Dosage and administration of fusion polypeptides for treating sickle cell disease

Abstract

The present disclosure provides methods for treating a human patient with sickle cell disease. The methods comprise administering to the patient a prelysin-binding antibody or antigen-binding fragment thereof.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing in XML format that has been submitted electronically, and the sequence listing is hereby incorporated by reference in its entirety. The XML copy was created on August 25, 2023, named 51196-032WO3_Sequence_Listing_8_25_23, and is 50,329 bytes in size. Background Art

[0003] Sickle cell disease (SCD) is the most common monogenic disease worldwide. In some forms, the disease is caused by mutations in the beta globin gene, for example, a single nucleotide mutation in the beta globin gene that causes glutamic acid at position 6 to be replaced by valine, which is also the cause of beta thalassemia (BT). Despite the widespread recognition of the underlying cause of the disease, there are few available treatment options for controlling SCD symptoms. The two main manifestations of SCD, anemia and vaso-occlusive crisis (VOC), affect the mortality, morbidity and quality of life of SCD patients. Although SCD patients have two approved treatment options, hydroxyurea and L-glutamine, they are generally considered to be suboptimal options for reducing disease symptoms. Therefore, there is a need for new treatments in the art. Summary of the invention

[0004] In a first aspect, the disclosure provides a method for treating a human patient with sickle cell disease, the method comprising administering to the patient a properdin binding antibody or antigen-binding fragment thereof, wherein the properdin binding antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 sequences shown as SEQ ID NOs: 2, 3, and 4, respectively. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a human serum albumin binding sequence. In some embodiments, the human serum albumin binding sequence is fused to the C-terminus of the properdin binding antibody or antigen-binding fragment thereof. In some embodiments, the human serum albumin binding sequence is fused to the C-terminus of the properdin binding antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the human serum albumin binding sequence comprises the CDR1, CDR2, and CDR3 sequences shown as SEQ ID NOs: 5, 6, and 7. In some embodiments, the antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 1 or a modification thereof. In some embodiments, the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

[0005] In some embodiments, the antibody or its antigen binding fragment is administered to the patient at a dose of 300 mg. In some embodiments, the antibody or its antigen binding fragment is administered to the patient once a week. In some embodiments, the antibody or its antigen binding fragment is administered to the patient for up to 13 weeks (e.g., 12 weeks). In some embodiments, the antibody or its antigen binding fragment is administered to the patient once every 2 weeks. In some embodiments, the antibody or its antigen binding fragment is administered up to 4 times.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof is administered to the patient at a dose of 600 mg. In some embodiments, the antibody or antigen-binding fragment thereof is administered to the patient once every 4 weeks. In some embodiments, the antibody or antigen-binding fragment thereof is administered to the patient up to 4 times.

[0007] In some embodiments, the patient has been clinically diagnosed with sickle cell disease. In some embodiments, the sickle cell disease is HbSS or HbSβ 0 -thalassemia. In some embodiments, the patient is further administered hydroxyurea. In some embodiments, the patient has received a stable dose of hydroxyurea for at least 3 months prior to administration of the antibody or antigen-binding fragment thereof. In some embodiments, the patient has not been administered hydroxyurea for at least 30 days prior to administration of the antibody or antigen-binding fragment thereof.

[0008] In some embodiments, the patient experienced no treatment-emergent adverse events after 12 weeks of treatment. In some embodiments, the patient experienced no serious adverse events after 12 weeks of treatment. In some embodiments, the patient experienced no adverse events after 12 weeks of treatment.

[0009] In some embodiments, the method further comprises measuring the change in serum concentration of the antibody or antigen-binding fragment thereof for up to 30 weeks after initiation of treatment. In some embodiments, the method further comprises measuring the change in blood concentration of the anti-drug antibody for up to 30 weeks after initiation of treatment. In some embodiments, the patient experiences a change in serum concentration of total properdin and free properdin relative to baseline after up to 30 weeks of treatment.

