WO2017149391A1

WO2017149391A1 - The use of antithrombin for coating organs during transplantation

Info

Publication number: WO2017149391A1
Application number: PCT/IB2017/000262
Authority: WO
Inventors: Yann Echelard
Original assignee: Laboratoire Francais Du Fractionnement Et Des Biotechnologies
Priority date: 2016-03-02
Filing date: 2017-03-02
Publication date: 2017-09-08

Abstract

The disclosure provides methods and compositions involving the use of antithrombin for preserving organs subject to transplantation.

Description

THE USE OF ANTITHROMBIN

FOR COATING ORGANS DURING TRANSPLANTATION

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/302,500, filed March 2, 2016, which is incorporated by reference herein in its entirety.

FIELD OF INVENTION

The present invention relates to methods and compositions involving the use antithrombin for coating organs, for example organs subjected to transplantation.

BACKGROUND OF INVENTION

Organ preservation is referred to as the "supply line" of organ transplantation.

(Guibert et al. (2011) Transfus. Med. Hemother. 38: 125-142; Southard et al. (1995) Annu. Rev. Med. 46:235-247.) The efficiency and efficacy of organ preservation are critical factors for successful organ transplantation. In the absence of such preservation methods, organs destined for transplantation may sustain extensive and irreparable cellular damage due to prolonged ischemia and subsequent reperfusion. Despite advances in organ preservation and transplantation methods, there remains a widening gap between the number of individuals waiting for an organ transplant and the number of available organs and transplants performed.

SUMMARY OF INVENTION

Aspects of the present disclosure provide methods comprising coating an organ with a biologically effective amount of antithrombin. In some embodiments, the organ is undergoing or will undergo transplantation from a first subject to a second subject. In some embodiments, the organ is selected from the group consisting of: kidney, liver, heart, lung, pancreas, pancreatic islets, and small bowel.

In some embodiments, the antithrombin has a high mannose glycosylation pattern. In some embodiments, the antithrombin has a high fucose glycosylation pattern. In some embodiments, the antithrombin comprises GalNac (N-acetylgalactosamine). In some embodiments, the antithrombin is recombinant antithrombin. In some embodiments, the antithrombin is transgenically produced antithrombin. In some embodiments, the

antithrombin is transgenically produced in a goat. In some embodiments, the antithrombin is ATryn®. In some embodiments, the antithrombin is at a concentration of 500 Ul/kg. In some embodiments, the coating is performed after the organ has been removed from the first subject.

In some embodiments, the antithrombin is in an organ preservation medium. In some embodiments, the coating is performed by immersing the organ in the organ preservation medium. In some embodiments, the organ preservation medium further comprises one or more additional thrombin inhibitors. In some embodiments, the organ preservation medium further comprises one or more components selected from the group consisting of: NaCl, KCl, NaC0₃H, glucose, phosphate, lactobionate, NaOH, KOH, MgS0_4i glutathione, adenosine, allopurinol, raffinose, penafraction (HES), insulin, dexamethasone, penicillin G, MgCl_2i glutamic acid, histidine, mannitol, CaCl_2i potassium hydrogen 2-ketoglutarate, MgCl_2i MgS0_4i histidine HC1, tryptophan, mannitol, PEG 25,000, adenosine, allopurinol, and raffinose.

In some embodiments wherein the organ is preserved during transplantation, the organ is subjected to cooling during preservation. In some embodiments, the organ is preserved for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more than 48 hours. In some embodiments, the organ is preserved using simple static cold storage (SCS). In some embodiments, the organ is preserved using machine perfusion (MP). In some embodiments, the machine perfusion is hypothermic machine perfusion (HMP).

In some embodiments, the second subject is administered antithrombin prior to transplantation of the organ. In some embodiments, the second subject is administered antithrombin after transplantation of the organ. In some embodiments, the antithrombin is administered to the second subject intravenously.

Other aspects of the disclosure provide compositions comprising an organ

preservation medium and a biologically effective amount of antithrombin. In some embodiments, the composition further comprises an organ that is undergoing or will undergo transplantation. In some embodiments, the organ is selected from the group consisting of: kidney, liver, heart, lung, pancreas, pancreatic islets, and small bowel.

In some embodiments, the antithrombin has a high mannose glycosylation pattern. In some embodiments, the antithrombin has a high fucose glycosylation pattern. In some embodiments, the antithrombin comprises GalNac (N-acetylgalactosamine). In some embodiments, the antithrombin is recombinant antithrombin. In some embodiments, the antithrombin is transgenically produced antithrombin. In some embodiments, the antithrombin is transgenically produced in a goat. In some embodiments, the antithrombin is ATryn®.

In some embodiments, the antithrombin is at a concentration of 500 Ul/kg. In some embodiments, the composition further comprises one or more additional thrombin inhibitors.

In some embodiments, the composition comprises one or more components selected from the group consisting of: NaCl, KCl, NaC0₃H, glucose, phosphate, lactobionate, NaOH, KOH, MgS0_4i glutathione, adenosine, allopurinol, raffinose, penafraction (HES), insulin, dexamethasone, penicillin G, MgCl_2, glutamic acid, histidine, mannitol, CaCl_2i potassium hydrogen 2-ketoglutarate, MgCl_2i MgS0_4i histidine HC1, tryptophan, mannitol, PEG 25,000, adenosine, allopurinol, and raffinose.

In some embodiments, the composition comprises: 10 mmol/L NaCl; 108 mmol/L KCl; 10 mmol/L NaC0₃H; 180 mmol/L glucose; 60 mmol/L phosphate; pH 7.3; and 340 mosmol/kg H₂0.

In some embodiments, the composition comprises: 100 mmol/L lactobionate; 25 NaOH mmol/L; 100 mmol/L KOH; 5 mM MgS0₄; 3 mmol/L glutathione; 5 mmol/L adenosine; 1 mmol/L allopurinol; 30 mmol/L raffinose; 50 g/L penafraction (HES); 25 mmol/L phosphate; 40 U/L insulin; 16 mg/L dexamethasone; 200,000 U/L penicillin G; pH 7.4; and 320 mosmol/kg H₂0.

In some embodiments, the composition comprises: 15 mmol/L KCl; 80 mmol/L lactobionate; 13 mmol/L MgCl₂; 100 mmol/L NaOH; 20 mmol/L glutamic acid; 3 mmol/L glutathione; 30 mmol/L histidine; 60 mmol/L mannitol; 0.25 mmol/L CaCl₂; pH 7.3; and 242-369 mosmol/kg H₂0.

In some embodiments, the composition comprises: 15 mmol/L NaCl; 9 mmol/L KCl; 1 mmol/L potassium hydrogen 2-ketoglutarate; 4 mmol/L MgCl₂; 5 mmol/L MgS0₄; 18 mmol/L histidine HC1; 180 mmol/L histidine; 2 mmol/L tryptophan; 30 mmol/L mannitol; 0.015 mmol/L CaCl₂; pH 7.02-7.2; and 310 mosmol/kg H₂0.

In some embodiments, the composition comprises: 100 mmol/L lactobionate; 0.03 mmol/L PEG 25,000; 5 mmol/L MgS0₄; 2 mmol/L glutathione; 5 mmol/L adenosine; 1 mmol/L allopurinol; 30 mmol/L raffinose; 0.03 mmol/L CaCl₂; 25 mmol/L phosphate; pH 7.4; and 320 mosmol/kg H₂0.

In some embodiments, the preservation medium is cooled during organ preservation.

Other aspects of the disclosure provide methods comprising removing an organ from a first subject; coating the organ with a biologically effective amount of antithrombin; and transplanting the organ to a second subject. In some embodiments, the organ is selected from the group consisting of: kidney, liver, heart, lung, pancreas, pancreatic islets, and small bowel.

antithrombin is transgenically produced in a goat. In some embodiments, the antithrombin is ATryn®.

In some embodiments, the antithrombin is at a concentration of 500 Ul/kg. In some embodiments, the coating is performed prior to removing the organ from the first subject. In some embodiments, the coating is performed after removing the organ from the first subject.

In some embodiments, the antithrombin is in an organ preservation medium. In some embodiments, the coating is performed by immersing the organ in the preservation medium. In some embodiments, the organ preservation medium further comprises one or more additional thrombin inhibitors.

