Extended interval administration of Anti-HPA-1a antibodies

Abstract

Provided is a regimen for administration of anti-HPA-la antibodies to a pregnant subject for prevention of maternal alloimmunization with HPA-la and fetal and neonatal alloimmune thrombocytopenia (FNAIT).

Description

EXTENDED INTERVAL ADMINISTRATION OF ANTI-HPA-la ANTIBODIESBACKGROUND

[0001] There are two predominant forms of human platelet antigen 1 (HPA-1) expressed on the surface of platelets: HPA-1 a and HP A- lb. Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disorder caused by a mismatch in the type of HPA-1 that is expressed by an expectant mother and her fetus. The incompatibility of HPA-1 is due to a Leu / Pro polymorphism at residue 33 of integrin (33 glycoprotein (GP Illa), which is present in the platelet membrane in complex with integrin all glycoprotein (GP lib) to function as a receptor for fibrinogen (Newman et al. , 1989). If the woman is negative for the HPA-la antigen (HPA-lb homozygous), fetal HPA-la positive platelets that have been inherited from the father (HPA-la homozygous or heterozygous) and that enter the maternal circulation can induce production of maternal anti-HPA-1 a antibodies in a process known as alloimmunization (Gohner et al. , 2017).

[0002] There are no currently available treatments for the prevention of alloimmunization of an HP A- la-negative pregnant woman with an incompatible (HP A- la-positive) fetus. Because there is no prophylactic treatment, pregnant women are not screened for HPA-1 incompatibility, and those who are at risk for FNAIT occurrence are generally not identified until after they have had a child bom with confirmed or suspected FNAIT (ACOG Practice Bulletin No. 207, 2019). In subsequent pregnancies, the current recommended therapy for at-risk women is human intravenous immune globulin (IVIG), beginning at Gestational Week 12 and continued throughout pregnancy (Pacheco et al., 2011). Importantly, IVIG does not prevent the occurrence of maternal alloimmunization, does not eliminate the risk of FNAIT occurrence in a subsequent pregnancy with maternal- fetal incompatibility and, in the doses administered, is accompanied by reports of poor tolerability (Vitiello etal., 2019).

[0003] Babies with FNAIT are typically diagnosed at the time of delivery by the presence of low- platelet counts, the presence of petechiae on the skin, or the manifestations of severe complications such as intracranial hemorrhage or gastrointestinal bleeding. In the neonate, platelet transfusion is the first line therapy for thrombocytopenia, although studies are too small to confirm whether the transfusions are effective at reducing neonatal morbidity or mortality (Lieberman et al., 2019). The aim of the transfusion is to maintain an acceptable platelet level within the first 72 to 96 hours of life (Espinoza et al., 2013). Intrauterine transfusion of platelets is performed rarely, due to the high risk of fetal morbidity andmortality associated with an intrauterine transfusion of platelets, as well as the need to perform the procedure frequently due to the short life span of transfused platelets (Regan et al., 2019; Brojer et a / ., 2016; Espinoza et al., 2013).

[0004] Prevention of alloimmunization in pregnant women not already alloimmunized will prevent FNAIT and all its consequences for the fetus / neonate since anti-HPA-la alloantibodies are the direct causative agent of FNAIT. Therefore, there is a need for a treatment that can prevent the maternal immune response that causes FNAIT.SUMMARY OF THE INVENTION

[0005] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, 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 different ways, and all such combinations are intended to fall within the scope of the present invention.

[0006] Provided are methods and compositions for preventing maternal alloimmunization with HPA-la and for preventing FNAIT caused by maternal alloimmunization with HPA- la comprising a regimen for administration of an anti-HPA-la antibody to a pregnant subject.

[0007] One embodiment is a method of preventing FNAIT caused by maternal alloimmunization with HPA-la in an HP A- la-positive fetus of an HP A- la-negative human subject, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an effective amount of an anti-HPA-la antibody, wherein the anti-HPA-la antibody does not bind HPA-lb; wherein an initial dose of the pharmaceutical composition is administered between gestational weeks 10 and 16; wherein maintenance doses of the pharmaceutical composition are administered at four weeks after the initial dose and once every four weeks thereafter during pregnancy; and wherein at least one dose is administered within 72 hours post-parturition. Also provided is a pharmaceutical composition comprising an effective amount of an anti-HPA-la antibody for use in the method of preventing FNAIT caused by maternal alloimmunization with HPA-la in an HP A- la-positive fetus of an HP A- la-negative human subject.

[0008] Another embodiment is a method of preventing alloimmunization with HPA-la in a subject, wherein the subject is an HP A- la-negative woman pregnant with an HPA-la-positive fetus, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an anti-HPA-la antibody, wherein the anti-HPA-la antibody does not bind HPA-lb; wherein an initial dose of the pharmaceutical composition is administered between gestational weeks 10 and 16 of pregnancy; wherein maintenance doses of the pharmaceutical composition are administered at four weeks after the initial dose and once every four weeks thereafter during pregnancy; and wherein at least one dose is administered within 72 hours postparturition. Also provided is a pharmaceutical composition comprising an effective amount of an anti-HPA-la antibody for use in the method of preventing alloimmunization with HPA-la in a subject, wherein the subject is an HP A- la-negative woman pregnant with an HP A- la-positive fetus.

[0009] In certain embodiments, the subject is HLA-DRB3*01:01 positive.

[0010] In one embodiment, the anti-HPA-la antibody is a polyclonal antibody. In a particular embodiment, the pharmaceutical composition is anti-HPA-1 a gamma globulin. In one embodiment, the anti-HPA-la antibody is a monoclonal antibody. In a particular embodiment, the monoclonal antibody is RLYB212.

[0011] In one embodiment, the pharmaceutical composition is administered via intravenous infusion. In a certain embodiment, the pharmaceutical composition is administered via subcutaneous injection. The pharmaceutical composition can be self-administered. In some embodiments, the pharmaceutical composition can be administered from a vial and syringe, a pre-filled syringe, a pen injector, or an autoinjector.

[0012] In a preferred embodiment, the steady-state plasma concentration of the anti-HPA-la antibody is between about 3.0 ng / mL and about 10 ng / mL.

[0013] In one embodiment, the initial dose is the same as the maintenance dose. In another embodiment, the initial dose is higher than the maintenance dose. In a particular embodiment, the maintenance dos is half the initial dose. For example, in one embodiment, the initial dose is 0. 12 mg and the maintenance dose is 0.06 mg.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A-1G show selected results of Phase 1 studies. FIG. 1A and FIG. IB show transfused platelets remaining in circulation versus time post-platelet transfusion. FIG.1C shows the mean pharmacokinetics (PK) profile for 0.09 mg and 0.29 mg subcutaneous (SC) doses of the anti-HPA-la monoclonal antibody RLYB212. with an antigen challenge at 7 days post-administration. FIG. ID shows HPA- la-positive platelet concentrationversus RLYB212 concentration at 6 hours post-platelet infusion. FIG. IE shows the area under the curve of HAP- la-positive platelet elimination over 24 hours. FIG. IF and FIG. 1G show mean PK and platelet elimination profiles after antigen challenge, respectively for the 0.09 mg and 0.29 mg dose groups.

[0015] FIG. 2 shows the two-compartment target-mediated drug disposition model described in Example 2.

