Methods and Compositions for Treating Autoimmune Conditions - Patent application

Abstract

The present disclosure provides methods and compositions for treating autoimmune and inflammatory conditions, including systemic lupus erythematosus and antiphospholipid syndrome.Certain aspects of the present disclosure relate to methods for treating autoimmune or inflammatory conditions, comprising administering a composition comprising a therapeutically effective amount of NAPc2 or NAPc2 / proline.Further aspects include pharmaceutical compositions comprising NAPc2 or NAPc2 / proline, and optionally one or more additional anti-inflammatory agents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 208,929, filed June 9, 2021, the contents of which are incorporated herein in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on June 8, 2021, is named ARCAP0067WO_Sequence_Listing.txt and is 1,943 bytes in size.

[0003] I. FIELD OF THEINVENTION Aspects of the invention relate to at least the fields of immunology, hematology, and medicine. [Background technology]

[0004] II. Background Although lipid-reactive antibodies appear transiently in infections, the clonal evolution of persistent autoimmune antiphospholipid antibodies (aPL) leads to antiphospholipid syndrome (APS), characterized by severe thromboembolic and microangiopathic complications, pregnancy morbidity, and fetal loss (miscarriage).Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of self-reactive autoantibodies, such as antiphospholipid and anti-dsDNA antibodies.

[0005] Recognized herein is a need for methods and compositions for treating subjects with autoimmune or inflammatory conditions, including systemic lupus erythematosus and antiphospholipid syndrome. Summary of the Invention

[0006] overview The present disclosure meets certain needs by providing methods and compositions for treating or preventing autoimmune or inflammatory conditions.Therefore, in an aspect of the present disclosure, methods and compositions are provided for treating subjects for autoimmune or inflammatory conditions, such as systemic lupus erythematosus or antiphospholipid syndrome.In a particular aspect, compositions comprising NAPc2 or NAPc2 / proline and methods of using such compositions in treating autoimmune and inflammatory conditions, including systemic lupus erythematosus and antiphospholipid syndrome, are disclosed.

[0007] The embodiments of the present disclosure include a method of treating a subject with an autoimmune or inflammatory condition, a method of treating a subject for systemic lupus erythematosus, a method of treating a subject for antiphospholipid syndrome, a method of evaluating the effectiveness of anti-inflammatory treatment, a pharmaceutical composition, a polynucleotide, and a nucleic acid. The method of the present disclosure may include at least one, two, three, or more of the following steps: diagnosing a subject for an autoimmune or inflammatory condition; measuring one or more symptoms of an autoimmune or inflammatory condition in a subject; detecting antiphospholipid antibodies in a biological sample from a subject; detecting anticardiolipin antibodies in a biological sample from a subject; administering NAPc2 to a subject; administering a NAPc2 variant to a subject; administering rNAPc2 to a subject; administering an anti-inflammatory agent to a subject; administering an anticoagulant to a subject; and administering a coagulation factor to a subject. In particular, it is believed that one or more of the above steps can be omitted in certain embodiments.

[0008] Disclosed herein, in some embodiments, is a method for treating a subject for an autoimmune or inflammatory condition, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising nematode anticoagulant protein c2 (NAPc2) or NAPc2 / proline. In some embodiments, the pharmaceutical composition comprises NAPc2. In some embodiments, the pharmaceutical composition comprises NAPc2 / proline. The pharmaceutical composition may comprise one or more additional therapeutic agents. In some embodiments, the method further comprises administering to the subject an additional anti-inflammatory agent. In some embodiments, the additional anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, the pharmaceutical composition does not comprise an additional therapeutic agent. The pharmaceutical composition may comprise one or more pharma- ceutical acceptable excipients.

[0009] In some embodiments, the subject has been diagnosed with an autoimmune or inflammatory condition. The subject may be or has been diagnosed with an autoimmune or inflammatory condition by any means known in the art. In some embodiments, the subject has been determined to have one or more symptoms of an autoimmune or inflammatory condition. The symptoms of an autoimmune or inflammatory condition may be, for example, fatigue, skin lesions, rash, fever, thrombosis (e.g., venous thrombosis, pulmonary embolism), thrombocytopenia, hypertension, renal failure, or recurrent miscarriage. One or more of these symptoms may be excluded from the aspects of the present disclosure. In some embodiments, the pharmaceutical composition is administered to the subject after the onset of symptoms. In some embodiments, the subject has not been diagnosed with an autoimmune or inflammatory condition. In some embodiments, the pharmaceutical composition is administered prior to the onset of symptoms of an autoimmune or inflammatory condition. For example, the pharmaceutical composition may be administered to a subject having or at risk of developing an autoimmune or inflammatory condition. In some embodiments, the subject has been determined to have antiphospholipid antibodies. In some embodiments, the method further comprises detecting the presence of antiphospholipid antibodies in the subject.

[0010] In some embodiments, the subject has been previously treated for the autoimmune or inflammatory condition with a previous treatment (e.g., a previous anti-inflammatory agent). In some embodiments, the subject has been determined to be resistant to a previous treatment. In some embodiments, the subject has not been previously treated for the autoimmune or inflammatory condition. In some embodiments, the subject is treated with a pharmaceutical composition comprising NAPc2 or NAPc2 / proline together with 1, 2, 3, 4, 5, 6, 7, or more additional therapeutic agents (e.g., anti-inflammatory agents, anticoagulants, etc.).

[0011] In some embodiments, the pharmaceutical composition is administered via subcutaneous injection. In some embodiments, the pharmaceutical composition is administered via intravenous infusion. In some embodiments, the pharmaceutical composition is administered to the subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, or 14 days. In some embodiments, the pharmaceutical composition is administered to the subject every other day. In some embodiments, the pharmaceutical composition is administered on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, and / or 14th day. In some embodiments, the pharmaceutical composition is administered on the 1st, 3rd, and 5th day.

[0012] In some embodiments, NAPc2 or NAPc2 / proline is at least, at most, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 , 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15.0 μg / kg. In some embodiments, NAPc2 or NAPc2 / proline is administered at a dose of 5 μg / kg to 10 μg / kg. In some embodiments, NAPc2 or NAPc2 / proline is administered at a dose of about 10 μg / kg. In some embodiments, NAPc2 or NAPc2 / proline is administered at a dose of about 7.5 μg / kg. In some embodiments, NAPc2 or NAPc2 / proline is administered at a dose of about 5 μg / kg. In some embodiments, NAPc2 or NAPc2 / proline is administered on days 1, 3, and 5. In some embodiments, NAPc2 or NAPc2 / proline is administered at a dose of about 7.5 μg / kg on day 1, about 5 μg / kg on day 3, and about 5 μg / kg on day 5.