[0010] In some embodiments, the patient experiences a change in serum concentration of complement component Ba (Ba), complement component C3a (C3a), or soluble complement component C5B-9 (sC5B9) relative to baseline after 12 weeks of treatment. In some embodiments, the patient experiences a change in blood or serum concentration of hemoglobin, nitric oxide, an inflammatory marker, or a cell adhesion marker relative to baseline after 12 weeks of treatment. In some embodiments, the inflammatory marker comprises interleukin-1. In some embodiments, the cell adhesion marker comprises soluble P-selectin.

[0011] In some embodiments, the patient experiences a change in hemoglobin level relative to baseline after 12 weeks of treatment. In some embodiments, the patient experiences a change in serum LDH level, indirect bilirubin, haptoglobin, or hemopexin relative to baseline after 12 weeks. In some embodiments, the patient experiences a change in reticulocyte level relative to baseline after 12 weeks.

[0012] In some embodiments, the patient experiences a reduced rate of vaso-occlusive crises after 12 weeks of treatment compared to baseline. In some embodiments, the patient experiences an increased time to first vaso-occlusive crisis after 12 weeks of treatment compared to baseline.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof is formulated for subcutaneous administration.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is formulated at a concentration of 150 mg / mL at pH 5.4 in an aqueous solution comprising 20 nM sodium acetate, 250 mM sucrose, and 0.05% polysorbate-80. In some embodiments, the human patient is between 18 and 65 years old. In some embodiments, the human patient weighs ≥ 40 kg.

[0015] In another aspect, the disclosure provides an antibody or antigen-binding fragment for use in treating a human patient with sickle cell disease, wherein the use comprises administering to the patient a properdin binding antibody or antigen-binding fragment thereof, wherein the properdin binding antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease further comprises a human serum albumin binding sequence. In some embodiments, the human serum albumin binding sequence is fused to the C-terminus of the properdin binding antibody or antigen-binding fragment thereof. In some embodiments, the human serum albumin binding sequence is fused to the C-terminus of the properdin binding antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the human serum albumin binding sequence comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 5, 6, and 7. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease comprises the sequence of SEQ ID NO: 1 or a modification thereof. In some embodiments, the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease is administered to the patient at a dose of 300 mg. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease is administered to the patient once a week. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease will be administered to the patient for up to 13 weeks. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease will be administered to the patient once every two weeks. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease will be administered up to four times. In some embodiments, the antibody or antigen-binding fragment thereof is administered to the patient at a dose of 600 mg. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease will be administered to the patient once every 4 weeks. In some embodiments, the antibody or antigen-binding fragment thereof for use in treating a human patient with sickle cell disease will be administered to the patient up to 4 times. In some embodiments, the patient has been clinically diagnosed with sickle cell disease. In some embodiments, the sickle cell disease is HbSS or HbSβ 0-thalassemia. In some embodiments, hydroxyurea will be further administered to the patient. In some embodiments, the patient has received a stable dose of hydroxyurea for at least 3 months prior to administration of the antibody or antigen-binding fragment thereof. In some embodiments, the patient has not been administered hydroxyurea for at least 30 days prior to administration of the antibody or antigen-binding fragment thereof. In some embodiments, the patient has not experienced adverse events during treatment after 12 weeks of treatment. In some embodiments, the patient has not experienced serious adverse events after 12 weeks of treatment. In some embodiments, the patient has not experienced adverse events after 12 weeks of treatment. In some embodiments, the patient has not experienced adverse events after 12 weeks of treatment. In some embodiments, the method further comprises measuring the change in serum concentration of the antibody or antigen-binding fragment thereof for up to 30 weeks after initiation of treatment. In some embodiments, the method further comprises measuring the change in blood concentration of anti-drug antibodies for up to 30 weeks after initiation of treatment. In some embodiments, the patient experiences a change in serum concentration of total properdin and free properdin relative to baseline after up to 30 weeks of treatment. In some embodiments, the patient experiences a change in serum concentration of complement component Ba (Ba), complement component C3a (C3a), or soluble complement component C5B-9 (sC5B9) relative to baseline after 12 weeks of treatment. In some embodiments, the patient experiences a change in blood or serum concentration of hemoglobin, nitric oxide, an inflammatory marker, or a cell adhesion marker relative to baseline after 12 weeks of treatment. In some embodiments, the inflammatory marker comprises interleukin-1. In some embodiments, the cell adhesion marker comprises soluble P-selectin. In some embodiments, the patient experiences a change in hemoglobin level relative to baseline after 12 weeks of treatment. In some embodiments, the patient experiences a change in serum LDH level, indirect bilirubin, haptoglobin, or hemoglobin relative to baseline after 12 weeks. In some embodiments, the patient experiences a change in reticulocyte level relative to baseline after 12 weeks. In some embodiments, the patient experiences a reduced rate of vaso-occlusive crises after 12 weeks of treatment compared to baseline. In some embodiments, the patient experiences an increased time to first vaso-occlusive crisis after 12 weeks of treatment compared to baseline. In some embodiments, the antibody or antigen-binding fragment thereof is formulated for subcutaneous administration. In some embodiments, the antibody or antigen-binding fragment thereof is formulated at a concentration of 150 mg / mL at pH 5.4 in an aqueous solution comprising 20 nM sodium acetate, 250 mM sucrose, and 0.05% polysorbate-80. In some embodiments, the human patient is between 18 and 65 years old. In some embodiments, the human patient weighs ≥ 40 kg.

