et al., 2000, *Stem Cells* 18:19-39; and references cited therein. No. 6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions. Delivery routes include, for example, systemic administration and in situ administration. Well-known viral delivery techniques include the use of adenoviral, retroviral, lentiviral, foamy virus, herpes simplex virus, vaccinia virus, and adeno-associated virus vectors.

[0160] a. Viral vectors Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with nucleic acid constructs carrying nucleic acid sequences encoding epitopes and targeting sequences of interest. Preferred viruses for certain embodiments encompassed by the present invention are adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses already approved for human use in gene therapy. Furthermore, preferred vectors for tolerization do not contain immunostimulatory sequences.

[0161] Adenovirus vectors One exemplary method for in vivo delivery of one or more nucleic acid sequences involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs that contain sufficient adenovirus sequences to (a) assist in packaging the construct and (b) express a polynucleotide cloned therein, in either a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesized. In certain embodiments, the delivery vector involves the commercially available ORF of cytochrome b5 reductase 3 (CYB5R3), transcript variant 1, in the adenovirus vector pAd, with a C-terminal Flag and His tag (Vigene Biosciences product code AH889428). WIPO Patent Application WO / 2015 / 050364 also teaches vectors with an expression construct containing the Cyb5r3 gene.

[0162] Because adenoviral vectors are highly immunogenic, they are less preferred for administration to induce tolerance by presenting antigens or in the case of autoimmune diseases, but these vectors can be used to induce immunity in the treatment of infectious diseases such as influenza, HBV, HCV, and HIV.

[0163] Adeno-associated viral vector (AAV) AAV is a good choice of delivery vehicle due to its safety, i.e., the genetically engineered (recombinant) gene is not integrated into the host genome. Similarly, AAV is not pathogenic and is not associated with any disease. By removing the viral coding sequence, immune responses to viral gene expression are minimized, so rAAV does not induce inflammatory responses. According to certain embodiments, AAV vectors containing epitope sequences comprising the nucleic acid constructs described herein are useful for transducing APCs.

[0164] Typically, viral vectors containing epitopes containing nucleic acid constructs are assembled from polynucleotides encoding the desired epitopes, suitable regulatory elements, and elements required for epitope expression that mediate cell transduction.In one embodiment, adeno-associated virus (AAV) vectors are used.In more specific embodiments, the AAV vector is AAV1, AAV6, or AAV8.

[0165] AAV expression vectors carrying a DNA molecule of interest bounded by AAV ITRs can be constructed by directly inserting a selected sequence(s) into the AAV genome with the major AAV open reading frame ("ORF") excised therefrom. Examples of constitutive promoters that can be included in the AAVs of the present invention include, but are not limited to, the exemplified CMV immediate early enhancer / chicken β-actin (CBA) promoter.

[0166] In eukaryotic cells, expression control sequences typically include a promoter, an enhancer such as those derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, which may contain splice donor and acceptor sites. The polyadenylation sequence is generally inserted after the transgene sequence and before the 3' ITR sequence. In one embodiment, bovine growth hormone polyA may be used.

[0167] Selection of these and other common vectors and regulatory elements is routine, and many such sequences are available. See, e.g., Sambrook et al., and the references cited therein, e.g., pages 3.18-3.26 and 16.17-16.27, and Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York. Naturally, not all vectors and expression control sequences function equally well to express all of the transgenes of the present invention. However, one of skill in the art can make a selection from among these expression control sequences without departing from the scope of the present invention. Suitable promoter / enhancer sequences can be selected by one of skill in the art using the guidance provided by this application. Such selection is a routine matter and does not limit the molecule or construct.

[0168] Retroviral vectors In certain embodiments, the viral vector may be a retroviral vector. A "retrovirus" is a virus having an RNA genome. In certain embodiments, a retroviral vector contains all of the cis-acting sequences necessary for packaging and integration of the viral genome, namely, (a) long terminal repeats (LTRs) or portions thereof at each end of the vector; (b) primer binding sites for minus-strand and plus-strand DNA synthesis; and (c) a packaging signal necessary for incorporating genomic RNA into virions. Further details regarding retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302; Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; Miller et al., 1993, Meth. Enzymol. 217:581-599; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

[0169] "Gammaretrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0170] Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., 1992, J. Virol. 66:2731-2739; Johann et al., 1992, J. Virol. 66:1635-1640; Sommerfelt et al., 1990, Virol. 176:58-59; Wilson et al., 1989, J. Virol. 63:2374-2378; Miller et al., 1991, J. Virol. 65:2220-2224; and PCT / US94 / 05700).

[0171] Lentiviral vectors are a genus of retroviral vectors that can infect dividing and non-dividing cells and typically produce high viral titers. Some examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0172] In certain embodiments, other retroviral vectors can be used. These include, for example, vectors based on human foamy virus (HFV) or other viruses of the spumavirus genus. Foamy virus (FV) is the largest retrovirus known today and is widespread in a variety of mammals, including all non-human primate species, but absent from humans. This complete non-pathogenicity qualifies FV vectors as ideal gene transfer vehicles for gene therapy in humans and clearly distinguishes them as gene delivery systems from HIV-derived and gammaretrovirus-derived vectors.

[0173] Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are replication-deficient retroviruses (i.e., capable of directing synthesis of desired proteins but unable to manufacture infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including incorporating foreign genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retrovirus using the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles) are known to those skilled in the art.

[0174] The retroviral genome contains three genes, gag, pol, and env, which encode capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream of the gag gene contains a signal for packaging the genome into virions. Retroviral vectors are gene transfer plasmids in which a heterologous nucleic acid resides between two retroviral long terminal repeats (LTRs). Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into viral virions in an appropriate packaging cell line (see, e.g., U.S. Pat. No. 4,650,764). These two long terminal repeat (LTR) sequences are located at the 5' and 3' ends of the viral genome. They contain strong promoter and enhancer sequences and are also required for integration into the host cell genome (Coffin, 1990). To construct a retroviral vector, a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in place of specific viral sequences to produce a replication-deficient virus. Also included are episomal or non-integrating forms of retroviral vectors based on lentiviruses (eg, a type of retrovirus).

[0175] Lentiviral vectors are useful when stable expression is required, but they can be immunogenic and have other undesirable effects. Therefore, while lentiviral vectors are convenient for research, caution is required when using them in humans, especially when the goal is to induce tolerance rather than immunity. Lentiviruses are suitable for manipulating T cells or dendritic cells or other antigen-presenting cells ex vivo for cancer therapy, but mRNA electroporation is safer. However, two recent advances have made the use of lentiviruses safer and more clinically translatable. First, coexpression of a suicide gene with an antigen, whose product becomes functional upon drug administration. A classic example is herpes simplex virus thymidine kinase (HSV-Tk). Cells expressing these genes can metabolize the drug ganciclovir into cytotoxic products that induce cell death. Therefore, if some transduced cells become malignant, they can be eradicated. Approximately 12 such systems exist (Duarte et al., 2012, Cancer Letters 324:160-170). Secondly, there are non-integrating lentiviral vectors currently under development, which are therefore non-oncogenic (Nightingale et al., 2006, Mol. Ther. 13:1121-1132). These methods can be used with the present invention according to the judgment of those skilled in the art.

[0176] Retroviral vectors suitable for use herein are described, for example, in U.S. Patent Nos. 5,399,346 and 5,252,479; WIPO Publications WO92 / 07573, WO90 / 06997, WO89 / 05345, WO92 / 05266, and WO92 / 14829, which provide descriptions of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors. Other retroviral vectors include, for example, mouse mammary tumor virus vectors (e.g., Shackleford et al., 1998, Proc. Natl. Acad. Sci. USA 85:9655-9659), lentiviruses, and the like. An exemplary viral vector is plentilox-IRES-GFP.

[0177] Additional retroviral viral delivery systems that can be readily adapted for delivery of transgenes encoding anti-CD39 antibody agents include, by way of example only, published PCT applications WO / 2010 / 045002, WO / 2010 / 148203, WO / 2011 / 126864, WO / 2012 / 058673, WO / 2014 / 066700, WO / 2015 / 021077, WO / 2015 / 148683, WO / 2017 / 040815, the specifications and drawings of each of which are incorporated herein by reference.

[0178] In certain embodiments, the retrovirus is a recombinant, replication-competent retrovirus comprising: a nucleic acid sequence encoding a retroviral GAG protein; a nucleic acid sequence encoding a retroviral POL protein; a nucleic acid sequence encoding a retroviral envelope; an oncoretroviral polynucleotide sequence comprising long terminal repeat (LTR) sequences at the 5' and 3' ends of the oncoretroviral polynucleotide sequence; a cassette comprising an internal ribosome entry site (IRES) operably linked to a coding sequence for an anti-CD39 antibody agent (the cassette is located 5' to the U3 region of the 3' LTR and 3' to the sequence encoding the retroviral envelope); and cis-acting sequences for reverse transcription, packaging and integration into a target cell.

[0179] In certain embodiments, the retrovirus is a recombinant, replication-competent retrovirus comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising a long terminal repeat (LTR) sequence at the 3' end of the retroviral polynucleotide sequence, a promoter sequence (a promoter suitable for expression in mammalian cells) at the 5' end of the retroviral polynucleotide, a gag nucleic acid domain, a pol nucleic acid domain, and an env nucleic acid domain; a cassette comprising the coding sequence of an anti-CD39 antibody agent operably linked to a heterologous polynucleotide (the cassette is positioned 5' to and operably linked to the 3' LTR and 3' to the env nucleic acid domain encoding the retroviral envelope); and cis-acting sequences required for reverse transcription, packaging, and integration into a target cell.

[0180] In certain preferred embodiments of the recombinant replication-competent retrovirus, the envelope is selected from one of amphotropic, polytropic, xenotropic, 10A1, GALV, baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles, or influenza virus envelopes.

[0181] In certain preferred embodiments of the recombinant replication-competent retrovirus, the retroviral polynucleotide sequence is engineered from a virus selected from the group consisting of murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), feline-derived retrovirus RD114, squirrel monkey retrovirus, xenotropic murine leukemia virus-related virus (XMRV), avian reticuloendotheliosis virus (REV), or gibbon ape leukemia virus (GALV).

[0182] In certain preferred embodiments of the recombinant replication-competent retrovirus, the retrovirus is a gammaretrovirus.

[0183] Certain preferred embodiments of recombinant replication-competent retroviruses include, for example, a second cassette downstream of the cassette that includes a coding sequence for a second therapeutic protein, such as another checkpoint inhibitor polypeptide, a costimulatory polypeptide, and / or an immunostimulatory cytokine (by way of example only). In certain examples, the second cassette can include an internal ribosome entry site (IRES) or a minipromoter or pol III promoter operably linked to the coding sequence for the second therapeutic protein.

[0184] In certain preferred embodiments of the recombinant replication-competent retrovirus, it is a non-lytic, amphotropic retroviral replicating vector that preferably selectively infects and replicates in cells of the inflamed tissue microenvironment.

[0185] Other viral vectors as expression constructs Other viral vectors can be used as expression constructs in the present invention to deliver oligonucleotides or polynucleotide sequences to host cells. Vectors derived from viruses such as vaccinia virus, poliovirus, and herpesvirus can be used. These offer several attractive functions in various mammalian cells. Hepatitis B virus is also included.

[0186] b. Non-viral vectors Plasmid vectors Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., 1989, cited above. For the past several years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes to cells in vivo. Plasmid vectors are particularly advantageous for such delivery because they do not have the same safety concerns as many viral vectors. However, these plasmids have promoters compatible with the host cell and can express peptide epitopes encoded by the nucleic acid within the plasmid. Other plasmids are well known to those skilled in the art. Furthermore, plasmids can be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered via a variety of parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or other routes. They can also be administered via nasal sprays or drops, rectal suppositories, and orally. They can also be administered to epidermal or mucosal surfaces using a gene gun. The plasmid may be obtained in aqueous solution and dried onto gold particles, or may be obtained in association with another DNA delivery system, including, but not limited to, liposomes, dendrimers, cochleates, and microencapsulation.

[0187] Thus, in one embodiment, a plasmid is provided for expression of an epitope-containing nucleic acid construct comprising an expression cassette, also referred to as a transcription unit. When the plasmid is placed in an environment suitable for epitope expression, the transcription unit expresses a polynucleotide comprising a sequence encoding the epitope, an ETS and an MHC II activator sequence, or a sequence encoding the epitope and a secretory signal sequence, and other elements encoded in the construct. The transcription unit comprises a transcriptional control sequence transcriptionally linked to a cellular immune response element-encoding sequence. The transcriptional control sequence may comprise a promoter / enhancer sequence, such as a cytomegalovirus (CMV) promoter / enhancer sequence. However, those skilled in the art will recognize that a variety of other promoter sequences suitable for expression in eukaryotic cells are known and can similarly be used in the constructs disclosed herein. The level of expression of the nucleic acid product will depend on the associated promoter and the presence and activation of associated enhancer elements.