[0013] In some embodiments, the method further comprises administering to the subject an additional anticoagulant. In some embodiments, the additional anticoagulant is a VKORC1 inhibitor, a thrombin inhibitor, or a factor Xa inhibitor. In some embodiments, the additional anticoagulant is warfarin, heparin or its synthetic analog, rivaroxaban, dabigatran, apixaban, or edoxaban. The method can comprise administering one, two, three, four, five, or more additional anticoagulants.

[0014] In some embodiments, the method does not include administering additional anticoagulant to the subject.For example, in some embodiments, the method does not include administering VKORC1 inhibitor, thrombin inhibitor, or factor Xa inhibitor.In some embodiments, the method does not include administering warfarin, heparin or its synthetic analog, rivaroxaban, dabigatran, apixaban, or edoxaban.

[0015] Also disclosed herein, in some embodiments, is a method for treating a subject for antiphospholipid syndrome, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising nematode anticoagulant protein c2 (NAPc2) or NAPc2 / proline. Furthermore, in some embodiments, is a method for treating a subject for systemic lupus erythematosus, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising nematode anticoagulant protein c2 (NAPc2) or NAPc2 / proline. In some embodiments, the pharmaceutical composition comprises NAPc2. In some embodiments, the pharmaceutical composition comprises NAPc2 / proline. In some embodiments, the method further comprises detecting the presence of antiphospholipid antibodies in a biological sample from the subject prior to administering the pharmaceutical composition. Antiphospholipid antibodies can be detected from the biological sample by any method known in the art. In some embodiments, detecting antiphospholipid antibodies comprises enzyme-linked immunosorbent assay (ELISA).

[0016] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error in any measuring or quantitating method.

[0017] The use of the words "a" or "an" when used in conjunction with the term "comprising" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," or "one or more than one."

[0018] The term "and / or" means "and" or "or." By way of example, A, B, and / or C includes: A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B and C in combination. In other words, "and / or" functions as an inclusive or.

[0019] The words "comprising" (and any form of comprising, such as "comprise", "comprises"), "having" (and any form of having, such as "have", "has"), "including" (and any form of including, such as "includes", "include"), or "containing" (and any form of containing, such as "contains", "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The compositions and methods of use may "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this specification. Compositions and methods that "consist essentially of" any of the disclosed components or steps limit the claims to particular materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. It is contemplated that embodiments described herein in the context of the term "comprising" may also be practiced in the context of the terms "consisting of" or "consisting essentially of."

[0021] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as the "use of" any compound, composition, or agent discussed herein to achieve or produce the described therapeutic, diagnostic, or physiological purpose or effect.

[0022] In particular, it is believed that limitations discussed with respect to one aspect of the invention may also be applied to other aspects of the invention. Moreover, the compositions of the invention may be used in the methods of the invention, and the methods of the invention may be used to prepare or utilize the compositions of the invention. An aspect discussed with respect to one aspect of the disclosure may also be applied to other aspects of the disclosure, and vice versa. For example, a step of a method described herein may also be applied to other methods. Furthermore, a method described herein may exclude any step or combination of steps. Aspects of an aspect described in an example may also be implemented in another example or in the context of an aspect discussed elsewhere in this application, such as in the "Summary", "Detailed Description", "Claims", or "Brief Description of the Figures".

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description, however, it should be understood that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, and that various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Next-generation sequencing analysis of aPL HL5B-induced activation of monocytic MM1 cells and its inhibition by rNAPc2 and anti-TF antibody 10H10 is shown. [Diagram 2] Figure 1 shows the effect of rNAPc2 treatment of MRL / lpr mice on aPL titers. Starting at 11 weeks of age, mice were treated with 0.5 mg / kg every other day; ***P<0.001; ****P<0.0001. [Diagram 3] Effect of rNAPc2 treatment of MRL / lpr mice on anticardiolipin (FIG. 3A) and β2GPI (FIG. 3B) titers. Starting at 11 weeks of age, mice were treated with 0.5 mg / kg every other day; **P<0.01; ****P<0.0001. [Figure 4] Figure 4A shows the effect of rNAPc2 treatment on circulating B cells reacting with fluorescently labeled phospholipids in MRL / lpr mice. Cells were stained in the presence of unlabeled competitors sEPCR / PC, sEPCR / LBPA, or β2GPI. Figure 4B shows the results of staining B cells with fluorescently labeled β2GPI in the presence or absence of sEPCR-PC or sEPCR-LBPA. [Diagram 5] 1 shows the results of an analysis of albuminuria in MRL / lpr mice that were sham-treated or rNAPc2-treated starting at 11 weeks of age. [Figure 6] Glomerular and tubular histology was assessed by semi-quantitative scoring as described in [1]. Pooled data from two cohorts treated with rNAPc2 versus saline are shown; ****P<0.0001. [Figure 7] Assessment of renal immune cell infiltration scored on sections stained for macrophages, T cells, and B cells as described in [1]. Pooled data from two cohorts treated with rNAPc2 versus saline are shown; *P<0.05; **P<0.01. [Figure 8A] Figure 8 shows the effect of NAPc2 treatment on the development of aPL in lupus-prone mice on heparin therapy. Figures 8A and 8B show antibody reactivity with cardiolipin or β2GPI in serum samples obtained from mice treated as indicated. Figures 8C and 8D show staining of peripheral blood B cells for phospholipid or β2GPI reactivity in the absence or presence of excess β2GPI, sEPCR, or sEPCR-LBPA. The percentage of CD19 positive cells is shown. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A] Figure 9 shows the results of next-generation sequencing analysis of monocytic MM1 cells stimulated with aPL HL5B or HL7G, which cross-reacts with β2GPI in plasma, in the presence and absence of NAPc2 inhibition. Figure 9A shows the heat map of differentially regulated transcripts induced by HL5 stimulation. Figure 9B shows the transcripts still induced in aPL HL5B stimulated cells in the presence of NAPc2. Figure 9C shows the transcripts still induced in aPL HL7G stimulated cells in the presence of NAPc2. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description The present disclosure is based, at least in part, on the discovery that NAPc2 abolishes antiphospholipid (aPL)-induced proinflammatory activation of monocytes, suppresses the increase of cardiolipin- and β2 glycoprotein I-reactive autoantibodies in mouse models of systemic lupus erythematosus (SLE), and prevents kidney disease in preclinical SLE models. Thus, as disclosed herein, NAPc2 not only acts as an inhibitor of TF-dependent thrombosis, but also prevents the development of pathogenic autoantibodies and organ pathology in autoimmune diseases. Accordingly, aspects of the present disclosure are directed to methods for treating a subject for an autoimmune or inflammatory condition, such as systemic lupus erythematosus and antiphospholipid syndrome, comprising administering to the subject NAPc2 or a variant thereof (e.g., NAPc2 / proline).