In some embodiments, the organ preservation medium further comprises one or more components selected from the group consisting of: NaCl, KCl, NaC0₃H, glucose, phosphate, lactobionate, NaOH, KOH, MgS0_4i glutathione, adenosine, allopurinol, raffinose,

penafraction (HES), insulin, dexamethasone, penicillin G, MgCl_2, glutamic acid, histidine, mannitol, CaCl_2, potassium hydrogen 2-ketoglutarate, MgCl_2, MgS0_4i histidine HC1, tryptophan, mannitol, PEG 25,000, adenosine, allopurinol, and raffinose.

In some embodiments, the method further comprises preserving the organ and subjecting the organ to cooling. In some embodiments, the organ is preserved for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more than 48 hours. In some embodiments, preserving the organ involves simple static cold storage (SCS). In some embodiments, preserving the organ involves machine perfusion (MP). In some embodiments, the machine perfusion is hypothermic machine perfusion.

In some embodiments, the method further comprises administering antithrombin to the second subject prior to transplantation of the organ. In some embodiments, the method further comprises administering antithrombin to the second subject after transplantation of the organ. In some embodiments, the antithrombin is administered to the second subject intravenously. Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. The figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: Figures 1A and IB show the concentration of fibrin in blood samples recovered after

0 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours following the intraportal administration of the islets. Figure 1A presents the concentration of fibrin monomers. Figure IB presents the concentration of fibrin D-dimers (DDI). The samples that received ATryn® concomitantly administered with the pancreatic islets are compared with the samples that did not receive ATryn®.

Figures 2A and 2B show the concentration of fibrin in blood samples recovered after 0 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours following the intraportal administration of the islets. Figure 2A presents the concentration of fibrin monomers. Figure 2B presents the concentration of fibrin D-dimers. The samples in which ATryn® was concomitantly administered with the pancreatic islets are compared with the samples in which ATryn® was administered alone to the recipient prior to and after the islet transplant.

DETAILED DESCRIPTION

The present disclosure provides methods of coating an organ with a biologically effective amount of antithrombin. In some embodiments, the organ is subject to

transplantation from a first subject to a second subject. Also provided are compositions comprising an organ preservation medium and antithrombin. In some embodiments, the antithrombin used in the methods and compositions disclosed herein is recombinant antithrombin. In some embodiments, the antithrombin used in the methods and compositions disclosed herein is ATryn®. In some embodiments, the antithrombin used in the methods and compositions disclosed herein is ACOALAN®. In some embodiments, the antithrombin used in the methods and compositions disclosed herein is plasmatic antithrombin (plasma- derived). In some embodiments, the antithrombin used in the methods and compositions disclosed herein is ACLOTINE®. In some embodiments, the antithrombin used in the methods and compositions disclosed herein is BAXALTA®. In some embodiments, the antithrombin used in the methods and compositions disclosed herein is THROMBATE III®.

Current methods for preserving or preparing organs for transplantation include modulating temperature of the organ and use of various preservation solutions; however, cellular damage, reperfusion injury, and organ rejection remain substantial hurdles in organ transplantation (Guibert et al. (2011) Transfus. Med. and Hemother. 38: 125-142).

It is surprisingly demonstrated herein that an organ preservation medium comprising antithrombin was able to preserve pancreatic islets, in particular by enhancing their viability. Results provided herein reveal that the coating of pancreatic islets with antithrombin allowed for a significant reduction in the concentration of fibrin degradation products, corresponding to a reduction of the level of activation of coagulation. These results indicate that the coating of an organ, such as pancreatic islets, with antithrombin, can improve aspects of organ transplantation, for example by reducing the level of activation of coagulation.

Aspects of the invention are not limited in their application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. Aspects of the invention are capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Organ transplantation

Aspects of the disclosure provide methods of coating an organ that is undergoing or will undergo transplantation. As used herein, the term "organ" refers to a differentiated structure comprising cells and tissues that performs a specific function in an organism. In some embodiments, the organ is a heart, lung, kidney, liver, pancreas, intestine, thymus, bone, eye, stomach, testis, tendons, cornea, skin, heart valves, nerves, blood vessels (veins/arteries), salivary gland, esophagus, gall bladder, ureter, bladder, small bowel, urethra, ovary, trachea, diaphragm, cartilage, ligaments, or eye. In some embodiments, a whole organ is transplanted from a first subject to a second subject. In other embodiments, a portion of an organ (e.g., a tissue or a cluster of cells obtained from an organ) is transplanted from a first subject to a second subject. In some embodiments, the organ is a portion of a liver, a portion of a pancreas, a lobe of a lung, the cornea of an eye, or the valve of a heart. In some embodiments, the organ is pancreatic islets or islets of Langerhans. In some embodiments, the organ is a limb of the body, such as a hand or foot.

In general, organ transplantation involves removal of an organ from a first subject and providing (e.g., implanting) the organ into a second subject. In some embodiments, the first or second subject is a human. In some embodiments, the first or the second subject is a non- human mammal. In some embodiments, the first and second subjects are the same subject (e.g., the organ or tissue is transplanted from one location in a subject's body to another location in the same subject's body). Such transplantations within the same subject are referred to as autografts. In some embodiments, the first and the second subject is a human. In some embodiments, the first and second subjects are different subjects but belong to the same species (e.g., first subject and second subject are humans). Transplantations between subjects that belong to the same species but are not genetically identical are referred to as allografts. Transplantations between subjects that belong to the same species and are genetically identical (e.g. , identical twins) are referred to as isografts.

In some embodiments, the first or second subject is non-human primate (e.g., a baboon, a chimpanzee, etc.) and the first or second subject is a human. For example, in some embodiments, the first subject is a non-human primate (e.g., a baboon, a chimpanzee, etc.) and the second subject is a human. Alternatively, in some embodiments, the first subject is a human and the second subject is a non-human primate (e.g., a baboon, a chimpanzee, etc.). In some embodiments, the first or second subject is a human and the first or second subject is a non-human mammal. For example, in some embodiments, the first subject is a human and the second subject is a non-human mammal. Alternatively, in some embodiments, the first subject is a non-human mammal and the second subject is a human. In some embodiments, the first subject is a pig and the second subject is a human. Transplantations between subjects belonging to different species are referred to as xenografts.

In some embodiments, the first subject is referred to as a "donor" and the second subject is referred to a "recipient." An organ for transplantation may be removed, such as by surgical removal, from a subject. For human subjects, an organ for transplantation may be removed from any willing subject following the consent of the subject or through the consent of a family member, guardian, or personal representative, in cases in which the human subject is not competent, is incapacitated, and/or is unable to make such decisions. In some embodiments, the first subject is deceased. In some embodiments, the first subject has been pronounced brain dead. In some embodiments, the first subject died due to circulatory death. In some embodiments, the first subject is a living donor, meaning the first subject has not died or been incapacitated prior to organ donation. Organs are generally removed from a deceased subject within 24 hours of the last heartbeat of the subject. In some embodiments, the first subject is a non- heart-beating donor. In some embodiments, the organ is removed after cardiac death of the first subject. Depending on the organ, immediate transplantation into another subject may be necessary or the organ may be able to be stored in a preservation medium for a period of time. In some embodiments, the organ is stored in a preservation medium for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours or more than approximately 48 hours. In some embodiments, the organ is stored in a preservation medium for approximately 60, 72, 84, 96 hours or more than approximately 96 hours. In some embodiments, the organ is stored in a preservation medium for approximately 120, 144, 168 hours or more than approximately 168 hours. In other embodiments, the organ is stored in a preservation medium for less than one hour.

