ALPHA- 1 ANTI-TRYPSIN

RELATED APPLICATION DATA

The present application claims priority from Australian Patent Application No. 2017900154 entitled "Method of Preventing an Immune Response" filed on 19 January 2017. The entire contents of which is hereby incorporated by reference.

SEQUENCE INFORMATION

The present application is filed with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD

The present application relates to a method for preventing an immune response against a therapeutic protein.

BACKGROUND

A large and increasing number of therapeutic proteins have been approved for clinical use. One of the primary limitations of therapeutic treatment using biological proteins is the immune response which the body generates in response to the presence of foreign substances in the body. This immune response is especially problematic when foreign substances must be repeatedly administered in order to be optimally effective. Virtually all therapeutic proteins elicit some level of immune response, which in some cases can lead to potentially serious side effects.

One example of such a situation is the repeated administration of agents for the treatment of bleeding disorders, such as Factor VIII deficiency diseases (e.g., classic haemophilia A and von Willebrand's disease) or Factor IX deficiency, also known as haemophilia B. Between 10 and 25 percent of patients with haemophilia develop an immune response to Factor VIII. These patients develop inhibitors, usually IgG antibodies, which neutralize Factor VIII activity and, thus, prevent effective therapy.

A number of strategies have been used to reduce immunogenicity of protein therapies. Such strategies include use of human proteins, deimmunization or humanization and use of human cells or cells that produce human-like proteins (e.g., glycoproteins). In the case of antibodies technologies such as phage display from human libraries, or the use of mice carrying human immunoglobulin gene repertoires are also used. Although useful, these procedures cannot guarantee that patients do not still react against unique features of the therapeutic protein.

Additional strategies to reduce the immunogenicity of protein therapeutics have been employed and include the prediction of epitopes that bind to MHC II, T cell receptors or B cell receptors, followed by their removal or alteration; PEGylation and sialylation. Each of these approaches has been shown to reduce immunogenicity of proteins however the resultant loss of functional activity has been a concern.

It will be clear to the skilled person from the foregoing, that there is a need in the art for preventing an immune response against therapeutic proteins.

SUMMARY

The present disclosure is based on the inventors identification of a method of preventing an immune response against a therapeutic protein in a subject.

In producing the present disclosure, the inventors studied the effects of the protease inhibitor, alpha- 1 anti-trypsin, in an accepted mouse model of haemophilia A. The inventors studied the effects of this inhibitor on the immune response against a therapeutic protein by administering alpha- 1 anti-trypsin in addition to the therapeutic protein. The inventors were able to prevent development of an immune response against the therapeutic protein, e.g., production of alloantibodies and inhibitors. The inventors also showed that administering the alpha- 1 anti-trypsin prior to, and throughout the course of administration of the therapeutic protein further reduced the immune response against the therapeutic protein. The inventors have demonstrated this method is applicable to preventing an immune response against a blood clotting factor, e.g., Factor VIII.

The findings by the inventors provide the basis for methods for preventing an immune response against a therapeutic protein in a subject by administering alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein. The present disclosure is broadly directed to a method for preventing an immune response against a therapeutic protein, comprising administering a protease inhibitor and the therapeutic protein to a subject.

In one example, the protease inhibitor is a serine protease inhibitor (also known as a serpin). For example, the serine protease inhibitor is a mammalian, plant, fungal, bacterial or viral serine protease inhibitor.

In one example, the serine protease inhibitor is a mammalian serine protease inhibitor. For example, the mammalian serine protease inhibitor is selected from the group consisting of serpinA, serpinB, serpinC, serpinD, serpinE, serpinF, serpinG, serpinH, serpinl, serpinJ, serpinK, serpinL, serpinM, serpinN, serpinO and serpinP. In one example, the mammalian serine protease inhibitor is a serpinA, for example, alpha- 1 anti-trypsin. In one example, the serine protease inhibitor is alpha- 1 anti-trypsin or functional variant thereof.

In one example, the serine protease inhibitor is a plant serine protease inhibitor. For example, the plant serine protease inhibitor is barley serpin Zx (BSZx).

In one example, the serine protease inhibitor is a fungal serine protease inhibitor. For example, the fungal serine protease inhibitor is celpin (e.g., derived from Piromyces spp. strain E2).

In one example, the serine protease inhibitor is a bacterial serine protease inhibitor.

In one example, the serine protease inhibitor is a viral serine protease inhibitor. For example, the viral serine protease inhibitor is selected from the group consisting of Serp-1, crma and Serp-2.

In one example, the disclosure provides a method for preventing an immune response against a therapeutic protein, comprising administering alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein to a subject. For example, administration of alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein delays an immune response against a therapeutic protein. For example, administration of alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein reduces the incidence or likelihood of an immune response against the therapeutic protein. In one example, the alpha- 1 anti-trypsin or functional variant thereof is plasma derived or recombinant. For example, the alpha- 1 anti-trypsin or functional variant thereof is alpha- 1 anti-trypsin protein. In one example, the alpha- 1 anti-trypsin is mammalian alpha- 1 anti-trypsin, e.g., human alpha- 1 anti-trypsin.

In one example, the alpha- 1 anti-trypsin or functional variant thereof is

PEGylated. For example, the alpha- 1 anti-trypsin or functional variant thereof is PEGylated recombinant alpha- 1 -anti-trypsin.

In one example, the alpha- 1 anti-trypsin or functional variant thereof is administered by administering a nucleic acid encoding alpha- 1 anti-trypsin or functional variant thereof. For example, the alpha- 1 anti-trypsin or functional variant thereof is administered by administering a recombinant adeno-associated virus comprising a nucleic acid encoding alpha- 1 antitrypsin or functional variant thereof.

In one example, the alpha- 1 anti-trypsin or functional variant thereof is administered by administering a cell expressing and/or secreting alpha- 1 antitrypsin or functional variant thereof.

In one example, the subject is suspected of having, predisposed to, or at risk of developing an immune response against a therapeutic protein.

In one example, the disclosure provides a method of prophylactically treating an immune response against a therapeutic protein in a subject comprising selecting a subject suspected of having, predisposed to, or at risk of developing an immune response against a therapeutic protein and administering alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein to the subject.

For example, a subject at risk of developing an immune response against a therapeutic protein includes, but is not limited, to those with a mutation, deletion or rearrangement in a gene, e.g., a gene encoding the therapeutic protein, e.g., a Factor VIII gene, or has a genetic polymorphism associated with increased cytokine production. In one example, the subject has a relative that has developed an immune response against a therapeutic protein.

In one example, the disclosure provides a method for preventing an inhibitory immune response against a therapeutic protein. For example, administration of the alpha- 1 anti-trypsin or functional variant thereof prevents, or reduces, or delays production of inhibitory antibodies, e.g., IgG antibody production. For example, the antibody titre is less than about 5.0 Bethesda Units (BU), or less than about 1.0 BU, or less than about 0.6 BU.

Methods for measuring the production of inhibitory antibodies will be apparent to the skilled person and include, for example, a Bethesda clotting assay.

In one example, the subject is in need of treatment with the therapeutic protein. In one example, the subject has not previously received treatment with the therapeutic protein. In one example, the subject has not previously had an immune response against the therapeutic protein or another therapeutic protein. In one example, the subject has previously had an immune response against another therapeutic protein.

In one example, the subject has not previously received treatment with alpha- 1 anti-trypsin or functional variant thereof. In one example, the subject is not in need of treatment with alpha- 1 anti-trypsin or functional variant thereof. In another example, the subject is in need of treatment with alpha- 1 anti-trypsin or functional variant thereof.

In one example, the method of the disclosure comprises administering to the subject the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein at separate time points. In one example, the method of the disclosure comprises administering to the subject the alpha- 1 anti-trypsin or functional variant thereof before administering the therapeutic protein. For example, the alpha- 1 anti-trypsin or functional variant thereof is administered to the subject up to 7 days before administering the therapeutic protein. For example, the alpha- 1 anti-trypsin or functional variant thereof is administered to the subject about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days before administering the therapeutic protein. For example, the alpha- 1 anti-trypsin or functional variant thereof is administered to the subject up to 24 hours before administering the therapeutic protein. For example, the alpha- 1 anti-trypsin or functional variant thereof is administered to the subject about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 6 hours, or about 8 hours, or about 10 hours, or about 12 hours, or about 14 hours, or about 16 hours, or about 18 hours, or about 20 hours, or about 22 hours, or about 24 hours before administering the therapeutic protein.

In one example, the method of the disclosure comprises administering to the subject the therapeutic protein a plurality of times. For example, the therapeutic protein is administered about 2 times, or about 3 times, or about 4 times, or about 5 times, or about 6 times, or about 7 times, or about 8 times, or about 9 times, or about 10 times.