[0026] I. Proteins As used herein, a "protein" or "polypeptide" refers to a molecule that comprises at least four amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a natural molecule present in an organism. In some embodiments, a wild type version of a protein or polypeptide is used, but in many embodiments of the present disclosure, a modified protein or polypeptide is used to generate an immune response. The above terms can be used interchangeably. A "modified protein" or "modified polypeptide", or "mutant", refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to the wild type protein or polypeptide. In some embodiments, a modified / mutated protein or polypeptide has at least one altered activity or function (with the recognition that a protein or polypeptide may have multiple activities or functions). Specifically, it is believed that a modified / mutated protein or polypeptide may be altered with respect to one activity or function, but retain wild type activity or function in other respects, such as immunogenicity.

[0027] When a protein is specifically mentioned herein, it is generally a reference to a natural (wild type) or recombinant protein, or optionally a protein with the signal sequence removed. A protein can be directly isolated from the organism in which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments encoding polypeptides, and recombinant vectors incorporating nucleic acid sequences encoding polypeptides. The term "recombinant" can be used in conjunction with a polypeptide or the name of a particular polypeptide, and generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro, or from a nucleic acid molecule that is a product of replication of such a molecule.

[0028] In certain embodiments, the size of the protein or polypeptide (wild type or modified) is, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 109, 110, 111, 112, 113, 114, 115, , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 77 The domain may comprise 5, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequence described or referenced herein. It is contemplated that the polypeptides can be mutated by truncation to be shorter than the corresponding wild type, and can also be modified by fusing or conjugating with heterologous protein or polypeptide sequences having a specific function (e.g., function for targeting or localization, enhancing immunogenicity, purification purposes, etc.). As used herein, the term "domain" refers to a distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids having a structure or function recognized by those skilled in the art.

[0029] Polypeptides of the disclosure can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more variant amino acids, or can include any of the variant amino acids set forth in SEQ ID NO:1 and / or SEQ ID NO:2. At least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 6 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 pieces or more consecutive amino acids or nucleic acids (or any range derivable therein).Polynucleotides of the disclosure can encode a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more variant amino acids, or can encode a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more variant amino acids. At least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or more In some embodiments, the nucleic acid sequence may encode a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous to a sequence of ...

[0030] In some embodiments, the protein or polypeptide comprises amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 12 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 (or any range derivable therein).

[0031] In some embodiments, the protein or polypeptide is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122 It may include 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 (or any range derivable therein) contiguous amino acids.

[0032] In some embodiments, a polypeptide or protein is a polypeptide or protein that is at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous to one of SEQ ID NO:1 and / or SEQ ID NO:2. At least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, It may include 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 (or any range derivable therein) contiguous amino acids.

[0033] In some aspects, the amino acid sequence of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 of SEQ ID NO:2 and / or SEQ ID NO:3 is , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, or 83, and At least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 (or any range derivable therein) consecutive amino acids or nucleotides.

[0034] Nucleotide sequences and protein, polypeptide and peptide sequences of various genes have been disclosed before and can be found in widely recognized computerized databases.Two commonly used databases are the Genbank and GenPept databases (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web) of the National Center for Biotechnology Information.The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or known to those skilled in the art.

[0035] A. Mutant Polypeptides The following is a discussion of changing the amino acid subunits of proteins to create the next generation of equivalent or even improved mutant polypeptides or peptides. For example, in a protein or polypeptide sequence, certain amino acids can be replaced with other amino acids with or without significant loss of interactive binding ability with structures such as binding sites on substrate molecules. Since it is the interactive ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in a protein sequence and its corresponding DNA coding sequence to still generate a protein with similar or desirable properties. Therefore, it is contemplated herein that various changes can be made in the DNA sequence of a gene encoding a protein without significantly impairing its biological usefulness or activity.

[0036] As used herein, the term "functionally equivalent codons" refers to codons that code for the same amino acid, such as the six different codons for arginine. Also contemplated is a "neutral substitution" or "neutral mutation," which refers to an alteration of a codon that codes for a biologically equivalent amino acid.

[0037] The amino acid sequence variants of the disclosure can be substitution, insertion, or deletion variants. Mutations in the polypeptides of the disclosure can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more non-contiguous or contiguous amino acids of a protein or polypeptide compared to the wild type. Variants can include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical (including all values ​​and ranges therebetween) to the sequences provided or referenced herein. The variant can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0038] It will also be understood that the amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, but still be essentially identical to those shown in one of the sequences disclosed herein, so long as they meet the above criteria, including maintenance of the biological protein activity to which protein expression pertains. The addition of terminal sequences applies particularly to nucleic acid sequences, e.g., nucleic acid sequences can include various non-coding sequences adjacent to the 5' or 3' portions of the coding region.

[0039] Deletion mutants usually lack one or more residues of the native or wild-type protein. Individual residues can be deleted or several consecutive amino acids can be deleted. A stop codon can be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate a truncated protein.

[0040] Insertional mutants usually involve the addition of amino acid residues to non-terminal positions of a polypeptide. This includes the insertion of one or more amino acid residues. Terminal additions can also be produced, and terminal additions can include fusion proteins that are multimers or concatamers of one or more peptides or polypeptides described or referenced herein.

[0041] Substitution variants usually involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, that is, an amino acid is replaced with one of similar chemical properties. A "conservative amino acid substitution" can involve the exchange of a member of an amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are usually incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other inverted or reversed forms of amino acid moieties.

[0042] Alternatively, substitutions may be "non-conservative" such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing an amino acid residue with one that is chemically dissimilar, for example, replacing a polar or charged amino acid with a non-polar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0043] B. Anticoagulant proteins and NAPc2 extracted from C. elegans Aspects of the present disclosure are directed to compositions comprising one or more Nematode-extracted Anticoagulant Proteins (NAPs) and methods of use thereof. In some embodiments, a method of treatment is disclosed that comprises administering to a subject a pharmaceutical composition comprising one or more NAPs. In some embodiments, the NAP of the present disclosure is one or more of the NAPs described in U.S. Pat. No. 5,866,542, which is incorporated herein by reference in its entirety. In some embodiments, the disclosed methods and compositions comprise NAPc2. In some embodiments, the disclosed methods and compositions comprise NAPc2 / proline.

