Compositions and methods for modulating inter-alpha-trypsin inhibitor h4 isoform (ITIH4) activity

Abstract

The invention features compositions and methods of treatment for immune-driven conditions that target ITIH4 activity.

Description

[0001] PATENT ATTORNEY DOCKET NO.: 51523-005WO3

[0002] COMPOSITIONS AND METHODS FOR MODULATING INTER-ALPHA-TRYPSIN INHIBITOR H4 ISOFORM (ITIH4) ACTIVITY

[0003] Sequence Listing

[0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 4, 2025, is named “51523-005WO3_Sequence_Listing_9_4_25” and is 11 ,132 bytes in size.

[0005] Background of the Invention

[0006] The interaction between endogenous proteases and protease inhibitors plays a critical role in regulating the immune system and maintaining self-tolerance. This interaction is especially crucial in regulating the immune system and maintaining self-tolerance during pregnancy. This type of regulation ensures the mother’s immune system can protect against infections while tolerating the semi-allogeneic fetus. Some of these regulated immune responses have been attributed to the activity of the plasma protein, inter-alpha-trypsin inhibitor H4 isoform (ITIH4). As such, the ability to monitor and modulate ITIH4 activity is of diagnostic and therapeutic value for immune-driven conditions.

[0007] Summary of the Invention

[0008] The present invention is directed at mechanisms and reagents that can be used for modulating ITIH4 proteolysis for treating immune-driven conditions.

[0009] In one aspect, the present invention is a method of diagnosing cancer in a subject, said method comprising the steps of:

[0010] (i) determining the ITIH4 activation in the subject; and

[0011] (ii) based on the level of ITIH4 activation, determining if the subject has cancer.

[0012] In some embodiments, the level of ITIH4 activation in the subject is determined by:

[0013] (i) exposing a plasma sample from the subject to recombinant ITIH4 protein;

[0014] (ii) determining ITIH4 proteolysis; and

[0015] (iii) determining the level of ITIH4 activation based on the amount of ITIH4 proteolysis.

[0016] In some embodiments, the recombinant ITIH4 protein is a tagged ITIH4 protein. In specific embodiments, the tagged ITIH4 protein is tagged with His, Flag, Myc, GST, CBP, glu-glu, HAT, T7 or V5.

[0017] In some embodiments, the recombinant ITIH4 protein is a labeled ITIH4 protein. In specific embodiments, the labeled ITIH4 protein is fluorescently labeled or biotinylated. In other specific embodiments, the labeled ITIH4 protein is labeled with HRP, POD, or alkaline phosphate.

[0018] In some embodiments, the recombinant ITIH4 protein is a fusion protein. In specific embodiments, the fusion protein comprises the PRR domain of the ITIH4 protein.

[0019] In some embodiments, the ITIH4 protein is a polypeptide comprising of gakipkpeas fsprrgwnrq agaagsrmnf (SEQ ID No.: 6). In specific embodiments, said polypeptide comprising SEQ ID No.: 6 is tagged. In other specific embodiments, said polypeptide comprising SEQ ID No.: 6 is labeled.

[0020] In some embodiments, the ITIH4 protein is a polypeptide comprising of glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr (SEQ ID No.: 7). In specific embodiments, said polypeptide comprising SEQ ID No.: 7 is tagged. In other specific embodiments, said polypeptide comprising SEQ ID No.: 7 is labeled. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 In some embodiments, the ITIH4 protein is a polypeptide of fewer than 50, 40, 35, 30, or 25 amino acid residues.

[0021] In some embodiments, the ITIH4 protein is a polypeptide of comprising of gesrnrnvhs gstffkyylq gakipkpeas fsprrgwnrq agaagsrmnf rpgvlssrql glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr vmnmkieett mttqtpapiq (SEQ ID No.: 5). In specific embodiments, said polypeptide comprising SEQ ID No.: 5 is tagged. In other specific embodiments, said polypeptide comprising SEQ ID No.: 5 is labeled.

[0022] In some embodiments, the level of ITIH4 activation in the subject is determined by:

[0023] (i) providing a plasma sample from the subject;

[0024] (ii) determining the levels of metabolites arising from ITIH4 proteolysis in vivo; and

[0025] (iii) on the basis of step (ii), diagnosing the subject.

[0026] In some embodiments, the metabolites are selected from OV40, OV63, OV67, or OV180.

[0027] In some embodiments, the cancer is breast cancer, colon cancer, renal cell cancer, lung cancer, hepatocellular carcinoma, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, bladder cancer, head and neck cancer, glioblastoma, diffuse large B-cell lymphoma, leukemia, or melanoma.

[0028] In another aspect, the invention is a method of diagnosing a subject with a specific type of cancer, said method comprising the steps of:

[0029] (i) determining the ITIH4 activation in the subject;

[0030] (ii) identifying the subject as having cancer based on the level of ITIH4 activation;

[0031] (iii) identifying the presence of ITIH4 PRR fragments associated with a specific type of cancer; and

[0032] (iv) based on step (iii), determining the type of cancer of the subject.

[0033] In some embodiments, the specific type of cancer is breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, or prostate cancer.

[0034] In another aspect, the invention is a method of determining the level of ITIH4 activation in a subject, said method comprising of identifying the ITIH4 PRR fragments in plasma of patients.

[0035] In some embodiments, the presence of said PRR region fragments comprising, ssrql glpgppdvpd haayhpf (SEQ ID No.: 8) or gesrnrnvhs gstffkyylq gakipkpeas fsprrgwnrq agaagsrmnf rpgvlssrql glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr vmnmkieett mttqtpapiq (SEQ ID No.: 5), is indicative of ITIH4 activation.

[0036] In another aspect, the invention is a method of treating an immunosuppressive condition by increasing level of complement activity in a subject, said method comprising the steps of:

[0037] (i) identifying a subject who has been diagnosed with an immunosuppressive condition; and

[0038] (ii) following step (i), administering to the subject an ITIH4 proteolysis inhibitor.

[0039] In some embodiments, the ITIH4 proteolysis inhibitor is one or more proteins.

[0040] In some embodiments, the ITIH4 proteolysis inhibitor comprises of an antibody.

[0041] In some embodiments, the ITIH4 proteolysis inhibitor comprises of an ITIH4 fusion protein.

[0042] In some embodiments, the ITIH4 proteolysis inhibitor comprises of the products of ITIH4 proteolysis.

[0043] In some embodiments, the immunosuppressive condition is a type of cancer, said method comprising the steps of: PATENT ATTORNEY DOCKET NO.: 51523-005WO3

[0044] (i) identifying a subject who has been diagnosed with cancer based upon the level of ITIH4 activation; and

[0045] (ii) following step (i), administering to the subject a protease inhibitor.

[0046] In some embodiments, the subject was diagnosed with a specific type of cancer.

[0047] In some embodiments, the protease inhibitor administered is selective for a specific cancer type.

[0048] In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, or prostate cancer.

[0049] In some embodiments, treating the cancer in the subject comprises administering to the subject an additional anticancer therapy.

[0050] In another aspect, the invention is a method of promoting immunosuppression in a subject with an immune-driven condition, the method comprising of administering a modulator of ITIH4 activity.

[0051] In some embodiments, the ITIH4 activity is modulated by administering an ITIH4 proteolysis promoter to the subject.

[0052] In some embodiments, the ITIH4 proteolysis promoter is one or more proteins.

[0053] In some embodiments, the ITIH4 proteolysis promoter comprises of an antibody.

[0054] In some embodiments, the ITIH4 proteolysis promoter comprises of an ITIH4 fusion protein.

[0055] In some embodiments, ITIH4 activity is modulated by administering activated ITIH4 to the subject.

[0056] In some embodiments, the activated ITIH4 comprises of at least one cleavage product of ITIH4 proteolysis.

[0057] In some embodiments, the activated ITIH4 comprises of: mpknvvfvid ksgsmsgrki qqtrealiki Iddlsprdqf nlivfsteat qwrpslvpas aenvnkarsf aagiqalggt nindamlmav qlldssnqee rlpegsvsli illtdgdptv getnprsiqn nvreavsgry slfclgfgfd vsyaflekla Idngglarri hedsdsalql (SEQ ID No.: 2).

[0058] In some embodiments, the activated ITIH4 comprises of: vevt gqyerekagf swievtfknp Ivwvhaspeh vvvtrnrrss aykwketlfs vmpglkmtmd ktg llllsdp dkvtigl Ifw dgrgeglrll Irdtdrfssh vggtlgqfyq evlwgspaas ddgrrtlrvq gndhsatrer rldyqegppg veiscwsv (SEQ ID No.: 3).

[0059] In some embodiments, the activated ITIH4 comprises of: apsailplpg qsverlcvdp rhrqgpvnll sdpeqgvevt gqyerekagf swievtfknp Ivwvhaspeh vvvtrnrrss aykwketlfs vmpglkmtmd ktgll llsdp dkvtigllfw dgrgeglrll Irdtdrfssh vggtlgqfyq evlwgspaas ddgrrtlrvq gndhsatrer rldyqegppg veiscwsvel (SEQ ID No.: 4).

[0060] In some embodiments, the immune-driven condition is inflammation.

[0061] In some embodiments, the immune-driven condition is an autoimmune disease.

[0062] In some embodiments, the immune-driven condition is the rejection of a tissue, cell, graft, or organ transplant from a donor by a recipient human subject in need thereof.

[0063] In some embodiments, the subject was administered an ITIH4 activity modulator prior to, simultaneously with, or after tissue, cell, graft, or organ transplantation. In specific embodiments, the organ transplant is a kidney transplant, a cornea transplant, a bone marrow transplant, a liver transplant, a lung transplant, or a heart transplant.

[0064] In some embodiments, the immune-driven condition is an adverse immune response resulting from administration of a therapeutic agent.

[0065] In another aspect, the invention is a method of determining the efficacy of a treatment for an immune-driven condition, said method comprising the steps of: PATENT ATTORNEY DOCKET NO.: 51523-005WO3

[0066] (i) determine the ITIH4 activity in a plasma sample from a subject previously identified as having an immune-driven condition;

[0067] (ii) subjecting the plasma sample to a treatment option;

[0068] (iii) determine the change in ITIH4 activity; and

[0069] (iv) based on the change in ITIH4 activity, make a determination about the efficacy of the treatment.

[0070] In some embodiments, the immune-driven condition is cancer.

[0071] In some embodiments, the treatment option is a protease inhibitor.

[0072] In some embodiments, the immune-driven condition is poor pregnancy health.

[0073] In some embodiments, the immune-driven condition is graft-versus-host disease.

[0074] In some embodiments, the treatment option is an ITIH4 activator.

[0075] In some embodiments, the treatment option is activated ITIH4.

[0076] In another aspect, the invention is a method for assessing pregnancy health of a pregnant subject, the method comprising

[0077] (i) identifying the level of ITIH4 activation of the pregnant subject;

[0078] (ii) based on the level of ITIH4 activation, assessing the pregnancy health of the subject.

[0079] In some embodiments, the level of ITIH4 activation in the subject is determined by:

[0080] (i) providing a plasma sample from the subject;

[0081] (ii) determining the levels of metabolites arising from ITIH4 proteolysis in vivo; and

[0082] (iii) on the basis of step (ii), providing the prognosis to the subject.

[0083] In some embodiments, the metabolites are selected from OV40, OV63, OV67, or OV180. In specific embodiments, the presence of said metabolites is indicative of a good prognosis for pregnancy health.

[0084] In another aspect, the invention is a method for modulating the pregnancy health in a pregnant subject, the method comprising administering an ITIH4 activity modulator.

[0085] In some embodiments, the method comprises administering an effective amount of ITIH4 inhibitor to the pregnant subject to terminate pregnancy. In specific embodiments, the ITIH4 inhibitor is a serpin A5 or serpin G1 . In particular embodiments, the serpin is serpin A5 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 9. In other particular embodiments, the serpin is serpin G1 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 10. In other particular embodiments, the serpin A5 or serpin G1 is part of a pharmaceutical composition, e.g., as described herein.

[0086] In some embodiments, the method comprises administering an effective amount of ITIH4 promoter to protect pregnancy. In specific embodiments, the ITIH4 promoter is an inhibitor of serpin protein activity. In further specific embodiments, the ITIH4 promoter is an inhibitor of serpin A5 or serpin G1 activity.

[0087] In another aspect, the invention is a kit for obtaining the data needed to diagnose a subject with cancer by a method described hereinabove.

[0088] In another aspect, the invention is a kit for obtaining the data needed to diagnose a subject with a specific type of cancer by a method described hereinabove.

[0089] In another aspect, the invention is a kit for determining the efficacy of a treatment for an immune- driven conditions by a method described hereinabove. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 In another aspect, the invention is a kit for obtaining the data needed to determine the prognosis for pregnancy health of a subject by a method described hereinabove.

[0090] In another aspect, the invention is a pharmaceutical composition comprising (i) serpin A5, or a conjugate or fusion protein thereof, in a concentration of from 1 mg / mL to 200 mg / mL; and (ii) a pharmaceutically acceptable excipient, wherein the serpin A5 comprises the amino acid sequence at least 85% identical to SEQ ID No.: 9.

[0091] In some embodiments, the serpin A5 is isolated or purified from human plasma.

[0092] In some embodiments, the serpin A5 is recombinantly produced.

[0093] In some embodiments, the serpin A5 is purified recombinant human serpin having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (e.g., 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical) to SEQ ID No.: 9, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma derived human serpin.

[0094] In some embodiments, the serpin A5 is part of a conjugate (e.g., the serpin protein is covalently attached to a chemical group such as a carbohydrate, lipid, another protein, antibody, small molecule, or metal ion) or fusion protein. In specific embodiments, the serpin A5 fusion protein comprises another protein (e.g., a fusion protein comprising two proteins). In further specific embodiments, the serpin A5 fusion protein comprises another serpin A5 protein, e.g., a serpin A5 dimer or a serpin of formula “serpin A5n” wherein n is at least 2. In specific embodiments, the serpin A5 fusion protein comprises an antibody. In specific embodiments, the fusion protein comprises a linker. In more specific embodiments, the linker comprises 1 to 100 amino acid(s). In specific embodiments, the serpin A5 fusion protein comprises an IgG or fragment of IgG. In specific embodiments, the serpin A5 fusion protein comprises IgG 1 , lgG2, or lgG3. In particular embodiments, the serpin A5 fusion protein comprises lgG3. In further specific embodiments, the IgG in the serpin A5 fusion protein is human IgG. In further specific embodiments, the serpin A5 fusion protein is fused to the IgG Fc domain, e.g., a serpin A5 of formula “serpin A5n-Fc” wherein n is at least 1 . In specific embodiments, the serpin A5 fusion protein comprises an immunoglobulin Fc polypeptide modified to enhance FcRn binding. In specific embodiments, the serpin A5 fusion protein comprises a small molecule.

[0095] In some embodiments, the serpin A5 is tagged.

[0096] In some embodiments, the pharmaceutical composition described hereinabove is an injectable composition (e.g., for parenteral administration).

[0097] In some embodiments, the pharmaceutical composition described hereinabove is formulated for oral administration (e.g., a tablet or aerosol).

[0098] In another aspect, the invention is a method of treating an immunosuppressive condition by increasing level of complement activity in a subject (e.g., promoting immunoenhancement), said method comprising the steps of:

[0099] (i) identifying a subject who has been diagnosed with an immunosuppressive condition; and

[0100] (ii) following step (i), administering to the subject a therapeutic comprising either:

[0101] (a) serpin A5, or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 9, or an expression construct (e.g., cDNA or mRNA) or viral vector (e.g., Adeno or Lenti) enabling production of serpin A5 in the subject (e.g., with genetic material such as a gene or an expression construct such as cDNA or mRNA); or PATENT ATTORNEY DOCKET NO.: 51523-005WO3 (b) serpin G1 , or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 10, or an expression construct (e.g., cDNA or mRNA) or viral vector (e.g., Adeno or Lenti) enabling production of serpin G1 in the subject (e.g., with genetic material such as a gene or an expression construct such as cDNA or mRNA), in an amount effective to treat the immunosuppressive condition.

[0102] In some embodiments, the therapeutic comprising the serpin is serpin A5 having an amino acid sequence at least 85% identical to SEQ ID No.: 9. In some embodiments, therapeutic comprising the serpin A5 administered is part of a pharmaceutical composition as described herein above.

[0103] In some embodiments, the therapeutic comprising the serpin is serpin G1 having an amino acid sequence at least 85% identical to SEQ ID No.: 10.

[0104] In some embodiments, the disease or condition is a cancer susceptible to treatment with immunotherapy.

[0105] In some embodiments, the cancer is a solid tumor cancer.

[0106] In some embodiments, the cancer is melanoma, renal cell carcinoma (RCC), colorectal cancer, Metastatic colorectal cancer, Gl malignancies, hepatocellular carcinoma (HCC), HNSCC, Malignant Pleural Mesothelioma, bladder cancer, sarcoma, osteosarcoma, Kaposi’s sarcoma, soft tissue sarcoma, lymphoma, mantle cell, follicular, small lymphocytic, B-cell malignancies, anaplastic large cell lymphoma, Merkel cell carcinoma, MSI-high cancer, gastric cancer, gastroesophageal cancer, cervical cancer, PMBCL, endometrial carcinoma, esophagus cancer, cutaneous squamous-cell carcinoma, breast cancer, Metastatic breast cancer, TMB-high cancer, SCLC, basal cell carcinoma, multiple myeloma, testicular cancer, pancreatic cancer, prostate cancer, ovarian cancer, thyroid cancer, leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia, acute promyelocytic leukemia (APL), head and neck cancers, lung cancer, non-small cell lung cancer (NSCLC), melanoma, gastrointestinal stromal tumors (GIST), solid tumors, malignant pleural mesothelioma, myelofibrosis, polycythemia vera, Waldenstrom’s macroglobulinemia, glioblastoma, urothelial carcinoma, or myelodysplastic syndromes (MDS).

[0107] In some embodiments, the therapeutic comprising the serpin administered (e.g., serpin A5 or serpin G1 ) activates the immune system.

[0108] In some embodiments, the therapeutic comprising the serpin administered is selected based on the type of cancer.

[0109] In some embodiments, the method of treating cancer is a combination therapy. In specific embodiments, the combination therapy comprises: a) a therapeutically effective amount of the therapeutic of claim 20, and b) a therapeutically effective amount of an additional anticancer therapy.

