WO2024239123A1 - Monovalent antibody blocking fc receptor - Google Patents

Abstract

There is provided an antibody or a fragment thereof comprising six complementarity-determining regions (CDRs) having the sequences of SEQ ID Nos: 1, 2, 3, 4, 5 and 6 as described herein. The antibody or fragment thereof is useful for treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcγRIIIA with HLA, and/or HPA-1a.

Description

MONOVALENT ANTIBODY BLOCKING FC RECEPTOR

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This disclosure claims priority from U. S. provisional application 63/504249 filed on May 25, 2023, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] This disclosure relates to monovalent antibodies specific for an activating Fc receptor as well as chimeric proteins comprising same for the use in the prevention, treatment and/or the alleviations of symptoms associated with an alloimmune or an auto-immune inflammatory condition or disorder in a subject.

BACKGROUND OF THE ART

[0003] Antibody-mediated pathological destruction of (self) cells or tissues is a major concern in the prevention and treatment of various auto-immune inflammatory conditions, such as, autoimmune-immune thrombocytopenia (ITP), rheumatoid arthritis, multiple sclerosis, type I diabetes, lupus erythematosus and hemolytic anemias. Antibodies which specifically recognize and bind to self-structures (such as cells and tissues) are recognized by the Fc receptor which is found on the surface of certain immune cells (among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils and mast cells). The formation of a complex between auto-antibodies, the self-structure and the Fc receptor contribute to the destruction of such self-structures by stimulating phagocytosis or antibody-dependent cell- mediated cytotoxicity against the “self structures.

[0004] It has long been considered that Fc gamma receptor (FcyR) blockade can potentially be an effective strategy to ameliorate a number of autoimmune diseases and inflammatory states where intravenous immunoglobulin (IVIg) is effective. Although the mechanism(s) of IVIg itself remains speculative, many consider IVIg to work in large part by mediating FcyR blockade. In addition, work going back many years has demonstrated that FcyR blockade can be an effective therapy in ITP. The conundrum with FcyR blockade is that the antibodies that block FcyRs have had serious adverse events that have precluded development of a therapeutic. It was then found that the adverse events were due to FcyR cross-linking via the bivalent nature of FcyR blocking antibodies which can be overcome using a monovalent FcyR blocker. [0005] Immune thrombocytopenia (ITP) has been used as a model for studying antibody- mediated destruction of cells and tissues occurring in auto-immune conditions and disorders. In ITP, auto-immune anti-platelet antibodies cause the destruction of platelets. Antibody-mediated platelet destruction in the majority of ITP patients involves Fc-mediated phagocytosis by macrophages via the Fc gamma receptors (FcyRs). Activating Fc gamma receptors (FcyRs) are normally a key component of immune defense mechanisms; however, they can also be involved in mediating tissue destruction in several autoimmune diseases, inflammatory diseases, and alloimmune conditions. Inhibition or interference with activating FcyRs has been demonstrated to be beneficial in the treatment of the autoimmune disease immune thrombocytopenia (ITP). One of the major activating FcyRs implicated in platelet depletion is the FcyRIIIA, also a therapeutic target. The first FcyRIIIA-specific monoclonal antibody (mAb) 3G8 was described in 1982, and was later investigated clinically in ITP patients. Encouragingly, more than 50% of ITP patients refractory to other treatments responded with significantly improved platelet counts. However, its continued therapeutic application was stalled by adverse events, including vomiting, nausea and fever. Unfortunately, while successful inhibition of FcyR activity has proven beneficial to alleviate tissue destruction, engagement of FcyRs by FcyR blocking antibodies able to induce cross-linking of the FcyRs, drives adverse inflammatory reactions limiting further development in this area.

[0006] In addition to their potential for use in the autoimmune disease ITP where autoantibodies engage platelets, platelet destruction can also occur due to alloantibodies. Indeed there are a number of important unmet needs which could potentially benefit from FcyR blockade. Patients with antibodies to human platelet antigens such as HPA-1 a, which can occur in the context of fetal and neonatal alloimmune thrombocytopenia (FNAIT) or platelet transfusion, can likewise suffer from platelet destruction due to platelet phagocytosis mediated by macrophages. Additionally, patients with antibodies reactive with HLA molecules on the surface of platelets, which can occur in the context of platelet transfusion, FNAIT, and tissue transplantation, can also suffer from platelet destruction due to platelet phagocytosis.

[0007] It would be desirable to be provided with alternative monoclonal antibodies with similar or improved efficacy compared to the currently used antibodies such as 3G8. It would also be desirable if the alternative monoclonal antibodies have reduced side effects for the treatment or alleviation of symptoms of auto-immune inflammatory disorders or conditions caused or maintained by auto-antibodies which recognize and engage an activating Fc receptor.

SUMMARY [0008] In one aspect, there is provided an antibody or a fragment thereof comprising six complementarity-determining regions (CDRs) having the sequences of SEQ ID Nos: 1 , 2, 3, 4, 5 and 6. The antibody or fragment thereof is a competitive inhibitor of the activating Fc receptor 11 IA and may be a single chain variable fragment (scFv) or a fragment antigen-binding (Fab) for example. The antibody or fragment thereof can be used for treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA- 1 a, and/or for preventing or limiting a phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a. More particularly, the antibody or the fragment thereof can be used in the treatment, prevention or alleviation of symptomps of immune thrombocytopenia (ITP), autoimmune hemolytic anemia (AHA), platelet transfusion induced immune refractoriness, fetal and neonatal alloimmune thrombocytopenia (FNAIT), conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, hereditary hemorrhagic telangiectasia (HHT), graft versus host disease (GvHD), or allograft rejection.

[0009] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.

DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a schematic representation of the procedure performed to obtain 17C02-scFv an antibody that binds to FcyRIIIA.

[0011] FIG. 2A is a graph showing the optical density for different serum dilutions obtained from three BALB/c mice labeled 1 , 2, and 3.

[0012] FIG. 2B is a graph showing the optical density for different serum dilutions obtained from three BALB/c mice labeled 4, 5, and 6.

[0013] FIG. 2C is a graph showing the optical density for different serum dilutions obtained from three BALB/c mice labeled 7, 8, and 9.

[0014] FIG. 3 is a graph showing the normalized optical density through five rounds of screening (R1 , R2A, R2B, R3A and R3B) as described in the Example section. The dashed line represents the cutoff value (OD450 nm = 0.4). The selection criteria also included an OD₄50 nm equal or higher than that detected for 3G8-scFv or 3G8-scFv-albumin. The selection criteria for each round of screening are described at the bottom of the graph. Data are representative of two independent assays. The percentage hit corresponds to the percent of data points above the cutoff line.

[0015] FIG. 4A is a graph showing the geomean fluorescence intensity indicative of the binding of semipurified scFvs to FcyRIIIA expressed on NK cells from healthy human donors.

[0016] FIG. 4B is a graph showing the percentage of inhibition of hlgG-FcyRIIIA interaction mediated by semipurified scFvs.

[0017] FIG. 5A is a graph of the optical density in function of the concentration of the antibodies and purified scFvs tested in a well plate coated with FcyRIIIA.

[0018] FIG. 5B is a graph of the optical density in function of the concentration of the antibodies and purified scFvs tested in a well plate coated with cynoFcyRIIIA.

[0019] FIG. 5C is a graph of the optical density in function of the concentration of the antibodies and purified scFvs tested in a well plate coated with FcyRI I IB

[0020] FIG. 5D is a graph of the optical density in function of the concentration of the antibodies and purified scFvs tested in a well plate coated with FcyRI.

[0021] FIG. 5E is a graph of the optical density in function of the concentration of the antibodies and purified scFvs tested in a well plate coated with FcyRI IA.

[0022] FIG. 6 is a graph showing inhibition curves of hlgG-FcyRIIIA interaction mediated by antibodies and purified scFvs and measured by Homogeneous Time Resolved Fluorescence for each purified scFv.

[0023] FIG. 7A is a sensogram showing the binding capacity of the antibodies, human albumin, and scFvs used as controls to the human FcylllA.

[0024] FIG. 7B is sensogram as per FIG. 7A with the addition of purified scFvs under evaluation and Her-scFv as negative control.

[0025] FIG. 8A is a schematic representation of the 17C02 amino acid sequence in a fusion protein with human albumin (17C02-albumin). [0026] FIG. 8B is a schematic representation of mouse lgG2a containing the amino acid sequence of 17C02 in the variable region (17C02-lgG2a).

[0027] FIG. 8C is a schematic representation of human lgG1 one-armed antibody containing the amino acid sequence of 17C02 in the variable region, the 17C02-lgG1 one-armed antibody (17C02-lgG1_OA).

[0028] FIG. 9A is a graph of the mean fluorescent intensity (MFI) showing the binding of 17C02-albumin to human FcyRIIIA expressed on THP-1-CD16A cells. Statistical analysis, *: p< 0.05; **: p<0.01.

[0029] FIG. 9B is a graph of MFI showing the binding of 17C02-albumin to human FcyRIIIA expressed on peritoneal macrophages isolated from FcyR-humanized mice.

[0030] FIG. 9C is a graph of MFI showing the binding of 17C02-albumin to human FcyRIIIA expressed on Natural killer cells from healthy human donors. Statisitical analysis, *: p< 0.05; **: p<0.01.

[0031] FIG. 9D is a graph of MFI showing the binding of 17C02-albumin to human FcyRIIIA expressed on Neutrophils from healthy human donors.

[0032] FIG. 10A is a graph of the mean fluorescent intensity (MFI) showing the binding of 17C02-lgG2a to human FcyRIIIA expressed on THP-1-CD16A cells. Statisitical analysis, *: p< 0.05.

[0033] FIG. 10B is a graph of MFI showing the binding of 17C02-lgG2a to human FcyRIIIA expressed on peritoneal macrophages isolated from FcyR-humanized mice.

[0034] FIG. 10C is a graph of MFI showing the binding of 17C02-lgG2a to human FcyRIIIA expressed on Natural killer cells from healthy human donors. Statisitical analysis, *: p< 0.05.

[0035] FIG. 10D is a graph of MFI showing the binding of 17C02-lgG2a to human FcyRIIIA expressed on Neutrophils from healthy human donors.

[0036] FIG. 11 is a graph showing the inhibition of FcyRIIIA-mediated phagocytosis of IgG- opsonized erythrocytes by 17C02-based molecules. Statistical analysis, ***: p <0.001. [0037] FIG. 12 is a graph showing the platelet count for different treatment conditions in an antibody-mediated model of immune thrombocytopenia. Two hours after treatment, mice were then intravenously injected with 15 pl of a rabbit anti-platelet serum to induce immune thrombocytopenia. Two hours post-treatment with the anti-platelet serum, mice were bled for enumeration of platelet counts. Statistical analysis, ***: p <0.001 .

[0038] FIG. 13A is a graph of the body temperature of mice in function of time after different treatments. Statistical analysis, ***: p <0.001 .

[0039] FIG. 13B is a bar graph showing the cell count of immune cells in the blood (error bar is the standard deviation (n=3)).

[0040] FIG. 13C is a bar graph showing the cell count of immune cells in the spleen (error bar is the standard deviation (n=3)).

[0041] FIG. 13D is a bar graph showing the cell count of immune cells in the bone marrow (error bar is the standard deviation (n=3)).

[0042] FIG. 13E is a graph showing the platelet count of the mice of Fig. 13A 2 h posttreatment, to determine the ability of the antibody to cause thrombocytopenia on its own. Statistical analysis, *: p<0.05; **: p<0.01 .

[0043] FIG. 14A is a graph showing the platelet counts for FcyR-humanized mice intravenously injected with 333.3 pM of full-length 17C02, 3G8, or 17C02-albumin and measured 2 hours post-treatment to determine the ability of the antibody to cause thrombocytopenia on its own. Statisitical analysis, ns: no statistical difference; *: p<0.05.

[0044] FIG. 14B is a graph showing the platelet counts of the mice of Fig. 14A which were intravenously injected with 15 pl of a rabbit anti-platelet serum two hours after the antibody injection to induce immune thrombocytopenia. Two hours post-treatment with the anti-platelet serum, mice were bled for enumeration of platelet counts. Statistical analysis, ns: no statistical difference; *: p<0.05; **: p<0.01.

[0045] FIG. 14C is a graph showing the body temperature of the mice of Fig. 14A over time.

Statistical analysis, ***: p<0.001 . [0046] FIG. 15 is a graph showing the phagocytic index calculated as the number of platelets engulfed per 100 macrophages in the phagocytosis of anti-HPA1a sera-opsonized human platelets for THP-1-CD16A cells differentiated to macrophages by treatment with PMA (100 ng/mL). Opsonization: (+) indicates platelets were non-opsonized (incubated with normal human serum), opsonized with the monoclonal antibody A2A9 (5 pg/mL) as positive control, or anti-HPA- 1 a patient serum. Statistical analysis, *: p<0.05; **: p<0.01 .

[0047] FIG. 16 is a graph showing the phagocytic index calculated as the number of platelets engulfed per 100 macrophages in the phagocytosis of anti-HLA IgG-opsonized human platelets for THP-1-CD16A cells were differentiated to macrophages by treatment with PMA (100 ng/mL). Opsonization: (+) indicates platelets were non-opsonized (incubated with normal human serum), opsonized with the monoclonal antibody A2A9 (5 pg/mL) as positive control, or anti-HLA patient serum. Statistical analysis, *: p<0.05; **: p<0.01.

[0048] FIG. 17 is a graph showing the number of 5-chloromethylfuorescein diacetate (CMFDA) platelets overtime post injection.

[0049] FIG. 18A is a graph showing the MFI in function of antibody concentration for THP-1 - CD16A and THP-1 cells.

[0050] FIG. 18B is a graph showing the MFI in function of antibody concentration for Natural killer cells.

[0051] FIG. 18C is a graph showing the MFI in function of antibody concentration for neutrophils.

[0052] FIG. 19A is a graph showing the phagocytic index of 17C02-lgG1oA for the phagocytosis of anti-HPA-1a sera-opsonized human platelets using THP-1 -CD16A cells.

[0053] FIG. 19B is a graph showing the phagocytic index of 17C02-lgG1oA for the phagocytosis of anti-HLA sera-opsonized human platelets using THP-1 -CD16A cells.

[0054] FIG. 20A is a graph showing the body temperature over time for FcyR-humanized mice that are untreated or treated with 17C02-lgG1oA.

[0055] FIG. 20B is a graph showing the platelet count for untreated FcyR-humanized mice, ITP model mice, and mice treated with 17C02-lgG1oA. [0056] FIG. 20C is a graph showing the platelet count for mice that were intravenously injected with nothing (Nil) or with 17C02-lgG1oA for 2 hours followed by 15 pl of a rabbit antiplatelet serum (ITP) for 2 hours to induce thrombocytopenia and mice were bled for enumeration of platelet counts to assess the ability of 17C02-lgG1oA to ameliorate thrombocytopenia.

DETAILED DESCRIPTION

[0057] The present disclosure relates to an antibody or a fragment thereof that blocks FcyRIIIA, inhibiting the ability of IgG specific for human leukocyte antigen (HLA) and/or human platelet antigen 1 a (HPA-1a) which can mediate decreases in platelets in vivo and mediate platelet phagocytosis (e.g. in vitro). The specific target of the antibody allows it to be used in the treatment of ITP as well as in alloimmune diseases where platelets are destroyed due to immunoglobulin (Ig) G that is reactive with the platelet. As demonstrated in the Example section below, the antibody of the present disclosure, in some embodiments, is a high-potency monovalent FcyRIIIA blocking therapeutic agent with demonstrated efficacy in vitro by blocking ITP sera-opsonized human platelet phagocytosis. It was also shown to ameliorate ITP in FcyR- humanized mice at a very low dosage (2 mg/kg body weight).

