WO2022212930A1 - Method of administering anti-hpa-1a gamma globulin - Google Patents

Abstract

Methods are described for administering to a subject anti-human platelet antigen (HPA)-1a gamma globulin. The methods include parenterally administering to the subject a pharmaceutical composition comprising anti-HPA-la gamma globulin in an amount effective to achieve a maximum plasma concentration of anti-HPA-la gamma globulin of about 0.01 lU/mL to about 10 IU/mL in the subject.

Description

Method of Administering Anti-HPA-la Gamma Globulin

BACKGROUND

[0001] Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs when the mother’s immune system attacks her fetus’ platelets. This response by the mother’s immune system is due to proteins, inherited from the father, that are located on the surface of the fetus’ platelets but absent from the mother’s platelets. If fetal platelets enter the mother’s circulation, the mother’s immune system produces antibodies to fight against the platelets in an alloimmune response. These antibodies can pass through the placenta and bind to and destroy the fetus’ platelets, resulting in fetal thrombocytopenia.

[0002] The consequences of FNAIT can be severe. While in some cases there are no symptoms, fetuses and newborns with FNAIT can experience mucosal bleeding, hematomas, retinal bleeding, and intracranial hemorrhage (ICH), which may lead to intrauterine death or lifelong disability (Radder et al. 2003; Kjeldesen-Kragh and Skogen 2013).

[0003] Retrospective studies have shown that alloimmunization and the development of FNAIT can occur during the antenatal period of the first pregnancy. In a cohort study that assessed pregnancies of children diagnosed with FNAIT who had a confirmed ICH, 70% of the confirmed ICH cases occurred in the first-born, wherein in 54% of the cases bleeding initiated prior to gestational Week 28, and in 46% of the cases there were first or second trimester miscarriages that preceded the first-born with FNAIT (Tiller et al. 2013). A more recent study similarly reported that 71% of ICH cases occurred during first-affected pregnancies, and of those cases intrauterine fetal death due to ICH occurred between 22 and 38 weeks of gestation (Jin et al. 2019).

[0004] The most common platelet protein that can cause FNAIT is human platelet antigen (HPA)-la, which accounts for over 80% of FNAIT cases (Ghevaert et al. 2007; Peterson et al. 2013). The propensity to develop HPA-la antibodies is closely linked to a certain human leukocyte antigen (HLA) type (Kjeldesen-Kragh et al. 2007). The risk for women who are HPA- la negative to become HPA-la immunized is approximately 25 times higher in those who are positive for the HLA-DRB3*01:01 allele than in women who do not carry this allele (Kjeldesen- Kragh and Olsen 2019).

[0005] However, there is no accepted treatment available for the prevention of alloimmunization in HPA-la negative women. For women with known pregnancies at risk for developing FNAIT (women who previously gave birth to an FNAIT-affected child), the general approach is weekly maternal intravenous administration of human immune globulin (IGIV), with or without the addition of glucocorticoids, but there is no consensus as to the optimal dosing strategy. Moreover, IGIV does not prevent alloimmunization and, in the doses administered, is accompanied by reports of poor tolerability (Vitiello et al. 2019; Rossi et al. 2015).

[0006] Thus, there remains a need for an effective treatment to prevent alloimmunization to HP A- la-positive platelets in the mother.

SUMMARY OF THU INVENTION

[0007] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Example, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various ways, and all such combinations are intended to fall within the scope of the present invention.

[0008] Accordingly, the disclosure provides a method of administering HPA-la gamma globulin to a subject that is HPA-la negative, in which the method comprises parenterally administering to the subject a pharmaceutical composition comprising a preparation of the anti- HP A- la gamma globulin. The disclosure also provides a pharmaceutical composition comprising a preparation of HPA-la gamma globulin for use in parenterally administering the anti-HP A-la gamma globulin to a subject. The anti-HP A-la gamma globulin is administered in an amount effective to achieve a maximum plasma concentration of the anti -HPA-la gamma globulin of about 0.01 IU/mL to about 10 IU/mL in the subject.

[0009] In some embodiments, the maximum plasma concentration of the anti-HPA-la gamma globulin is about 0.05 IU/mL to about 5 IU/mL, or about 0.1 IU/mL to about 3 IU/mL. In certain embodiments, the maximum plasma concentration of the anti-HPA-la gamma globulin is about 0.5 IU/mL to about 3 IU/mL.

[0010] In some embodiments, the anti-HPA-la gamma globulin is administered in an amount of about 50 IU to about 10,000 IU, or about 100 IU to about 10,000 IU. In certain embodiments, the anti-HPA-la gamma globulin is administered in an amount of about 1000 IU to about 10,000 IU.

[0011] In some embodiments, the pharmaceutical composition is administered intravenously.

In alternative embodiments, the pharmaceutical composition is administered subcutaneously.

[0012] In some embodiments, the subject is an HPA-la negative woman. In certain embodiments, the subject is pregnant. In particular embodiments, the subject is HLA- DRB3*01:01 positive.

[0013] In embodiments of the invention, the administration of the anti-HPA-la gamma globulin achieves clearance of HPA-la positive platelets in the subject within about 10 hours, or within about 5 hours, of administering the anti-HPA-la gamma globulin to the subject. In certain embodiments, administration of the anti-HPA-la gamma globulin achieves clearance of HPA-la positive platelets in the subject within about 3 hours of administering the anti-HPA-la gamma globulin to the subject. In some embodiments, half-life of the HPA-la positive platelets is reduced by about 150-250 fold in the subject relative to a subject who has not been administered the anti-HPA-la gamma globulin.

[0014] In certain embodiments, the administration of the anti-HPA-la gamma globulin prevents an alloimmune response to HP A- la-positive platelets in the subject. In particular embodiments, the administration of the anti-HPA-la gamma globulin induces antibody-mediated immune suppression of an immune response to HP A- la-positive platelets in the subject.

[0015] In addition, embodiments of the invention include those in which the pharmaceutical composition comprises a stabilizing agent and a surfactant. In some embodiments, the pharmaceutical composition comprises maltose and polysorbate 80. In certain embodiments, the pharmaceutical composition comprises maltose in an amount of about 10% (w/w) and polysorbate 80 in an amount of about 0.03% (w/w).

[0016] In particular embodiments, the pharmaceutical composition has a pH of about 5.0-6.5. [0017] Further aspects, features, and advantages of the present invention will be better appreciated upon a reading of the following detailed description of the invention and claims.