[0110] In some embodiments of the aforementioned combination therapy, the additional anticancer therapy is a chemotherapy. In specific embodiments, the chemotherapy is a cytotoxic chemotherapy agent selected from: an alkylating agent, a platinum-based compound, a antimetabolite, a antitumor antibiotic, or a plant alkaloid. In specific embodiments, the cytotoxic chemotherapy agent is selected from a group consisting of: cyclophosphamide (Cytoxan), infosfamide (Ifex), melphalan (Alkeran), chlorambucil (Leukeran), bendamustine (Tranda, Bendeka), cisplatin, carboplatin, oxaliplatin (Eloxatin), methotrexate, 5-fluorouracil (5-FU), capecitabine (Xeloda), gemcitabine (Gemzar), pemetrexed (Alimta), cytarabine (ara- C), and 6-mercaptopurine (6-MP), doxorubicin, daunorubicin, epirubicin, bleomycin, and mitomycin PATENT ATTORNEY DOCKET NO.: 51523-005WO3 (Mutamycin), vincristine, vinblastine, vinorelbind, paclitaxel (Taxol), docetaxel (Taxotere), cabazitaxel (Jevtana), etopside (VP-16), camptothecins, irinotecan (Camptosar), and topotecan (Hycamtin).

[0111] In some embodiments of the aforementioned combination therapy, the additional anticancer therapy is a targeted therapy. In specific embodiments, the targeted therapy is a tyrosine kinase inhibitor (TKI), a monoclonal antibody (mAb), or a antibody-drug conjugate (ADC). In specific embodiments, the targeted therapy is selected from a group consisting of: imatinib (Gleevec), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif), ponatinib (Iclusig), erlotinib (Tarceva), gefitinib (Iressa), afatinib (Gilotrif), osimertinib (Tagrisso), sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), axitinib (Inlyta), cabozantinib (Cabometyx), dabrafenib (Tafinlar), vemurafenib (Zelboraf), trametinib (Mekinist), cobimetinib (Cotellic), ruxolitinib (Jakafi), ibrutinib (Imbruvica), acalabrutinib (Calquence), idelalisib (Zydelig), duvelisib (Copiktra), rituximab (Rituxan), obinutuzumab (Gazyva), ofatumumab (Arzerra), trastuzumab (Herceptin), pertuzumab (Perjeta), cetuximab (Erbitux), panitumumab (Vectibix), bevacizumab (Avastin), daratumumab (Darzalex), isatuximab (Sarclisa), elotuzumab (Empliciti), inotuzumab ozogamicin (Besponsa), T-DM1 I ado-trastuzumab emtansine (Kadcyla), brentuximab vedotin (Adcetris), sacituzumab govitecan (Trodelvy), and trastuzumab deruxtecan (Enhertu).

[0112] In some embodiments of the aforementioned combination therapy, the additional anticancer therapy is an immunotherapy. In specific embodiments, the immunotherapy is an immune checkpoint inhibitor. In specific embodiments, the immune checkpoint inhibitor is anti-CTLA-4, anti-PD-1 , or anti- PDpL1 . In specific embodiments, the immunotherapy is selected from a group consisting of: ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), durvalumab (Imfinzi), and avelumab (bavencio).

[0113] In some embodiments of the aforementioned combination therapy, the additional anticancer therapy is a hormonal therapy. In specific embodiments, the hormonal therapy is a selective estrogen receptor modulator (SERM), an aromatase inhibitor, a selective estrogen receptor degrader (SERD), an anti-androgen, a GnRH (LHRH) agonist, or a GnRH (LHRH) antagonist. In specific embodiments, the hormonal therapy is selected from a group consisting of: tamoxifen, anastrozole (Arimidex), letrozole (Femara), exemestane (Aromasin), fulvestrant (Faslodex), bicalutamide (Casodex), flutamide, nilutamide, enzalutamide (Xtandi), apalutamide (Erleada), darolutamide (Nubeqa), leuprolide (Lupron), goserelin (Zoladex), degarelix (Firmagon), and Relugolix (Orgovyx).

[0114] In some embodiments of the aforementioned combination therapy, the additional anticancer therapy is selected from a group consisting of: lenalidomide (Revlimid), thalidomide (Thalomid), pomalidomide (Pomalyst), bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro), venetoclax (Venclexta), olaparib (Lynparza), rucaparib (Rubraca), niraparib (Zejula), talazoparib (Talzenna), everolimus (Afinitor), temsirolimus (Torisel), arsenic trioxide (Trisenox), all-trans retinoic acid (ATRA), trabectedin (Yondelis), L-asparaginase (Elspar, Erwinia asparaginase, Pegaspargase), and selinexor (Xpovio).

[0115] In some embodiments of the aforementioned combination therapy, the administration of the combination therapy comprises administering simultaneously (e.g., co-administered or concurrent therapy): a) a therapeutically effective amount of a immunoenhancing therapeutic (e.g., a therapeutic agent comprising serpin A5 or serpin G1 ), and b) a therapeutically effective amount of an additional anticancer therapy. In specific embodiments, the immunoenhancing therapeutic (e.g., a therapeutic agent comprising serpin A5 or serpin G1 ) and the anticancer therapy are formulated and administered together. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 In some embodiments of the aforementioned combination therapy, the administration of the combination therapy comprises administering sequentially: a) a therapeutically effective amount of a immunoenhancing therapeutic (e.g., a therapeutic agent comprising serpin A5 or serpin G1 ), and b) a therapeutically effective amount of an additional anticancer therapy. In specific embodiments, the sequential administration comprises administering the immunoenhancing therapeutic (e.g., a therapeutic agent comprising serpin A5 or serpin G1 ) and administering the additional anticancer therapy within one week of each other (e.g., 1 week between each administration, 6 days between each administration, 5 days between each administration, 4 days between each administration, 3 days between each administration, 2 days between each administration, or 1 day between each administration). In specific embodiments, the sequential administration comprises administering immunoenhancing therapeutic (e.g., a therapeutic agent comprising serpin A5 or serpin G1 ) and the anticancer therapy within 24 hours of each other (e.g., 24 hours between each administration, 22 hours between each administration, 20 hours between each administration, 18 hours between each administration, 16 hours between each administration, 14 hours between each administration, 12 hours between each administration, 10 hours between each administration, 8 hours between each administration, 6 hours between each administration, 4 hours between each administration, 2 hours between each administration, 1 hour between each administration, 30 minutes between each administration, 15 minutes between each administration, 5 minutes between each administration, or 1 minute between each administration).

[0116] In some embodiments of the aforementioned combination therapy, the administration of the combination therapy comprises administering the immunoenhancing therapeutic (e.g., a therapeutic agent comprising serpin A5 or serpin G1 ) before the additional anticancer agent.

[0117] In some embodiments of the aforementioned combination therapy, the administration of the combination therapy comprises administering the additional anticancer agent before the immunoenhancing therapeutic (e.g., a therapeutic agent comprising serpin A5 or serpin G1 ).

[0118] In some embodiments, the therapeutic agent administered comprises a viral vector (e.g., Adeno or Lenti). In specific embodiments, the viral vector comprises a gene that enables the production of serpin A5 or serpin G1 .

[0119] In some embodiments, the therapeutic agent administered comprises cDNA encoding serpin A5 or serpin G1 .

[0120] In some embodiments, the therapeutic agent administered comprises mRNA encoding serpin A5 or serpin G1 .

[0121] In some embodiments of any of the aforementioned methods, the serpin administered has a molecular weight of 45 to 52 kDa (e.g., 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, 51 kDa, 52 kDa).

[0122] In some embodiments of any of the aforementioned methods, the serpin administered has a molecular weight of 100 to 110 kDa (e.g., 100 kDa, 101 kDa, 102 kDa, 103 kDa, 104 kDa, 105 kDa, 106 kDa, 107 kDa, 108 kDa, 109 kDa, 110 kDa).

[0123] In some embodiments of any of the aforementioned methods, the serpin administered is isolated or purified from human plasma.

[0124] In some embodiments of any of the aforementioned methods, the serpin administered is recombinantly produced. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 In some embodiments of any of the aforementioned methods, the serpin is purified recombinant human serpin having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (e.g., 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical) to SEQ ID No.: 9 or human serpin having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (e.g., 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical) to SEQ ID No.: 10, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma derived human serpin.

[0125] In some embodiments of any of the aforementioned methods, the serpin administered is part of a fusion protein.

[0126] In some embodiments of any of the aforementioned methods, the serpin fusion protein comprises two serpin protein (e.g., two serpin A5 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 9 or two serpin G1 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 10).

[0127] In some embodiments of any of the aforementioned methods, the serpin fusion protein comprises of a linker (e.g., a group of atoms or molecules that join components of the fusion protein). In specific embodiments, the linker comprises of 1 to 100 amino acid(s).

[0128] In some embodiments of any of the aforementioned methods, the serpin is fused with a IgG or fragment of IgG.

[0129] In some embodiments of any of the aforementioned methods, the IgG is selected from: IgG 1 , lgG2, or lgG3.

[0130] In some embodiments of any of the aforementioned methods, the IgG is lgG3.

[0131] In some embodiments of any of the aforementioned methods, the IgG is human IgG.

[0132] In some embodiments of any of the aforementioned methods, the serpin is fused to the IgG Fc domain.

[0133] In some embodiments of any of the aforementioned methods, the serpin fusion protein comprises an immunoglobulin Fc polypeptide modified to enhance FcRn binding.

[0134] In some embodiments of any of the aforementioned methods, the serpin is fused with an antibody.

[0135] In some embodiments of any of the aforementioned methods, the serpin is fused with a small molecule.

[0136] In some embodiments of any of the aforementioned methods, the serpin is tagged.

[0137] In some embodiments of any of the aforementioned methods, the serpin is administered by parenteral or oral administration.

[0138] In another aspect, the present invention is a method of treating an immune-driven condition characterized by adverse immunoenhancement by decreasing level of complement activity in a subject, said method comprising the steps of:

[0139] (i) identifying a subject who has been diagnosed with said condition; and

[0140] (ii) following step (i), administering to the subject an effective amount of an ITIH4 proteolysis promoter to treat the immune-driven condition.

[0141] In some embodiments, the condition is graft-versus-host disease.

[0142] In some embodiments, the condition is an autoimmune condition. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 In some embodiments, the condition is poor pregnancy health.

[0143] In specific embodiments, the ITIH4 proteolysis promoter inhibits activity of serpin A5. In specific embodiments, the ITIH4 proteolysis promoter inhibits activity of serpin G1 .

[0144] Definitions

[0145] The terms, “ITIH4 activation” and “ITIH4 proteolysis” are used interchangeably to refer to the process in which ITIH4 becomes cleaved via proteolysis by human protease and the products can inhibit functions in the immune system.

[0146] The term “administration” refers to the administration of a composition to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be an orally absorbed pill, or by injection including, but not limited to, intravenous, intramuscular, subcutaneous, and intradermal.

[0147] The term, “autoimmune disorder” refers to an immune-driven condition in which the immune system produces an immune response (for instance, a B cell or a T cell response) against an endogenous antigen, with consequent injury to tissues. This includes, but is not limited to, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, renal, pulmonary, hepatic fibroses, Addison's disease, type I diabetes, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease.

[0148] The term, “cancer” refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas. Certain cancer types can be characterized by a low white blood cell count, e.g., leukemia. Certain cancer types can be characterized by a low level of complement activity.

[0149] The term, “complement-dependent cytotoxicity” (CDC) refers to a complement system pathway. An example of an activation of this pathway includes activation by IgG and IgM antibodies when they are bound to surface antigens on target cells.

[0150] The term, “effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, an effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be “refractory” to an “effective amount.” To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable. In some embodiments, reference to an effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). Those of ordinary skill in the art will appreciate that, in some embodiments, an effective amount may be formulated and / or administered in a single dose. In some embodiments, an effective amount may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 The term, “graft-versus-host disease”, refers to an immune-driven condition wherein rejection is a consequence of organ or tissue transplantation caused by the transplant recipient's (host's) immune response to the transplanted organ / tissue which can damage or destroy it (see also, e.g., “immune-driven condition” and “rejection” definitions). Ordinarily, the immune response protects the body from potentially harmful substances (antigens) such as microorganisms, toxins, and cancer cells. The immune system distinguishes “self” from “foreign” by reacting to proteins on the surfaces of cells. It reacts against substances it recognizes as foreign (antigens). The presence of foreign blood or tissue in the body triggers an immune response that can result in blood transfusion reactions and transplant rejection when antibodies are formed against foreign antigens on the transplanted or transfused material.

[0151] The term “injectable composition” as used herein refers to an injectable mixture which has low enough viscosity for injection. Preferably, the injectable composition of the present invention can be injected subcutaneously or intramuscularly.

[0152] The term, “immune-driven condition” refers to a condition, disease, or disorder in which the subject's immune system is not functioning in normal capacity. This includes subjects that are immunocompromised, meaning an immune system that is functioning below normal levels. Those subjects who can be considered to be immunocompromised include, but are not limited to, subjects with AIDS (or HIV positive), subjects with severe combined immune deficiency (SCID), diabetics, and those who are receiving chemotherapy for cancer. Immunocompromised subjects also include subjects with most forms of cancer (other than skin cancer), sickle cell anemia, cystic fibrosis, those who do not have a spleen, pregnant subjects, subjects with end stage kidney disease (dialysis), and those who have been taking corticosteroids on a frequent basis by pill or injection within the last year. Subjects with severe liver, lung, or heart disease also can be immunocompromised. Immune-driven conditions also include oestrogen-related conditions. Subjects with immune-driven conditions also include subjects with immune systems that perform undesired immune response activity. For example, although immune recognition of foreign proteins is essential to avoid and eliminate infection, under some conditions this immune response can be adverse. Such conditions include, but are not limited to, subjects with allergies, rejection (e.g., rejection of organ post-transplant), or those with autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, or insulin dependent diabetes mellitus. This can also include subjects with inflammatory diseases and disorders, in which an abnormal or otherwise deregulated inflammatory response contributes to the etiology or severity of disease. Examples include autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, Celiac disease, and diabetes, infectious diseases such as tuberculosis and various forms of meningitis and encephalitis, including West Nile Virus encephalitis as well as ischemia reperfusion diseases such as myocardial infarction and transplant reperfusion injury. Additionally, this includes when there is an increase in inflammation observed after surgery, injury, or other tissue trauma has deleterious effects on a patient's recovery.

[0153] The terms, “immunosuppressive”, “immunosuppression”, and “suppress immune function” are used interchangeably herein and refer to a decrease in the immune response. This includes any condition in which there is a deficiency in the production of humoral and / or cell-mediated immunity.

[0154] The term, “immunoenhancement” and “enhance immune function” are used interchangeably herein and refer to an increase in the immune response, which means there is an increase in the organism’s capacity to respond to foreign or disease-specific antigens (e.g., cancer antigens) i.e. , those PATENT ATTORNEY DOCKET NO.: 51523-005WO3 cells primed to attach such antigens are increased in number, activity, and ability to detect and destroy those antigens. Strength of immune response can be measured by standard tests including: direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays (see, e.g., Provinciali M. et al (1992, J. Immunol. Meth. 155: 19-24), cell proliferation assays (see, e.g., Vollenweider, I. And Groseurth, P. J. (1992, J. Immunol. Meth. 149: 133-135), immunoassays of immune cells and subsets (see, e.g., Loeffler, D. A., et al. (1992, Cytom. 13: 169-174); Rivoltini, L., et al. (1992, Can. Immunol. Immunother34: 241 -251 ); or skin tests for cell-mediated immunity (see, e.g., Chang, A. E. et al. (1993, Cancer Res. 53: 1043-1050). Any statistically significant increase in strength of immune response as measured by the foregoing tests is considered “immunoenhancement” as used herein. Enhanced immune response can also indicated by physical manifestations such as fever and inflammation, as well as healing of systemic and local infections, and reduction of symptoms in disease, i.e. , decrease in tumor size, alleviation of symptoms of a disease or condition including, but not restricted to, leprosy, tuberculosis, malaria, naphthous ulcers, herpetic and papillomatous warts, gingivitis, artherosclerosis, the concomitants of AIDS such as Kaposi's sarcoma, bronchial infections, and the like. Such physical manifestations also define “immunoenhancement” as used herein.

[0155] The term, “modulator” refers to increasing or decreasing, either directly or indirectly, the level and / or functional activity. In certain embodiments, “modulation” or “modulating” means that a desired / selected response is more efficient upon the addition of a modulating agent (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or more than 60%). For example, a modulating agent (e.g., an immunotherapy) that inhibits ITIH4 proteolysis will result in a more efficient immune response (e.g., immunoenhancement or increase in complement activity). In certain embodiments, “modulation” or “modulating” means that a desired / selected response is less efficient upon the addition of a modulating agent (e.g., at least 10% less efficient, at least 20% less efficient, at least 30% less efficient, at least 40% less efficient, at least 50% less efficient, at least 60%, or more than 60% less efficient). For example, a modulating agent (e.g., an immunotherapy) that promotes ITIH4 proteolysis will result in a decreased immune response (e.g., immunosuppression or decrease in complement activity). In the present invention, a modulator of immune function includes serpin proteins, e.g., serpin A5 or serpin G1 .

[0156] The term, “pregnancy health” refers to the condition of the pregnancy of a pregnant subject. The methods of the invention can be used to evaluate pregnancy health based upon ITIH4 activity of a pregnant subject. Good pregnancy health refers to a pregnancy in which the fetus is considered in a healthy condition based upon ITIH4 activity. Bad pregnancy health refers to a pregnancy in which the fetus is considered in poor health based upon ITIH4 activity. A bad pregnancy health can lead to a rejection of the fetus by the body of the pregnant subject resulting in loss of pregnancy.

[0157] The term, “rejection” refers to an immune-driven condition in which the immune system recognizes a foreign substance (e.g., cell, tissue, organ, therapeutic agent) and attempts to destroy the foreign substance just as the immune system would attempt to destroy infectious microorganisms such as bacteria and viruses. For example, rejection of transplanted organs and tissues can often result in damage to and / or rejection of the transplant. Tissue rejection, also called graft-versus-host disease, is a consequence of organ or tissue transplantation caused by the transplant recipient's (host's) immune response to the transplanted organ / tissue which can damage or destroy it. No two people (except identical twins) have identical tissue antigens. Therefore, in the absence of an immunosuppressive agent, PATENT ATTORNEY DOCKET NO.: 51523-005WO3 transplantation would almost always cause an immune response against the foreign tissue (rejection), which would result in destruction of the transplant.

[0158] As used herein, the term “pharmaceutical composition” refers to an active agent (e.g., a therapeutic agent of the present invention such as a serpin protein or derivative thereof as described herein), or pharmaceutically acceptable salt thereof, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent or salt is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. In other embodiments, pharmaceutical compositions include those formulated for oral administration (“oral dosage forms”). Oral dosage forms can be, for example, in the form of tablets, capsules, a liquid solution or suspension, a powder, or liquid or solid crystals, which contain the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like. Compositions for oral administration may also be presented as chewable tablets, as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules where the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

[0159] The term, “subject,” as used herein, represents a human or non-human animal (e.g., a mammal).