[0044] As used herein, NAPc2 (SEQ ID NO:1) refers to a single-chain, non-glycosylated, 85 amino acid protein (MW=9732 Da). "rNAPc2" refers to recombinant NAPc2 protein. Without wishing to be bound by theory, rNAPc2 is understood to inhibit the activity of the TF:Factor VIIa (FVIIa) complex, which initiates the TF pathway and other important pathways in coagulation via the formation of a quaternary complex after binding to the zymogen FX. Also disclosed herein are mutants of rNAPc2. In some embodiments, the disclosed therapeutic compositions comprise a protein having at least or at most 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% sequence identity (or any range or value derivable therein) to NAPc2 (SEQ ID NO:1) or NAPc2 / proline (SEQ ID NO:2). In some embodiments, compositions comprising NAPc2 / proline are disclosed. "NAPc2 / proline" (SEQ ID NO:2) refers to a variant of NAPc2 modified to add a proline residue to the C-terminus of the sequence of NAPc2.

[0045] Table 1. Sequences of NAPc2 and NAPc2 mutants TIFF2024522626000001.tif45166

[0046] II. Autoimmune and Inflammatory Conditions An aspect of the present disclosure is directed to a method for treating autoimmune or inflammatory conditions.As used herein, the terms "autoimmune disease", "autoimmune condition" and "inflammatory condition" are used interchangeably.In some embodiments, a method for treating autoimmune or inflammatory conditions is disclosed, comprising administering NAPc2 or NAPc2 / proline to a subject.

[0047] Autoimmune or inflammatory conditions suitable for treatment may include, but are not limited to, the following conditions: diabetes (e.g., type 1 diabetes), transplant rejection, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, schizophrenia, and osteoarthritis. rheumatoid arthritis, spondyloarthritis, systemic early-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, chronic primary polyarthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis (such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis), atopy (including atopic diseases such as hay fever and Job's syndrome), dermatitis (contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis), x-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, urticaria (including chronic autoimmune urticaria, such as chronic allergic urticaria and chronic idiopathic urticaria), myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic sclerosis), multiple sclerosis (including systemic sclerosis), multiple sclerosis (including MSC), multiple sclerosis (including glaucoma ... S), such as spino-optical MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, diffuse sclerosis, ataxic sclerosis, etc.), neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, ulcerative colitis, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis).colitis, as well as autoimmune inflammatory bowel disease, enteritis, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome (including adult or acute respiratory distress syndrome (ARDS)), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage such as meningitis, herpes gestationis, pregnancy pemphigoid, scrotal pruritus, autoimmune premature ovarian failure, sudden deafness due to autoimmune conditions, IgE-mediated diseases (such as anaphylaxis, allergic and atopic rhinitis), encephalitis (such as Rasmussen's encephalitis, limbic and / or brainstem encephalitis), uveitis (anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, etc.)uveitis, posterior uveitis, or autoimmune uveitis), glomerulonephritis (GN) with and without nephrotic syndrome, e.g. chronic or acute glomerulonephritis (primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous proliferative GN (MPGN) including types I and II, and rapidly progressive GN, proliferative nephritis), autoimmune polyendocrine deficiency, balanitis (including plasma cell balanoposthitis, balanoposthitis), erythema annulare centrifugally, erythema dyschromicus perstans, erythema multiforme, granuloma annulare, lichen sclerosus atrophicus, lichen simplex chronicus, lichen spinous, lichen planus, ichthyosis lamellar, ichthyosa-like erythroderma, precancerous keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reactions, eczema (allergic or atopic) eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema), asthma (such as bronchial asthma, autoimmune asthma), conditions with T cell infiltration and chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood groups during pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus (including lupus nephritis, lupus encephalitis, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, lupus alopecia, systemic lupus erythematosus (SLE) (such as cutaneous SLE or subacute cutaneous SLE), neonatal lupus syndrome (NLE), disseminated lupus erythematosus), juvenile-onset (Type I) diabetes (including childhood insulin-dependent diabetes mellitus (IDDM)), as well as adult-onset diabetes (Type II diabetes) and autoimmune diabetes.