[0188] In certain embodiments, sequences encoding desired epitopes and targeting sequences can be cloned into expression plasmids containing regulatory elements (i.e., transcriptional control sequences) for transcription, translation, RNA stability, and replication. Such expression plasmids are well known in the art, and those skilled in the art can design appropriate expression constructs containing polynucleotides containing sequences encoding cellular immune response elements or fragments thereof so that the cellular immune response elements can be expressed. There are many examples of suitable expression plasmids into which polynucleotides containing certain sequences can be cloned, such as pCI-neo, pUMVC, or pcDNA3.

[0189] Large quantities of bacterial hosts carrying plasmids for expression of cellular immune response elements or fragments thereof can be fermented, and the plasmids can be purified for subsequent use. Current human clinical trials using plasmids utilize this approach (Recombinant DNA Advisory Committee Data Management Report, 1994, Human Gene Therapy 6:535-548). Current DNA isolation methods known in the art involve removing contaminating lipopolysaccharides (endotoxins) from the bacteria used to propagate the plasmids. Because endotoxins act as powerful adjuvants and can cause unwanted immune stimulation, this step is most preferably performed for the use of tolerogenic DNA vaccines.

[0190] The purpose of a plasmid is to efficiently deliver a nucleic acid sequence to a cell or tissue and express a therapeutic epitope in the cell or tissue. In particular, the purpose of a plasmid may be to achieve high copy number, avoid potential sources of plasmid instability, and provide a means for plasmid selection. With regard to expression, a nucleic acid cassette contains the elements necessary for expression of the nucleic acid within the cassette. Expression involves efficient transcription by the plasmid of the inserted gene, nucleic acid sequence, or nucleic acid cassette. The expression product may be a protein, polypeptide, or RNA. The nucleic acid sequence may be contained in the nucleic acid cassette. Expression of the nucleic acid may be continuous or regulated.

[0191] Mini Circle Nucleic acid constructs described herein may be prepared in the form of minicircle DNA. Minicircle DNA refers to small (2-4 kb) circular plasmid derivatives that are free of all prokaryotic vector components. Because minicircle DNA vectors do not contain bacterial DNA sequences, they are less likely to be recognized as foreign and destroyed (typical transgene delivery methods involve plasmids containing foreign DNA). As a result, these vectors can be expressed for longer periods (on the order of weeks or months) compared to conventional plasmids (days to weeks). The smaller size of minicircles expands cloning capacity and facilitates their delivery into cells. Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). Information regarding minicircle DNA is provided in Dietz et al., 2013, Vector Engineering and Delivery Molecular Therapy 21(8):1526-1535 and Hou et al., 2015, Molecular Therapy - Methods & Clinical Development, Article number: 14062 doi:10.1038 / mtm.2014.62. Further information regarding minicircles is provided in Chen et al., 2003 September, Mol. Ther. 8(3):495-500, which demonstrates that minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcription levels (Gracey Maniar et al., 2013 January, Mol. Ther. 21(1):131-8).

[0192] The first step in ultimately achieving expression of a product encoded by a nucleic acid is cellular uptake of the nucleic acid. Cellular uptake of nucleic acids depends on several factors, one of which is the length of time the nucleic acid remains in close proximity to the cell surface. For example, intramuscular (im) administration of plasmid DNA in a buffer solution followed by muscle massage resulted in a significant decrease in gene expression, likely due to leakage of DNA from the muscle directly or via lymphatics (Human Gene Therapy 4:151-159 (1993)). Therefore, it may be desirable to formulate nucleic acids with compounds that slow the rate at which nucleic acids diffuse or transport them away from the site where cellular uptake of the nucleic acid is desired. Furthermore, these compounds may be suitable for administration to an organism by means such as injection, while maintaining or restoring the physical properties necessary to increase cellular uptake of nucleic acids.

[0193] To effect expression of the oligonucleotide or polynucleotide sequences, the expression construct must be delivered to a cell. In certain embodiments encompassed by the present invention, the expression construct containing one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or a plasmid.

[0194] To stimulate immunity, any type of DNA vaccine vector can be engineered to be CpG-rich (to stimulate TLR9 on immune cells) or conversely, to remove CpGs, replacing CpG motifs with GpG motifs if possible (Ho et al., 2003, J. Immunol. 71(9):4920-6; Ho et al., 2005, J. Immunol. 175(9):6226-34). DNA vaccines can be designed to contain antigen(s) / epitope(s) and can also include additional genes for co-expression with the antigen (multiple promoter vectors) to act as adjuvants or immunomodulators. These DNA vaccines have been shown to be clinically safe, for example, in T1D patients (Roep et al., 2013, Sci. Transl. Med. 5(191):191ra82).

[0195] mechanical delivery system Additional non-viral delivery methods include, but are not limited to, mechanical delivery systems that can be used in vitro, such as the approach described in Woffendin et al., 1994, Proc. Natl. Acad. Sci. USA 91(24):11581; deposition of photopolymerized hydrogel materials or the use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92 / 11033); the use of handheld gene transfer particle guns (see, e.g., U.S. Pat. No. 5,149,655); and the use of ionizing radiation to activate transferred genes (see, e.g., U.S. Pat. No. 5,206,152 and WO 92 / 11033). Delivery devices may also be biocompatible and biodegradable. The formulation preferably provides a relatively constant level of release of the active ingredient. On the other hand, a more rapid release rate immediately after administration may be desired. The formulation of such compositions is well within the level of one of ordinary skill in the art using known techniques.

[0196] Physical methods for enhancing delivery include electroporation (short pulses of high voltage carry nucleic acids across membranes), gene guns (DNA is loaded onto gold particles, forcing the DNA into cells), sonoporation, magnetofection, and hydrodynamic delivery, all of which are known to those skilled in the art. DNA can also be encapsulated in liposomes, preferably cationic liposomes, or polymersomes (synthetic liposomes), which can interact with cell membranes and undergo fusion or endocytosis, resulting in DNA transfer into cells. DNA can also be complexed with polymers (polyplexes) or dendrimers, which can release their payload directly into the cell cytoplasm.

[0197] Exemplary carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran, and the like. Other exemplary delayed-release carriers include supramolecular biovectors comprising a non-liquid hydrophilic core (e.g., cross-linked polysaccharides or oligosaccharides) and, optionally, an outer layer comprising an amphiphilic compound such as a phospholipid (see, e.g., U.S. Pat. No. 5,151,254, and PCT applications WO94 / 20078, WO94 / 23701, and WO96 / 06638). The amount of active agent contained in the sustained-release formulation depends on the implantation site, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

[0198] Biodegradable microspheres (e.g., polylactic acid polyglycolate) can be used as the carrier of the composition. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344; 5,407,609; and 5,942,252. Modified hepatitis B core protein carrier systems, such as those described in WO / 99 / 40934 and the references cited therein, may also be useful in many applications. Another exemplary carrier / delivery system uses a carrier comprising a microparticulate protein complex, such as that taught in U.S. Patent No. 5,928,647.

[0199] Biodegradable polymeric nanoparticles facilitate the delivery of nonviral nucleic acids into cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self-assembly of cationic, hydrolyzable poly(beta-amino ester) and plasmid DNA.

[0200] Polynucleotides can also be administered to cells by direct microinjection, transient cell permeabilization (eg, co-administration of a repressor and / or activator with a cell permeabilizing agent), fusion to a membrane-translocating peptide, or the like.

[0201] In certain embodiments of the present disclosure, gene constructs are introduced into target cells via electroporation. Electroporation involves exposing cells (or tissues) and DNA (or DNA complexes) to a high-voltage electrical discharge. In vivo electroporation is a gene delivery technique that has been successfully used to efficiently deliver plasmid DNA to many different tissues. Systemic and local expression of genes or cDNAs encoded by plasmids can be achieved by performing in vivo electroporation. The use of in vivo electroporation has been shown to enhance the uptake of plasmid DNA in target inflamed tissues, resulting in expression within the inflamed tissue, and to deliver plasmids to muscle tissues, resulting in systemic expression of anti-CD39 antibodies (see, e.g., US8026223). Exemplary techniques, vectors, and devices for electroporating anti-CD39 antibody agent transgenes into cells in vivo include PCT Publications WO / 2017 / 106795, WO / 2016 / 161201, WO / 2016 / 154473, WO / 2016 / 112359, and WO / 2014 / 066655.

[0202] U.S. Patent No. 7,245,963 describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues within a body or plant. The modular electrode system includes multiple needle electrodes, a hypodermic needle, an electrical connector providing a conductive link from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes attached to a support structure and firmly insert them into selected tissues of a living organism or plant. The biomolecules are then delivered to the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated, applying constant current electrical pulses to the multiple needle electrodes. The applied constant current electrical pulses facilitate the introduction of biomolecules into cells between the multiple electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0203] U.S. Patent Publication No. 2005 / 0052630 describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues within a body or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between electrodes in an array based on user control and input of pulse parameters, and allows for the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk with an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk (see U.S. Patent Publication No. 2005 / 0052630, incorporated herein by reference).

[0204] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 are adapted for deep penetration into tissues such as muscle, as well as other tissues or organs. The electrode array configuration also allows the injection needle (for delivering a selected biomolecule) to be inserted completely into the target organ, and the injection is administered perpendicular to the target tissue in a region pre-delineated by the electrodes.

[0205] Typically, the electric field required for cell electroporation in vivo is generally similar in magnitude to that required for cells in vitro. In one embodiment, the electric field magnitude ranges from about 10 V / cm to about 1500 V / cm, preferably from about 300 V / cm to 1500 V / cm, and preferably from about 1000 V / cm to 1500 V / cm. Alternatively, pulse lengths are longer at lower electric field strengths (about 10 V / cm to 100 V / cm, more preferably from about 25 V / cm to 75 V / cm). For example, when the nominal electric field is about 25 to 75 V / cm, a pulse length of about 10 milliseconds is preferred.

[0206] The pulse length can be from about 10 seconds to about 100 milliseconds. Any desired number of pulses can be used, typically 1 to 100 pulses per second. The delay between pulse sets can be any desired time, such as 1 second. The waveform, field strength, and pulse duration can also depend on the type of cell and the type of molecule to be introduced into the cell via electroporation.

[0207] Also included are electroporation devices incorporating electrochemical impedance spectroscopy ("EIS"). Such devices provide real-time information in vivo, particularly inflamed tissue electroporation efficiency, allowing for optimization of conditions. Examples of electroporation devices incorporating EIS can be found in, for example, WO2016 / 161201, which is incorporated herein by reference.

[0208] The uptake of non-viral delivery vectors encompassed by the present invention can also be enhanced by plasma electroporation, also known as avalanche transfection. Briefly, a microsecond electrical discharge generates cavitation microbubbles on the electrode surface. The mechanical force created by the collapsing microbubbles combined with a magnetic field helps to increase the transport efficiency across the cell membrane compared to the diffusion-mediated transport associated with conventional electroporation. The plasma electroporation technique is described in U.S. Patent Nos. 7,923,251 and 8,283,171. This technique can also be used in vivo for cell transformation (Chaiberg et al., 2006, Investigative Ophthalmology & Visual Science 47:4083-4090; Chaiberg et al., United States Patent No. 8,101 169, Issued January 24, 2012).

[0209] Other alternative electroporation techniques are also contemplated. In vivo plasmid delivery can be performed using cold plasma. Plasma is one of the four fundamental states of matter; the others are solid, liquid, and gas. Plasma is an electrically neutral medium of unbound positive and negative particles (i.e., the overall charge of plasma is near zero). Plasma can be created by heating a gas or by subjecting it to a strong electromagnetic field using a laser or microwave generator. This increases or decreases the number of electrons, generating positively or negatively charged particles called ions (Luo et al., 1998, Phys. Plasma 5:2868-2870), accompanied by the dissociation of molecular bonds, if any exist.