[0016] FIG. 3A-3B show the median simulated PK of RLYB212 and HPA- la positive platelet following SC administration of RLYB212 to pregnant women at a loading dose of 1 mg and maintenance doses of 0.5 mg once every four weeks (Q4W), without (FIG. 3A) and with (FIG. 3B) 10-billion HPA- la-positive platelet challenges at 19 and 34 weeks gestation.

[0017] FIG. 4A-4B show the median simulated PK of RLYB212 and HPA- la positive platelet following SC administration of RLYB212 to pregnant women at a loading dose of 0.12 mg and maintenance doses of 0.06 mg Q4W, without (FIG. 4A) and with (FIG. 4B) 10-billion HP A- la-positive platelet challenges at 19 and 34 weeks gestation.

[0018] FIG. 5A-5B show the median simulated PK of RLYB212 and HPA- la positive platelet following SC administration of RLYB212 to pregnant women at a loading dose of 0. 1 mg and maintenance doses of 0.05 mg Q4W, without (FIG. 5A) and with (FIG. 5B) 10-billion HP A- la-positive platelet challenges at 19 and 34 weeks gestation.

[0019] FIG. 6A-6B show the median simulated PK of RLYB212 and HPA- la positive platelet following SC administration of RLYB212 to pregnant women at a loading dose of 0.02 mg and maintenance doses of 0.01 mg Q4W, without (FIG. 6A) and with (FIG. 6B) 10-billion HP A- la-positive platelet challenges at 19 and 34 weeks gestation.DETAILED DESCRIPTION OF THE INVENTION

[0020] The practice of the present invention can employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry', cell biology, cell culture, molecular biology, microbiology, recombinant DNA, immunology, 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 byreference 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.I. Definitions

[0024] The phraseology7or terminology7in 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 otherw ise. The terms “a” (or “an”) as w ell 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 (z.e., intermediate)encompassed by the range, including integers and fractions. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth.

[0029] Unless otherwise indicated, the terms "at least’ ’ or “about” preceding a series of elements is to be understood to refer to even’ element in the series. The term “about” preceding a numerical value includes ± 10% of the recited value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0030] The terms “polypeptide,” “peptide.” and “protein” are used interchangeably to refer to polymers of amino acids of any length, and their salts. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g. , for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Groups or strings of amino acid abbreviations are used to represent peptides. Except where specifically indicated, peptides are indicated with the N-terminus of the left and the sequence is written from the N-terminus to the C-terminus.

[0031] An “isolated” molecule is one that is in a form not found in nature, including those which have been purified.

[0032] The human amino acid sequence of GPIIIa (integrin (33) is set forth in GenBank accession no. AAA52589. 1, which includes a 26-amino acid signal peptide. HPA-1 is a polymorphism at position 33 of the mature integrin (33 chain. Individuals who have a Leu at position 33 in one or more copies of ITGB3 (z.e., the gene that encodes integrin (33) or in any of their integrin (33 are “HPA-la positive,” “positive for HPA-la,” “HPA-la,” or “HPA-lab.” while individuals who do not have a Leu at position 33 (e.g., have a Pro at position 33) in all copies of ITGB3 or in all of their integrin (33 are “HPA-1 a negative” or “negative for HPA-la.” An individual who is HPA-la positive can be homozy gous (HPA- la / a) or heterozygous (HPA-la / b) for HPA-la An individual who is HPA-la negative is homozygous for HPA-1 b / b.

[0033] The term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigenrecognition site within the variable region of the immunoglobulin molecule. The terms “antibody” or “immunoglobulin” are used interchangeably herein.

[0034] A ty pical antibody is 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 can mediate the binding of the antibody to host tissues or factors. Immunoglobulin molecules can be divided into classes 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 subclasses. IgG is the main type of antibody found in blood and extracellular fluid, and it plays a central role in the humoral immune response.

[0035] A “monoclonal antibody” (mAb) refers to a homogeneous antibody population that is involved in the highly specific recognition and binding of a single antigenic determinant (epitope). “Polyclonal antibodies” are a mixture of monoclonal antibodies directed against different epitopes of the same antigen. The term “monoclonal” can apply to full-length monoclonal antibodies, as well as to antigen-binding fragments, fusion proteins comprising an antigen-binding region, and any other modified immunoglobulin molecule comprising an antigen recognition site.

[0036] The term “antigen-binding fragment” refers to a portion of an intact antibody comprising the complementarity determining regions of the antibody. Examples of antigen-binding fragments include Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, single chain antibodies (e.g., ScFvs), and multi-specific antibodies formed from antibody fragments.

[0037] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions 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.). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair. The affinity' of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity binding partners generally bind slowly and tend todissociate readily, whereas high-affinity binding partners generally bind faster and tend to remain bound longer.

[0038] The affinity or avidity of a molecule for its binding partner can be determined experimentally using any suitable method known in the art, e.g. flow cytometry, enzyme- linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA® or BIACORE™ or OCTET® analysis). Direct binding assays as well as competitive binding assay formats can be readily employed. (See, e.g., Berzofsky et al., “Antibody -Antigen Interactions,” in Fundamental Immunology, Paul, W. E., ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: New York, N.Y. (1992)). The measured affinity of a particular binding pair interaction can vary if measured under different conditions (e.g, salt concentration, pH, temperature). Thus, measurements of affinity and other binding parameters (e.g. KD or Kd, Kon, Kofi) are made with standardized solutions of binding partners and a standardized buffer, as known in the art.

[0039] An “active agent” is an ingredient that is intended to furnish biological activity. The active agent can be in association with one or more other ingredients. An active agent that is a peptide can also be referred to as an “active peptide.”

[0040] 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 va7based on the substance that is being measured. For anti-HPA- la antibodies, the amount of anti-HPAla 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-HPA-1 a Standard (100 IU)).

[0041] As used herein, “WHO Standard” refers to the WHO International Standard of anti- HPA- la antibodies, prepared by pooling human plasma collected from six donors immunized against HPA-la (see WHO International Standard: Anti-HPA- la Standard (100 IU)).

[0042] An “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.

[0043] 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 apharmaceutically 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.

[0044] A “subject’’ or “individual” or “animal” or “patient” or “mammal,” 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.

[0045] 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.

[0046] With respect to the presence of a cell type, such as platelets, the terms “clear,” “clearance,” “eliminate,” and “elimination” are used interchangeably, and refer to achieving an undetectable level of the cell type. 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).

[0047] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. 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. An “inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule.

[0048] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder if the patient shows total, partial, or transient alleviation or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder.

[0049] “Prevent” or “prevention” refers to prophylactic or preventative measures that prevent and / or slow' the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those at risk of or susceptible to developing the disorder.

[0050] '‘Pharmacokinetics” or “PK” refers to the study of how an administered agent is processed by the body of a subject. PK determinations include how7the agent enters the blood circulation (absorption), is dispersed or disseminated throughout the fluids and tissues of the body (distribution), is recognized and transformed by the body (metabolism), and is removed from the body (excretion). The agent can be an active agent, e.g., a therapeutic antibody. Pharmacokinetics can be evaluated using various metrics, many of which are calculated based on the quantity of the agent in the body (e.g., in the plasma) at various time points following the administration of the agent.

[0051] “Cmax” or “Cp” is the peak plasma concentration of an agent after administration.“Cpss” is the trough plasma concentration of an agent at steady -state. ‘'Cthreshoid” is the target plasma concentration (exposure threshold) of an agent.