[0017] Each embodiment is combinable, unless the context clearly implies otherwise. Each embodiment is applicable to each aspect of the invention, unless the context clearly implies otherwise.

[0018] Specific embodiments of the invention will become apparent from the following more detailed description of certain preferred embodiments and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of the study presented in Example 1.

[0020] Figure 2 is a schematic diagram of the study presented in Example 2.

[0021] Figure 3 is a graph showing the mean serum complement activity pathways over time for each cohort described in Example 2.

[0022] definition

[0023] To facilitate understanding of the present disclosure, a plurality of terms are defined below. The terms defined herein have meanings commonly understood by those of ordinary skill in the art related to the present disclosure. Terms such as "a, an" and "the" are not intended to refer to a single entity, but rather to general categories that can be illustrated using specific examples. The terms herein are used to describe specific embodiments, but their use does not limit the present disclosure unless outlined in the claims.

[0024] As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0025] As used herein, any value provided in a range of values ​​includes the upper and lower limits, and any value contained within the upper and lower limits.

[0026] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single-chain versions thereof. In a preferred embodiment, an "antibody" refers to a glycoprotein or an antigen-binding portion thereof, which comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region consists of one domain, CL. H and V L The V region can be further subdivided into regions of hypervariability, also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V LIt is composed of three CDRs and four FRs, which are arranged in the following order from amino-terminal to carboxyl-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant regions of these antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0027] As used herein, "effective treatment" refers to treatment that produces a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement relative to a baseline, i.e., an improvement relative to a measurement or observation made prior to initiating therapy according to the method. Effective treatment can refer to a reduction in at least one symptom of sickle cell disease.

[0028] As used herein, the term "fused to" refers to the preparation of a polypeptide by combining more than one sequence, typically by cloning one sequence (e.g., a coding sequence) in-frame with one or more second coding sequences into an expression vector so that the two (or more) coding sequences are transcribed and translated into a single continuous polypeptide. In addition to being prepared by recombinant techniques, portions of a polypeptide can be "fused to" each other by chemical reactions or means known in the art for preparing conventional polypeptides.

[0029] The term "heavy chain antibody" refers to an antibody composed of two heavy chains and lacking the two light chains found in conventional antibodies. Camelids (biologically members of the Camelidae family, the only extant family in the Tylopoda suborder; extant camelids include dromedary camels, Bactrian camels, wild or undomesticated camels, llamas, alpacas, llamas and guanacos) are the only mammals with single-chain VHH antibodies. About 50% of antibodies in camelids are heavy chain antibodies, and the other 50% are common or conventional mammalian heavy chain / light chain antibody types.

[0030] A "VHH domain" refers to the variable domain present in naturally occurring heavy chain antibodies, thereby distinguishing it from the heavy chain variable domain present in conventional four-chain antibodies (referred to herein as a "VH domain") and the light chain variable domain present in conventional four-chain antibodies (referred to herein as a "VL domain").