[0210] Cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed, high-voltage signal to suitable electrodes. Cold plasma devices can take the form of gas jet devices or dielectric barrier discharge (DBD) devices. Low-temperature plasma has attracted much enthusiasm and interest due to its ability to provide plasma at relatively low gas temperatures. Providing plasma at such temperatures is of interest for a variety of applications, including wound healing, antibacterial processes, and various other medical treatments and sterilization. As previously mentioned, cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed, high-voltage signal to suitable electrodes. Cold plasma devices can take the form of gas jet devices, dielectric barrier discharge (DBD) devices, or multi-frequency harmonic-rich power sources.

[0211] Dielectric barrier discharge devices rely on a different process to generate cold plasma. Dielectric barrier discharge (DBD) devices include at least one conductive electrode covered with a dielectric layer. An electrical return path is formed by a ground, which may be provided by the target substrate undergoing cold plasma processing, or by providing a built-in ground to the electrode. Energy for a dielectric barrier discharge device can be provided by a high-voltage power supply, such as those described above. More commonly, energy is input to a dielectric barrier discharge device in the form of a pulsed DC voltage to form a plasma discharge. The dielectric layer isolates the discharge from the conductive electrode, reducing electrode etching and gas heating. The pulsed DC voltage can be varied in amplitude and frequency to achieve various operating regimes. Any device incorporating such principles of cold plasma generation (e.g., a DBD electrode device) is within the scope of various embodiments encompassed by the present invention.

[0212] In certain exemplary embodiments, a transgene construct encoding an anti-CD39 antibody agent encompassed by the present invention is delivered using an electroporation device comprising: an applicator; a plurality of electrodes extending from the applicator, the electrodes associated with a coverage area; a power source in electrical communication with the electrodes, the power source configured to generate one or more electroporation signals to cells in the coverage area; and a guide member coupled to the electrodes, the guide member configured to adjust the coverage area of ​​the electrodes. At least a portion of the electrodes can be arranged within the applicator in a conical configuration. The one or more electroporation signals can each be associated with an electric field. The device can further comprise a potentiometer coupled to the power source and the electrodes. The potentiometer can be configured to maintain the electric field substantially within a predetermined range. The one or more electroporation signals may each be associated with an electric field. The device may further include a potentiometer coupled to the power source and the electrodes. The potentiometer may be configured to maintain the electric field within a predetermined range to substantially prevent permanent damage to cells within the covered area and / or substantially minimize pain. For example, the potentiometer may be configured to maintain the electric field at approximately 1300 V / cm.

[0213] The power source may provide a first electrical signal to the first electrode and a second electrical signal to the second electrode. The first electrical signal and the second electrical signal may combine to generate a wave having a beat frequency. The first electrical signal and the second electrical signal may each have at least one of a unipolar waveform and a bipolar waveform. The first electrical signal may have a first frequency and a first amplitude. The second electrical signal may have a second frequency and a second amplitude. The first frequency may be different from or the same as the second frequency. The first amplitude may be different from or the same as the second amplitude.

[0214] In certain embodiments, the present invention provides a method for treating a subject having an inflammatory condition, the method comprising injecting an effective amount of a plasmid encoding an anti-CD39 antibody agent into the inflamed tissue (or tissue proximal thereto) and administering electroporation therapy to the target tissue. In certain embodiments, the electroporation therapy further comprises administering at least one voltage pulse of about 200 V / cm to about 1500 V / cm with a pulse width of about 100 microseconds to about 20 milliseconds.

[0215] In certain embodiments, the plasmid (or the second electroporated plasmid) further encodes at least one or more additional immunosuppressive biologic(s), such as adalimumab, certolizumab, etanercept, golimumab, infliximab, risankizumab, and ustekinumab.

[0216] Lipids and polycationic molecules for delivery of nucleic acid constructs encoding anti-CD39 antibodies Lipid-mediated nucleic acid delivery and expression of exogenous nucleic acids, such as mRNA, in vitro and in vivo have been highly successful. Lipid-based nonviral formulations offer an alternative to adenoviral gene therapy. Current in vivo lipid delivery methods use subcutaneous, intradermal, pulmonary, gastrointestinal, submucosal, intrasynovial, intrathecal, or intracranial injections. Advances in lipid formulations have improved the efficiency of in vivo gene transfer (see PCT Application WO98 / 07408). For example, lipid formulations composed of an equimolar ratio of 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) and cholesterol can significantly improve systemic in vivo gene transfer. DOTAP:cholesterol lipid formulations form unique structures called "sandwich liposomes." These formulations have been reported to "sandwich" DNA between an invaginated bilayer or "vase" structure. Beneficial features of these lipid structures include positive p, colloidal stabilization by cholesterol, two-dimensional nucleic acid packing, and increased serum stability.

[0217] Cationic liposome technology is based on the ability of amphipathic lipids, which possess positively charged head groups and hydrophobic lipid tails, to bind negatively charged DNA or RNA, forming particles that typically enter cells via endocytosis. Some cationic liposomes also contain neutral co-lipids, which are thought to enhance liposome uptake by mammalian cells. Similarly, other polycations, such as poly-l-lysine and polyethyleneimine, complex with nucleic acids through charge interactions and assist in the condensation of DNA or RNA into nanoparticles, which then become substrates for endosome-mediated uptake. Several of these cationic nucleic acid complex technologies, including complexes with various forms of plasmid DNA (pDNA), oligodeoxynucleotides, and synthetic RNA, are being developed as potential clinical products.

[0218] The nucleic acid constructs disclosed herein may be associated with polycationic molecules, which aid in enhanced cellular uptake. Complexing the nucleic acid construct with polycationic molecules also aids in packaging the construct, such as reducing its size, which is believed to aid cellular uptake. Upon entry into endosomes, the low pH allows the complex to dissociate, allowing the polycationic molecules to disrupt the endosomal membrane, facilitating the escape of DNA into the cytoplasm before it can be degraded. Preliminary data indicate that embodiments of the nucleic acid construct, when complexed with polycationic molecules such as polylysine or polyethyleneimine, exhibit enhanced uptake in SCs compared to DCs.

[0219] One example of a polycationic molecule useful for complexing with nucleic acid constructs is a cell-penetrating peptide (CPP), which includes polylysine (see above), polyarginine, and Tat peptide. Cell-penetrating peptides (CPPs) are small peptides that can bind to DNA and, upon release, penetrate the cell membrane, facilitating DNA escape from endosomes to the cytoplasm. Another example of a CPP is a 27-residue chimeric peptide, termed MPG, which was recently shown to bind ss-oligonucleotides and ds-oligonucleotides in a stable manner, resulting in noncovalent complexes that protect the nucleic acid from DNase degradation and efficiently deliver the oligonucleotides to cells in vitro (Mahapatro et al., 2011, J Nanobiotechnol 9:55). When different peptide:DNA ratios were tested, as well as ratios of 10:1 and 5:1 (150 nm and 1 um, respectively), the complexes formed small particles ranging from approximately 150 nm to 1 um. Another CPP is a modified tetrapeptide (tetralysine containing a guanidinocarbonylpyrrole (GCP) group (TL-GCP)) that has been reported to bind with high affinity to 6.2 kb of plasmid DNA, producing positively charged aggregates of 700–900 nm (Li et al., 2015, Agnew Chem Int Ed Enl, 54(10):2941–4). RNA can also be complexed with such polycationic molecules for in vivo delivery.

[0220] Other examples of polycationic molecules that may form complexes with the nucleic acid constructs described herein include the polycationic polymers commercially available as JETPRIME® and in vivoJET (Polypus-transfection, SA, Illkirch, France).

[0221] VI. Methods of Use and Pharmaceutical Compositions The anti-CD39 antibodies encompassed by the present invention are useful in a variety of applications, including, but not limited to, therapeutics such as the treatment of inflammatory diseases and disorders, and conditions generally characterized by eosinophilia. In certain embodiments, the anti-CD39 antibodies described herein are useful for inactivating or otherwise reducing eosinophil-mediated aspects of inflammation and immune responses, as well as systemic or local eosinophilia or drug-induced eosinophilia.

[0222] In general, "eosinophils" encompass a class of hematopoietic cells. In specific embodiments, eosinophils encompassed by the present invention (i) are CD39+ eosinophils; (ii) co-express one or more cell surface markers selected from the group consisting of CD45, CD11b, Siglec-8, the alpha subunit of the IL-5 receptor (IL-5Rα or CD125), the alpha subunit of the IL-3 receptor (IL-3Rα or CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), PIRA, PIRB, L-selectin, EMR1, CCR3 (CD193), and CRTh2 (CD294); (iii) are CD45+CD11b+ eosinophil cells; and / or (iv) are CD45+CD11b+Siglec-8+ eosinophil cells.

[0223] In certain embodiments, the CD39-targeted eosinophil-depleting agents of the present invention can be used to treat gastrointestinal inflammatory conditions. The present disclosure provides methods for administering pharmaceutical compositions comprising CD39-targeted eosinophil-depleting agents to treat, prevent, ameliorate, or delay symptoms and / or inflammation associated with gastrointestinal inflammatory diseases. In some embodiments, the gastrointestinal inflammatory disorder is in the esophagus. In some embodiments, the gastrointestinal inflammatory disorder is eosinophilic esophagitis. In some embodiments, patients exhibit substantial improvements in esophageal function and morphology, including a reduction in the esophageal groove, a reduction in esophageal focal narrowing, an increase in esophageal diameter, an increase in esophageal compliance, an increase in esophageal body distensibility, an increase in ease of swallowing, a reduction in edema, an improvement in angiogenesis, a reduction in rings, a reduction or elimination of exudates, and / or elimination of narrowing.

[0224] For example, CD39-targeted eosinophil-depleting agents can be used as part of the treatment for eosinophilic esophagitis (EoE), an allergic / immune condition in which subjects suffer from inflammation and / or swelling of the esophagus, affecting the patient's ability to swallow food and resulting in malnutrition and growth retardation. While eosinophils are not typically found in the esophagus, in EoE these cells accumulate, causing swelling and a reduction in the esophageal diameter, making swallowing and feeding extremely difficult. Patients often experience episodes of food impaction, which can cause food to become lodged in the patient's esophagus, requiring emergency medical treatment. Due to difficulty swallowing and fear of food impaction, many EoE patients limit themselves to eating soft foods such as yogurt, soups, and smoothies. In severe cases of EoE, patients receive parenteral nutrition (e.g., intravenous nutrition). While this can provide necessary nutrition, it can limit the patient's activity and lead to increased infections at the catheter site.

[0225] In certain embodiments, the present invention provides methods for administering pharmaceutical compositions containing CD39-targeted eosinophil-depleting agents to treat symptoms and / or inflammation associated with eosinophilic esophagitis. The pharmaceutical compositions can be delivered locally to the esophagus, including, by way of example only, via submucosal injection, and systemically. In the case of nucleic acids encoding CD39-targeted eosinophil-depleting agents, the nucleic acids can be transfected into esophageal tissue containing eosinophil infiltrates or tissue adjacent thereto.

[0226] In certain embodiments, the CD39-targeted eosinophil-depleting agent may be part of a treatment that includes one or more glucocorticosteroids, leukotriene antagonists, mast cell stabilizers, immunomodulators, and proton pump inhibitors (PPIs).

[0227] In one embodiment, an antibody or antigen-binding fragment thereof or formulation according to the present disclosure is used to treat a chronic inflammatory condition, which is a condition associated with inappropriate inflammation, including, but not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, vasculitis, Sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma, fibrotic diseases (including cirrhosis of the liver), pulmonary fibrosis, as well as UV damage and psoriasis.

[0228] In certain embodiments, CD39-targeted eosinophil-depleting agents can be part of a treatment for chronic inflammatory diseases or disorders. Chronic inflammation is a debilitating and severe condition associated with many of the diseases described above and is characterized by persistent inflammation at sites of infection or injury, or persistent inflammation of unknown cause, or persistent inflammation associated with altered immune responses in autoimmune diseases.

[0229] Thus, in one embodiment, CD39-targeted eosinophil-depleting agents are used to treat chronic inflammatory conditions, which are associated with any condition associated with inappropriate inflammation, including, but not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, vasculitis, Sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma, fibrotic diseases (including cirrhosis of the liver), pulmonary fibrosis, UV damage, and psoriasis.

[0230] In certain embodiments, the CD39-targeted eosinophil-depleting agent is used to treat a condition selected from axial spondyloarthropathy, primary biliary cholangitis, and allergies, e.g., food allergies such as peanut allergies, or pollen allergies.