BRIEF DESCRIPTION OF THE FTGTTRES

[0018] FIG. 1A-1B show the proportion of transfused HPA-la positive platelets in circulation after administration of anti-HP A-la gamma globulin or placebo in cohorts 1 and IB over 7 days (FIG. 1A) and 24 hours (FIG. IB). Data are normalized at 100% for baseline samples collected prior to anti-HP A-la gamma globulin administration (cohort 1) or at the end of platelet infusion (cohort IB). Time is measured from anti-HP A-la gamma globulin administration (cohort 1) or the end of platelet infusion (cohort IB). Each line represents a different subject. Triangles denote cohort 1; circles denote cohort IB.

[0019] FIG. 2A-2B show results of platelet saturation studies. FIG. 2A shows flow cytometric analysis of the binding of human IgG (left panel) or anti-HP A- la gamma globulin (right panel) to isolated human platelets from donors heterozygous for HPA-1. FIG. 2B shows flow cytometry results of two experiments in which HPA-1 a/b platelets were incubated with the indicated antibody concentration in anti -HPA-1 a gamma globulin, measured in mean fluorescence intensity (MFI).

PET ATT, ED DESCRIPTION OF THE INVENTION

[0020] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, immunology, hematology, cell biology, molecular biology, clinical pharmacology, and clinical practice, which are within the skill of the art.

[0021] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0022] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. [0023] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.

Definitions

[0024] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0025] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0026] Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone).

Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0027] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are included.

[0028] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10. Where a numeric term is preceded by “about,” the term includes the stated number and values ±10% of the stated number. [0029] The term “antibody” refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having one “heavy” chain and one “light” chain. Each chain is comprised of a variable region, which forms the antibody binding site, and a constant region, which may mediate the binding of the antibody to host tissues or factors. Immunoglobulin molecules can be divided into subclasses depending on the constant region of the heavy chain. The classes are immunoglobulin gamma (IgG), immunoglobulin mu (IgM), immunoglobulin delta (IgD), immunoglobulin epsilon (IgE), and immunoglobulin alpha (IgA). The heavy chain constant regions differ structurally and antigenically among the classes. IgG is the main type of antibody found in blood and extracellular fluid, and it plays a central role in the humoral immune response.

[0030] Glycoprotein Ilb/IIIa (GPIIb/IIIa), also known as integrin al Ibp3 , is a platelet membrane glycoprotein that binds fibrinogen and von Willebrand factor, and plays a role in platelet activation. Human platelet antigen-1 (HPA-1) is a polymorphism at position 33 on the b3 chain of GPIIb/IIIa. In particular, individuals who have a Leu at position 33 are “HPA-la positive,” “positive for HPA-la,” or “HPA-la,” while individuals who have a Pro at position 33 are “HPA-la negative,” “negative for HPA-la,” “HPA-lb homozygous,” or “HPA-lbb.”

[0031] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g, acetate, phosphate, or citrate buffer), a surfactant (e.g, polysorbate), a stabilizing agent (e.g, polyol or amino acid), a preservative (e.g, sodium benzoate), and/or other conventional solubilizing or dispersing agents.

[0032] A “subject” or “individual” or “patient” is any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g, humans, non-human primates, canines, felines, porcines, bovines, equines, rodents, including rats and mice, rabbits, etc. [0033] The term “international unit” or “IU” is a unit of measurement of the amount of a substance as determined by its activity. The mass or volume that constitutes one international unit of a substance will vary based on the substance that is being measured. For anti-HP A-la antibodies, the quantity of antibodies in one IU is set to an international standard (Allen et al. 2005) adopted by the World Health Organization ( see WHO International Standard: Anti-HP A- la Standard (100 IU)).

[0034] An “effective amount” of a therapy is an amount sufficient to carry out a specifically stated purpose, such as to elicit a desired biological or medicinal response in a subject.

[0035] The terms “clear” or “clearance” or “eliminate” or “elimination” are used interchangeably and refer to achieving an undetectable level of a cell type, for example, HP A-la positive platelets. Detection of the cell type can be carried out by known methods, including, for example, immunohistochemistry or flow cytometry, such as fluorescence-activated cell sorting (FACS).

[0036] The terms “inhibit,” “block,” “suppress” and “reduce” are used interchangeably and refer to any statistically significant decrease in occurrence or activity or extent or volume, including full blocking or complete elimination of the occurrence or activity or extent or volume. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%,

70%, 80%, 90% or 100% in activity or occurrence. As another example, “reduction” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in extent or volume.

[0037] “Binding” generally refers to the non-covalent interaction between a single binding site of a molecule and its binding partner ( e.g ., a receptor and its ligand, an antibody and its antigen, two monomers that form a dimer, etc.). In the case of binding between an antibody and its antigen, the interaction can, for example, prevent other molecules from binding to or recognizing the antigen, can initiate the destruction of the antigen, or can alter the structure or functionality of the antigen.

[0038] The terms “alloimmune response” or “alloimmunization” is an immune response to non-self antigens that are from the same species. As a result, the body produces antibodies against the non-self antigens. [0039] The term “antibody-mediated immune suppression” or “AMIS” refers to a process in which an acquired antibody inhibits sensitization to a given antigen or otherwise modulates the development of an immune response to the antigen.

Anti-HP A-la Gamma Globulin

[0040] The invention relates to the use of a preparation comprising polyclonal anti-HPA-la antibodies of the gamma isotype. The preparation is referred to herein as “anti-HPA-la gamma globulin.”

[0041] The preparation is produced from pooled plasma of donors with anti-HPA-la antibodies. In one embodiment, the donor is an HP A-la negative subject who has been alloimmunized with HPA-la, for example, as a result of a previous pregnancy with an HP A- la- positive fetus. In another embodiment, the donor is an HPA-la negative subject who has been deliberately immunized with HPA-la positive platelets or with a purified or recombinant preparation HPA-la antigen. The preparation contains the total IgG from the pooled source plasma.

[0042] The preparation of anti-HPA-la gamma globulin may be manufactured by a process comprising: purification of the IgG from the source plasma containing antibodies to HPA-la, clearance of viruses from the purified IgG, and concentration of the purified IgG. Purification of the IgG is performed preferably using anion-exchange chromatography, although other suitable techniques may be used, such as alcohol fractionation (see, e.g. , Cohn el al. 1946; Oncley el al. 1949; Kistler and Nitschmann 1962) and polyethylene glycol (PEG) precipitation (see, e.g., Poison and Ruiz-Bravo 1972).