Selection of an appropriate first subject from which the organ or tissue is obtained will be evident to one of ordinary skill in the art and may include considerations such as species, health, age, sex, and blood serotype. In some embodiments, the first subject and the second subject undergo blood serotyping prior to organ transplantation. In some

embodiments, the first subject and the second subject have the same blood serotype. In some embodiments, the first subject and the second subject have substantially similar blood serotypes such that the risk of an adverse transplant rejection is deemed low. Organ transplantation between subjects with compatible blood serotypes, including the same blood serotype or substantially similar blood serotypes, is referred to as ABO compatible transplantation. In some embodiments, the first subject and the second subject do not have compatible serotypes. Transplantation between subjects that are not serologically compatible is referred to as ABO incompatible transplantation. Antithrombin

In one aspect, the disclosure provides methods of preparing an organ that is undergoing or will undergo transplantation, for example, coating an organ that is undergoing or will undergo transplantation with a biologically effective amount of antithrombin. In some embodiments, the antithrombin is plasmatic antithrombin (plasma-derived

antithrombin). In some embodiments, the antithrombin has a high mannose glycosylation pattern. In some embodiments, the antithrombin has a high fucose glycosylation pattern. In some embodiments, the antithrombin comprises GalNac (N-acetylgalactosamine). In some embodiments, the antithrombin is recombinant antithrombin. In some embodiments, the antithrombin is transgenically produced antithrombin. In some embodiments, the

antithrombin is transgenically produced in a goat. In some embodiments, the antithrombin is ATryn®. ATryn® was approved by the U.S. Food and Drug Administration in 2009 for the prevention of peri-operative and peri-partum thromboembolic events in hereditary antithrombin deficient patients. (ATryn, Antithrombin (Recombinant) US Package Insert at p. 1.)

Traditionally, the term "antithrombin" relates to a family of closely related proteins that includes antithrombin I, antithrombin II, antithrombin III and antithrombin IV.

However, antithrombin III is the only member of the antithrombin family that has been associated with a significant physiological function, and the current literature often uses the terms antithrombin and antithrombin III interchangeably. Antithrombin, as used herein, refers to antithrombin III, unless specified differently. However, it should be appreciated that members of the antithrombin family other than antithrombin III that have an activity similar to antithrombin III also can be used in the methods disclosed herein.

Generally, antithrombin is a protein of 432 amino acids with a molecular weight of about 58 kDA. However, some non-human antithrombins are 433 amino acids in length.

Antithrombin is a serine protease inhibitor that inhibits thrombin and Factor Xa and is naturally found in the serum of mammals including humans. The physiological level of antithrombin in human serum from a healthy individual is about 14-20 mg/dL. Antithrombin is a glycoprotein that includes four glycosylation sites: Asn96, Asnl35, Asnl55 and Asnl92. Antithrombin occurs both in an alpha form (alpha-antithrombin) and in a beta form (beta- antithrombin), with the alpha form being the most prevalent. The beta form of antithrombin can be distinguished from the alpha form because the beta form is not glycosylated at Asnl35. In some embodiments, antithrombin, as used in the methods disclosed herein, includes both the major alpha form of antithrombin and the minor beta form of antithrombin. In some embodiments, antithrombin, as used in the methods disclosed herein, is alpha- antithrombin. In other embodiments, the antithrombin is beta-antithrombin.

Antithrombin is conserved between mammalian species with only minor differences in amino acid sequence. In some embodiments, the species of antithrombin used in the treatment of a subject according to the methods disclosed herein is the same species as the subject. Thus, for instance, human antithrombin (according to its amino acid sequence) is used in methods of treatment in humans. In other embodiments, the species of antithrombin used in the treatment of a subject is from a different species as the subject.

It should further be appreciated that in addition to the amino acid sequence, the glycosylation of antithrombin can also be species specific. Thus, for instance, human antithrombin isolated from human plasma (plasma-derived human antithrombin), has a different glycosylation pattern than goat antithrombin isolated from goat plasma. However, as explained below, human antithrombin, for example, may be produced in a goat, providing human antithrombin (antithrombin with a human amino acid sequence) with a glycosylation pattern that mimics the glycosylation of goat antithrombin.

Aspects of the invention relate to the use of plasma-derived antithrombin or plasmatic antithrombin, for example, in coating an organ according to the methods described herein. The plasma-derived antithrombin used in the methods described herein can be produced by any of a variety of methods known in the art.

In some embodiments, plasma-derived antithrombin used in the methods and compositions described herein is ACLOTINE®. In some embodiments, plasma-derived antithrombin is ANTITHROMBIN III IMMUNO®. ANTITHROMBIN III IMMUNO® is indicated for prophylaxis and treatment of thrombotic and thromboembolic disorders in patients with hereditary antithrombin III deficiency. In some embodiments, plasma-derived antithrombin is THROMBATE III®. THROMBATE III® is an antithrombin concentrate purified from human plasma, approved by the FDA for the treatment of patients with hereditary antithrombin III (AT) deficiency in connection with surgical or obstetrical procedures or thromboembolism.

In some embodiments, the antithrombin used in the methods disclosed herein has a high mannose glycosylation pattern. Antithrombin with a high mannose glycosylation pattern, as used herein, refers to an antithrombin in which one or more of the glycosylation side chains comprise an oligomannose or a hybrid type oligosaccharide (in contrast to side chains comprising bi-antennary complex oligosaccharides, which are the predominant side chain structure found in plasma-derived human antithrombin). In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% of the glycosylation side chains comprise an oligomannose or hybrid type oligosaccharide. In some embodiments, the antithrombin used in the methods disclosed herein has a high fucose glycosylation pattern. A high fucose glycosylation pattern, as used herein, refers to antithrombin that has fucose on its proximal GlcNac on a majority of the glycosylation sites that have complex oligosaccharides. In some embodiments, the recombinant antithrombin used in the methods disclosed herein comprises GalNac (N- acetylgalactosamine). In some embodiments, the recombinant antithrombin used in the methods disclosed herein has a high mannose pattern, a high fucose pattern and includes GalNac. In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% of the glycosylation sites that have complex oligosaccharides comprise fucose. Human antithrombin (antithrombin with a human amino acid sequence) that is transgenically produced in goats generally has a high mannose pattern, a high fucose pattern and includes GalNac, while plasma-derived human antithrombin generally does not have these glycosylation patterns (See e.g., U.S. Patent 5,843,705, U.S. Patent 6,441, 145, U.S. Patent 7,019, 193, and U.S. Patent 7,928,064, incorporated by reference herein).

Aspects of the invention relate to the use of recombinant antithrombin , for example, in coating an organ according to the methods described herein. The recombinant

antithrombin used in the methods disclosed herein can be produced through a variety of methods. In some embodiments, the recombinant antithrombin used in the methods disclosed herein is transgenically produced. In some embodiments, the transgenically produced antithrombin is produced in a non-human mammal. In some embodiments, the transgenically produced antithrombin is produced in a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama. In some embodiments, the transgenically produced antithrombin is produced in an ungulate. In some embodiments, the transgenically produced antithrombin is produced in a goat. It should be appreciated that the antithrombin produced in a first species can be an antithrombin from a second species. Thus, for instance, human

antithrombin can be transgenically produced in mice and goats. Similarly, bovine antithrombin can also be transgenically produced in mice and goats. In addition,

antithrombin can also be transgenically produced in the species of origin. Thus, goat antithrombin can be transgenically produced in goats.

In some embodiments, the recombinant antithrombin used in the methods disclosed herein is transgenically produced. In some embodiments, the transgenically produced antithrombin has a glycosylation pattern that is different from plasma-derived antithrombin. In general, the glycosylation pattern of the antithrombin depends on the species of animal the antithrombin is produced in. Thus, for instance, antithrombin transgenically produced in mice is expected to have a different glycosylation pattern than antithrombin produced in goats.

In some embodiments, the recombinant antithrombin used in the methods disclosed herein has the glycosylation pattern of antithrombin transgenically produced in goats. In some embodiments, human antithrombin is produced in a goat and has the glycosylation pattern of antithrombin produced in goats.

It should be appreciated that the glycosylation pattern of transgenically produced antithrombin can also depend on the nature of the organ, or body part, of the transgenic animal in which the protein is produced. Thus, the glycosylation pattern of antithrombin produced in the mammary gland is expected to be different from antithrombin produced in the blood, even if produced in the same species. In some embodiments, the recombinant antithrombin used in the methods disclosed herein is produced in the mammary gland of a non-human mammal. In some embodiments, the recombinant antithrombin used in the methods disclosed herein is produced in the mammary gland of goats.