[0231] The CD39-targeted eosinophil-depleting agents of the present invention are useful in the treatment of inflammatory or obstructive airway diseases, resulting in, for example, a reduction in tissue damage, airway inflammation, bronchial hyperresponsiveness, remodeling, or disease progression. Inflammatory or obstructive airway diseases to which the present invention is applicable include asthma of any type or origin, including both intrinsic (non-allergic) and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchial asthma, exercise-induced asthma, occupational asthma, and asthma induced after bacterial infection. Treatment of asthma also encompasses the treatment of subjects who exhibit symptoms of wheezing and have been or can be diagnosed as "wheezing infants," an established patient category of major medical concern, but who are now often identified as having paroxysmal or early-stage asthma, e.g., subjects under the age of 4 or 5.

[0232] The preventive effect in the treatment of asthma is evidenced by a reduction in the frequency or severity of symptomatic attacks, such as acute asthma attacks or bronchoconstriction attacks, an improvement in lung function, or an improvement in airway hyperresponsiveness. Furthermore, it can be evidenced by a reduction in the need for other symptomatic treatments, such as anti-inflammatory or bronchodilator therapy, which are intended to limit or discontinue symptomatic attacks when they occur. The preventive effect on asthma may be particularly evident in subjects prone to "morning dip." "Morning dip" is a recognized asthma syndrome common to a significant proportion of asthma patients, characterized by an asthma attack occurring for approximately 4 to 6 hours, i.e., a time substantially separated from the previously administered symptomatic asthma treatment.

[0233] The CD39-targeted eosinophil-depleting agents of the present invention can be used to treat other inflammatory or obstructive airway diseases and conditions to which the present invention is applicable, including acute lung injury (ALI), adult / acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary, airway, or lung disease (COPD, COAD, or COLD), including chronic bronchitis or related respiratory distress, pneumonia, and exacerbation of airway hyperresponsiveness due to other medications, particularly other inhaled medications. The present invention is also applicable to the treatment of bronchitis of any type or cause, including, but not limited to, acute, arachidic, catarrhal, croupus, chronic, or tuberculous bronchitis. Further inflammatory or obstructive airways diseases to which the present invention is applicable include pneumoconiosis of all types and causes (an inflammatory lung disease, generally occupational, caused by repeated inhalation of dust, often associated with chronic or acute airway obstruction) such as aluminosis, anthrax, asbestosis, chalazion, miliary, siderosis, silicosis, tobacco disease, and byssinosis.

[0234] With regard to their anti-inflammatory activity, and particularly with regard to inhibition of eosinophil activity, the CD39-targeted eosinophil-depleting agents of the invention are also useful in treating eosinophil-associated disorders, such as eosinophilia, and particularly eosinophil-associated disorders of the airways (e.g., involving pathological eosinophil infiltration of lung tissue), including eosinophilia affecting the airways and / or lungs, as well as eosinophil-associated disorders of the airways resulting from or occurring concomitantly with Loeffler's syndrome, eosinophilic pneumonia, parasitic (especially metazoan) infections (including tropical eosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa (including Churg-Strauss syndrome), eosinophilic granulomas, and eosinophil-associated disorders affecting the airways caused by drug reactions.

[0235] The CD39-targeted eosinophil-depleting agents of the invention are also useful in the treatment of inflammatory or allergic conditions of the skin, such as psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema, dermatitis herpetica, scleroderma, vitiligo, hypersensitivity vasculitis, urticaria, bullous pemphigus, lupus erythematosus, systemic lupus erythematosus, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, epidermolysis bullosa, acne vulgaris, and other inflammatory or allergic conditions of the skin.

[0236] The CD39-targeted eosinophil-depleting agents of the present invention may also be used to treat diseases or conditions that have an inflammatory component, e.g., ocular diseases and conditions such as ocular allergy, conjunctivitis, keratoconjunctivitis sicca, and vernal conjunctivitis, inflammatory diseases that involve an autoimmune response or have an autoimmune component or etiology, diseases affecting the nose, including allergic rhinitis, including autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, red blood cell anemia, idiopathic thrombocytopenia), systemic lupus erythematosus, rheumatoid arthritis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, STDs, and rheumatoid arthritis. Bowen-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), irritable bowel syndrome, celiac disease, periodontitis, hyaline membrane disease, kidney disease, glomerular disease, alcoholic liver disease, multiple sclerosis, endocrine ophthalmopathy, Graves' disease, sarcoidosis, alveolitis, chronic hypersensitivity pneumonitis, multiple sclerosis, primary biliary cirrhosis, uveitis (anterior and posterior), Sjogren's syndrome, keratoconjunctivitis sicca and vernal conjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, systemic juvenile idiopathic arthritis, cryopyrin-associated periodic syndrome, nephritis, vasculitis, diverticulitis, interstitial bladder disease Cystitis, glomerulonephritis (with or without nephrotic syndrome, including idiopathic nephrotic syndrome or minimal change nephropathy), chronic granulomatous disease, endometriosis, leptospirosis, kidney disease, glaucoma, retinal disease, aging, headache, pain, complex regional pain syndrome, cardiac hypertrophy, muscle atrophy, catabolic disorders, obesity, fetal growth retardation, hypercholesterolemia, heart disease, chronic heart failure, mesothelioma, anhidrotic ectodermal dysplasia, Behçet's disease, incontinentia pigmenti, Paget's disease, pancreatitis, hereditary periodic fever syndromes, asthma (allergic and non-allergic, mild, moderate, severe, bronchitis, exercise-induced), acute lung injury, acute Respiratory distress syndrome, eosinophilia, hypersensitivity, anaphylaxis, sinusitis, eye allergies, silica-induced disease, COPD (reduction of damage, airway inflammation, bronchial hyperresponsiveness, remodeling or disease progression), lung disease, cystic fibrosis, acid-induced lung injury, pulmonary hypertension, polyneuropathy, cataracts, myositis associated with systemic sclerosis, inclusion body myositis, myasthenia gravis, thyroiditis, Addison's disease, lichen planus, type 1 or type 2 diabetes, appendicitis, atopic dermatitis, asthma, allergies, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, chronic transplant rejection, colitis,Conjunctivitis, Crohn's disease, cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, enteritis, epicondylitis, epididymitis, fasciitis, fibrosis, gastritis, gastroenteritis, Henoch-Schönlein purpura, hepatitis, hidradenitis suppurativa, immunoglobulin A nephropathy, interstitial lung disease, laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis, nephritis, oophoritis, It may be used to treat other diseases or conditions, such as orchitis, osteitis, otitis media, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, phlebitis, pneumonia, pneumonia, polymyositis, proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, ulcerative colitis, uveitis, vaginitis, vasculitis, or vulvitis.

[0237] In some embodiments, the inflammatory disease that can be treated according to the methods of the present invention is a skin disease, hi some embodiments, the inflammatory disease of the skin is selected from contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforme, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity vasculitis, urticaria, bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, epidermolysis bullosa acquisita, and other inflammatory or allergic diseases of the skin.

[0238] In some embodiments, inflammatory diseases that may be treated according to the methods of the present invention are selected from acute and chronic gout, chronic gouty arthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile idiopathic arthritis (SJIA), cryopyrin-associated periodic syndromes (CAPS), and osteoarthritis.

[0239] In certain embodiments, the CD39-targeted eosinophil-depleting agents of the invention can be used to treat drug-induced eosinophilia, such as eosinophilic asthma and hypereosinophilic disorders, secondary to ICI therapy and / or other drugs, including, but not limited to, antimalarials (e.g., pyrimethamine and dapsone), penicillins, glycopeptides, cephaloporins, sulfonamides, tetracyclines (particularly minocycline), nitrofurantoin, antituberculosis therapy, ACE inhibitors, tryptophan, anticonvulsants (e.g., phenytoin, carbamazepine, and phenobarbitone), NSAIDs, gold, H2 receptor antagonists, proton pump inhibitors, aminosalicylates, and chlorpropamide.

[0240] The present invention provides compositions comprising a CD39-targeted eosinophil-depleting agent, such as an anti-CD39 antibody described herein. The present invention also provides pharmaceutical compositions comprising an anti-CD39 antibody described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in immunotherapy. In some embodiments, the pharmaceutical compositions are used in inflammatory and / or autoimmune diseases. In some embodiments, the pharmaceutical compositions are used in the treatment of human patients.

[0241] Formulations are prepared for storage and use by combining a purified agent encompassed by the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those skilled in the art generally consider pharmaceutically acceptable carriers, excipients, and / or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

[0242] In some embodiments, the anti-CD39 antibodies are lyophilized and / or stored in lyophilized form. In some embodiments, formulations comprising the anti-CD39 antibodies described herein are lyophilized.

[0243] Suitable pharmaceutically acceptable vehicles include non-toxic buffers, such as phosphate, citric acid, and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight polypeptides (fewer than about 10 amino acid residues); proteins, such as serum albumin, Examples of suitable surfactants include, but are not limited to, albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.)

[0244] The pharmaceutical compositions encompassed by the present invention can be administered in any number of ways for either local or systemic treatment: topically, by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders; pulmonary, by inhalation or insufflation of powders or aerosols, such as with a nebulizer, intratracheal, and intranasal; or parenterally, such as intravenously, intraarterially, intracavernously, subcutaneously, intraperitoneally, intramuscularly (e.g., by injection or infusion), or intracranially (e.g., intrathecally or intraventricularly).

[0245] Therapeutic formulations can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets, the primary active ingredient is mixed with a pharmaceutical carrier. Standard tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and diluents (e.g., water). These can be used to form solid preformulation compositions containing a homogeneous mixture of the compounds encompassed by the present invention or their non-toxic pharmaceutically acceptable salts. The solid preformulation composition is then subdivided into unit dosage forms of the type described above. Tablets or pills of the formulation or composition can be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, a tablet or pill can include an inner composition surrounded by an outer component. Moreover, the two components can be separated by an enteric layer, which serves to resist disintegration and permits the inner component to pass intact into the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0246] Anti-CD39 antibodies can also be encapsulated in microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, as described in Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.

[0247] In certain embodiments, the pharmaceutical formulation comprises an anti-CD39 antibody complexed with a liposome. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be produced by reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0248] In certain embodiments, sustained-release formulations comprising anti-CD39 antibodies can be produced. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing anti-CD39 antibodies, which matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0249] In certain embodiments, in addition to administering an anti-CD39 antibody, the method or treatment further comprises administering at least one additional immune response stimulator. In some embodiments, the additional immune response stimulator includes, but is not limited to, a colony-stimulating factor (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-colony-stimulating factor (G-CSF), stem cell factor (SCF)), interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), checkpoint inhibitor, antibody that blocks immunosuppressive function (e.g., anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), toll-like receptor (e.g., TLR4, TLR7, TLR9), or member of the B7 family (e.g., CD80, CD86). The additional immune response stimulator can be administered before, simultaneously with, and / or after administration of the anti-CD39 antibody. Also provided are pharmaceutical compositions comprising an anti-CD39 antibody and an immune response stimulator(s). In some embodiments, the immune response stimulator comprises one, two, three, or more immune response stimulators.

[0250] In certain embodiments, in addition to administering an anti-CD39 antibody, the method or treatment further comprises administering at least one additional therapeutic agent. The additional therapeutic agent can be administered before, simultaneously with, and / or after administration of the anti-CD39 antibody. Pharmaceutical compositions comprising an anti-CD39 antibody and additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises one, two, three, or more additional therapeutic agents.

[0251] Combination therapy with two or more therapeutic agents often, but not necessarily, uses agents that act via different mechanisms of action. Combination therapy using agents with different mechanisms of action may result in additive or synergistic effects. Combination therapy may allow for lower doses of each agent than those used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the anti-CD39 antibody. In some embodiments, combination therapy includes a therapeutic agent that may inhibit immune activation, inhibit immune stimulatory signals, or promote normal tissue regeneration at sites of inflammatory damage.