[0160] The term, “therapeutic agent” refers to a biological agent, such as a protein (e.g., a serpin protein), lipid, or enzyme, and / or may be part of a complex with another biological agent or chemical agent (e.g., a small molecule) intended to result in the treatment of a subject. For example, in the present invention, a therapeutic agent may include a therapy intended to modulate an immune response, e.g., an immunotherapy that modulates immune response via either activation or suppression of the immune system. For example, a therapeutic agent of the present invention includes serpin proteins which will function as immunotherapies by activating immune function (see, e.g., Examples herein). Conversely, a therapeutic agent of the present invention will inhibit serpin protein function in order to suppress immune function. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 The term, “treatment” (also “treat” or “treating”), in its broadest sense, refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be administered to a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, in some embodiments, treatment may be administered to a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0161] Other features and advantages of the invention will be apparent from the following Detailed Description, the Drawings, and the claims.

[0162] Brief Description of the Drawings

[0163] FIG. 1 is a Venn diagram depicting the metabolomic profiling discussed in Example 1 for nonpregnant healthy women, pregnant healthy women, and women with ovarian cancer.

[0164] FIG. 2A and FIG. 2B are principal component analysis (PCA) of metabolites in plasma discussed in Example 1 . The data shows the metabolic profile comparison between a normal heathy female to a female with ovarian cancer. FIG. 2A is a 2D Scatter plot of samples from subjects (healthy female subjects and female subjects with ovarian cancer) and the principal components where scores are derived from projecting the original data onto the principal components. FIG. 2B is a loading plot showing how each metabolite contributes to specific principal components.

[0165] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D are graphs of several comparative analysis to assess fold changes in specific metabolites during trimester 1 (T1 ), 2 (T2), and 3 (T3) of pregnancy. FIG. 3A depicts fold changes in OV40 during pregnancy T1 -T3. FIG. 3B depicts fold Change in OV67 during pregnancy T1 -T3. FIG. 3C depicts fold Change in OV63 during pregnancy T1 -T3. FIG 3D depicts fold Change in OV180 during pregnancy T 1 -T3. The results show greater than 1000-fold increases in ITIH4 (OV67 and OV180) in a pregnancy term (see, Example 1 ).

[0166] FIG. 4 displays the NCBI reference sequence NP_002209.2 for ITIH4 1 precursor. The regions in the boxes indicate particular fragments of interest when ITIH4 is activated (see, Examples 3-5). The pattern recognition receptor (PRR) region in the sequence of ITIH4 1 precursor is highlighted (see, Example 1 ).

[0167] FIG. 5A and FIG. 5B show the state of activation of endogenous ITIH4 in plasma: ITIH4 protein is activated in plasma from pregnant woman. The two images of gels include: FIG. 5A sheep anti human ITIH4 polyclonal, and FIG. 5B mouse anti human ITIH4 HRP conjugated.

[0168] FIG. 6A and FIG. 6B show the ability of plasma from pregnant donor to activate exogenous ITIH4-His protein in plasma: ITIH4-His protein is activated in plasma from pregnant woman. The two images of gels include: FIG. 6A mouse anti human ITIH4 HRP conjugated, and FIG. 6B adding ITIH4-His to the plasma. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show the screening for inhibitors of ITIH-4 activity: Serpin A5 and Serpin G1 inhibit activation of ITIH-4-His protein in plasma from pregnant woman (see, Example 6). The four images of gels include: FIG. 7A table with the legend for the gel data, FIG. 7B anti-His, FIG. 7C anti-ITI H4, and FIG. 7D anti-His Serpin control.

[0169] Detailed Description of the Invention

[0170] The interaction between endogenous proteases and protease inhibitors is crucial for the precise regulation of immune responses and the maintenance of self-tolerance. This balance ensures that the immune system can effectively combat pathogens while avoiding damage to host tissues and preventing autoimmunity.

[0171] The immune system produces desired immune responses in the body of an organism when it protects the organism from harm through identifying and destroying foreign substances. The complement system is considered an integral part of the innate immune system and is an example of such a system. Complement is considered to be activated by proteases and regulated by protease inhibitors in either the lectin pathway (LP), classical pathway (CP), or the alternative pathway (AP). Human plasma contains factors which determine the activity of the complement system to produce a desired immune response. For example, in the plasma of pregnant humans, activity is regulated in order to avoid an immune response that would harm the fetus. Alternatively, complement system activity (e.g., complementdependent cytotoxicity (CDC)) could be promoted to destroy harmful cells (e.g., bacterial or viral infected cells). In immune-driven conditions, undesired immune responses can arise, which is when the immune system (e.g., complement system) does not modulate its activity appropriately to protect the organism from harm. For example, some types of cancers may produce inhibitors in the organism that decrease the complement system activity in order to suppress the immune system and allow the cancerous cells to avoid CDC. This results in an undesired immune tolerance of cancer cells. Alternatively, an immune- driven condition can result in an undesired immunoenhancement, in which immune tolerance may be lost, as is seen in autoimmune disease and graft-versus-host disease. In both conditions it can be hypothesized that the presence or absence of specific complement regulation factors in the human plasma to be the cause.

[0172] The ITIH4 protein is hypothesized to be a significant contributor to these immune-driven conditions. As such, tracking the activity of ITIH4 proteolysis could provide a means, a litmus test for measuring the level of activity of the complement, that can give insights into immune-driven condition diagnoses and treatment effectiveness. Additionally, immune system modulators that are directed to controlling the ITIH4 activity appropriately could prove to be a useful method of treatment for immune- driven conditions. Specifically, the serine protease inhibitor (serpin) family of proteins could prove useful as ITIH4 activity modulators and therapeutic agents for subjects suffering from immune-driven conditions.

[0173] Activated ITIH4

[0174] Activated ITIH4 may comprise of the entire ITIH4 protein or any one of the components resulting from an ITIH4 activation cleavage event (e.g., ITIH4 proteolysis products). Activated ITIH4 can comprise of at least one of its components in a complex with another molecule (e.g., a complex with a biomolecule or as part of a fusion protein). In the present invention, activated ITIH4 may serve as a biomarker for PATENT ATTORNEY DOCKET NO.: 51523-005WO3 diagnosis or prognosing a condition or as a treatment option. There are several ITIH4 components of particular importance to the present invention (see, e.g., Figure 4).

[0175] In one aspect, ITIH4 proteolysis provides cleavage products of particular importance (see, e.g., the boxed regions in Figure 4): 1 . a signal peptide comprising of reference sequence, mkpprpvrtc skvlvllsll aihqttta (SEQ ID No.: 1 ); 2. a protein comprising of reference sequence, mpknvvfvid ksgsmsgrki qqtrealiki Iddlsprdqf nlivfsteat qwrpslvpas aenvnkarsf aagiqalggt nindamlmav glldssngee rlpegsvsli illtdgdptv getnprsiqn nvreavsgry slfclgfgfd vsyaflekla Idngglarri hedsdsalql (SEQ ID No.: 2); 3. a protein comprising of reference sequence, vevt gqyerekagf swievtfknp Ivwvhaspeh vvvtrnrrss aykwketlfs vmpglkmtmd ktgll llsdp dkvtig llfw dgrgeglrll Irdtdrfssh vggtlgqfyq evlwgspaas ddgrrtlrvq gndhsatrer rldyqegppg veiscwsv (SEQ ID No.: 3); and 4. a protein comprising of reference sequence apsailplpg qsverlcvdp rhrqgpvnll sdpeqgvevt gqyerekagf swievtfknp Ivwvhaspeh vvvtrnrrss aykwketlfs vmpglkmtmd ktgll llsdp dkvtigllfw dgrgeglrll Irdtdrfssh vggtlgqfyq evlwgspaas ddgrrtlrvq gndhsatrer rldyqegppg veiscwsvel (SEQ ID No.: 4). In some embodiments of the invention, activated ITIH4 is administered to the subject for the treatment of an immune-driven condition. Activated ITIH4 may comprise of any one of these cleavage product components, any combination of components, or all of them. In some embodiments, the activated ITIH4 administered may include at least one of the cleavage products in combination with another molecule (e.g., fused or in a complex). In some embodiments, the activated ITIH4 administered is capable of forming a complex in the plasma of the subject, e.g., a complex with a protease or plasma kallikrein.

[0176] In another aspect, activated ITIH4 is part of a complex administered in a method of treating an immune-driven condition. In some embodiments, treatment comprises administering a fusion protein comprising of at least one activated ITIH4 component described herein above. In some embodiments, the treatment comprises administering a complex of at least one activated ITIH4 component described hereinabove with a protease, e.g., MASP-1 . In some embodiments, the treatment comprises of activated ITIH4 comprising of all of the components described hereinabove, wherein the activated ITIH4 can be part of a fusion protein or in a complex with another molecule, e.g., a protease.

[0177] In another aspect, the present invention provides a method for determining the ITIH4 activity in a subject through an assay for the ITIH4 proteolysis products, as described herein. In some embodiments, any of the cleavage products as described hereinabove are recognized as indicators of ITIH4 activity. In some embodiments, the formation of a complex that comprises of at least one of the components of activated ITIH4 is an indicator of ITIH4 activity (e.g., a complex with MASP-1 , MASP-2, or plasma kallikrein).

[0178] Another important region of ITIH4 for the present invention is the PRR region (see, e.g., Figure 4). In one aspect, the PRR region of ITIH4 is used as a diagnostic probe. For example, the PRR region will be used to diagnose a specific type of cancer as described herein. In another aspect, the PRR region can be used as a probe for determining the ITIH4 activity in a subject. For example, the presence of the PRR region could be indicative of the presence of activated ITIH4. In particular embodiments, the PRR region has reference sequence, gesrnrnvhs gstffkyylq gakipkpeas fsprrgwnrq agaagsrmnf rpgvlssrql glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr vmnmkieett mttqtpapiq (SEQ ID No.: 5) (see Figure 4). In other embodiments, the region of interest comprises of fewer amino acid residues, e.g., gakipkpeas fsprrgwnrq agaagsrmnf (SEQ ID No.: 6), glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr (SEQ ID No.: 7), and ssrql glpgppdvpd haayhpf (SEQ IS No.: 8). PATENT

[0179] ATTORNEY DOCKET NO.: 51523-005WO3

[0180] Serpin Protein Therapeutics

[0181] Serpin Family Proteins

[0182] The serpins (serine protease inhibitors) are a protein superfamily defined as having a structure ranging from about 330 to 500 amino acid residues (for reviews, see for example, Bird, Results Probl Cell Differ 24:63-89 (1998); Pemberton, Cancer J 10(1 ):1 -11 (1997); Worrall et al., Biochem Soc Trans 27 (4):746-50 (1999); and Irving et al., Genome Res 10:1845-64 (2000)). The human serpin proteins are organized into two main clades: (1 ) the extracellular clade A; and (2) the intracellular clade B. The clade A serpins are located on chromosomes 1 , 14, and X, and the clade B serpins are located on chromosomes 18 and 6. Some nonlimiting exemplary roles of plasma serpins in humans include: coagulation and fibrinolysis, immune and inflammatory responses, hormone transport, nerve growth, and blood pressure regulation. Other nonlimiting exemplary roles of serpins include: tissue protection against protease activation, regulation of angiogenesis, cell or organ development, and apoptosis and extracellular matrix remodeling. Some of the serpin’s functions relate to protease inhibition.

[0183] In one aspect of the present invention, serpin A5 and serpin G1 proteins are modulators of ITIH4 activity, e.g., ITIH4 proteolysis inhibitors (see, e.g., Example 6 and FIGs. 5A, 5B, 6A, 6B, 7A, 7B, 7C, and 7D). In another aspect, serpin proteins are the target of therapeutics directed towards promoting ITIH4 proteolysis and, in turn, suppressing the immune system.

[0184] Serpin A5

[0185] Serpin A5 (SEQ ID No.: 9), also known as protein C inhibitor (PCI), is a serine protease inhibitor, encoded by the SERPINA5 gene. Serpin A5 has a molecular weight of around 45 - 52 kDa. It belongs to the clade A family of serpins, which primarily inhibit serine proteases. Serpin A5 primarily acts as an anticoagulant by inhibiting protein C, which is a critical regulator of blood coagulation. It also inhibits other proteases involved in the coagulation cascade, such as thrombin, kallikrein, and factor Xa, and plays roles in reproductive biology by protecting sperm and facilitating fertilization. Serpin A5 was found to be an inhibitor of ITIH4 activity (see, e.g., Example 6 and FIGs. 7A, 7B, 7C, and 7D). As such, serpin A5, or derivatives thereof, or other similar serpin family proteins, could function as a proinflammatory or an immune system activator in a therapeutic agent, e.g., immunotherapy. These therapeutic agents could serve to treat immune-driven conditions with adverse immunosuppression by promoting immunoenhancement.

[0186] SEQ ID No.: 9:

[0187] MQLFLLLCLVLLSPQGASLHRHHPREMKKRVEDLHVGATVAPSSRRDFTFDLYRALASAAPSQSI FFSPVSISMSLAMLSLGAGSSTKMQILEGLGLNLQKSSEKELHRGFQQLLQELNQPRDGFQLSL GNALFTDLVVDLQDTFVSAMKTLYLADTFPTNFRDSAGAMKQINDYVAKQTKGKIVDLLKNLDSN AVVIMVNYIFFKAKWETSFNHKGTQEQDFYVTSETVVRVPMMSREDQYHYLLDRNLSCRVVGVP YQGNATALFILPSEGKMQQVENGLSEKTLRKWLKMFKKRQLELYLPKFSIEGSYQLEKVLPSLGI SNVFTSHADLSGISNHSNIQVSEMVHKAVVEVDESGTRAAAATGTIFTFRSARLNSQRLVFNRPF LMFIVDNNILFLGKVNRP

[0188] Serpin G1 PATENT ATTORNEY DOCKET NO.: 51523-005WO3 Serpin G1 (SEQ ID No.: 10), also known as C1 -inhibitor (C1 -INH), is a serine protease inhibitor. Encoded by the SERPING1 gene, serpin G1 has a molecular weight of approximately 104 kDa. It is part of clade G and is a glycoprotein with several glycosylation sites that affect its size and function, serpin G1 functions as an inhibitor of the complement system, specifically inhibiting C1 r and C1 s (components of the C1 complex) in the classical pathway, as well as kallikrein in the contact activation pathway. It plays a key role in controlling inflammation and is crucial in preventing excessive complement activation. It was found that serpin G1 showed effect on pregnancy mediated activation of ITIH4 (see, e.g., Example 6 and FIGs. 7A, 7B, 7C, and 7D). As serpin G1 was shown to inhibit ITIH4 activation, the protein could be used to activate the immune system of a subject. Serpin G1 could be utilized as a therapeutic agent, e.g., immunotherapy, for treating an immune-driven condition, e.g., as described herein.

[0189] SEQ ID No.: 10:

[0190] MASRLTLLTLLLLLLAGDRASSNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLP TTNSTTNSATKITANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSD LESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILS YPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINT WVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKK YPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTL LTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAAS AISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA

[0191] Preparation of Serpin for Therapeutics

[0192] A serpin suitable for the present invention may be produced by any available means. For example, the serpin may be derived from plasma, e.g., human plasma. The serpin obtained may be purified or further purified, e.g., by filtration, cell lysis, centrifugation, affinity chromatography, ion exchange chromatography, and size exclusion chromatography. Alternatively, the serpin may be recombinantly produced by utilizing a host cell system engineered to express a serpin protein-encoding nucleic acid. The purified recombinant human serpin A5 or serpin G1 of the present invention will have an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the naturally occurring serpin, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma-derived human serpin. Alternatively, or additionally, a serpin may be produced by activating endogenous genes. Alternatively, or additionally, a serpin may be partially or fully prepared by chemical synthesis. Any expression system can be used for the recombinantly produced serpin proteins. A few non limiting examples of known expression systems include: E. coli, egg, baculovirus, plant, yeast, or mammalian cells.

[0193] The serpin suitable for the present invention may be a fragment of the protein. For example, the serpin may be less than 90% of the total residues of a serpin herein (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%).

[0194] The serpin A5 or serpin G1 suitable for the present invention (e.g., as described herein) may be in a complex (e.g., fusion protein, tagged protein, covalently bound to a biomolecules) with another molecule or molecules (e.g., proteins, antibodies, tags, or small molecules). The additional molecule or PATENT ATTORNEY DOCKET NO.: 51523-005WO3 molecules included in a serpin protein complex may improve bioavailability, half-life, efficacy, or reduce potential side-effects.

[0195] The serpin A5 or serpin G1 can be a fusion protein of formula: “serpinn-Lm-Rm” where n is 1 or 2, m is 0, 1 , or 2, L is a peptide sequence linker including from 1 to 100 amino acids or a small molecule (e.g., a molecule with a molecular weight of 1000 Da or less), and R is a protein, antibody, protein tag (e.g., His, Flag, Myc, GST, CBP, glu-glu, HAT, T7, or V5 tag), or small molecule. In some embodiments, the serpin protein is of formula: “serpin2-Lm-Rm” where two of the same serpins are in the fusion protein (e.g., two serpin A5 of SEQ ID No.: 9 or two serpin G1 of SEQ ID No.: 10). In specific embodiments of serpin A5 or serpin G1 fusion proteins with formula: “serpin2-Lm-Rm”, the serpin proteins are fused together. Nonlimiting examples of L include: peptide sequences of 1 to 100 amino acids(e.g., 1 to 95 amino acids, 1 to 90 amino acids, 1 to 85 amino acids, 1 to 80 amino acids, 1 to 75 amino acids, 1 to 70 amino acids) or small molecules such as sugars or metabolites. Nonlimiting examples of R include: immunoglobulin G (IgG) such as IgG 1 , lgG2, or lgG3, Fc polypeptide, a cytokine targeting polypeptide sequence (e.g., those that target TNF-alpha (Tumor Necrosis Factor alpha), IgE, or IL-1 alpha (lnterleukin-1 alpha), IL-1 beta (lnterleukin-1 beta), IL-2 (lnterlukin-2), IL-4 (lnterleukin-4), IL-6 (lnterleukin-6), IL-7 (lnterleukin-7), IL-10 (Interleukin-10), IL-12 (Interleukin-12), IL-13 (Interleukin-13), IL- 18 (Interleukin-18), IL-27 (Interleukin-27), or IL-32 (Interleukin-32) receptors), sugars (including linear or cyclic forms) such as glucose, metabolites, or small molecule therapeutics. In some embodiments, the fusion protein comprises at least one human serpin A5 or serpin G1 polypeptide operably linked to a modified human immunoglobulin (Ig) Fc polypeptide (e.g., a serpin-Fc fusion protein). In specific embodiments, the serpin A5 or serpin G1 fusion protein is fused with lgG3. In specific embodiments, the fused serpin protein is serpin A5 fused with lgG3 (e.g., attached to the Fc domain). In some embodiments, the serpin fusion proteins are mutated to modulate proteolytic cleavage, e.g., by mutating at least one proteolytic cleavage site. In some embodiments, the fusion proteins are modified to modulate an Fc effector function of the fusion protein and retain binding and / or inhibitory function of the modified serpin fusion protein. Nonlimiting examples of Fc effector functions include: Fc receptor binding, promoting of proinflammatory mediator release upon binding to the Fc receptor, phagocytosis, modified antibody-dependent cell-mediated cytotoxicity (ADCC), modified complement-dependent cytotoxicity (CDC), modified glycosylation at Asn297 residue (from EU index of Kabat numbering used herein, see, e.g., Kabat et al 1991 Sequences of Proteins of Immunological Interest) of the Fc polypeptide. In some embodiments, the serpin A5 or serpin G1 fusion proteins are modified to influence Fc receptor binding (e.g., via mutation). In some embodiments of the serpin A5 or serpin G1 fusion proteins that include the Fc polypeptide, the Fc polypetide is mutated to enhance FcRn binding. Nonlimiting examples of Fc polypeptide mutations that enhance binding to FcRn include: Met252Tyr, Ser254Thr, Thr256Glu, (Kabat number scheme: M252Y, S256T, T256E) or Met428Leu and Asn434Ser (Kabat number scheme: M428L, N434S) (Zalevsky et al. 2010, Nature Biotech, Vol. 28(2): 157-159). In some embodiments of the serpin A5 and serpin G1 fusion proteins, the Fc polypeptide portion will be modified (e.g., mutating residues on the dimerization face) to disrupt Fc-mediated dimerization in order to achieve monomeric Fc fusions (Ying et al. 2012, J. Biol Chem 287(23): 19399-19408).