It should further be appreciated that antithrombin with the glycosylation pattern of antithrombin produced in goats can also be provided by producing or obtaining the antithrombin, for example, in a species other than the goat, and modifying the glycosylation pattern, such as in downstream processing. For instance, glycosylated antithrombin may be produced in mice and the glycosylation pattern of the mice-produced antithrombin may be altered to generate the glycosylation pattern of goat antithrombin by in vitro modification. For instance, the mouse-produced antithrombin may be altered through the action of glycosylases or transferases. In addition, the glycosylation pattern may be modified by nonenzymatic (e.g., synthetic) methods. Antithrombin with the glycosylation pattern of goat- produced antithrombin may also be provided by producing antithrombin in cells (e.g., insect cells, bacterial cells) and adding or modifying the glycosylation pattern in downstream processing. Alternatively, recombinant antithrombin with the glycosylation pattern of goat- produced antithrombin may be provided by isolation from plasma from a non-goat species (or a different tissue of a goat) and the glycosylation pattern may subsequently be modified in downstream processing.

In some embodiments, the recombinant antithrombin used in the methods disclosed herein is ATryn®. ATryn® is a transgenic human alpha- antithrombin that is produced in the goat mammary gland (See e.g., US Patent 5,843,705, US Patent 6,441,145, US Patent 7,019,193, and US Patent 7,928,064, which are incorporated by reference herein). ATryn® is approved by the FDA for the prevention of peri-operative and peri-partum thromboembolic events in hereditary antithrombin deficient patients. In Europe, ATryn® is approved for use in surgical patients with congenital antithrombin deficiency for the prophylaxis of deep vein thrombosis and thromboembolism in clinical risk situations.

The glycosylation pattern of the goat-produced (human) antithrombin ATryn® differs from the glycosylation pattern of plasma-derived human antithrombin. Because the glycosylation pattern is different, ATryn® has some physiological properties that are different from plasma-derived human antithrombin. For instance, the clearance rate of ATryn® is higher than the clearance rate of plasma-isolated antithrombin (See e.g., US Patent 7,019,193, incorporated by reference herein).

In some embodiments, the recombinant antithrombin used in the methods and compositions disclosed herein is produced in a hamster ovary-derived CHO cell. In some embodiments, the CHO cell has been modified such that the cell has reduced or absent activity of an enzyme relating to synthesis of an intracellular carbohydrate nucleotide, GDP- fucose. In some embodiments, the CHO cell has been modified such that the cell has reduced or absent activity of an enzyme relating to the modification of a carbohydrate chain in which 1 -position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through a-bond in a complex type N-glycoside-linked carbohydrate chain. See, e.g., US 7,691,810, incorporated by reference herein. In some embodiments, the recombinant antithrombin is ACOALAN®. ACOALAN® is a preparation containing a recombinant antithrombin produced in FUT 8 Knock Out CHO cells. It was developed by the Japanese company Kyowa Hakko Kirin (KW-3357) and has been approved by the Japanese Ministry of Health, Labor and Welfare for thrombophilia due to congenital antithrombin deficiency (CAD) and disseminated intravascular coagulation (DIC) accompanied by a decrease in antithrombin.

Subject

In one aspect, the disclosure provides methods of preparing an organ that is undergoing or will undergo transplantation, e.g., coating an organ that is undergoing or will undergo transplantation from a first subject to a second subject with a biologically effective amount of antithrombin. A "subject", as used herein, is a human or other vertebrate mammal including, but not limited to, mouse, rat, dog, cat, horse, cow, pig, sheep, goat, or non-human primate. In some embodiments, the first or second subject is a human. In some embodiments, the first and the second subject is a human. In some embodiments, the first subject is a pig and the second subject is a human. In some embodiments, the first subject is non-human primate (e.g., a baboon, a chimpanzee, etc.) and the second subject is a human. In some embodiments, the first subject is a human and the second subject is a non-human mammal. Transplantations between subjects belonging to different species are referred to as xenografts.

In some embodiments, the second subject is in need of an organ transplant. In some embodiments, the second subject has a condition that necessitates an organ transplantation. Non-limiting examples of conditions that may result in a need for an organ transplant include cardiomyopathy, heart failure, myocarditis, heart disease, a birth defect in an organ, a hereditary defect in an organ, organ failure or dysfunction, a chronic infection, organ or tissue damage, diabetes, pancreas failure, cancer, cystic fibrosis, pulmonary hypertension, emphysema, pulmonary edema, high blood pressure, cystic kidney disease, twisted or blocked intestines, short-gut syndrome, trauma (such as burn trauma), degenerative joint disease, and arthritis.

Biologically effective amount

In one aspect, the disclosure provides methods of preparing an organ that is undergoing or will undergo transplantation, e.g., coating an organ that is undergoing or will undergo transplantation with a biologically effective amount of antithrombin. As used herein, a "biologically effective amount" refers to an amount of antithrombin that improves or enhances at least one aspect of organ transplantation, such as by enhancing the viability of the organ, reducing organ damage, reducing reperfusion injury, reducing the level of activation of coagulation in the organ and/or in the organ recipient during or after

transplantation, reducing rejection of the organ by the second subject and/or reducing mortality of the second subject. In some embodiments, the biologically effective amount of antithrombin increases the time an organ may be maintained outside of a body (i.e., ischemic time). In some embodiments, the organ is coated with antithrombin in a biologically effective amount to reduce blood clotting. In some embodiments, the organ is coated with antithrombin in a biologically effective amount to reduce inflammation.

When blood supply is severed during removal of the organ from a first subject, cells of the organ begin anaerobic respiration, quickly utilizing the cellular glucose stores and accumulating toxic metabolites. In general, the amount of organ damage correlates with the time an organ spends without blood supply (i.e., ischemic time). In some embodiments, the biologically effective amount of antithrombin enhances the viability of the organ to be transplanted. For example, in some embodiments, an organ that is coated with a biologically effective amount of antithrombin is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% more viable than an organ that was not coated in antithrombin. Alternatively or in addition, the biologically effective amount of antithrombin may reduce the effects of ischemia, such as organ damage. In some embodiments, an organ that is coated with a biologically effective amount of antithrombin has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% less cell or organ damage than an organ that was not coated in antithrombin. The viability and/or damage of an organ may be assessed by any routine method in the art, such as assessing cell membrane integrity, toxic metabolite concentrations, potassium and sodium ion concentrations, and cell viability assays.

In some embodiments, the biologically effective amount of antithrombin may reduce the amount of reperfusion injury experienced by an organ. In some embodiments, an organ that is coated with a biologically effective amount of antithrombin experiences least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% less reperfusion injury than an organ that was not coated in

antithrombin.

Coating an organ with a biologically effective amount of antithrombin following removal from a first subject may increase the amount of time an organ can remain outside of a body (i.e., ischemic time). In some embodiments, the ischemic time is increased by at least 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or at least 10.0 hours as compared to the ischemic time of an organ that was not coated with antithrombin. In some embodiments, the ischemic time is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95%, as compared to the ischemic time of an organ that was not coated with antithrombin.

In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to reduce blood clotting in an organ (e.g., prior to, during, or after transplantation) by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to blood clotting in an organ prior to or in the absence of coating with antithrombin. In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to reduce the amount of one or more blood clotting factor(s)(e.g., in the organ or in a blood sample collected from a recipient, during or after transplantation) by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the amount of the blood clotting factor(s) in the organ prior to or in the absence of coating with antithrombin, or to the amount of the blood clotting factor(s) in a blood sample collected, during or after transplantation, from a subject receiving an organ that is not coated with antithrombin.

In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to maintain one or more blood clotting factors (e.g., as measured in a blood sample collected from a recipient during or after transplantation) at a level that corresponds to an increase of no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10% or lower, from the corresponding level measured in the recipient prior to transplantation.

In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to reduce inflammation, e.g., after transplantation in the organ and/or in the subject. In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to reduce the quantity of pro -inflammatory or inflammatory factors, for example pro-inflammatory or inflammatory cytokines (e.g. , interleukin 1 (IL- 1), interleukin 6 (IL-6), TNFa). In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to reduce the quantity of IL- 1 and/or IL-6 and/or TNFa, e.g., after transplantation by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as compared to the quantity of IL- 1 and/or IL-6 and/or TNFa in an organ prior to or in the absence of coating with antithrombin.