[0048] Additional autoimmune and inflammatory conditions contemplated herein include sarcoidosis, granulomatosis (including lymphomatoid granulomatosis, Wegener's granulomatosis), granulocytopenia, vasculitis, large vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis (Churg-Strauss vasculitis or syndrome (CSS)). ) and ANCA-associated small vasculitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, hemolytic anemia or immune hemolytic anemia (including autoimmune hemolytic anemia (AIHA)), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte leakage, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes (e.g., secondary to sepsis, trauma, or hemorrhage), antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome ( also known as "antiphospholipid syndrome"), allergic neuritis, Behçet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid (including bullous pemphigoid and cutaneous pemphigoid), pemphigus (including pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigus, and pemphigus erythematous), autoimmune polyendocrine syndrome, Reiter's disease or syndrome, burns, preeclampsia, immune complex disorders (including immune complex nephritis), antibody-mediated nephritis, polyneuropathy, chronic neuropathy (IgM polyneuropathy) autoimmune or immune-mediated thrombocytopenia (such as chronic or acute idiopathic thrombocytopenic purpura (ITP)), scleritis (such as idiopathic keratoscleritis, episcleritis), autoimmune disease of the testes and ovaries (including autoimmune orchitis and oophoritis), primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases (including thyroiditis such as autoimmune thyroiditis), Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyendocrine syndrome,autoimmune polyendocrine syndromes (or polyendocrine syndromes), paraneoplastic syndromes (including paraneoplastic neurological syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome), stiff-man syndrome or stiff-person syndrome, encephalomyelitis (such as allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis), myasthenia gravis (such as thymoma-associated myasthenia gravis), cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory neuropathy, Multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) and NSIP (non-specific interstitial pneumonia), Guillain-Barré syndrome, Burger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis (primary biliary cirrhosis and pneumonocirrhosis, etc.), autoimmune bowel syndrome, celiac disease, Chronic pulmonary disease, Celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease (autoimmune inner ear disease (AIED), autoimmune hearing loss, etc.), polychondritis (refractory or relapsing or relapsing polychondritis, etc.), pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, herpes zoster-associated pain, amyloidosis, noncancerous lymphocytosis, primary lymphocytosis (monoclonal B cell Lymphocytosis (including, e.g., monoclonal gammopathy, monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic neurological syndromes, channelopathies (such as epilepsy, migraine, arrhythmias, myopathy, hearing loss, blindness, periodic paralysis, and CNS channelopathies), autism, inflammatory myopathy, focal or segmental glomerular sclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune liver disorders, fibromyalgia, multiple endocrine deficiencies, Schmidt syndrome, adrenal inflammation, gastric atrophy, presenile dementia,Demyelinating diseases (including autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy), Dressler syndrome, alopecia areata, alopecia totalis, CREST syndrome (subcutaneous calcification, Raynaud's phenomenon, esophageal hypoperistalsis, digital scleroderma, and telangiectasia), male and female autoimmune infertility (e.g., due to antisperm antibodies), mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, and post-cardiotomy syndrome syndrome, Cushing's syndrome, bird fancier's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis (including allergic alveolitis and fibrosing alveolitis), interstitial lung disease, transfusion reactions, leprosy, malaria, parasitic diseases (including leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis), aspergillosis, Sampter's syndrome syndrome), Kaplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum, fetal erythroblastosis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis (chronic cyclitis, heterochromic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis cyclitis), Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal dysfunction, Sydenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, hyperthyroidism, spinal cord Phlegmon, choroiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemic reperfusion injury, transplanted organ reperfusion injury, autoimmune retinopathy, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, aphtha, aphthous stomatitis, arteriosclerotic disease, azoospermia, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, phacosensitivity endophthalmitis, allergic enterocolitis, leprosy erythema nodosum, idiopathic facial paralysis, chronic fatigue syndrome,Rheumatic fever, Hamman-Rich disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, focal ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, granulomatous orchitis, pancreatitis, acute multiple granulomas, pyoderma gangrenosum, Kervan thyroiditis, acquired splenic atrophy atrophy, nonmalignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions with T cell infiltration, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases with leukocyte leakage, multiple organ dysfunction syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia ophthalmia), rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, polyglandular autoimmune syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathies (dilated cardiomyopathy, etc.), epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid sinusitis, frontal sinusitis, maxillary sinusitis or sphenoid sinusitis, eosinophil-related disorders (e.g., eosinophilia, pulmonary infiltrative eosinophilia, eosinophilia-myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma, or eosinophil-containing granuloma), anaphylaxis, seronegative spondyloarthritis, polyglandular autoimmune disease, sclerosing cholangitis, scleritis, episcleritis, chronic mucocutaneous candidiasis, Bruton's syndrome syndrome), transient infantile hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vascular ectasia, autoimmune diseases associated with collagen diseases, rheumatism, neurological diseases, lymphadenitis, decreased blood pressure response, vascular insufficiency, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases with angiogenesis, allergic hypersensitivity, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, reperfusion injury of the myocardium or other tissues, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with an acute inflammatory component, multiple organ failure, bullous diseases, renal cortical necrosis,Acute purulent meningitis or other central nervous system inflammatory diseases, ocular and orbital inflammatory diseases, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, narcolepsy, severe acute inflammation, chronic refractory inflammation, pyelitis, intimal hyperplasia, peptic ulcer, valvular disease, graft-versus-host disease, cytokine storm, contact hypersensitivity, asthmatic airway hyperresponsiveness, and endometriosis.

[0049] In some embodiments, the autoimmune or inflammatory condition is systemic lupus erythematosus. In some embodiments, the autoimmune or inflammatory condition is antiphospholipid syndrome (APS; also referred to as "antiphospholipid syndrome").

[0050] III. Treatment of Autoimmune and Inflammatory Conditions Aspects of the present disclosure are directed to methods for treating a subject having an autoimmune or inflammatory condition, e.g., an autoimmune or inflammatory condition disclosed herein, such as systemic lupus erythematosus or antiphospholipid syndrome. In some embodiments, methods of treating a subject having an autoimmune or inflammatory condition are disclosed, comprising providing a therapeutically effective amount of NAPc2 or a variant thereof (e.g., NAPc2 / proline).

[0051] Certain embodiments of the present disclosure are directed to treating a subject with one or more symptoms of an autoimmune or inflammatory condition. In an embodiment where the autoimmune or inflammatory condition is systemic lupus erythematosus, the symptoms may include, but are not limited to, fatigue, skin lesions, skin rash, renal failure, and fever. In an embodiment where the autoimmune or inflammatory condition is antiphospholipid syndrome, the symptoms may include, but are not limited to, thrombosis (e.g., venous thrombosis, pulmonary embolism), thrombocytopenia, hypertension, renal failure, and recurrent miscarriage. In some embodiments, the subject has been diagnosed with an autoimmune or inflammatory condition. In some embodiments, the subject has not been diagnosed with an autoimmune or inflammatory condition. In some embodiments, the subject has or is at risk of developing an autoimmune or inflammatory condition.

[0052] In some embodiments, the subject has been previously treated for an autoimmune or inflammatory condition. In some embodiments, a composition comprising NAPc2 or NAPc2 / proline is provided to a subject with an autoimmune or inflammatory condition, where the subject has previously suffered from an autoimmune or inflammatory condition and has been treated with one or more anti-inflammatory agents. In some embodiments, the one or more anti-inflammatory agents do not include NAPc2 or NAPc2 / proline. In some embodiments, the subject has been determined to be resistant to one or more anti-inflammatory agents previously provided.

[0053] In some embodiments, a subject being treated for an autoimmune or inflammatory condition is at least, at most, or is the following ages: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 112, 113, 11 8, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 years of age, or any range derivable therein.

[0054] In some embodiments, the subject is administered a pharmaceutical composition comprising NAPc2 or a variant thereof (e.g., NAPc2 / proline). The pharmaceutical composition may be administered in a therapeutically effective amount. In some embodiments, NAPc2 or NAPc2 / proline is at least, at most, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15.0 μg / kg or mg / kg, or any range or value derivable therein. The pharmaceutical composition may be administered to the subject daily, every other day, every third day, or every third day. In some embodiments, the pharmaceutical composition is administered to the subject on days 1, 3, and 5. NAPc2 or a variant thereof (e.g., NAPc2 / proline) may be administered at the same dose or at different doses on each day. In some embodiments, NAPc2 or a variant thereof (e.g., NAPc2 / proline) is provided at a first dose on day 1 and at a second dose on each subsequent treatment day. In some embodiments, NAPc2 or a variant thereof (e.g., NAPc2 / proline) is provided at a first dose on day 1 and at a second dose on days 3 and 5. In some embodiments, NAPc2 or a variant thereof (e.g., NAPc2 / proline) is provided at a dose of about 7.5 μg / kg on day 1, about 5.0 μg / kg on day 3, and about 5.0 μg / kg on day 5.