[0252] In certain embodiments, the present invention provides methods for treating an inflammatory disease, disorder, or condition, comprising administering to a patient in need thereof a CD39-targeted eosinophil-depleting agent and one or more additional therapeutic agents. Such additional therapeutic agents may be small molecules or recombinant biological agents, such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen, etodolac (Lodine®) and celecoxib, colchicine (Colcrys®), corticosteroids such as prednisone, prednisolone, methylprednisolone, hydrocortisone, probenecid, allopurinol, febuxostat (Uoric®), and the like. ®), sulfasalazine (Azulfidine®), antimalarials such as hydroxychloroquine (Plaquenil®) and chloroquine (Aralen®), methotrexate (Rheumatrex®), gold salts such as gold thioglucose (Solganal®), gold thiomalate (Myochrysine®), and auranofin (Ridaura®), D-penicillamine (Depen®), or Cuprimine®), azathioprine (Imuran®), cyclophosphamide (Cytoxan®), chlorambucil (Leukeran®), cyclosporine (Sandimmune®), leflunomide (Arava®), and "anti-TNF" agents such as etanercept (Enbrel®), infliximab (Remicade®), golimumab (Simponi®), cephalosporin (Campanil ... lutolizumab pegol (Cimzia®) and adalimumab (Humira®), "anti-IL-1" agents such as anakinra (Kineret®) and rilonacept (Arcalyst®), canakinumab (Ilaris®), "anti-Jak" inhibitors such as tofacitinib, antibodies such as rituximab (Rituxan®), "anti-T cell" agents such as abatacept (Orencia®), "anti-IL-6" agents such asTocilizumab (Actemra®), diclofenac, cortisone, hyaluronic acid (Synvisc® or Hyalgan®), monoclonal antibodies such as tanezumab, anticoagulants such as heparin (Calcinparine®), Liquaemin® and warfarin (Coumadin®), antidiarrheals such as diphenoxylate (Lomotil®) and loperamide (Imodium®), bile acid binders such as cholestyramine, allotrone (Lotronex®), lubiprostone (Amitiza®), laxatives such as Milk of Magnesia, polyethylene glycol (MiraLax®), Dulcolax®, and Seconcot®, anticholinergics or anticonvulsants such as dicyclomine (Bentyl®), Singulair®, beta 2 agonists such as albuterol (Ventolin® HFA, Proventil® HFA), levalbuterol (Xopenex®), metaproterenol (Alupent®), pirbuterol acetate (Maxair®), terbutaline sulfate (Brethaire®), salmerol xinaformate (Serevent®) and formoterol (Foradil®), anticholinergics such as benzodiazepine bromide ipratropium (Atrovent®) and tiotropium (Spiriva®), inhaled corticosteroids such as beclomethasone dipropionate (Beclovent®, Qvar®, and Vanceril®), triamcinolone acetonide (Azmacort®), mometasone (Asthmanex®), budesonide (Pulmocort®), and flunisolide (Aerobid®), Afviar®, Symbicort®, Dulera®, cromolyn sodium (Intal®), methylxanthines such as theophylline (Theo-Dur®, Theolair®, Slo-bidt,Uniphyl®, Theo-24®), and aminophylline, IgE antibodies such as omalizumab (Xolair®), nucleoside reverse transcriptase inhibitors such as zidovudine (Retrovir®), abacavir (Ziagen®), abacavir / lamivudine (Epzicom®), abacavir / lamivudine / zidovudine (Trizivir®), didanosine (Videx®), emtricitabine (Emtriva®), lamivudine (Epivir®), nucleotide reverse transcriptase inhibitors, such as tenofovir (Viread®), amprenavir (Agenerase®), thiazolinone (Thiazolinone ... (R), protease inhibitors such as atazanavir (Reyataz®), darunavir (Prezista®), fosamprenavir (Lexiva®), indinavir (Crixivan®), lopinavir and ritonavir (Kaletra®), nelfinavir (Viracept®), ritonavir (Norvir®), sabiquinavir (Fortovase® or Invirase®), and tipranavir (Aptivus®), Intracellular endothelial growth factor receptor 1 (EOTR) inhibitors, such as enfuvirtide (Fuzeon®) and maraviroc (Selzentry®), integrase inhibitors, such as raltegravir (Isenpress®), doxorubicin (hydrodaunorubicin®), vincristine (Oncovin®), bortezomib (Velcade®), and dexamethasone (Decadron®) in combination with lenalidomide (Revlimid®), or any combination(s) thereof.

[0253] In another embodiment, the present invention provides a method of treating gout, comprising administering to a patient in need thereof a CD39-targeted eosinophil-depleting agent and one or more additional therapeutic agents selected from nonsteroidal anti-inflammatory drugs (NSAIDS), such as aspirin, ibuprofen, naproxen, etodolac (Lodine®) and celecoxib, colchicine (Colcrys®), corticosteroids, such as prednisone, prednisolone, methylprednisolone, hydrocortisone, probenecid, allopurinol, and febuxostat (Uloric®).

[0254] In another embodiment, the present invention provides a method of treating rheumatoid arthritis, comprising the combination of a CD39-targeted eosinophil-depleting agent with a nonsteroidal anti-inflammatory drug (NSAIDS), such as aspirin, ibuprofen, naproxen, etodolac (Lodine®) and celecoxib; a corticosteroid, such as prednisone, prednisolone, methylprednisolone, hydrocortisone, sulfasalazine (Azulfidine®); an antimalarial, such as hydroxychloroquine; methotrexate (Rheumatrex®), gold salts such as gold thioglucose (Solganal®), gold thiomalate (Myochrysine®), and auranofin (Ridaura®), D-penicillamine (Depen® or Cuprimine®), azathioprine (Imuran®), , cyclophosphamide (Cytoxan®), chlorambucil (Leukeran®), cyclosporine (Sandimmune®), leflunomide (Arava®), and "anti-TNF" agents such as etanercept (Enbrel®), infliximab (Remicade®), golimumab (Simponi®), certolizumab pegol (Cimzia®), and adalimumab (Humil®). a®), "anti-IL-1" agents, e.g., anakinra (Kineret®) and rilonacept (Arcalyst®), antibodies, e.g., rituximab (Rituxan®), "anti-T cell" agents, e.g., abatacept (Orencia®), and "anti-IL-6" agents, e.g., tocilizumab (Actemra®), to a patient in need thereof.

[0255] In some embodiments, the present invention provides methods of treating osteoarthritis, comprising administering to a patient in need thereof a CD39-targeted eosinophil-depleting agent and one or more additional therapeutic agents selected from acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen, etodolac (Lodine®) and celecoxib, diclofenac, cortisone, hyaluronic acid (Synvisc® or Hyalgan®), and monoclonal antibodies such as tanezumab.

[0256] In some embodiments, the present invention provides methods of treating lupus, comprising administering to a patient in need thereof a CD39-targeted eosinophil-depleting agent and one or more additional therapeutic agents selected from acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen, etodolac (Lodine®) and celecoxib, corticosteroids such as prednisone, prednisolone, methylprednisolone, hydrocortisone, etc., anti-inflammatory agents such as hydroxychloroquine (Plaquenil®) and chloroquine (Alalen®), cyclophosphamide (Cytoxan®), methotrexate (Rheumatrex®), azathioprine (Imuran®), and anticoagulants such as heparin (Calcinparine® and Liquaemin®), warfarin (Coumadin®).

[0257] In some embodiments, the present invention provides methods of treating inflammatory bowel disease, comprising administering to a patient in need thereof a CD39-targeted eosinophil-depleting agent and one or more additional therapeutic agents selected from mesalamine (Asacol®), sulfasalazine (Azulfidine®), antidiarrheal agents such as diphenoxylate (Lomotil®) and loperamide (Imodium®), bile acid binders such as cholestyramine, allotrone (Lotronex®), lubiprostone (Amitiza®), laxatives such as Milk of Magnesia, polyethylene glycol (MiraLax®), Dulcolax®, and Seconcot®, anticholinergics or anticonvulsants such as dicyclomine (Bentyl®), anti-TNF therapy, steroids, and antibiotics such as Flagyl or ciprofloxacin.

[0258] In some embodiments, the present invention provides a method of treating asthma, comprising administering a CD39-targeted eosinophil-depleting agent in combination with a beta-2 agonist, such as Singulair®, albuterol (Ventolin® HFA, Proventil® HFA), levalbuterol (Xopenex®), metaproterenol (Alupent®), pirbuterol acetate (Maxair®), terbutaline sulfate (Brethaire®), salmerol xinaformate (Serevent®), or a combination of these agents. anticholinergics such as ipratropium bromide (Atrovent®) and tiotropium (Spiriva®), inhaled corticosteroids such as beclomethasone dipropionate (Beclovent®, Qvar®, and Vanceril®), triamcinolone acetonide (Azmacort®), mometasone (Asthmanex®), budesonide (Pulmocort®), and one or more additional therapeutic agents selected from flunisolide (Aerobid®), Afviar®, Symbicort®, Dulera®, cromolyn sodium (Intal®), methylxanthines such as theophylline (Theo-Dur®, Theolair®, Slo-bidt, Uniphyl®, Theo-24®), and aminophylline, and IgE antibodies such as omalizumab (Xolair®) to a patient in need thereof.

[0259] In some embodiments, the present invention provides a method of treating COPD, comprising the combination of a CD39-targeted eosinophil-depleting agent with a beta-2 agonist, such as albuterol (Ventolin® HFA, Proventil® HFA), levalbuterol (Xopenex®), metaproterenol (Alupent®), pirbuterol acetate (Maxair®), terbutaline sulfate (Brethaire®), salmerol xinaformate (Serevent®), and formoterol (Foradil®), an anticholinergic, such as ipratropium bromide (Atrovent®) and tiotropium (Spiriva®), a methylxanthine, such as theophylline (Theo-Dur and one or more additional therapeutic agents selected from aminophylline, inhaled corticosteroids such as prednisone, prednisolone, beclomethasone dipropionate (Beclovent®, Qvar®, and Vanceril®), triamcinolone acetonide (Azmacort®), mometasone (Asthmanex®), budesonide (Pulmocort®), flunisolide (Aerobid®), Afviar®, Symbicort®, and Dulera® to a patient in need thereof.

[0260] For the treatment of disease, the appropriate dosage of an anti-CD39 antibody will depend on the type of disease being treated, the severity and course of the disease, the disease's response, whether the anti-CD39 antibody is being administered for therapeutic or prophylactic purposes, previous therapy, and the patient's clinical history, all of which will be determined by the treating physician. The anti-CD39 antibody can be administered once or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease is achieved. The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body and will vary depending on the relative efficacy of each individual agent. The administering physician can determine the optimal dosage, method of administration, and repetition rate. In certain embodiments, the dosage is from 0.01 μg to 100 mg / kg body weight, 0.01 μg to 10 mg / kg body weight, 0.1 μg to 100 mg / kg body weight, 0.1 μg to 10 mg / kg body weight, 1 μg to 100 mg / kg body weight, 1 μg to 10 mg / kg body weight, 0.01 mg to 100 mg / kg body weight, 0.01 mg to 50 mg / kg body weight, 0.01 mg to 25 mg / kg body weight, 0.01 mg to 10 mg / kg body weight The dose of the anti-CD39 antibody is about 0.01 mg to about 10 mg / kg body weight, 0.01 mg to 5 mg / kg body weight, 0.1 mg to 100 mg / kg body weight, 0.1 mg to 50 mg / kg body weight, 0.1 mg to 25 mg / kg body weight, 0.1 mg to 10 mg / kg body weight, 0.1 mg to 5 mg / kg body weight, 1 mg to 100 mg / kg body weight, 1 mg to 50 mg / kg body weight, 1 mg to 25 mg / kg body weight, 1 mg to 10 mg / kg body weight, or 1 mg to 5 mg / kg body weight. In certain embodiments, the dose of the anti-CD39 antibody is about 0.01 mg to about 10 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 0.01 mg / kg body weight.

[0261] In some embodiments, the dosage of the anti-CD39 antibody is about 0.025 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.05 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.1 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.25 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 1 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 1.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 2 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 2.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 7.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 10 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 25 mg / kg body weight. In some embodiments, the dosage is within a range bounded by the dosages described herein, e.g., 0.025 mg / kg to 25 mg / kg body weight, or any range therebetween, e.g., 2 to 25 mg / kg body weight, 5 to 10 mg / kg, etc. In specific embodiments, the dosage can be administered once or more daily, three times per week, twice per week, once per week, once per month, or once per year. In specific embodiments, the anti-CD39 antibody is administered once per week, once every two weeks, once every three weeks, or once every four weeks.

[0262] In some embodiments, the anti-CD39 antibody may be administered at an initial, higher "loading" dose, followed by one or more subsequent lower doses. The frequency of administration may also vary in some embodiments. In some embodiments, the dosing regimen may include administering an initial dose, followed by additional doses (or "maintenance" doses) once per week, once every two weeks, once every three weeks, or once per month. For example, the dosing regimen may include administering an initial loading dose, followed by weekly maintenance doses, e.g., half the initial dose. In some embodiments, the dosing regimen may include administering an initial loading dose, followed by alternate weekly maintenance doses, e.g., half the initial dose. In some embodiments, the dosing regimen may include administering three initial doses over three weeks, followed by alternate weekly maintenance doses, e.g., of the same amount.