[0031] VHH domains have many unique structural features and functional properties, making isolated VHH domains (as well as sdAbs, which are based on VHH domains and share these structural features and functional properties with naturally occurring VHH domains) and proteins containing VHH domains very useful for use as functional antigen-binding domains or proteins. For example, VHH domains and sdAbs that bind to antigens in the absence of VL can act as single, relatively small functional antigen-binding structural units, domains or proteins. The small size of these molecules distinguishes VHH domains from the VH and VL domains of conventional four-chain antibodies. The use of VHH domains and sdAbs as single antigen-binding proteins or as antigen-binding domains (e.g., as part of a larger protein or polypeptide) provides many significant advantages over the use of conventional VH and VL domains and scFv or conventional antibody fragments (such as Fab or F(ab')2 fragments). Only a single domain is needed to bind antigen with high affinity and high selectivity, for example, so that there is no need for two separate domains, nor is it necessary to ensure that the two domains exist in a specific spatial conformation and configuration (for example, by using a specific linker, as in scFv). VHH domains and sdAbs can also be expressed by a single gene, and no post-translational folding or modification is required. VHH domains and sdAbs can be easily engineered into multivalent and multispecific forms. VHH domains and sdAbs are also highly soluble and do not have a tendency to aggregate (Ward, E. et al., Nature [Nature], 341: 544-6, 1989), and they are highly stable to heat, pH, proteases and other denaturants or conditions (Ewert, S. et al., Biochemistry [Biochemistry], 41: 3628-36, 2002). The preparation of VHH domains and sdAbs is relatively easy and cheap, even on the scale required for production. For example, VHH domains, sdAbs, and polypeptides containing VHH domains or sdAbs can be produced using microbial fermentation using methods known in the art, and do not require the use of mammalian expression systems, such as conventional antibody fragments. Compared with conventional four-chain antibodies and antigen-binding fragments thereof, VHH domains and sdAbs are relatively small (about 15 kDa, or 10 times smaller than conventional IgG), and therefore show higher tissue permeability (including but not limited to solid tumors and other dense tissues) than conventional four-chain antibodies and antigen-binding fragments thereof. VHH domains and sdAbs can show so-called "cavity binding" properties (due to, for example, their extended CDR3 loops), and can approach targets and epitopes that conventional four-chain antibodies and antigen-binding fragments thereof cannot approach.For example, VHH domains and sdAbs have been shown to inhibit enzymes (WO 97 / 49805; Transue, T. et al., Proteins, 32:515-22, 1998; Lauwereys, M. et al., EMBO J., 17:3512-20, 1998).

[0032] As used herein, the term "single domain antibody" or "sdAb" is an antibody or fragment thereof consisting of a single monomeric variable antibody domain. It is not limited to a specific biological source or a specific method of preparation. sdAbs can be obtained, for example, by the following methods: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) "camelization" of a naturally occurring VH domain from any animal species, particularly a mammalian species, such as a human, or by expression of a nucleic acid encoding such a camelized VH domain; (5) "camelization" of a "domain antibody" ("Dab") or by expression of a nucleic acid encoding such a camelized VH domain; (6) preparing an engineered polypeptide or fusion protein using synthetic or semisynthetic techniques; (7) preparing a nucleic acid encoding an sdAb using nucleic acid synthesis techniques, followed by expression of the nucleic acid thus obtained; and / or (8) any combination of the above.

[0033] The fusion polypeptides or fusion proteins described herein may comprise, for example, an amino acid sequence of a naturally occurring VHH domain that has been "humanized," for example, by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence with one or more amino acid residues present at the corresponding position in a VH domain from a human.

[0034] The fusion polypeptides or fusion proteins described herein may comprise, for example, an amino acid sequence of a naturally occurring VH domain that has been "camelized", i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain with one or more amino acid residues present in the corresponding positions of, for example, a VHH domain of a camel antibody. This can be performed in a manner known in the art. Such camelization may preferentially occur at amino acid positions present at the VH-VL interface and at the so-called "Camelidae Hallmark Residues" (WO 94 / 04678). The VH domain or sequence used as a parent sequence or starting material for generating or designing a camelized sequence may be, for example, a VH sequence from a mammal, and in certain embodiments, a human VH sequence. However, it should be noted that such camelized sequences can be obtained in any suitable manner known in the art, and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring parent VH domains.