[0058] Immune thrombocytopenia (ITP) is an autoimmune platelet disease and was originally called idiopathic thrombocytopenic purpura (ITP). To keep the name consistent over time the abbreviation ITP remained but the name of the disease was changed in 2009 to immune thrombocytopenia (ITP). In the present disclosure, henceforth, when referring to ITP, the reference is meant to be for the autoimmune disease immune thrombocytopenia (ITP). However, immune thrombocytopenias can also be due to alloantibodies (for example due to HPA1 a antibodies or anti-HLA class I antibodies). These other cases are also referred to herein as immune thrombocytopenias, and can be more specifically labeled as anti-HPA1 a thrombocytopenia or as anti-HLA thrombocytopenia respectively.

[0059] Alloimmune diseases include fetal and neonatal alloimmune thrombocytopenia (FNAIT) which is a severe disorder that affects about 1 in 1000 newborns. It is characterized by the destruction of fetal platelets by maternal antibodies directed against paternally inherited platelet antigens. The major causative antigen in Caucasians is the human platelet antigen 1a (HPA-1 a). The most feared complication is intracranial hemorrhage (ICH) and this usually occurs during the late stages of pregnancy. Non-fatal cases can be affected by life-long sequela. Due to the lack of a population-based screening program, diagnosis is mostly made after birth through the observation of neonatal thrombocytopenia and bleeding complications impacting treatment options. Treatment has been aimed at two outcomes - treating FNAIT-affected newborns and preventing intra-cranial hemorrhage (ICH) in subsequent pregnancies. The main goal of natal treatment is to prevent severe bleeding and ICH. Similar to the ability of anti-D to effectively prevent hemolytic disease of the newborn (HDFN) it is possible to use pooled HPA-1 a⁺ IgG (called NAITgam or RLYB211) to prevent FNAIT. Other possible approaches to prevent or treat FNAIT include monoclonal antibodies specific for HPA-1 a that do not have Fc function which would sterically block the HPA-1 a epitope. In addition to platelet count, the factors that contribute to ICH may involve effects on endothelial cells, complement activation and effects on the placenta. Placental dysfunction, miscarriages and effects on endothelial cells mediated by the FNAIT IgG are generally related to uterine Natural Killer (NK) cells. The depletion of NK cells, and importantly, the in vivo blocking of FcyRIII both ameliorate pregnancy loss and ameliorate the FNAIT. Thus, FcyR blockade of FcyRIII is desired since NK cells express FcyRIIIA as the only activating Fc receptor and therefore, targeting this receptor may be helpful in preventing pregnancy loss.

[0060] HPA-1 a antibodies are known to trigger phagocytosis and antibodies to platelet glycoprotein (GPIIb/llla) induce platelet phagocytosis largely dependent on FcyRIIIA. FNAIT and bleeding severity are linked to the ability of the pathogenic IgG involved to have an Fc region with high affinity for FcyRIIIA based on Fc fucosylation status. Thus, the blockade of FcyRIIIA in FNAIT is a beneficial treatment and specifically the blockade of FcyRIIIA prevents platelet phagocytosis due to HPA-1 a antibodies.

[0061] In addition to the presence of HPA-1 a antibodies in FNAIT, it is not uncommon that HLA class I IgG antibodies are also found in cases of FNAIT with roughly half of the mothers displaying HLA antibodies. In addition, some studies have reported that HLA antibodies can actually cause FNAIT.

[0062] HLA class I antibodies can also be implicated in immune platelet transfusion refractoriness (PTR). Immune PTR can be a major problem for patients with hematologic malignancy with these patients having poorer survival, increased length of hospitalization, increased bleeding, and increased utilization of blood products. In addition, the complexities (and costs) of provision of HLA-matched products are significant as a very large donor pool is required for patients with multiple antibody specificities and sometimes compatible donors are not available. [0063] Although in FNAIT the role of HLA antibodies is still being elucidated, in PTR, it is clear that binding of HLA antibodies to donor platelets results in the rapid clearance of platelets from the circulation. As to the mechanism of this platelet clearance, the ability of HLA antibodies to trigger platelet phagocytosis or platelet clearance has been suggested. Some HLA IgG antibodies can mediate FcyRIIA-dependent platelet activation and subsequent phagocytosis by macrophages, and HLA antibody-mediated internalization of human platelets by macrophages can correlate with HLA antigen expression. It is demonstrated in the Example section below that FcyRIII is involved in the macrophage-mediated phagocytosis of platelets in vitro due to human HLA-reactive alloantibodies.

[0064] The antibody or a fragment thereof described herein that targets FcyRIIIA, comprises three light chain complementarity-determining regions (CDRs) having an amino acid sequence as per SEQ ID Nos: 1 , 2, and 3 (see below) and three heavy chain CDRs having an amino acid sequence as per SEQ ID Nos: 4, 5, and 6 (see below).

• SEQ ID No: 1 = KASQDVSTAVA

• SEQ ID No: 2 = SASYRYT

• SEQ ID No: 3 = QQHYSTPPT

• SEQ ID No: 4 = TSGLGVG

• SEQ ID No: 5 = QIWWDDDKYYNPAL

• SEQ ID No: 6 = ISLYHMGAMDY

[0065] An “antibody”, as used in the context of the present disclosure, refers to an immunoglobulin polypeptide having at least three complementary determining regions (CDRs) and, in some embodiments, up to twelve CDRs. A “complementary determining region” refers to a region of the immunoglobulin polypeptide located in the variable parts of the polypeptide and involved in specifically binding the epitope. The combination of CDRs constitutes the paratope of the antibody.

[0066] As used herein, a “fragment” of an antibody (which can be, for example, a monoclonal antibody) is a portion of an antibody that is capable of specifically recognizing the same epitope as the full version of the antibody. Antibody fragments include, but are not limited to, the antibody light chain, antibody heavy chain, single chain antibodies, Fv, Fab, Fab' and F(ab')2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can be used to generate Fab or F(ab')2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding the heavy chain of an F(ab')2 fragment can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain.

[0067] The process of identifying and isolating an antibody having the CDRs of SEQ ID Nos: 1-6 is provided in detail in the Example section below but is also summarized in Fig. 1. As illustrated in Fig. 1 , BALB/c mice were intraperitoneally injected with seven doses, once weekly (10 pg the first dose and 5 pg each remaining dose) of the recombinant human Fc gamma receptor 11 IA (FcyRIIIA). Mice with a stronger antibody response were selected, from which the total splenic RNA was isolated. The genes encoding for the variable heavy (VH) and light (VL) chains of the antibody repertoire were amplified by polymerase chain reaction (PCR) and cloned into a phage vector. The resulting constructions were then transformed into Escherichia coli (E. coli) and a single-chain variable fragment (scFv) phage display library was obtained. Five rounds of screening (R1 , R2A, R2B, R3A, and R3B) were performed to select phages bound to FcyRIIIA with minimal cross-reactivity with FcyRIIA. Periplasm preparations from the selected phages were obtained and scFv selection was based on binding to FcyRIIIA expressed on NK cells (flow cytometry) and inhibition of hlgG-FcyRIIIA interaction (Homogeneous Time Resolved Fluorescence (HTRF)). Finally, purified scFv were screened again analyzing binding to FcyRIIIA by enzyme-linked immunosorbent assay (ELISA) and Octet, minimal cross-reactivity with the other human receptors (FcyRIA, FcyRIIA, and FcyRIIIB), and inhibition of hlgG-FcyRIIIA interaction. The final antibody fragment, was selected from 10 candidates based in part on a sequencing analysis on glycosylation, oxidation, aggregation, deamidation/isomerization, and proteolytic sites to exclude scFv molecules with low biochemical stability (see Table 4).

[0068] Antibodies of the present disclosure further include antibody derivatives, such as, for example chimeric and humanized antibodies. Accordingly, the antibody or fragment thereof can be optionally combined with a carrier to form a chimeric protein. The terms “chimeric protein” or “chimera” refer to a first proteinaceous entity (e.g., a monovalent antibody moiety) which is associated with another (second) entity, which may be proteinaceous as well. The first proteinaceous entity does not naturally occur in association with the second entity. The first proteinaceous entity is modified (via genetic or chemical means) to be capable of associating or be associated with the second entity. The first and second entity may be derived from the same species or the same genera or can be derived from different species or different genera. The first and second entity can be derived from the genera or the species intended to receive the monovalent antibody or the chimeric protein. For example, the first and/or the second entity can be derived from humans if the monovalent antibody or the chimeric protein are intended to be administered to humans.

[0069] The chimeric protein comprises at least two components or entities: an antibody moiety and a carrier. The two entities can be associated together prior to the administration to a recipient. The two entities can also be associated only after the antibody moiety is administered to the recipient. The association between the two moieties can be covalent or non-covalent and can occur prior to, during or after administration.

[0070] In an embodiment, the carrier is a protein or polypeptide, such as, for example, a plasma protein. Plasma proteins include, but are not limited to serum albumin, immunoglobulins fragments (provided that these fragments do not directly bind the activating Fc receptor or cause the chimeric protein to simultaneously bind to more than one site on the activating Fc receptor), alpha-1 -acid glycoprotein, transferrin, or lipoproteins. In some instances, it is contemplated that a human protein, such as a human plasma protein be used as the carrier. This embodiment is particularly useful when designing therapeutics for the treatment of humans or for making a chimeric protein in which the monovalent antibody moiety is derived (directly or indirectly) from a human antibody or a humanized antibody. In an embodiment, the carrier is immunoglobulin fragment, such as a monovalent antibody moiety of an antibody, for example the anti-neonatal FcR (FcRn) antibody. In such embodiment, the antibody-binding region of the anti-FcRn antibody is associated with the monovalent antibody in order to allow the recognition and binding of the carrierto the FcRn. In another embodiment, the carrier is not proteinaceous in nature, but is rather a chemical polymer. Such polymers include, but are not limited to, polyethylene glycol (PEG).

[0071] In some instances, the chimeric protein is exclusively made of amino acids and is produced by a living organism using a genetic recombination technique. The chimeric protein can consist of an antibody moiety (preferably specific for the Fey receptor), albumin as a carrier and an amino acid linker (such as, for example, a multi-glycine linker (G6 linker)).

[0072] In the chimeric protein, the antibody moiety can be associated directly to the carrier. Alternatively, the antibody moiety can be associated indirectly to the carrier by using one or more linkers between the monovalent antibody moiety and the carrier. Preferably a single linker is used to indirectly associate the monovalent antibody moiety and the carrier. In the context of the present disclosure, the linker must be selected so as not to cause the production of specific antibodies or be recognized by existing antibodies upon the administration to the subject. In an embodiment, the linker is composed of one or more amino acid residues located between the monovalent antibody moiety and the carrier. This embodiment is especially useful when the chimeric protein is intended to be produced in a living organism using a genetic recombinant technique. The amino acid linker can comprise one or more amino acid residues. For example, the amino acid linker can comprises one or more glycine residues such as an hexa-glycine linker. The present chimeric protein also includes those using a non-amino acid linker, such as a chemical linker.

[0073] The antibody moiety can be associated with the linker or the carrier at any amino acid residue(s), provided that the association does not impede the antibody moiety from binding to the activating Fc receptor. In some instances, the linker or the carrier is associated to one or more amino acid residue(s) of the antibody moiety that is (are) not involved in specifically binding the activating Fc receptor. In some instances, the linker or the carrier is associated to a single amino acid residue of the antibody moiety. The linker or the carrier can be associated with any amino acid residue of the antibody moiety, including the amino acid residue located at the aminoterminus of the antibody moiety or at the carboxyl-terminus of the antibody moiety. In instances in which the linker and the carrier are also of proteinaceous nature, the antibody moiety can be associated to any amino acid residue of the linker or the carrier, including the amino acid residue located at the amino-terminus of the linker or the carrier or the amino acid residue located at the carboxyl-terminus of the linker or the carrier. In an embodiment, the amino acid residue located at the amino-terminus of the linker or the carrier is associated to the amino acid residue located at the carboxyl-terminus of the antibody moiety. In still another embodiment, when the linker is present and is of a protaneicous nature, its amino terminus is associated to the carboxyl terminus of monovalent antibody and its carboxyl terminus is associated with the amino terminus of the carrier.

[0074] In instances where a covalent association is sought between the monovalent antibody moiety and the carrier, the association between the two entities can be a peptidic bond. Such embodiment is especially useful for chimeric proteins wherein the at least two entities are both proteinaceaous and are intended to be produced as a fusion protein in an organism (prokaryotic or eukaryotic) using a genetic recombinant technique. Alternatively, the covalent association between the two moieties can be mediated by any other type of chemical covalent bounding. In some instances, the chimeric proteins are designed so as not to be susceptible of being cleaved into the two moieties in the general circulation (for example in plasma).

[0075] The association between the two entities can be non-covalent. Exemplary non- covalent associations include, but are not limited to the biotin-streptavidin/avidin system. In such system, a label (biotin) is covalently associated to one entity/moiety while a protein (streptavidin or biotin) is covalently associated with the other entity/moiety. In such embodiment, the biotin can be associated to the antibody moiety or to the carrier, providing that the other entity in the system is associated with streptavidin or avidin.

[0076] In a further system of non-covalent association, the first entity is designed to be non- covalently associated to the second entity only upon its administration into the intended recipient. This embodiment is especially useful when the carrier is a protein present in the blood of the recipient. For example, the antibody moiety may be associated (in a covalent or a non-covalent fashion) with a second antibody, a lectin or a fragment thereof (referred to herein as an antibody- derived linker) which is capable of non-covalently binding the carrier once administrated to the intended recipient. For example, the second antibody, lectin or fragment thereof can be specific for any blood/plasma protein present in the intended recipient (such as, for example, serum albumin, immunoglobulins fragments (provided that these fragments do not directly bind the activating Fc receptor or cause the chimeric protein to simultaneously bind to more than one site on the activating Fc receptor), alpha-1 -acid glycoprotein, transferrin, or lipoproteins). The second antibody, lectin or fragment thereof can be associated, preferably in a covalent manner, with the antibody moiety at any amino acid residue of the antibody moiety, but preferably at the amino- or carboxyl-end of the antibody moiety. In such embodiment, the second antibody, lectin or fragment thereof is akin to a linker between the antibody moiety and the carrier. Upon the administration of this embodiment of the antibody moiety in the recipient, the carrier (a blood or plasma protein for example) associates with the second antibody, lectin or fragment thereof to form, in vivo, the chimeric protein. In a specific embodiment, the second antibody is an antibody specifically recognizing albumin (such as, for example, an antibody specifically recognizing human albumin).

[0077] In the present disclosure, the antibody, a fragment thereof, or a chimera comprising same is a competitive inhibitor of the activating Fc receptor. More specifically, the monovalent antibody moiety can compete with a binding site used by the activating Fc receptor ligand. The Fc receptor ligands are the Fc region of antibodies. Upon the binding of the Fc receptor ligands to the activating Fc receptor, the activating Fc receptor cross-links and mediates internal signaling leading to a pro-inflammatory immune response in an immune cell. The antibody, a fragment thereof, or a chimera comprising the same, compete forthe activating Fc receptor ligand’s binding site(s) and either prevents the activating Fc receptor ligand from binding to the activating Fc receptor or limits the amount of the Fc receptor ligand that can bind to the activating Fc receptor.