[0043] Viral clearance in the purified IgG may be performed by virus removal (for example, by phase partitioning or PEG precipitation, affinity chromatography, ion exchange or gel exclusion chromatography, filtration, etc.), by virus inactivation (for instance, by cold ethanol fractionation, heating, solvent/detergent, exposure to an acidic environment, etc.), or a combination thereof. In preferred embodiments, viral clearance may be performed by both nanofiltration and exposing the purified IgG to a solvent detergent such as tri-n-butyl phosphate. Use of a solvent detergent to clear viruses from the purified IgG may be followed by a step of removing the solvent detergent, for example, by reverse-phase chromatography. [0044] Following viral clearance, the purified IgG may be concentrated using, for example, ultrafiltration. In addition, diafiltration may be used to remove microsolutes such as salts from the preparation.

[0045] Additional steps may be included in manufacturing the preparation of the anti-HPA-la gamma globulin. For instance, prior to purification, plasma from different sources ( e.g ., from different persons) may be pooled together. The pooled plasma may be diluted, and dextran sulphate may be added the pooled plasma to remove lipids. Further, after viral clearance, a step may be performed to reduce procoagulant factors (e.g., such as Factor XI and activated Factor XI) using, for instance, affinity chromatography.

[0046] In some embodiments, the anti-HPA-la gamma globulin may be formulated in a pharmaceutical composition. The pharmaceutical composition may comprise one or more carriers, diluents, excipients, or other additives. For example, the composition can comprise one or more stabilizing agents (e.g, dextran 40, glycine, lactose, mannitol, trehalose, maltose), one or more buffers (e.g, acetate, citrate, histidine, lactate, phosphate, Tris), one or more pH adjusting agents (e.g, hydrochloric acid, nitric acid, potassium hydroxide, sodium hydroxide), one or more surfactants (polysorbate, sodium lauryl sulfate, polyethylene glycol-fatty acid esters, lecithins), and/or one or more diluents (e.g, water, physiological saline). The pH of the composition is preferably between about 3.0 and 8.0. In certain embodiments, the pH is between about 4.0 and 7.0, or between about 5.0 and 6.5. In certain embodiments, the composition does not comprise any preservatives. In certain embodiments, the composition does not comprise mercury.

[0047] In certain embodiments, the pharmaceutical composition comprises anti-HPA-la gamma globulin; maltose in an amount of about 5% to 15%, or about 10% (w/w); and polysorbate 80 in an amount of about 0.1% to about 0.05%, or about 0.03% (w/w).

[0048] In certain embodiments, the total protein concentration in the pharmaceutical composition is about 10 mg/mL to about 150 mg/mL, or about 20 mg/mL to about 100 mg/mL, or more particularly about 30 mg/mL to about 100 mg/mL, or about 30 mg/mL to about 92 mg/mL. [0049] In certain embodiments, the pharmaceutical composition contains no more than about 60 pg/mL, or no more than about 50 pg/mL, or no more than about 40 pg/mL, of immunoglobulin A.

Methods of Administering Anti-HP A-la Gamma Globulin

[0050] Aspects of the invention relate to methods of administering the preparation of anti- HP A- la gamma globulin to a subject that is HPA-la negative. These methods may provide delivery of the anti-HPA-la gamma globulin preparation to the subject in an effective and safe amount. Accordingly, the methods of the invention comprise parenterally administering to the subject a pharmaceutical composition comprising a preparation of anti-HPA-la gamma globulin, in which the anti-HPA-la gamma globulin is administered to the subject in an amount effective to achieve a maximum plasma concentration of anti-HPA-la gamma globulin of about 0.1 IU/mL to about 5 IU/mL in the subject.

[0051] In some embodiments, the anti-HPA-la gamma globulin is administered in an amount effective to achieve a maximum plasma concentration of anti-HPA-la gamma globulin of about 0.01 IU/mL to about 10 IU/mL, or about 0.05 IU/mL to about 5 IU/mL, or about 0.1 IU/mL to about 4 IU/mL, or about 0.1 IU/mL to about 3 IU/mL, or about 0.5 IU/mL to about 3 IU/mL, in the subject. For example, the anti-HPA-la gamma globulin is administered in an amount effective to achieve a maximum plasma concentration of anti-HPA-la gamma globulin of about 0.01 IU/mL, or about 0.02 IU/mL, or about 0.03 IU/mL, or about 0.04 IU/mL, or about 0.05 IU/mL, or about 0.06 IU/mL, or about 0.07 IU/mL, or about 0.08 IU/mL, or about 0.09 IU/mL, or about 0.1 IU/mL, or about 0.2 IU/mL, or about 0.3 IU/mL, or about 0.4 IU/mL, or about 0.5 IU/mL, or about 0.6 IU/mL, or about 0.7 IU/mL, or about 0.8 IU/mL, or about 0.9 IU/mL, or about 1 IU/mL, or about 1.5 IU/mL, or about 2 IU/mL, or about 2.5 IU/mL, or about 3 IU/mL, or about 3.5 IU/mL, or about 4 IU/mL, or about 4.5 IU/mL, or about 5 IU/mL, or about 6 IU/mL, or about 7 IU/mL, or about 8 IU/mL, or about 9 IU/mL, or about 10 IU/mL, or any value in between, in the subject. These amounts can also serve as endpoints for a range of maximum plasma concentrations of anti-HPA-la gamma globulin to be achieved in the subject by administering the anti-HPA-la gamma globulin, for example, about 0.2 IU/mL to about 3.5 IU/mL, about 0.7 IU/mL to about 1.5 IU/mL, etc. [0052] In some embodiments, the anti-HPA-la gamma globulin may be administered to the subject in an amount of between about 50 IU to about 10,000 IU, or between about 100 IU to about 10,000 IU, or between about 500 IU and about 10,000 IU, or between about 1000 IU and about 10,000 IU, or between about 2000 IU and about 10,000 IU, or between about 3000 IU and about 10,000 IU, or between about 4000 IU and about 10,000 IU, or between about 5000 IU and about 10,000 IU. Examples of the amount of the anti-HPA-la gamma globulin that may be administered include about 50 IU, or about 100 IU, or about 150 IU, or about 200 IU, or about 250 IU, or about 300 IU, or about 350 IU, or about 400 IU, or about 450 IU, or about 500 IU, or about 550 IU, or about 600 IU, or about 650 IU, or about 700 IU, or about 750 IU, or about 800 IU, or about 850 IU, or about 900 IU, or about 950 IU, or about 1000 IU, or about 1,100 IU, or about 1, 200 IU, or about 1,300 IU, or about 1,400 IU, or about 1,500 IU, or about 1,600 IU, or about 1,700 IU, or about 1,800 IU, or about 1,900 IU, or about 2000 IU, or about 2500 IU, or about 3000 IU, or about 3500 IU, or about 4000 IU, or about 4500 IU, or about 5000 IU, or about 5500 IU, or about 6000 IU, or about 6500 IU, or about 7000 IU, or about 7500 IU, or about 8000 IU, or about 8500 IU, or about 9000 IU, or about 9500 IU, or about 10,000 IU.