[0035] Both "humanization" and "camelization" can be achieved by providing a construct encoding a naturally occurring VHH domain or a V H The nucleotide sequence of the VHH domain or VH domain can be designed based on the amino acid sequence or nucleotide sequence of the naturally occurring VHH domain or VH domain, and then one or more codons in the nucleotide sequence can be changed in a manner known to those skilled in the art so that the new nucleotide sequence encodes a humanized or camelized sequence, respectively. In addition, based on the amino acid sequence or nucleotide sequence of the naturally occurring VHH domain or VH domain, a nucleotide sequence encoding a desired humanized or camelized sequence can be designed and synthesized de novo using nucleic acid synthesis techniques known in the art, and the nucleotide sequence thus obtained can then be expressed in a manner known in the art.

[0036] As used herein, the term "antigen" or "antigenic target" refers to a molecule or portion of a molecule that can be bound by an antibody, one or more Ig binding domains, or other immunological binding moiety, including, for example, an engineered polypeptide or fusion polypeptide disclosed herein. An antigen can be used in an animal to produce an antibody that can bind to an epitope of the antigen. An antigen can have one or more epitopes.

[0037] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. Such "fragments" are, for example, between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, i.e., a fragment consisting of a V L 、V H (ii) a F(ab')2 fragment, i.e. a bivalent fragment having two Fab fragments connected by a disulfide bridge in the hinge region; (iii) a monovalent fragment consisting of V H and CH1 domains; (iv) a V fragment consisting of a single arm of the antibody L and V H Fv fragments composed of domains, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which are composed of V H and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains of the Fv fragment, V L and V H, are encoded by separate genes, but they can be joined using recombinant methods via synthetic linkers, allowing them to be made into a single protein chain, with V L and V H The single-chain Fv (sFv) domains are paired to form monovalent molecules (called single-chain Fv (sFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0038] As used herein, the term "binding domain" refers to a portion of a protein or antibody that contains amino acid residues that interact with an antigen. Binding domains include, but are not limited to, antibodies (e.g., full-length antibodies), and antigen-binding portions thereof. The binding domain confers specificity and affinity to the binding agent for the antigen. The term also encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0039] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope can be formed by either continuous amino acids or non-continuous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by continuous amino acids are generally retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are generally lost when treated with denaturing solvents. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or continuous peptides from an antigen are tested for reactivity with a given antibody. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, e.g., X-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, ed. (1996)).

[0040] The term "bispecific" refers to a fusion polypeptide of the present disclosure that is capable of binding to two antigens.

[0041] The term "effective amount" refers to an amount of an agent that provides a desired biological, therapeutic and / or preventive result. The result can be reduction, amelioration, alleviation, mitigation, delay and / or alleviation of one or more signs, symptoms or causes of a disease, or any other desired change in a biological system. In one example, an "effective amount" is an amount of a fusion polypeptide or fragment thereof that is clinically demonstrated to alleviate at least one symptom of sickle cell disease. An effective amount can be administered once or multiple times.

[0042] As used herein, "effective treatment" refers to treatment that produces a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement relative to a baseline, i.e., an improvement relative to a measurement or observation made prior to initiating therapy according to the method. Effective treatment can refer to a reduction in at least one symptom of sickle cell disease.

[0043] As used herein, the term "fused to" refers to the preparation of a polypeptide by combining more than one sequence, typically by cloning one sequence (e.g., a coding sequence) in-frame with one or more second coding sequences into an expression vector so that the two (or more) coding sequences are transcribed and translated into a single continuous polypeptide. In addition to being prepared by recombinant techniques, portions of a polypeptide can be "fused to" each other by chemical reactions or means known in the art for preparing conventional polypeptides.

[0044] As used herein, the term "peptide linker" refers to one or more amino acid residues inserted or included between the engineered polypeptides of a fusion polypeptide. For example, a peptide linker can be inserted or included at the transition between the engineered polypeptides of a fusion polypeptide at the sequence level.

[0045] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when administered to a patient.

[0046] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing delivery of the engineered polypeptides or fusion polypeptides of the disclosure.