[0078] In some embodiments, the antibody is a monovalent antibody moiety. The monovalent antibody moiety can be derived (directly or indirectly) from a multivalent antibody. The monovalent antibody moiety does not include a functional crystallizable fragment (Fc fragment) of the multivalent antibody it is derived from. The monovalent antibody moiety can be derived (directly or indirectly) from antibodies of any isotypes including IgA, IgD, IgE, IgG, IgM, IgW or IgY. The monovalent antibody can be derived from more than one antibody or from more than one genera or species and, in such instances, is characterized as being a chimeric monovalent antibody moiety. In some instances, the monovalent antibody moiety is derived (directly or indirectly) from the IgG antibody and preferably from a human IgG antibody. The antibody moiety is considered to be “monovalent” because it contains a single antigen binding site. The monovalent antibody moiety has no more than three variable light domains (VL) associated (covalently or not) and no more than three corresponding variable heavy domains (VH). This contrasts with multivalent full- length antibodies which comprises at least two antigen binding sites and more than three VH and more than three VL domains. The monovalent antibody moiety can be fully or partially glycosylated, when compared to the parent multivalent antibody it can be derived from but if glycosylated will have the Fc region functionally impaired. In some instances, the monovalent antibody moiety is not glycosylated. The monovalent antibody moiety can be a humanized or a chimeric monovalent antibody moiety.

[0079] In some instances, the monovalent antibody is a single-chain variable fragment (scFv) derived from one or more multivalent antibody. The scFv is single molecular entity (a fusion protein) consisting of a single antigen-binding region and having no more than three VH and no more than three VL domains from a multivalent antibody which are connected with a linker (e.g., usually a short peptide linker). As such, the scFv consists of a single antigen-binding region and comprises three VL and three VH domains. The scFv can be obtained from screening a synthetic library of scFvs, such as, for example, a phage display library of scFvs.

[0080] In other instances, the monovalent antibody moiety is the fragment antigen-binding region (Fab) of a multivalent antibody. The Fab fragment comprises two molecular entities (a light chain fragment and a heavy chain fragment), consists of a single antigen-binding site and comprises one constant and one variable domain from each heavy and light chain of the antibody which are associated to one another by disulfide bonds. The Fab includes three VL and three VH domains.

[0081] In preferred embodiments, the antibody or fragment thereof according to the present invention is in the form of a monovalent scFv-albumin fusion protein, a monovalent one-armed human lgG1 antibody or an Fc region impaired lgG2a antibody.

[0082] The Fc receptor is a receptor present on the surface of various immune cells such as, for example, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, monocytes, neutrophils, eosinophils, basophils and mast cells. The antibody specifically binds to and recognizes a single antigen or epitope on the activating Fc receptor being FcyRIIIA polypeptide. In some embodiments, even though the antibody may lack a Fc region, the antibody can bind to the activating Fc receptor portion which does recognize the Fc portion of the Fc receptor ligands (antibodies). The antibody or a fragment thereof can be used in the treating, preventing or alleviating the symptoms of an autoimmune or alloimmune disease caused by antibodies which bind FcyRIIIA at their Fc region, bridging the Fc receptor with IgG specific for HLA and/or HPA-1a. More specifically, can be used to prevent platelet phagocytosis and platelet clearance in the context of ITP, HPA-1a alloantibodies, and HLA class I alloantibodies. The antibody was also found to be effective in preventing erythrocyte phagocytosis due to alloimmune IgG specific for erythrocytes and this could also be useful in treating patients with erythrocytespecific alloantibodies. This would include patients who receive multiple red cell (RBC) transfusions and make RBC-reactive alloantibodies, including patients with sickle cell disease or patients with hereditary hemorrhagic telangiectasia (HHT), and cardiac surgery patients (see Example section).

[0083] The antibody or a fragment thereof is capable of limiting or avoiding the activation of an immune cell induced by IgG antibodies which bind FcyRIIIA via their Fc region and bridge the phagocytic cell with HLA and/or HPA-1a present on the platelet. In some embodiment, the antibody or a fragment thereof is capable of preventing signaling from a component of the activating FcyRIIIA receptor complex. This can be achieved by the ability of the antibody or a fragment thereof to prevent or limit the binding of the activating FcyRIIIA with an autoantibody or alloantibody specific for HLA and/or HPA-1 a with their corresponding activating Fc receptor, to prevent or limit signaling associated with a trigger of phagocytosis by the cell comprising the activating Fc receptor.

[0084] In the example provided below, in a disease model of ITP, it was shown that the administration of an embodiment of the antibody as described herein provided a therapeutic response superior to the known and standard 3G8 antibody. The antibody can be used in the treatment, prevention or alleviation of symptoms of FNAIT and immune PTR, but also in autoimmune hemolytic anemia or patients with alloantibodies directed to erythrocytes. Indeed, in the Example provided below, it was shown that the administration of an embodiment of the antibody described herein provided a better treatment response than the standard 3G8 antibody against human leukocyte antigen (HLA) and HPA-1 a alloantibody-mediated phagocytosis. These results show that the antibody can treat platelet transfusion induced immune refractoriness. As such, the present disclosure concerns the use of the antibody, a fragment thereof or a chimera comprising same forthe prevention, treatment or alleviation of symptoms associated with an autoimmune disease which is caused, induced or maintained by the presence of antibodies that bind to FcyRIIIA via their Fc region. Auto-immune conditions which are maintained, mediated or induced by the antibodies that bind to FcyRIIIA via their Fc region, including IgG specific for HLA and/or HPA-1 a include, but are not limited to immune thrombocytopenia, platelet transfusion induced immune refractoriness, FNAIT, conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, HHT, graft versus host disease (GvHD), autoimmune hemolytic anemia and allograft rejection. The antibody, a fragment thereof or a chimera comprising same can successfully be used to treat, prevent or reduce the symptoms of these diseases alone or in combination with other known therapeutic agents.

[0085] The antibody, a fragment thereof or a chimera comprising the same can be formulated for administration with an excipient. An excipient or “pharmaceutical excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more chimeric protein to a subject, and is typically liquid. A pharmaceutical excipient is generally selected to provide for the desired bulk, consistency, etc., when combined with components of a given pharmaceutical composition, in view of the intended administration mode. Typical pharmaceutical excipients include, but are not limited to binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0086] In addition, the term “pharmaceutically effective amount” or “therapeutically effective amount” refers to an amount (dose) effective in treating a subject afflicted by or suspected to be afflicted by an auto-immune inflammatory condition or disorder. It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents.

EXAMPLE

Cell culture, Fc gamma receptors, antibodies, and mice

[0087] THP-1-CD16A cells (American Type Culture Collection (ATCC), CRL3575) were maintained in a 37°C, 5% CO2 environment in complete medium of Roswell Park Memorial Institute (RPMI) 1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM Gluta-Plus (Wisent Bioproducts, Canada), 100 U/mL penicillin, and 100 pg/mL streptomycin (Wisent Bioproducts, Canada). This medium was also supplemented with 5 pg/mL puromycin (Wisent Bioproducts, Canada). Culturing was performed in 75 cm² Nunc cell culture treated EasYFIasks™ (Thermo Fisher Scientific, Denmark).

[0088] The extracellular domain of human FcyRs fused with a polyhistidine tag at the C- terminus were purchased from Sino Biological (Pennsylvania, USA). FcyRI (CD64, Cat: 10256- H08H), FcyRIIIA (CD16A, Cat: 10389-H27H) and FcyRIIIB (CD16B, Cat: 11046-H08C). Biotinylated-FcyRIIA (Cat: 10374-H27H-B) and -FcyRIIIA (Cat: 10389-H27H1-B) were also purchased from Sino Biological. Recombinant Cynomolgus FcyRIIIA (cynoFcyRIIIA, Cat: 9224- FC), was purchased from R&D (Minneapolis, USA).

[0089] Antibodies were obtained from commercial suppliers. Normal mouse IgG (mlgG) and IgG from human serum (hlgG) were purchased from Millipore Sigma (Oakville, Canada). Mouse anti-human CD16A (clone 3G8), mouse anti-human CD64 (clone 10.1), BV421 -conjugated mouse anti-human CD16A (clone 3G8), PE/Cy7-conjugated anti-mouse F4/80 (clone BM8), PE/Cy7-conjugated mouse lgG1 isotype control (clone MOPC-21), BV605-conjugated antihuman CD56 (clone HCD56) and AF674-conjugated anti-c-myc (clone 9E10) were purchased from BioLegend (California, USA). Mouse anti-human CD32 (clone AT10) was purchased from Novus Biologicals Canada (Ontario, Canada). Mouse anti-human CD32 (clone IV3) was purchased from Stemcell Technologies Inc (British Columbia, Canada). Mouse anti human CD16B (clone 2D2G5B9) was purchased from SinoBiological US Inc. (Pennsylvania, USA). Purified mouse lgG1 (clone MOPC-21), lgG2a (clone N/A-CP150), and lgG2b (clone MPC-11) isotype controls were purchased from BioXCell (New Hampshire, USA). Alexa Fluor 647- conjugated mouse anti-human CD42a (clone GRP-P) was purchased from BioRad Laboratories (California, USA). APC-conjugated F(ab)’2 goat anti-mouse IgG-Fcy specific, AF674-conjugated goat anti-mouse IgG (H+L), and AF674 donkey anti-human IgG (H+L) were purchased from Jackson ImmunoResearch (Baltimore, USA). FITC-conjugated goat anti-human serum albumin was purchased from Bethyl Laboratories (Massachusetts, USA).

[0090] FcyR humanized (H-2^d) mice (Smith et al., 2012) were a kind gift from Dr. Jeffery Ravetch from the Rockefeller University, USA. These mice were bred in-house, and both male and female mice aged 7-14 weeks were used for the experimentation. Female BALB/c (H-2^d) mice (aged 4-5 weeks) and female NSG-HLA-A2/HHD mutant mice which express human HLA class I heavy and light chains (NOD. C -Prkdc^SGId H2rg^tm1w^ Tg(HLA-A/H2-D/B2M)1 Dvs/SzJ) were purchased from Jackson Laboratories (California, USA). All mice were maintained in a specific pathogen-free facility at Keenan Research Centre for Biomedical Science, St. Michael's Hospital. All studies were approved by the St. Michael’s Hospital Institutional Animal Care and Use Committee.

BALB/c mice immunization and assessment of the humoral immune response by ELISA

[0091] Mice were intraperitoneally injected with seven doses, once weekly (10 pg the first dose and 5 pg each remaining dose) of the recombinant human Fc gamma receptor IIIA (FcyRIIIA) (SinoBiological US Inc, USA) adjuvanted with aluminum hydroxide (alum) (Alhydrogel) (Brenntag Biosector, Denmark) at a final concentration of 4.68 mg/mL and 40 pg/mL of CpG ODN (Integrated DNA Technology, USA) in a volume of 0.2 mL. Animals were bled on weeks 4 and 7, and the anti- FcyRIIIA antibody response from these bleeds was evaluated by ELISA.

[0092] Briefly, the antibody response was assessed using polystyrene 96-well plates (Corning, USA) that were coated with 8 pg/mL FcyRIIIA in phosphate-buffered saline (PBS) without calcium and magnesium (Gibco, USA) at 4°C for 16 hours. The plates were blocked with 2% bovine serum albumin (BSA) (Millipore Sigma, Canada) in phosphate buffered saline (PBS) for 1 h at room temperature (RT). After three washes with distilled water, the immune response from each mouse was tested by adding to the plates, serial dilutions of sera (1 :400 to 1 :6553600) in PBS with 5% sodium azide for 1 .5 hours at RT. Normal mouse IgG and antibody 3G8, both at 100 nM, were used as negative and positive controls respectively. Plates were then washed three times with distilled water and incubated with 0.2 pg/mL of goat anti-mouse IgG-peroxidase, Fey fragment specific (Jackson ImmunoResearch, USA) in PBS for 1 hour at RT. After that, plates were washed, and incubated with Enhanced K-Blue™ Substrate (TMB) (Neogen, USA/Canada) for 30 min at RT. The reaction was stopped with 1 M HCI and absorbance at 450 nm was read on Spectramax plate reader (Molecular Devices, USA).

Construction of a single-chain variable fragment (scFv) display phage library, screening strategy, and preparation of individual scFv clones.

[0093] FcyRIIIA-immunized mice were splenectomized and the splenocytes were isolated under aseptic conditions, eliminating erythrocytes by lysis in ACK lysing buffer (Gibco, USA). Total mRNA was purified using Dynabeads™ mRNA Purification Kit (Thermo Fisher Scientific, Canada) and cDNA was obtained by reverse transcription based on the protocol of Coronella et al., 2000. Afterwards, universal primers, specific for mouse germline sequences, were used to amplify the genes that encode the heavy and light chain variable regions (VH and VL) by PCR (Coronella et al., 2000). PCR products were recovered and further amplified separately to introduce a G4S sequence as a linker for the VH and VL. Finally, the VH- and VL-linkers were joined by overlap PCR and the final constructions were cloned into the phagemid vector pADL-23c (Antibody Design Laboratories, USA; Cat: PD0111), which were transformed into Escherichia coli (TG1 electrocompetent cells; Agilent, USA) to generate the scFv display phage library.

[0094] The phage library was screened to select FcyRIIIA blocking scFvs following an insolution screening strategy. Five rounds were performed (Table 1) and all of them concluded with a selection step. Rounds R2B and R3B had a de-selection step before its selection step (Table 1). Briefly, 150 pL of the phage library in 1 .5 mL tubes was blocked with 950 pL of blocking buffer (PBS containing 3% skimmed milk) for 1 hour at 4°C. Fifty pL of the blocked phages were stored at -80°C as “Input phages” until the samples were ready for titration. Concurrently, streptavidin magnetic beads (New England Biolabs, UK) were treated with blocking buffer (PBS containing 3% skimmed milk) for 1 hour at 4°C. After that, the blocked streptavidin magnetic beads were mixed with 950 pL of the blocked phages for 1 hour at RT. The mixture was then centrifuged, after which the supernatant was incubated for 2 hours at RT with the concentration of biotinylated FcyR for selection or de-selection as specified in Table 1 . Afterwards, the mixture of phage and FcyR was incubated with a fresh set of blocked streptavidin magnetic beads for 15 min at RT, centrifuged, and the supernatant discarded (selection steps) or collected (de-selection steps). For rounds R2B and R3B (Table 1) the collected supernatant was then incubated for 2 hours at RT with the concentration of biotinylated FcyR for their selection step. Later, the mixture of phage and FcyR was incubated with a fresh set of blocked streptavidin magnetic beads for 15 min at RT, centrifuged, and the supernatant discarded. For all rounds, the elution of phages was performed as follows. Beads were washed 5 times with 1 mL PBS 0.1 % Tween-20™ and then 5 times more with 1 mL only PBS. Finally, the bound phages were eluted by treating the beads with 1 mL of 100 mM triethylamine (Sigma Millipore, Canada) for 10 min at RT. Eluted phages were transferred to a tube containing 500 pL sterile 1 M Tris-HCI, pH 7.5 to neutralize the triethylamine. Fifty pL of eluted phages were stored at -80°C as “Output phages” until ready for performing titration. Rounds R2A and R2B started with the eluted phages from round R1. Rounds R3A and R3B started with the eluted phages from both (a mixture) of rounds R2A and R2B.