These amounts can also serve as endpoints for a range of amounts to be administered, for example, about 2000 IU to about 9000 IU, about 4000 IU to about 8000 IU, etc.

[0053] In embodiments of the invention, administration of the anti-HPA-la gamma globulin according to embodiments of the present invention achieves clearance of HP A- la positive platelets in the subject. HPA-la positive platelets may be present in the subject for reasons that include, but are not limited to, the subject received a transfusion of HPA-la positive platelets, and HPA-la positive platelets were introduced to the subject during pregnancy (e.g., fetal HPA- la positive platelets entered the subject’s circulation). In some embodiments, such administration of the anti-HPA-la gamma globulin achieves accelerated clearance of HPA-la positive platelets in the subject. For example, clearance may be achieved within about 10 hours of administering the anti-HPA-la gamma globulin to the subject, such as within about 10 hours, or about 9 hours, or about 8 hours, or about 7 hours, or about 6 hours, or about 5 hours, or about four hours, or about 3 hours, or about 2 hours, or about 1 hour, of administering the anti-HPA-la gamma globulin to the subject. In some embodiments, clearance may be achieved within about lhour to about 10 hours, or about 1 hour to about 5 hours, or about 2 hours to about 4 hours, or about 2 hours to about 3 hours, of administering the anti-HPA-la gamma globulin to the subject. [0054] In some embodiments, administration of the anti-HPA-la gamma globulin according to the present invention prevents an alloimmune response to HP A- la-positive platelets in the subject. In some embodiments, administration of the anti-HPA-la gamma globulin according to the present invention induces antibody-mediated immune suppression of an immune response to HP A- la-positive platelets in the subject. In some embodiments, half-life of the HPA-la positive platelets is reduced by about 150-250 fold in the subject relative to a subject who has not been administered the anti-HPA-la gamma globulin.

[0055] The pharmaceutical composition comprising the preparation of the anti-HPA-la gamma globulin is administered parenterally. Parenteral routes of administration include intravenous, intramuscular, intraperitoneal, intrathecal, and subcutaneous. In a preferred embodiment, the pharmaceutical composition is administered via intravenous infusion or subcutaneously.

[0056] In embodiments of the invention, the subject is a woman who is HPA-la negative and is pregnant. In some embodiments, the subject is a woman who is HPA-la negative and had a previous pregnancy in which the fetus was HPA-la positive. In other embodiments, the subject is a pregnant woman who is HPA-la negative and is carrying an HPA-la positive fetus.

[0057] In some embodiments, the subject may carry the HLA-DRB3*01 :01 allele, i.e., is HLA- DRB3*01:01 positive. In other embodiments, the subject lacks the HLA-DRB3*01:01 allele.

[0058] As provided herein, the pharmaceutical composition may be administered without inducing a severe adverse event in the subject. As used herein, a severe adverse event encompasses any event that is fatal or immediately life-threatening; that requires inpatient hospitalization or prolongation of existing hospitalization; that results in persistent disability /incapacity; or that is a congenital anomaly/birth defect.

EXAMPLES

[0059] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

Example 1: Anti-HP A-la Gamma Globulin Dose-Finding Study

[0060] A randomized, single-center, placebo-controlled, single-blind study was conducted to establish the safety and efficacy of a single dose of anti-HP A-la gamma globulin needed to eliminate HP A-la positive platelets transfused to HP A-la- and HLA-A2 negative subjects.

Objectives and Endpoints

[0061] The objectives and endpoints of the study are presented in Table 1.

Table 1. Objectives and endpoints of the study.

Overall Design

[0062] The study was designed with four cohorts (1, IB, 2, and 3). In Cohorts 1, 2, and 3 (n =

8 for each cohort), three different anti-HPA-la gamma globulin doses are assessed. Participants are randomized to receive a study drug (either anti-HPA-la gamma globulin or placebo (3:1)) in a blinded manner, and are allocated randomly using randomization blocks of eight. For the first four participants in a cohort, the randomization is arranged to include one placebo participant as number 1 or 2, and one placebo participant as number 3 or 4, while the rest of the participants in the cohort receives anti-HPA-la gamma globulin. Data from Cohorts 1 and 2 are used to support the primary endpoint (platelet clearance), and data from Cohort 3 are used to establish pharmacokinetics of anti-HPA-la gamma globulin. Platelet transfusion for Cohort 1 is performed one hour prior to study drug administration. Platelet transfusion for Cohort 2 is performed one hour prior to study drug administration or seven days after study drug administration — the timing will be determined after completion of Cohort IB.

[0063] In Cohort IB (n = 4), participants are recruited upon completion of the safety review by the Safety Review Team of Cohort 1 and precedes Cohorts 2 and 3. Participants are randomized 1 : 1 to receive a study drug (anti-HPA-la gamma globulin or placebo) in a blinded manner. The randomization is arranged to include one placebo participant as number 1 or 2, and one placebo participant as number 3 or 4. Platelet transfusion is performed seven days following study drug administration. Data from Cohort IB are intended to provide an assessment of the duration of treatment effect of anti-HPA-la gamma globulin on platelet clearance, including whether the platelet clearance effect of anti-HPA-la gamma globulin persists for up to seven days. [0064] A maximum of two participants are treated in parallel and are monitored for three days before the next two participants may be exposed.

[0065] Following completion of each study cohort, the Safety Review Team evaluates safety data before opening enrollment to the subsequent cohorts. Major safety events include any severe adverse event (SAE) with suspected relation to study drug (especially hypersensitivity and severe hypotension [< 90/50 mmHg] or thrombotic event); and any other severe reaction possibly related to study drug according to the treating physician observed in more than one participant. After all of the participants in each cohort have completed seven days of follow-up after study drug administration, the Safety Review Team evaluates the cumulated data and either decides to open enrollment for the subsequent cohort or to terminate the study.