[0047] An antibody, immunoglobulin or immunologically functional immunoglobulin fragment, or an engineered polypeptide or fusion polypeptide disclosed herein is said to "specifically" bind to an antigen when the molecule preferentially recognizes its antigenic target in a complex mixture of proteins and / or macromolecules. As used herein, the term "specifically binds" refers to the ability of an antibody, immunoglobulin or immunologically functional immunoglobulin fragment or engineered polypeptide or fusion polypeptide disclosed herein to bind to an antigen containing an epitope with a specific affinity of at least about 10 -6 M, 10 -7 M, 10 - 8 M, 10 -9 M, 10-10 M, 10 -11 M, 10 -12 M or higher K D The ability to bind to, and / or bind to, an epitope with an affinity that is at least two times greater than its affinity for a nonspecific antigen.

[0048] As used herein, the term "subject" or "patient" is a human patient (eg, a patient with sickle cell disease). As used herein, the terms "subject" and "patient" are interchangeable.

[0049] As used herein, the term "treatment" or "treat" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those suffering from the disorder, as well as those at risk of the disorder, or those in whom the disorder is to be prevented. DETAILED DESCRIPTION

[0050] The present disclosure provides methods of treating sickle cell disease (SCD) in a subject in need thereof by administering a fusion polypeptide comprising an engineered polypeptide that specifically binds human properdin fused to a polypeptide that specifically binds human serum albumin via a peptide linker.

[0051] Fusion polypeptide specifically binding to albumin and properdin

[0052] Fusion proteins and formulations thereof are described herein. In some embodiments, the fusion protein is a bispecific antibody in which two antigen-binding polypeptides are connected (e.g., by a linker, such as a linker). Such bispecific constructs may include an anti-properdin binding polypeptide (e.g., a monovalent VHH antibody or a VHH variable domain) connected by a linker to a second polypeptide (e.g., a second monovalent antibody or a VHH variable domain). The second polypeptide may, for example, enhance the in vivo stability of the bispecific construct, target different therapeutic targets, or place two antigens in close proximity (e.g., thereby targeting a first binding antigen to a second binding antigen). In some embodiments, the second polypeptide is an albumin binding molecule, an albumin binding peptide, or an anti-albumin antibody (e.g., a monovalent antibody) or a modified form thereof (e.g., a variable domain of a llama antibody that specifically binds to human serum albumin). In addition to the present disclosure, albumin binding peptides are known in the art (WO 2007 / 106120 (see Tables 1 to 9); Dennis, M. et al., J. Biol. Chem., 277:35035-43, 2002; the disclosures of which are hereby incorporated by reference).

[0053] The antibodies described herein can inhibit, for example, the binding of properdin to C3b, C3Bb, and C3bBb. Inhibition of properdin results in a reduction in alternative pathway complement activation, suggesting a therapeutic benefit for patients suffering from alternative pathway dysregulated diseases in which the alternative pathway is overactivated.

[0054] The anti-properdin antibodies described herein can be prepared by using full-length properdin, properdin polypeptides and / or using peptides carrying antigenic properdin epitopes (e.g., fragments of properdin polypeptides). Properdin peptides and polypeptides can be isolated as natural polypeptides, recombinant or synthetic recombinant polypeptides and used to produce antibodies. All antigens that can be used to produce anti-properdin antibodies can be used to produce monovalent antibodies. Suitable monovalent antibody formats and methods for preparing them are known in the art (WO 2007 / 048037 and WO 2007 / 059782, the entire contents of which are incorporated herein by reference).

[0055] The anti-properdin antibody may be a monoclonal antibody or may be derived from a monoclonal antibody. Suitable monoclonal antibodies against a selected antigen may be prepared by known techniques ("Monoclonal Antibodies: A manual of techniques," Zola (CRC Press, 1988); "Monoclonal Hybridoma Antibodies: Techniques and Applications," Hurrell (CRC Press, 1982), the entire contents of which are incorporated herein by reference).