[0052] “Steady state” is achieved when the plasma concentration of an agent is maintained at a therapeutically effective level by administration of regular doses of the agent to balance the amount of drug being cleared. Once steady state is reached, the plasma concentration of the agent ranges from a peak (Cmax) to a trough (Cmin) concentration.

[0053] Time after administration is measured from To, which is the time that administration of a single dose of the agent is administered. “Tmax” refers to the time of after administration of the agent (To) to reach maximum plasma concentration (Cmax or Cp) of the agent. “T1 / 2” refers to the half-life of the agent, i. e. , the time required for the concentration of the agent to reach half of its original value.II. Antibodies and Compositions

[0054] The administration regimens provided herein can employ any anti-HPA-la antibody that binds specifically to HPA-la, such as a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof. Anti-HPA-la antibodies administered in a regimen of the invention are “specific for HPA-la,” which means that they do not display- detectable binding to HP A- lb.

[0055] The polyclonal antibody can be “anti-HPA-la gamma globulin,” which refers to a preparation produced from pooled plasma of donors with anti-HPA-la antibodies. A polyclonal antibody preparation can also be produced from the plasma of a single donor with anti-HPA-la antibodies. In one embodiment, the donor is an HPA-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 HP A-1 a negative subject who has been deliberately immunized with HPA-1 a positive platelets or with a purified or recombinant preparation HPA-1 a antigen. The preparation contains the total IgG from the pooled source plasma. See, e.g, US Patent No. 9,834,613.

[0056] Examples of monoclonal anti -HP A- la antibodies include, for instance, mAb 26.4 (Eksteen etal., 2015) and RLYB212, both of which are human monoclonal antibodies that bind specifically to the HPA-la isoform of integrin |33 and do not display detectable binding to the HPA-lb isoform of either recombinant or native integrin P3. RLYB212 differs from mAb 26.4 by a single amino acid substitution in the heavy chain, with the replacement of methionine at position 96 with valine to eliminate a potential site of oxidation. The CDRs of mAb 26.4 and RLYB212, as designated by the International ImMunoGeneTics (IMGT) method (Lefranc et al. , 2003), are set forth in SEQ ID NO: 3-8. The amino acid sequences of RLYB212's heavy chain variable and light chain variable regions are set forth in SEQ ID NO: 1 and 2. respectively. The amino acid sequences of RLYB212’s heavy chain and light chain are set forth in SEQ ID NO: 9 and 10, respectively. In a preferred embodiment, the anti-HPA-la antibody is RLYB212.

[0057] The anti-HPA-la antibody is formulated in a pharmaceutical composition. The pH of the composition can be 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 one embodiment, the pH is about 6.0.

[0058] The pharmaceutical composition can 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). In certain embodiments, the composition does not comprise mercury. In certain embodiments, the composition does not comprise a preservative.

[0059] In one embodiment, the pharmaceutical composition comprises anti-HPA-la gamma globulin, maltose, and polysorbate 80. In one embodiment, the pharmaceutical composition comprises RLYB212 succinate, arginine, sucrose, tryptophan, and water for injection.III. Administration and Methods of Prevention

[0060] Mismatch between fetal and maternal HPA-1 alloantigens, where an HP A- la-negative woman carries a fetus having a paternally-inherited HPA-la allotype, can lead to maternal production of anti-HPA- la antibodies. These antibodies can traverse the placenta, bind fetal platelets, and accelerate platelet destruction, causing fetal neonatal alloimmune thrombocytopenia (FNAIT). There is currently no prophylactic treatment for FNAIT.

[0061] A subject “at risk” of having an FNAIT pregnancy is an HPA-1 a negative woman who becomes pregnant with an HPA-la positive fetus. Women at “higher FNAIT risk” are HPA-la negative, indicating FNAIT risk, and are HLA-DRB3*01:01 positive; indicating ~25-fold higher alloimmunization risk compared to those without this human leukocyte antigen (HLA) allele.

[0062] Administration of polyclonal or monoclonal anti-HPA-1 a antibodies to an HPA-la negative mother has been contemplated as a potential prophylaxis for FNAIT (Kjeldsen- Kragh et al., 2012; Tiller et al.. 2012; Eksteen et al., 2015). The prior art identified the 72 hours before and after birth as the critical timeframe for administration of anti-HPA-1 a antibodies, because maternal alloimmunization was believed to be largely the result of fetal platelets entering the mother’s circulation in association with delivery (Kjeldsen- Kragh et a / ., 2012; Tiller et al.. 2012; Eksteen et al. , 2015). However, in addition to being expressed on the membrane of platelets, integrin (33 is also expressed on the membrane of other cell types, creating additional sources of potential antigenic stimulation for the maternal immune system (Zhou etal., 1997).

[0063] Contrary to the theory' that a single administration of anti-platelet antibodies after antigenic challenge would prevent alloimmunization, we have developed a multi-dose administration regimen in which a composition comprising an antibody specific for HPA- la is administered to a woman at risk for FNAIT early in pregnancy, before exposure to the HPA-la antigen, and is continued throughout the course of the pregnancy. The anti- HPA- la antibody is administered at doses well below the threshold known to cause adverse clinical sequelae in the fetus or neonate, can safety and effectively prevent maternal alloimmunization.

[0064] One embodiment is a method of preventing FNAIT caused by maternal alloimmunization with HPA-la in an HP A- la-positive fetus of an HP A- la-negative subject, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an effective amount of an anti-HPA- 1 aantibody, wherein the anti-HPA- la antibody does not bind HPA-l b; wherein an initial dose of the pharmaceutical composition is administered between gestational weeks 10 and 16; wherein maintenance doses of the pharmaceutical composition are administered after the initial dose, at a regular dose interval until delivery; and wherein at least one dose is administered within 72 hours post-parturition. Also included is a pharmaceutical composition comprising an effective amount of an antibody specific for HPA-la for use in the method.

[0065] Another embodiment is a method of preventing alloimmunization with HPA-la in a subject, wherein the subject is an HPA-la-negative woman pregnant with an HPA-la- negative fetus, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an anti-HPA-la antibody, wherein the anti-HPA-la antibody does not bind HPA-lb; wherein an initial dose of the pharmaceutical composition is administered between gestational weeks 10 and 16; w herein maintenance doses of the pharmaceutical composition are administered after the initial dose, at a regular dose interval until delivery ; and wherein at least one dose is administered within 72 hours post-parturition. Also included is a pharmaceutical composition comprising an effective amount of an antibody specific for anti-HPA-la for use in the method.

[0066] Gestational age is determined by known methods, including menstrual history, clinical examination, and / or ultrasonography. “Delivery ” and “parturition” are used interchangeably in reference to childbirth.

[0067] “Initial dose,” “loading dose.” and “induction dose” are used interchangeably and refer to the first dose of the pharmaceutical composition comprising an anti-HPA-la antibody administered to the subject. “Maintenance dose” and “repeat dose” are used interchangeably and refer to the doses of the pharmaceutical composition administered to the subject subsequent to the initial dose.

[0068] The pharmaceutical composition comprising an anti-HPA-la antibody is administered parenterally. Parenteral routes of administration include intravenous, intramuscular, intraperitoneal, intrathecal, and subcutaneous. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously. The pharmaceutical composition can be administered, for example, via a vial and syringe, a pre-filled syringe, a pen injector, or an autoinjector. The pharmaceutical composition can be selfadministered. “Self-administration” means that the pharmaceutical composition isadministered by the subject, and can also include administration by someone else, such as a family member or friend.