[0263] As known to those skilled in the art, the administration of any therapeutic agent can result in side effects and / or toxicity. In some cases, the side effects and / or toxicity are so severe that it becomes impossible to administer a particular agent at a therapeutically effective dose. In some cases, it may be necessary to discontinue drug therapy and try other drugs. However, many drugs in the same therapeutic class often exhibit similar side effects and / or toxicity. This means that patients will need to discontinue treatment or, if possible, will suffer from unpleasant side effects associated with the therapeutic agent.

[0264] In some embodiments, the administration schedule may be limited to a specific number of administrations or "cycles." In some embodiments, the anti-CD39 antibody is administered for 2, 3, 4, 5, 6, 7, 8, or more cycles. For example, the anti-CD39 antibody is administered every 2 weeks for 6 cycles, the anti-CD39 antibody is administered every 3 weeks for 6 cycles, the anti-CD39 antibody is administered every 2 weeks for 4 cycles, or the anti-CD39 antibody is administered every 3 weeks for 4 cycles. The administration schedule can be determined and subsequently modified by one of skill in the art.

[0265] Thus, the present invention provides methods of administering an anti-CD39 antibody described herein to a subject, the methods comprising using an intermittent dosing strategy to administer one or more agents that can reduce side effects and / or toxicity associated with the administration of the anti-CD39 antibody, an anti-inflammatory agent, etc. In some embodiments, methods are provided for treating a disease or condition associated with unwanted eosinophil activity in a human subject, the method comprising administering to the subject a therapeutically effective dose of an anti-CD39 antibody in combination with a therapeutically effective dose of an anti-inflammatory agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every two weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every three weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of an anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every four weeks, hi some embodiments, the anti-CD39 antibody is administered using an intermittent dosing strategy and the anti-inflammatory agent is administered weekly.

[0266] VII. Anti-CD39 antibody complex The anti-CD39 antibodies disclosed herein can also be conjugated to a cytotoxic moiety, hi some embodiments, the bispecific anti-CD39 antibodies disclosed herein are conjugated to a cytotoxic moiety to further improve specificity.

[0267] In certain embodiments, anti-CD39 antibodies (e.g., bispecific anti-CD39 antibodies described herein) can induce cytotoxicity in CD39-expressing cells (e.g., eosinophils) through internalization of the antibody conjugated to or associated with a cytotoxic moiety. Cytotoxic moieties include, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracin dione; tubulin inhibitors, for example, maytansine or an analogue or derivative thereof; mitotic inhibitors, for example, monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; antimetabolites, for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, flucloxin, fluoxetine ... Darabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; alkylating agents, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives, such as Cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or analogs or derivatives thereof; antibiotics, such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB);Diphtheria toxin and related molecules, e.g., diphtheria A chain and its active fragments and hybrid molecules, ricin toxin such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, Shiga-like toxins, e.g., SLTI, SLT II, ​​SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas aeruginosa exotoxin, allorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alphasarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins, e.g., PAPI, PAPII, and PAP-S, momordica charantia inhibitors, curcin, sable, sapaonaria officinalis inhibitors, gelonin, mitogenin, restrictocin, phenomycin, and enomycin toxins; ribonucleases (RNases); DNases I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0268] In one embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to an auristatin or a peptide analog, derivative, or prodrug thereof. Auristatins interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., 2001, Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer activity (US Pat. No. 5,663,149) and antifungal activity (Pettit et al., 1998, Antimicrob. Agents and Chemother. 42:2961-2965). For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugating auristatins to Abs, are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588, and 6,214,345, and International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968, and WO205082023.

[0269] In another embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or an analog, derivative, or prodrug thereof. Suitable PDBs and PDB derivatives, as well as related techniques, are described, for example, in Sagnou et al., 2000, Bioorg Med Chem Lett 10(18):2083-2086; Antonow et al., 2008, Cancer J 14(3):154-169; Howard et al., 2009, Bioorg Med Chem Lett 19:6463-6466; and Hartley et al., 2010, Cancer Res 70(17):6849-6858.

[0270] In another embodiment, the anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, a maytansine, a calicheamicin, a duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or an analog, derivative, or prodrug of any of them.

[0271] In certain embodiments, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to an anthracycline, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to maytansine, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to calicheamicin, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to duocarmycin, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to rachelmycin (CC-1065), or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to dolastatin 10, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to dolastatin 15, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to monomethyl auristatin E, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to monomethyl auristatin F, or an analog, derivative, or prodrug thereof. In another specific embodiment, an anti-CD39 antibody (e.g., a bispecific anti-CD39 antibody described herein) is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine, or an analog, derivative, or prodrug thereof.In another specific embodiment, the anti-CD39 antibody (eg, a bispecific anti-CD39 antibody described herein) is conjugated to irinotecan, or an analog, derivative, or prodrug thereof.

[0272] VIII. Pharmaceutical Compositions The anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention can be formulated into compositions, particularly pharmaceutical compositions. Such compositions comprise a therapeutically or prophylactically effective amount of the anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors, mixed with a suitable carrier, e.g., a pharmaceutically acceptable agent. Typically, the anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention are sufficiently purified for administration to animals before being formulated into a pharmaceutical composition.

[0273] Pharmaceutically acceptable agents for use in the pharmaceutical compositions of the present invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluents, emulsifiers, suspending agents, solvents, fillers, extenders, buffers, delivery vehicles, tonicity agents, co-solvents, wetting agents, complexing agents, buffering agents, antibacterial agents, and surface active agents.

[0274] Neutral buffered saline or saline mixed with serum albumin are exemplary suitable carriers. Pharmaceutical compositions can include antioxidants such as ascorbic acid; low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics, and polyethylene glycol (PEG). Suitable tonicity enhancers include, for example, alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and the like. Hydrogen peroxide may also be used as a preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. Buffers can be standard buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffers can be about pH 4-5.5, and Tris buffers can be about pH 7-8.5. Additional pharmaceutical agents are described in Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990.

[0275] The composition may be in liquid or lyophilized or freeze-dried form and may include one or more cryoprotectants, excipients, surfactants, high molecular weight structural additives, and / or bulking agents (see, e.g., U.S. Patent Nos. 6,685,940, 6,566,329, and 6,372,716). In one embodiment, a cryoprotectant is included, and the cryoprotectant is a non-reducing sugar, such as sugar, lactose, or trehalose. The amount of cryoprotectant typically included is such that, upon reconstitution, the resulting formulation is isotonic, although hypertonic or slightly hypotonic formulations may also be suitable. Furthermore, the amount of cryoprotectant should be sufficient to prevent unacceptable amounts of protein degradation and / or aggregation during lyophilization. Exemplary cryoprotectant concentrations of sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 mM to about 400 mM.In another embodiment, nonionic surfactants and ionic surfactants, such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine, are used. Examples of surfactants include linoleyl betaine, myristyl betaine, or cetyl betaine; lauroamidopropyl betaine, cocamidopropyl betaine, hydroxypropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methylisothiazolinone taurate; and surfactants such as the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and ethylene and propylene glycol copolymers (e.g., Pluronics, PF68, etc.). Exemplary amounts of surfactants that may be present in the pre-lyophilized formulation are about 0.001-0.5%.High molecular weight structural additives (e.g., fillers, binders) may include, for example, acacia gum, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylosesulfonate, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfate, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylate, povidone, sodium alginate, tragacanth, microcrystalline cellulose, starch, and zein. Exemplary concentrations of the high molecular weight structural additives are 0.1% to 10% by weight. In other embodiments, bulking agents (eg, mannitol, glycine) may be included.

[0276] The composition may be suitable for parenteral administration.Exemplary compositions are suitable for injection or infusion into animals by any route available to those skilled in the art, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracerebroparenchymal), intraventricular, intramuscular, intraocular, intraarterial, or intralesional route.Parenteral formulations are typically sterile pyrogen-free isotonic aqueous solutions, and optionally contain pharmaceutically acceptable preservatives.

[0277] Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases. See generally Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980, incorporated herein by reference.

[0278] The pharmaceutical compositions described herein can be formulated for sustained or extended release in a manner that results in increased local concentration of the product (e.g., bolus, depot effect) and / or stability or half-life in a particular local environment. The compositions can include formulations of the anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention in particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, and other agents, as well as biodegradable agent matrices, injectable microspheres, microcapsule particles, microcapsules, bioerodible particle beads, liposomes, and other implantable delivery devices that provide sustained or extended release of the active agent, such as depot injections. Techniques for formulating such sustained or extended release delivery vehicles are known, and a variety of polymers have been developed and used for sustained release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, such as those formed by complexation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature- or pH-sensitive properties, may be desirable for creating a drug depot effect due to the mild aqueous conditions associated with entrapment of bioactive protein agents (e.g., antibodies). See, for example, PCT Publication WO 93 / 15722, which describes sustained-release porous polymeric microparticles for delivery of pharmaceutical compositions.

[0279] Materials that are suitable for this purpose include polylactide (see, e.g., U.S. Pat. No. 3...

Claims

1. A composition comprising an anti-CD39 antibody or an antigen-binding fragment thereof, wherein the composition is for reducing the function of eosinophil cells or eosinophils in a target, and the anti-CD39 antibody or its antigen-binding fragment is (i) At a site where the anti-CD39 antibody forms a stable immune complex, at least one antigen-binding domain that binds to ectonucleoside triphosphate diphosphohydrolase-1 (CD39), (ii) The composition comprising an FcγRIIIa binding moiety that binds to the FcγRIIIa receptor and confers to the anti-CD39 antibody a) antibody-dependent cell-mediated cytotoxicity (ADCC) and / or b) antibody-dependent cell-mediated phagocytosis (ADCP) activity against CD39+ cells.

2. The eosinophil cells mentioned above are CD39+ eosinophil cells. In some cases, The aforementioned CD39+ eosinophil cells, (i) Co-expressing one or more cell surface markers selected from the group consisting of CD45, CD11b, Siglect-8, α-subunit of the IL-5 receptor (IL-5Rα or CD125), α-subunit of the IL-3 receptor (IL-3Rα or CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), PIRA, PIRB, L-selectin, EMR1, CCR3 (CD193), and CRTh2 (CD294); (ii) CD45 + CD11b + eosinophil cells; and / or, (iii) CD45 + CD11b + Siglec-8 + eosinophil cells, Preferably, The anti-CD39 antibody or its antigen-binding fragment (i) Stable immune complex formation when incubated with HCC1739BL cells, characterized by a loss of less than 30% of immune complexes after 24 hours (optionally, the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody); (ii) CD39+ eosinophil depletion; (iii) Binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 30; (iv) Binding to CD39 in a non-competitive or partially competitive manner with monoclonal antibody clone A1 that binds to CD39; (v) Depletion of CD39+ eosinophils via ADCC-mediated killing and / or ADCP-mediated killing; and / or (vi) Exhaustion of CD39+ eosinophils in the form of an antibody-drug conjugate that is taken up by the CD39+ eosinophils and is toxic to the CD39+ eosinophils. To promote more, The composition according to claim 1, wherein the FcγRIIIIa binding portion is selected from the group consisting of an Fc domain, an antibody or fragment thereof that binds to FcγRIIIIa, and an FcγRIIIIa-binding peptide.

3. The antigen-binding domain is Fab, Fab', F(ab') ２ A selection from the group consisting of Fv or single-stranded Fv (scFv), Fav, dsFv, sc(Fv)2, Fde, sdFv, single-domain antibody (dAb), and diabody fragment, wherein the antigen-binding domain is an scFv containing the sequence of SEQ ID NO:

40. In some cases, The composition according to claim 1 or 2, wherein the anti-CD39 antibody or antigen-binding fragment is monoclonal.

4. The anti-CD39 antibody or its antigen-binding fragment comprises a VH domain having an amino acid sequence that can be encoded by a nucleic acid that hybridizes to the nucleic acid of SEQ ID NO: 1 under stringent conditions, and a VL domain having an amino acid sequence that can be encoded by a nucleic acid that hybridizes to the nucleic acid of SEQ ID NO: 3 under stringent conditions. Preferably, The anti-CD39 antibody or its antigen-binding fragment comprises a heavy chain having a CDR that is at least 60% identical to one of the CDRs of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a light chain having a CDR that is at least 60% identical to one of the CDRs of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, more, The composition according to claim 1 or 2, comprising a variable heavy chain (VH) in which the anti-CD39 antibody or its antigen-binding fragment is at least 60% identical to any one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a variable light chain (VL) in which the anti-CD39 antibody or its antigen-binding fragment is at least 60% identical to any one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.