In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to reduce apoptosis of cells of the organ, e.g., prior to, during, and/or after transplantation. In some embodiments, the biologically effective amount of antithrombin is an amount sufficient to reduce apoptosis of the organ, e.g., prior to, during, and/or after transplantation by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as compared to apoptosis of an organ prior to or in the absence of coating with antithrombin. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as the size of the organ (e.g., weight, surface area), the age of the organ, the age of the first and/or second subject, or the severity of the disease or disorder of the second subject, an effective method can be selected which does not cause substantial toxicity and yet is effective to achieve the desired outcome (e.g, enhance organ viability, reduce organ damage, etc.). One of ordinary skill in the art can empirically determine the effective amount of antithrombin without necessitating undue experimentation. In some embodiments, it is preferred that a maximum amount be used, that is, the highest safe dose according to some medical judgment.

The amount of antithrombin (e.g., ATryn®) to be used is generally expressed in mg/kg or in international units (UI). International units are referred to herein interchangeably as "UI" or "units." In some embodiments, the amount of antithrombin is expressed as units of antithrombin per kg of organ. For example, in some embodiments, antithrombin is applied or administered at a dose of 1 unit per kg or more, 2 units per kg or more, 5 units per kg day or more, 10 units per kg or more, 20 units per kg or more, 30 units per kg or more, 40 units per kg or more, 50 units per kg or more, 100 units per kg or more, 150 units per kg or more, 200 units per kg or more, 250 units per kg or more, 300 units per kg or more, 350 units per kg or more, 400 units per kg or more, 450 units per kg or more, 500 units per kg or more, 600 units per kg or more, 700 units per kg or more, 800 units per kg or more, 900 units per kg or more, or 1000 units per kg or more. In some embodiments, the antithrombin is administered at a dose of 6 units per kg. In some embodiments, the antithrombin is administered at a dose of 50 units per kg. In some embodiments, the antithrombin is administered at a dose of 80 units per kg. In some embodiments, the antithrombin is administered at a dose of 100 units per kg. In some embodiments, the antithrombin is administered at a dose of 295 units per kg. In some embodiments, the antithrombin is administered at a dose of 500 units per kg.

In some embodiments where an organ is coated by immersing the organ in an organ preservation medium comprising antithrombin, the concentration of antithrombin in the organ preservation medium may be 0.5 UI/mL, 1 UI/mL, 2 UI mL, 3 UI/mL, 4 UI/mL, 5 UI/mL, 6 UI/mL, 7 UI/mL, 8 UI/mL, 9 UI mL, 10 UI/mL, 15 UI/mL, 20 UI/mL, or more.

The amount of recombinant antithrombin (e.g., ATryn®) to be used in the methods and compositions disclosed herein can vary depending on the desired biological goal. The amount of antithrombin necessary to achieve a particular biological goal may be a different amount of antithrombin that is necessary to achieve a different biological goal. One or more doses of antithrombin can also be administered to a subject receiving an organ transplant. In some embodiments, the antithrombin is administered to the subject intravenously. The one or more doses of antithrombin can be administered to the subject prior to, during, or after transplantation. In some embodiments, the one or more doses of antithrombin is administered to the subject prior to transplantation. In some embodiments, the one or more doses of antithrombin is administered to the subject after transplantation.

In some embodiments, the amount of antithrombin is expressed as units of antithrombin per kg of the subject, e.g., the subject receiving an organ transplant. For example, in some embodiments, antithrombin is administered at a dose of 1 unit per kg or more, 2 units per kg or more, 5 units per kg day or more, 10 units per kg or more, 20 units per kg or more, 30 units per kg or more, 40 units per kg or more, 50 units per kg or more, 100 units per kg or more, 150 units per kg or more, 200 units per kg or more, 250 units per kg or more, 300 units per kg or more, 350 units per kg or more, 400 units per kg or more, 450 units per kg or more, 500 units per kg or more, 600 units per kg or more, 700 units per kg or more, 800 units per kg or more, 900 units per kg or more, or 1000 units per kg or more. In some embodiments, the antithrombin is administered at a dose of 6 units per kg. In some embodiments, the antithrombin is administered at a dose of 50 units per kg. In some embodiments, the antithrombin is administered at a dose of 80 units per kg. In some embodiments, the antithrombin is administered at a dose of 100 units per kg. In some embodiments, the antithrombin is administered at a dose of 295 units per kg. In some embodiments, the antithrombin is administered at a dose of 500 units per kg.

In some embodiments, the organ preservation medium also contains one or more additional thrombin inhibitors. In some embodiments, the biologically effective amount of antithrombin is less when administered in the presence of or more additional thrombin inhibitors than the biologically effective amount of antithrombin when administered in the absence of one or more additional thrombin inhibitors.

Organ Preservation During Transplantation

Organ preservation methods during transplantation generally involve static preservation methods or dynamic preservation methods. Static organ preservation methods, such as static cold storage (SCS) relies on hypothermic conditions to reduce the metabolic rate of the cells of the organ. SCS may be performed using a cold organ preservation medium after perfusion, such as cold perfusion, of the organ (e.g., with an organ preservation medium). Alternatively, dynamic organ preservation methods, such as machine perfusion (MP), involve continuously circulating an organ preservation medium through the organ to provide nutrients and remove toxic metabolites. MP may be performed under hypothermic conditions or normothermic conditions. (Guibert et al. (2011) Transfus. Med. Hemother. 38: 125-142; Yuan et al. (2010) Transplant. Int. 23: 561-570).

Aspects of the disclosure relate to methods of coating an organ that is undergoing or will undergo transplantation with a biologically effective amount of antithrombin. As used herein, the term "coat" or "coating" an organ with antithrombin refers to contacting the organ with a composition comprising antithrombin such that a substantial portion of the surface of the organ is contacted at least temporarily with the composition comprising antithrombin. The organ may be partially or completely coated with antithrombin. In some embodiments, both the external surfaces of the organ and the internal surfaces {e.g., blood vessels, lumen of the organ) of the organ are coated with antithrombin. In some embodiments, the external surfaces of the organ are coated with antithrombin and the internal surfaces are not coated with antithrombin.

In some embodiments, the antithrombin is in an organ preservation medium. In some embodiments, the organ preservation medium comprising the antithrombin is used to coat the organ. In some embodiments, the organ preservation medium also contains one or more additional thrombin inhibitors. The organ may be coated with the antithrombin using any method known in the art. In some embodiments, the surfaces (external and/or internal) are brushed, sprayed, or rinsed with antithrombin or an organ preservation medium containing antithrombin. In some embodiments, the organ may be removed from the first subject and immersed or submerged (partially or completely) in antithrombin or an organ preservation medium containing antithrombin. In some embodiments, the organ may be subjected to perfusion, such as retrograde perfusion, with a medium comprising antithrombin. In some embodiments, the organ is subjected to perfusion with a medium comprising antithrombin prior to removal of the organ from the first subject.

In some embodiments, the organ is preserved in an organ preservation medium containing antithrombin until transplantation into a second subject. In some embodiments, the organ is preserved in an organ preservation medium containing antithrombin for at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours or more than approximately 48 hours. In some embodiments, the organ is preserved in an organ preservation medium containing antithrombin for at least approximately 60, 72, 84, 96 hours or more than approximately 96 hours. In some embodiments, the organ is preserved in an organ preservation medium containing antithrombin for at least approximately 120, 144, 168 hours or more than approximately 168 hours. In other embodiments, the organ is stored in a preservation medium for less than one hour.

Decreasing the temperature of the organ may reduce the metabolic rate of the cells of the organ and enhance viability and/or reduce organ damage. In some embodiments, the temperature of the organ subject to transplantation is reduced following removal from the first subject. In some embodiments, the temperature of the organ subject to transplantation is reduced by exposing the organ to a cold or cool medium, such as an organ preservation medium. In some embodiments, the temperature of the organ is reduced to about 30 °C, 29 °C, 28 °C, 27 °C, 26 °C, 25 °C, 24 °C, 23 °C, 22 °C °C 21 °C, 20°C, 19 °C, 18 °C, 17 °C, 16 °C, 15 °C, 14 °C, 13 °C, 12 °C, 11 °C, 10 °C, 9 °C, 8 °C, 7 °C, 6 °C, 5 °C, or about 4 °C. In some embodiments, the organ preservation medium containing the antithrombin is at about 30 °C, 29 °C, 28 °C, 27 °C, 26 °C, 25 °C, 24 °C, 23 °C, 22 °C °C 21 °C, 20°C, 19 °C, 18 °C, 17 °C, 16 °C, 15 °C, 14 °C, 13 °C, 12 °C, 11 °C, 10 °C, 9 °C, 8 °C, 7 °C, 6 °C, 5 °C, or about 4 °C. In some embodiments, the organ is maintained at body temperature (e.g., approximately 37°C). In some embodiments, the coating of the organ is performed under normothermic conditions.