[0196] In other embodiments of serpin fusion proteins of serpin A5 or serpin G1 , the fusion proteins may include: lgG4, Elafin (Trapppin-2), human serum albumin, a WAP domain-containing protein (e.g., secretory leukocyte protease inhibitor (SLPI), Elafin, or Eppin), or an anti-TNFa antibody. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 The serpin A5 or serpin G1 can be tagged with a protein tag (e.g., His, Flag, Myc, GST, CBP, glu- glu, HAT, T7, or V5 tag).

[0197] The serpin A5 or serpin G1 can be complexed with a small molecule or other biomolecule (e.g., a protease).

[0198] Pharmaceutical Compositions

[0199] The serpin A5 or serpin G1 of the present invention may serve as a therapeutic agent, e.g., an immunotherapy, in a pharmaceutical composition of the present invention. In some embodiments, the concentration of the serpin protein therapeutic agent in the pharmaceutical composition is from 1 mg / mL tO 200 mg / mL (e.g., 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, 150 mg / mL, 160 mg / mL, 165 mg / mL, 170 mg / mL, 175 mg / mL, 180 mg / mL, 185 mg / mL, 190 mg / mL, 195 mg / mL, 200 mg / mL). In some other embodiments, the concentration of the serpin protein therapeutic agent in the pharmaceutical composition is from 10 lU / mL to 10,000 lU / mL (wherein IU (international unit) is the biological activity of a substance). The additional components of the pharmaceutical composition may improve bioavailability, half-life, efficacy, or reduce potential side-effects. Additional components may include, but are not limited to, compound(s) used to adjust the tonicity (e.g., render the composition hypertonic or hypotonic) or compound(s) for increasing or decreasing the mass or viscosity of the composition.

[0200] The composition may further include one or more additional pharmaceutically acceptable ingredients such as carriers, excipients, diluents, stabilizers, buffers, and the like, which may be incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. See also: Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2) :210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1 -2):1 -60 (2000), Charman W N “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations.

[0201] An injectable pharmaceutical composition of the present invention may be ready for injection from a storage vial or reconstituted by adding sterile water to obtain a solution (e.g., from 1 mg / mL to 500 mg / mL). Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

[0202] In some embodiments, the serpin A5 or serpin G1 therapeutic agent is administered as part of a combination therapy (e.g., with chemotherapy, radiation therapy, or targeted therapy). The therapeutic of the present invention may be administered simultaneously or sequentially with the other therapy. In some embodiments, the serpin therapeutic agent is administered before the other therapeutic agent (e.g., within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day, or within 12 hours). In some embodiments, the serpin therapeutic agent is administered after the other therapeutic PATENT ATTORNEY DOCKET NO.: 51523-005WO3 agent (e.g., within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day, or within 12 hours). In some embodiments, the serpin therapeutic agent and the other therapeutic are co-administered (e.g., concurrent therapy). In some embodiments, the additional therapeutic agent is formulated with the serpin therapeutic agent (e.g., as described herein) prior to administration.

[0203] In some embodiments, the serpin A5 or serpin G1 therapeutic agent may be formulated with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation (e.g., implants and microencapsulated delivery systems).

[0204] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0205] Gene Therapy

[0206] In one aspect, a therapeutic agent of the present invention (e.g., a ITIH4 activity modulator as described herein) will be administered using gene therapy (e.g., a gene delivery system). In some particular embodiments, the gene therapy is anticancer gene therapy. In some embodiments, a gene therapy of the present invention uses expression constructs (e.g., cDNA or mRNA) or viral vectors (e.g., Adeno or Lenti) for delivering the therapeutic agents into cells (e.g., cells of a subject in need of a treatment for an immune-driven condition as described herein).

[0207] In some embodiments, the therapeutic agent is a serpin A5 of serpin G1 protein. In some embodiments, a viral vector is used to deliver genetic material (e.g., a gene) to enable production of the serpin A5 or serpin G2 proteins in the subject. In some embodiments, a viral vector comprising of a serpin A5 or serpin G1 therapeutic (e.g., a serpin A5 or serpin G1 therapeutic as described herein), is administered to the subject. In some embodiments, expression constructs, such as cDNA or mRNA, will be used as a means of getting expression of the serpin A5 or serpin G1 proteins, and increasing plasma levels of the serpin A5 or serpin G1 proteins in the subject.

[0208] Dosing Regimen

[0209] In some embodiments, the treatment with the serpin therapeutic is in an injectable dosage form (e.g., parenteral administration, i.e., intravenous, intradermal, subcutaneous) or an oral dosage form (e.g., a tablet or aerosol), wherein the ITIH4 proteolysis promoter (e.g., a serpin A5 or serpin G1 inhibitor) dose or ITIH4 proteolysis inhibitor (e.g., serpin A5, serpin G1 , or a derivative thereof) dose is from 0.0001 mg / kg to 500 mg / kg (e.g., from 0.1 mg / kg to 250 mg / kg, from 0.1 mg / kg to 100 mg / kg, or from 1 mg / kg to 20 mg / kg, or 1 mg / kg, 6 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg), depending on subject age (e.g., a child age 6 to 11 , an adolescent age 12 to 18, or an adult at least 18 years old) and bodyweight (e.g., <84 kg, 84 kg, >84 kg). In some embodiments, the dose is administered as a single dose on a dosing schedule. In some embodiments, the dosing schedule can include administration at a frequency of once a day to once every six months. In other embodiments, the dosing schedule can include administration of a dose at least twice a day. In yet another embodiment, the dosing schedule can include a dose titration. In specific embodiments, the titration dosing schedule may include giving a higher dose for a period of time, e.g., day 1 or week 1 , followed by a lower dose for a period of time or the remainder of treatment. The higher dose may be achieved through a higher quantity of the therapeutic in a single dose or more frequent PATENT ATTORNEY DOCKET NO.: 51523-005WO3 administration of doses. In other specific embodiments, the dosing schedule may include a lower initial dose for a period of time, e.g., day 1 or week 1 , followed by a higher dose for a period of time or the remainder of treatment. The lower dose may be achieved through a lower quantity of the therapeutic in a single dose or less frequent administration of doses. In some embodiments, the dose may be administered by intravenous infusion over a period of time greater than 1 minute (e.g., over 1 minute, over 5 minutes, over 10 minutes, over 15 minutes, over 25 minutes, over 30 minutes, over 35 minutes, over 40 minutes, over 45 minutes, over 50 minutes, over 55 minutes, or over 60 minutes).

[0210] Immune-driven Conditions

[0211] The present invention features compositions and methods for features methods for diagnosing, prognosing, and treating immune-driven conditions (e.g., immunotherapy). Exemplary conditions include AIDS (or HIV positive), severe combined immune deficiency (SCID), diabetics, immunocompromised from receiving chemotherapy for cancer, most forms of cancer, sickle cell anemia, cystic fibrosis, no spleen, pregnancy, ophthalmology diseases (e.g., age-related macular degeneration (AMD), uveitis, scleritis, conjunctivitis, blepharitis, or choroiditis) end stage kidney disease (dialysis), immunocompromised from corticosteroid use on a frequent basis by pill or injection, and severe liver, lung, or heart disease. Exemplary conditions also include inflammatory conditions such as asthma (e.g., aspirin sensitive / exacerbated asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, corticosteroid naive asthma, chronic asthma, corticosteroid resistant asthma, corticosteroid refractory asthma, newly diagnosed and untreated asthma, asthma due to smoking, asthma uncontrolled on corticosteroids, etc.), airway hyperreactivity, airway hyperresponsiveness, rhinosinusitis, rhinosinusitis with polyps, nasal polyposis, arthritis (e.g., osteoarthritis, rheumatoid arthritis, arthritic joints as a result of injury, etc.), seronegative enthesopathy and arthropathy (SEA) syndrome, osteoporosis, eosinophilic esophagitis, scleroderma, dermatitis, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, cartilage inflammation, polymyalgia rheumatic, polyarteritis nodossa, Wegener’s granulamatosis, Behcet’s disease, myolitis, polymyolitis, dermatomyolitis, dermatomyositis, vasculitis, arteritis, diabetic nephropathy, interstitial cystitis, graft versus host disease (GVHD), gastrointestinal inflammatory conditions (e.g., inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn’s disease (CD), colitis (e.g., colitis caused by environmental insults (e.g., caused by or associated with a therapeutic regimen, such as chemotherapy, radiation therapy, etc.), infectious colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis in conditions such as chronic granulomatous disease or celiac disease, food allergies, gastritis, infectious gastritis or enterocolitis (e.g., Helicobacter pylori- infected chronic active gastritis), and other forms of gastrointestinal inflammation caused by an infectious agent), and inflammatory pulmonary conditions (e.g., chronic obstructive pulmonary disease (COPD)), infection-induced pulmonary conditions (including those associated with viral (e.g., influenza, parainfluenza, rotavirus, human metapneumovirus, and respiratory syncytial virus), bacterial, fungal (e.g., Aspergillus), parasitic, or prion infection, allergen-induced pulmonary conditions, pollutant-induced pulmonary conditions (e.g., asbestosis, silicosis, or berylliosis), gastric aspiration-induced pulmonary conditions, immune dysregulation, inflammatory conditions with genetic predisposition such as cystic fibrosis, physical trauma-induced pulmonary conditions (e.g., ventilator injury), emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangiomyomatosis, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, pneumonia (e.g., community-acquired pneumonia, PATENT ATTORNEY DOCKET NO.: 51523-005WO3 nosocomial pneumonia, ventilator-associated pneumonia, viral pneumonia, bacterial pneumonia, and severe pneumonia), airway exacerbations, and acute respiratory distress syndrome (ARDS)). Exemplary conditions also include immune disorders such as those mediated at least in part by mast cells, such as asthma, eczema, itch, allergic rhinitis, allergic conjunctivitis, as well as autoimmune disorders including rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, pancreatitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, paraneoplastic autoimmune diseases, autoimmune hepatitis, bullous pemphigoid, myasthenia gravis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, celiac disease, thyroiditis (e.g., Graves’ disease), Sjogren’s syndrome, Guillain-Barre disease, Raynaud’s phenomenon, Addison’s disease, liver diseases (e.g., primary biliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis), and diabetes (e.g., type I diabetes).

[0212] Assays for ITIH4 Proteolytic Activation

[0213] ITIH4 plays a central role in immune responses (see, e.g., Pihl et al., Sci. Adv. 2021 ; 7:1 -17). One mechanism by which ITIH4 may control immune responses is through inhibition of proteases associated with innate inflammatory responses, such as, MASP-1 , MASP-2, Kallikrein, MMP-7, MMP-13, thrombin, papain, and plasmin. In this process, the ITIH4 becomes activated by a human protease within a protease-susceptible region, wherein the ITIH4 is cleaved. The products of the ITIH4 activation function as protease inhibitors, thus allowing the products to serve as modulators of the immune system. This can result in undesired immune responses that are hallmarks of immune-driven conditions. For example, an increase in ITIH4 proteolysis may be associated with immunosuppressive conditions, e.g., cancer. Alternatively, a decrease in ITIH4 proteolysis, resulting from a decrease in ITIH4 activation, may be associated with adverse immunoenhancement conditions, e.g., graft-versus-host disease.

[0214] Since the ITIH4 proteolytic activity is correlated to immune responses, ITIH4 proteolysis and its products can serve as a biomarker for immune-driven conditions. For example, blood plasma from a subject believed to have an immune-driven condition, e.g., cancer, can by subjected to recombinant ITIH4 protein and monitored for ITIH4 proteolysis. The amount of ITIH4 proteolysis observed is positively corrected to the level of ITIH4 activation. One approach to determine the level of ITIH4 activation can be determined by the identity and quantity of metabolites expected to be present based on the amount of ITIH4 proteolysis occurring in blood. The present invention identifies the relevant metabolite biomarkers expected based on subject populations with conditions leading to different immune responses.

[0215] One aspect of the present invention is directed to an assay for the level of ITIH4 activation. The assay will include subjecting blood plasma samples from healthy subjects (individuals with normal immune responses), healthy subjects with suppressed immune responses (e.g., pregnant subjects), and subjects with cancer (individuals with suppressed immune responses) to recombinant ITIH4 protein in order to determine the level of ITIH4 activation and the metabolites that would be expected from groups of subjects with different immune responses. In some embodiments, the recombinant ITIH4 protein is tagged for detection and analysis with His (polyhistidine), FLAG (hydrophilic 8-amino acid peptide SEQ: DYKDDDDK), Myc (c-Myc protein), GST (glutathione S-transferase), CBP (calmodulin binding protein), glu-glu (glu-glu epitope), HAT (histidine affinity tag), T7 (T7 epitope tag), or V5 (V5 epitope tag). In some embodiments, the recombinant ITIH4 protein is labeled for detection and analysis. In some embodiments, the ITIH4 protein is fluorescently labeled or biotinylated or labeled with a chromogenic, fluorogenic, or PATENT ATTORNEY DOCKET NO.: 51523-005WO3 chemiluminescent molecule. In other embodiments the ITIH4 protein is labeled with peroxidase (POD), horseradish peroxidase (HRP), or alkaline phosphate. In some embodiments, the recombinant ITIH4 protein capable of detection may comprise of a fusion protein or an antibody. In specific embodiments, the fusion protein comprises the PRR region of the ITIH4 protein. In some embodiments of the present invention, the means for the detection of ITIH4 activity, e.g., labeled proteins, or fusion proteins comprising the PRR region of the ITIH4 protein are included as part of a kit for the ITIH4 proteolysis assay.

[0216] The present ITIH4 proteolysis and biomarker assays could further be used to diagnose subjects suspected of immune-driven conditions based on the level of ITIH4 activation. In some embodiments of the present invention, the assay will diagnosis cancer. The present assay could be used to diagnose any cancer type in which ITIH4 activation is shown when testing the plasma of the subject. In specific embodiments, a specific type of cancer can be diagnosed based on the assay including, but not limited to, breast cancer, colon cancer, renal cell cancer, lung cancer, hepatocellular carcinoma, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, bladder cancer, head and neck cancer, glioblastoma, diffuse large B-cell lymphoma, leukemia, or melanoma. In another embodiment of the present invention, the ITIH4 proteolysis and biomarker assays could be used to provide a prognosis for pregnancy health. The immunosuppressive response needed to ensure the health of the fetus can be evaluated via the level of ITIH4 activation in the plasma sample of a healthy subject.

[0217] In another aspect of the present invention, the ITIH4 proteolysis and biomarker assays can be used to identify activators as well as inhibitors of ITIH4 proteolytic activity. For example, the change in ITIH4 proteolysis in an in vitro sample subjected to a potential immune system modulator (e.g., a ITIH4 proteolysis promoter or inhibitor) can be compared to the ITIH4 proteolysis in a control sample. This screening process could be useful in determining a treatment for immune-driven conditions. For example, the inhibition of ITIH4 activation may allow for the treatment of immunosuppressive conditions, e.g., cancer. On the other hand, the promotion of ITIH4 activation may allow for the treatment of immunoenhancement conditions, e.g., graft-versus-host disease. In some embodiments of the present invention, this ITIH4 proteolysis and biomarker assay could be used to determine the efficacy of a treatment for an immune-driven condition. For example, a plasma sample from a subject previously diagnosed with an immune-driven condition, e.g., cancer or graft-versus-host disease, can be screened for responsiveness to ITIH4 modulation by an ITIH4 modulator.

[0218] ITIH4 Activity Modulators for Treatment

[0219] Modulators for Immunosuppression

[0220] One aspect of the present invention includes compositions and methods of treatment for immune- driven conditions wherein the subject could benefit from suppression of the immune system (e.g., subjects who have an immune-driven condition that can be characterized by adverse immunoenhancement). The desired immunosuppressive response could be obtained by promoting ITIH4 proteolysis in the subject in vivo, resulting in protease inhibition. In the present invention, the compounds and compositions used to promote ITIH4 proteolysis are referred to as “ITIH4 proteolysis promoters”. In preferred embodiments, the ITIH4 proteolysis promoters block the function of serpin A5 or serpin G1 .

[0221] Subjects that have an immune-driven condition, where the immune-driven condition is characterized by adverse immunoenhancement, may be treated with ITIH4 proteolysis promoters or PATENT ATTORNEY DOCKET NO.: 51523-005WO3 ITIH4 proteolysis products in vitro or in vivo. In some embodiments, the subject’s immune-driven condition is an autoimmune disease, endangered pregnancy, or rejection of a tissue, cell, graft, or organ transplant from a donor. In specific embodiments, the subject was exposed to ITIH4 proteolysis promoters or ITIH4 proteolysis products prior to, simultaneously with, or after tissue, cell, graft, or organ transplant. In other specific embodiments, the organ transplant can be a kidney transplant, a cornea transplant, a bone marrow transplant, a liver transplant, a lung transplant, or a heart transplant. In other embodiments, the subject’s immune-driven condition is either arthritis, allergy, asthma, pollinosis, or atopy. In other embodiments, the subject’s immune-driven condition is an autoimmune disease. In other embodiments, the subject’s immune-driven condition is a result of an adverse immune response to a separate therapeutic treatment.

[0222] Modulators for Immunoenhancement

[0223] Another aspect of the present invention includes compositions and methods of treatment for immune-driven conditions where the subject could benefit from enhancement of the immune system (e.g., subjects who have an immune-driven condition that can be characterized by adverse immunosuppression or are immunocompromised). In some embodiments, subjects with adverse immunosuppression include, but are not limited to, subjects with AIDS (or HIV positive), subjects with severe combined immune deficiency (SCID), diabetics, those who are receiving chemotherapy for cancer, and those characterized as having a low white blood cell count. Immunocompromised subjects also include subjects with most forms of cancer (other than skin cancer), sickle cell anemia, cystic fibrosis, those who do not have a spleen, subjects with end stage kidney disease (dialysis), and those who have been taking corticosteroids on a frequent basis by pill or injection within the last year. Subjects with severe liver, lung, or heart disease also have adverse immunosuppression.