[0069] Maintenance doses of the pharmaceutical composition are administered at a regular dose interval, meaning that the time between doses is fixed. In one embodiment, the pharmaceutical composition is administered about every two weeks (Q2W), about every four weeks (Q4W), about every six weeks (Q6W), or about every 8 weeks (Q8W). In one embodiment, the pharmaceutical composition is administered once monthly, once every two months, or once every three months. At least one dose is administered within about 72 hours of delivery. Subsequent post-parturition doses can be administered about 4, 5. or 6 days after delivery, or about 1, 2, 3, 4, 5, 6, 7, or 8 weeks after delivery.

[0070] The initial dose is selected to result in a maximum plasma concentration of anti-HPA- la antibody of about 3 ng / mL to about 10 ng / mL, preferably about 8-10 ng / mL. In some embodiments, the initial dose of the anti-HPA-la antibody is the same as the maintenance dose, while in other embodiments, the initial dose of the anti-HPA-la antibody is higher than the maintenance dose. For example, the initial dose can be about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the maintenance dose. In preferred embodiments, the initial dose is 3 or 4 times a Q2W maintenance dose or about 2 times a Q4W maintenance dose. In one embodiment, the initial dose of the anti-HPA-la antibody is about 5-400 pg or about 30- 350 pg or about 60-270 pg or about 90-180 pg or about 100-140 pg. In one embodiment, the initial dose is about 0.02 mg or about 0. 1 mg or about 0. 12 mg or about 0. 14 mg or about 0.2 mg or about 0.6 mg or about 1 mg.

[0071] In one embodiment, the maintenance dose of the anti-HPA-la antibody is about 5-400 pg or about 10-200 pg or about 15-120 pg or about 20-90 pg or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 pg. In one embodiment, the maintenance dose is about 0.01, 0.05, 0.06, 0.07, 0. 1, 0.3, or 0.5 mg.

[0072] In one embodiment in which the anti-HPA-la antibody is RLYB212, the initial dose is about 0.02-1 mg or about 0.06-0.18 mg, and the maintenance dose is about 0.01-0.5 mg or about 0.03-0.09 mg. In a particular embodiment in which the anti-HPA-la antibody is RLYB212, the initial dose is about 0. 12 mg and the maintenance dose is about 0.03 mg Q2W or about 0.06 mg Q4W.

[0073] The methods of the invention involve administration of an amount of antibody specific for HPA-la that is effective to prevent maternal alloimmunization or FNAIT caused by maternal alloimmunization. An effective amount of a given anti-HPA-la antibody can bedetermined, for example, by the ability of the antibody to clear HPA-1 a positive platelets in the circulation of an HPA-1 a-negative subject. In some embodiments, HPA-1 a positive platelets are cleared in a subject within about 6-24 hours of administering the anti-HPA-la antibody, or within about 2 or 3 hours of administering the anti-HPA-la antibody. In certain embodiments, HPA-1 a positive platelets are cleared in a population of subjects within a mean of about 6-24 hours of administering the anti-HPA-la antibody to the subjects, or within about 2 or 3 hours of administering the anti-HPA-la antibody.

[0074] In certain embodiments, the effective amount is determined by the dose required to achieve a particular target plasma concentration (Cthreshoid) of the anti-HPA-la antibody. Preferably, the Cthreshoid is about 0.3-0.7 lU / mL or about 3-10 ng / mL. In one embodiment, the Cthreshoid is 0.5 lU / mL. In one embodiment, the Cthreshoid is about 5-8 ng / mL. Concentration of an anti-HPA-la antibody can be determined, for example, using a monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay (Kiefel et al., 1987; Campbell etal., 2007) or other ELISA-based method.

[0075] In certain embodiments, the frequency and dosage of the maintenance dose is adjusted during pregnancy to achieve a Cthreshoid of the anti-HPA-la antibody of about 3-10 ng / mL. For example, due to changing metabolic factors, weight, and / or blood volume of the subject during pregnancy, it may be necessary to increase or decrease the dosage and / or frequency of maintenance doses. In one embodiment, the subject is administered an initial dose betw een gestational w eeks 10-16, is administered a first maintenance dose four weeks later, and subsequent maintenance doses are administered every four weeks or every two weeks or even- week, as needed to maintain a steady state plasma concentration of anti-HPA-la antibody of about 3-10 ng / mL. Preferably, the maintenance dose is about 40-60% of the initial dose for Q4W dosing, about 20-30% of the initial dose for Q2W dosing, or about 10-15% of the initial dose for QW dosing.

[0076] In certain embodiments, the anti-HPA-la antibody prevents FNAIT caused by maternal alloimmunization by outcompeting alloantibody binding to fetal platelets. For example, the anti-HPA-la antibody can be an effectorless antibody. The Cthreshoid in such embodiments can be 10,000, 20,000, 30,000, 40,000, or 50,000 lU / mL.

[0077] In some embodiments, the antibody drives clearance of fetal-derived antigen but does not bind neonatal Fc receptor (FcRn), and therefore, does not cross the placenta. In such embodiments, there is no risk to the fetus of pathological effects from the treatment,because there is no fetal exposure to the anti-HPA-1 a antibody. The Cthreshoid in such embodiments can be 10,000, 20,000, 30,000, 40,000, or 50,000 lU / mL.

[0078] In embodiments in which the anti-HPA-1 a antibody is administered intravenously, the Tmax is at the end of infusion. In some embodiments in which the anti-HPA-1 a antibody is administered subcutaneously, the Tmaxof the anti-HPA-1 a antibody is about 5-15 days, or about 8-12 days or about 9-11 days after administration of the initial dose. In embodiments wherein the initial dose is higher than the maintenance dose, the Tmaxcan be achieved, for example, after the first maintenance dose, or after a subsequent maintenance dose. In one embodiment, the Tmax of the anti-HPA-1 a antibody is about 3-7 days after administration of the final maintenance dose, for example, about 5 days after the final maintenance dose. Each maintenance dose will achieve a Cmax before falling to a trough concentration (Cmin). In some embodiments, Cmax of the anti-HPA-1 a antibody is reached 3-7 days after each maintenance dose. In one embodiment, Tmax of the anti-HPA-1 a antibody for an administration regimen of the invention is 3-7 days, preferably about 5 days, after administration of the final maintenance dose. In certain embodiments, at steady state, the plasma concentration of anti-HPA-1 a antibody is between about 3 ng / mL and about 10 ng / mL or between about 5.0 ng / mL and 8.5 ng / mL.IV. Methods of Preparation

[0079] Monoclonal antibodies can be prepared by methods known in the art. For example, an anti-HPA-1 a antibody can be prepared from memory B cells isolated from an HPA-la alloimmunized subject according to the method described by Eksteen et al. (2015). Alternatively, a recombinant anti-HPA-1 a antibody, for example, mAb 26.4 or RLYB212, can be expressed in host cells. In another embodiment, anti-HPA-la antibodies can be raised in mice or other mammals immunized with human HPA-la, produced using hybridoma technology (Kohler et al., 1975), and preferably humanized.

[0080] Polyclonal antibodies can be prepared by producing a mixture of two or more monoclonal antibodies. Alternatively, polyclonal antibodies can be prepared from plasma of one or more donors with anti-HPA-la antibodies. The manufacturing process can comprise purification of IgG from source plasma containing antibodies to HPA-la, clearance of viruses from the purified IgG, and concentration of the purified IgG. Purification of the IgG can be performed using anion-exchange chromatography, althoughother suitable techniques can be used, such as alcohol fractionation and polyethylene glycol (PEG) precipitation.