Table 1 . Rounds of in-solution selection and its steps

Selection refers to the process of retaining phages that were bound to the FcyR specified while deselection refers to the process of collecting the phages that were not bound to the FcyR.

[0095] Input and output phage titers refer to the number of colony-forming units/mL at the beginning and end of the round. Both titers were determined by infecting TG1 electrocompetent cells with 10-fold serial dilution of the phages, as previously described (Hoogenboom, 1997). As an example, phages were diluted in blocking buffer (PBS containing 3% skimmed milk) and 10 pL of each dilution (from 10¹ to 10⁹) were incubated at 37°C for 30 min with 100 pL of a suspension of TG1 cells with an optical density (ODeoonm) of 0.53. Ten pL of the total volume was spread onto 2xTY agarose plates (Millipore Sigma, Canada) supplemented with 2% glucose (Millipore Sigma, Canada) and 100 pg/mL carbenicillin (Millipore Sigma, Canada). The plates were then incubated at 37°C for 16 hours. The plate with the highest dilution where the number of colonies was countable was selected to calculate the titer.

In-Solution Input Titer Calculation tion . . .. , x 110 /zL - x 10⁶ x 1 mL

In Solution Output Titer Calculation

# of phage in 1.5

1.5 mL

[0096] The plates used for determining the output phage titers of each round were stored at 4°C and then used for picking individuals colonies to create master glycerol stocks. Briefly, 120 pL of 2xYT medium (Millipore Sigma, Canada) supplemented with 2% glucose (Millipore Sigma, Canada) and 100 pg/mL carbenicillin (Millipore Sigma, Canada) were added to 96-well U bottom plates (Greinier-Bio-One, USA). Each well was inoculated with an individual colony using filter tips. Plates were sealed with breathable seal, incubate 16 hours at 37°C, 80% humidity at 150 rpm for allowing bacterial growth. Next day, 60 pL of 50% glycerol was added to each well and plates were then sealed with adhesive foil and stored at -80°C. Each well represents an individual scFv clone.

Obtaining periplasmic prep (periprep)

[0097] For each scFv clone, 5 pL of master glycerol stock was inoculated into 900 pL of 2xYT medium (Millipore Sigma, Canada) containing 0.1 % glucose (Millipore Sigma, Canada) and 100 pg/mL carbenicillin (Millipore Sigma, Canada) in 96-well polypropylene DeepWell plates (Thermo Fisher Scientific, Denmark) using Biomek Liquid Handling Automation (Beckman Coulter, Canada). Plates were sealed with breathable seals and incubated in MaxQ8000^TM incubator (Thermo Fisher Scientific, Canada) at 200 rpm, 37°C for 5 hours. After the initial culture, the expression of scFv was induced with isopropyl-p-D-thiogalactopyranoside (IPTG) (Millipore Sigma, Canada) 0.2 mM (final concentration) during 16 hours at 30°C. Plates were then centrifuged at 2500 rpm for 10 min to remove supernatant and cells were lysed with 150 pL periprep buffer 1 (50 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA), 20% sucrose, pH 8), vigorously mixed, diluted with 150 pL periprep buffer 2 (periprep buffer 1 diluted 1 :5) and incubated on ice for 30 min. Plates were centrifuged at 4000 rpm for 15 min at 4°C and 200 pL of supernatant was collected and blocked with 200 pL of 6% skimmed milk, 2% BSA in PBS for 1 hour at RT (blocked periprep).

Expression and purification of scFV clones

[0098] For each scFV clone, 2 pL of master glycerol stock was inoculated into 10 mL of 2xYT medium containing 0.1 % glucose, 100 pg/mL carbenicillin using loosely capped 50 mL falcon tubes and grown in MaxQ8000^TM incubator at 200 rpm, 37°C, for 4 hours. After initial culture, scFv expression was induced with IPTG af final concentration of 0.2 mM in MaxQ8000 ^TM incubator at 200 rpm, 30°C for 16 hours. Five cultures of each clone were combined, and cells pelleted at 2500 rpm for 10 min to remove the supernatant. Cells were lysed with 300 pL periprep buffer 1 (50 mM Tris, 1 mM EDTA, 20% sucrose, pH 8), vigorously mixed, diluted with 300 pL periprep buffer 2 (periprep buffer 1 diluted 1 :5) and incubated on ice for 30 min. After the incubation, cell debris was pelleted at 4000 rpm for 15 min at 4°C, supernatant was collected and spiked with MgCh at a final concentration of 10 mM.

[0099] His MultiTrap HP plates (GE Healthcare, USA) were initially equilibrated with 500 pL distilled water and two times 500 pL binding buffer (20 mM sodium phosphate, 500 mM NaCI, 20 mM imidazole (pH 7.45)). Samples were loaded onto plates, incubated for 3 min, and then centrifuged at 100 xg for 4 min. Flow through was re-loaded back onto plates, incubated for 3 min, and centrifuged at 100 xg for 4 min. Plates were washed three times with 600 pL binding buffer and centrifuged at 500 xg for 2 min between washes. Samples were eluted by two incubations with 200 pL elution buffer (20 mM sodium phosphate, 500 mM NaCI, 500 mM imidazole (pH 7.46)) and centrifugation at 500x g for 2 min. Eluted samples were buffer exchanged into PBS with Amicon Ultra Filter Device (10 kDa MWCO) (Millipore Sigma, Canada) according to manufacturer’s protocol and quantified using Thermo Scientific™ Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, Canada).

Binding of anti-FcyRIIIA antibodies to human FcyRs and cynomolgus FcyRIIIA by ELISA

[0100] MaxiSort™ 96-well plates (Thermo Fisher Scientific, Canada) were coated with 100 pL/well of FcyRIA (2.5 pg/mL), FcyRIIIA (10 pg/mL), cynoFcyRIIIA (10 pg/mL), or FcyRIIIB (2.5 pg/mL) diluted in PBS at 4°C for 16 hours. For the specific case of FcyRIIA, polystyrene 96-well plates (Corning, USA) were precoated with 10 pg/mL neutravidin protein (Thermo Fisher Scientific, Canada) diluted in PBS at 4°C for 16 hours prior to coating the plate with biotinylated- FcyRIIA (2.5 pg/mL) for an additional hour. All plates were blocked for 1 hour at RT with PBS containing 3% skimmed milk, 1 % BSA. The plates were then washed three times with distilled water and incubated with blocked periprep, purified scFv (0.07-700 nM), or controls for 1 hr at RT. Controls for each FcyR are described in Table 2. Afterwards, the plates were washed as described above, except for the periprep samples where plates were washed five times with PBS 0.1 % tween-20. The plates were then incubated with the appropriate HRP-conjugated secondary antibody (Table 2) for 1 hour at RT. Plates were washed as described above, followed by a 30- minute incubation with Enhanced K-Blue™ Substrate (TMB) at RT. The reaction was stopped with 1 M HCI and the absorbance was read at 450 nm on a Spectramax™ plate reader.

Table 2. Controls and secondary antibodies for FcyR binding assay

mlgG1: mouse lgG1; mlgG2a: mouse lgG2a; HSA: human serum albumin (Abeam, Canada)

Binding to human NK cells by Flow Cytometry

[0101] Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll-Paque™ Plus (Amersham Biosciences AB, Sweden) density gradient centrifugation as previously described (Lan et al., 2007). Cells were seeded in a 96-well v bottom plate (Corning, USA) at 50000 cells per well and incubated with blocked peripreps, 70 nM 3G8, 70 nM mlgG1 (isotype control), 700 nM 3G8-scFv or 700 nM Her-scFv for 1 hour on ice. Cells were washed with PBS and incubated for 30 min on ice with a mixture of BV605-conjugated anti-human CD56 (1 :100) and propidium iodide (1 :1000) in PBS 1 % FBS plus AF674-conjugated anti-c-myc (1 :1000) for detection of blocked peripreps or scFv, or 2 pg/mL AF647-conjugated anti-mouse IgG for detection of full- length antibodies. Cells were washed, resuspended in PBS, 1 % FBS and analyzed on the Intellicyt iQue™ Screener Plus (Sartorius, Germany) using Forecyt™ Software (Sartorius, Germany).

Inhibition of the interaction between FcyRIHA and human IgG

[0102] The capacity of blocked peripreps and scFvto inhibit the interaction between FcyRIHA and human IgG (hlgG) was evaluated by Homogeneous Time Resolved Fluorescence (HTRF) essentially using a method previously reported (Degorce et al., 2009). Briefly, equal volumes of 1 :62.5 streptavidin-XL665 (CisBio, Canada), 0.25 mg/mL biotinylated-FcyRI I IA, 1 :31.25 europium-conjugated anti-hlgG (CisBio, Canada), and 0.8 mg/mL hlgG were mixed in reconstitution buffer (CisBio, Canada). Then, 10 pL of the mixture were plated in each well of 384- well low volume polystyrene microplates (Corning, USA). After that, 10 pL of blocked peripreps, or working stocks (0.06 - 1000 nM) of purified scFV, Her-scFV, 3G8-HSA or HSA, or 0.6-100 nM 3G8 or mlgG isotype control were added and incubated for 3 hours at RT in the dark. As negative control, 10 pL of reconstitution bufferwas used (as a no competition control). Afterthis incubation, samples were analyzed on a Synergy 4 plate reader using Gen5 Software (Filter wheels 330/80 and 620/10).

[0103] The following summarizes final concentrations for HTRF reagents:

• 1 :500 streptavidin-XL665,

• 20 nM biotinylated-FcyRII IA,

• 1 :250 europium-conjugated anti-hlgG,

• 20 nM hlgG, and

• Competing reagent: blocked peripreps, 0.03-500 nM scFV or 3G8-HSA or 0.3-

50 nM IgG. [0104] For screening, the concentration used was 200 nM scFV or 3G8-HSA and 20 nM human IgG (hlgG).

[0105] The following formulas were used to calculate the percentage of inhibition for each sample (Background = 1 :250 europium-conjugated anti-hlgG in reconstitution buffer):

Delta Ratio = Sample Ratio — Background Ratio

Binding analysis to FcyRIIIA by Octet

[0106] The biomolecular interaction of purified scFv to the human FcyRIIIA was evaluated using streptavidin biosensors (ForteBio, USA) on ForteBio Octet Red96e (ForteBio, USA) with Data Acquisition 11.1 software. Data was analyzed using Data Analysis HT 11.1 software as essentially described (Kamat & Rafique, 2017). Briefly, the biosensor was dipped into buffer assay (PBS pH 7.4, 0.02% Tween-20^TM, 0.5% BSA) to establish the baseline. Later, the biosensor was dipped into buffer assay containing the biotinylated-FcyRI HA at 1.5 pg/mL to immobilize the receptor and another baseline step was performed by dipping the biosensor into assay buffer. For the association step, the biosensor containing the Fc receptor of interest, or a control protein was then dipped into assay buffer containing the analyte (e.g., 500 nM purified scFv) and finally the dissociation is allowed by dipping biosensor into assay buffer. Conditions are described in Table 3. The following molecules were used as controls: 3G8 or mouse lgG1 (mlgG1), 3G8-scFv or Her-scFv, and 3G8-HSA or HSA.

Table 3. Conditions for the binding of purified scFv to FcyRIIIA by Octet

Expression of the selected scFv 17C02 as an albumin fusion molecule, a mouse lgG2a, and a human lgG1 one-armed antibody

[0107] The gene that encodes for the selected scFv 17C02 with additional genetic sequences encoding human albumin and linkers as well as a 6xHis tag (Figs. 8A-8C) were cloned into the mammalian expression vector pCEP4 (Thermo Fisher Scientific, Canada). For the expression of the molecule, 30 pg of the construction (pCEP4-VH-Vi_-HSA-6His) was incubated with polyethylenimine hydrochloride 40K (PEI) (Polysciences, USA) in a 1 :3 (DNA: PEI) ratio for 20 min at RT. Afterwards, the mixture was added to 30x10⁶ Expi293F cells (Thermo Fisher Scientific, Canada) at a concentration of 10⁶ cells/mL for a final ratio of 1 pg of DNA per 1 million cells. These cells were cultured with 0.1 % Pluronic F68 (Avantor, Canada) and 0.5 M valproic acid (Sigma Millipore, Canada) for 7 days, after which the supernatant was collected to check for antibody expression and then purified using Ni-NTA Superflow Columns (Qiagen, USA).

[0108] In addition, the genes that encode for VL and VH of the select scFv 17C02 were cloned into the plasmids pFUSE2-CLIg-mK (Catalogue code: pfuse2-mclkm; InvivoGen, USA) and pFUSE-CHIg-mG2a (Catalogue code: pfuse-mchg2a; InVivoGen, USA) respectively, to express the molecule as a full-length mouse lgG2a antibody. Both genes were synthesized with IDT (Integrated DNA Technologies, USA) and an II-2 leading sequence was added to both. Finally, the gene encoding the VH region from 17C02 was cloned into the plasmid pFUSE-CHIg-mG2a vector using the restriction sites EcoR I and Eco47lll. The gene encoding the VL portion from 17C02 was cloned into the plasmid pFUSE2-CLIg-mK using the restriction sites Agel and BstAPI. For expression of the antibody, 20 pg of both constructions (pFUSE2-CLIg-mK-VL and pFUSE- CHIg-mG2a-VH) were incubated with PEI (Polysciences, USA) in a 1 :3 (DNA: PEI) ratio for 20 min at RT. Afterwards, the mixture was added to 40x10⁶ Expi293F cells (Thermo Fisher Scientific, Canada) (at a concentration of 10⁶ cells/mL) for a final ratio of 1 pg of DNA per 1 million cells. Cells were cultured with 0.1 % Pluronic F68 (Avantor, Canada) and 0.5 M valproic acid (Sigma Millipore, Canada) for 7 days, after which the supernatant was collected to check for antibody expression and then purified using a Protein G Sepharose matrix (GE Healthcare, USA).

[0109] Finally, the genes encoding the VL and VH of the select scFv 17C02 were used to express a human lgG1 one-armed antibody using the “knob into hole” strategy. Briefly, the plasmid pEmiLC (Addgene, USA) was modified to express the heavy chain of the human lgG1 with LALA mutations (L234A and L235A) to avoid antibody glycosylation and thus Fc region function (e.g., engagement with FcyRs and complement). In addition, the sequence WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) was introduced in the 3' untranslated region of the coding sequence to enhance the expression of the gene. The CH3 region was also modified to create the “hole” site. A second pEmiLC vector was modified to express the kappa light chain of the human antibody. The sequence WPRE was also introduced in the 3' untranslated region of the coding sequence. A third pEmiLC vector was modified to express the CH2-CH3 portion of a human lgG1 with the “knob” site. The WPRE sequence was also incorporated into the 3' untranslated region of the coding sequence. Forthe expression of the one-armed antibody, 13 pg of each of the vectors were incubated with PEI (Polysciences, USA) in a 1 :3 (DNA: PEI) ratio for 20 min at RT. Afterwards, the mixture was added to 40x106 Expi293F cells (Thermo Fisher Scientific, Canada) (at a concentration of 106 cells/mL) for a final ratio of 1 pg of DNA per 1 million cells. Cells were cultured with 0.1 % Pluronic F68 (Avantor, Canada) and 0.5 M valproic acid (Sigma Millipore, Canada) for 7 days, after which the supernatant was collected to check for antibody expression and then purified using a Protein A Sepharose matrix (GE Healthcare, USA).