In one example, the method of the disclosure comprises administering to the subject the alpha- 1 anti-trypsin or functional variant thereof a plurality of times. For example, the alpha- 1 anti-trypsin or functional variant thereof is administered about 2 times, or about 3 times, or about 4 times, or about 5 times, or about 6 times, or about 7 times, or about 8 times, or about 9 times, or about 10 times.

In one example, the method of the disclosure comprises administering to the subject the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein a plurality of times. In one example, the alpha- 1 anti-trypsin or functional variant thereof is always administered before administration of the therapeutic protein.

In one example, the method of the disclosure comprises administering to the subject the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein a plurality of times at separate time points. For example, each administration of the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein is separated by a period of hours. For example, each administration of the alpha- 1 antitrypsin or functional variant thereof and therapeutic protein is separated by the period of about 1 hour, or about 2 hours, or about 3 hours, or about 4 hours, or about 6 hours, or about 8 hours, or about 12 hours, or about 16 hours, or about 20 hours, or about 24 hours. For example, each administration of the alpha- 1 anti-trypsin or functional variant thereof and therapeutic protein is separated by a period of days. For example, each administration of the alpha- 1 anti-trypsin or functional variant thereof and therapeutic protein is separated by the period of about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days. In one example, the length of time between administration of the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein is the same throughout the course of administration. In one example, the length of time between administration of the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein is different throughout the course of administration. In one example, the length of time between administration of the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein is variable.

In one example, the method of the disclosure comprises administering to the subject the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein concomitantly. For example, the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein are formulated for administration at the same time, or nearly the same time.

In one example, the alpha- 1 anti-trypsin or functional variant thereof and/or the therapeutic protein is administered parenterally. For example, the alpha- 1 anti-trypsin or functional variant thereof and/or the therapeutic protein are administered intravenously, or subcutaneously, or intramuscularly, or intraperitoneally.

In one example, the subject is at risk of developing a bleeding disorder. For example, a subject at risk of developing a bleeding disorder includes, but is not limited, to those with a mutation, deletion or rearrangement in a blood clotting factor, e.g., Factor VIII, or a platelet disorder. In one example, the subject has a relative that has developed a bleeding disorder. For example, the bleeding disorder is inherited. In one example, the bleeding disorder is acquired.

In one example, the alpha- 1 anti-trypsin or functional variant thereof and the therapeutic protein are administered after the onset of symptoms of a bleeding disorder.

Symptoms of a bleeding disorder will be apparent to the skilled person and include, for example:

• Easy bruising;

· Bleeding gums;

• Heavy bleeding from small cuts or dental work;

• Unexplained nosebleeds;

• Heavy menstrual bleeding;

• Bleeding into joints; and/or

· Excessive bleeding following surgery. In one example, the bleeding disorder is caused by a blood coagulation disorder. For example, the blood coagulation disorder is haemophilia, von Willebrand disease, Factor I deficiency, Factor II deficiency, Factor V deficiency, combined Factor V/Factor VIII deficiency, Factor VII deficiency, Factor X deficiency, Factor XI deficiency or Factor XIII deficiency. In one example, the haemophilia is haemophilia A or haemophilia B. In one example, the subject has a condition requiring prophylactic treatment.

In one example, the therapeutic protein is a blood coagulation factor. In one example, the blood coagulation factor is plasma derived. In one example, the blood coagulation factor is a recombinant protein. For example, the blood coagulation factor is selected from the group consisting of Factor I, Factor II, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII and Factor XIII. In one example, the blood coagulation factor is a modified form thereof, e.g., a PEGylated form thereof or a fusion protein with, e.g., albumin. In one example, the blood coagulation factor is Factor VIII, including recombinant forms thereof, such as single chain forms thereof.

In one example, the therapeutic protein is an antibody or antigen binding fragment thereof, an antibody mimetic, a domain antibody, a fusion protein, a cytokine or an enzyme. For example, the therapeutic protein is a protein that induces an immune response in a subject.

In one example of any method described herein, the therapeutic protein is an antibody mimetic. For example, the therapeutic protein is a protein comprising an antigen binding domain of an immunoglobulin, e.g., an IgNAR or a camelid antibody.

In one example of any method described herein, the therapeutic protein is a domain antibody (e.g., comprising only a heavy chain variable region or only a light chain variable region) or a heavy chain only antibody (e.g., a camelid antibody or an IgNAR) or variable region thereof.

In one example of any method described herein, the therapeutic protein is a protein comprising a Fv. For example, the protein is selected from the group consisting of:

(i) a single chain Fv fragment (scFv); (ii) a dimeric scFv (di-scFv); or

(iv) a diabody;

(v) a triabody;

(vi) a tetrabody;

(vii) a Fab;

(viii) a F(ab')2;

(ix) a Fv; or

(x) one of (i) to (ix) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CH3.

In another example of any method described herein, the therapeutic protein is an antibody. Exemplary antibodies are full-length and/or naked antibodies. For example, the antibody induces an immune response in the subject. Exemplary antibodies known to induce an immune response in a subject include, but are not limited to, adalimumab

(Humira®), bevacizumab (Avastin®), golimumab (Simponi®), panitumumab (Vectibix®) and alemtuzumab (Lemtrada®).

In one example of any method described herein, the therapeutic protein is a protein that is recombinant, chimeric, CDR grafted, humanized, synhumanized, primatized, deimmunized or human.

In one example of any method described herein, the therapeutic protein is an enzyme For example, the therapeutic enzyme induces an immune response in the subject. Exemplary therapeutic enzymes suitable for use in the present disclosure are known in the art and/or described herein.

In one example of any method described herein, the therapeutic protein is a cytokine. For example, the cytokine induces an immune response in the subject. Exemplary cytokines suitable for use in the present disclosure are known in the art and/or described herein.

In one example, the alpha- 1 anti-trypsin or functional variant thereof is administered in an amount sufficient to reduce the production of immunoglobulins against epitopes on the therapeutic protein.

In one example, the therapeutic protein and/or alpha- 1 anti-trypsin or functional variant thereof are in the form of a composition. For example, the composition comprises alpha- 1 anti-trypsin or functional variant thereof and/or a therapeutic protein. In one example, the composition further comprises a pharmaceutical carrier and/or excipient.

In one example of any method described herein, the subject is a mammal, for example a primate, such as a human.

Methods of treatment described herein can additionally comprise administering a further compound.

In one example, the present disclosure provides a composition comprising alpha- 1 anti-trypsin or functional variant thereof, for use in preventing an immune response against a therapeutic protein in a subject in need thereof.

In one example, the present disclosure provides use of alpha- 1 anti-trypsin or functional variant thereof in the manufacture of a medicament for preventing an immune response against a therapeutic protein in a subject in need thereof.

The present disclosure also provides a kit comprising at alpha- 1 anti-trypsin or functional variant thereof packaged with instructions for use in preventing an immune response against a therapeutic protein in a subject in need thereof. Optionally, the kit additionally comprises a therapeutic protein.

Exemplary effects of therapeutic proteins and alpha- 1 antitrypsin or functional variant thereof are described herein and are to be taken to apply mutatis mutandis to the examples of the disclosure set out in the previous four paragraphs.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graphical representation showing the effect of prophylactic alpha- 1 antitrypsin treatment prior to, and throughout the course of rFVIII therapeutic protein treatment. (A) Dosing schedule of alpha- 1 antitrypsin and rFVIII treatment. Alpha- 1 antitrypsin treatment prior to, and during the course of rFVIII treatment significantly reduced antibody formation (B, C). Mean + SEM; n=4-6/group; *: P<0.05 compared to control, Mann-Whitney test.

Figure 2 is a graphical representation showing the effect of prophylactic alpha- 1 antitrypsin priming treatment during rFVIII therapeutic protein treatment. (A) Dosing schedule of alpha- 1 antitrypsin and rFVIII treatment. Alpha- 1 antitrypsin treatment prior to, and during the course of rFVIII treatment significantly reduced antibody formation (B, C) whilst treatment with alpha- 1 antitrypsin prior to rFVIII treatment only (priming) did not significantly reduce antibody formation. Mean + SEM; n=6- 9/group; *: P<0.05 compared to control, Mann Whitney test.

Figure 3 is a graphical representation showing the effect of prophylactic alpha- 1 antitrypsin treatment prior to, and throughout the course of rFVIII therapeutic protein treatment. (A) Dosing schedule of alpha- 1 antitrypsin and rFVIII treatment. Alpha- 1 antitrypsin treatment prior to, and during the course of rFVIII treatment significantly reduced antibody formation (B, C). Mean + SEM; n=10/group; *: P<0.05 compared to control, unpaired T-test.