[0081] Viral clearance in purified IgG can 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 example, by cold ethanol fractionation, heating, solvent / detergent, exposure to an acidic environment, etc.; or by a combination thereof. In some embodiments, viral clearance is performed by nanofiltration and exposure of the purified IgG to a solvent detergent, such as tri-n-butyl phosphate. Use of a solvent detergent to clear viruses from purified IgG can be followed by removal of the solvent detergent, for example, by reverse-phase chromatography.

[0082] Following viral clearance, the purified IgG can be concentrated using, for example, ultrafiltration. In addition, diafiltration can be used to remove microsolutes such as salts from the preparation.

[0083] Additional steps can be included. For instance, prior to purification, plasma can be diluted and dextran sulphate can be added to plasma to remove lipids. Plasma from different sources (e.g., from different persons) can be pooled together. After viral clearance, a step can be performed to reduce procoagulant factors, such as Factor XI and activated Factor XI, for example, by affinity chromatography.EXAMPLES

[0084] 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. RLYB212 Clinical Studies

[0085] We have conducted two Phase 1 clinical studies involving administration ofRLYB212. The first is a single-blind, placebo-controlled study investigating the safety and pharmacokinetics of RLYB212 in HPA-lb / b healthy volunteers. The primary objective of this ongoing study is to evaluate the safety of single and multiple doses of RLYB212. The secondary’ objectives are to evaluate the immunogenicity of single and multiple doses of RLYB212 and establish the pharmacokinetic (PK) profile of RLYB212 following subcutaneous (SC) administration. Participants are enrolled to 1 of 2 cohorts, with 8 participants in each cohort randomized 3: 1 to receive RLYB212 or placebo in a blinded manner. In Cohort 1. participants received placebo or a single dose (0.21 mg) of RLYB212on Day 1 . In Cohort 2, participants receive study drug on Day 1 and then every 2 weeks for an additional 10 weeks. PK samples were collected and analyzed for inclusion in the modeling described in Example 2.

[0086] The second is a single-blind, placebo-controlled, single-center study investigating the capacity of RLYB212 to eliminate HPA-1 a positive platelets transfused to HPA-lb / b male volunteers. The primary objective was to establish the ability of RLYB212 to markedly (10-fold or greater) accelerate the elimination of HPA-1 a positive platelets transfused to HPA-lb / b volunteers. The secondary obj ectives were: as to characterize kinetic parameters of the elimination of HPA-1 a positive platelets transfused to HPA-lb / b volunteers; to characterize PK parameters of RLYB212; to characterize the pharmacokinetic / pharmacodynamic (PK / PD) profile of RLYB212; to evaluate the safety of RLYB212; to monitor alloimmune response to HPA-la positive platelets; and to evaluate the immunogenicity of a single dose of RLYB212. Participants are randomized 4: 1 to receive a single dose of RLYB212 or placebo in a single-blinded manner. Participants received placebo or a single SC dose (0.09 mg or 0.29 mg) of RLYB212 on Day 1. On Day 8, participants received an intravenous transfusion of exogenous HPA-1 a positive platelets. PK and dynamic HP A- 1 a positive platelet data was collected and analyzed for inclusion in the modeling described in Example 2.

[0087] In the second study, RLYB212 drove rapid and complete elimination of HPA-la positive platelets in a concentration-dependent manner, meeting the prespecified proof-of- concept criterion of >90% mean reduction in platelet elimination half-life vs placebo. SC RLYB212 was well tolerated with no reports of serious or drug-related adverse events. All subjects receiving RLYB212 displayed accelerated platelet elimination (FIG. 1A-1B), displaying a biphasic elimination profile with an initial lag-phase lasting from 2-6 hours before the elimination phase.

[0088] The PK profile of RLYB212 was generally consistent with that of a subcutaneously administered IgG, with an apparent x between 7 and 14 days (FIG. 1C). RLYB212 absorption parameters prior to the platelet transfusion increased with dose, while the terminal elimination parameters were generally similar for both dose groups. Immediately after the transfusion of the HPA-la positive platelet challenge, there was a rapid decline in RLYB212 concentrations over the next 2-24 hours.

[0089] RLYB212 concentrations at the time of the antigen challenge ranged from 1.92 ng / mL to 4.27 ng / mL in the low dose group, and from 8.68 to 20.55 ng / mL in the high dosegroup. All parameters of platelet elimination kinetics were found to be dependent on the concentration of RLYB212, with a dynamic response at RLYB212 concentrations below about 5 ng / mL and saturation of maximal effect at concentrations above about 5 ng / mL (FIG. ID- IE)

[0090] The rapid decline in RLYB212 concentrations after the platelet challenge corresponds to a first order clearance rate and was concurrent with the initial lag phase prior to the accelerated elimination phase of the platelets (FIG. 1F-1G). This indicates that RLYB212 accumulation on HPA- la positive platelets must reach a sufficient threshold density before phagocytosis can begin to drive platelet elimination. Once the HPA-la platelets were eliminated (within 12-24 hours), RLYB212 concentrations began to rise at the same absorption rate prior to the platelet challenge.

[0091] The estimated amount of RLYB212 consumed during platelet elimination can be calculated as:(RLYB212 pretransfusion - RLYB212 Cmtn within 24 hrs. of transfusion) x est. blood volume

[0092] The estimated amount of RLYB212 consumed during the platelet elimination ranged from 7.6 to 20.4 pg across all subjects, w ith geometric means of 8.2 pg for the 0.09 mg dose group and 14.0 pg for the 0.29 mg dose group.

[0093] One can further estimate the ratio of RLYB212 molecules consumed to the total number of HPA- la-positive integrin alip3 receptors on the platelets administered during the antigen challenge (assuming 40,000 HPA-la receptors per HPA-la / b platelet), with geometric means ranging from 8.3 % for the 0.09 mg dose group and 14.2 % for the 0.29 mg dose group. An estimate of about 10% of receptor binding to drive phagocytosis is consistent with the previously reported clinical results with anti-RhD therapeutics and preclinical efficacy studies with RLYB212, wherein the reported minimal threshold of 5- 15% receptor binding was required to drive phagocytosis of antigen-positive cells. Furthermore, these results demonstrate that this receptor binding threshold is both necessary and sufficient to engage macrophage and effect phagocytosis.

[0094] Taken together, the totality of the data suggests that concentrations of RLYB212 greater than about 3 ng / mL should be sufficient to drive rapid and complete elimination of fetal antigen from maternal circulation, even in the event of a rare large volume fetal- maternal hemorrhage.Example 2. Target-Mediated Drug Disposition Model

[0095] A target-mediated drug disposition (TMDD) model was developed to characterize the PK of RLYB212 and HPA-la positive platelet dynamics. This modeling informs dose and regimen for pregnant women receiving RLYB212. which requires consideration of the demographic differences between the subjects in the Phase 1 studies described in Example 1 and pregnant women, including the dynamic weight and blood volume changes that occur with gestational age. Between-subject variability7from the model and from a metaanalysis of monoclonal antibodies, simple allometry, and gestational age-dependent scaling were performed and utilized for simulation of drug PK and effect in pregnant women. The final PK / PD model parameters were physiologically based, plausible, and mechanistically described drug-target binding, which impacts both PK and PD.