In some embodiments, coating of an organ with antithrombin is performed prior to removal of the organ from the first subject. In other embodiments, coating of an organ with antithrombin is performed after the organ has been removed from the first subject.

Without being limited to a particular theory, it is hypothesized that antithrombin improves at least one aspect of organ transplantation by its anti-coagulation, antiinflammatory and/or anti-apoptotic properties exerted to the organ itself by means of interactions with the cells at the external and internal surfaces of the organ and/or interactions with the coagulation, inflammation or apoptosis factors.

Some anti-inflammatory properties of AT are mediated by its anticoagulation actions, such as thrombin inhibition. Thrombin activates platelets and endothelial cells, which contribute to local inflammation. Antithrombin neutralizes coagulation enzymes including thrombin, plasmin, and Factors IXa, Xa, XIa, and Xlla and, in particular, forms an enzyme- inhibitor complex with thrombin that can rapidly be removed from the circulation in the organ undergoing or that will undergo transplantation. Antithrombin also prevents Factor Xa-induced production of IL-6. Diminishing activation of coagulation and local

inflammation in the organ can enhance the integrity of the organ and the health of the recipient. In addition, antithrombin appears to possess potent anti-inflammatory properties that are independent of its anticoagulation activity, for example properties mediated by direct interaction of antithrombin with leucocytes and lymphocytes. Antithrombin could reduce inflammation in the organ and reduce organ damage, through prostacyclin-mediated inhibition of leucocyte-endothelial cell interactions thus preventing leukocyte activation in the organ or in the recipient.

Organ preservation medium

Also within the scope of the present disclosure are organ preservation media, such as organ preservation media containing antithrombin. The terms "organ preservation medium" and "organ preservation solution" are used interchangeably herein and refer to any medium that may be used to store an organ subject to transplantation. In some embodiments, the organ is removed from a first subject and immediately placed into the organ preservation medium. In some embodiments, the organ may be placed into a solution prior to being placed into the organ preservation medium. In some embodiments, the organ may be washed or perfused with a solution prior to being placed into the organ preservation medium.

Organ preservation media are formulated to maintain the viability of an organ subject to transplantation. In some embodiments, the organ preservation media are formulated to enhance the viability of an organ subject to transplantation. In some embodiments, the organ preservation media are formulated to reduce and/or prevent the effects of ischemia. In general, organ preservation media contain a buffer or buffering agent to maintain the pH of the media, large molecules to maintain the osmotic potential, and electrolytes. In some embodiments, the organ preservation media contains a buffer comprising lOg/L glycine, 7.9 g/L NaCl, 2.5 gL sodium citrate, at pH 7. In some embodiments, the electrolytes of the organ preservation medium are similar to those of intracellular fluid. In some embodiments, the organ preservation medium contains a relatively high concentration of potassium and a relatively low concentration of sodium. In some embodiments, the organ preservation medium contains a relatively low concentration of potassium and a relatively high concentration of sodium.

The organ preservation medium may be formulated to maintain or enhance viability of the organ, reduce organ damage, reduce or prevent reperfusion injury, reduce or prevent rejection of the organ by the second subject (recipient), maintain or enhance the health of the recipient, and/or increase the time an organ may be maintained outside of a body {i.e., ischemic time). In some embodiments, the organ preservation medium is formulated to allow cells to regenerate adenosine triphosphate (ATP) and/or maintain cell membrane integrity. Examples of organ preservation media include, without limitation, Euro-Collins, University of Wisconsin solution (Viaspan®, CoStorSol®), University of Wisconsin Modified,

Celsior®, Custodiol®, IGL-1®, Ross-Marshall citrate solution, hyperosmolar citrate solution, Bretschneider histidine tryptophan ketoglutarate solution, phosphate buffered sucrose solution, Kyoto ET solution, Perfadex®. Examples of organ preservation media also include, without limitation, vitamin rich CMRL medium, for example CMRL-1969 or CMRL-1066, optionally supplemented with Human Serum Albumin. Components of several non-limiting examples of organ preservation media are provided in Tables 1 and 2. It should be appreciated that any organ preservation medium known to one of ordinary skill in the art may be compatible with aspects of the invention. It should also be appreciated that one of ordinary skill in the art could modify or optimize any of the known organ preservation media without undue experimentation. Table 1: Components of several non-limiting examples of organ preservation media (Guibert et al. (2011) Transfus. Med. Hemother. 38: 125-142)

mosmol/kg

Custodiol 15 mmol/L NaCl, 9 mmol/L KC1, 1 mmol/L potassium hydrogen 2-ketoglutarate, 4 mmol/L MgC12, 5 mmol/L MgS04, 18 mmol/L histidine HC1, 180 mmol/L histidine, 2 mmol/L tryptophan, 30 mmol/L mannitol, 0.015 mmol/L CaC12, pH 7.02-7.2, 310 mosmol/kg H20

IGL-1 100 mmol/L lactobionate, 0.03 mmol/L PEG

25,000, 5 mmol/L MgS04, 2 mmol/L glutathione, 5 mmol/L adenosine, 1 mmol/L allopurinol, 30 mmol/L raffinose, 0.03 mmol/L CaC12, 25 mmol/L phosphate, pH 7.4, 320 mosmol/kg H20

Table 2: Components of CMRL-1066

NADP 1.00

UTP 1.00

L-Alanine 25.00

L-Arginine x HCl 70.00

L-Aspartic acid 30.00

L-Cysteine free base 199.66

L-Cystine 20.00

L-Glutamine 100.00

L- Glutamic acid 75.00

Glycine 50.00

L-Histidine x HCl x H20 20.00

L-Hydroxyproline 10.00

L-Isoleucine 20.00

L-Leucine 60.00

L-Lysine x HCl 70.00

L-Methionine 15.00

L-Phenylalanine 25.00

L-Proline 40.00

L-Serine 25.00

L-Threonine 30.00

L-Tryptophan 10.00

L-Tyrosine 40.00

L- Valine 25.00 p-Aminobenzioc acid 0.05

L-Ascorbin acid 50.00

D(+)-Biotine 0.01

D-Calcium pantothenate 0.01

Choline chloride 0.50

Folic acid 0.01 myo-Inositol 0.05

Nicotinic acid 0.025

Nicotinamide 0.025 Pyridoxal x HC1 0.025

Pyridoxide x HC1 0.025

Riboflavin 0.01

Thiamine x HC1 0.01

- Deoxy adenosine 10.00

- Deoxycytidine x HC1 11.60

- Deoxyguanosine 10.00

- Deoxy thymidine 10.00

In some embodiments, the organ preservation medium comprises one or more additional thrombin inhibitors. In some embodiments, the organ preservation medium comprises 1, 2, 3, 4, 5 or more additional thrombin inhibitors. Non-limiting examples of thrombin inhibitors include, hirudin, bivalirudin, lepirudin, desirudin, argatroban, melagatran, dabigatran, TLCK hydrochloride, PPACK dihydrochloride, p-APMSF hydrochloride, benzamidine hydrochloride, ximelagatran, methanesulfonyl fluoride, rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, otamixaban, fondaparinux, idraparinux, enoxaparin, dalteparin, warfarin, coumarin, acenocoumarol, coumatetralyl, dicoumarol, ethyl

biscoumacetate, phenprocoumon, 1,3-indandione, clorindione, diphenadione, phenindione, bemiparin sodium, certoparin, nadroparin, parnaparin, reviparin, tinzaparin, danaparoid sodium, and sulodexide.

Kits

In one aspect, the disclosure provides kits comprising antithrombin (e.g., ATryn®).