KEY TO SEQUENCE LISTING

SEQ ID NO: 1 is an amino acid sequence of human alpha- 1 antitrypsin

SEQ ID NO: 2 is an amino acid sequence of human FVIII

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309- 320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

Any discussion of a protein or antibody herein will be understood to include any variants of the protein or antibody produced during manufacturing and/or storage. For example, during manufacturing or storage an antibody can be deamidated (e.g., at an asparagine or a glutamine residue) and/or have altered glycosylation and/or have a glutamine residue converted to pyroglutamate and/or have a N-terminal or C-terminal residue removed or "clipped" and/or have part or all of a signal sequence incompletely processed and, as a consequence, remain at the terminus of the antibody. It is understood that a composition comprising a particular amino acid sequence may be a heterogeneous mixture of the stated or encoded sequence and/or variants of that stated or encoded sequence.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Selected Definitions

Alpha- 1 anti-trypsin (A1AT or AAT) is a protease inhibitor belonging to the serpin superfamily. It is also known as serum trypsin inhibitor and alpha- 1 proteinase inhibitor (A1PI). For the purposes of nomenclature only and not limitation exemplary sequences of human alpha- 1 anti-trypsin are set out in NCBI Reference Sequence NP_001121179, protein accession number P01009 and in SEQ ID NO: 1. Exemplary alpha- 1 anti-trypsin proteins include, but are not limited to, Aralast® (Baxter Healthcare Corporation), Prolastin-C® (Talecris Bio therapeutics, Inc.), Zemaira® (CSL Behring) or Glassia® (Kamada Ltd.). Exemplary recombinant alpha- 1 antitrypsin proteins in the form of Fc fusion proteins include, but are not limited to, those described in WO2013106589 and WO2012178102. The use of the term alpha- 1 anti- trypsin also includes functional variants thereof, e.g., as is known in the art and/or described herein. Exemplary functional variants of alpha- 1 anti-trypsin proteins include, but are not limited to, those described in US8,715,649. Additional sequence of alpha- 1 anti-trypsin can be determined using sequences provided herein and/or in publically available databases and/or determined using standard techniques (e.g., as described in Ausubel et ah, (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

Coagulation Factor VIII (FVIII) is a blood-clotting protein, also known as anti- haemophilic factor (AHF). For the purposes of nomenclature only and not limitation exemplary sequences of human FVIII are set out in NCBI Reference Sequence NP_000123, protein accession number NM_000132.3 and in SEQ ID NO: 2. Exemplary FVIII include, but are not limited to, FEIBA®, Monoclate-P®, Biostate®, Advate®, Eloctate®, Recombinate®, Kogenate Fs®, Helixate® Fs, Helixate®, Xyntha®/Refacto Ab®, Hemofil-M®, Monarc-M®, Alphanate®, Koate-Dvi®, Nuwiq® or Hyate:C®. Additional sequence of FVIII can be determined using sequences provided herein and/or in publically available databases and/or determined using standard techniques (e.g., as described in Ausubel et ah, (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

As used herein, the term "immune response" includes any type of immune response which is initiated by or dependent upon co- stimulatory signals, e.g., a cellular or a humoral response, that can occur in a subject in response to a foreign antigen.

As used herein, the term "inhibitory immune response" shall be taken to mean an immune response by a subject against the therapeutic protein that reduces or prevents the therapeutic activity of that protein. For example, immunoglobulin production against the epitopes of a therapeutic protein and reduce or prevent activity of that protein upon administration to a subject.

Reference to a "functional variant" of alpha- 1 anti-trypsin should be understood as a reference to a variant of alpha- 1 anti-trypsin which have retained and exhibit immunomodulatory activity (i.e., ability to prevent an immune response to a therapeutic protein). A functional variant may comprise or consist of a fragment, derivative or analogue of alpha- 1 anti-trypsin or combinations thereof. A functional variant of alpha- 1 anti-trypsin may have undergone mutation, truncation and/or substitution of one or more amino acids using well known techniques for site directed mutagenesis or any other conventional method. For example, the functional variant is a functional fragment of alpha- 1 anti-trypsin.

The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

As used herein, the term "therapeutic protein" shall be taken to mean a protein that is capable of preventing, alleviating, treating or curing a disease, disorder or injury. Exemplary proteins will be apparent to the skilled person or described herein. In one example, the therapeutic protein is not alpha- 1 antitrypsin.

As used herein, the term "preventing", "prevent" or "prevention" includes providing prophylaxis with respect to occurrence or recurrence of an immune response against a therapeutic protein in a subject, delaying an immune response against a therapeutic protein and reducing the incidence, likelihood, extent or risk of an immune response against the therapeutic protein. A subject may be predisposed to or at risk of developing the immune response but has not yet developed the immune response.

As used herein, the term "treating", "treat" or "treatment" shall be taken to mean reducing or eliminating at least one symptom of a specified condition or disease.

As used herein, a mammal "at risk" of developing a disease or condition or relapse thereof or relapsing may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment according to the present disclosure. "At risk" denotes that a mammal has one or more risk factors, which are measurable parameters that correlate with development of the disease or condition, as known in the art and/or described herein. An "effective amount" or "prophylactically effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, (i.e., prevention of an immune response against a therapeutic protein). An effective amount can be provided in one or more administrations. The effective amount may vary according to the disease or condition to be treated and also according to the weight, age, racial background, sex, health and/or physical condition and other factors relevant to the mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a "dosage" range) that can be determined through routine trial and experimentation by a medical practitioner. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

As used herein, the term "concomitant" as in the phrase "concomitant administration" includes administering a second agent while a first agent is still providing a therapeutic/prophylactic effect to a subject. A concomitant therapeutic treatment method includes methods in which the first and second or additional agents are co-administered. A concomitant therapeutic treatment method may be executed step-wise by different actors. For example, one actor may administer to a subject a first agent and as a second actor may administer to the subject a second agent and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and/or additional agents) is after administration in the presence of the second agent (and/or additional agents) and both agents are acting in the subject at the same time. The actor and the subject may be the same entity (e.g. a human).

As used herein, the term "condition" refers to a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.

As used herein, the term "bleeding condition" refers to a condition in which there is abnormal blood coagulation, e.g., reduced or insufficient blood coagulation capability and/or abnormal bleeding (internal and/or external), e.g., excessive bleeding.

As used herein, the term "blood coagulation factor" refers to a factor that is associated with the formation of a blot clot, i.e., blood coagulation. The term "recombinant" shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody variable region, this term does not encompass an antibody naturally-occurring within a subject's body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody variable region. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody variable region. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

The term "immunoglobulin" will be understood to include any antigen binding protein comprising an immunoglobulin domain. Exemplary immunoglobulins are antibodies. Additional proteins encompassed by the term "immunoglobulin" include domain antibodies, camelid antibodies and antibodies from cartilaginous fish (i.e., immunoglobulin new antigen receptors (IgNARs)). Generally, camelid antibodies and IgNARs comprise a VH, however lack a VL and are often referred to as heavy chain immunoglobulins. Other "immunoglobulins" include T cell receptors.

The skilled artisan will be aware that an "antibody" is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a light chain variable region (V _L ) and a polypeptide comprising a heavy chain variable region (V _H ). An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain. A V _H and a V _L interact to form a Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a κ light chain or a λ light chain and a heavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

The term "antibody" also encompasses humanized antibodies, primatized antibodies, human antibodies, synhumanized antibodies and chimeric antibodies. The terms "full-length antibody," "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild- type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

An "antigen binding fragment" of an antibody comprises one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

As used herein, "variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDRl, CDR2, and CDR3, and framework regions (FRs). Exemplary variable regions comprise three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. In the case of a protein derived from an IgNAR, the protein may lack a CDR2. V _H refers to the variable region of the heavy chain. V _L refers to the variable region of the light chain.

As used herein, the term "complementarity determining regions" (syn. CDRs; i.e., CDRl, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDRl, CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems in the performance of this disclosure, e.g., the canonical numbering system of Chothia and Lesk J. Mol Biol. 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and/or Al-Lazikani et al., J Mol Biol 273: 927-948, 1997; the IMGT numbering system of Lefranc et al., Devel. And Compar. Immunol., 27: 55-77, 2003; or the AHO numbering system of Honnegher and Plukthun J. Mol. Biol., 309: 657-670, 2001. "Framework regions" (FRs) are those variable domain residues other than the CDR residues.