5. The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain variable domain comprising a complementarity-determining region (CDR) H1 having an amino acid sequence at least 80% identical to SEQ ID NO: 29, CDRH2 having an amino acid sequence at least 80% identical to SEQ ID NO: 30, and CDRH3 having an amino acid sequence at least 80% identical to SEQ ID NO: 31, (ii) A light chain variable domain comprising CDRL1 having an amino acid sequence at least 80% identical to SEQ ID NO: 32, CDRL2 having an amino acid sequence at least 80% identical to SEQ ID NO: 33, and CDRL3 having an amino acid sequence at least 80% identical to SEQ ID NO: 34, In some cases, The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain variable domain comprising CDRH1 having the sequence of SEQ ID NO: 29, CDRH2 having the amino acid sequence of SEQ ID NO: 30, and CDRH3 having the sequence of SEQ ID NO: 31, (ii) A light chain variable domain comprising CDRL1 having the sequence of SEQ ID NO: 32, CDRL2 having the sequence of SEQ ID NO: 33, and CDRL3 having the sequence of SEQ ID NO: 34, Preferably, The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain variable domain containing the sequence of Sequence ID No. 2; (ii) A light chain variable domain containing the sequence of sequence number 4, including, more, The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain containing the sequence of sequence number 36; (ii) A light chain containing the sequence of sequence number 38, A composition according to claim 1 or 2, comprising:

6. The anti-CD39 antibody or its antigen-binding fragment comprises a heavy chain having a CDR selected from the group consisting of one of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54; a light chain having a CDR selected from the group consisting of one of SEQ ID NOs: 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56; and a human framework sequence, forming a humanized heavy chain and light chain having an antigen-binding site that can specifically bind to human CD39. In some cases, The composition according to claim 1 or 2, wherein the anti-CD39 antibody or its antigen-binding fragment comprises an Fc domain of an IgG1 or IgG3 isotype.

7. The Fc domain is human. In some cases, The anti-CD39 antibody or its antigen-binding fragment is low-fucosylated or defucosylated. Furthermore, in some cases, The anti-CD39 antibody or its antigen-binding fragment is human or humanized. Preferably, The anti-CD39 antibody or its antigen-binding fragment is bispecific, comprising at least one additional antigen-binding site for eosinophil antigens. more, The additional antigen-binding site binds to one or more targets selected from the group consisting of Siglect-8, IL-5Rα (CD125), IL-3Rα (CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), PIRA, PIRB, L-selectin, EMR1, CCR3 (CD193), and CRTh2 (CD294). or The aforementioned additional antigen-binding site binds to an antigen that is upregulated by activated eosinophils. or The aforementioned additional antigen-binding sites are CD3, CD4, γdTCR, CD9, CD28, CD29, CD40, CD44, CD45, CD45RO, CD48, CD58, CD63 (lysosome-binding membrane protein 3), CD66b (CEACAM 8), CD66e (CEACAM 5), CD67, CD69, CD80, CD86, C5αR (CD88), CD101, CD122, CD137 (tumor necrosis factor receptor superfamily member 9, induced by lymphocyte activation, 4-1BB), CD274 (programmed death ligand 1), α ＩＩｂ Integrin (CD41), α2-integrin (CD49b), α4-integrin (CD49d), αL-integrin (CD11a), αM-integrin (CD11b), αX-integrin (CD11c), αD-integrin, β2-integrin (CD18), aminopeptidase N (CD13), FcαRI (CD89), FcγRIIII (CD16), FcγRII (CD32), FcεRII (CD23), granulocyte-monocyte colony-stimulating factor Rα (CD116), HLA-DR, intercellular adhesion molecule-1 (CD 54) The composition according to claim 1 or 2, which binds to interleukin (IL)-2Rα (CD25), IL-17RA, IL-17RB, galectin-3, neuropeptide S receptor, P-selectin glycoprotein ligand-1 (CD162), semaphorin 7A (CD108), thymic interstitial lymphocyte neogenesis factor protein receptor (TSLPR), activated αM integrin, activated β1 integrin (CD29), activated β2 integrin, activated FcγRII, or activated CRTh2 (CD294).

8. The anti-CD39 antibody or its antigen-binding fragment reduces eosinophils. or The anti-CD39 antibody or its antigen-binding fragment is CD39 ｈｉｇｈ Reduce inducible eosinophils and / or activated eosinophils, and optionally, the CD39 ｈｉｇｈ Inducible eosinophils and / or activated eosinophils are presented in i) pathological conditions such as asthma, vasculitis, dermatitis, or sinusitis; and / or ii) located in a space selected from the group consisting of blood, bone marrow, lesions, and / or combinations thereof. In some cases, Eosinophil-related diseases suitable for treatment with the aforementioned anti-CD39 antibody include CD39 in a space selected from the group consisting of blood, bone marrow, lesions, and / or combinations thereof. ｈｉｇｈ The composition according to claim 1 or 2, determined by the presence of inducible eosinophils and / or activated eosinophils.

9. The subject has a disease or condition accompanied by undesirable eosinophil activity, Preferably, The aforementioned undesirable eosinophil activity is caused by abnormal or excessive activation of eosinophils. In some cases, The aforementioned disease or condition is an inflammatory disorder or an autoimmune disease. Furthermore, in some cases, The aforementioned inflammatory disorder is a gastrointestinal inflammatory disorder. Preferably, The aforementioned gastrointestinal inflammatory disorder is eosinophilic esophagitis and / or Crohn's disease. more, The method further comprises administering to the subject one or more agents selected from the group consisting of glucocorticosteroids, leukotriene antagonists, mast cell stabilizers, immunomodulators, and proton pump inhibitors (PPIs). In some cases, The aforementioned inflammatory disorder is a chronic inflammatory state. Furthermore, in some cases, The aforementioned chronic inflammatory state is selected from the group consisting of rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, vasculitis, Sjögren's disease, diabetes mellitus, lupus erythematous, asthma, fibrous disease, UV-induced injury, and psoriasis. Preferably, The aforementioned fibrosis is selected from the group consisting of pulmonary fibrosis, hepatic fibrosis, heart disease, arthral fibrosis, Dupuytren's contracture, keloid fibrosis, mediastinal fibrosis, myelofibrosis, nephrogenic systemic fibrosis, retroperitoneal fibrosis, and scleroderma. more, The pulmonary fibrosis is cystic fibrosis, idiopathic pulmonary fibrosis, or progressive nodular fibrosis. In some cases, The aforementioned liver fibrosis is liver fibrosis, cirrhosis, or primary biliary cirrhosis. Furthermore, in some cases, The aforementioned heart disease is atrial fibrosis, intramyocardial fibrosis, or aged myocardial infarction. Preferably, The inflammatory bowel disease is more preferably ulcerative colitis or Crohn's disease. The aforementioned disease or condition is an inflammatory or obstructive airway disease. In some cases, The inflammatory or obstructive airway disease is selected from the group consisting of asthma, acute lung injury (ALI), adult / acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease, airway or lung disease (COPD, COAD, or COLD), pulmonary activoma, exacerbation of airway hyperreactivity due to other drug therapies, bronchitis, and pneumonia. Furthermore, in some cases, The aforementioned disease or condition is an inflammatory or allergic condition of the skin. Preferably, The aforementioned inflammatory or allergic condition of the skin is selected from the group consisting of psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforme, herpetiform dermatitis, scleroderma, vitiligo, hypersensitivity vasculitis, urticaria, bullous pemphigus, lupus erythematous, lupus erythematous systemicus, pemphigus vulgaris, pemphigus phyllodes, paraneoplastic pemphigus, acquired epidermolysis bullosa, and acne vulgaris. more, The aforementioned disease or condition is inflammatory pneumonia, axial spondyloarthropathy, primary biliary cholangitis, allergic rhinitis, chronic lung disease, allergy, or eosinophilia. or The disease or condition is treated with immune checkpoint inhibitors (ICIs) and / or antimalarial agents (e.g., pyrimethamine and dapsone), penicillin, glycopeptides, cephaloporines, sulfonamides, tetracyclines (especially minocycline), nitrofurantoin, antituberculosis therapy, ACE inhibitors, tryptophan, anticonvulsants (e.g., phenytoin, carbamazepine, and phenobarbiton), NSAIDs, gold, H ２ Drug-induced eosinophilia, such as eosinophilic asthma and hypereosinophilic disorders, secondary to receptor antagonists, proton pump inhibitors, aminosalicylates, and other drugs including but not limited to chlorpropamide. In some cases, The composition according to claim 1 or 2, wherein the disease or condition is related to the respiratory system, digestive system, cardiovascular system, endocrine system, cutaneous system, muscular system, or nervous system, or is a non-neoplastic hematological disorder.

10. The aforementioned disease or condition is tissue graft rejection. Preferably, The composition according to claim 1 or 2, wherein the tissue graft is autologous or allogeneic.

11. The subject is a mammal. Depending on the situation, The composition according to claim 1 or 2, wherein the mammal is a human or a rodent.

12. The anti-CD39 antibody or its antigen-binding fragment is administered to the subject together with one or more pharmaceutically acceptable excipients, buffers, or solutions. and / or The anti-CD39 antibody or its antigen-binding fragment is administered to the subject at a dose of 0.01 to 10 mg / kg, and optionally, the administration is provided by a sustained slow delivery platform to avoid / limit antibody-mediated target cytosis (or antigen modulation or antigen shaving) for optimal ADCC-mediated and / or ADCP-mediated eosinophil depletion efficacy. and / or The anti-CD39 antibody or its antigen-binding fragment is administered to the subject at least once a day, three times a week, twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks, with the administration being optional, once a week. and / or The anti-CD39 antibody or its antigen-binding fragment is administered to the subject for a period of at least two to six treatment cycles, or administered to the subject monthly for lifelong use. and / or The composition according to claim 1 or 2, wherein the anti-CD39 antibody or its antigen-binding fragment is administered to the subject via parenteral administration, submucosal hydrogel administration, transpulmonary administration, or topical application, and the parenteral administration is by subcutaneous administration, intravenous administration, or intramuscular administration.

13. A composition comprising an anti-CD39 antibody or an antigen-binding fragment thereof, wherein the composition is for treating a disease or condition related to undesirable eosinophilic activity in a subject, and the anti-CD39 antibody or the antigen-binding fragment thereof (i) At a site where the anti-CD39 antibody forms a stable immune complex, at least one antigen-binding domain that binds to ectonucleoside triphosphate diphosphohydrolase-1 (CD39), (ii) The composition comprising an FcγRIIIa binding moiety that binds to the FcγRIIIa receptor and confers to the anti-CD39 antibody a) antibody-dependent cell-mediated cytotoxicity (ADCC) and / or b) antibody-dependent cell-mediated phagocytosis (ADCP) activity against CD39+ cells.

14. The disease or condition associated with the unwanted eosinophil activity is caused by 1) abnormal activation or eosinophilia, 2) inflammatory disorders, and / or 3) drug-induced eosinophilia. In some cases, The eosinophil activity is derived from CD39+ eosinophil cells, and optionally, the CD39+ eosinophil cells are (i) Co-expressing one or more cell surface markers selected from the group consisting of CD45, CD11b, Siglect-8, α-subunit of the IL-5 receptor (IL-5Rα or CD125), α-subunit of the IL-3 receptor (IL-3Rα or CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), PIRA, PIRB, L-selectin, EMR1, CCR3 (CD193), and CRTh2 (CD294); (ii) CD45 + CD11b + eosinophil cells; and / or, (iii) CD45 + CD11b + Siglec-8 + eosinophil cells, Preferably, The anti-CD39 antibody or its antigen-binding fragment (i) Stable immune complex formation when incubated with HCC1739BL cells, characterized by a loss of less than 30% of immune complexes after 24 hours (optionally, the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody); (ii) CD39+ eosinophil depletion; (iii) Binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 30; (iv) Binding to CD39 in a non-competitive or partially competitive manner with monoclonal antibody clone A1 that binds to CD39; (v) The anti-CD39 antibody or its antigen-binding fragment depletes CD39+ eosinophils via ADCC-mediated killing and / or ADCP-mediated killing; and / or (vi) The anti-CD39 antibody or its antigen-binding fragment is taken up by the CD39+ eosinophils, and in the form of an antibody-drug conjugate that is toxic to the CD39+ eosinophils, depleting the CD39+ eosinophils. To promote more, The composition according to claim 13, wherein the FcγRIIIIa binding portion is selected from the group consisting of an Fc domain, an antibody or fragment thereof that binds to FcγRIIIIa, and an FcγRIIIIa binding peptide.