[0224] The desired immunoenhancement response (e.g., an increase complement activity) could be obtained by inhibiting ITIH4 proteolysis in the subject through the administration of an ITIH4 proteolysis inhibitor (e.g., a serpin protein therapeutic comprising serpin A5 or serpin G1 as described herein). In particular embodiments, the ITIH4 proteolysis inhibitor is selected from serpin A5 (SEQ ID No.: 9) or serpin G1 (SEQ ID No.: 10), or a derivative thereof, e.g., serpin A5 fragment of less than 45 kDa, serpin G1 fragment of less than 100 kDa, a serpin as described herein, a fusion protein comprising a serpin (e.g., a serpin A5 or serpin G1 fusion protein of the formula: “serpinn-Lm-Rn”, or a fragment thereof, as described herein), or a tagged protein comprising a serpin (e.g., a serpin A5 or serpin G1 protein tagged with a tag selected from His, Flag, Myc, GST, CBP, glu-glu, HAT, T7, or V5 tags).

[0225] Cancer

[0226] The complement system is considered an immune surveillance system against cancer. However, tumor cells develop inhibitory mechanisms to prevent CDC. Further, complement activation within the tumor environment can promote tumor growth, which may lead to side effects such as inflammation, immunosuppression, angiogenesis, and the initiation of other cancer-related signaling pathways.

[0227] One aspect of the present invention is a method for treating cancer by administering a therapeutic agent, e.g., an immunotherapy, that provides immunoenhancement, e.g., a proinflammatory. In specific embodiments, said therapeutic agent is a serpin that functions as an ITIH4 proteolysis inhibitor, e.g., serpin A5 or serpin G1 . In some embodiments, the serpin A5 or serpin G1 is part of a serpin fusion PATENT ATTORNEY DOCKET NO.: 51523-005WO3 protein (e.g., as described herein), in which the additional components aid in the anti-cancer activity and / or reduce the adverse side effects that may arise from using a serpin A5 or serpin G1 protein therapeutic with certain cancer types.

[0228] In some embodiments of the present invention, the efficacy of the serpin ITIH4 proteolysis inhibitor (e.g., a serpin A5 or serpin G1 as described herein) is determined through screening for the inhibitory activity in a plasma sample from the subject in need of a treatment for cancer before treatment. In some specific embodiments, the therapeutic agent is a composition comprising of serpin A5 (SEQ ID No.: 9) or derivatives thereof, e.g., serpin A5 fragment of less than 45 kDa, a fusion protein with serpin A5 or a fragment thereof, and / or a tagged serpin A5, e.g., His, Flag, Myc, GST, CBP, glu-glu, HAT, T7, or V5 tags. In other specific embodiments, the therapeutic agent is serpin G1 (SEQ ID No.: 10), or derivatives thereof, e.g., serpin G1 fragment of less than 100 kDa, a fusion protein with serpin G1 or a fragment thereof, or a tagged serpin G1 , e.g., His, Flag, Myc, GST, CBP, glu-glu, HAT, T7, or V5 tags.

[0229] In another embodiment of the present invention, the serpin ITIH4 proteolysis inhibitor is administered to a subject having a cancer susceptible to treatment by activating immune function, e.g., a cancer that can be treated by activating immunotherapy. Nonlimiting examples of cancers susceptible to treatment with immunotherapy include: Melanoma, renal cell carcinoma (RCC), colorectal cancer (e.g., KRAS wild-type, MSI-H), Metastatic colorectal cancer, or other Gl malignancies, hepatocellular carcinoma (HCC), HNSCC, Malignant Pleural Mesothelioma, bladder cancer (e.g., intravesical), sarcomas (e.g., osteosarcoma, Kaposi’s sarcoma, soft tissue sarcomas), lymphomas (e.g., Hodgkin, Non-Hodgkin, indolent B-cell non-Hodgkin, diffuse large B-cell (DLBCL), mantle cell, follicular, small lymphocytic, B-cell malignancies, anaplastic large cell), Merkel cell carcinoma, MSI-high cancer, Gastric cancer (e.g., HER2- positive), gastroesophageal cancer, cervical cancer, PMBCL, endometrial carcinoma, esophagus cancer, cutaneous squamous-cell carcinoma, breast cancer (e.g., HER2-possitive, HER2-low, triple negative, ERpositive (adjuvant or metastatic), hormone receptor positive), Metastatic breast cancer, TMB-high cancer, SCLC, basal cell carcinoma, multiple myeloma, testicular cancer, pancreatic cancer, prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., BRCA-mutated), thyroid cancer, leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia, acute promyelocytic leukemia (APL), head and neck cancers, lung cancer, non-small cell lung cancer (NSCLC) (e.g., non-squamous, EGFR-mutant, ALK- positive), melanoma, gastrointestinal stromal tumors (GIST), solid tumors, malignant pleural mesothelioma, myelofibrosis, polycythemia vera, Waldenstrom’s macroglobulinemia, glioblastoma, urothelial carcinoma, and myelodysplastic syndromes (MDS).

[0230] Anti-Cancer Combination Therapies

[0231] The serpin A5 or serpin G1 therapeutic agent can also be administered as part of a combination therapy for the treatment of cancer. In the present invention, treating cancer with a combination therapy requires administering to a patient a therapeutically effective amount of: i) a serpin A5 or serpin G1 therapeutic (e.g., as described herein), and ii) an anticancer therapy (e.g., chemotherapy, target therapy, immunotherapy, hormonal therapy). In some embodiments, the serpin therapeutic agent is administered before the anticancer therapeutic agent (e.g., within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day, or within 12 hours). In some embodiments, the serpin therapeutic agent is administered after the anticancer therapeutic agent (e.g., within 1 week, within 6 PATENT ATTORNEY DOCKET NO.: 51523-005WO3 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day, or within 12 hours). In some embodiments, the serpin therapeutic agent and the anticancer therapeutic agent are co-administered (e.g., concurrent therapy). In some embodiments, the anticancer therapeutic agent is formulated with the serpin therapeutic agent prior to administration.

[0232] The anticancer therapy utilized in the combination therapy may be a cytotoxic chemotherapy agent such as an alkylating agent, a platinum-based compound, a antimetabolite, a antitumor antibiotic, or a plant alkaloid. Nonlimiting examples of alkylating agents include: cyclophosphamide (Cytoxan), infosfamide (Ifex), melphalan (Alkeran), chlorambucil (Leukeran), and bendamustine (Tranda, Bendeka). Nonlimiting examples of platinum-based compounds include: cisplatin, carboplatin, oxaliplatin (Eloxatin). Nonlimiting examples of antimetabolites include: methotrexate, 5-fluorouracil (5-FU), capecitabine (Xeloda), gemcitabine (Gemzar), pemetrexed (Alimta), cytarabine (ara-C), and 6-mercaptopurine (6-MP). Nonlimiting examples of antitumor antibiotics include: doxorubicin, daunorubicin, epirubicin, bleomycin, and mitomycin (Mutamycin). Nonlimiting examples of plant alkaloids include: vica alkaloids such as vincristine, vinblastine, and vinorelbind; taxanes such as paclitaxel (Taxol), docetaxel (Taxotere), and cabazitaxel (Jevtana); epipodophyllotoxins (Topoisomerase II inhibitors) such as etopside (VP-16); and camptothecins (Topoisomerase I inhibitors) such as irinotecan (Camptosar) and topotecan (Hycamtin).

[0233] The anticancer therapy utilized in the combination therapy may be a targeted therapy such as a tyrosine kinase inhibitor (TKI), a monoclonal antibody (mAb), or a antibody-drug conjugate (ADC). Nonlimiting examples of tyrosine kinase inhibitors include: imatinib (Gleevec), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif), ponatinib (Iclusig), erlotinib (Tarceva), gefitinib (Iressa), afatinib (Gilotrif), osimertinib (Tagrisso), sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), axitinib (Inlyta), cabozantinib (Cabometyx), dabrafenib (Tafinlar), vemurafenib (Zelboraf), trametinib (Mekinist), cobimetinib (Cotellic), ruxolitinib (Jakafi), ibrutinib (Imbruvica), acalabrutinib (Calquence), idelalisib (Zydelig), and duvelisib (Copiktra). Nonlimiting examples of monoclonal antibodies include: rituximab (Rituxan), obinutuzumab (Gazyva), ofatumumab (Arzerra), trastuzumab (Herceptin), pertuzumab (Perjeta), cetuximab (Erbitux), panitumumab (Vectibix), bevacizumab (Avastin), daratumumab (Darzalex), isatuximab (Sarclisa), elotuzumab (Empliciti), and inotuzumab ozogamicin (Besponsa). Nonlimiting examples of antibody-drug conjugates include: T-DM1 I ado-trastuzumab emtansine (Kadcyla), brentuximab vedotin (Adcetris), sacituzumab govitecan (Trodelvy), and trastuzumab deruxtecan (Enhertu).

[0234] The anticancer therapy utilized in the combination therapy may be an immunotherapy such as an immune checkpoint inhibitor. Nonlimiting examples of immune checkpoint inhibitors include: anti-CTLA-4 such as ipilimumab (Yervoy); anti-PD-1 such as pembrolizumab (Keytruda), nivolumab (Opdivo), and cemiplimab (Libtayo); and anti-PDpL1 such as atezolizumab (Tecentriq), durvalumab (Imfinzi), and avelumab (bavencio).

[0235] The anticancer therapy utilized in the combination therapy may be a hormonal (endocrine_ therapy such as a selective estrogen receptor modulator (SERM), an aromatase inhibitor, a selective estrogen receptor degrader (SERD), a anti-androgen, or a GnRH (LHRH) agonist or antagonist. Nonlimiting examples of SERM include: tamoxifen. Nonlimiting examples of aromatase inhibitors include: anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin). Nonlimiting examples of SERD include: fulvestrant (Faslodex). Nonlimiting examples of anti-androgens include: bicalutamide (Casodex), flutamide, nilutamide, enzalutamide (Xtandi), apalutamide (Erleada), and darolutamide (Nubeqa). PATENT ATTORNEY DOCKET NO.: 51523-005WO3 Nonlimiting examples of GnRH (LHRH) agonists include: leuprolide (Lupron) and goserelin (Zoladex). Non limiting examples of GnRH (LHRH) antagonists include: degarelix (Firmagon) and Relugolix (Orgovyx).

[0236] Other anticancer therapy agents that may be utilized in the combination therapy include, but are not limited to: lenalidomide (Revlimid), thalidomide (Thalomid), pomalidomide (Pomalyst), bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro), venetoclax (Venclexta), olaparib (Lynparza), rucaparib (Rubraca), niraparib (Zejula), talazoparib (Talzenna), everolimus (Afinitor), temsirolimus (Torisel), arsenic trioxide (Trisenox), all-trans retinoic acid (ATRA), trabectedin (Yondelis), L- asparaginase (Elspar, Erwinia asparaginase, Pegaspargase), and selinexor (Xpovio).

[0237] Pregnancy

[0238] The ITIH4 activity is altered in a subject during the course of pregnancy. As this activity is related to immune function, the ITIH4 proteolytic activity has been observed to be enhanced in pregnant subjects in order to suppress the immune function to protect the fetus. Therefore, monitoring the ITIH4 activity will allow for insights into the pregnancy health of a pregnant subject. Moreover, modulating the ITIH4 activity through the administration of ITIH4 promoters or ITIH4 inhibitors could result in a change to the pregnancy health of the subject.

[0239] One aspect of the present invention is a method for treating poor pregnancy health. As stated, if the ITIH4 activity is not increased appropriately to suppress the immune system, the fetus could be harmed. The poor pregnancy health resulting from an undesired immune response can be addressed by administering an ITIH4 proteolysis promoter to suppress immune function. In some embodiments, the effects of the treatment can be monitored by analyzing the change in the ITIH4 activity in blood plasma samples from the subject before and after treatment. In some embodiments, the treatment is administered until the desired ITIH4 activity is reached. In other embodiments, the treatment is administered for the entire pregnancy term.

[0240] In particularly preferred embodiments, the ITIH4 proteolysis promoter administered inhibits the activity of a serpin protein (e.g., serpin A5 (protein C inhibitor; SEQ ID No.: 9) or serpin G1 (SEQ ID No.: 10)). As described herein, e.g., Example 6, the serpin A5 and serpin G1 inhibit the activation of ITIH4 protein in the plasma of pregnant women. As such, inhibiting the function of serpin A5, serpin G1 , or another serpin described herein, may allow for an increase in ITIH4 activity.

[0241] Another aspect of the present invention is a method for terminating pregnancy. As ITIH4 activity results in the immunosuppression needed for a healthy pregnancy, the ITIH4 activity can be modified to encourage rejection of the fetus (e.g., increasing complement activity for immunoenhancement via administering a complement activity promotor such as a proinflammatory). In some embodiments, the termination of pregnancy will include administering an effective amount of an inhibitor of ITIH4 proteolysis in order to enhance the immune response to promote rejection. In some embodiments, the treatment will be administered until the desired ITIH4 activity is achieved. In other embodiments, the treatment will be administered until the pregnancy is terminated. As described in Example 6, serpin A5 and serpin G1 show the desired ITIH4 inhibition activity in the plasma of pregnant women (see, e.g., FIGs. 7A, 7B, 7C, and 7D). As such, in particularly preferred embodiments, the ITIH4 proteolysis inhibitor is serpin A5 or a derivative thereof, e.g., a fusion protein, a tagged protein, or a fragment of SEQ ID No.: 9. In other PATENT ATTORNEY DOCKET NO.: 51523-005WO3 particularly preferred embodiments, the ITIH4 proteolysis inhibitor is serpin G1 or a derivative thereof, e.g., a fusion protein, a tagged protein, or a fragment of SEQ ID No.: 10.

[0242] Dosing Regimen

[0243] In some embodiments, the treatment is in an injectable dosage form or an oral dosage form, wherein the ITIH4 proteolysis promoter dose or ITIH4 proteolysis product dose or ITIH4 proteolysis inhibitor dose is from 0.0001 mg / kg to 100 mg / kg. In some embodiments, the dose is administered as a single dose on a dosing schedule. In some embodiments, the dosing schedule can include administration at a frequency of once a day to once every six months. In other embodiments, the dosing schedule can include administration of a dose at least twice a day. In yet another embodiment, the dosing schedule can include a dose titration. In specific examples, the titration dosing schedule may include giving a higher dose for a period of time, e.g., day 1 or week 1 , followed by a lower dose for a period of time or the remainder of treatment. The higher dose may be achieved through a higher quantity of the therapeutic in a single dose or more frequent administration of doses. In other specific embodiments, the dosing schedule may include a lower initial dose for a period of time, e.g., day 1 or week 1 , followed by a higher dose for a period of time or the remainder of treatment. The lower dose may be achieved through a lower quantity of the therapeutic in a single dose or less frequent administration of doses.

[0244] Kits

[0245] Also featured are kits comprising of requisites for performing diagnostic analysis, prognostic analysis, and for determining the efficacy of treatments directed to ITIH4 activity. Such kits for performing diagnostic analysis include, e.g., those for diagnosing a subject with cancer. In some instances, the kit would also include materials for identifying the PRR region of the ITIH4 sequence that can be used to determine a specific cancer type. In other instances, a kit comprises the materials needed to provide a prognosis for pregnancy health. In certain related instances, the kit comprises the materials needed to monitor the pregnancy health of the subject throughout the pregnancy term. A kit of the present invention also may comprise of the materials needed to evaluate the efficacy of treatments for immune-driven conditions. In some embodiments, a kit for determining the efficacy of treatment may comprise a means for monitoring the ITIH4 activity in a plasma sample in order to determine the change in ITIH4 activity due to administering an ITIH4 activity modulator, such as, a ITIH4 proteolysis promoter for immunosuppression or a ITIH4 proteolysis inhibitor for immunoenhancement.

[0246] Kits of the present invention may optionally include a means for detecting the ITIH4 activity. Such a kit may comprise of recombinant ITIH4 protein capable of detection. The recombinant ITIH4 proteins capable of detection may comprise of ITIH4 labeled proteins, for example, a fluorescent label, HRP, POD, or alkaline phosphate label. The recombinant ITIH4 proteins capable of detection may comprise of ITIH4 tagged proteins, for example, His, Flag, Myc, GST, CBP, glu-glu, HAT, T7 or V5 tags. A kit may optionally include one or more protease inhibitors. A kit may optionally include one or more ITIH4 proteolysis promoters. Any one of the kits of the present invention may optionally comprise of the buffer.

[0247] Examples

[0248] The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood PATENT ATTORNEY DOCKET NO.: 51523-005WO3 that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure or scope of the appended claims.

[0249] Example 1. Identification of a Metabolite Biomarker Assay for the ITIH4 Proteolysis Product

[0250] Methods

[0251] Using the Sciex X500R QTOF to analyze the plasma metabolic profiles of the samples collected from subjects. MARKERVIEW™ software from Sciex was used for the analysis.

[0252] The accurate molecular ion masses were used for composition calculation and collection of data dependent MS / MS data. MS / MS data was collected for structural deduction and database searches. The combined monitored ion profile confirmed the PCA-directed selection process.

[0253] Metabolome Comparative Analysis

[0254] One study utilized three human subject populations: 1 . non-pregnant healthy females, 2. pregnant healthy females, 3. females with ovarian cancer. As pregnant females represent a population with altered ITIH4 proteolysis activity (e.g., to promote the survival of a fetus in a pregnant woman), this population allows for a representation of a healthy, but immunosuppressed population. Blood plasma samples were obtained from each subject population. The aim was to identify the metabolic similarities and differences in these subject populations. Principal component analysis (PCA) of the metabolites revealed four common metabolites between the pregnant healthy female population and the females with ovarian cancer (FIG. 1 ).

[0255] Another related study included a patient population with ovarian cancer. The study again utilizes pregnant female subjects, as pregnant females represent a population with altered ITIH4 proteolysis activity for a tolerogenic response (e.g., to promote the survival of a fetus in a pregnant woman). PCA analysis revealed common specific metabolites between pregnant females and those with ovarian cancer (FIG. 2). This suggests the immunosuppressive responses seen in healthy pregnant females and those with cancer both involve modulating the ITIH4 activity. Specific biomarkers OV67 and OV180 were identified as undergoing >1 OOO-fold increase during the pregnancy term, leading to a hypothesis that these biomarkers are a result of an essential immune response process (e.g., ITIH4 proteolysis is activated in vivo resulting in an immunosuppressive response necessary for a healthy fetus). The relevant metabolites from the study were characterized by MS and are described in Table 1 .