[0055] Aspects of the present disclosure are directed to the administration of one or more anti-inflammatory agents. In some embodiments, the anti-inflammatory agent of the present disclosure is NAPc2 or a variant thereof (e.g., NAPc2 / proline). In some embodiments, the anti-inflammatory agent is NAPc2. In some embodiments, the anti-inflammatory agent is NAPc2 / proline. Additional anti-inflammatory agents are known in the art and contemplated herein, examples of which include corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), TNFα inhibitors (e.g., adalimumab, certolizumab, etanercept, golimumab, infliximab), and other biologic anti-inflammatory agents (e.g., belimumab).

[0056] IV. Administration of Therapeutic Compositions The treatments provided herein may include administration of a single therapeutic agent (e.g., NAPc2, NAPc2 / proline), or a combination of therapeutic agents, such as NAPc2 (or NAPc2 / proline) and an additional anti-inflammatory agent. The treatments may be administered in any suitable manner known in the art. For example, each of the first and second treatments may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second treatments are administered in separate compositions. In some embodiments, the first and second treatments are administered in the same composition.

[0057] Aspects of the present disclosure relate to compositions and methods, including therapeutic compositions. The therapeutic compositions may include a single therapeutic agent (e.g., NAPc2, NAPc2 / proline) or multiple different therapeutic agents. The different therapeutic agents may be administered in one composition or in two or more compositions, e.g., two compositions, three compositions, or four compositions. Combinations of various therapeutic agents may be employed.

[0058] The therapeutic agents of the present disclosure (e.g., NAPc2, NAPc2 / proline) can be administered by the same or different routes of administration. In some embodiments, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the therapeutic agent (e.g., NAPc2, NAPc2 / proline) is administered subcutaneously. In some embodiments, the therapeutic agent (e.g., NAPc2, NAPc2 / proline) is administered intravenously. The appropriate dosage can be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0059] The treatment can include various "unit doses." A unit dose is defined as comprising a predetermined amount of a therapeutic composition. The amount to be administered and the particular route and formulation are within the judgment skills of one of ordinary skill in the clinical arts. A unit dose need not be administered as a single injection, but may comprise a continuous infusion over a defined period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0060] The amount administered, both according to the number of treatments and the unit dose, depends on the desired therapeutic effect. It is understood that an effective dose refers to the amount required to achieve a specific effect. In the implementation of certain embodiments, it is believed that a dose ranging from 1 μg / kg to 200 μg / kg can affect the protective capacity of these agents. Doses are contemplated to include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, mg / day, or any range of doses derivable therein. In some embodiments, an effective dose is at least, at most, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0 μg / kg. Furthermore, such doses can be administered multiple times in a day and / or over multiple days, weeks, or months.

[0061] The precise amount of therapeutic composition to be administered also depends on the physician's judgment and is specific to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (palliation of symptoms or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other treatments the subject may be undergoing.

[0062] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg (per kg body weight) can be converted and expressed in comparable concentration units of μg / ml or mM (blood concentration). It will also be understood that uptake into the body is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions for uptake and concentration measurements are well known, and those skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding doses, efficacy and results as described herein.

[0063] V. General Pharmaceutical Compositions In some embodiments, the pharmaceutical composition is administered to the subject. Various aspects may include administering an effective amount of the composition to the subject. In some embodiments, NAPc2 (or NAPc2 / proline) can be administered to the subject to prevent or treat a condition (e.g., an autoimmune or inflammatory condition). Such compositions can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0064] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not cause adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infectives and vaccines, can also be incorporated into the compositions.

[0065] The active compound can be formulated for parenteral administration, for example, for injection by intravenous, intramuscular, subcutaneous or intraperitoneal route.Generally, such compositions can be prepared as liquid solutions or suspensions;Also, solid dosage forms suitable for adding liquid to prepare liquid solutions or suspensions before injection can be prepared;And, the preparation can be emulsified.

[0066] Pharmaceutical dosage forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations, for example, containing aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0067] Proteinaceous compositions can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Also, salts formed with free carboxyl groups can be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, etc., and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0068] The pharmaceutical compositions may contain solvents or dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coating agents such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0069] Sterile injectable solution can be prepared by placing the required amount of active compound in a suitable solvent containing various other ingredients as listed above as necessary, and then carrying out filtration sterilization or equivalent procedures.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from those listed above.For the sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces powder of active ingredient containing additional desired ingredients from the solution previously sterilized by filtration.

[0070] The administration of the composition is usually via a common route, including, but not limited to, oral or intravenous administration. Alternatively, or additionally, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions will generally be administered as pharma- ceutically acceptable compositions that include physiologically acceptable carriers, buffers, or other pharmaceutical excipients.

[0071] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. EXAMPLES

[0072] The following examples are provided to demonstrate certain aspects of the present invention.Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that have been found to work well in the implementation of the present invention, and therefore are considered to constitute certain modes for its implementation.However, those skilled in the art should recognize in light of this disclosure that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain the same or similar results.

[0073] Example 1 – Effect of rNAPc2 on aPL-induced gene expression in monocytes The effect of recombinant NAPc2 / proline (rNAPc2) on proinflammatory activation of monocytic MM1 cells by antiphospholipid antibodies (aPL) was evaluated using an established in vitro assay method [1]. MM1 cells were suspended in human plasma-containing medium to provide a source of FX recognized by rNAPc2 to inhibit TF-FVIIa. Cells were then stimulated with aPL HL5B or IgG control for 1 h and processed for genome-wide transcriptional profiling by next-generation sequencing (NGS). In separate reactions, the inhibitory effect of rNAPc2 and that of anti-TF 10H10, an established inhibitor of aPL-induced TF activation and endosomal signaling [2, 3], were evaluated. Induction of procoagulant (TF, F3) and proinflammatory responses was blocked by rNAPc2 as efficiently as the TF antibody 10H10 (Figure 1). Thus, NAPc2 is an effective inhibitor of aPL-induced signaling in monocytic cells.