[0096] In order to develop a TMDD model that described drug-target interaction, it was necessary to transform the HPA-la positive platelet data into a molar estimate. To do so, the assumption was made that the percentage of transfused HPA-la positive platelets to endogenous host platelets in the platelet sample at 10 min w as representative of all platelets in the system (100%). Each patient was injected 10 billion total heterozygous HPA-la / b platelets. It has been reported that there are about 80.000 integrin (33 subunits per platelet (Wagner et al., 1996), and it was also assumed that half of these are HPA-la positive.where Pi = individual platelet count in molar units; PSi = individual platelet signal; NA = Avogadro’s constant.

[0097] Nonlinear mixed-effects modeling software (NONMEM®; ICON, Hanover, MD, US) version 7.3.0, a software package for nonlinear mixed-effects analysis, w as used to analyze RLYB212 concentration-time data The first-order conditional estimation method of NONMEM with interaction (FOCEI) was used for PK / PD model development. R version 4.2. 1 (R Core Team; Vienna, Austria) was used for model diagnostics, plotting, and simulations. Perl-speaks-NONMEM® (PsN; Department of Pharmacy, Uppsala University7, Uppsala, Sweden) was also used for the facilitation of NONMEM tasks, such as covariate testing. The R packages RxODE version 1.1.6 and Rtools version 4.2 w ere used to perform simulations using the final model.

[0098] A target-mediated drug disposition (TMDD) model was developed to provide fitting to RLYB212 concentration data, while also providing HP A- la positive platelet dynamics in HP A- Ib / b volunteers (FIG. 2). One- and two-compartment structural models with first- order elimination were tested for the base RLYB212 PK model. Consistent with the distribution of other large molecule drugs (e.g, monoclonal antibodies), the two- compartment PK model structure resulted in better fit of the PK data, demonstrated by less trend in diagnostic fittings, lower objective function and acceptable parameter estimate precision. This PK model was utilized as a starting point for model development. One- and two-compartment models were also tested for HPA- la positive platelets. Consistent with the distribution of platelets in literature, the two-compartment PD model structure was utilized (Peters, 1985). Furthermore, it resulted in better fit of the PD data, lower objective function, and acceptable parameter estimate precision.

[0099] Simultaneous fitting of the data is necessary as both RLYB212 PK and HPA- la positive platelet dynamics are dependent on concentration and binding of each to their counterpart. RLYB212 PK was described by two compartment distribution model, with a respective central volume (VI) and a peripheral volume (V2), featuring linear clearance (CL) from central compartment. HPA- la positive platelets were also described by two compartment distribution model, with a respective central volume (VI) and a peripheral volume (V3). Elimination of HPA- la-positive platelets was described by first-order degradation constant, kdeg.

[0100] HPA- la positive platelets are not produced endogenously in the targeted HPA-lb / b patient population; as a result, no synthesis rate of HP A- la positive platelets was incorporated into the final model. The interaction between RLYB212 and HPA- la positive platelets was assumed to occur in the central compartment, by the second-order binding constant, kon, and the first-order dissociation constant, koff. Tw o koir estimates w ere fixed and tested for the interaction between RLYB212 and HPA- la-positive platelets-2 -4 individually: 1) 1.58x10 / s, identified from a BI Acore Assay Report, and 2) 1.0x10 / h, utilized to describe no disassociation. Consistent with reports that anti-03 -integrin antibodies engage in bivalent binding to platelet surfaces (Kutok etal., 1994). and after testing both koff estimates, it was concluded following binding of RLYB212 and HPA-la- positive platelets, that there will not be dissociation; thus, 1.0xl0’4 / h w as utilized for kOff. Furthermore, konof 3.97xlO5 / M s, identified by the BIAcore Assay Report was fixed within the model.

[0101] Phagocytic elimination of platelets rate (kphagocytosis) was dependent on a minimum threshold for percent coating of platelets (THRES). The minimum threshold was initially estimated; however, the model was unstable when between-subject variability was incorporated. Thus, minimum threshold for percent receptor occupancy of platelets was fixed at 10%. The assumption of 10% was supported by: 1) threshold estimation ranged from 3-22% when all parameters were being estimated with and without between-subject variability' and 2) estimates of about 10% HPA- la binding is necessary' and sufficient to drive phagocytosis of transfused platelets and prevention of alloimmunization in a murine model of FNA1T (Zhi et al., 2022), and 3) literature describing prophylactic treatment with anti- Rhesus D (RhD) IgG to prevent alloimmunization to RhD antigen, where polyclonal anti-RhD is estimated to require 5% antigen binding and monoclonal anti-RhD is estimated to require 10-15% antigen binding on RhD positive erythrocytes (Brine et al., 2009).

[0102] SC absorption is assumed to occur from the SC space by first-order rate constant, ka.

[0103] Simulations w ere performed to inform dose and regimen for a Phase 2 study in pregnant women with RLYB212. Simple allometry using the equation below with assumed exponents of 1 and 0.85 were used to scale volume and linear clearances, respectively (Deng et al., 2011). To account for physiologic changes in blood volume associated with pregnancy, time-dependent scaling on central volume w as performed via gestational age-based scaling as previously described (Abduljalil et al., 2012). The median healthy volunteer weight in the modeling dataset (91.5 kg) and a median female of childbearing age weight (69.5 kg) were used to scale parameters that vary over gestational age (Fryar et al., 2021). To account for weight gain during pregnancy, time-dependent scaling on body weight w as conducted via gestational age-based scaling with mean w eight gain based on recommendations by the American College of Obstetricians and Gynecologists (Gynecologists TACoOa, 2013).where pw = pregnant w oman; hv = healthy volunteer; BW = body w eight.

[0104] Simulations were performed using the allometrically scaled population and timedependent scaling estimates for selected PK parameters as described in Table 1. Simulations utilized 500 subjects and were performed as described in Table 2. TheRLYB212 concentration and HPA-1 a positive platelet amount for each simulation were plotted over gestational age (GA) Week 15 through Week 40.Table 1. Parameters and Scaling used for SimulationSee Deng et al., 2011; Abduljalil et al., 2012; Gynecologists TACoOa, 2013.Abbreviations: CL = clearance; F = bioavailability; GA = gestational age; ka = absorption rate; kdeg = degradation rate; kpliagocy tosis = phagocytosis rate; koff = dissociation rate of DR; kon = formation rate of DR; Prop.RE = proportional residual error; Q = inter-compartment transfer rate constant between central and peripheral compartment of drug; QP = inter-compartment transfer rate constant between central and peripheral compartment of platelet; RSE% = percent relative standard error; SC = subcutaneous; THRES = receptor occupancy threshold; VI = central volume of distribution of drug; V2 = peripheral volume of distribution of drug V3 = peripheral volume of distribution of platelet; WT = weight.*Median weight of a female of childbearing age (18-49 years old) is 69.5 kg (Fryar et al., 2021).Table 2 Simulation ScenariosAbbreviations: Q4W = every 4 weeks, SC = subcutaneous.

[0105] Simulations were conducted of Q4W SC dosing with and without loading dose as well as with and without HPA-1 a positive platelet challenges. Dosing of RLYB212 was initiated at Week 15 and continued through Week 40, resulting in a total of 7 doses simulated. Loading dose was double that of the maintenance dose. HPA-1 a positive platelet challenges were simulated twice, one after the first dose prior to the next dose (Week 19) and another at steady -state prior to the next dose (Week 35). Results are shown in FIG. 3A-6B

[0106] Simulations using 0.06 mg Q4W with loading dose of 0. 12 mg, with and without two HPA-la positive platelet challenges, are projected to achieve near steady state concentrations after the first dose, and to maintain median concentrations below the defined target upper boundary (10 ng / mL or about 0.6 lU / mL) and above the concentration show n to markedly accelerate the clearance of exogenously administered HPA-1 a / b platelets (FIG. 4A-4B).