[0256] Table 1 :

[0257] The metabolites identified in these experiments can be used as biomarkers for cancers that suppress immune function. PATENT ATTORNEY DOCKET NO.: 51523-005WO3

[0258] Activation of ITIH4 as an Indicator for Pregnancy Health

[0259] Testing for the activation of ITIH4 in the plasma of female subjects could provide a means of assessing the pregnancy health of the subject as well as identifying and circumventing fertility problems. The similar immunosuppressive pathway observed between pregnant females and those with ovarian cancer provides insight into the relevant biomarkers. The immunosuppressive response of pregnant females is desirable for the health of the fetus. As such, the assay for ITIH4 activity will indicate if the correct immunosuppressive response is occurring in the pregnant subject. If the immunosuppressive response has not occurred, this could be indicative of problems in the pregnancy. By providing the prognosis of pregnancy health to the subject, a treatment plan could be established wherein a ITIH4 modulator is administered in order to suppress the immune system to improve pregnancy health.

[0260] Activation of ITIH4 as a Diagnostic Test for Cancer

[0261] Early diagnosis for a variety of cancers could be achieved through a method of testing for the activation of ITIH4 in plasma samples in vitro. The method for diagnosing cancer will include obtaining a plasma sample from a subject followed by inducing ITIH4 proteolysis. The induction of ITIH4 proteolysis could be achieved through the addition of recombinant ITIH4 protein, wherein the level of activation of the recombinant ITIH4 protein can be monitored. The level of ITIH4 activation data will be used for the diagnosis of the subject.

[0262] Identification of Fragments from Proline-Rich Region (PRR) of ITIH4

[0263] The ITIH4 proteolysis results in ITIH4 PRR fragments that are associated with specific cancer type disease conditions. One method of identifying the PRR fragments in subjects’ plasma samples is through SELDI-based immunoassay (see, e.g., Song et al, Clin. Chem. 2006; 52(6): 1045-1053). The identification of the specific PRR fragments will allow for a diagnosis of the specific cancer types (see FIG 4).

[0264] Activation of ITIH4 as a Screening for Anticancer Agents

[0265] A method for determining the effectiveness of treatments will be developed. The method includes obtaining plasma samples from a cancer patient for treatment analysis. A control study will be conducted to determine the level of ITIH4 activation currently experienced by the subject. The method includes subjecting the sample to recombinant ITIH4 protein wherein the level of activation of the recombinant ITIH4 protein can be determined by the identity and quantity (e.g., 0 or >0) of the metabolites present post proteolysis. The data in the control study will establish the baseline level of ITIH4 activation for the patient. The effectiveness of a treatment will be determined by subjecting a patient plasma sample to: (1 ) recombinant ITIH4 protein; (2) a modulating agent; after (2), (3) identifying the identity and quantity of the metabolites present post ITIH4 proteolysis; and (4) comparing the metabolite profile to a control sample. Plasma samples from cancer patients can be used to test the validity of an ITIH4 activation inhibitor. This methodology will be utilized as a means for identifying and developing agents for complement restoring activity, which will also be used in identification of targeted cancer therapies. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 Example 2. Treatment of Cancer with Protease Inhibitor

[0266] A challenge with the use of protease inhibitors for cancer treatment is the variation in effectiveness depending on the type of cancer. One method of treatment will utilize the ITIH4 PRR fragment information to determine the appropriate protease inhibitor for the cancer patient. Another method of treatment will utilize the metabolite information resulting from ITIH4 proteolysis.

[0267] Example 3. Graft-Versus-Host Disease

[0268] A therapeutic identified as an ITIH4 proteolysis promoter will be administered to a patient with graft-versus-host disease in order to inhibit the undesired immune response. The therapeutic can be activated ITIH4, which comprises of at least one of the ITIH4 cleavage products (see, e.g., FIG. 4). In some examples, parenteral administration will be utilized, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. In other examples, oral administration will be utilized.

[0269] Example 4. Pregnancy

[0270] Improve pregnancy health

[0271] A therapeutic identified as an ITIH4 proteolysis promoter will be administered to a patient with an endangered pregnancy in order to inhibit the undesired immune response through immunosuppression. The therapeutic will be administered either orally or via parenteral administration either until the desired immune response is reached or through the term of the pregnancy.

[0272] Terminate pregnancy

[0273] A therapeutic identified as an ITIH4 proteolysis inhibitor will be administered to a patient to terminate pregnancy via the activation of the immune system to promote rejection. The therapeutic will be administered either orally or via parenteral administration until the desired immune response is observed or until pregnancy is terminated.

[0274] Example 5. Modulators of Inflammatory Responses

[0275] A test for evaluating the complement LP modulating activity of various therapeutics with the appropriate assays will be developed. As a representative example, one therapeutic of interest includes ITIH4 fusion proteins (e.g., Ig and IgM). A complement blood test for the activity of C2 and C4 proteins will be used to determine the immune response compared with a control sample. Other therapeutics that will be analyzed include antibodies to ITIH4, products of ITIH4 proteolysis (e.g., activated ITIH4 as described herein), and ITIH4 activating proteins. The effect of MASP-1 , MASP-2, and kallikrein activators and inhibitors on ITIH4 mediated regulation of the LP activity can also be evaluated.

[0276] Example 6. Identification of a ITIH4 Activity Inhibitor

[0277] Methods

[0278] Using the Sciex X500R QTOF to analyze the plasma metabolic profiles of the samples collected from subjects. MARKERVIEW™ software from Sciex was used for the analysis. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 Activation and Inhibition of ITIH4

[0279] ITIH4 protein was found to be activated when subjected to the plasma of pregnant women compared to plasma from non-pregnant subjects (see FIGs. 5A, 5B, 6A, and 6B). To screen for inhibitors of ITIH4 activity, several members of the Serpin family were screened in plasma samples from pregnant women to compare activation (see FIGs. 7A, 7B, 7C, and 7D). It was found that Serpin A5 and Serpin G1 inhibit the activation of ITIH4 protein in the plasma from pregnant women.

[0280] Example 7. Cancer

[0281] Efficacy of Treatment

[0282] Numerous types of cancer, e.g., the types of cancers described herein, can be characterized as producing immunosuppressive responses. Subjects in need of a treatment for a cancer susceptible to treatment with an immunotherapy will be candidates for treatment with a serpin A5 or serpin G1 therapeutic as described herein. In some instances, subjects will have a sample of their blood plasma utilized to determine the efficacy of different ITIH4 inhibiting therapeutic agents before treatment. These agents include protein therapeutics derived from serpin A5 (SEQ ID No.: 9) or serpin G1 (SEQ ID No.: 10).

[0283] Treatment

[0284] A protein therapeutic agent of the present invention that will provide the desired immunoenhancement response will be selected. In some instances, the therapeutic agent selected is based on the type of cancer and / or the efficacy of treatment screening as described above.

[0285] Alternatively, a subject with a cancer known to be susceptible to treatment with an immunotherapy will be administered a therapeutic agent comprising serpin A5 or serpin G1 . The therapeutic agent may be part of a pharmaceutical composition as described herein.

[0286] Example 8. Pregnancy

[0287] Improve pregnancy health

[0288] A therapeutic identified as an ITIH4 proteolysis promoter, e.g., an agent that inhibits the activity of serpin A5 or serpin G1 , will be administered to a patient with an endangered pregnancy in order to inhibit the undesired immune response through immunosuppression. The therapeutic will be administered either orally or via parenteral administration either until the desired immune response is reached or through the term of the pregnancy.

[0289] Terminate pregnancy

[0290] A therapeutic comprising of serpin A5 or serpin G1 (e.g., as described herein) will be administered to a patient to terminate pregnancy via the inhibition of ITIH4 to promote rejection. The therapeutic will be administered either orally or via parenteral administration until the desired immune response is observed or until pregnancy is terminated. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 Enumerated Embodiments

[0291] E1 . A pharmaceutical composition comprising (i) serpin A5, or a conjugate or fusion protein thereof, in a concentration of from 1 mg / mL to 200 mg / mL; and (ii) a pharmaceutically acceptable excipient, wherein the serpin A5 comprises the amino acid sequence at least 85% identical to SEQ ID No.: 9.

[0292] E2. The pharmaceutical composition of embodiment E1 , wherein the serpin A5 is isolated or purified from human plasma.

[0293] E3. The pharmaceutical composition of embodiment E1 , wherein the serpin A5 is recombinantly produced.

[0294] E4. The pharmaceutical composition of embodiment E3, wherein the serpin A5 is purified recombinant human serpin A5 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 9, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma derived human serpin.

[0295] E5. The pharmaceutical composition of any one of embodiments E1 -E4, wherein the serpin A5 is a fusion protein.

[0296] E6. The pharmaceutical composition of embodiment E5, wherein the serpin A5 fusion protein comprises an antibody.

[0297] E7. The pharmaceutical composition of embodiment E5 or E6, wherein the serpin A5 fusion protein comprises a small molecule.

[0298] E8. The pharmaceutical composition of any one of embodiments E5-E7, wherein the serpin A5 fusion protein comprises a linker.

[0299] E9. The pharmaceutical composition of embodiments E8, wherein the linker comprises of 1 to 100 amino acid(s).

[0300] E10. The pharmaceutical composition of any one of embodiments E5-E9, wherein the serpin A5 fusion protein comprises two serpin A5 proteins.

[0301] E11 . The pharmaceutical composition of any one of embodiments E5-E11 , wherein the serpin A5 fusion protein comprises an IgG or fragment of IgG.

[0302] E12. The pharmaceutical composition of embodiment E11 , wherein the IgG is selected from: lgG1 , lgG2, and lgG3.

[0303] E13. The pharmaceutical composition of embodiment E11 or E12, wherein the IgG is lgG3. PATENT ATTORNEY DOCKET NO.: 51523-005WO3

[0304] E14. The pharmaceutical composition of any one of embodiments E11 -E13, wherein the IgG is human IgG.

[0305] E15. The pharmaceutical composition of any one of embodiments E5-E14, wherein the serpin A5 is fused to the IgG Fc domain.

[0306] E16. The pharmaceutical composition of any one of embodiments E5-E15, wherein the serpin A5 fusion protein comprises an immunoglobulin Fc polypeptide modified to enhance FcRn binding.

[0307] E17. The pharmaceutical composition of any one of embodiments E1 -E16, wherein the serpin A5 is tagged.

[0308] E18. The pharmaceutical composition of any one of embodiments E1 -E17, wherein the pharmaceutical composition is an injectable composition.

[0309] E19. The pharmaceutical composition of any one of embodiments E1 -E18, wherein the pharmaceutical composition is formulated for oral administration.

[0310] E20. A method of treating an immunosuppressive condition by increasing level of complement activity in a subject, said method comprising the steps:

[0311] (i) identifying a subject who has been diagnosed with an immunosuppressive condition; and

[0312] (ii) following step (i), administering to the subject a therapeutic comprising: serpin A5, or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 9, or an expression construct or viral vector enabling production of serpin A5 in the subject.

[0313] E21 . The method of embodiment E20, wherein the therapeutic comprises serpin A5 having an amino acid sequence at least 85% identical to SEQ ID No.: 9.

[0314] E22. The method of embodiment E20 or E21 , wherein the serpin A5 administered is part of a pharmaceutical composition according to any one of embodiments E1 -E19.

[0315] E23. The method of any one of embodiments E20-E22, wherein the serpin A5 administered is isolated or purified from human plasma.

[0316] E24. A method of treating an immunosuppressive condition by increasing level of complement activity in a subject, said method comprising the steps:

[0317] (i) identifying a subject who has been diagnosed with an immunosuppressive condition; and

[0318] (ii) following step (i), administering to the subject a therapeutic comprising: PATENT

[0319] ATTORNEY DOCKET NO.: 51523-005WO3 serpin G1 , or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 10, or an expression construct or viral vector enabling production of serpin G1 in the subject, in an amount effective to treat the immunosuppressive condition.

[0320] E25. The method of embodiment E24, wherein the therapeutic comprises serpin G1 having an amino acid sequence at least 85% identical to SEQ ID No.: 10.

[0321] E26. The method of embodiment E25, wherein the serpin G1 administered is part of a pharmaceutical composition, wherein the serpin G1 concentration is 1 mg / mL to 200 mg / mL.

[0322] E27. The method of any one of embodiments E24-E26, wherein the serpin G1 administered is isolated or purified from human plasma.

[0323] E28. The method of any one of embodiments E20-E27, wherein the serpin A5 or serpin G1 administered is a serpin fusion protein.

[0324] E29. The method of embodiment E28, wherein the serpin fusion protein comprises an antibody.

[0325] E30. The method of embodiment E28 or E29, wherein the serpin fusion protein comprises a small molecule.

[0326] E31 . The method of any one of embodiments E28-E30, wherein the fusion protein comprises a linker.

[0327] E32. The method of embodiment E31 , wherein the linker comprises of 1 to 100 amino acid(s).

[0328] E33. The method of any one of embodiments E28-E32, wherein the serpin fusion protein comprises either two serpin A5 proteins or two serpin G1 proteins.

[0329] E34. The method of any one of embodiments E28-E33, wherein the serpin fusion protein comprises an IgG or fragment of IgG.

[0330] E35. The method of embodiment E34, wherein the IgG is selected from: IgG 1 , lgG2, and lgG3.

[0331] E36. The method of embodiment E34 or E35, wherein the IgG is lgG3.

[0332] E37. The method of any one of embodiments E34-E36, wherein the IgG is human IgG.

[0333] E38. The method of any one of embodiments E34-E36, wherein the fusion protein is fused to the IgG

[0334] Fc domain. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E39. The method of any one of embodiments E20-E38, wherein the serpin further comprises an immunoglobulin Fc polypeptide modified to enhance FcRn binding.

[0335] E40. The method of any one of embodiments E20-E39, wherein the serpin is tagged.

[0336] E41 . The method of any one of embodiments E20-E40, wherein the serpin is administered by parenteral or oral administration.

[0337] E42. The method of any one of embodiments E20-E41 , wherein the disease or condition is a cancer susceptible to treatment with immunotherapy.

[0338] E43. The method of embodiment E42, wherein the cancer is a solid tumor cancer.

[0339] E44. The method of embodiment E42 or E43, wherein the cancer is melanoma, renal cell carcinoma

[0340] (RCC), colorectal cancer, Metastatic colorectal cancer, Gl malignancies, hepatocellular carcinoma (HCC), HNSCC, Malignant Pleural Mesothelioma, bladder cancer, sarcoma, osteosarcoma, Kaposi’s sarcoma, soft tissue sarcoma, lymphoma, mantle cell, follicular, small lymphocytic, B-cell malignancies, anaplastic large cell lymphoma, Merkel cell carcinoma, MSI-high cancer, gastric cancer, gastroesophageal cancer, cervical cancer, PMBCL, endometrial carcinoma, esophagus cancer, cutaneous squamous-cell carcinoma, breast cancer, Metastatic breast cancer, TMB-high cancer, SCLC, basal cell carcinoma, multiple myeloma, testicular cancer, pancreatic cancer, prostate cancer, ovarian cancer, thyroid cancer, leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia, acute promyelocytic leukemia (APL), head and neck cancers, lung cancer, non-small cell lung cancer (NSCLC), melanoma, gastrointestinal stromal tumors (GIST), solid tumors, malignant pleural mesothelioma, myelofibrosis, polycythemia vera, Waldenstrom’s macroglobulinemia, glioblastoma, urothelial carcinoma, or myelodysplastic syndromes (MDS).

[0341] E45. The method of any one of embodiments E20-E44, wherein the serpin A5 or serpin G1 administered activates the immune system.

[0342] E46. The method of any one of embodiments E42-E45, wherein the method comprises administering to the subject a combination therapy comprising: a) the therapeutic of embodiment E20, and b) an additional anticancer therapy, each in an amount that together is effective for the treatment of cancer.

[0343] E47. The method of any one of embodiments E42-E45, wherein the method comprises administering to the subject a combination therapy comprising: a) the therapeutic of embodiment E20, and b) chemotherapy, each in an amount that together is effective for the treatment of cancer.

[0344] E48. The method of embodiment E47, wherein the chemotherapy is a cytotoxic chemotherapy agent selected from: an alkylating agent, a platinum-based compound, a antimetabolite, a antitumor antibiotic, or a plant alkaloid. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E49. The method of embodiment E47 or E48, wherein the cytotoxic chemotherapy agent is selected from a group consisting of: cyclophosphamide (Cytoxan), infosfamide (Ifex), melphalan (Alkeran), chlorambucil (Leukeran), bendamustine (Tranda, Bendeka), cisplatin, carboplatin, oxaliplatin (Eloxatin), methotrexate, 5-fluorouracil (5-FU), capecitabine (Xeloda), gemcitabine (Gemzar), pemetrexed (Alimta), cytarabine (ara-C), and 6-mercaptopurine (6-MP), doxorubicin, daunorubicin, epirubicin, bleomycin, and mitomycin (Mutamycin), vincristine, vinblastine, vinorelbind, paclitaxel (Taxol), docetaxel (Taxotere), cabazitaxel (Jevtana), etopside (VP-16), camptothecins, irinotecan (Camptosar), and topotecan (Hycamtin).

[0345] E50. The method of any one of embodiments E42-E45, wherein the method comprises administering to the subject a combination therapy comprising: a) the therapeutic of embodiment E20, and b) a targeted therapy, each in an amount that together is effective for the treatment of cancer.

[0346] E51 . The method of embodiment E50, wherein the targeted therapy is a tyrosine kinase inhibitor (TKI), a monoclonal antibody (mAb), or an antibody-drug conjugate (ADC).

[0347] E52. The method of embodiment E50 or E51 , wherein the targeted therapy is selected from a group consisting of: imatinib (Gleevec), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif), ponatinib (Iclusig), erlotinib (Tarceva), gefitinib (Iressa), afatinib (Gilotrif), osimertinib (Tagrisso), sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), axitinib (Inlyta), cabozantinib (Cabometyx), dabrafenib (Tafinlar), vemurafenib (Zelboraf), trametinib (Mekinist), cobimetinib (Cotellic), ruxolitinib (Jakafi), ibrutinib (Imbruvica), acalabrutinib (Calquence), idelalisib (Zydelig), duvelisib (Copiktra), rituximab (Rituxan), obinutuzumab (Gazyva), ofatumumab (Arzerra), trastuzumab (Herceptin), pertuzumab (Perjeta), cetuximab (Erbitux), panitumumab (Vectibix), bevacizumab (Avastin), daratumumab (Darzalex), isatuximab (Sarclisa), elotuzumab (Empliciti), inotuzumab ozogamicin (Besponsa), T-DM1 I ado- trastuzumab emtansine (Kadcyla), brentuximab vedotin (Adcetris), sacituzumab govitecan (Trodelvy), and trastuzumab deruxtecan (Enhertu).

[0348] E53. The method of any one of embodiments E42-E45, wherein the method comprises administering to the subject a combination therapy comprising: a) the therapeutic of embodiment E20, and b) an immunotherapy, each in an amount that together is effective for the treatment of cancer.

[0349] E54. The method of embodiment E53, wherein the immunotherapy is an immune checkpoint inhibitor.