In some embodiments, the antithrombin and/or other components are in sterile container(s). In some embodiments, the kit comprises a pharmaceutical carrier and instructions for administration of the kit components. In some embodiments, the kit further comprises an organ preservation solution and/or another thrombin inhibitor. In some embodiments, the kit includes a pharmaceutical preparation vial, a pharmaceutical preparation diluent vial, and the antithrombin. The diluent vial may contain a diluent such as physiological saline for diluting what could be a concentrated solution or lyophilized powder of a composition of the disclosure. In some embodiments, the instructions include instructions for mixing a particular amount of the diluent with a particular amount of a concentrated pharmaceutical composition, whereby a final formulation such as for injection or infusion is prepared. In some embodiments, the instructions include instructions for use in a syringe or other administration device. In some embodiments, the instructions include instructions for treating a patient with an effective amount of a composition of the disclosure. It also will be understood that the containers contained within a kit, with or without containing the preparations, whether the container is a bottle, a vial with a septum, an ampoule with a septum, an infusion bag, and the like, may contain indicia such as conventional markings which change color when the preparation has been autoclaved or otherwise sterilized.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

Examples

Example 1 : Organ preservation medium containing antithrombin

An organ preservation medium is prepared using the CMRL-1066 medium (Table 2) supplemented with 2% Human Serum Albumin (HAS), a buffer (10 g/L glycine, 7.9 g/L NaCl, 2.5 g/L sodium citrate, at pH 7) and 5 UI mL of recombinant antithrombin {e.g., ATryn®).

Once pancreatic islets were recovered from a donor pig, they were preserved in the organ preservation medium containing antithrombin for 24 hours.

After 24 hours, the viability of the pancreatic islets was measured. A Trypan Blue

Solution was used to assess cell viability using the dye exclusion test. The dye exclusion test is based upon the concept that viable cells do not take up impermeable dyes (such as Trypan Blue), but dead cells are permeable and take up the dye. The percentage of islets recovered was evaluated by counting the cells. The results showed that the organ preservation medium was able to preserve the pancreatic islets, in particular by enhancing their viability.

Example 2: Pig model of pancreatic islet transplantation

The porcine model of islet graft used in the experiments is a model of acute coagulation activation followed by inflammatory conditions compatible with pathological conditions observed in organ transplantation in humans. Intraportal administration and the subsequent systemic diffusion of the pancreatic islets instantly induces disseminated intravascular coagulation (DIC). This model is thus an appropriate model to assess the antithrombin properties {e.g., anti-coagulant, anti-inflammatory or anti-apoptotic properties) that would be representative of its properties in organ transplantation in humans, in particular when DIC occurs.

After removal of the pancreas from donor pigs, the pancreas was digested, and the pancreatic islets were recovered following the Ricordi semi-automatic method (Ricordi et al, (1988) "Automated Method for Isolation of Human Pancreatic Islets," Diabetes 37:4 413- 420). The pancreatic islets could be preserved 24 hours in an organ preservation medium, such as the organ preservation medium described in Example 1.

Each recipient group received an intraportal administration of pancreatic islets of 0.15 mL/kg by continuous infusion for 25-30 minutes, with an administration rate of 2 mL/minute.

For each animal, a blood sample was recovered in standard sterile tubes at 0 minutes,

30 minutes, 1 hour, 2 hours, 4 hours and 24 hours following initiation of the intraportal administration (T=0 minutes). The blood samples were centrifuged (4000g, 15 minutes) and the plasma aliquots were stored at -80°C before analysis.

The concentration of coagulation, inflammatory and/or apoptotic markers was then measured in each of the samples. For example, the concentration of fibrin degradation products (FDP) in the blood samples was measured, since the FDP are markers of a coagulation condition due to their presence in the blood after a blood clot is degraded by fibrinolysis. The concentration of fibrin D-dimers (DDI, contains two crosslinked D fragments of the fibrin protein) and fibrin monomers was measured at 0 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours after the intraportal administration of the islets using a standard ELISA Kit for D-dimers or sFMC (Soluble Fibrin Monomer Complex). Animals were sacrificed at 24 hours by intravenous injection of potassium chloride (15%).

Example 3: Coating of pancreatic islets with antithrombin Two groups of 7 pigs were subject to an intraportal administration of pancreatic islets according to the following protocol:

Group (1) Control: pancreatic islets (0.15ml/kg) + NaCl 0.9 %

Group (2) Pancreatic islets (0.15ml/kg) + 500 Ul/kg of ATryn®

Group (3) Positive control: Pancreatic islets (0.15ml/kg) + 25 Ul/kg of Heparin

(standard anticoagulant used in islet graft for diabetic patients)

At 0 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours after the initiating the intraportal administration of the pancreatic islets, a blood sample was recovered from each animal and the concentration of fibrin D-dimers and fibrin monomers was measured.

Results:

The concentrations of fibrin monomers and fibrin D-dimers are presented in Figures 1A and IB. The results show that there was a greater reduction in fibrin degradation products in the blood samples when the pancreatic islets were coated by antithrombin prior to and during administration to the recipient pig, as compared to when pancreatic islets were administered alone. The activity of antithrombin was at least equal to the activity of Heparin. Example 4: Coating of pancreatic islets with antithrombin prior to administration compared with antithrombin injection of the recipient after transplantation

Two groups of 7 pigs were subject to an intraportal administration of pancreatic islets according to the following protocol:

Group (1): Continuous infusion of the preparation containing pancreatic islets over 25-30 minutes combined with 500 Ul/kg of ATryn®, as described in Example 3 (Group 2 of Example 3, listed above);

Group (2): Continuous infusion of the preparation containing pancreatic islets over 25-30 minutes. Group (2) received 500 Ul kg ATryn® as follows: 50% of the ATryn® was injected 30 minutes prior to administration of the islets, and the remaining 50% was injected 30 minutes after administration of the islets, in a 2 hours timeframe.

At 0 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours after the initiation of intraportal administration of the pancreatic islets, a blood sample was recovered from each animal and the concentration of fibrin D-dimers and fibrin monomers was measured. Results:

The concentrations of fibrin monomers and fibrin D-dimers are presented in Figures 2A and 2B. The results showed that the coating of pancreatic islets with antithrombin allowed for a significant reduction in the concentration of fibrin degradation products, corresponding to a reduction of the level of activation of coagulation, in comparison with an administration of antithrombin to the second subject (the recipient) prior to and after the islet transplant.

In conclusion, these results showed that the coating of an organ, such as pancreatic islets, with antithrombin, can improve aspects of organ transplantation, for example by reducing the level of activation of coagulation in the organ, and in the recipient after transplantation.

Equivalents

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the examples provided, since the examples are intended as an illustration of certain aspects and embodiments of the invention. Other functionally equivalent embodiments are within the scope of the invention.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents, disclosed herein are incorporated by reference in their entirety.

Claims

CLAIMS What is claimed is:

1. A method comprising,

coating an organ with a biologically effective amount of antithrombin.

2. The method of claim 1, wherein the organ is undergoing or will undergo

transplantation from a first subject to a second subject.

3. The method of claim 1 or 2, wherein the organ is selected from the group consisting of: kidney, liver, heart, lung, pancreas, pancreatic islets, and small bowel.

4. The method of any one of claims 1-3, wherein the antithrombin has a high mannose glycosylation pattern.

5. The method of any one of claims 1-4, wherein the antithrombin has a high fucose glycosylation pattern.

6. The method of any one of claims 1-5, wherein the antithrombin comprises GalNac (N-acetylgalactosamine).

7. The method of any one of claims 1-6, wherein the antithrombin is recombinant antithrombin.

8. The method of any one of claims 1-7, wherein the antithrombin is transgenically produced antithrombin.

9. The method of claim 8, wherein the antithrombin is transgenically produced in a goat.

10. The method of any one of claims 1-9, wherein the antithrombin is ATryn®.

11. The method of any one of claims 1-10, wherein the antithrombin is at a concentration of 500 Ul/kg.

12. The method of any one of claims 2-11, wherein the coating is performed after the organ has been removed from the first subject.

13. The method of any one of claims 1-12, wherein the antithrombin is in an organ preservation medium.

14. The method of claim 13, wherein the coating is performed by immersing the organ in the organ preservation medium.

15. The method of claim 13 or 14, wherein the organ preservation medium further comprises one or more additional thrombin inhibitors.