[0107] Simulations without the HPA-la positive platelet challenges predict RLYB212 concentrations at steady state for the median ( I (f'- O111prediction interval) Cmaxand Cminrange from 8 ng / mL (5.8-11) to 5.8 ng / mL (3.6-8.8), respectively. Thus, for all simulations, the simulated dosing regimen is projected to remain above the lowest concentration shown to meet the proof-of-concept criteria for accelerated elimination of exogenously administered HPA-1 a / b platelets in the Phase 1 platelet-challenge study (3.57 ng / mL) and below the defined target upper boundary (10 ng / mL or about 0.6 lU / mL) for all but brief excursions to 11 ng / mL (~ 0.65 lU / mL) in the 90thprediction interval in the absence of TMDD. This regimen is predicted to result in a ~ 93% reduction in platelet exposure relative to no-drug control.SEQUENCESSEQ ID NO: 1 - RLYB212 Heavy Chain Variable (VH) RegionGin Vai Gin Leu Gin Gin Ser Gly Pro Gly Leu Vai Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Ala He Ser Gly Asp Ser Vai Ser Ser Asn Ser Ala Ala Trp Asn Trp He Arg Gin Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Phe Arg Ser Asn Trp Tyr Asn Asp Tyr Ala Ala Ser Vai Lys Ser Arg lie Thr lie Asn Gin Asp Thr Ser Lys Asn Gin Leu Ser Leu Gin Leu Asn Ser Vai Thr Pro Glu Asp Thr Ala Vai Tyr Tyr Cys Ala Arg Asp Gly Ala Trp Gly Gly Ser Ser Trp Trp Pro Gly Leu Pro His His Tyr Tyr Ser Gly Met Asp Vai Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser SerSEQ ID NO: 2 - RLYB212 Light Chain Variable (VL) RegionGlu He Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie Tyr Asp Ala Ser Lys Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr He Arg Ser Leu Glu Pro Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asp Trp Gin Gly Leu Thr Phe Gly Gly Gly Thr Lys Vai Glu He LysSEQ ID NO: 3 - RLYB212 VH Complementarity Determining Region (CDR) 1Gly Asp Ser Vai Ser Ser Asn Ser Ala AlaSEQ ID NO: 4 - RLYB212 VH CDR 2Thr Tyr Phe Arg Ser Asn Trp Tyr AsnSEQ ID NO: 5 - RLYB212 VH CDR 3Ala Arg Asp Gly Ala Trp Gly Gly Ser Ser Trp Trp Pro Gly Leu Pro His His Tyr Tyr Ser Gly Met Asp VaiSEQ ID NO: 6 - RLYB212 VLCDR 1Gin Ser Vai Ser Ser TyrSEQ ID NO: 7 - RLYB212 VLCDR 2Asp Ala SerSEQ ID NO: 8 - RLYB212 VLCDR 3Gin Gin Arg Ser Asp Trp Gin Gly Leu ThrSEQ ID NO: 9 - RLYB212 Heavy ChainGin Vai Gin Leu Gin Gin Ser Gly Pro Gly Leu Vai Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Ala He Ser Gly Asp Ser Vai Ser Ser Asn Ser Ala Ala Trp Asn Trp He Arg Gin Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Phe Arg Ser Asn Trp Tyr Asn Asp Tyr Ala Ala Ser Vai Lys Ser Arg lie Thr lie Asn Gin Asp Thr Ser Lys Asn Gin Leu Ser Leu Gin Leu Asn Ser Vai Thr Pro Glu Asp Thr Ala Vai Tyr Tyr Cys Ala Arg Asp Gly Ala Trp Gly Gly Ser SerTrp Trp Pro Gly Leu Pro His His Tyr Tyr Ser Gly Met Asp Vai Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly LysSEQ ID NO: 10 - RLYB212 Light ChainGlu He Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie Tyr Asp Ala Ser Lys Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr He Arg Ser Leu Glu Pro Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asp Trp Gin Gly Leu Thr Phe Gly Gly Gly Thr Lys Vai Glu He Lys Arg Thr Vai Ala Ala Pro Ser Vai Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser Phe Asn Arg Gly Glu CysREFERENCESAbdulj alii K. et al. Anatomical, physiological and metabolic changes with gestational age during normal pregnancy: a database for parameters required in physiologically based pharmacokinetic modelling. Clin. Pharmacokinet. 2012:51 (6): 365-96.ACOG Practice Bulletin No. 207: Thrombocytopenia in Pregnancy. Obstet Gynecol.2019; 133(3) : e 181 -el 93. doi: 10. 1097 / AOG.0000000000003100.Allen D, et al. Collaborative study to establish the first international standard for quantitation of anti -HP A- la. Vox Sang. 2005;89: 100-104.Baker EK, et al. A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro. Proc Natl Acad Sci U S A. 1997:94(5): 1973-1978.Bertrand G, et al. Predictive value of sequential maternal anti-HPA-1 a antibody concentrations for the severity of fetal alloimmune thrombocytopenia. J Thromb Haemost. 2006;4(3):628-637.Brine D, Lazarus A. Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn. Hematology Am Soc Hematol. Educ. Program. 2009: 185-91.Brojer E, et al. Fetal / Neonatal Alloimmune Thrombocytopenia: Pathogenesis, Diagnostics and Prevention. Arch Immunol Ther Exp (Warsz). 2016;64(4):279-290.Campbell K. et al. A modified rapid monoclonal antibody-specific immobilization of platelet antigen assay for the detection of human platelet antigen (HP A) antibodies: a multicentre evaluation. Vox Sanguinis 2007;93:289-97.Coller BS, et al. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins lib and / or Illa. J Clin Invest. 1983;72(l):325-338.Coller BS, et al. Abolition of in vivo platelet thrombus formation in primates with monoclonal antibodies to the platelet GPIIb / IIIa receptor. Correlation with bleeding time, platelet aggregation, and blockade of GPIIb / IIIa receptors. Circulation. 1989;80: 1766-1774.Defilippi P, et al. Differential distribution and modulation of expression of alpha 1 / beta 1 integrin on human endothelial cells. J Cell Biol. 1991 ; 114(4):855-863.Deng R, et al. Projecting human pharmacokinetics of therapeutic antibodies from nonclinical data: what have we learned? MAbs. 201 l;3(l):61-6.Eksteen M. el al. Characterization of a human platelet antigen-la- specific monoclonal antibody derived from a B cell from a woman alloimmunized in pregnancy. J Immunol. 2015;194(12):5751-5760.Espinoza JP, et al. Fetal and neonatal alloimmune thrombocytopenia. Rev Obstet Gynecol. 2013;6(l):el5-e21.Fryar CD, et al. Anthropometric reference data for children and adults: United States. 2015- 2018. 2021:3(46).Gohner C, et al. Immune-modulatory effects of syncytiotrophoblast extracellular vesicles in pregnancy and preeclampsia. Placenta. 2017;60 Suppl LS41S51.Gold HK, et al. Pharmacody namic study of F(ab')2 fragments of murine monoclonal antibody 7E3 directed against human platelet glycoprotein Ilb / IIIa in patients with unstable angina pectoris. J Clin Invest. 1990;86:651-659.Gynecologists TACoOa. Committee Opinion No. 548: Weight Gain During Pregnancy. Obstetrics & Gynecology. 2013; 121( l):210-2.Kiefel V. et al. Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies. Blood 1987;70: 1722-26.Killie MK, et al. A prospective study of maternal anti- HPA la antibody level as a potential predictor of alloimmune thrombocytopenia in the newborn. Haematologica. 2008;93(6):870- 877.Kjeldsen-Kragh J, et al. Towards a prophylactic treatment of HPA-related foetal and neonatal alloimmune thrombocytopenia. Curr Opin Hematol. 2012;19(6):469474.Kohler, et al. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495.Kutok J, et al. Partial inhibition of platelet aggregation and fibrinogen binding by a murine monoclonal antibody to GPIIIa: requirement for antibody bivalency. Thromb. Haemost. 1994;72(6): 964-72.Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. 2003;27:55-77.Lieberman L, et al. Fetal and neonatal alloimmune thrombocytopenia: recommendations for evidence-based practice, an international approach. Br J Haematol. 2019;185(3):549-562.Newman PJ, et al. The human platelet alloantigens, Pl Al and P1A2, are associated with a Ieucine33 / proline33 amino acid polymorphism in membrane glycoprotein Illa, and are distinguishable by DNA typing. J Clin Invest. 1989;83(5): 1778-1781.Pacheco LD. et al. Fetal and neonatal alloimmune thrombocytopenia: a management algorithm based on risk stratification. Obstet Gy necol. 2011 ; 118(5): 1157-1163.Peters A. Platelet Kinetics. In: Thakur ML, et al., editors. Radiolabeled Cellular Blood Elements: Pathophysiology, Techniques, and Scintigraphic Applications. Boston. MA: Springer US; 1985. p. 111-33.Regan F, et al. Prenatal Management of Pregnancies at Risk of Fetal Neonatal Alloimmune Thrombocytopenia (FNAIT): Scientific Impact Paper No. 61. BJOG. 2019;126(10):el73- el85.Riches AC, et al. Blood volume determination in the mouse. J Physiol, 1973;228(2):279-284.Tiller H, et al. Toward a prophylaxis against fetal and neonatal alloimmune thrombocytopenia: induction of antibody -mediated immune suppression and prevention of severe clinical complications in a murine model. Transfusion. 2012;52(7): 1446-1457.Vetlesen A, et al. Recovery, survival, and function of transfused platelets and detection of platelet engraftment after allogeneic stem cell transplantation. Transfusion 2012;52: 1321- 1332.Vitiello G, et al. Intravenous immunoglobulin therapy: a snapshot for the internist. Intern Emerg Med. 2019;14(7):1041-1049.Wagner C, et al. Analysis of GPIIb / IIIa receptor number by quantification of 7E3 binding to human platelets. Blood. 1996;88(3):907-14.WHO International Standard: Anti-HPA-la Standard (100 IU). NIBSC code: 03 / 152. National Institute for Biological Standards and Control, Hertfordshire, United Kingdom.Zhi H, et al. High-resolution mapping of the polyclonal immune response to the human platelet alloantigen HPA-la (P1(A1)). Blood Adv. 2018; 2(21): 3001-3011.Zhi H, et al. An Authentic Murine Model of Fetal / Neonatal Alloimmune Thrombocytopenia. Blood 2019; 134 (Suppl. 1):97.Zhi H, et al. Prophylactic administration of HPA-la- specific antibodies prevents fetal / neonatal alloimmune thrombocytopenia in mice. Blood. 2022;140(20):2146-53.Zhou Y, et al. Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion? J Clin Invest. 1997;99(9):2139-2151.***The present invention is further described by the following claims.