[0350] E55. The method of embodiment E53 or E54, wherein the immune checkpoint inhibitor is anti-CTLA-4, anti-PD-1 , or anti-PDpL1 .

[0351] E56. The method of any one of embodiments E53 to E55, wherein the immunotherapy is selected from a group consisting of: ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), durvalumab (Imfinzi), and avelumab (bavencio). PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E57. The method of any one of embodiments E42-E45, wherein the method comprises administering to the subject a combination therapy comprising: a) the therapeutic of embodiment E20, and b) hormonal therapy, each in an amount that together is effective for the treatment of cancer.

[0352] E58. The method of embodiment E57, wherein the hormonal therapy is a selective estrogen receptor modulator (SERM), an aromatase inhibitor, a selective estrogen receptor degrader (SERD), an antiandrogen, a GnRH (LHRH) agonist, or a GnRH (LHRH) antagonist.

[0353] E59. The method of embodiment E57 or E58, wherein the hormonal therapy is selected from a group consisting of: tamoxifen, anastrozole (Arimidex), letrozole (Femara), exemestane (Aromasin), fulvestrant (Faslodex), bicalutamide (Casodex), flutamide, nilutamide, enzalutamide (Xtandi), apalutamide (Erleada), darolutamide (Nubeqa), leuprolide (Lupron), goserelin (Zoladex), degarelix (Firmagon), and Relugolix (Orgovyx).

[0354] E60. The method of any one of embodiments E42-E45, wherein the method comprises administering to the subject a combination therapy comprising: a) the therapeutic of embodiment E20, and b) an additional anticancer therapy is selected from a group consisting of: lenalidomide (Revlimid), thalidomide (Thalomid), pomalidomide (Pomalyst), bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro), venetoclax (Venclexta), olaparib (Lynparza), rucaparib (Rubraca), niraparib (Zejula), talazoparib (Talzenna), everolimus (Afinitor), temsirolimus (Torisel), arsenic trioxide (Trisenox), all-trans retinoic acid (ATRA), trabectedin (Yondelis), L-asparaginase (Elspar, Erwinia asparaginase, Pegaspargase), and selinexor (Xpovio)each in an amount that together is effective for the treatment of cancer.

[0355] E61 . The method of any one of embodiments E46 to E60, wherein the administration of the combination therapy comprises administering the therapeutic of embodiment E20 and the additional anticancer therapy simultaneously.

[0356] E62. The method of embodiment E61 , wherein the therapeutic of embodiment E20 and the additional anticancer therapy are formulated and administered together.

[0357] E63. The method of any one of embodiments E46 to E60, wherein the administration of the combination therapy comprises administering the therapeutic of claim 20 and the additional anticancer therapy sequentially.

[0358] E64. The method of embodiment E63, wherein the sequential administration comprises administering the therapeutic of claim 20 and administering the additional anticancer therapy within one week of each other.

[0359] E65. The method of embodiment E63 or E64, wherein the sequential administration comprises administering the therapeutic of embodiment E20 and administering the additional anticancer therapy within 24 hours of each other. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E66. The method of any one of embodiments E63-E65, wherein the therapeutic of embodiment E20 is administered before the additional anticancer agent.

[0360] E67. The method of any one of embodiment E63-E65, wherein the therapeutic of embodiment E20 is administered after the additional anticancer agent.

[0361] E68. The method of any one of embodiments E20 to E67, wherein the therapeutic agent administered comprises of a viral vector.

[0362] E69. The method of embodiment E68, wherein the viral vector comprises a gene that enables the production of serpin A5 or serpin G1 .

[0363] E70. The method of any one of embodiments E20 to E69, wherein the therapeutic administered to the subject comprises cDNA encoding serpin A5 or serpin G1 .

[0364] E71 . The method of any one of embodiments E20 to E70, wherein the therapeutic administered to the subject comprises mRNA encoding serpin A5 or serpin G1 .

[0365] E72. A method of terminating pregnancy, the method comprises administering an effective amount of ITIH4 inhibitor to the pregnant subject to terminate pregnancy.

[0366] E73. The method of embodiment E72, wherein the ITIH4 inhibitor is a therapeutic comprising (a) serpin A5, or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 9 or (b) serpin G1 , or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 10.

[0367] E74. A method of terminating pregnancy, the method comprises administering an effective amount of ITIH4 inhibitor to the pregnant subject to terminate pregnancy, wherein the ITIH4 inhibitor is serpin A5, or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 9.

[0368] E75. The method of embodiment E74, wherein the therapeutic comprising serpin A5 is part of a pharmaceutical composition according to any one of claims 1 -19.

[0369] E76. A method of terminating pregnancy, the method comprises administering an effective amount of ITIH4 inhibitor to the pregnant subject to terminate pregnancy, wherein the ITIH4 inhibitor is serpin G1 , or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 10.

[0370] E77. The method of embodiment E76, wherein the therapeutic comprising serpin G1 administered is part of a pharmaceutical composition, wherein the serpin G1 concentration is 1 mg / mL to 200 mg / mL. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E78. The method of any one of embodiments E73-E75, wherein the therapeutic comprising serpin A5 administered is isolated or purified from human plasma.

[0371] E79. The method of any one of embodiments E73-E77, wherein the therapeutic comprising serpin G1 administered is isolated or purified from human plasma.

[0372] E81 . The method of any one of embodiments E73, E76, or E77, wherein the therapeutic comprising serpin A5 administered is recombinantly produced.

[0373] E82. The method of any one of embodiments E73, E76, or E77, wherein the therapeutic comprising serpin G1 administered is recombinantly produced.

[0374] E83. The method of embodiment E81 or E82, wherein the therapeutic comprising serpin A5 or serpin G1 comprises purified recombinant human serpin A5 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 9 or serpin G1 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 10, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma derived human serpin.

[0375] E84. The method of any one of embodiments E73-E83, wherein the therapeutic comprising serpin A5 or serpin G1 administered comprises a serpin fusion protein.

[0376] E85. The method of embodiment E84, wherein the serpin fusion protein comprises an antibody.

[0377] E86. The method of embodiment E84 or E85, wherein the serpin fusion protein comprises a small molecule.

[0378] E87. The method of any one of embodiments E84-E86, wherein the serpin fusion protein comprises a linker.

[0379] E88. The method of embodiment E87, wherein the linker comprises of 1 to 100 amino acid(s).

[0380] E89. The method of any one of embodiments E84-E88, wherein the serpin fusion protein comprises two serpin A5 proteins or two serpin G1 proteins.

[0381] E90. The method of any one of embodiments E84-E89, wherein the serpin fusion protein comprises an IgG or fragment of IgG.

[0382] E91 . The method of embodiment E90, wherein the IgG is selected from: IgG 1 , lgG2, and lgG3.

[0383] E92. The method of embodiment E90 or E91 , wherein the IgG is lgG3. PATENT

[0384] ATTORNEY DOCKET NO.: 51523-005WO3

[0385] E93. The method of any one of embodiments E90-E92, wherein the IgG is human IgG.

[0386] E94. The method of any one of embodiments E90-E93, wherein the serpin is fused to the IgG Fc domain.

[0387] E95. The method of any one of embodiments E73-E94, wherein the therapeutic comprising serpin A5 or serpin G1 further comprises an immunoglobulin Fc polypeptide modified to enhance FcRn binding.

[0388] E96. The method of any of embodiments E73-E95, wherein the serpin is tagged.

[0389] E97. The method of any one of embodiments E73-E95, wherein the therapeutic comprising serpin A5 or serpin G1 is administered by parenteral or oral administration.

[0390] E98. A method of treating an immune-driven condition characterized by adverse immunoenhancement by decreasing level of complement activity in a subject, said method comprising the steps of:

[0391] (i) identifying a subject who has been diagnosed with said condition; and

[0392] (ii) following step (i), administering to the subject an effective amount of an ITIH4 proteolysis promoter to treat the immune-driven condition.

[0393] E99. The method of embodiment E98, wherein the condition is graft-versus-host disease.

[0394] E100. The method of embodiment E98, wherein the condition is an autoimmune condition.

[0395] E101 . The method of embodiment E98, wherein the condition is poor pregnancy health.

[0396] E102. The method of any one of embodiments E98-E101 , wherein the ITIH4 proteolysis promoter inhibits activity of serpin A5.

[0397] E101 . The method of any one of embodiments E98-E101 , wherein the ITIH4 proteolysis promoter inhibits activity of serpin G1 .

[0398] E102. A method of treating graft-versus-host disease in a subject, said method comprising the steps of:

[0399] (i) identifying a subject who has been diagnosed with said condition; and

[0400] (ii) following step (i), administering to the subject an effective amount of an ITIH4 proteolysis promoter to treat the immune-driven condition.

[0401] E103. A method of treating an autoimmune condition in a subject, said method comprising the steps of:

[0402] (i) identifying a subject who has been diagnosed with said condition; and

[0403] (ii) following step (i), administering to the subject an effective amount of an ITIH4 proteolysis promoter to treat the immune-driven condition.

[0404] E104. A method of treating poor pregnancy health in a subject, said method comprising the steps of: PATENT ATTORNEY DOCKET NO.: 51523-005WO3

[0405] (i) identifying a subject who has been diagnosed with said condition; and

[0406] (ii) following step (i), administering to the subject an effective amount of an ITIH4 proteolysis promoter to treat the immune-driven condition.

[0407] E105. A method of diagnosing cancer in a subject, said method comprising the steps of:

[0408] (i) determining the ITIH4 activation in the subject; and

[0409] (ii) based on the level of ITIH4 activation, determining if the subject has cancer.

[0410] E106. The method of embodiment E105, wherein the level of ITIH4 activation in the subject is determined by:

[0411] (i) exposing a plasma sample from the subject to recombinant ITIH4 protein;

[0412] (ii) determining ITIH4 proteolysis; and

[0413] (iii) determining the level of ITIH4 activation based on the amount of ITIH4 proteolysis.

[0414] E107. The method of embodiment E106, wherein the recombinant ITIH4 protein is a tagged ITIH4 protein.

[0415] E108. The method of embodiment E107, wherein the tagged ITIH4 protein is tagged with His, Flag, Myc, GST, CBP, glu-glu, HAT, T7 or V5.

[0416] E109. The method of embodiment E108, wherein the recombinant ITIH4 protein is a labeled ITIH4 protein.

[0417] E110. The method of embodiment E109, wherein the labeled ITIH4 protein is fluorescently labeled or biotinylated.

[0418] E111 . The method of embodiment E110, wherein the labeled ITIH4 protein is labeled with HRP, POD, or alkaline phosphate.

[0419] E112. The method of embodiment E106, wherein the recombinant ITIH4 protein is a fusion protein.

[0420] E113. The method of embodiment E112, wherein the fusion protein comprises the PRR domain of the ITIH4 protein.

[0421] E114. The method of any one of embodiments E105-E113, wherein the ITIH4 protein is a polypeptide comprising of gakipkpeas fsprrgwnrq agaagsrmnf (SEQ ID No.: 6), wherein the protein is tagged or labeled.

[0422] E115. The method of any one of embodiments E105-E113, wherein the ITIH4 protein is a polypeptide comprising of glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr (SEQ ID No.: 7), wherein the protein is tagged or labeled. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E116. The method of embodiment E114 or E115, wherein the ITIH4 protein is a polypeptide of fewer than 50 amino acid residues.

[0423] E117. The method of embodiment E114 or E115, wherein the ITIH4 protein is a polypeptide of comprising of gesrnrnvhs gstffkyylq gakipkpeas fsprrgwnrq agaagsrmnf rpgvlssrql glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr vmnmkieett mttqtpapiq (SEQ ID No.: 5), wherein the protein is tagged or labeled.

[0424] E118. The method of embodiment E105, wherein the level of ITIH4 activation in the subject is determined by:

[0425] (i) providing a plasma sample from the subject;

[0426] (ii) determining the levels of metabolites arising from ITIH4 proteolysis in vivo; and

[0427] (iii) on the basis of step (ii), diagnosing the subject.

[0428] E119. The method of embodiment E118, wherein the metabolites are selected from OV40, OV63, OV67, or OV180.

[0429] E120. The method of embodiment E105, wherein the cancer is breast cancer, colon cancer, renal cell cancer, lung cancer, hepatocellular carcinoma, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, bladder cancer, head and neck cancer, glioblastoma, diffuse large B-cell lymphoma, leukemia, or melanoma.

[0430] E121 . A method of diagnosing a subject with a specific type of cancer, said method comprising the steps of:

[0431] (i) determining the ITIH4 activation in the subject;

[0432] (ii) identifying the subject as having cancer based on the level of ITIH4 activation;

[0433] (iii) identifying the presence of ITIH4 PRR fragments associated with a specific type of cancer; and

[0434] (iv) based on step (iii), determining the type of cancer of the subject.

[0435] E122. The method of embodiment E121 , wherein the specific type of cancer is breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, or prostate cancer.

[0436] E123. A method of determining the level of ITIH4 activation in a subject, said method comprising of identifying the ITIH4 PRR fragments in plasma of patients

[0437] E124. The method of embodiment E123, wherein the presence of said PRR region fragments comprising, ssrql glpgppdvpd haayhpf (SEQ ID No.: 8), or gesrnrnvhs gstffkyylq gakipkpeas fsprrgwnrq agaagsrmnf rpgvlssrql glpgppdvpd haayhpfrrl ailpasappa tsnpdpavsr vmnmkieett mttqtpapiq (SEQ ID No.: 5), is indicative of ITIH4 activation.

[0438] E125. A method of treating an immunosuppressive condition by increasing level of complement activity in a subject, said method comprising the steps of: PATENT ATTORNEY DOCKET NO.: 51523-005WO3

[0439] (i) identifying a subject who has been diagnosed with an immunosuppressive condition; and

[0440] (ii) following step (i), administering to the subject an ITIH4 proteolysis inhibitor.

[0441] E126. The method of embodiment E125, wherein the ITIH4 proteolysis inhibitor is one or more proteins.

[0442] E127. The method of embodiment E125, wherein the ITIH4 proteolysis inhibitor comprises of an antibody.

[0443] E128. The method of embodiment E125, wherein the ITIH4 proteolysis inhibitor comprises of an ITIH4 fusion protein.

[0444] E129. The method of embodiment E125, wherein the ITIH4 proteolysis inhibitor comprises of the products of ITIH4 proteolysis.

[0445] E130. A method of embodiment E125, wherein the immunosuppressive condition is a type of cancer, said method comprising the steps of:

[0446] (i) identifying a subject who has been diagnosed with cancer based upon the level of ITIH4 activation; and

[0447] (ii) following step (i), administering to the subject a protease inhibitor.

[0448] E131 . The method of embodiment E130, wherein the subject was diagnosed with a specific type of cancer.

[0449] E132. The method of embodiment E131 , wherein the protease inhibitor administered is selective for a specific cancer type.

[0450] E133. The method of embodiment E132, wherein the cancer is breast cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, or prostate cancer.

[0451] E134. The method of any one of embodiments E130-E133, wherein treating the cancer in the subject comprises administering to the subject an additional anticancer therapy.

[0452] E135. A method of promoting immunosuppression in a subject with an immune-driven condition, the method comprising of administering a modulator of ITIH4 activity.

[0453] E136. The method of embodiment E135, wherein ITIH4 activity is modulated by administering an ITIH4 proteolysis promoter to the subject.

[0454] E137. The method of embodiment E136, wherein the ITIH4 proteolysis promoter is one or more proteins.

[0455] E138. The method of embodiment E136, wherein the ITIH4 proteolysis promoter comprises of an antibody. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E139. The method of embodiment E136, wherein the ITIH4 proteolysis promoter comprises of an ITIH4 fusion protein.

[0456] £140. The method of embodiment E135, wherein ITIH4 activity is modulated by administering activated ITIH4 to the subject.

[0457] E141 . The method of embodiment E O, wherein the activated ITIH4 comprises of at least one cleavage product of ITIH4 proteolysis.

[0458] E 2. The method of embodiment EMO, wherein the activated ITIH4 comprises of: mpknvvfvid ksgsmsgrki qqtrealiki Iddlsprdqf nlivfsteat qwrpslvpas aenvnkarsf aagiqalggt nindamlmav qlldssnqee rlpegsvsli illtdgdptv getnprsiqn nvreavsgry slfclgfgfd vsyaflekla Idngglarri hedsdsalql (SEQ ID No.: 2).

[0459] E 3. The method of embodiment EMO, wherein the activated ITIH4 comprises of: vevt gqyerekagf swievtfknp Ivwvhaspeh vvvtrnrrss aykwketlfs vmpglkmtmd ktgllllsdp dkvtig llfw dgrgeglrl I Irdtdrfssh vggtlgqfyq evlwgspaas ddgrrtlrvq gndhsatrer rldyqegppg veiscwsv (SEQ ID No.: 3).

[0460] EM4. The method of embodiment EMO, wherein the activated ITIH4 comprises of: apsailplpg qsverlcvdp rhrqgpvnll sdpeqgvevt gqyerekagf swievtfknp Ivwvhaspeh vvvtrnrrss aykwketlfs vmpglkmtmd ktgllllsdp dkvtigllfw dgrgeglrll Irdtdrfssh vggtlgqfyq evlwgspaas ddgrrtlrvq gndhsatrer rldyqegppg veiscwsvel (SEQ ID No.: 4).

[0461] EM5. The method of any one of embodiments E135-EM4, wherein the immune-driven condition is inflammation.

[0462] EM6. The method of any one of embodiments E135-EM4, wherein the immune-driven condition is an autoimmune disease.

[0463] EM7. The method of any one of embodiments E135-EM4, wherein the immune-driven condition is the rejection of a tissue, cell, graft, or organ transplant from a donor by a recipient human subject in need thereof.

[0464] EM8. The method of embodiment EM7, wherein the subject was administered an ITIH4 activity modulator prior to, simultaneously with, or after tissue, cell, graft, or organ transplantation.

[0465] EM9. The method of embodiment EM7, wherein the organ transplant is a kidney transplant, a cornea transplant, a bone marrow transplant, a liver transplant, a lung transplant, or a heart transplant.

[0466] E150. The method of embodiment E135, wherein the immune-driven condition is an adverse immune response resulting from administration of a therapeutic agent. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E151 . A method of determining the efficacy of a treatment for an immune-driven condition, said method comprising the steps of:

[0467] (i) determine the ITIH4 activity in a plasma sample from a subject previously identified as having an immune-driven condition;

[0468] (ii) subjecting the plasma sample to a treatment option;

[0469] (iii) determine the change in ITIH4 activity; and

[0470] (iv) based on the change in ITIH4 activity, make a determination about the efficacy of the treatment.

[0471] E152. The method of embodiment E151 , wherein the immune-driven condition is cancer.

[0472] E153. The method of embodiment E152, wherein the treatment option is a protease inhibitor.

[0473] E154. The method of embodiment E151 , wherein the immune-driven condition is poor pregnancy health.

[0474] E155. The method of embodiment E151 , wherein the immune-driven condition is graft-versus-host disease.

[0475] E156. The method of embodiment E154 or E155, wherein the treatment option is an ITIH4 activator.