As used herein, the term "Fv" shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a V _L and a V _H associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen. The V _H and the V _L which form the antigen binding site can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding sites which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the V _H is not linked to a heavy chain constant domain (C _H ) 1 and/or the V _L is not linked to a light chain constant domain (C _L ). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., C _H 2 or C _H 3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a V _H and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab' fragment can also be produced by recombinant means. A "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A "Fab2" fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a C _H 3 domain. A "single chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

As used herein, the term "subject" shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

Preventing an Immune Response against a Therapeutic Protein

As discussed herein, the present disclosure provides a method of preventing an immune response against a therapeutic protein. In one example, the method comprises administering alpha- 1 anti-trypsin or functional fragment thereof and the therapeutic protein to a subject in need thereof.

Immune responses to a therapeutic protein can be manifested in the production of immunoglobulins against epitopes on the therapeutic protein.

In one example, the immune response is an inhibitory immune response.

In one example, the immune response against the therapeutic protein is the production of immunoglobulins against the therapeutic protein. For example, the alpha- 1 antitrypsin or functional fragment thereof is administered in an amount sufficient to reduce the production of antibodies against the therapeutic protein.

In one example, administration of alph-1 antitrypsin or functional fragment thereof induces antigen specific tolerance to the therapeutic protein.

Methods for assessing immune responses and induction of antigen specific tolerance are known to the skilled person in the art and include, for example, measuring secondary immune responses (e.g., cellular or humoral responses) to an antigen.

In one example, the subject suffers from a bleeding disorder, but the onset of an immune response to a therapeutic protein has not yet occurred. The bleeding disorder can be inherited or acquired. For example, a subject suffering from a bleeding disorder has suffered a clinically acceptable symptom of a bleeding disorder, such as:

• Easy bruising;

• Bleeding gums;

· Heavy bleeding from small cuts or dental work;

• Unexplained nosebleeds; • Heavy menstrual bleeding;

• Bleeding into joints; and/or

• Excessive bleeding following surgery.

In one example, the subject is at risk of developing a bleeding disorder, but the onset of the bleeding disorder has not yet occurred. A subject is at risk if he or she has a higher risk of developing a bleeding disorder than a control population. The control population may include one or more subjects selected at random from the general population (e.g., matched by age, gender, race and/or ethnicity) who have not suffered from or have a family history of angina, stroke and/or heart attack. A subject can be considered at risk for a bleeding disorder if a "risk factor" associated with a bleeding disorder is found to be associated with that subject. A risk factor can include any activity, trait, event or property associated with a given disorder, for example, through statistical or epidemiological studies on a population of subjects. A subject can thus be classified as being at risk for a bleeding disorder even if studies identifying the underlying risk factors did not include the subject specifically. For example, a subject who has excessive bleeding is at risk of developing a bleeding disorder because the frequency of a bleeding disorder is increased in a population of subjects who have excessive bleeding as compared to a population of subjects who do not.

The methods of the present disclosure can be readily applied to any form of bleeding disorder in a subject.

In one example, the present disclosure provides a method of treating a bleeding disorder, the method comprising administering to a subject suffering from a bleeding disorder alpha- 1 antitrypsin or functional fragment thereof and a blood coagulation factor. For example, the alpha- 1 antitrypsin or functional fragment thereof is administered in an amount sufficient to prevent an immune response against the blood coagulation factor.

In one example, the bleeding disorder is haemophilia, for example, haemophilia

A.

In one example, the blood coagulation factor is Factor VIII, such as a single chain recombinant factor VIII, e.g., as described in Zollner et ah, Thromb Res. 32:280-287, 2013 or US9394353. In one example, the single chain recombinant factor VIII is CSL627, e.g., as described in US9394353.

In one example, the present disclosure provides a method of treating haemophilia A, the method comprising administering to a subject suffering from haemophilia A alpha- 1 antitrypsin or functional fragment thereof and Factor VIII (e.g., a single chain form of Factor VIII). For example, the alpha- 1 antitrypsin or functional fragment thereof is administered in an amount sufficient to prevent an immune response against the blood coagulation factor. Therapeutic Proteins

As discussed here, therapeutic proteins of the present disclosure can take various forms, e.g., protein-based therapeutics. In one example, the protein-based therapeutic is a blood coagulation factor or an antibody or antigen binding fragment thereof. Exemplary protein-based therapeutics are discussed herein.

Blood coagulation factors

In one example, the therapeutic protein of the present disclosure is a blood coagulation factor.

Blood coagulation occurs through a cascade of stages involving release of several coagulation factors, ultimately resulting in the formation of a blood clot containing insoluble fibrin. Exemplary blood coagulation factors include, but are not limited to, Factor I (Fibrinogen), Factor II (Prothrombin), Factor III (Tissue factor), Factor V (Labile factor), Factor VII (Proconvertin), Factor VIII (Antihaemophilic factor), Factor IX (Christmas factor), Factor X (Stuart-Prower factor), Factor XI (Plasma thromboplastin antecedent), Factor XII (Hageman (contact) factor) and Factor XIII (Fibrin-stabilizing factor/Prekallikrein (Fletcher) factor/ HMWK (Fitzgerald) factor).

Blood coagulation factors of the present disclosure may be plasma derived from a donor. Suitable methods for the isolation of blood coagulation factors from donor plasma are available to those skilled in the art. In one example, the blood coagulation factor is a recombinant protein. For example, the blood coagulation factor is selected from the group consisting of Factor I, Factor II, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII and Factor XIII. In one example, the blood coagulation factor is Factor VIII, such as a single chain recombinant factor VIII, e.g., as described in Zollner et ah, Thromb Res. 32:280-287, 2013 or US9394353.

In one example, the blood coagulation factor is a Factor Vila. For example, the Factor Vila product is NovoSeven®.

In one example, the blood coagulation factor is FVIII. For example, the blood coagulation factor is a plasma derived Factor VIII product, such as FEIBA®, Monoclate-P®, or Biostate®. For example, the blood coagulation factor is a recombinant Factor VIII product, such as Afstyla®, Advate®, Eloctate®, Recombinate®, Kogenate Fs®, Helixate® Fs, Helixate®, Xyntha®/Refacto Ab®, Hemofil-M®, Monarc-M®, Alphanate®, Koate-Dvi®, Nuwiq® or Hyate:C®.

In one example, the blood coagulation factor is a Factor IX. For example, the blood coagulation factor is a plasma derived Factor IX product such as, Berinin® P, MonoFIX® or Mononine®. For example, the blood coagulation factor is a recombinant Factor IX product such as Alphanine SD®, Alprolix®, Bebulin®, Bebulin VH®, Benefix®, Ixinity®, , Profilnine SD®, Proplex T®, or Rixubis®.

In one example, the blood coagulation factor is a Factor XIII. For example, the

Factor XIII product is Fibrogammin® P, Corifact®, Cluvot® or Cluviat®.

In one example, the blood coagulation factor is a von Willebrand factor/FVIII complex. For example, the von Willebrand factor/FVIII product is Humate-P®, Haemate®-P, Biostate® or Voncento®.

In one example, the blood coagulation factor is a prothrombin complex. For example, the prothrombin complex factor is Beriplex® P/N, Confidex® or Kcentra®.

In one example, the blood coagulation factor is a fibrinogen. For example the fibrinogen product is RiaSTAP®, Haemocomplettan® P. Antibodies

In one example, the therapeutic protein of the present disclosure is an antibody, or antigen binding fragment thereof. In one example, the therapeutic protein of the present disclosure is an antibody or antigen binding fragment thereof that induces an immune response in a subject.

Exemplary therapeutic antibodies are known to the skilled person and include, but are not limited to, Abagovomab; Abciximab; Abituzumab; Abrilumab; Actoxumab;

Adalimumab; Adecatumumab; Aducanumab; Afelimomab; Afutuzumab; Alacizumab pegol; Alemtuzumab; Alirocumab; Altumomab pentetate; Amatuximab; Anatumomab mafenatox; Anetumab ravtansine; Anifrolumab; Anrukinzumab; Apolizumab;

Arcitumomab; Ascrinvacumab; Aselizumab; Atezolizumab; Atinumab; Atlizumab

(tocilizumab); Atorolimumab; Bapineuzumab; Basiliximab; Bavituximab;

Bectumomab; Begelomab; Belimumab; Benralizumab; Bertilimumab; Besilesomab;

Bevacizumab; Bezlotoxumab; Biciromab; Bimagrumab; Bimekizumab; Bivatuzumab mertansine; Blinatumomab; Blosozumab; Bococizumab; Brentuxim abvedotin;

Briakinumab; Brodalumab; Brolucizumab; Brontictuzumab; Canakinumab;

Cantuzumab mertansine; Cantuzumab ravtansine; Caplacizumab; Capromab pendetide;

Carlumab; Catumaxomab; cBR96-doxorubicin immunoconjugate; Cedelizumab;