Study Duration

[0066] The study consists of a screening period (up to 28 days), a single dose administration of the study drug (one day), and a 24-week follow-up period. In total, participants are in this study for approximately 28 weeks.

Participants

[0067] Twenty-eight eligible HPA-la negative (and in Cohorts 1, IB, and 2: human leukocyte antigen [HLA]-A2 negative) healthy, male participants are included in this study. For the determination of the optimal dose of anti-HP A-l a gamma globulin, at least six evaluable participants in Cohorts 1 and 2 who received HPA-la positive platelets and anti-HPA-la gamma globulin dose are needed. Non-evaluable participants may be replaced (up to two replacements per cohort).

[0068] In Cohorts 1, IB, and 2, study participants must be both HPA-la negative and HLA-A2 negative. They are transfused with HPA-la positive platelets as the anti-HPA-la gamma globulin’s target. Additionally, differences in the HLA antigens between platelet donor and recipient are used to distinguish transfused platelets from endogenously produced platelets. Platelet Selection and Dose for Transfusion

[0069] In Cohorts 1, IB, and 2, the study participants must be HPA-la negative, because they receive a standardized transfusion of HPA-la positive platelets. As HLA class I antigens are expressed on platelets, differences in the HLA antigens between platelet donor and recipient are used to distinguish transfused platelets from endogenously produced platelets.

[0070] To avoid that anti-A and/or anti-B in plasma of the study participants sensitize transfused platelets, which reduces platelet survival, transfusion of platelets follows general rules to prevent major ABO mismatch, i.e., recipients of blood group O can be transfused with platelets from blood group O donors; recipients of blood group A can be transfused with platelets from blood donors of group O or blood group A; recipients of blood group B can be transfused with platelets from blood donors of group O or blood group B, and recipients of blood group AB can be transfused with platelets from donors of all ABO groups.

[0071] To ensure HLA discrepancy between platelet donor and recipient, which is necessary for detecting the transfused platelets by flow cytometry, the donors’ platelets are positive for HLA-A2, which is not expressed on the recipient’s platelets.

[0072] The dose of HPA-la positive (and HLA-A2 positive) platelets being transfused is 10 x 10⁹, which corresponds to the approximate number of platelets in 30 mL of fetal blood.

[0073] The platelets being transfused are obtained from platelet apheresis products from platelet donors. The platelet units are manufactured according to standard practice. The donors all have the platelet type HPA-la/b (heterozygous) and are HLA-A2 positive (homozygous) and do not have HLA antibodies.

[0074] In Cohort 1, the platelet transfusion is performed 60 minutes prior to treatment with anti-HP A-la gamma globulin. In Cohort IB, the platelet transfusion is performed seven days after treatment with anti-HP A-la gamma globulin. In Cohort 2, the platelet transfusion is performed either 60 minutes prior to treatment with anti-HPA-la gamma globulin, or seven days after treatment with anti-HPA-la gamma globulin.

[0075] During transfusion, a peripheral venous catheter is placed into a peripheral vein. Before administration of the platelet concentrate, the platelet unit is thoroughly, but gently mixed. An ordinary transfusion set, suitable for platelet transfusions, is coupled to the platelet unit. A syringe (10 mL) is attached to the luer-lock of the transfusion set and used to withdraw the appropriate volume from the platelet concentrate. The syringe for transfusion (after calculation of the appropriate volume of the platelets and withdrawal of another 10 mL blood from the peripheral venous catheter) is then immediately connected to the peripheral venous catheter and the platelets are transfused over a period of one to two minutes. After completing the transfusion, a syringe with attached needle is used to withdraw approximately 10 mL from a vial containing 50 mL of saline (sodium chloride 0.9%). The peripheral venous catheter is then flushed with 10 mL saline. Another 6 mL of the remaining platelet apheresis product is collected and used as a positive control for the flow cytometry measurements. Unused platelet concentrate is discarded at the study site. Time zero (To) is recorded as the time the saline flush is complete.

Study Drug and Administration

[0076] Participants are administered either anti-HPA-la gamma globulin or a placebo. The anti-HP A-la gamma globulin is provided in 6 mL vials, in which each vial contains 200 IU/mL of the anti-HPA-la gamma globulin. The anti-HPA-la gamma globulin is delivered as a one time administration at the following dosages:

Cohort 1: 1,000 IU

Cohort IB: 1,000 IU

Cohort 2: 4,000 IU

Cohort 3: 10,000 IU

[0077] The placebo is a sodium chloride injection, 0.9% (saline).

[0078] In all cohorts, the study drug is administered as a slow injection through the peripheral venous catheter (approximately 10 mL/60 sec). After administration of study drug, the peripheral venous catheter is flushed with 10 mL saline (0.9% NaCl solution). The participant is carefully observed during the injection and continuously after the administration (including continuous monitoring of vital signs and electrocardiogram).

End of Study

[0079] A participant is considered to have completed the study if he/she has completed all phases of the study including the last scheduled procedure. [0080] The primary completion date is defined as the date when the last participant is assessed or receives an intervention for the final collection of data for the primary endpoint(s) for the purposes of conducting the primary analysis. This date is defined as when the last participant has completed assessments for Day 1.

[0081] The end of the study is defined as the date of the last visit of the last participant in the study or last scheduled procedure for the last participant in the study.

Eligibility Criteria

[0082] Inclusion criteria are as follows:

1. Capable of giving signed informed consent, which includes compliance with the requirements and restrictions listed in the informed consent form (ICF) and in this protocol.

2. Healthy male participants, > 18 and < 65 years of age.

3. Body mass index (BMI) < 35 kg/m².

4. HP A- la negative

5. Cohorts 1, IB, and 2 only: HLA-A2 negative.

[0083] Exclusion criteria are as follows

1. History of hypersensitivity to platelet concentrates or human plasma proteins.

2. IgA levels < 0.06 g/L.

3. Blood transfusion received within 3 weeks of screening.

4. Platelet counts < 150 x 10⁹/L or > 450 x 10⁹/L.

5. Any type of known platelet function disorder.

6. Treatment with non-steroidal anti-inflammatory drugs (NSAIDs, e.g, acetylsalicylic acid) or selective serotonin reuptake inhibitors within 7 days prior to screening.

7. Chronic or ongoing active infectious disease requiring systemic treatment including, but not limited to, chronic renal infection, chronic chest infection with bronchiectasis, and tuberculosis. 8. Individuals with increased risk of thrombotic events ( e.g ., history of pulmonary embolism or thrombosis).