[0476] E157. The method of embodiment E154 or E155, wherein the treatment option is activated ITIH4.

[0477] E158. A method for assessing pregnancy health of a pregnant subject, the method comprising

[0478] (i) identifying the level of ITIH4 activation of the pregnant subject;

[0479] (ii) based on the level of ITIH4 activation, assessing the pregnancy health of the subject.

[0480] E159. The method of embodiment E158, wherein the level of ITIH4 activation in the subject is determined by:

[0481] (i) providing a plasma sample from the subject;

[0482] (ii) determining the levels of metabolites arising from ITIH4 proteolysis in vivo; and

[0483] (iii) on the basis of step (ii), providing the prognosis to the subject.

[0484] E160. The method of embodiment E159, wherein the metabolites are selected from OV40, OV63, OV67, or OV180.

[0485] E161 . The method of embodiment E160, wherein the presence of said metabolites is indicative of a good prognosis for pregnancy health.

[0486] E162. A method for modulating the pregnancy health in a pregnant subject, the method comprising administering an ITIH4 activity modulator. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 E163. The method of embodiment E162, the method comprising administering an effective amount of ITIH4 inhibitor to the pregnant subject to terminate pregnancy.

[0487] E164. The method of embodiment E162, the method comprising administering an effective amount of ITIH4 promoter to protect pregnancy.

[0488] E165. A kit for obtaining the data needed to diagnose a subject with cancer by the method described in any one of embodiments E105-E120.

[0489] E166. A kit for obtaining the data needed to diagnose a subject with a specific type of cancer by the method described in embodiments E121 or E122.

[0490] E167. A kit for determining the efficacy of a treatment for an immune-driven conditions by the method described in any one of embodiments E151 -E157.

[0491] E168. A kit for obtaining the data needed to determine the prognosis for pregnancy health of a subject by the method described in any one of embodiments E158-E161 .

[0492] Other Embodiments

[0493] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

1. PATENT ATTORNEY DOCKET NO.: 51523-005WO3 What is claimed is:CLAIMS1 . A pharmaceutical composition comprising (i) serpin A5, or a conjugate or fusion protein thereof, in a concentration of from 1 mg / mL to 200 mg / mL; and (ii) a pharmaceutically acceptable excipient, wherein the serpin A5 comprises the amino acid sequence at least 85% identical to SEQ ID No.: 9.

2. The pharmaceutical composition of claim 1 , wherein the serpin A5 is isolated or purified from human plasma.

3. The pharmaceutical composition of claim 1 , wherein the serpin A5 is recombinantly produced.

4. The pharmaceutical composition of claim 3, wherein the serpin A5 is purified recombinant human serpin A5 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 9, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma derived human serpin.

5. The pharmaceutical composition of any one of claims 1 -4, wherein the serpin A5 is a fusion protein.

6. The pharmaceutical composition of claim 5, wherein the serpin A5 fusion protein comprises an antibody.

7. The pharmaceutical composition of claim 5 or 6, wherein the serpin A5 fusion protein comprises a small molecule.

8. The pharmaceutical composition of any one of claims 5-7, wherein the serpin A5 fusion protein comprises a linker.

9. The pharmaceutical composition of claim 8, wherein the linker comprises of 1 to 100 amino acid(s).

10. The pharmaceutical composition of any one of claims 5-9, wherein the serpin A5 fusion protein comprises two serpin A5 proteins.11 . The pharmaceutical composition of any one of claims 5-10, wherein the serpin A5 fusion protein comprises an IgG or fragment of IgG.

12. The pharmaceutical composition of claim 11 , wherein the IgG is selected from: IgG 1 , lgG2, and lgG3.

13. The pharmaceutical composition of claim 11 or 12, wherein the IgG is lgG3.PATENTATTORNEY DOCKET NO.: 51523-005WO314. The pharmaceutical composition of any one of claims 1 1 -13, wherein the IgG is human IgG.

15. The pharmaceutical composition of any one of claims 5-14, wherein the serpin A5 is fused to the IgG Fc domain.

16. The pharmaceutical composition of any one of claims 5-15, wherein the serpin A5 fusion protein comprises an immunoglobulin Fc polypeptide modified to enhance FcRn binding.

17. The pharmaceutical composition of any one of claims 1 -16, wherein the serpin A5 is tagged.

18. The pharmaceutical composition of any one of claims 1 -17, wherein the pharmaceutical composition is an injectable composition.

19. The pharmaceutical composition of any one of claims 1 -18, wherein the pharmaceutical composition is formulated for oral administration.

20. A method of treating an immunosuppressive condition by increasing level of complement activity in a subject, said method comprising the steps:(i) identifying a subject who has been diagnosed with an immunosuppressive condition; and(ii) following step (i), administering to the subject a therapeutic comprising either:(a) serpin A5, or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 9, or an expression construct or viral vector enabling production of serpin A5 in the subject; or(b) serpin G1 , or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 10, or an expression construct or viral vector enabling production of serpin G1 in the subject, in an amount effective to treat the immunosuppressive condition.21 . The method of claim 20, wherein the therapeutic comprises serpin A5 having an amino acid sequence at least 85% identical to SEQ ID No.: 9.

22. The method of claim 20 or 21 , wherein the serpin administered is part of a pharmaceutical composition according to any one of claims 1 -19.

23. The method of claim 20, wherein the therapeutic comprises serpin G1 having an amino acid sequence at least 85% identical to SEQ ID No.: 10.

24. The method of claim 23, wherein the serpin G1 administered is part of a pharmaceutical composition, wherein the serpin G1 concentration is 1 mg / mL to 200 mg / mL.PATENT ATTORNEY DOCKET NO.: 51523-005WO325. The method of any one of claims 20-24, wherein the serpin administered is isolated or purified from human plasma.

26. The method of any one of claims 20-24, wherein the serpin administered is recombinantly produced.

27. The method of claim 26, wherein the serpin A5 or serpin G1 is purified recombinant human serpin A5 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 9 or serpin G1 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 10, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma derived human serpin.

28. The method of any one of claims 20-27, wherein the serpin A5 or serpin G1 administered is a serpin fusion protein.

29. The method of claim 28, wherein the serpin fusion protein comprises an antibody.

30. The method of claim 28 or 29, wherein the serpin fusion protein comprises a small molecule.31 . The method of any one of claims 28-30, wherein the fusion protein comprises a linker.

32. The method of claim 31 , wherein the linker comprises of 1 to 100 amino acid(s).

33. The method of any one of claims 28-32, wherein the serpin fusion protein comprises either two serpin A5 proteins or two serpin G1 proteins.

34. The method of any one of claims 28-33, wherein the serpin fusion protein comprises an IgG or fragment of IgG.

35. The method of claim 34, wherein the IgG is selected from: IgG 1 , lgG2, and lgG3.

36. The method of claim 34 or 35, wherein the IgG is lgG3.

37. The method of any one of claims 34-36, wherein the IgG is human IgG.

38. The method of any one of claims 34-36, wherein the fusion protein is fused to the IgG Fc domain.

39. The method of any one of claims 20-38, wherein the serpin further comprises an immunoglobulinFc polypeptide modified to enhance FcRn binding.

40. The method of any one of claims 20-39, wherein the serpin is tagged.PATENT ATTORNEY DOCKET NO.: 51523-005WO341 . The method of any one of claims 20-40, wherein the serpin is administered by parenteral or oral administration.

42. The method of any one of claims 20-41 , wherein the disease or condition is a cancer susceptible to treatment with immunotherapy.

43. The method of claim 42, wherein the cancer is a solid tumor cancer.

44. The method of claim 42 or 43, wherein the cancer is melanoma, renal cell carcinoma (RCC), colorectal cancer, Metastatic colorectal cancer, Gl malignancies, hepatocellular carcinoma (HCC), HNSCC, Malignant Pleural Mesothelioma, bladder cancer, sarcoma, osteosarcoma, Kaposi’s sarcoma, soft tissue sarcoma, lymphoma, mantle cell, follicular, small lymphocytic, B-cell malignancies, anaplastic large cell lymphoma, Merkel cell carcinoma, MSI-high cancer, gastric cancer, gastroesophageal cancer, cervical cancer, PMBCL, endometrial carcinoma, esophagus cancer, cutaneous squamous-cell carcinoma, breast cancer, Metastatic breast cancer, TMB-high cancer, SCLC, basal cell carcinoma, multiple myeloma, testicular cancer, pancreatic cancer, prostate cancer, ovarian cancer, thyroid cancer, leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia, acute promyelocytic leukemia (APL), head and neck cancers, lung cancer, non-small cell lung cancer (NSCLC), melanoma, gastrointestinal stromal tumors (GIST), solid tumors, malignant pleural mesothelioma, myelofibrosis, polycythemia vera, Waldenstrom’s macroglobulinemia, glioblastoma, urothelial carcinoma, or myelodysplastic syndromes (MDS).

45. The method of any one of claims 20-44, wherein the serpin A5 or serpin G1 administered activates the immune system.

46. The method of any one of claims 42-45, wherein the method comprises administering to the subject a combination therapy comprising: a) the therapeutic of claim 20, and b) an additional anticancer therapy, each in an amount that together is effective for the treatment of cancer.

47. The method of claim 46, wherein the additional anticancer therapy is a chemotherapy.

48. The method of claim 47, wherein the chemotherapy is a cytotoxic chemotherapy agent selected from: an alkylating agent, a platinum-based compound, a antimetabolite, a antitumor antibiotic, or a plant alkaloid.

49. The method of claim 47 or 48, wherein the cytotoxic chemotherapy agent is selected from a group consisting of: cyclophosphamide (Cytoxan), infosfamide (Ifex), melphalan (Alkeran), chlorambucil (Leukeran), bendamustine (Tranda, Bendeka), cisplatin, carboplatin, oxaliplatin (Eloxatin), methotrexate, 5-fluorouracil (5-FU), capecitabine (Xeloda), gemcitabine (Gemzar), pemetrexed (Alimta), cytarabine (ara- C), and 6-mercaptopurine (6-MP), doxorubicin, daunorubicin, epirubicin, bleomycin, and mitomycin (Mutamycin), vincristine, vinblastine, vinorelbind, paclitaxel (Taxol), docetaxel (Taxotere), cabazitaxel (Jevtana), etopside (VP-16), camptothecins, irinotecan (Camptosar), and topotecan (Hycamtin).PATENTATTORNEY DOCKET NO.: 51523-005WO350. The method of claim 46, wherein the additional anticancer therapy is a targeted therapy.51 . The method of claim 50, wherein the targeted therapy is a tyrosine kinase inhibitor (TKI), a monoclonal antibody (mAb), or an antibody-drug conjugate (ADC).

52. The method of claim 50 or 51 , wherein the targeted therapy is selected from a group consisting of: imatinib (Gleevec), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif), ponatinib (Iclusig), erlotinib (Tarceva), gefitinib (Iressa), afatinib (Gilotrif), osimertinib (Tagrisso), sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), axitinib (Inlyta), cabozantinib (Cabometyx), dabrafenib (Tafinlar), vemurafenib (Zelboraf), trametinib (Mekinist), cobimetinib (Cotellic), ruxolitinib (Jakafi), ibrutinib (Imbruvica), acalabrutinib (Calquence), idelalisib (Zydelig), duvelisib (Copiktra), rituximab (Rituxan), obinutuzumab (Gazyva), ofatumumab (Arzerra), trastuzumab (Herceptin), pertuzumab (Perjeta), cetuximab (Erbitux), panitumumab (Vectibix), bevacizumab (Avastin), daratumumab (Darzalex), isatuximab (Sarclisa), elotuzumab (Empliciti), inotuzumab ozogamicin (Besponsa), T-DM1 I ado- trastuzumab emtansine (Kadcyla), brentuximab vedotin (Adcetris), sacituzumab govitecan (Trodelvy), and trastuzumab deruxtecan (Enhertu).

53. The method of claim 46, wherein the additional anticancer therapy is an immunotherapy.

54. The method of claim 53, wherein the immunotherapy is an immune checkpoint inhibitor.

55. The method of claim 53 or 54, wherein the immune checkpoint inhibitor is anti-CTLA-4, anti-PD-1 , or anti-PDpL1 .

56. The method of any one of claims 53 to 55, wherein the immunotherapy is selected from a group consisting of: ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), durvalumab (Imfinzi), and avelumab (bavencio).

57. The method of claim 46, wherein the additional anticancer therapy is a hormonal therapy.

58. The method of claim 57, wherein the hormonal therapy is a selective estrogen receptor modulator (SERM), an aromatase inhibitor, a selective estrogen receptor degrader (SERD), an anti-androgen, a GnRH (LHRH) agonist, or a GnRH (LHRH) antagonist.

59. The method of claim 57 or 58, wherein the hormonal therapy is selected from a group consisting of: tamoxifen, anastrozole (Arimidex), letrozole (Femara), exemestane (Aromasin), fulvestrant (Faslodex), bicalutamide (Casodex), flutamide, nilutamide, enzalutamide (Xtandi), apalutamide (Erleada), darolutamide (Nubeqa), leuprolide (Lupron), goserelin (Zoladex), degarelix (Firmagon), and Relugolix (Orgovyx).PATENT ATTORNEY DOCKET NO.: 51523-005WO360. The method of claim 46, wherein the additional anticancer therapy is selected from a group consisting of: lenalidomide (Revlimid), thalidomide (Thalomid), pomalidomide (Pomalyst), bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro), venetoclax (Venclexta), olaparib (Lynparza), rucaparib (Rubraca), niraparib (Zejula), talazoparib (Talzenna), everolimus (Afinitor), temsirolimus (Torisel), arsenic trioxide (Trisenox), all-trans retinoic acid (ATRA), trabectedin (Yondelis), L- asparaginase (Elspar, Erwinia asparaginase, Pegaspargase), and selinexor (Xpovio).61 . The method of any one of claims 46 to 60, wherein the administration of the combination therapy comprises administering the therapeutic of claim 20 and the additional anticancer therapy simultaneously.

62. The method of claim 61 , wherein the therapeutic of claim 20 and the additional anticancer therapy are formulated and administered together.

63. The method of any one of claims 46 to 60, wherein the administration of the combination therapy comprises administering the therapeutic of claim 20 and the additional anticancer therapy sequentially.

64. The method of claim 63, wherein the sequential administration comprises administering the therapeutic of claim 20 and administering the additional anticancer therapy within one week of each other.

65. The method of claim 63 or 64, wherein the sequential administration comprises administering the therapeutic of claim 20 and administering the additional anticancer therapy within 24 hours of each other.

66. The method of any one of claims 63-65, wherein the therapeutic of claim 20 is administered before the additional anticancer agent.

67. The method of any one of claims 63-65, wherein the therapeutic of claim 20 is administered after the additional anticancer agent.

68. The method of any one of claims 20 to 67, wherein the therapeutic agent administered comprises of a viral vector.

69. The method of claim 68, wherein the viral vector comprises a gene that enables the production of serpin A5 or serpin G1 .

70. The method of any one of claims 20 to 69, wherein the therapeutic administered to the subject comprises cDNA encoding serpin A5 or serpin G1 .71 . The method of any one of claims 20 to 70, wherein the therapeutic administered to the subject comprises mRNA encoding serpin A5 or serpin G1 .

72. A method of terminating pregnancy, the method comprises administering an effective amount of ITIH4 inhibitor to the pregnant subject to terminate pregnancy.PATENTATTORNEY DOCKET NO.: 51523-005WO373. The method of claim 72, wherein the ITIH4 inhibitor is a therapeutic comprising (a) serpin A5, or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 9 or (b) serpin G1 , or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 10.

74. The method of claim 73, wherein the ITIH4 inhibitor is serpin A5, or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 9.

75. The method of claim 74, wherein the therapeutic comprising serpin A5 is part of a pharmaceutical composition according to any one of claims 1 -19.

76. The method of claim 73, wherein the ITIH4 inhibitor is serpin G1 , or a conjugate or fusion protein thereof, having an amino acid sequence at least 85% identical to SEQ ID No.: 10.

77. The method of claim 76, wherein the therapeutic comprising serpin G1 administered is part of a pharmaceutical composition, wherein the serpin G1 concentration is 1 mg / mL to 200 mg / mL.

78. The method of any one of claims 73-77, wherein the therapeutic comprising serpin A5 or serpin G1 administered is isolated or purified from human plasma.

79. The method of any one of claims 73-77, wherein the therapeutic comprising serpin A5 or serpin G1 administered is recombinantly produced.

80. The method of claim 79, wherein the therapeutic comprising serpin A5 or serpin G1 comprises purified recombinant human serpin A5 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 9 or serpin G1 having an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No.: 10, wherein the purified recombinant human serpin has a half-life similar to or longer than plasma derived human serpin.81 . The method of any one of claims 73-77, wherein the therapeutic comprising serpin A5 or serpin G1 administered comprises a serpin fusion protein.

82. The method of claim 81 , wherein the serpin fusion protein comprises an antibody.

83. The method of claim 81 or 82, wherein the serpin fusion protein comprises a small molecule.

84. The method of any one of claims 81 -83, wherein the serpin fusion protein comprises a linker.

85. The method of claim 84, wherein the linker comprises of 1 to 100 amino acid(s).PATENTATTORNEY DOCKET NO.: 51523-005WO386. The method of any one of claims 81 -85, wherein the serpin fusion protein comprises two serpin A5 proteins or two serpin G1 proteins.

87. The method of any one of claims 81 -86, wherein the serpin fusion protein comprises an IgG or fragment of IgG.

88. The method of claim 87, wherein the IgG is selected from: IgG 1 , lgG2, and lgG3.

89. The method of claim 87 or 88, wherein the IgG is lgG3.

90. The method of any one of claims 87-89, wherein the IgG is human IgG.91 . The method of any one of claims 87-90, wherein the serpin is fused to the IgG Fc domain.

92. The method of any one of claims 73-91 , wherein the therapeutic comprising serpin A5 or serpinG1 further comprises an immunoglobulin Fc polypeptide modified to enhance FcRn binding.

93. The method of any of claims 73-92, wherein the serpin is tagged.

94. The method of any one of claims 73-92, wherein the therapeutic comprising serpin A5 or serpin G1 is administered by parenteral or oral administration.

95. A method of treating an immune-driven condition characterized by adverse immunoenhancement by decreasing level of complement activity in a subject, said method comprising the steps of:(i) identifying a subject who has been diagnosed with said condition; and(ii) following step (i), administering to the subject an effective amount of an ITIH4 proteolysis promoter to treat the immune-driven condition.

96. The method of claim 95, wherein the condition is graft-versus-host disease.

97. The method of claim 95, wherein the condition is an autoimmune condition.

98. The method of claim 95, wherein the condition is poor pregnancy health.

99. The method of any one of claims 95-98, wherein the ITIH4 proteolysis promoter inhibits activity of serpin A5.

100. The method of any one of claims 95-98, wherein the ITIH4 proteolysis promoter inhibits activity of serpin G1 .

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