[0074] Example 2 – Inhibition of autoantibody production and autoimmune pathology in the MRL / lpr mouse model of SLE by rNAPc2 Inhibition of lupus pathology was evaluated in a mouse model genetically prone to autoimmune disease. MRL / lpr mice develop an SLE-like syndrome with the appearance of high titers of aPL and β2 glycoprotein I (β2GPI) autoantibodies [1]. MRL / lpr mice develop aPL at 5–6 weeks of age, and aPL titers persist during the development of a severe SLE-like syndrome, necessitating euthanasia of the mice before they reach 15–16 weeks of age. MRL / lpr mice were treated at 11 weeks of age with 0.5 mg / kg rNAPc2 or saline control every other day for 3 weeks. At the time of randomization to treatment, both groups had similar anticardiolipin aPL titers, but aPL titers rapidly declined in rNAPc2-treated mice within 1 week of treatment and did not reappear for the duration of the experiment (Figure 2).

[0075] In a second cohort, MRL / lpr mice were similarly randomized to treatment and the reactivity of serum samples to cardiolipin or β2GPI was measured during treatment with rNAPc2. Whereas sham-treated mice showed a marked increase in antibody titers over time, mice randomized to receive rNAPc2 treatment continued to produce very low titers of autoantibodies to both cardiolipin (Figure 3A) and β2GPI (Figure 3B).

[0076] Autoimmune aPL is produced by B1 cells circulating in the blood [1]. At the end of the above experiments, the effect of rNAPc2 treatment on the reactivity of circulating B1 cells was measured using either fluorescently labeled phospholipid vesicles or β2GPI. As expected from the aPL titers measured in these mice, sham-treated MRL / lpr mice showed a high percentage of phospholipid- and β2GPI-reactive circulating B cells (Figures 4A and 4B). Staining of B cells with phospholipid vesicles was not blocked by phosphatidylcholine-loaded soluble EPCR (sEPCR-PC) but was prevented by pathogenic targets of aPL, LBPA-loaded sEPCR, or unlabeled β2GPI (Figure 4A). Consistent with the measured antibody titers, no phospholipid-reactive B cells were detected in MRL / lpr mice treated with rNAPc2. Similarly, β2GPI-reactive B cells were only found in sham-treated mice, but not in rNAPc2-treated MRL / lpr mice that exhibited lupus-like pathology (Figure 4B). β2GPI staining was specifically blocked by sEPCR-LBPA, indicating that aPL in this autoimmune mouse model have dual reactivity with EPCR-LBPA and β2GPI, and that rNAPc2 treatment effectively blocked the development of autoimmune antibodies.

[0077] Renal function was assessed by measuring urinary albumin during treatment with rNAPc2. Sham-treated control mice developed progressive albuminuria over the observation period, whereas urinary albumin concentrations did not increase in rNAPc2-treated mice (Figure 5). One mouse with high baseline albuminuria was randomized to the rNAPc2 treatment group, and this mouse showed a decrease in albuminuria at the end of the experiment. These data indicated that renal function was preserved in rNAPc2-treated MRL / lpr mice.

[0078] Renal pathology was assessed by histology in the two treatment cohorts. Renal pathology score, a composite measure of glomerular and tubular damage, was significantly improved in rNAPc2-treated mice compared to control mice (Figure 6). Additionally, immune cell infiltration was semiquantitatively scored for macrophages (CD68), T cells (CD4), and B cells (B220) on sections processed for immunohistochemistry. Treatment with rNAPc2 significantly reduced immune cell infiltration in the kidney (Figure 7).

[0079] These data demonstrate the inhibitory effect of rNAPc2 on aPL signaling in monocytes and prevention of autoimmune disease in a relevant preclinical SLE model. Suppression of B cell proliferation, which is responsible for the production of aPL in response to both cardiolipin and β2GPI, provides a rationale for therapeutic intervention in patients with severe antiphospholipid syndrome, and attenuation of renal pathology in a mouse model of SLE-like pathology supports the broad therapeutic potential of rNAPc2 in preventing and reversing end organ damage in autoimmune disease.

[0080] Example 3 – NAPc2 outperforms the anticoagulant effect of heparin in suppressing the development of aPL in lupus-prone mice The therapeutic effect of anticoagulation with low molecular weight heparin (LMWH) on the development of aPL and activation of reactive circulating B1 cells was compared with EPCR-LBPA versus NAPc2 treatment. MRL / lpr mice were randomized at 5 weeks of age to receive NAPc2 or a therapeutic dose of LMWH for 19 days. NAPc2 suppressed the development of antibodies reactive with β2GPI as well as cardiolipin, whereas heparin therapy had no effect on the antibody titers developed in MRL / lpr mice (Figures 8A and 8B). Peripheral blood was collected from these mice and stained for phospholipid and β2GPI reactivity of B1 cells. NAPc2, but not heparin, suppressed the emergence of these B cells, and staining was specifically blocked by EPCR-LBPA but not by unmodified EPCR (Figures 8C and 8D). These data demonstrate that NAPc2, but not standard heparin anticoagulation, blocks the emergence of B cells that recognize EPCR-LBPA, the pathogenic target of lupus-associated antiphospholipid syndrome.

[0081] Example 4 – NAPc2 acts as a modulator of aPL signaling in monocytes NAPc2 was shown to reprogram monocyte responses to aPL in a unique manner. Stimulation of monocytic MM1 cells in plasma with human monoclonal antiphospholipid antibodies HL5B or HL7G resulted in similar transcriptional responses (Fig. 9A). Inhibition of aPL signaling under these experimental conditions confirmed that NAPc2 effectively suppressed the aPL proinflammatory response and the upregulation of procoagulant TF (Fig. 9B). However, induction of interferon signaling was not inhibited, as indicated by the upregulation of IRF1 and IRF7 (Fig. 9C). NAPc2 showed the same inhibitory profile when the response was induced by the aPL HL7G, which cross-reacts with β2GPI and EPCR-LBPA (Fig. 2D, E). These data indicate that NAPc2 does not simply act as a suppressor of aPL signaling, but rather as a response modifier with profound anti-inflammatory properties.