Certolizumab pegol; Cetuximab; Citatuzumab bogatox; Cixutumumab; Clazakizumab; Clenoliximab; Clivatuzumab tetraxetan; Codrituzumab; Coltuximab ravtansine;

Conatumumab; Concizumab; Crenezumab; Dacetuzumab; Daclizumab; Dalotuzumab;

Dapirolizumab pegol; Daratumumab; Dectrekumab; Demcizumab; Denintuzumab mafodotin; Denosumab; Derlotuximab bio tin; Detumomab; Dinutuximab;

Diridavumab; Dorlimomab aritox; Drozitumab; Duligotumab; Dupilumab; Durvalumab; Dusigitumab; Ecromeximab; Eculizumab; Edobacomab; Edrecolomab;

Efalizumab; Efungumab; Eldelumab; Elgemtumab; Elotuzumab; Elsilimomab;

Emactuzumab; Emibetuzumab; Enavatuzumab; Enfortumab vedotin; Enlimomab pegol; Enoblituzumab; Enokizumab; Enoticumab; Ensituximab; Epitumomab cituxetan; Epratuzumab; Erlizumab; Ertumaxomab; Etanercept; Etaracizumab; Etrolizumab; Evinacumab; Evolocumab; Exbivirumab; Fanolesomab; Faralimomab;

Farletuzumab; Fasinumab; Felvizumab; Fezakinumab; Ficlatuzumab; Figitumumab; Firivumab; Flanvotumab; Fletikumab; Fontolizumab; Foralumab; Foravirumab;

Fresolimumab; Fulranumab; Futuximab; Galiximab; Ganitumab; Gantenerumab;

Gavilimomab; Gemtuzumab ozogamicin; Gevokizumab; Girentuximab;

Glembatumumab vedotin; Golimumab; Gomiliximab; Guselkumab; Ibalizumab; Ibritumomab tiuxetan; Icrucumab; Idarucizumab; Igovomab; Imalumab; Imciromab;

Imgatuzumab; Inclacumab; Indatuximab ravtansine; Indusatumab vedotin; Infliximab;

Inolimomab; Inotuzumab ozogamicin; Intetumumab; Ipilimumab; Iratumumab;

Isatuximab; Itolizumab; Ixekizumab; Keliximab; Labetuzumab; Lambrolizumab;

Lampalizumab; Lebrikizumab; Lemalesomab; Lenzilumab; Lerdelimumab; Lexatumumab; Libivirumab; Lifastuzumab vedotin; Ligelizumab; Lilotomab satetraxetan; Lintuzumab; Lirilumab; Lodelcizumab; Lokivetmab; Lorvotuzumab mertansine; Lucatumumab; Lulizumab pegol; Lumiliximab; Lumretuzumab;

Mapatumumab; Margetuximab; Maslimomab; Matuzumab; Mavrilimumab;

Mepolizumab; Metelimumab; Milatuzumab; Minretumomab; Mirvetuximab soravtansine; Mitumomab; Mogamulizumab; Morolimumab; Motavizumab;

Moxetumomab pasudotox; Muromonab-CD3; Nacolomab tafenatox; Namilumab;

Naptumomab estafenatox; Narnatumab; Natalizumab; Nebacumab; Necitumumab;

Nemolizumab; Nerelimomab; Nesvacumab; Nimotuzumab; Nivolumab; Nofetumomab merpentan; Obiltoxaximab; Obinutuzumab; Ocaratuzumab; Ocrelizumab; Odulimomab; Ofatumumab; Olaratumab; Olokizumab; Omalizumab; Onartuzumab;

Ontuxizumab; Opicinumab; Oportuzumab monatox; Oregovomab; Orticumab;

Otelixizumab; Otlertuzumab; Oxelumab; Ozanezumab; Ozoralizumab; Pagibaximab;

Palivizumab; Panitumumab; Pankomab; Panobacumab; Parsatuzumab; Pascolizumab;

Pasotuxizumab; Pateclizumab; Patritumab; Pembrolizumab; Pemtumomab; Perakizumab; Pertuzumab; Pexelizumab; Pidilizumab; Pinatuzumab vedotin;

Pintumomab; Placulumab; Polatuzumab vedotin; Ponezumab; Priliximab;

Pritoxaximab; Pritumumab; Quilizumab; Racotumomab; Radretumab; Rafivirumab;

Ralpancizumab; Ramucirumab; Ranibizumab; Raxibacumab; Refanezumab;

Regavirumab; Reslizumab; Rilotumumab; Rinucumab; Rituximab; Robatumumab; Roledumab; Romosozumab; Rontalizumab; Rovelizumab; Ruplizumab; Sacituzumab govitecan; Samalizumab; Sarilumab; Satumomab pendetide; Secukinumab; Seribantumab; Setoxaximab; Sevirumab; Sibrotuzumab; Sifalimumab; Siltuximab; Simtuzumab; Siplizumab; Sirukumab; Sofituzumab vedotin; Solanezumab; Solitomab; Sonepcizumab; Sontuzumab; Stamulumab; Sulesomab; Suvizumab; Tabalumab; Tacatuzumab tetraxetan; Tadocizumab; Talizumab; Tanezumab; Taplitumomab paptox; Tarextumab; Tefibazumab; Telimomab aritox; Tenatumomab; Teneliximab; Teplizumab; Teprotumumab; Tesidolumab; Tetulomab; Ticilimumab; Tigatuzumab; Tildrakizumab; Tocilizumab; Toralizumab; Tosatoxumab; Tositumomab; Tovetumab; Tralokinumab; Trastuzumab; Tregalizumab; Tremelimumab; Trevogrumab; Tucotuzumab celmoleukin; Tuvirumab; Ublituximab; Ulocuplumab; Urelumab; Urtoxazumab; Ustekinumab; Vandortuzumab vedotin; Vantictumab; Vanucizumab; Vapaliximab; Varlilumab; Vatelizumab; Vedolizumab; Veltuzumab; Vepalimomab; Vesencumab; Visilizumab; Volociximab; Vorsetuzumab mafodotin; Votumumab; Zalutumumab; Zanolimumab; Zatuximab; Ziralimumab; Zolimomab aritox. Deimmunized, Chimeric, Humanized, Svnhumanized, Primatized and Human Antibodies or Antigen Binding Fragments

The antibodies or antigen binding fragments of the present disclosure may be may be humanized.

The term "humanized antibody" shall be understood to refer to a protein comprising a human-like variable region, which includes CDRs from an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into FRs from a human antibody (this type of antibody is also referred to a "CDR- grafted antibody"). Humanized antibodies also include antibodies in which one or more residues of the human protein are modified by one or more amino acid substitutions and/or one or more FR residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found in neither the human antibody or in the non-human antibody. Any additional regions of the antibody (e.g., Fc region) are generally human. Humanization can be performed using a method known in the art, e.g., US5225539, US6054297, US7566771 or US5585089. The term "humanized antibody" also encompasses a super-humanized antibody, e.g., as described in US7732578. A similar meaning will be taken to apply to the term "humanized antigen binding fragment".

The antibodies or antigen binding fragments thereof of the present disclosure may be human antibodies or antigen binding fragments thereof. The term "human antibody" as used herein refers to antibodies having variable and, optionally, constant antibody regions found in humans, e.g. in the human germline or somatic cells or from libraries produced using such regions. The "human" antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve conservative substitutions or mutations in a small number of residues of the protein, e.g. in 1, 2, 3, 4 or 5 of the residues of the protein). These "human antibodies" do not necessarily need to be generated as a result of an immune response of a human, rather, they can be generated using recombinant means (e.g., screening a phage display library) and/or by a transgenic animal (e.g., a mouse) comprising nucleic acid encoding human antibody constant and/or variable regions and/or using guided selection (e.g., as described in US5565332). This term also encompasses affinity matured forms of such antibodies. For the purposes of the present disclosure, a human antibody will also be considered to include a protein comprising FRs from a human antibody or FRs comprising sequences from a consensus sequence of human FRs and in which one or more of the CDRs are random or semi-random, e.g., as described in US6300064 and/or US6248516. A similar meaning will be taken to apply to the term "human antigen binding fragment".

The antibodies or antigen binding fragments thereof of the present disclosure may be synhumanized antibodies or antigen binding fragments thereof. The term "synhumanized antibody" refers to an antibody prepared by a method described in WO2007/019620. A synhumanized antibody includes a variable region of an antibody, wherein the variable region comprises FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region.

The antibody or antigen binding fragment thereof of the present disclosure may be primatized. A "primatized antibody" comprises variable region(s) from an antibody generated following immunization of a non-human primate (e.g., a cynomolgus macaque). Optionally, the variable regions of the non-human primate antibody are linked to human constant regions to produce a primatized antibody. Exemplary methods for producing primatized antibodies are described in US6113898.