Antibody deglycosylation

[0110] Antibodies 3G8, 10.1 , AT10, IV.3, 17C02-lgG2a, and isotypes controls (lgG1 and lgG2a) at 0.5 mg/mL in PBS (Gibco, USA) were fully Fc region deglycosylated using glycerol-free recombinant PNGase-F (NEB, USA) with 8 units PNGase-F/pL antibody and incubated for 48 hours in a 37° CO2 incubator using a method previously described (Norris et al., 2021). Antibody was separated from the glycans and PNGase F using a 50 kDa molecular weight cut-off column concentrator (Millipore Sigma, Canada) with repeated washing with PBS.

Isolation of peritoneal macrophages and splenocytes from FcyR-humanized mice

[0111] Peritoneal macrophages were prepared by 2 mL-injection of 3% brewer’s thioglycollate medium (Sigma-Aldrich, USA) into the peritoneum of mice. Peritoneal cells were collected four days later as a terminal procedure as described (Zhang et al., 2008). Animals were also splenectomized and spleen cells were obtained under aseptic conditions, eliminating erythrocytes by lysis in ACK buffer (Gibco, USA). Both primary cells were washed with cold PBS and re-suspended in complete RPMI medium. Cells were cultured in 25 cm² Nunc cell culture treated EasYFIasks™ (Thermo Fisher Scientific, Canada) at 37°C and 5% CO2 for 4 hours, followed by extensive washing with cold PBS to remove non-adherent cells. Adherent cells were then incubated in complete RPMI medium at 37°C, 5% CO2 for 16 hours. The purity of the PerM0 and the percentage of splenic macrophages (SpM0) was determined by flow cytometry using a fluorescently labeled anti-F4/80 antibody.

Flow cytometric analysis

[0112] The binding capacity of 17C02-albumin, 17C02-lgG2a, and 17C02-lgG one-armed antibody (17C02-lgG1oA) to different sources of cells expressing the FcyRIIIA was analyzed by flow cytometry using 3G8 (positive control), 3G8-albumin (positive control), and albumin, mlgG1 , mlgG2a, and hlgG1 as negative controls. Molecules were diluted (to concentrations of 274-0.055 pM) in phosphate-buffered saline (PBS) 1 % BSA solution and incubated with cells for 30 minutes on ice. Cells were then washed two times with PBS and then incubated with the corresponding secondary antibody: FITC-conjugated goat anti-albumin (dilution 1 :200), APC-conjugated F(ab)’2 goat anti-mouse IgG-Fcy specific (dilution 1 :300) or AF647-conjugated donkey anti-human IgG (H+L) (dilution 1 :300). After this incubation, cells were washed two times with PBS and analyzed by flow cytometry using a BD LSRFortessa™ X-20 (Beckton Dickson, USA). Data analysis was performed using FlowJo v10 (Beckton Dickson, USA).

Phagocytosis of antibody-opsonized erythrocytes

[0113] Phagocytosis of antibody-opsonized erythrocytes was essentially carried out as described (Norris et al., 2021). Briefly, THP-1-CD16A cells were seeded into wells of 24-well polystyrene plate at 2x10⁵ cells/well in complete RPMI medium also containing 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) (BioShop, Canada) for macrophage differentiation, and incubated at 37°C, 5% CO2. Twenty-four hours later, medium was replaced by fresh complete RPMI medium, and cells were maintained under the same condition overnight. Human erythrocytes expressing RhD (RhD⁺) were washed three times with PBS by centrifugation at 170xg for 2 minutes without break and finally resuspended in 0.5 mL PBS. Erythrocyte concentration was determined using a Guava easyCyte™ flow cytometer (Luminex Corporation, USA) and adjusted to 5x10⁸ cells/mL. Erythrocytes were opsonized with a polyclonal anti-human RhD antibody (WinRho SDF^TM) for 30 minutes at room temperature and washed by centrifugation. Concurrently, macrophages were treated with deglycosylated FcyR-blocking antibodies or isotypes controls at a concentration of 10 pg/mL diluted in complete RPMI medium for 30 minutes at 37°C, 5% CO2, followed by two washes with PBS. Complete RPMI was added back to all wells and opsonized erythrocytes were added to macrophages (1x10⁷ erythrocytes per well, ~20:1 ratio erythrocyte per macrophage). Phagocytosis was allowed to proceed for 30 minutes at 37°C, 5% CO2 and the reaction stopped on ice. Wells were washed with PBS before performing a 60-second hypotonic lysis using water followed by 10X PBS to return to normal osmolarity. Wells were washed with PBS and macrophages were fixed with 4% formaldehyde solution in PBS. Macrophages were imaged by microscopy using an inverted microscope near the center of each well. At least four images were taken per well for >500 cells counted. Phagocytic index (PI) was calculated as:

Phagocytosis of Antibody-Opsonized Platelets

[0114] Phagocytosis of antibody-opsonized platelets was essentially carried out as described (Norris et al., 2021). Briefly, THP-1 -CD16A cells were seeded on sterile glass coverslips (Thermo Fisher Scientific, Canada) inside wells of a 24-well polystyrene plate at a concentration of 2x10⁵ cells/well in complete RPMI and incubated overnight at 37°C, 5% CO2. PMA-induced differentiation of THP-1 CD16A cells was performed as described in the previous section. The following day, whole blood was collected into a tube containing anticoagulant citrate-dextrose solution (BD, USA). Platelet-rich plasma was collected by centrifugation (400xg, 8 minutes, slow break) and platelets were counted using a Multisizer 3 particle counter (Beckman Coulter, Canada). Platelets were kept in the presence of 100 ng/mL of Prostaglandin E1 (Sigma-Aldrich, USA) for all manipulations to prevent activation. PBS-EDTA 0.1 % was added, and platelets were centrifuged to remove plasma (800xg, 10 minutes, slow break). Platelets were fluorescently labelled by resuspending to 4x10⁸ platelets/mL in PBS with 20 pM 5-chloromethylfuorescein diacetate (CMFDA) (Thermo Fisher Scientific, USA) and incubated for 45 minutes, room temperature, protected from light under constant gentle agitation. Platelets were washed by centrifugation (800xg, 10 minutes) and opsonized with thrombocytopenic patient serum or normal human serum at 1 :1 ratio (serum:platelet) for 30 minutes at room temperature. Platelets were washed by centrifugation and resuspended in PBS before addition to macrophages. Macrophages were treated with FcyR-blocking antibodies or controls at a concentration of 10 pg/mL in complete RPMI for 30 minutes at 37°C, 5% CO2, and washed two times with PBS. Antibody-opsonized platelets were added to the macrophages at a ratio of 100:1 (platelets:macrophage) in complete RPMI. Phagocytosis was allowed to proceed for 60 minutes at 37°C before stoppage on ice, PBS washing, and formaldehyde fixation (4% solution in PBS). After fixation, wells were washed with PBS and surface-bound platelets were stained for 30 minutes with an anti-CD42a (GPIX)-AlexaFluor 647 (GP5) to distinguish them from internalized platelets. Wells were washed two times with PBS. Coverslips were mounted onto glass slides (Thermo Fisher Scientific, USA) with Dako Fluorescence Mounting Medium (Agilent Technologies, USA). Macrophages were observed by spinning-disc confocal microscopy under 63x objective oil immersion (numerical aperture 1 .47) with differential interference contrast (DIC) and laser fluorescence (488 nm, 647 nm excitation) on a Quorum multi-modal imaging system (Quorum Technologies, Canada) equipped with 50 micrometer pinhole spinning disc and ORCA- Flash 4.0 V2 PLUS sCMOS camera. At least four different images were taken near the centre of each well for >500 cells imaged, with Z-stacking every 0.33 pm with >30 stacks. Z-stacked images were 3D reconstructed for analysis using Imaris v8.0.2 (Bitplane, UK). Surface-bound (nonphagocytosed) platelets were identified by staining positively for AlexaFluor 647. Phagocytic index (PI) was calculated as:

PI = [(Total number of platelets internalized) I (Total number of macrophages counted)] x 100

In vivo evaluation of 17C02-based molecules in a passive model of ITP

[0115] Immune thrombocytopenia was passively induced with a rabbit anti-platelet serum (Cedarlane, CLA31440) in FcyR-humanized mice using a method previously described in detail (Crow et al., 2015). Briefly, all treatments were administered intravenously via the lateral tail vein. To examine the in vivo effect of 17C02-albumin and 17C02-lgG2a, mice were treated with an equimolar amount (540 or 333.3 pM/mouse) of these molecules as well as human albumin as a negative control. For clarity, two different concentrations of these FcyRIIIA blockers as well as the albumin control were used in the blocking studies. The same equimolar amounts of 3G8-albumin and 3G8 (mouse lgG1) were also evaluated as references. Body (rectal) temperature was then monitored 15, 30 and 45 minutes after treatment (e.g., injection of mice with 17C02-albumin) using a Digi-Sense Type J/K/T Thermocouple Thermometer rectal temperature probe (Kent Scientific, USA) coated with Vaseline (Healthcare Plus, Canada) to assess adverse events. After two hours, the animals were bled via the saphenous vein to count platelets (as described in detail; (Beeton et al., 2007)) and ITP was induced with 15 pL of the rabbit anti-platelet serum. Animals were bled again two hours later via the saphenous vein and the platelet number was enumerated by a Multisizer 3 particle counter (Beckman Coulter, Canada) as described in detail (Crow et al., 2015).

In vivo evaluation of 17C02-albumin to block the clearance of platelets due to anti-HLA-A2 alloantibody-mediated model of immune thrombocytopenia

[0116] A passive model of alloimmune thrombocytopenia was induced in FcyR-humanized transgenic mice using ex vivo sensitized HLA-A2⁺ mouse platelets. NSG-HLA-A2/HHD mutant mice were anesthetized using 5% isoflurane in an oxygen atmosphere. Cardiac puncture procedure was performed using 1 mL syringe with a 25-gauge needle tip containing 200 pL 1 :1 (anticoagulant buffer: BSGC) with 1 pg/mL of carbaxyclin (Santa Cruz Biotechnology, USA). Blood from these mice was then expelled into a tube with 3 mL Buffered Saline Glucose Citrate buffer (BSGC) with 1 pg/mL of carbaxyclin, and then centrifuged at 300xg for 3 minutes (8 acceleration, low brake). Platelet-rich plasma was collected, and platelets were enumerated using a Multisizer 3 particle counter (Beckman Coulter, Canada). Platelets were fluorescently labelled by resuspending to 4x10⁸ platelets/mL in PBS, 1 % EDTA, 1 pg/mL of carbaxyclin, with 20 pM CMFDA and incubated for 45 minutes, room temperature, protected from light under constant gentle agitation. Platelets were washed by centrifugation (800xg, 10 minutes) and opsonized with anti-HLA-A2 patient serum or normal human serum at 1 :1 ratio (serum:platelet) for 30 minutes at room temperature. Fluorescent anti-HLA-A2-sensitized platelets were washed by centrifugation and resuspended in PBS at 5x10⁸ platelets/mL priorto the injection in FcyR-humanized transgenic mice.

[0117] Mice (FcyR-humanized transgenic mice) were then either injected with 5x10⁷ nonsensitized fluorescent platelets (Control), NHS-sensitized fluorescent platelets (NHS), or anti- HLA-A2-sensitized fluorescent platelets. The number of CMFDA-labeled platelets in the blood circulation of the recipient mice was followed for 4 hours using a Guava easyCyte™ flow cytometer (Luminex Corporation, USA). An anti-FcyRI I IA therapeutic intervention was evaluated injecting a group of mice with 540 pM of 17C02-albumin 2 hours before the administration of a nti- HLA-A2-opsonized CMFDA-labeled platelets.

• Anticoagulant buffer: 130 mM trisodium citrate dihydrate, 10 mM disodium EDTA dihydrate, 10 mM Theophyllin.

• BSGC buffer: 13.6 mM trisodium citrate dihydrate, 116 mM NaCI, 8.6 mM Na₂HPO₄ heptahydrate, 0.9 mM disodium EDTA dihydrate, 11.1 mM glucose, pH 6.8.

Statistical analysis

[0118] Prism version 8.00 for Windows (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Data normality was verified using D’Agostino-Pearson’s test, and homogeneity of variance was checked using Bartlett’s test. Non-parametric tests were used for further analysis whenever data was not normally distributed even after transformation. Parametric analyses of more than two groups were performed using a one-way or two-way analysis of variance with Tukey’s post-test or Sidak's post-test. Non-parametric analyses of more than two groups were performed using the Kruskal-Wallis test and Dunn’s Multiple Comparison test.

A single-chain variable fragment (scFv) display phage library against the human FcyRIIIA

[0119] FcyRs are associated with protection against infectious diseases but also pathogenicity in several antibody-mediated autoimmune and inflammatory disorders. FcyRIIIA is an activating and inflammatory receptor involved in the pathogenesis of several antibody- mediated inflammatory disorders, such arthritis, glomerulonephritis, IgG-dependent anaphylaxis, IgG-mediated hemolytic anemia, and immune thrombocytopenia. Thus, biological therapies targeting this receptor are of interest. Phage display libraries of human single-chain variable fragments (scFv) are a reliable source of developing human antibodies and antibody-like therapies for scientific and clinical applications.

[0120] To develop a scFv display phage library against the human FcyRIIIA (overview of the strategy is shown in Fig. 1), nine BALB/c mice were intraperitoneally injected with seven doses of the recombinant human FcyRIIIA once weekly (10 pg for the first dose and 5 pg each remaining dose) adjuvanted with aluminum and CpG ODN. [0121] Fig. 1 shows that scFvs are obtained which bind and block FCYRIIIA. This resulted in the generation of the 17C02 antibody. BALB/c mice were intraperitoneally injected with seven doses, once weekly (10 pg the first dose and 5 pg each remaining dose) of the recombinant human Fc gamma receptor 111 A (FcyRIIIA). Mice with a stronger antibody response were selected, from which the total splenic RNA was isolated. The genes encoding for the variable heavy (VH) and light (VL) chains of the antibody repertoire were amplified by PCR and cloned into a phage vector. The resulting constructions were then transformed into Escherichia coli (E. coli) and a single-chain variable fragment (scFv) phage display library was obtained. Five round of screening (R1 , R2A, R2B, R3A, and R3B) were performed to select phages bound to FcyRIIIA with minimal cross-reactivity with FcyRIIA. Periplasm preparations from the selected phages were obtained and scFv selection was based on binding to FcyRIIIA expressed on NK cells (flow cytometry) and inhibition of hlgG-FcyRIIIA interaction (Homogeneous Time Resolved Fluorescence (HTRF)). Finally, purified scFv were screened again, analyzing binding to FcyRIIIA by ELISA and Octet, minimal cross-reactivity with the other human receptors (FcyRIA, FcyRIIA, and FcyRIIIB), and inhibition of hlgG-FcyRIIIA interaction (i.e., FcyRIIIA blockers). The final antibody fragment, 17C02-scFv, was selected from 10 candidates based on sequencing analysis on glycosylation, oxidation, aggregation, deamidation/isomerization, and proteolytic sites to exclude scFv molecules with low biochemical stability. Details regarding the selection of 17C02 are presented further below.

[0122] Nine BALB/c mice were immunized with seven doses (40 pg total) of the human recombinant FcyRIIIA adjuvanted in Alum and CpG ODNs. Animals were bled at week 4 and week 7, and the anti-FcyRIIIA antibody response was evaluated by ELISA. Figs. 2A-2C shows that all mice developed an antibody response against the antigen. Four weeks of immunization appeared to be sufficient to induce this response (solid lines, B1) with a moderate booster effect observed after the seven period for mice 1 , 2, 6, and 7.