9. Current participation in any other interventional clinical study (or within 90 days prior to screening).

10. Participants known or suspected of not being able to comply with this study protocol (e.g., due to alcoholism, drug dependency, or psychological disorder).

11. Presence of HLA class I-antibodies at screening; for participants, not previously transfused, this is defined as mean fluorescence intensity (MFI) value > 3,000 on the One-Lambda’s LAB Screen Single Antigen assay.

12. Ongoing active infection with HIV and/or hepatitis B and/or C virus.

13. Vaccination received within 1 month of screening.

14. Current diagnosis of diabetes mellitus or increased hemoglobin Ale (HbAlc) at screening.

15. Body mass index (B MI) > 35 kg/m2.

Efficacy Assessment

[0084] Flow cytometry is used to monitor survival of the transfused platelets by detecting circulating platelets expressing the relevant HLA antigens. Blood samples are analyzed consecutively. If transfused platelets cannot be detected in two consecutive samples, samples from subsequent time points are not obtained or examined.

[0085] Flow cytometry is a well-proven method for determining the exact size of minute cell populations in peripheral blood and has been used for years to examine patients for minimal residual disease after treatment for hematological malignancies (Bene and Kaeda 2009). A new flow cytometry -based method was developed which directly assesses the survival of transfused platelets (Vetlesen et al. 2012). As platelets express HLA class I molecules on their surface, the flow cytometry -based method takes advantage of discrepancy in HLA class I molecules between donor and recipient. By using fluorochrome-conjugated anti-HLA antibodies, it is possible to distinguish between populations of platelets with different HLA types. [0086] Platelets are collected at specified timepoints after platelet transfusion and/or after study drug administration.

Safety Assessments

[0087] The safety assessment includes physical examinations, measurements of vital signs, electrocardiograms, and clinical safety laboratory assessments scheduled at screening, on the day of study drug administrations, and for specified periods thereafter. The clinical safety laboratory assessment includes platelet, red blood cell, and white blood cell counts; clinical chemistry measurements that include blood urea nitrogen, creatine, glucose, potassium, calcium, alanine transaminase/serum glutamic-pyruvic, aspartate transaminase/serum glutamic-oxaloacetic transaminase; alkaline phosphatase, total and direct bilirubin, and total protein; urinalysis measurements that includes specific gravity, pH, glucose, protein, blood, ketones, bilirubin, urobilinogen, nitrite, and leukocyte esterase by dipstick; serology measurements that include HIV antibody, hepatitis B surface antigen [HBsAg], and hepatitis C virus antibody; and coagulation measurements that include prothrombin time and international normalized ratio.

[0088] The occurrence of adverse events and serious adverse events are also monitored. An adverse event is any untoward medical occurrence in a patient or clinical study participant, temporally associated with the use of study intervention, whether or not considered related to the study intervention. Examples of events that meet the definition of an adverse event includes any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments ( e.g ., ECG, radiological scans, vital signs measurements), including those that worsen from baseline, considered clinically significant in the medical and scientific judgment of the investigator; exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition; any new condition detected or diagnosed after study intervention administration even though it may have been present before the start of the study; and signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction.

[0089] A serious adverse event is defined as any untoward medical occurrence that, at any dose, results in death, is immediately life threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent disability/incapacity, is a congenital anomaly/birth defect, or another medically important serious event that may jeopardize the participant or may require medical or surgical intervention to prevent one of the other outcomes listed in this definition.

Pharmacokinetic Assessments

[0090] Anti-HPA-la gamma globulin pharmacokinetics are assessed for the participants in Cohort 3 only. Serum samples will be collected for measurement of concentrations of the study drug at the following timepoints:

^■ 15 min prior to study drug administration

^■ 20 min, 40 min, 1 hr, 4 hr, and 24 hr after study drug administration

^■ Days 3 and 7, and Weeks 2, 4, 8, 12, 16, 20, and 24.

Measurement of Anti-HPA-la Antibodies

[0091] Antibodies to HPA-la platelets will be evaluated in blood samples collected from all participants to assess exogenous anti-HPA-la gamma globulin (to support anti-HPA-la gamma globulin pharmacokinetic endpoints via Cohort 3) or endogenously developed anti-HPA-la antibodies (safety monitoring in all cohorts). In some embodiments, anti-HPA-la antibodies may be identified as exogenous anti-HPA-la gamma globulin or endogenously developed anti- HPA-la antibodies based on their immunoglobulin class — exogenous anti-HPA-la gamma globulin consists entirely of IgG, while endogenously developed anti-HPA-la antibodies are of the IgM class during the first phase of the immune response and subsequently switch to the IgG class. Samples collected for detection of antibodies may also be evaluated for HPA-la platelets serum concentration to enable interpretation of the antibody data.

[0092] Blood samples will be screened for antibodies binding to HPA-la platelets and the antibody levels in confirmed positive samples will be reported. Other analyses may be performed to verify the stability of antibodies to HPA-la platelets and/or further characterize the immunogenicity of HPA-la platelets.

Example 2: Anti-HPA-la gamma globulin Clears HPA-la Positive Platelets

Administration to Cohorts 1 and IB

[0093] As described in Example 1, participants of Cohort 1 (n=8) were both HPA-la negative and HLA-A2 negative. They were randomized (3 : 1) to receive the study drug (anti-HPA-la gamma globulin) or placebo, in a blinded manner and were allocated randomly using randomization blocks of eight. For the first four participants, the randomization was arranged to include one placebo participant as number 1 or 2, and one placebo participant as number 3 or 4, while the rest of the participants received anti-HPA-la gamma globulin.

[0094] On Day 1, the participants received a transfusion of HP A- la positive (and HLA-A2 positive) platelets at a dose of 10 ^c 10^9, equivalent to the number of platelets in 30 mL of fetal blood (Commission 2021). Sixty minutes after completion of the transfusion, the study drug was administered. Participants administered the anti-HPA-la gamma globulin (n=6) received a dose of 1,000 IU, while participants administered the placebo (n=2) received a sodium chloride injection, 0.9% (saline), with a follow-up period of 24 weeks.

[0095] As described in Example 1, after randomization, subjects in cohort IB (n=4) received either IV anti-HPA-la gamma globulin (1000 IU of anti-HPA-la; n=3) or placebo (n=l), followed 7 days later by transfusion of 10 ^c 10⁹ HP A- lab positive and HLA-A2 positive platelets (day 1), with a 12-week follow-up period.