In one example an antibody or antigen binding fragment thereof of the disclosure is a chimeric antibody or fragment. The term "chimeric antibody" or "chimeric antigen binding fragment" refers to an antibody or fragment in which one or more of the variable domains is from a particular species (e.g., murine, such as mouse or rat) or belonging to a particular antibody class or subclass, while the remainder of the antibody or fragment is from another species (such as, for example, human or non- human primate) or belonging to another antibody class or subclass. In one example, a chimeric antibody comprising a V _H and/or a V _L from a non-human antibody (e.g., a murine antibody) and the remaining regions of the antibody are from a human antibody. The production of such chimeric antibodies and antigen binding fragments thereof is known in the art, and may be achieved by standard means (as described, e.g., in US6331415; US5807715; US4816567 and US4816397).

The present disclosure also contemplates a deimmunized antibody or antigen binding fragment thereof, e.g., as described in WO2000/34317 and WO2004/108158. De-immunized antibodies and fragments have one or more epitopes, e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a subject will raise an immune response against the antibody or protein. For example, an antibody of the disclosure is analyzed to identify one or more B or T cell epitopes and one or more amino acid residues within the epitope is mutated to thereby reduce the immunogenicity of the antibody.

Antibody Fragments

Single-Domain Antibodies

In some examples, an antigen binding fragment of an antibody of the disclosure is or comprises a single-domain antibody (which is used interchangeably with the term "domain antibody" or "dAb"). A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody. Diabodies, Triabodies, Tetrabodies

In some examples, an antigen binding fragment of the disclosure is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in WO98/044001 and/or WO94/007921.

For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure V _L -X-V _H or V _H -X-V _L , wherein X is a linker comprising insufficient residues to permit the V _H and V _L in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the V _H of one polypeptide chain binds to a V _L of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The V _L and V _H can be the same in each polypeptide chain or the V _L and V _H can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).

A diabody, triabody, tetrabody, etc capable of inducing effector activity can be produced using an antigen binding fragment capable of binding to IL-3Ra and an antigen binding fragment capable of binding to a cell surface molecule on an immune cell, e.g., a T cell (e.g., CD3).

Single Chain Fv ( scFv ) Fragments

The skilled artisan will be aware that scFvs comprise V _H and V _L regions in a single polypeptide chain and a polypeptide linker between the V _H and V _L which enables the scFv to form the desired structure for antigen binding (i.e., for the V _H and V _L of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly ₄ Ser) ₃ being one of the more favored linkers for a scFv.

The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of V _H and a FR of V _L and the cysteine residues linked by a disulfide bond to yield a stable Fv.

Alternatively, or in addition, the present disclosure encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.

The present disclosure also contemplates a dimeric scFv capable of inducing effector activity. For example, one scFv binds to IL-3Ra and another scFv binds to a cell surface molecule on an immune cell, e.g., a T cell (e.g., CD3 or CD19). In one example, the dimeric protein is a combination of a dAb and a scFv. Examples of bispecific antibody fragments capable of inducing effector function are described, for example, in US7235641. Other Antibodies and Antibody Fragments

The present disclosure also contemplates other antibodies and antibody fragments, such as:

(i) "key and hole" bispecific proteins as described in US5731168;

(ii) heteroconjugate proteins, e.g., as described in US4676980;

(iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4676980; and

(iv) Fab ₃ (e.g., as described in EP19930302894).

Immunoglobulins and Immunoglobulin Fragments

An example of a therapeutic protein of the present disclosure is a protein comprising a variable region of an immunoglobulin, such as a T cell receptor or a heavy chain immunoglobulin (e.g., an IgNAR, a camelid antibody).

Heavy Chain Immunoglobulins

Heavy chain immunoglobulins differ structurally from many other forms of immunoglobulin (e.g., antibodies), in so far as they comprise a heavy chain, but do not comprise a light chain. Accordingly, these immunoglobulins are also referred to as "heavy chain only antibodies". Heavy chain immunoglobulins are found in, for example, camelids and cartilaginous fish (also called IgNAR).

The variable regions present in naturally occurring heavy chain immunoglobulins are generally referred to as "V _HH domains" in camelid Ig and V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "V _H domains") and from the light chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "V _L domains").

Heavy chain immunoglobulins do not require the presence of light chains to bind with high affinity and with high specificity to a relevant antigen. This means that single domain binding fragments can be derived from heavy chain immunoglobulins, which are easy to express and are generally stable and soluble.

A general description of heavy chain immunoglobulins from camelids and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in the following references WO94/04678, WO97/49805 and WO 97/49805.

A general description of heavy chain immunoglobulins from cartilaginous fish and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in WO2005/118629.

Cytokines

In one example, the therapeutic protein of the present disclosure is a cytokine. In one example, the therapeutic protein of the present disclosure is a cytokine that induces an immune response in a subject.

Exemplary therapeutic cytokines are known to the skilled person and include but are not limited to, interleukin-1 receptor antagonist (IL-IRa; anakinra (Kineret®)), interleukin-2 (IL-2; aldesleukin (Proleukin®)), interleukin-7 (IL-7), interleukin-10 (IL- 10), interleukin- 11 (IL-11; Neumega; Oprelvekin), interleukin-12 (IL-12), interleukin- 15 (IL-15), interleukin-21 (IL-21); interferon alpha (IFN-a; Infergen®, Intron-A®; Roferon A®); interferon beta (IFN-β; Avonex®, Betaseron®, Extavia®, Rebif®), interferon gamma (IFN-γ; Actinmmune®; Imukin®), IFN-a2a (peginterferon alpha-2a (Pegasys®)); IFN-a2b (peginterferon alfa-2b (Pegintron®; Sylatron®); Albinterferon alfa-2b), granulocyte colony stimulating factor (G-CSF; lenograstim (Granocyte®); filgrastim (Neupogen®); pegfilgrastim (Neulasta®)), granulocyte macrophage colony stimulating factor (GM-CSF; molgramostim (Leucomax®); sargramostim (Leukine®)), tumour necrosis factor alpha (TNF-a; tasonermin (Beromun®)), TNF-related activation protein (CD40L; TRAP), TNF-related apoptosis inducing ligand (TRAIL).

Enzymes

In one example, the therapeutic protein of the present disclosure is a therapeutic enzyme. In one example, the therapeutic protein of the present disclosure is a therapeutic enzyme that induces an immune response in a subject.

Exemplary therapeutic enzymes are known to the skilled person and include, but are not limited to, uricase, imiglucerase, taliglucerase, velaglucerase, agalsidase alfa, agalsidase beta, alglucosidase alfa, sebelipase alfa, mucopolysaccharidose I (laronidase), mucopolysaccharidose II (idursulfase), mucopolysaccharidose IVA (elosulfase), mucopolysaccharidose VI (galsulfase), alteplase, reteplase, tenecteplase, streptokinase; asfotase alfa, dornase alfa, pegloticase, rasburicase, L-asparaginase, collagenase, pegademase bovine, glucarpidase), ocriplasmin and hyaluronidase.

Constant Regions

The present disclosure encompasses compounds (e.g., antibodies and antigen binding fragments thereof) comprising a constant region of an antibody and/or a Fc region of an antibody.

Sequences of constant regions and/or Fc regions useful for producing the immunoglobulins, antibodies or antigen binding fragments of the present disclosure may be obtained from a number of different sources. In some examples, the constant region, Fc or portion thereof of the compound is derived from a human antibody. The constant region, Fc or portion thereof may be derived from any antibody class, including IgA, IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgGl, IgG2, IgG3 and IgG4. In one example, the constant region or Fc is human isotype IgGl or human isotype IgG2 or human isotype IgG3 or a hybrid of any of the foregoing.

In one example, the constant region or Fc region is capable of inducing an effector function. For example, the constant region or Fc region is a human IgGl or IgG3 Fc region. In another example, the constant region or Fc region is a hybrid of an IgGl and an IgG2 constant region or Fc region or a hybrid of an IgGl and an IgG3 constant region or Fc region or a hybrid of an IgG2 and an IgG3 constant region or Fc region. Exemplary hybrids of human IgGl and IgG2 constant region or Fc regions are described in Chappel et al, Proc. Natl Acad. Set USA, 88: 9036-9040, 1991. Screening Assays

Methods for assessing immune responses and induction of antigen specific tolerance are known to the skilled person in the art and include, for example, measuring secondary immune responses (e.g., cellular or humoral responses) to an antigen.

Similarly, amounts and timing of administration of alpha- 1 antitrypsin suitable for use in a method described herein can be determined or estimated using techniques known in the art, e.g., as described below.