Claims

CLAIMS1. A method of preventing alloimmunization with HPA-la in a subject, wherein the subject is an HP A- la-negative woman pregnant with an HP A- la-positive fetus, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an anti-HPA-1 a antibody, wherein the anti-HPA-la antibody does not bind HPA-lb: wherein an initial dose of the pharmaceutical composition is administered between gestational weeks 10 and 16 of pregnancy; wherein maintenance doses of the pharmaceutical composition are administered at four weeks after the initial dose and once every four weeks thereafter during pregnancy; and wherein at least one dose is administered within 72 hours post-parturition.

2. A method of preventing fetal and neonatal alloimmune thrombocytopenia (FNAIT) caused by maternal alloimmunization with HPA-la in an HP A- la-positive fetus of an HP A- la-negative human subject, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an effective amount of an anti-HPA-la antibody, wherein the anti-HPA-la antibody does not bind HPA-lb; wherein an initial dose of the pharmaceutical composition is administered between gestational weeks 10 and 16; wherein maintenance doses of the pharmaceutical composition are administered at four weeks after the initial dose and once every four weeks thereafter during pregnancy; and wherein at least one dose is administered within 72 hours post-parturition.

3. The method of claim 1. wherein the anti-HPA-la antibody is a polyclonal antibody.

4. The method of claim 3, wherein the pharmaceutical composition is anti-HPA- la gamma globulin.

5. The method of claim 1, wherein the anti-HPA-la antibody is a monoclonal antibody.

6. The method of claim 5, wherein the monoclonal antibody is RLYB212.

7. The method of claim 1, wherein the pharmaceutical composition is administered via subcutaneous injection.

8. The method of claim 1 , wherein the initial dose is the same as the maintenance dose.

9. The method of claim 1, wherein the initial dose is higher than the maintenance dose.

10. The method of claim 9, wherein the maintenance dose is half the initial dose.

11. The method of claim 1. wherein the steady-state plasma concentration of the anti-HPA-1 a antibody is between about 3.0 ng / mL and about 10 ng / mL.

12. The method of claim 5. wherein the initial dose of the monoclonal antibody is 0.12 mg.

13. The method of claim 5, wherein the maintenance dose of the anti-HPA-1 a antibody is 0.06 mg.

14. The method of claim 1, wherein the subject is HLA-DRB3*01:01 positive.

15. A pharmaceutical composition comprising an effective amount of an anti- HPA-1 a antibody for use in a method of preventing alloimmunization w ith HPA-la in a subject, wherein the subject is an HP A- la-negative woman pregnant with an HP A- la- positive fetus, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an anti-HPA-la antibody, wherein an initial dose of the pharmaceutical composition is administered betw een gestational weeks 10 and 16; wherein maintenance doses of the pharmaceutical composition are administered at four weeks after the initial dose and once every four weeks thereafter during pregnancy; and wherein at least one dose is administered within 72 hours post-parturition.

16. A pharmaceutical composition comprising an effective amount of an anti- HPA-la antibody for use in a method of preventing fetal and neonatal alloimmune thrombocytopenia (FNAIT) caused by maternal alloimmunization with HPA-la in an HPA- la-positive fetus of an HP A- la-negative human subject, the method comprising parenterally administering to the subject multiple doses of a pharmaceutical composition comprising an effective amount of an anti-HPA-la antibody. w herein an initial dose of the pharmaceutical composition is administered between gestational w eeks 10 and 16; wherein maintenance doses of the pharmaceutical composition are administered at four weeks after the initial dose and once every four weeks thereafter during pregnancy; and wherein at least one dose is administered within 72 hours post-parturition.

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