[0096] Cohort 1 was designed to assess the ability of anti-HPA-la gamma globulin to eliminate HPA-lab platelets immediately upon its administration. Cohort IB, in contrast, was designed to simulate prophylactic administration of anti-HPA-la gamma globulin prior to fetal- maternal transfer of HPA-lab platelets.

Platelet and Plasma Preparation

[0097] Platelets to be transfused were obtained by plateletpheresis from existing ABO- compatible platelet donors at the German Red Cross Blood Donor Service Baden-Wurttemberg- Hessen, Frankfurt, Germany. Platelets were obtained 20 to 24 hours before transfusion. All platelet donors were HPA-la/b heterozygous and HLA-A2 homozygous. None of the platelet donors had HLA antibodies.

[0098] Platelet-rich plasma prepared from whole blood collected from the recipients was used to determine the proportion of HLA-A2 positive platelets. Platelet-rich plasma was prepared from the anticoagulant citrate dextrose (ACD-A) plasma collected from the study subjects, and platelets were immediately preserved with ThromboFix Platelet Stabilizer (Beckman Coulter) according to the manufacturer’s instructions. Data Collection

[0099] The proportion of transfused HPA-lab platelets in circulation after administration of anti-HPA-la gamma globulin or placebo was determined by flow cytometry using the HLA-A2 discrepancy between donor (HLA-A2 positive) and recipient (HLA-A2 negative). Proof of concept was prospectively defined as elimination of HP A- la positive platelets by 10-fold or greater based on platelet half-life.

[00100] Both endogenous and transfused platelets in cohort 1 were analyzed 15 minutes before platelet transfusion; 15 minutes before study drug administration ( i.e ., 1 hour later); 10, 20, 30, 40, and 50 minutes and 1, 2, 3, 4, and 24 hours after study drug administration; on day 3; and on day 7, if transfused platelets were still detectable on day 3.

[00101] For cohort IB, in which study drug was administered 7 days before platelet transfusion, platelet analysis took place 15 minutes before platelet transfusion; 10, 20, 30, 40, and 50 minutes and 1, 2, 3, 4, and 24 hours after platelet transfusion; on day 3; and on day 7, if platelets were detected in a transfused individual on day 3.

Flow Cytometry

[00102] A validated flow cytometry-based method was used to determine the frequency of HLA-A2 positive platelets in the recipient at specified time points after administration of anti- HPA-la gamma globulin (Vetlesen et al. 2012; Kjaer et al. 2018). Optimization and validation of this method were performed on mixtures of low frequencies of HLA-A2 positive platelets into HLA-A2 negative platelets, and expected frequencies were compared with observed frequencies. The lower limit of quantification (LLOQ) was 0.015%, and linearity was 0.97. Approximately 1.5 x 10⁶ platelets were double-stained with 6 fluorescein isothiocyanate (FITC)-conjugated HLA-A2 antibodies (3.6%; clone H0037; ONE Lambda Inc.) and 12 PC5-conjugated CD41 antibodies (0.7%; clone P2; Beckman Coulter). After 20 minutes of incubation in the dark at room temperature, 600 pL of fixation buffer (0.2% paraformaldehyde in phosphate-buffered saline) was added.

[00103] One million events were collected at the lowest collection rate using a Canto II flow cytometer ( Becton Dickinson). FACSDiva software (BD Bioscience) was used to determine the proportion of transfused platelets. Platelets were identified on scatter plots showing the forward scatter properties versus PC5 fluorescence, and the frequency of transfused platelets was assessed on scatter plots showing FITC fluorescence versus side scatter. Blood samples were analyzed consecutively. If transfused platelets could not be detected in 2 consecutive samples, samples from subsequent time points were not obtained or examined.

Analysis of HPA-la Antibodies

[00104] To determine whether infusion of HP A- la-incompatible platelets had induced HPA- la antibodies, blood was drawn to quantify HPA-la antibodies, as described by Mortberg el al. The World Health Organization International Standard anti-HP A-la (100 IU; NIBSC code 03/152) was used as a calibrator (Mortberg et al. 2016). The five-parameter model was used for curve fitting and for calculating concentrations in study samples, using SoftMaxPro v. 7.1 software (Molecular Devices).

Statistical Analysis

[00105] Data were tabulated and presented graphically. Platelet kinetic analysis was performed on all subjects for whom sufficient data were available to derive at least one of the platelet kinetic endpoints. The safety analysis set included all subjects who received platelets and/or study drug (anti-HP A-la gamma globulin or placebo) and was used for reporting of safety, demographic characteristics, and exposure to treatment.

[00106] Individual plots of the primary endpoint were normalized to the baseline assessment, which was defined as the first post-infusion flow cytometry data point. Actual sampling time points relative to baseline were used for derivation of the noncompartmental analysis (NCA) and on the individual plots of platelet versus time. Flow cytometry-assessed platelet concentration values that were below the highest pre-baseline value were excluded from the NCA. Concentration values below the LLOQ and missing values were also excluded from the NCA.

No formal analysis of “outliers” was performed.

[00107] For the primary endpoint, the terminal elimination half-life (ti_/2) rate was calculated by NCA. The elimination phase was determined by visual inspection of the individual concentration curves. At least 3 points above LLOQ belonging to the elimination phase were used for estimating the slope (l_z) of the log concentration-versus-time curve. Results

[00108] Rapid and complete platelet elimination was achieved in all subjects after administration of anti-HPA-la gamma globulin (“study drug”). The proportion of transfused HPA-la positive platelets in circulation after administration of anti-HPA-la gamma globulin or placebo is shown for cohorts 1 and IB over 7 days in FIG. 1A, and over 4 hours in FIG. IB. In both cohorts, administration of anti-HPA-la gamma globulin markedly accelerated the elimination of HPA-la positive platelets compared with placebo. For cohort 1, nearly all transfused platelets were eliminated after 2 hours, and the platelet elimination profile was consistent across all subjects, with little interindividual variation (Table 2).

Table 2. Summary of Individual Subjects.

[00109] The ability of anti-HPA-la gamma globulin to rapidly eliminate transfused platelets was also observed 7 days after a single dose administration. In cohort IB, 2 out of 3 subjects receiving anti-HPA-la gamma globulin achieved near-total elimination of platelets after 2 to 3 hours, and in the third subject, 36% of the transfused platelets could still be detected after 4 hours, resulting in a slightly longer platelet half-life. As no samples were scheduled between 4 and 24 hours after platelet transfusion, it was not possible to determine the exact platelet half-life for this subject. A conservative estimate assuming it took 24 hours to eliminate all transfused platelets would result in a platelet half-life of 2.6 hours, compared with 0.42 and 0.59 for the other 2 subjects in cohort IB. Despite the longer platelet half-life in this one subject, all anti- HPA-la gamma globulin-treated subjects met the proof-of-concept criterion of elimination of HPA-la positive platelets by 10-fold or greater versus placebo, as defined by platelet half-life.