In vivo Animal Models

In one example, the method of preventing an immune response to a therapeutic protein is tested in an animal model of a bleeding disorder.

Animal models of bleeding disorders are known in the art and/or exemplified herein. Exemplary animal models include, for example, HaemA (FVIII knockout) mouse model.

Alpha- 1 antitrypsin inhibitor can be administered to such an animal model. Simultaneously or following alpha- 1 antitrypsin administration, a therapeutic protein is also administered. Prevention of an immune response against the therapeutic protein in the animal model in the presence of alpha- 1 antitrypsin compared to in the absence of the alpha- 1 antitrypsin indicates that the alpha- 1 antitrypsin is useful in preventing an immune response against the therapeutic protein.

In one example, the animal model is a model of a bleeding disorder, for example, haemophilia. For example, the mouse model is a HaemA (FVIII knockout) mouse model.

Binding Assays

To determine whether administration of alpha- 1 antitrypsin prevents the development of an immune response to a therapeutic protein an in vitro assay to detect the presence of antibodies can be used. For example, effective inhibition of antibody formation against a therapeutic protein can be identified.

In one example, an in vitro assay involves an enzyme-linked immunosorbent assay (ELISA). Methods of performing an ELISA are known to the skilled person in the art and/or described herein.

In one example, the therapeutic protein is immobilized on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support. A sample (e.g., plasma sample) treated with the therapeutic protein is then brought into physical relation with the therapeutic protein, and the protein to which said therapeutic binds is bound or 'captured'. The bound protein is then detected using a second labeled compound that binds to a different protein or a different site in the same protein. Alternatively, a third labeled antibody can be used that binds the second (detecting) antibody.

In one example, the ELISA involves immobilizing a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide). A therapeutic protein is brought into direct contact with the immobilized biological sample, and forms a direct bond with any of its target protein present in said sample. This antibody is generally labeled with a detectable reporter molecule, such as for example, an enzyme (e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP) or β-galactosidase) in the case of an ELISA, or alternatively a second labeled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody the label is detected through the addition of a substrate, such as for example 5-bromo-4-chloro-3-indol-beta- D-galaotopyranoside (x-gal).

Bethesda Assay

In one example, the development of inhibitors against a therapeutic protein can be determined using an in vitro clotting assay, e.g., using a commercially available kits, such as the Bethesda assay (Affinity Biologicals) and/or FVIII Inhibitor Reagent Kit (Technoclone).

Pharmaceutical Compositions and Methods of Preventing an Immune Response

The present disclosure provides a method of preventing an immune response against a therapeutic protein in a subject comprising administering alpha- 1 antitrypsin or functional fragment thereof and a therapeutic protein to a subject in need thereof.

In one example, the therapeutic protein is useful for parenteral, topical, oral, or local administration, aerosol administration, or transdermal administration. In one example, the therapeutic protein is administered parenterally. In one example, the therapeutic protein is administered subcutaneously, intraperitoneally or intravenously. For example, the therapeutic protein is administered intravenously.

In one example, the alpha- 1 antitrypsin or functional fragment thereof is useful for parenteral, topical, oral, or local administration, aerosol administration, or transdermal administration. In one example, the alpha- 1 antitrypsin or functional fragment thereof is administered parenterally. In one example, the alpha- 1 antitrypsin or functional fragment thereof is administered subcutaneously, intraperitoneally or intravenously. For example, the alpha- 1 antitrypsin or functional fragment thereof is administered intraperitoneally.

Formulation of a compound to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected. An appropriate pharmaceutical composition comprising compound to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The compound can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.

The dosage ranges for the administration of the alpha- 1 antitrypsin or functional fragment thereof of the disclosure are those large enough to produce the desired effect. For example, the composition comprises an amount of alpha- 1 antitrypsin sufficient to prevent an immune response against the therapeutic protein.

As used herein, the term "effective amount" shall be taken to mean a sufficient quantity of the alpha- 1 antitrypsin or functional fragment thereof to inhibit or prevent an immune response against a therapeutic protein in a subject. The skilled artisan will be aware that such an amount will vary depending on, for example, the therapeutic protein and/or the particular subject and/or the type and/or the severity of the immune response to be treated. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number of alpha- 1 antitrypsin.

The dosage should not be so large as to cause adverse side effects, such as hyper viscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

Dosage of the alpha- 1 antitrypsin can vary from about 0.1 mg/kg to about 300 mg/kg, e.g., from about 0.2 mg/kg to about 200 mg/kg, such as, from about 10 mg/kg to about 80 mg/kg, for example about 60mg/kg in one or more dose administrations, for one or several days. In one example, the dosage of the alpha- 1 antitrypsin is between about lOmg/kg and about 150mg/kg, for example about 60mg/kg or about 120mg/kg in one or more dose administrations, for one or several days. In one example, the dosage is 60mg/kg, e.g., 60mg/kg on a weekly basis. In another example, the dosage is

120mg/kg, e.g., 120mg/kg on a weekly basis.

In some examples, the alpha- 1 antitrypsin or functional fragment thereof is administered at an initial (or loading) dose which is higher than subsequent (maintenance doses). For example, the alpha- 1 antitrypsin or functional fragment thereof is administered at an initial dose of between about 40mg/kg to about 80mg/kg.

The alpha- 1 antitrypsin is then administered at a maintenance dose of between about 10 mg/kg to about 40mg/kg. The maintenance doses may be administered every 3-100 days, such as, every 3 or 7 or 14 or 28 or 56 or 84 days.

In some examples, a dose escalation regime is used, in which alpha- 1 antitrypsin or functional fragment thereof is initially administered at a lower dose than used in subsequent doses. This dosage regime is useful in the case of subject's initially suffering adverse events

In the case of a subject that is not adequately responding to treatment, multiple doses in a week may be administered. Alternatively, or in addition, increasing doses may be administered.

A subject may be retreated with the alpha- 1 antitrypsin, by being given more than one exposure or set of doses, such as at least about two exposures of the compound, for example, from about 2 to 60 exposures, and more particularly about 2 to 40 exposures, most particularly, about 2 to 20 exposures.

In another example, any retreatment may be given at defined intervals. For example, subsequent exposures may be administered at various intervals, such as, for example, about 24-28 weeks or 48-56 weeks or longer. For example, such exposures are administered at intervals each of about 24-26 weeks or about 38-42 weeks, or about 50-54 weeks.

A method of the present disclosure may include co-administration of the alpha- 1 antitrypsin or functional fragment thereof together with the administration of the therapeutic protein for the prevention of an immune response against a therapeutic protein in a subject.

As will be apparent from the foregoing, the present disclosure provides methods of concomitant treatment of a subject, comprising administering to a subject in need thereof an effective amount of a first agent and a second agent, wherein said first agent is a therapeutic protein, and the second agent is alpha- 1 antitrypsin or functional fragment thereof for the prevention of an immune response against the therapeutic protein.

In one example, the disclosure also provides a method for preventing an immune response against a therapeutic protein in a subject, the method comprising administering to the subject a first pharmaceutical composition comprising alpha- 1 antitrypsin or functional fragment thereof and a second pharmaceutical composition comprising one or more therapeutic proteins.

In one example, a method of the disclosure comprises administering alpha- 1 antitrypsin or functional fragment thereof to a subject suffering from a bleeding disorder and receiving another treatment.

Kits and other compositions of matter

Another example of the disclosure provides kits containing alpha- 1 antitrypsin or functional fragment thereof useful for the prevention of an immune response against a therapeutic protein as described above.

In one example, the kit comprises (a) a container comprising alpha- 1 antitrypsin or functional fragment thereof, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for preventing an immune response against a therapeutic protein in a subject.

In one example, the kit comprises (a) a container comprising a therapeutic protein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions to administer alpha- 1 antitrypsin or functional fragment thereof for preventing an immune response against a therapeutic protein in a subject.

In accordance with this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for treating atherosclerosis and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the alpha- 1 antitrypsin. The label or package insert indicates that the composition is used for treating a subject eligible for treatment, e.g., one having or predisposed to developing an immune response against a therapeutic protein, with specific guidance regarding dosing amounts and intervals of alpha- 1 antitrypsin and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate -buffered saline, Ringer's solution, and/or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kit optionally further comprises a container comprising a second medicament, wherein the alpha- 1 antitrypsin or functional fragment thereof is a first medicament, and which article further comprises instructions on the package insert for treating the subject with the second medicament, in an effective amount. The second medicament may be a therapeutic protein set forth above.

The present disclosure includes the following non-limiting Examples.