[0123] Mice 4, 8, and 9 were splenectomised and the splenocytes were isolated to purify total mRNA using Dynabeads™ mRNA Purification Kit. Afterwards, cDNA was obtained by reverse transcription and the genes that encode the heavy and light chain variable regions (VH and VL) were amplified by PCR using universal primers specific for mouse germline sequences. Regions VH and VL were then linked using a G4S sequence through an overlap PCR and the final constructions were cloned into the phagemid vector pADL-23c, which were transformed into Escherichia coli (TG1 strain) to generate the scFv display phage library. The size of the library obtained was 1 ,61x10⁹ colony-forming (CFU), as was confirmed by titration using TG1 cells. After determining the sequence of 100 clones from the library, it was observed that 49% of the clones were in frame and 43% had a unique CDR3 sequence. Therefore, the size of the library was adjusted to 6.92x10⁸ CFU.

Phage library screening to select FcyRIIIA binding clones with reduced FcyRIIA crossreactivity

[0124] Five rounds of screening were performed (Table 1). The first round (R1) screened the entire phage library for phages bound to 100 nM of FcyRIIIA, after which 9.3x10⁴ CFU were selected. The selected phages were amplified by infecting TG1 cells, after which 3.41x10¹³ CFU were used to perform a second (R2A) and third (R2B) round of screening. R2A selected phages bound to 10 nM of FcyRIIIA, which yielded 3.22x10⁶ CFU. R2B deselected the phages bound to 30 nM of FcyRIIA, and the unbound phages were used for an additional selection step using 10 nM of FcyRIIIA. As a result, a total of 6.1x10⁶ CFU were obtained after R2B. Phages collected from R2A and R2B were amplified by infecting TG1 cells and 2.31x10¹² CFU were used for the following rounds of selection. The fourth round (R3A) selected phages bound to 1 nM of FcyRIIIA, and the fifth round (R3B) selected phages bound to 1 nm of FcyRIIIA after deselecting phages attached to 3 nM of FcyRIIA. After phage titration, R3A and R3B yielded 3.45x10⁷ and 2.07x10⁷ CFU respectively.

[0125] Phages obtained from each round of screening were used to infect TG1 cells, which were cultured onto 2xYT agarose plates as described above. Individual colonies were selected to prepare scFv master glycerol stocks that were later used to obtain preparations of individual scFvs secreted into the periplasm of the bacteria (peripreps). The expression of an scFv was induced with ITPG, taking advantage of the lactose operon in the parental plasmid.

Selection of clones (peripreps) which bind FcyRIIIA on NK cells and inhibit hlgG-FcyRIIIA interactions

[0126] One hundred peripreps obtained from each round of screening were used to select for those that bound to FcyRIIIA and possessed the capacity to inhibit binding between human IgG (hlgG) and FcyRIIIA. The one hundred peripreps were evaluated for their binding capacity to FcyRIIIA by ELISA. The monoclonal antibody 3G8, a single chain of this antibody (3G8-scFv), and the single chain fused to human albumin (3G8-scFv-albumin) were included as positive controls. [0127] The selection criteria were a normalized optical density (OD450nm) higher than 0.4 and a similar or better binding capacity than 3G8-scFv or 3G8-albumin. Fig. 3 plots the normalized optical density at 450 nm (OD450 nm) for each sample calculated as the subtraction of the OD450 nm measured for a solution of PBS 5% BSA used as a negative control to the OD450 nm measured for peripreps. The dashed line represents the cut-off value for the peripreps that were considered a binder or “hit” (OD450 nm = 0.4). Fig. 3 demonstrates the percentage of “hits” identified after each round of screening, which also necessitated that the periprep have an OD450 nm equal or higher than that detected for 3G8-scFv or 3G8-scFv-albumin. Data are representative of two independent assays.

[0128] As a result, a total of 170 clones were selected (Fig. 3). Only 4% of peripreps from R1 met these selection criteria, whereas from R2A and R2B approximately half of the peripreps met the criteria. A lower percentage of peripreps from rounds R3A (35%) and R3B (40%) met the criteria (Fig. 3). As expected, the negative controls used in the assay did not show binding to FcyRIIIA (Fig. 3). Fourteen negative peripreps (i.e, poor/low-FcyRIIIA binders; including 19H02 which was used as a weak control) were also selected as controls (not shown) for the following steps.

[0129] The 170 positive clones were then evaluated for their binding capacity to FcyRIIIA on NK cells from human PBMC by flow cytometry. PBMC from human donors were isolated by density gradient centrifugation. Cells were then incubated with selected peripreps or 3G8-scFv, 3G8, Her-scFv or mlgG1 (isotype control) as controls. After washing, cells were incubated with a mixture of BV605-conjugated anti-human CD56 and propidium iodide plus AF674-conjugated anti-c-myc for the detection of either peripreps or scFv, or AF647-conjugated anti-mouse IgG for the detection of full-length antibodies. Stained cells were washed and analyzed on the Intellicyt iQue™ Screener Plus using Forecyt™ Software. The graph plots the geometric mean (geomean) of the fluorescent intensity value for each sample. Data are representative of two independent determinations. The dashed line represents the cutoff value defined as two times the geomean of 3G8-scFV. The capacity of peripreps to inhibit hlgG-FcyRIIIA interaction was measured by Homogeneous Time Resolved Fluorescence. Equal volumes of streptavidin-XL665, biotinylated- FcyRIIIA, europium-conjugated anti-hlgG, and hlgG were incubated with either the peripreps, 3G8, 3G8-scFv, 3G8-scFv-albumin (3G8-scFv-HSA), Her-scFV, albumin (HSA), or mlgG1 (isotype control). Reconstitution buffer (CisBio, Canada) was used as a “no competition” negative control. Samples were then analyzed on a Synergy 4 plate reader using the Gen5 Software (Filter wheels 330/80 and 620/10). Data in Figs. 4A-4B are representative of two independent assays. The dashed lines represent the cut-off values used for scFv selection.

[0130] A single chain of Herceptin (Her-scFv), a monoclonal antibody against the human epidermal growth factor receptor 2, was included as a non-binding scFv control. Non-viable cells were excluded using propidium iodide and NK cells were gated as CD56⁺ lymphocytes. The binding of clones was compared with the binding of 3G8-scFv. Eighteen clones showed a geometric fluorescent intensity (geomean) value greater than 4000, which was FcyRIIIA binding 2xthe geomean of the positive control, 3G8-scFv (Fig. 4A). The capacity of the initial 170 clones to inhibit the binding of hlgG to FcyRIIIA was also evaluated by Homogeneous Time Resolved Fluorescence (HTRF), as described above. For this assay 3G8, 3G8-scFv, 3G8-scFv-albumin, Her-scFv, human albumin (HSA), and mouse IgG (mlgG) were included as controls. The highest percentages of inhibition (approximately 70% inhibition) were detected for 3G8, 3G8-scFv, and 3G8-scFv-albumin (Fig. 4B). Ten of the assayed clones inhibited >30% of binding of hlgG to FcyRIIIA (above the dotted line) (Fig. 4B). All these 10 clones (16A12, 17D10, 17C02, 17F07, 14F04, 17F11 , 15B07, 17E03, 10E07, and 17A09) were selected for further steps. As expected, the negative controls (Her-scFv, HSA, and mlgG) did not inhibit the binding (Fig. 4B).

Selection of the best scFv (17C02) to express as an albumin fusion protein, a normal bivalent mlgG2a, and a hlgG1 one-armed antibody without Fc function

[0131] To select one scFv from the 10 clones previously selected, each scFv was purified using His MultiTrap HP plates (each scFv possessed the 6xHis-tag on the C-terminus). Two clones (10E07 and 17A09) had low protein recovery after the purification process and were thus excluded from further analysis. Clone 19H02 was then included as a “weak” control considering it did not meet the selection criteria but it did inhibit approximately 28% of binding of hlgG to FcyRIIIA.

[0132] Each purified scFv was assessed in terms of binding to FcyRIIIA, limited crossreactivity with FcyRIIIB and cynomolgus FcyRIIIA (cynoFcyRIIIA), and non-binding to FcyRI and FcyRII. Binding of purified scFv to FcyRIIIA, cynoFcyRIIIA, FcyRIIIB, FcyRI, and FcyRIIA, was measured by ELISA. Polystyrene 96-well plates were coated with FcyRIA (2.5 pg/mL), FcyRIIIA (10 pg/mL), cynoFcyRIIIA (10 pg/mL), or FcyRIIIB (2.5 pg/mL). For the specific case of FcyRIIA, plates were precoated with 10 pg/mL neutravidin protein prior to coating with biotinylated-FcyRIIA (2.5 pg/mL). Plates were then incubated with selected purified scFv candidates (0.07-700 nM), or controls (3G8-scFv, 3G8-scFv-albumin, Her-scFv, albumin, 3G8 or mlgG1 (isotype control)), after which the optical density at 450 nm (OD450 nm) was assessed for each scFv concentration. Antibodies against FcyRI (clone 10.1), FcyRIIA (clone IV.3) and FcyRIIIB (clone 2D2G5B9) were used as positive controls in the analysis of binding to these specific FcyRs. Data are representative of two independent determinations.

[0133] Four scFv (17D10, 19H02, 17F07, 14F04) showed binding capacity to FcyRIIIA (Fig. 5A). High level of binding to cynoFcyRIIIA was observed for 17D10, 17E03 and 17C02 (Fig. 5B). 14F04 showed high crossreactivity with FcyRI and FcyRIIA (Figs. 5D and 5E) while 17D10 and 19H02 bound to FcyRIIIB (Fig. 5C).

[0134] Importantly, HTRF was used to evaluate the capacity of selected scFvs to inhibit the binding of hlgG to FcyRIIIA. The ability of human IgG to bind to FcyRIIIA in the presence of different concentrations of each purified scFv was measured by Homogeneous Time Resolved Fluorescence. Equal volumes of streptavidin-XL665, biotinylated-FcyRIIIA, europium-conjugated anti-hlgG, and hlgG were mixed and incubated with a purified scFv candidate (0.03-500 nM), or 3G8, 3G8-scFv, 3G8-scFv-albumin, Her-scFV, albumin, or mlgGI (isotype control) for 3 hours at room temperature in the dark. Incubation with reconstitution buffer (CisBio, Canada) was used as a “no competition” negative control. Samples were then analyzed on a Synergy 4 plate reader using the Gen5 Software (Filter wheels 330/80 and 620/10) and the data were plotted as the percentage of hlgG-FcyRIIIA interaction inhibition. Data is presented as the mean ± standard deviation of two independent experiments. The strongest inhibitors were 3G8 and 3G8-scFv (Fig. 6). Two scFvs (15B07 and 17C02) showed a higher percentage of inhibition than 3G8-scFv- albumin, whereas 14F04, 17F07, and 17F11 had a similar percentage of inhibition as 3G8-scFV- albumin (Fig. 6). Interestingly, these results were not completely in accordance with the ELISA shown in Figs. 5A-5E, where 17D10, 17F07, and 14F04 appeared to have the greatest binding to FcyRIIIA, yet with HTRF, 15B07 and 17C02 had the greatest IgG-FcyRIIIA inhibitory capacity. As can be observed the 3G8 antibody was superior to all other formats tested. The 3G8 antibody is a bivalent antibody and likely had much better activity because of its bivalent nature. All of the other scFvs tested in this assay binded very roughly equivalently but better than the weak control. Thus this high throughput assay was not sufficient to resolve smaller differences in binding. Moreover, without wishing to be bound by theory, the secondary antibody in the HTRF was different from the one used in the ELISA and there may have been some interference I influence by these secondary antibodies. In addition, the HTRF was performed using semi-purified preparations while the ELISA used purified reagents. [0135] As a third assessment, the biomolecular interaction between each scFv and FcyRIIIA was evaluated using ForteBio Octet Red96e as described above, through which the associationdissociation curves for each scFv were recorded and compared using 3G8, 3G8-scFv, and 3G8- scFv-albumin as controls. Biotin-FcyRIIIA (3 pg/mL) was loaded onto Streptavidin Biosensors, followed by association and dissociation of the molecule of interest (Fig. 7 A: Association with 50 nM of 3G8 or mlgG1 isotype control or 500 nM of 3G8-scFv, 3G8-scFv-albumin, or albumin, and subsequent dissociation, and Fig. 7B: in addition to 3G8-scFv (dark blue line), the association and dissociation of the Herceptin single chain (Her-scFv; negative control) and other scFv antibodies obtained from the scFv phage display library were assessed. Binding was performed using Streptavidin Biosensors on ForteBio Octet Red96e with the Data Acquisition 11.1 Software and data analyzed using the Data Analysis HT 11 .1 Software.

[0136] The highest receptor association was observed for 3G8 (a standard antibody format), with the dissociation pattern indicating a stable interaction (Fig. 7A). 3G8-scFv-albumin showed a better association to the receptorthan 3G8-scFv, but a faster dissociation. In fact, the interaction with 3G8-scFv appeared to be more stable than 3G8-scFv-albumin considering the smaller negative slope of the dissociation curve. Four scFvs (17C02, 16A12, 17D10, and 17E03) showed a better association and more stable interaction with the receptor than 3G8-scFv, with 17C02 being the best candidate (Fig. 7B).

[0137] The fourth assessment to select the best scFv candidate was the analysis of their amino acid sequence to determine possible undesirable sites of deamidation/isomerization, proteolytic cleavage, oxidation, aggregation, and glycosylation. Table 4 shows the sequence of the complementarity-determining regions for the light (LCDR) and heavy (HCDR) chains of the 8 selected (16A12, 17D10, 17C02, 17F07, 14F04, 17F11 , 15B07 and 17E03) scFvs as well as the negative control (19H02). The sequences for 3G8 and its variants were also included. Apart from 17C02, all others scFvs possessed undesirable sites, which could compromise the stability of the molecule. Thus, 17C02 was selected as the final candidate of a potentially new FcyRIIIA-blocking therapeutic to treat autoantibody and alloantibody-mediated thrombocytopenias considering its association-dissociation curve, inhibitory capacity of hlgG-FcyRIIIA interaction, and lack of detectable crossreactivity with FcyRI and FcyRIIA.

Table 4. Amino acid (aa) sequence of the Complementarity-determining regions for the light (LCDR) and heavy (HCDR) chains of each scFv

Table 5. Sequences for 15B07, 16A12, 17C02, 17D10, 17E03 and 17F07

The sequence RGGGGSGGGGSGGGGSGGGGS (SEQ ID No: 43) represents the linker between VL and VH for these scFvs. 17CO2- the amino acids in this table in bold represent the variable region of the light chain. The amino acid sequences in this table in dotted underline represent the variable region of the heavy chain.

[0138] The amino acid sequence of 17C02 was then used to obtain three different constructs: an albumin fusion protein (17C02-albumin) (Fig. 8A), a mouse standard lgG2a antibody (17C02- lgG2a) (Fig. 8B) and a human lgG1 one-armed antibody (Fig. 8C). These three constructs were used to evaluate their in vitro and in vivo therapeutic capacity in models of human autoimmune and alloimmune thrombocytopenias. Fig. 8A shows the 17C02-albumin having the gene that encodes forthe 17C02-scFvwith a linker (connecting the VH with the VL chains) as shown in Table 5 (underlined amino acids) as well as another linker (amino acids: RGGGGSGGGGS (SEQ ID No: 44)) to connect the scFvto human albumin and a 6xHis tag afterthe albumin sequences were cloned into the mammalian expression vector pCEP4 to express the single chain as an albumin fusion protein. In Fig. 8B, the 17C02-lgG2a, the genes that encode for the VL and VH of 17C02 were cloned into the plasmids pFUSE2-CLIg-mK and pFUSE-CHIg-mG2a respectively to express the molecule as a full-length mouse lgG2a antibody.