16. The method of any one of claims 13-15, wherein the organ preservation medium further comprises one or more components selected from the group consisting of: NaCl, KCl, NaC0₃H, glucose, phosphate, lactobionate, NaOH, KOH, MgS0_4i glutathione, adenosine, allopurinol, raffinose, penafraction (HES), insulin, dexamethasone, penicillin G, MgCl_2, glutamic acid, histidine, mannitol, CaCl_2i potassium hydrogen 2-ketoglutarate, MgCl_2i MgS0₄ histidine HC1, tryptophan, mannitol, PEG 25,000, adenosine, allopurinol, and raffinose.

17. The method of any one of claims 2-16, wherein the organ is preserved during transplantation and wherein the organ is subjected to cooling during preservation.

18. The method of claiml7, wherein the organ is preserved for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more than 48 hours.

19. The method of claim 17 or 18, wherein the organ is preserved using simple static cold storage (SCS).

20. The method of claim 17 or 18, wherein the organ is preserved using machine perfusion (MP).

21. The method of claim 20, wherein the machine perfusion is hypothermic machine perfusion (HMP).

22. The method of any one of claims 2-21, wherein the second subject is administered antithrombin prior to transplantation of the organ.

23. The method of any one of claims 2-21, wherein the second subject is administered antithrombin after transplantation of the organ.

24. The method of claim 22 or 23, wherein the antithrombin is administered to the second subject intravenously.

25. A composition comprising

an organ preservation medium and a biologically effective amount of antithrombin.

26. The composition of claim 25, wherein the composition further comprises an organ that is undergoing or will undergo transplantation.

27. The composition of claim 26, wherein the organ is selected from the group consisting of: kidney, liver, heart, lung, pancreas, pancreatic islets, and small bowel.

28. The composition of any one of claims 25-27, wherein the antithrombin has a high mannose glycosylation pattern.

29. The composition of any one of claims 25-28, wherein the antithrombin has a high fucose glycosylation pattern.

30. The composition of any one of claims 25-29, wherein the antithrombin comprises GalNac (N-acetylgalactosamine).

31. The composition of any one of claims 25-30, wherein the antithrombin is recombinant antithrombin.

32. The composition of any one of claims 25-31, wherein the antithrombin is transgenically produced antithrombin.

33. The composition of claim 32, wherein the antithrombin is transgenically produced in a goat.

34. The composition of any one of claims 25-33, wherein the antithrombin is ATryn®.

35. The composition of any one of claims 25-34, wherein the antithrombin is at a concentration of 500 LWkg.

36. The composition of any one of claims 25-35, further comprising one or more additional thrombin inhibitors.

37. The composition of any one of claims 25-36, wherein the composition comprises one or more components selected from the group consisting of: NaCl, KCl, NaC0₃H, glucose, phosphate, lactobionate, NaOH, KOH, MgS0_4i glutathione, adenosine, allopurinol, raffinose, penafraction (HES), insulin, dexamethasone, penicillin G, MgCl_2, glutamic acid, histidine, mannitol, CaCl_2, potassium hydrogen 2-ketoglutarate, MgCl_2, MgS0_4i histidine HC1, tryptophan, mannitol, PEG 25,000, adenosine, allopurinol, and raffinose.

38. The composition of claim 37, wherein the composition comprises: 10 mmol/L NaCl; 108 mmol/L KCl; 10 mmol/L NaC0 H; 180 mmol/L glucose; 60 mmol/L phosphate; pH 7.3; and 340 mosmol/kg H₂0.

39. The composition of claim 37, wherein the composition comprises: 100 mmol/L lactobionate; 25 NaOH mmol/L; 100 mmol/L KOH; 5 mM MgS0₄; 3 mmol/L glutathione; 5 mmol/L adenosine; 1 mmol/L allopurinol; 30 mmol/L raffinose; 50 g/L penafraction (HES); 25 mmol/L phosphate; 40 U/L insulin; 16 mg/L dexamethasone; 200,000 U/L penicillin G; pH 7.4; and 320 mosmol/kg H₂0.

40. The composition of claim 37, wherein the composition comprises: 15 mmol/L KCl; 80 mmol/L lactobionate; 13 mmol/L MgCl₂; 100 mmol/L NaOH; 20 mmol/L glutamic acid; 3 mmol/L glutathione; 30 mmol/L histidine; 60 mmol/L mannitol; 0.25 mmol/L CaCl₂; pH 7.3; and 242-369 mosmol/kg H₂0.

41. The composition of claim 37, wherein the composition comprises: 15 mmol/L NaCl; 9 mmol/L KC1; 1 mmol/L potassium hydrogen 2-ketoglutarate; 4 mmol/L MgCl₂; 5 mmol/L

MgS0₄; 18 mmol/L histidine HC1; 180 mmol/L histidine; 2 mmol/L tryptophan; 30 mmol/L mannitol; 0.015 mmol/L CaCl₂; pH 7.02-7.2; and 310 mosmol/kg H₂0.

42. The composition of claim 37, wherein the composition comprises: 100 mmol/L lactobionate; 0.03 mmol/L PEG 25,000; 5 mmol/L MgS0₄; 2 mmol/L glutathione; 5 mmol/L adenosine; 1 mmol/L allopurinol; 30 mmol/L raffinose; 0.03 mmol/L CaCl₂; 25 mmol/L phosphate; pH 7.4; and 320 mosmol/kg H₂0.

43. The composition of any one of claims 25-42, wherein the preservation medium is cooled during organ preservation.

44. A method comprising,

removing an organ from a first subject;

coating the organ with a biologically effective amount of antithrombin; and transplanting the organ to a second subject.

45. The method of claim 44, wherein the organ is selected from the group consisting of: kidney, liver, heart, lung, pancreas, pancreatic islets, and small bowel.

46. The method of claim 44 or 45, wherein the antithrombin has a high mannose glycosylation pattern.

47. The method of any one of claims 44-46, wherein the antithrombin has a high fucose glycosylation pattern.

48. The method of any one of claims 44-47, wherein the antithrombin comprises GalNac (N- acetylgalacto samine) .

49. The method of any one of claims 44-48, wherein the antithrombin is recombinant antithrombin.

50. The method of any one of claims 44-49, wherein the antithrombin is transgenically produced antithrombin.

51. The method of claim 50, wherein the antithrombin is transgenically produced in a goat.

52. The method of any one of claims 44-51, wherein the antithrombin is ATryn®.

53. The method of any one of claims 44-52, wherein the antithrombin is at a

concentration of 500 Ul/kg.

54. The method of any one of claims 44-53, wherein the coating is performed prior to removing the organ from the first subject.

55. The method of any one of claims 44-53, wherein the coating is performed after removing the organ from the first subject.

56. The method of any one of claims 44-53, wherein the antithrombin is in an organ preservation medium.

57. The method of claim 56, wherein the coating is performed by immersing the organ in the preservation medium.

58. The method of claim 56 or 57, wherein the organ preservation medium further comprises one or more additional thrombin inhibitors.

59. The method of any one of claims 56-58, wherein the organ preservation medium further comprises one or more components selected from the group consisting of: NaCl, KCl, NaC0₃H, glucose, phosphate, lactobionate, NaOH, KOH, MgS0_4i glutathione, adenosine, allopurinol, raffinose, penafraction (HES), insulin, dexamethasone, penicillin G, MgCl_2i glutamic acid, histidine, mannitol, CaCl_2i potassium hydrogen 2-ketoglutarate, MgCl_2i MgS0_4i histidine HCl, tryptophan, mannitol, PEG 25,000, adenosine, allopurinol, and raffinose.

60. The method of any one of claims 44-59, further comprising preserving the organ and subjecting the organ to cooling.

61. The method of claim 60, wherein the organ is preserved for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more than 48 hours.

62. The method of claim 60 or 61, wherein preserving the organ involves simple static cold storage (SCS).

63. The method of any one of claims 60-62, wherein preserving the organ involves machine perfusion (MP).

64. The method of claim 63, wherein the machine perfusion is hypothermic machine perfusion.

65. The method of any one of claims 44-64, further comprising administering

antithrombin to the second subject prior to transplantation of the organ.

66. The method of any one of claims 44-64, further comprising administering

antithrombin to the second subject after transplantation of the organ.

67. The method of claim 65 or 66, wherein the antithrombin is administered to the second subject intravenously.