15. The antigen-binding domain is Fab, Fab', F(ab') ２ A selection from the group consisting of Fv or single-stranded Fv (scFv), Fav, dsFv, sc(Fv)2, Fde, sdFv, single-domain antibody (dAb), and diabody fragment, wherein the antigen-binding domain is an scFv containing the sequence of SEQ ID NO:

40. In some cases, The anti-CD39 antibody or antigen-binding fragment is monoclonal. Furthermore, in some cases, The anti-CD39 antibody or its antigen-binding fragment comprises a VH domain having an amino acid sequence that can be encoded by a nucleic acid that hybridizes to the nucleic acid of SEQ ID NO: 1 under stringent conditions, and a VL domain having an amino acid sequence that can be encoded by a nucleic acid that hybridizes to the nucleic acid of SEQ ID NO: 3 under stringent conditions. Preferably, The anti-CD39 antibody or its antigen-binding fragment comprises a heavy chain having a CDR that is at least 60% identical to SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a light chain having a CDR that is at least 60% identical to SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, more, The anti-CD39 antibody or its antigen-binding fragment comprises a variable heavy chain (VH) which is at least 60% identical to SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a variable light chain (VL) which is at least 60% identical to SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. In some cases, The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain variable domain comprising a complementarity-determining region (CDR) H1 having an amino acid sequence at least 80% identical to SEQ ID NO: 29, CDRH2 having an amino acid sequence at least 80% identical to SEQ ID NO: 30, and CDRH3 having an amino acid sequence at least 80% identical to SEQ ID NO: 31, (ii) A light chain variable domain comprising CDRL1 having an amino acid sequence at least 80% identical to SEQ ID NO: 32, CDRL2 having an amino acid sequence at least 80% identical to SEQ ID NO: 33, and CDRL3 having an amino acid sequence at least 80% identical to SEQ ID NO: 34, Furthermore, in some cases, The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain variable domain comprising CDRH1 having the sequence of SEQ ID NO: 29, CDRH2 having the amino acid sequence of SEQ ID NO: 30, and CDRH3 having the sequence of SEQ ID NO: 31, (ii) A light chain variable domain comprising CDRL1 having the sequence of SEQ ID NO: 32, CDRL2 having the sequence of SEQ ID NO: 33, and CDRL3 having the sequence of SEQ ID NO: 34, Preferably, The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain variable domain containing the sequence of Sequence ID No. 2; (ii) A light chain variable domain containing the sequence of sequence number 4, including, more, The anti-CD39 antibody or its antigen-binding fragment (i) A heavy chain containing the sequence of sequence number 36; (ii) A light chain containing the sequence of sequence number 38, including, In some cases, The anti-CD39 antibody or its antigen-binding fragment comprises a heavy chain having a CDR selected from the group consisting of one of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54; a light chain having a CDR selected from the group consisting of one of SEQ ID NOs: 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56; and a human framework sequence, forming a humanized heavy chain and light chain having an antigen-binding site that can specifically bind to human CD39. Furthermore, in some cases, The composition according to claim 13 or 14, wherein the anti-CD39 antibody or its antigen-binding fragment comprises an Fc domain of an IgG1 or IgG3 isotype.

16. The Fc domain is human. In some cases, The anti-CD39 antibody or its antigen-binding fragment is low-fucosylated or defucosylated. Furthermore, in some cases, The anti-CD39 antibody or its antigen-binding fragment is human or humanized. Preferably, The anti-CD39 antibody or its antigen-binding fragment is bispecific, comprising at least one additional antigen-binding site for eosinophil antigens. more, The additional antigen-binding site binds to one or more targets selected from the group consisting of Siglect-8, IL-5Rα (CD125), IL-3Rα (CD123), IL-4R, IL-9R, IL-13R, IL-14R, ST2 (IL-33R), PIRA, PIRB, L-selectin, EMR1, CCR3 (CD193), and CRTh2 (CD294). or The aforementioned additional antigen-binding site binds to an antigen that is upregulated by activated eosinophils. or The aforementioned additional antigen-binding sites are CD3, CD4, γdTCR, CD9, CD28, CD29, CD40, CD44, CD45, CD45RO, CD48, CD58, CD63 (lysosome-binding membrane protein 3), CD66b (CEACAM 8), CD66e (CEACAM 5), CD67, CD69, CD80, CD86, C5αR (CD88), CD101, CD122, CD137 (tumor necrosis factor receptor superfamily member 9, induced by lymphocyte activation, 4-1BB), CD274 (programmed death ligand 1), α ＩＩｂ Integrin (CD41), α2-integrin (CD49b), α4-integrin (CD49d), αL-integrin (CD11a), αM-integrin (CD11b), αX-integrin (CD11c), αD-integrin, β2-integrin (CD18), aminopeptidase N (CD13), FcαRI (CD89), FcγRIIII (CD16), FcγRII (CD32), FcεRII (CD23), granulocyte-monocyte colony-stimulating factor Rα (CD116), HLA-DR, intercellular adhesion molecule-1 (CD5 4) The composition according to claim 13 or 14, which binds to interleukin (IL)-2Rα (CD25), IL-17RA, IL-17RB, galectin-3, neuropeptide S receptor, P-selectin glycoprotein ligand-1 (CD162), semaphorin 7A (CD108), thymic interstitial lymphocyte neogenesis factor protein receptor (TSLPR), activated αM integrin, activated β1 integrin (CD29), activated β2 integrin, activated FcγRII, or activated CRTh2 (CD294).

17. The anti-CD39 antibody or its antigen-binding fragment reduces eosinophils. or The anti-CD39 antibody or antigen-binding fragment thereof binds to CD39 ｈｉｇｈ to reduce induced eosinophils and / or activated eosinophils, and optionally, the CD39 ｈｉｇｈ induced eosinophils and / or activated eosinophils are presented in a pathological condition such as asthma, vasculitis, dermatitis, or rhinitis; and / or ii) are located within a space selected from the group consisting of blood, bone marrow, lesions, and / or combinations thereof Furthermore, in some cases, Eosinophil-related diseases suitable for treatment with the aforementioned anti-CD39 antibody include CD39 in a space selected from the group consisting of blood, bone marrow, lesions, and / or combinations thereof. ｈｉｇｈ Determined by the presence of inducible eosinophils and / or activated eosinophils, Preferably, The aforementioned inflammatory disorder is a gastrointestinal inflammatory disorder. more, The aforementioned gastrointestinal inflammatory disorder is eosinophilic esophagitis and / or Crohn's disease. In some cases, The method further comprises administering one or more agents selected from the group consisting of glucocorticosteroids, leukotriene antagonists, mast cell stabilizers, immunomodulators, and proton pump inhibitors (PPIs) to the target. Furthermore, in some cases, The aforementioned inflammatory disorder is a chronic inflammatory state. Preferably, The aforementioned chronic inflammatory state is selected from the group consisting of rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, vasculitis, Sjögren's disease, diabetes mellitus, lupus erythematous, asthma, fibrous disease, UV-induced injury, and psoriasis. more, The aforementioned fibrosis is selected from the group consisting of pulmonary fibrosis, hepatic fibrosis, heart disease, arthral fibrosis, Dupuytren's contracture, keloid fibrosis, mediastinal fibrosis, myelofibrosis, nephrogenic systemic fibrosis, retroperitoneal fibrosis, and scleroderma. In some cases, The pulmonary fibrosis is cystic fibrosis, idiopathic pulmonary fibrosis, or progressive nodular fibrosis. Furthermore, in some cases, The aforementioned liver fibrosis is liver fibrosis, cirrhosis, or primary biliary cirrhosis. Preferably, The aforementioned heart disease is atrial fibrosis, intramyocardial fibrosis, or aged myocardial infarction. more, The aforementioned inflammatory bowel disease is ulcerative colitis or Crohn's disease. In some cases, The aforementioned inflammatory disorder is an inflammatory or obstructive airway disease. Furthermore, in some cases, The inflammatory or obstructive airway disease is selected from the group consisting of asthma, acute lung injury (ALI), adult / acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease, airway or lung disease (COPD, COAD, or COLD), pulmonary activoma, exacerbation of airway hyperreactivity due to other drug therapies, bronchitis, and pneumonia. Preferably, The aforementioned inflammatory disorder is an inflammatory or allergic condition of the skin. more, The aforementioned inflammatory or allergic condition of the skin is selected from the group consisting of psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforme, herpetiform dermatitis, scleroderma, vitiligo, hypersensitivity vasculitis, urticaria, bullous pemphigus, lupus erythematous, lupus erythematous systemicus, pemphigus vulgaris, pemphigus phyllodes, paraneoplastic pemphigus, acquired epidermolysis bullosa, and acne vulgaris. In some cases, The inflammatory disorder is acute and chronic gout, chronic gouty arthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile idiopathic arthritis (SJIA), cryopyrin-associated periodic syndromes (CAPS), or osteoarthritis. Furthermore, in some cases, The disease or condition is treated with immune checkpoint inhibitors (ICIs) and / or antimalarial agents (e.g., pyrimethamine and dapsone), penicillin, glycopeptides, cephaloporines, sulfonamides, tetracyclines (especially minocycline), nitrofurantoin, antituberculosis therapy, ACE inhibitors, tryptophan, anticonvulsants (e.g., phenytoin, carbamazepine, and phenobarbiton), NSAIDs, gold, H ２ The composition according to claim 13 or 14, which is a drug-induced eosinophilia such as eosinophilic asthma and hypereosinophilic disorder secondary to other drugs, including but not limited to receptor antagonists, proton pump inhibitors, aminosalicylic acids, and chlorpropamide.

18. The subject is a mammal. In some cases, The mammal in question is either a human or a rodent. Furthermore, in some cases, The anti-CD39 antibody or its antigen-binding fragment is administered to the subject together with one or more pharmaceutically acceptable excipients, buffers, or solutions. and / or The anti-CD39 antibody or its antigen-binding fragment is administered to the subject at a dose of 0.01 to 10 mg / kg, and optionally, the administration is provided by a sustained slow delivery platform to avoid / limit antibody-mediated target cytosis (or antigen modulation or antigen shaving) for optimal ADCC-mediated and / or ADCP-mediated eosinophil depletion efficacy. and / or The anti-CD39 antibody or its antigen-binding fragment is administered to the subject at least once a day, three times a week, twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks, with the administration being optional, once a week. and / or The anti-CD39 antibody or its antigen-binding fragment is administered to the subject for a period of at least 2 to 6 treatment cycles, or administered to the subject monthly for long-term use. and / or The anti-CD39 antibody or its antigen-binding fragment is administered to the subject via parenteral administration, submucosal hydrogel administration, transpulmonary administration, or topical application, and the parenteral administration is by subcutaneous administration, intravenous administration, or intramuscular administration. Preferably, The method includes administering at least one additional therapeutic agent. more, The at least one additional therapeutic agent is an anti-inflammatory agent, which is optionally selected from the group consisting of nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, leukotriene modifiers, and cytokine pathway blockers. In some cases, The composition according to claim 13 or 14, wherein the additional therapeutic agent is administered before, simultaneously with, and / or after the administration of the anti-CD39 antibody or its antigen-binding fragment.

19. The use of an anti-CD39 antibody or its antigen-binding fragment in the manufacture of a pharmaceutical product for reducing the function of eosinophil cells or eosinophils in a target, Anti-CD39 antibody or its antigen-binding fragment, (i) At a site where the anti-CD39 antibody forms a stable immune complex, at least one antigen-binding domain that binds to ectonucleoside triphosphate diphosphohydrolase-1 (CD39), (ii) A use comprising an FcγRIIIa binding moiety that binds to the FcγRIIIa receptor and confers to the anti-CD39 antibody a) antibody-dependent cell-mediated cytotoxicity (ADCC) and / or b) antibody-dependent cell-mediated phagocytosis (ADCP) activity against CD39+ cells.

20. Use of an anti-CD39 antibody or its antigen-binding fragment in the manufacture of a pharmaceutical product for treating a disease or condition associated with unwanted eosinophilic activity in a subject, Anti-CD39 antibody or its antigen-binding fragment, (i) At a site where the anti-CD39 antibody forms a stable immune complex, at least one antigen-binding domain that binds to ectonucleoside triphosphate diphosphohydrolase-1 (CD39), (ii) A use comprising an FcγRIIIa binding moiety that binds to the FcγRIIIa receptor and confers to the anti-CD39 antibody a) antibody-dependent cell-mediated cytotoxicity (ADCC) and / or b) antibody-dependent cell-mediated phagocytosis (ADCP) activity against CD39+ cells.

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