17C02-based molecules bind to FcvRIHA-bearing cells

[0139] Firstly, an in vitro evaluation of 17002-based molecule binding to FcyRIIIA on various cell types was performed. THP-1 transgenic cells expressing the CD16A molecule (THP-1-CD16A cells), peritoneal macrophages from FcyR-humanized mice, and PBMC from human donors were also used. In all cases, 3G8 and 3G8-albumin were used as positive controls, and two different secondary antibodies (FITC-conjugated goat anti-human serum albumin or AF674-conjugated goat anti-mouse IgG (H+L)) were used to detect the binding of the albumin constructs or the full- length antibodies, respectively.

[0140] In Figs. 9A-9D, 5x10⁵ cells were incubated with either 17C02-albumin, 3G8-albumin, or Albumin alone (negative control). In Figs. 10A-10D, 5x10⁵ cells were incubated with various concentrations of either 17C02-lgG2a or 3G8. Binding was detected using an APC-labelled goat F(ab’)2 anti-mouse IgG (Fc-specific). Binding was detected using a FITC-labelled monoclonal anti-HSA antibody. The following were the cell types used for each figure: Figs. 9A and 10A: THP- 1 -CD16A cells, Figs. 9B and 10B: Peritoneal macrophages isolated from FcyR-humanized mice, Figs. 9C and 10C: Natural killer cells from healthy human donors, and Figs. 9D and 10D: Neutrophils from healthy human donors. Stained cells were washed and analyzed by flow cytometry using the BD LSRFortessa™ X-20. Data analysis was performed using FlowJo v10. Data are presented as mean ± standard deviation from three independent experiments. MFI refers to the mean fluorescent intensity (arbitrary units). The dashed line represents the MFI value for the secondary antibody alone at 5 pg/mL. Statistical analysis was performed using a two-way ANOVA and Sidak's multiple comparisons test, comparing the MFI values of the three molecules at each antibody concentration (*: p< 0.05; **: p<0.01).

[0141] Both 17C02-albumin and 17C02-lgG2a (deglycosylated) bound to FcyRIIIA on THP- 1 -CD16A cells in a dose-dependent manner. 17C02-albumin and 17C02-lgG2a had greater binding capacities than 3G8-albumin and 3G8 respectively (Figs. 9A and 10A). These results suggest that 17C02-based molecules have a higher affinity to FcyRIIIA than the previously established 3G8-based molecules. However, when investigating FcyRIIIA expressed on peritoneal macrophages from FcyR-humanized mice, (p>0.05), a similar binding capacity was observed between the albumin constructs (17C02-albumin and 3G8-albumin) and the full-length antibodies (17C02-lgG2a and 3G8) (Figs. 9B and 10B). The difference in binding capacity to THP- 1 -CD16A cells vs peritoneal macrophages may be explained in part by the differential expression of FcyRIIIA on these cells (data not shown).

[0142] Using PBMCs from human donors, binding capacity to FcyRIIIA on NK cells and crossreactivity with FcyRIIIB on neutrophils were also evaluated. In line with previous results, 17002-based molecules showed higher binding to FcyRIIIA on NK cells than their 3G8 counterparts (Figs. 9C and 10C). In addition, both 17C02- and 3G8-based molecules demonstrated crossreactivity with FcyRIIIB on neutrophils (Figs. 9D and 10D). No statistical differences between these molecules were identified in binding to the receptor on neutrophils.

17C02-based molecules inhibit the phagocytosis of IgG-opsonized human red blood cells.

[0143] THP-1-CD16A macrophages can phagocytose anti-D-opsonized RhD⁺ red blood cells (RBCs), with notable contribution from FcRIIIA (Gil Gonzalez et al., 2022). Therefore, the capacity of 17002-based molecules to inhibit the phagocytosis of RBCs using THP-1-CD16A macrophages was evaluated.

[0144] THP-1-CD16A cells were differentiated to macrophages by treatment with PMA (100 ng/mL). Macrophages were incubated with erythrocytes that were incubated with PBS or opsonized with a polyclonal anti-human RhD antibody (opsonized). To isolate for the biological activity of FcyRIII and limit the use of other FcyRs, prior to incubation with erythrocytes, macrophages were also treated with a cocktail containing the following deglycosylated FcyR- blocking antibodies: anti-FcyRI (clone 10.1) and anti-FcyRIIA (clone IV.3) and anti-FcyRIIA/B/C (clone AT10) at a final concentration of 10 pg/mL each. Afterwards, the indicated concentrations of deglycosylated full-length 17C02, 3G8, or 17C02-albumin, or 3G8-albumin were added to the macrophages to analyze the contribution of FcyRIIIA in mediating phagocytosis. The upper horizontal dark-grey bar represents the phagocytic index range obtained when macrophages were incubated with opsonized-erythrocytes plus negative control deglycosylated mouse lgG1 (clone MOPC-21) and deglycosylated mouse lgG2a (clone N/A-CP150) and deglycosylated mouse lgG2b (clone MPC-11) isotype controls (final concentration of 30 pg/mL; i.e., 10 pg/mL each isotype control). The lower horizontal light-grey bar represents the phagocytic index range obtained when macrophages were incubated with opsonized erythrocytes plus the above blocking antibody cocktail alone. For clarity, the upper horizontal dark-grey bar shows RBC phagocytosis due to all Fc receptors while the lower horizontal gray bar shows the maximal phagocytosis due to Fc receptor I HA alone. The phagocytic index was calculated as the number of erythrocytes engulfed per 100 macrophages. Data in Fig. 11 is presented as mean ± standard deviation of five independent experiments. Statistical analysis was performed with a one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test (***: p <0.001).

[0145] Blockade of the aforementioned FcyRs inhibited approximately 60% of the phagocytosis of opsonized-RBCs, as indicated by the light grey horizontal bar (Fig. 11). The dark grey horizontal bar indicated the phagocytic index of IgG-opsonized RBCs in the presence of isotype control antibodies only.

[0146] 17C02-based or 3G8-based molecules were then added at various concentration (133, 13.3, and 1.33 pM) in addition to the FcyRI and Il-specific blocking antibody cocktail to examine the effect of these molecules on FcyRIIIA-dependent RBC phagocytosis. When comparing the full-length antibodies, 17C02-lgG2a caused a significantly greater decrease in phagocytosis compared to 3G8 at both 133 and 13.3 pM. Similarly, phagocytosis was significantly lower with 17C02-albumin than 3G8-albumin, though only at the 133 pM concentration (Fig. 11).

[0147] The addition of 17C02-lgG2a or 17C02-albumin significantly reduced the phagocytosis of the anti-D-opsonized RBCs, at least at the highest dose assayed (95% or 85%, respectively). The medium dose (13.3 pM) of 17C02-lgG2a also showed a significant inhibition (~77%) of the uptake of the anti-D-opsonized RBC (Fig. 11). The higher dose of 3G8 or 3G8-albumin also significantly inhibited of phagocytosis (80% or 73% respectively). However, 17C02-based molecules showed a higher inhibition of phagocytosis (p<0.001) compared to their 3G8-based molecule counterparts.

17C02-based molecules successfully ameliorate thrombocytopenia in a passive mouse model of ITP

[0148] FcyR-humanized mice (lack mouse FcyRI, FcyRllb, FcyRIII, and FcyRIV, and express human FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and FcyRIIIB) were used to evaluate the capacity of 17C02-based molecules to ameliorate ITP in comparison with 3G8 and 3G8-albumin (Fig. 12). A passive model of immune thrombocytopenia was induced in FcyR-humanized transgenic mice with 15 pl of a rabbit anti-platelet serum (Cedarlane, CLA31440). Mice were either left untreated (Untreated), treated with the anti-platelet serum alone (Nil), or treated with the rabbit anti-platelet serum in addition to an anti-FcyRI HA therapeutic intervention as indicated on the x-axis. Eighty- one pg (540 pM) of either deglycosylated 17C02 (degly-17C02-lgG2a), 3G8 (degly-3G8), or an equimolar amount of their albumin-fusion scFv counterparts (50 pg) were administered to examine the molecule’s ability to increase platelet counts 2 hours after administration of the antiplatelet serum. Data in Fig. 12 is presented as mean ± standard deviation from 3 independent experiments (n=6). The statistical analysis was performed by a one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test (***: p <0.001).

[0149] Human albumin and isotype controls (mlgG1 and mlgG2a) were also used as negative controls of ITP amelioration. Animals were treated with 540 pM/mouse of either deglycosylated 17C02-lgG2a, 17C02-albumin, deglycosylated 3G8, or 3G8-albumin to block FcyRIIIA 2 hours prior to administering rabbit anti-mouse platelet serum to cause thrombocytopenia. Two hours post-treatment with the anti-platelet serum, platelet counts were recorded. Animals that received the anti-platelet serum alone (Nil) developed thrombocytopenia compared to untreated animals (Fig. 12). However, treatment with 17C02-albumin successfully ameliorated ITP, significantly increasing platelet counts compared to animals in the Nil group (approximate 50% increase in platelet counts). Interestingly, despite the ability for 17C02-lgG2a and both 3G8-based molecules to inhibit phagocytosis, these compounds did not ameliorate thrombocytopenia (Fig. 12). The severe adverse event activity observed in animals injected with either deglycosylated 3G8 or deglycosylated 17C02-lgG2a may be the reason why these antibodies could not ameliorate thrombocytopenia as discussed below.

[0150] To evaluate potential adverse effects associated with the anti-FcyRIIIA molecules, changes in body temperature was assessed post-treatment. Mice were treated with an intravenous administration of either deglycosylated full-length 17C02 or 3G8 (81 pg/mouse; 540 pM/mouse), 17C02-albumin or 3G8-albumin (50 pg/mouse; equimolar amount), or albumin alone (35.1 pg/mouse; equimolar amount). Decreases in rectal temperature were evaluated as an indicator of an inflammatory adverse event comparing 0- (pre-treatment) to 15-, 30-, and 45- minutes post-treatment. Data is presented as mean ± standard deviation (n=5-7). The statistical analysis was performed by a two-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test (***: p <0.001). As expected, deglycosylated 3G8 provoked a decrease in body temperature (Fig. 13A). Similar adverse reactions were observed for deglycosylated 17C02- lgG2a, likely due to the bivalent nature of the molecule. However, both monovalent constructs that were fused to albumin (17C02-albumin and 3G8-albumin) did not cause a change in body temperatures (Fig. 13A). [0151] Immune cells were enumerated from blood, spleen, and bone marrow from mice treated with either 540 pM/mouse of 17C02-albumin or deglycosylated 17C02-lgG2a (degly- 17C02-lgG2a). Control data was obtained from untreated animals. Live cells were quantified, and the frequencies of B cells (CD19+), T cells (CD3+), NK cells (NK1.1 +), and neutrophils (CD11 b+/Ly-6G+) were assessed using a combination of absolute cell counts from a Guava cytometer coupled with flow cytometry. Analysis of select immune cell populations revealed that mice administered with 17C02-lgG2a exhibited a reduction in neutrophil counts both in the blood and spleen, along with a decrease in NK cell numbers in the blood (Figs. 13B-13D). Conversely, mice treated with 17C02-albumin maintained immune cell levels comparable to those observed in untreated mice (Figs. 13B-13D).

[0152] Considering the lack of ameliorative capacity observed with 3G8 and 17C02-lgG2a, platelet counts were assessed after the administration of these antibodies alone, prior to the induction of ITP. The ability of the full-length anti-FcyRI I IA antibodies vs the albumin-fused scFv counterparts to induce thrombocytopenia as an adverse event was evaluated 2 hours posttreatment in comparison with untreated mice, or animals treated with an isotype control antibody (equimolar amount), or rabbit anti-platelet serum alone (ITP, 15 pL/mouse). Data is presented as mean ± standard deviation (n=5). The statistical analysis was performed using Kruskal-Wallis and Dunn’s multiple comparison test (*: p<0.05; **: p<0.01). Treatment with either deglycosylated 3G8 or deglycosylated 17C02-lgG2a alone caused a significant decrease in platelet count compared to their respective isotype controls (p<0.05) (Fig. 13E). In fact, the level of thrombocytopenia was similar to that of animals injected with the rabbit anti-mouse platelet serum alone. However, the albumin constructs (3G8-albumin and 17C02-albumin) did not affect the platelet counts (Fig. 13E). The severe adverse event activity observed in animals injected with either deglycosylated 3G8 or deglycosylated 17C02-lgG2a may be the reason why these antibodies could not ameliorate thrombocytopenia. This observation is consistent with the finding that decreased body temperature drives thrombocytopenia in rodents (see for example de Vrij et al. 2014) In addition, LPS and other inflammatory mediators are also known to cause hypothermia in rodents as well as thrombocytopenia.

[0153] A lower dose of the FcyRIIIA blocking antibodies (50 pg or 333.3 pM vs. 81 pg or 540 pM) was then evaluated to investigate possible dose-dependent effects on causing thrombocytopenia (Figs. 14A-14C, (n=4-6)). FcyR-humanized mice were intravenously injected with 333.3 pM of full-length 17C02, 3G8, or 17C02-albumin and platelet counts were measured 2 hours post-treatment to determine the ability of the antibody to cause thrombocytopenia on its own (in comparison with 540 pM used in Figs. 13A-13E). Interestingly, the lower dose of deglycosylated 17C02-lgG2a (333.3 pM) did not cause apparent thrombocytopenia on its own, while deglycosylated 3G8 (333.3 pM) did (Fig. 14A). Mice were then intravenously injected with 15 pl of a rabbit anti-platelet serum to induce immune thrombocytopenia. Two hours posttreatment with the anti-platelet serum, mice were bled for enumeration of platelet counts. 17C02- albumin, similar to 17C02-lgG2a also did not affect platelet counts. Thus, in accordance with these data, both 17002-based molecules ameliorated ITP in animals treated with the rabbit antimouse platelet serum, whereas 3G8 at the same dose failed to significantly increase platelet counts (Fig. 14B). Upon injection with the anti-FcyRIIIA molecule (333.3 pM), body temperatures of mice were assessed until 45 minutes post-treatment to investigate the inflammatory nature of the molecules at, where timepoint “0 mins” indicates prior to any treatment. Although deglycosylated 17C02-lgG2a demonstrated therapeutic capacity, an associated decrease in body temperature was still present, similar to 3G8 (Fig. 14C). However, treatment with 17C02-albumin did not cause changes in body temperature while being able to ameliorate ITP. This last result supports the bivalent nature of the antibody which could be associated with FcyR crosslinking causing inflammation.

Alloantibody studies: 17C02-based molecules inhibit the phagocytosis of anti-HPA-1a- as well as anti-HLA-opsonized human platelets

[0154] Furthermore, having demonstrated the efficacy of 17C02-based molecules in an in vivo mouse model of ITP, their in vitro functionality in two other models relating to human thrombocytopenias was also evaluated. Firstly, fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare disorder in which maternal-fetal platelet incompatibility leads to the formation of maternal antibodies against human platelet antigen (HPA), most commonly anti-HPA-1 a, which results in fetal and neonatal thrombocytopenia. Secondly, platelet refractoriness (PTR) is a condition defined by a lack of adequate post-transfusion platelet count increments after multiple transfusions, which can be caused by immune and nonimmune factors. Immune factors, which play a role in 10-25% of patients with PTR, are caused by antibodies against HLA class I.