[0082] All methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described with respect to specific embodiments, it will be apparent to those skilled in the art that changes can be made in the methods and steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein with the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0083] References The following references, and references cited elsewhere in this specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024522626000002.tif91160

Claims

A pharmaceutical composition for use in a method for treating a subject for an autoimmune or inflammatory condition, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, the pharmaceutical composition comprising nematode anticoagulant protein c2 (NAPc2) or NAPc2 / proline. Claim 2. (a) The pharmaceutical composition comprises NAPc2, or NAPc2 / proline; and / or (b) The subject is (i) a subject determined to have symptoms of an autoimmune or inflammatory condition, and optionally, the pharmaceutical composition is administered to the subject after the onset of the symptoms; or (ii) does not have symptoms of an autoimmune or inflammatory condition; and / or (c) The pharmaceutical composition is administered before the onset of symptoms of an autoimmune or inflammatory condition; and / or (d) The subject is a subject previously treated for an autoimmune or inflammatory condition by a previous treatment, and optionally, the subject is determined to be resistant to the previous treatment; and / or (e) The pharmaceutical composition is administered via subcutaneous injection or via intravenous infusion; and / or (f) The pharmaceutical composition is administered to the subject every other day; and / or (g) NAPc2 or NAPc2 / proline is administered at a dose of 5 μg / kg to 10 μg / kg. The pharmaceutical composition according to claim 1. Claim 3. (a) NAPc2 or NAPc2 / proline is administered at a dose of about 10 μg / kg; or (b) NAPc2 or NAPc2 / proline is administered at a dose of about 7.5 μg / kg; or (c) NAPc2 or NAPc2 / proline is administered at a dose of about 5 μg / kg; or (d) The method includes the steps of administering NAPc2 or NAPc2 / proline to the subject at a dose of about 7.5 μg / kg on day 1, providing NAPc2 or NAPc2 / proline to the subject at a dose of about 5 μg / kg on day 3, and providing NAPc2 or NAPc2 / proline to the subject at a dose of about 5 μg / kg on day 5. The pharmaceutical composition according to claim 1.

4. (a) The method further includes the step of administering an additional anti-inflammatory agent to the subject, and optionally, the additional anti-inflammatory agent is a non-steroidal anti-inflammatory drug; and / or (b) (i) The method further includes the step of administering an anticoagulant to the subject, and optionally, the anticoagulant is (A) an inhibitor of vitamin K epoxide reductase complex 1 (VKORC1), a thrombin inhibitor, or a factor Xa inhibitor; or (B) warfarin, heparin or its synthetic analog, rivaroxaban, dabigatran, apixaban, or edoxaban; or (ii) The method does not include the step of administering an additional anticoagulant; or (iii) The pharmaceutical composition does not include an additional anticoagulant. The pharmaceutical composition according to claim 1.

5. (a) The autoimmune condition or inflammatory condition is systemic lupus erythematosus or antiphospholipid syndrome; and / or (b) (i) The subject is determined to have antiphospholipid antibodies; or (ii) The method further includes the step of detecting the presence of antiphospholipid antibodies in the subject before the step of administering the pharmaceutical composition to the subject, and optionally, the step of detecting antiphospholipid antibodies includes an enzyme-linked immunosorbent assay (ELISA). The pharmaceutical composition according to claim 1.

6. A pharmaceutical composition for use in a method for treating a subject for antiphospholipid syndrome, comprising NAPc2 or NAPc2 / proline, wherein said method comprises administering to the subject a therapeutically effective amount of said pharmaceutical composition.

7. (a) the pharmaceutical composition comprises NAPc2, or NAPc2 / proline; and / or (b) the pharmaceutical composition is administered via subcutaneous injection or via intravenous infusion; and / or (c) the pharmaceutical composition is administered to the subject every other day. The pharmaceutical composition according to claim 6.

8. (a) NAPc2 or NAPc2 / proline is administered at a dose of 5 μg / kg to 10 μg / kg; or (b) NAPc2 or NAPc2 / proline is administered at a dose of about 10 μg / kg; or (c) NAPc2 or NAPc2 / proline is administered at a dose of about 7.5 μg / kg; or (d) NAPc2 or NAPc2 / proline is administered at a dose of about 5 μg / kg; or (e) the method comprises administering NAPc2 or NAPc2 / proline at a dose of about 7.5 μg / kg on day 1, administering NAPc2 or NAPc2 / proline at a dose of about 5 μg / kg on day 3, and administering NAPc2 or NAPc2 / proline at a dose of about 5 μg / kg on day 5. The pharmaceutical composition according to claim 6.

9. (a) the method further comprises detecting the presence of antiphospholipid antibodies in the subject prior to the step of administering the pharmaceutical composition, and optionally, the step of detecting antiphospholipid antibodies comprises an enzyme-linked immunosorbent assay (ELISA); and / or (b) (i) the method does not comprise administering an additional anticoagulant; or (ii) the pharmaceutical composition does not comprise an additional anticoagulant. The pharmaceutical composition according to claim 6.

10. A pharmaceutical composition for use in a method for treating a subject for systemic lupus erythematosus, comprising NAPc2 or NAPc2 / proline, wherein said method comprises administering to the subject a therapeutically effective amount of said pharmaceutical composition.

11. (a) the pharmaceutical composition comprises NAPc2, or NAPc2 / proline; and / or (b) the pharmaceutical composition is administered via subcutaneous injection or via intravenous infusion; and / or (c) the pharmaceutical composition is administered to the subject every other day. The pharmaceutical composition according to claim 10.

12. (a) NAPc2 or NAPc2 / proline is administered at a dose of 5 μg / kg to 10 μg / kg; or (b) NAPc2 or NAPc2 / proline is administered at a dose of about 10 μg / kg; or (c) NAPc2 or NAPc2 / proline is administered at a dose of about 7.5 μg / kg; or (d) NAPc2 or NAPc2 / proline is administered at a dose of about 5 μg / kg; or (e) the method comprises administering NAPc2 or NAPc2 / proline at a dose of about 7.5 μg / kg on day 1, administering NAPc2 or NAPc2 / proline at a dose of about 5 μg / kg on day 3, and administering NAPc2 or NAPc2 / proline at a dose of about 5 μg / kg on day 5. The pharmaceutical composition according to claim 10.

13. (a) the method further comprises detecting the presence of antiphospholipid antibodies in the subject prior to the step of administering the pharmaceutical composition, and optionally, the step of detecting antiphospholipid antibodies comprises an enzyme-linked immunosorbent assay (ELISA); and / or (b) (i) the method does not comprise administering an additional anticoagulant; or (ii) the pharmaceutical composition does not comprise an additional anticoagulant. The pharmaceutical composition according to claim 10.

14. A pharmaceutical composition for use in a method for inhibiting anti-phospholipid antibody-induced signal transduction in cells, comprising NAPc2, wherein the method comprises administering an effective amount of the pharmaceutical composition to the cells.

15. A pharmaceutical composition for use in a method for inhibiting anti-phospholipid antibody-induced signal transduction in cells, comprising NAPc2 / proline, wherein the method comprises administering an effective amount of the pharmaceutical composition to the cells.

Images