[00110] Administration of anti-HPA-la gamma globulin showed acceptable safety and tolerability, with minimal adverse events observed and no serious adverse events. None of the subjects developed HPA-la antibodies at 12 weeks (n=12) or 24 weeks (n=8).

Example 3: In Vitro Binding of Anti-HPA-la Gamma Globulin to HPA-la/b Platelets

[00111] Human platelets heterozygous for HPA were incubated at a final concentration of 1 x 10⁶ cells/mL with human IgG (control) or anti-HPA-la gamma globulin at antibody concentrations of 0.0625-5000 pg/mL (0.00025-20 IU/mL). Samples were washed in PBS buffer containing 1% bovine serum albumin. Washed platelets were incubated with fluorescein (FITC) AffmiPure F(ab’ )2 donkey anti-human IgG (Jackson ImmunoResearch Inc., West Grove, PA) and analyzed by flow cytometry. Data were analyzed using FlowJo software (Tree Star Inc., Ashland OR). Results are shown in FIG. 2A-2B. FIG. 2A represents histogram plots of Alexa Fluor signal at varying concentrations of human IgG control (left panel) or anti-HPA-la gamma globulin (right panel). FIG. 2B represents fitted sigmoidal curves of two replicates of the anti- HPA-la gamma globulin binding isotherm. Binding above the lower asymptote is detectable at 0.16 IU/mL.

REFERENCES

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Claims

1. A method of administering anti -human platelet antigen (HPA)-la gamma globulin to a subject that is HPA-la negative, the method comprising parenterally administering to the subject a pharmaceutical composition comprising a preparation of the anti-HP A-la gamma globulin, wherein the anti-HP A-la gamma globulin is administered in an amount effective to achieve a maximum plasma concentration of the anti -HPA-la gamma globulin of about 0.01 IU/mL to about 10 IU/mL in the subject.

2. A pharmaceutical composition comprising a preparation of anti-human platelet antigen (HPA)-la gamma globulin for use in a method of parenterally administering the anti- HP A- la gamma globulin to a subject that is HPA-la negative, wherein the anti-HP A-la gamma globulin is administered in an amount effective to achieve a maximum plasma concentration of the anti -HPA-la gamma globulin of about 0.01 IU/mL to about 10 IU/mL in the subject.

3. The method or pharmaceutical composition of any preceding claim, wherein the maximum plasma concentration of the anti -HPA-la gamma globulin is about 0.05 IU/mL to about 5 IU/mL.

4. The method or pharmaceutical composition of any preceding claim, wherein the maximum plasma concentration of the anti -HPA-la gamma globulin is about 0.1 IU/mL to about 3 IU/mL.

5. The method or pharmaceutical composition of any preceding claim, wherein the maximum plasma concentration of the anti-HP A-la gamma globulin is about 0.5 IU/mL to about 3 IU/mL.

6. The method or pharmaceutical composition of any preceding claim, wherein the anti-HP A-la gamma globulin is administered in an amount of about 50 IU to about 10,000 IU.

7. The method or pharmaceutical composition of any preceding claim, wherein the anti-HP A-la gamma globulin is administered in an amount of about 100 IU to about 10,000 IU.

8. The method or pharmaceutical composition of any preceding claim, wherein the anti-HP A-la gamma globulin is administered in an amount of about 1000 IU to about 10,000 IU.

9. The method or pharmaceutical composition of any one of claims 1-8, wherein the pharmaceutical composition is administered intravenously.

10. The method or pharmaceutical composition of any one of claims 1-8, wherein the pharmaceutical composition is administered subcutaneously.

11. The method or pharmaceutical composition of any one of claims 1-8, wherein the pharmaceutical composition is administered intramuscularly.

12. The method or pharmaceutical composition of any preceding claim, wherein the subject is HPA-la negative.

13. The method or pharmaceutical composition of claim 12, wherein the subject is pregnant.

14. The method or pharmaceutical composition of claim 13, wherein the subject is carrying an HPA-la positive fetus.

15. The method or pharmaceutical composition of any preceding claim, wherein the subject is HLA-DRB3*01:01 positive.

16. The method or pharmaceutical composition of any preceding claim, wherein the administration of the anti-HP A-la gamma globulin achieves clearance of HPA-la positive platelets in the subject within about 10 hours of administering the anti -HPA-la gamma globulin to the subject.

17. The method or pharmaceutical composition of any preceding claim, wherein the administration of the anti-HP A-la gamma globulin achieves clearance of HPA-la positive platelets in the subject within about 5 hours of administering the anti -HPA-la gamma globulin to the subject.

18. The method or pharmaceutical composition of any preceding claim, wherein the administration of the anti-HP A-la gamma globulin achieves clearance of HPA-la positive platelets in the subject within about 3 hours of administering the anti -HPA-la gamma globulin to the subject.

19. The method or pharmaceutical composition of any preceding claim, wherein the administration of the anti-HP A-la gamma globulin prevents an alloimmune response to HPA-la- positive platelets in the subject.

20. The method or pharmaceutical composition of any preceding claim, wherein the administration of the anti-HP A-la gamma globulin induces antibody-mediated immune suppression of an immune response to HP A- la-positive platelets in the subject.

21. The method or pharmaceutical composition of any preceding claim, wherein the pharmaceutical composition further comprises a stabilizing agent and a surfactant.

22. The method or pharmaceutical composition of any preceding claim, wherein the pharmaceutical composition further comprises maltose and polysorbate 80.

23. The method or pharmaceutical composition of any preceding claim, wherein the pharmaceutical composition further comprises maltose in an amount of about 10% (w/w) and polysorbate 80 in an amount of about 0.03% (w/w).

24. The method or pharmaceutical composition of any preceding claim, wherein the pharmaceutical composition has a pH of about 5.0-6.5.

25. The method or pharmaceutical composition of any preceding claim, wherein half- life of the HP A- la positive platelets is reduced by about 150-fold to about 250-fold in the subject, relative to a subject who has not been administered the anti-HPA-la gamma globulin.