[0155] Sera pooled from patients with anti-HPA-1a or from 2 patients with any anti-HLA antibodies were used to opsonize human platelets and perform platelet phagocytosis assays using THP-1-CD16A macrophages. The HPA-1 a pooled plasma was obtained from HPA-1a- alloimmunized women due to previous pregnancy or transfusion (NAITgam). The anti-HLA antibodies were from two patients with ITP without detectable platelet autoantibodies but were broadly positive for anti-HLA IgG by luminex, likely due to transfusions, as ITP patients often receive platelet transfusions when their platelet count becomes dangerously low. FcyR utilization and the capacity of 17C02-based molecules to inhibit phagocytosis were then evaluated.

[0156] THP-1-CD16A cells were differentiated to macrophages by treatment with PMA (100 ng/mL). Opsonization: ((+) on Figs. 15-16) indicates platelets were non-opsonized (incubated with normal human serum), opsonized with the monoclonal antibody A2A9 (5 pg/mL) as positive control, or anti-HPA-1a pooled plasma (Fig. 15) or anti-HLA patient serum (Fig. 16). The HPA-1 a pooled plasma (called NAITgam) was obtained from HPA-1a-aloimmunized women due to previous pregnancy or transfusion. Anti-HLA sera were from 2 patients (not pooled) with ITP without detectable platelet autoantibodies but were positive for anti-HLA IgG by luminex, likely due to transfusions. The phagocytic index was calculated as the number of platelets engulfed per 100 macrophages. The contribution of each FcyR to phagocytosis was evaluated using Fc region deglycosylated blocking antibodies (final concentration of 10 pg/mL; 0.07 pM each): anti-FcyRI (clone 10.1), anti-FcyRIIA/B/C (clone AT10), or anti-FcyRI I IA (clone 3G8). The deglycosylated mouse lgG1 (clone MOPC-21) was used as an isotype control (final concentration of 0.07 pM). In addition, the blocking capacity of 17C02-based molecules was evaluated (17C02-albumin and deglycosylated 17C02-lgG2a) using the same final molar concentration. 3G8-albumin was included for comparison. Human albumin and the deglycosylated mouse lgG2a (clone N/A- CP150) were used as control (final concentration of 0.07 pM). Data is presented as the mean ± the standard deviation of four independent experiments. Three experiments were performed with ITP patient #1 and two experiments were with ITP patient #2. The statistical analysis was performed using Kruskal-Wallis and Dunn’s multiple comparison test (*: p<0.05; **: p<0.01).

[0157] It was found that sensitization of platelets with either anti-HPA-1 a or anti-HLA antibodies caused platelet phagocytosis (Figs. 15 and 16). The treatment of cells with various FcyR blocking antibodies showed that FcyRI (clone 10.1) and FcyRIIIA (clone 3G8) contribute to phagocytosis by HPA-1a and HLA antibodies (Figs. 15 and 16). In fact, the blockade of either of these receptors inhibited approximately 50% of phagocytosis relative to anti-HPA-1a (Fig. 15) or anti-HLA (Fig. 16) serum-opsonized platelets. Contrarily, approximately 20% of phagocytosis was inhibited with an FcyRI lA/B/C-blocking antibody (Figs. 15 and 16), suggesting this receptor is not utilized to the same extent. In accordance with these results, blocking FcyRIIIA with 17C02- albumin or 17C02-lgG2a led to a significant inhibition of platelet phagocytosis in both anti-HPA- 1 a and anti-HLA sera conditions (Figs. 15 and 16). Inhibition of platelet phagocytosis was achieved, albeit to a lesser extent when FcyRIIIA-blockade was achieved with 3G8-albumin or 3G8.

17C02-albumin ameliorates anti-HLA-A2 antibody-mediated thrombocytopenia

[0158] The therapeutic efficacy of 17C02-albumin was evaluated in an anti-HLA-A2 antibody- mediated model of immune thrombocypenia. The model was developed by injecting FcyR- humanized transgenic mice with mouse HLA-A2⁺ platelets obtained from NSG-HLA-A2/HHD mutant mice. More specifically, the passive model of immune thrombocytopenia was induced in FcyR-humanized transgenic mice using ex vivo sensitized HLA-A2⁺ mouse platelets. Platelets from HLA-A2 humanized transgenic mice were isolated, labeled with CMFDA, and then ex vivo opsonized with human anti-HLA-A2 serum or normal human serum (NHS). FcyR-humanized transgenic mice were either injected with 5x10⁷ non-opsonized platelets (Control), NHS- opsonized platelets (NHS), or anti-HLA-A2-opsonized platelets (anti-HLA-A2). The number of CMFDA-labeled platelets in the blood circulation of the recipient mice was followed for 4 hours. An anti-FcyRI I IA therapeutic intervention was evaluated injecting a group of mice with 540 pM of 17C02-albumin 2 hours before the administration of anti-HLA-A2-opsonized CMFDA-labeled platelets (17C02-albumin + anti-HLA-A2). Data are presented as mean ± standard deviation from one experiment (n=2 mice). Animals either receiving non-opsonized or NHS-opsonized platelets had a stable number of CMFDA-labeled platelets in their blood circulation. On the contrary, CMFDA-labeled platelets that were sensitized with the anti-HLA-A2 human serum started to be cleared 2 hours afterthe injection (Fig. 17), through FcyR-mediated phagocytosis (Fig. 16). At the end of the observation period more than 60% of platelets had been eliminated by the anti-HLA- A2 serum. However, the treatment of animals with 17C02-albumin to block the FcyRIIIA before the administration of platelets successfully ameliorated clearance of anti-HLA-A2-sensitized platelets (Fig. 17). At the end of the observation period animals receiving the anti-FcyRI I IA treatment had more than twice the number of platelets recorded as compared to the non- therapeutically treated mice.

17C02 expressed as a human lgG1 one-armed antibody binds to FcyRIIIA

[0159] Finally, the amino acid sequence of 17C02 was used to obtain a human lgG1 one- armed antibody (17C02-lgG1oA) as described above to demonstrate that the 17C02 sequence is independent of the specific backbone used. Another potential advantage of the one-armed antibody is its monovalent nature, which like the albumin construct limits FcyR crosslinking. The binding of this molecule to FcyRIIIA on various type cells, as well as its capacity to block IgG- opsonized platelet phagocytosis and thrombocytopenia in a mouse model of ITP were evaluated.

[0160] The genes encoding the VL and VH of 17C02 were used to express a human lgG1 one- armed antibody using the “knob into hole” strategy (Fig. 8C). Plasmid pEmiLC was used to obtain three different vectors: one vector expressing the heavy chain of a human lgG1 antibody containing the VH region of 17C02 and a “hole” site in the CH3 portion; a second vector to express the kappa light chain of the human lgG1 antibody containing the VL region of 17C02; and a third vector to express the CH2-CH3 portion of a human lgG1 antibody with the “knob” site. For binding, 5x10⁵ cells were incubated with various concentrations of 17C02-lgG1 oA. Binding was detected using an AF647-labelled donkey anti-human IgG (H+L). The following were the cell types used for figure: Fig. 18A: THP-1-CD16A and THP-1 cells, Fig. 18B: Natural killer cells from healthy human donors, and Fig. 18C: Neutrophils from healthy human donors. Stained cells were washed and analyzed by flow cytometry using the BD LSRFortessa X-20. Data analysis was performed using FlowJo v10. Data are presented as mean ± standard deviation from three independent experiments. The dashed line represents the MFI value for the secondary antibody alone at 5 pg/mL. As expected, 17C02-lgG1 oA showed a specific binding to THP-1 -CD16A cells with no binding to THP-1 cells, which lack FcyRIIIA and were used as negative control (Fig. 18A). Accordingly, the molecule bound to the receptor expressed on NK cells (Fig. 18B) and had high level of cross-binding with FcyRIIIB on human neutrophils (Fig. 18C).

17C02-lgG1oA inhibits the phagocytosis of IgG-opsonized human platelets

[0161] Human platelets sensitized with sera from thrombocytopenic patients with anti-HPA- 1 a as well as anti-HLA antibodies were used to perform phagocytosis assays using THP-1 -CD16A macrophages. The capacity of 17C02-lgG1 oA to inhibit this phagocytosis was evaluated by treating macrophages with the blocking antibody before incubation with the sensitized platelets. THP-1 -CD16A cells were differentiated to macrophages by treatment with PMA (100 ng/mL). Opsonization: (+) indicates platelets were non-opsonized (incubated with normal human serum), opsonized with the monoclonal antibody A2A9 (5 pg/mL) as positive control, or patient serum. Fig. 19A shows phagocytosis using anti-HPA-1 a patient serum and Fig. 19B shows phagocytosis using anti-HLA patient serum. The phagocytic index was calculated as the number of platelets engulfed per 100 macrophages. The blocking capacity of 17C02-lgG1 oA was evaluated by incubating macrophages with 0.07 pM of this antibody prior to feeding them with the opsonized platelets. Data is presented as the mean ± the standard deviation of three independent experiments. As observed in previous experiments, platelets sensitized with either anti-HPA-1 a or anti-HLA antibodies were successfully phagocytosed (Figs. 19A-19B). The blockade of FcyRIIIA with the one-armed antibody inhibited 68% of phagocytosis relative to anti-HPA-1 a serum-opsonized platelets (Fig. 19A) or 65% of phagocytosis relative to anti-HLA serum- opsonized platelets (Fig. 19B).

17C02-lgG1oA ameliorates thrombocytopenia in a mouse model of ITP

[0162] FcyR-humanized mice were treated with 540 pM/mouse of 17C02-lgG1 oA, the same equimolar amount previously used for the other 17C02-based molecules. To evaluate potential adverse events associated with this treatment, change in body temperature and induction of thrombocytopenia on its own were assessed post-administration.

[0163] In Fig. 20A FcyR-humanized mice were intravenously injected with 540 pM of 17C02- IgGl OA and body temperatures of mice were assessed for 45 minutes post-treatment to investigate the inflammatory nature of the molecule (time ‘0’ indicates prior to treatment). In Fig. 20B, mice were bled, and platelet counts assessed 2 hours post-17C02-lgG1 OA treatment to determine the ability of the antibody itself to cause thrombocytopenia. In Fig. 20C, mice were intravenously injected with nothing (Nil) or with 17C02-lgG1 oA for 2 hours followed by 15 pl of a rabbit anti-platelet serum (ITP) for 2 hours to induce thrombocytopenia and mice were bled for enumeration of platelet counts to assess the ability of 17C02-lgG1 oA to ameliorate thrombocytopenia. Data are presented as mean ± standard deviation (n=4 mice). Unfortunately, the molecule provoked a drop in body temperature compared to untreated mice (Fig. 20A). However, platelet counts were normal when they were evaluated 2 hours after the injection (Fig. 20B). Thrombocytopenia was then induced in the animals by administering rabbit anti-mouse platelet serum, and two after this injection, platelet counts were recorded again. Animals that received the anti-platelet serum alone (Nil) developed thrombocytopenia compared to untreated animals (Fig. 20C). However, treatment with 17C02-lgG1 oA successfully ameliorated ITP, increasing platelet counts by around 56% compared to animals in the Nil group (Fig. 20C). A decrease in rodent body temperature is associated with lower platelet counts. It was also previously demonstrated that cross-linking of FcyRIIIA decreases body temperature. Since nonglycosylated lgG1 can retain some degree of FcyRIIIA binding, it is likely that the one armed antibody Fc regions retains some level of binding to Fc receptors. Human IgG 1 without any glycan on the Fc region can still bind to human FcyRIIIA despite removing the glycosylation site. This is likely the reason why this one armed antibody triggers a drop in temperature. This is consistent with the observation made by de Vrij et al. 2014 that decreased body temperature drives thrombocytopenia in rodents independent of an antibody.

References

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Claims

WHAT IS CLAIMED IS:

1 . An antibody or a fragment thereof comprising six complementarity-determining regions (CDRs) having the sequences of SEQ ID Nos: 1 , 2, 3, 4, 5 and 6.

2. The antibody or the fragment according to claim 1 being a competitive inhibitor of the activating Fc receptor IIIA.

3. The antibody or the fragment according to claim 1 or 2 being a single chain variable fragment (scFv).

4. The antibody or the fragment according to claim 1 or 2 being a fragment antigen-binding (Fab).

5. The antibody or fragment thereof according to any one of claims 1 to 4 for use in treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

6. The antibody or fragment thereof according to any one of claims 1 to 4 for use in preventing or limiting a phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

7. The antibody or fragment thereof according to any one of claims 1 to 4 for use in the treatment, prevention or alleviation of symptomps of immune thrombocytopenia (ITP), auto-immune hemolytic anemia (AHA), platelet transfusion induced immune refractoriness, fetal and neonatal alloimmune thrombocytopenia (FNAIT), conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, hereditary hemorrhagic telangiectasia (HHT), graft versus host disease (GvHD), or allograft rejection.

8. The antibody or fragment thereof according to any one of claims 1 to 4 for use in the manufacture of a medicament fortreating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

9. The antibody or fragment thereof according to any one of claims 1 to 4 for use in the manufacture of a medicament for preventing or limiting a phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

10. The antibody or fragment thereof according to any one of claims 1 to 4 for use in the manufacture of a medicament for the treatment of ITP, AHA, platelet transfusion induced immune refractoriness, FNAIT, conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, HHT, GvHD or allograft rejection.

11. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

12. The pharmaceutical composition according to claim 11 , for use in treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

13. The pharmaceutical composition according to claim 11 , for use in preventing or limiting a phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

14. The pharmaceutical composition according to claim 11 , for use in the treatment of ITP, AHA, platelet transfusion induced immune refractoriness, FNAIT, conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, HHT, GvHD or allograft rejection.

15. The pharmaceutical composition according to claim 11 , for use in the manufacture of a medicament for treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

16. The pharmaceutical composition according to claim 11 , for use in the manufacture of a medicament for preventing or limiting phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

17. The pharmaceutical composition according to claim 11 , for use in the manufacture of a medicament for the treatment of ITP, AHA, platelet transfusion induced immune refractoriness, FNAIT, conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, HHT, GvHD or allograft rejection.

18. A method of treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 1 1 .

19. A method of preventing or limiting phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or fragment thereof as defined in any one of claims 1 to 4 orthe pharmaceutical composition as defined in claim 11 .

20. A method of treating, preventing or alleviating the symptoms of ITP, AHA, platelet transfusion induced immune refractoriness, FNAIT conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, HHT, GvHD or allograft rejection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 1 1 .

21. Use of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 11 for treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

22. Use of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 11 for preventing or limiting phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

23. Use of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 11 for treating ITP, AHA, platelet transfusion induced immune refractoriness, FNAIT, conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, HHT, GvHD or allograft rejection.

24. Use of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 1 1 in the manufacture of a medicament for treating, limiting, or avoiding the activation of an immune cell caused by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1 a.

25. Use of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 1 1 in the manufacture of a medicament for preventing or limiting a phagocytosis mediated by IgG antibodies bridging FcyRIIIA with HLA, and/or HPA-1a.

26. Use of the antibody or fragment thereof as defined in any one of claims 1 to 4 or the pharmaceutical composition as defined in claim 11 in the manufacture of a medicament for treating ITP, AHA, platelet transfusion induced immune refractoriness, FNAIT, conditions associated with alloreactive erythrocyte antibodies, sickle cell disease, HHT, GvHD or allograft rejection.