Examples

Example 1: Prophylactic treatment with alpha- 1 antitrypsin prior to and during the course of rFVIII treatment reduced rFVIII antibody formation

Adult male and female HaemA mice (FVIII KO, Jackson Lab) of 10-12 weeks of age were treated 4 times with 250 IU/kg of rFVIII (CSL627). Injections were given 7 days apart and intravenously. Alpha- 1 anti-trypsin inhibitor (AAT, Zemaira) was injected intraperitonealy at a dose of 60 mg/kg every 3-4 days for the duration of rFVIII treatment and treatment commenced 24 h prior to the first injection with rFVIII.

Seven days after the last injection with rFVIII, mice were euthanized with C0 ₂ for terminal plasma collection. Figure 1A is a schematic representation of the treatment strategy used.

Efficiency of AAT in preventing antibody formation against rFVIII was shown by testing the plasma in an ELISA coated with rFVIII. Efficiency of AAT in preventing inhibitor formation against rFVIII was shown by testing the plasma in a Bethesda clotting assay with chromogenic read-out.

The data was analyzed using Mann-Whitney test. Samples were analyzed individually and differences were regarded as significant if p<0.05.

Treatment with AAT prior to, and throughout the course of treatment with rFVIII reduced antibody formation against rFVIII in the ELISA (Figure IB ; Table 1) and significantly reduced inhibitor levels in the Bethesda clotting assay (Figure 1C; Table 1). Table 1: Reduction in antibody formation against rFVIII following prophylactic alpha- 1 antitrypsin treatment prior to, and throughout the course of rFVIII therapeutic protein treatment.

Treatment Anti-FVIII Ab FVIII inhibitors

group (Titer at 0.5) (BU/ml)

CSL627 only 3619 15.79

CSL627 only 289 5.61

CSL627 only 20775 71.93

CSL627 only 4528 18.77

Mean+SKM 7303+4582 28.02+ 14.90

AAT + CSL627 18 0.00

AAT + CSL627 33 0.00

AAT + CSL627 26 0.00

AAT + CSL627 53 0.00

AAT + CSL627 205 0.00

AAT + CSL627 6966 26.74

Mean+SKM 1217+1 150 4.46+4.46 Example 2: Priming with alpha- 1 antitrypsin 24 hours prior to rFVIII treatment did not reduce rFVIII antibody formation

Adult male and female HaemA mice (FVIII KO, Jackson Lab) of 10-12 weeks of age were treated 4 times with 120 IU/kg of rFVIII (CSL627). Injections were given 7 days apart and intravenously. Alpha- 1 anti-trypsin inhibitor (AAT, Zemaira) was injected intraperitonealy at a dose of 60 or 120mg/kg every 3-4 days, or only 24 h prior to rFVIII (prime only) for the duration of rFVIII treatment. Treatment commenced 24 h prior to the first injection with rFVIII. Figure 2A is a schematic representation of the treatment strategy.

Seven days after the last injection with rFVIII, mice were euthanized with C0 ₂ for terminal plasma collection.

Efficiency of AAT in preventing antibody formation against rFVIII was shown by testing the plasma in an ELISA coated with rFVIII. Efficiency of AAT in preventing inhibitor formation against rFVIII was shown by testing the plasma in a Bethesda clotting assay with chromogenic read-out.

The data was analyzed using Mann-Whitney test. Samples were analyzed individually and differences were regarded as significant if p<0.05.

Treatment with AAT prior to, and throughout the course of treatment with rFVIII significantly reduced antibody formation against rFVIII in the ELISA (Figure 2B; Table 2) and significantly reduced inhibitor levels in the Bethesda clotting assay (Figure 2C; Table 2). However, treatment with AAT only 24 hours prior to rFVIII did not significantly reduce rFVIII antibody formation (Figures 2B and 2C). Table 2: Reduction in antibody formation against rFVIII following prophylactic alpha- 1 antitrypsin treatment prior to, and throughout the course of rFVIII therapeutic protein treatment.

Treatment group Anti-FVIII Ab FVIII inhibitors

(Titer at 0.5) (BU/ml)

CSL627 only 1126 0.78

CSL627 only 181384 50.39

CSL627 only 304 0.40

CSL627 only 4623 13.16

CSL627 only 3554 8.86

CSL627 only 69 0.00

Mean+SKM 31843+29917 12.26+7.94

AAT (60mg/kg, 3-4x) + CSL627 1 8 0.00

AAT (60mg/kg, 3-4x) + CSL627 7054 4.21

AAT (60mg/kg, 3-4x) + CSL627 0 0.00

AAT (60mg/kg, 3-4x) + CSL627 9 0.00

AAT (60mg/kg, 3-4x) + CSL627 1369 0.34

AAT (60mg/kg, 3-4x) + CSL627 29 0.00

AAT (60mg/kg, 3-4x) + CSL627 17 0.00

AAT (60mg/kg, 3-4x) + CSL627 24 0.00

AAT (60mg/kg, 3-4x) + CSL627 35 0.43

Mean+SKM 951+777 0.55+0.46

AAT (60mg/kg, prime only) + CSL627 8185 10.12

AAT (60mg/kg, prime only) + CSL627 0 0.00

AAT (60mg/kg, prime only) + CSL627 12096 8.39

AAT (60mg/kg, prime only) + CSL627 0 0.00

AAT (60mg/kg, prime only) + CSL627 161 0.00

AAT (60mg/kg, prime only) + CSL627 541 1.56

AAT (60mg/kg, prime only) + CSL627 858 1.59

AAT (60mg/kg, prime only) + CSL627 24 0.57 Treatment group Anti-FVIII Ab FVIII inhibitors

(Titer at 0.5) (BU/ml)

AAT (60mg/kg, prime only) + C SL627 4143 14.47

Mean+S M 2890+1474 4.0X+I .X2

AAT (120mg/kg, prime only) + CSL627 298 0.32

AAT (120mg/kg, prime only) + CSL627 249 0.00

AAT (120mg/kg, prime only) + CSL627 221 0.00

AAT (120mg/kg, prime only) + CSL627 135 0.00

AAT (120mg/kg, prime only) + CSL627 22 0.00

AAT (120mg/kg, prime only) + CSL627 1518 2.70

AAT (120mg/kg, prime only) + CSL627 438 11.33

AAT (120mg/kg, prime only) + CSL627 8997 23.63

Mean+SKM 14X5+10X6 4.75+3.03

Example 3: Prophylactic treatment with alpha-1 antitrypsin prior to and during the course of rFVIII treatment reduced rFVIII antibody formation

Adult male and female HaemA mice (FVIII KO, Jackson Lab) of 10-12 weeks of age were treated 4 times with 120 IU/kg of rFVIII (CSL627). Injections were given 7 days apart and intravenously. Alpha-1 anti-trypsin inhibitor (AAT, Zemaira) was injected intraperitonealy at a dose of 120mg/kg every 3-4 days for the duration of rFVIII treatment. Treatment commenced 24 h prior to the first injection with rFVIII. Figure 3A is a schematic representation of the treatment strategy.

Seven days after the last injection with rFVIII, mice were euthanized with C0 ₂ for terminal plasma collection.

Efficiency of AAT in preventing antibody formation against rFVIII was shown by testing the plasma in an ELISA coated with rFVIII. Efficiency of AAT in preventing inhibitor formation against rFVIII was shown by testing the plasma in a Bethesda clotting assay with chromogenic read-out.

The data was analyzed using unpaired T-test. Samples were analyzed individually and differences were regarded as significant if p<0.05.

Treatment with AAT prior to, and throughout the course of treatment with rFVIII significantly reduced antibody formation against rFVIII in both the ELISA (Figure 3B; Table 3) and Bethesda clotting assay (Figure 3C; Table 3).

Table 3: Reduction in antibody formation against rFVIII following prophylactic alpha- 1 antitrypsin treatment prior to, and throughout the course of rFVIII therapeutic protein treatment.

Treatment Anti-FVIII Ab FVIII

group (Titer at 0.5) inhibitors

(BU/ml)

CSL627 only 23 0.42

CSL627 only 16553 30.05

CSL627 only 34 0.00

CSL627 only 132 0.40

CSL627 only 19564 55.46

CSL627 only 164 0.00

CSL627 only 2038 6.83

CSL627 only 24234 42.25

CSL627 only 13453 62.95

CSL627 only 125 4.33

Mean + SKM 7632+3067 20.27+7.95

AAT + CSL627 375 1.38

AAT + CSL627 34 0.33

AAT + CSL627 61 0.00

AAT + CSL627 33 0.70

AAT + CSL627 44 0.00

AAT + CSL627 92 0.96

AAT + CSL627 227 1.18

AAT + CSL627 565 1.54

AAT + CSL627 19 0.00

AAT + CSL627 5907 1.27

Mean + SKM 736+577 0.74+0.19