[0056] In other embodiments, the antibody may be a single domain antibody, such as a VHH. Such antibodies are naturally present in camelids and sharks (Saerens, D. et al., Curr. Opin. Pharmacol. [Contemporary Pharmacology Perspectives], 8: 600-8, 2008). Camelid antibodies are described in, for example, U.S. Patent Nos. 5,759,808, 5,800,988, 5,840,526, 5,874,541, 6,005,079 and 6,015,695, each of which is incorporated herein by reference in its entirety. The cloned and isolated VHH domain is a stable polypeptide characterized by having the full antigen binding ability of the original heavy chain antibody. The VHH domain combines the advantages of conventional antibodies (high target specificity, high target affinity and low inherent toxicity) with the important features of small molecule drugs (the ability to inhibit enzymes and approach receptor clefts) with its unique structural and functional properties. In addition, they are stable, can potentially be administered by means other than injection, are easier to manufacture, and can be humanized (U.S. Pat. No. 5,840,526; U.S. Pat. No. 5,874,541; U.S. Pat. No. 6,005,079, U.S. Pat. No. 6,765,087; EP 1589107; WO 97 / 34103; WO 97 / 49805; U.S. Pat. No. 5,800,988; U.S. Pat. No. 5,874,541 and U.S. Pat. No. 6,015,695, the entire contents of each of which are incorporated herein by reference).

[0057] The anti-properdin component of the bispecific antibodies described herein comprises CDR sequences including a CDR-H1 having an amino acid sequence at least 90% identical to GRISSIIHMA (SEQ ID NO:2); a CDR-H2 having an amino acid sequence at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) identical to RVGTTVYADSVKG (SEQ ID NO:3); and a CDR-H3 having an amino acid sequence at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) identical to LQYEKHGGADY (SEQ ID NO:4). The bispecific antibody described herein may comprise: a CDR-H1 having an amino acid sequence of GRISSIIHMA (SEQ ID NO: 2); a CDR-H2 having an amino acid sequence of RVGTTVYADSVKG (SEQ ID NO: 3); and a CDR-H3 having an amino acid sequence of LQYEKHGGADY (SEQ ID NO: 4).

[0058] In addition, the engineered fusion proteins described herein can specifically bind to serum albumin in the following manner, i.e., when the engineered protein is bound to or otherwise associated with the serum albumin molecule, the binding of the serum albumin molecule to FcRn is not significantly reduced or inhibited compared to the binding of the serum albumin molecule to FcRn when the polypeptide is not bound thereto. In this embodiment, "not significantly reduced or inhibited" means that the binding affinity of serum albumin to FcRn (as measured using a suitable assay such as, for example, SPR) is reduced by no more than 50%, or no more than 30%, or no more than 10%, or no more than 5%, or not reduced at all. In this embodiment, "not significantly reduced or inhibited" also means that the half-life of the serum albumin molecule is not significantly reduced. Specifically, the engineered polypeptide can bind to the amino acid residues on serum albumin that are not involved in the binding of serum albumin to FcRn. More specifically, the engineered polypeptide can bind to the amino acid residues or sequences of serum albumin that do not form part of domain III of serum albumin, for example, the engineered polypeptide can bind to the amino acid residues or sequences of serum albumin that form part of domain I and / or domain II.

[0059] The anti-albumin component of the bispecific antibodies described herein may comprise CDR sequences including a CDR-H1 having an amino acid sequence at least 87% identical to GRPVSNYA (SEQ ID NO: 5); a CDR-H2 having an amino acid sequence at least 87% identical to INWQKTAT (SEQ ID NO: 6); and a CDR-H3 having an amino acid sequence at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) identical to AAVFRVVAPKTQYDYDY (SEQ ID NO: 7). The bispecific antibodies described herein may comprise: a CDR-H1 having an amino acid sequence of GRPVSNYA (SEQ ID NO: 5); a CDR-H2 having an amino acid sequence of INWQKTAT (SEQ ID NO: 6); and a CDR-H3 having an amino acid sequence of AAVFRVVAPKTQYDYDY (SEQ ID NO: 7).

[0060] In some embodiments, the fusion protein comprises an anti-properdin binding portion and an anti-albumin binding portion. In some embodiments, when the anti-properdin binding domain has an exposed N-terminus, the N-terminal glutamine can be converted to a cyclized pyroglutamate. Such modifications are known in the art (see, e.g., Liu et al., The Journal of Biological Chemistry 286 (13: 11211-11217, 2011)). The portion encoding the anti-properdin binding portion can have at least 90% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) identity with the following amino acid sequence:

[0061] EVQ LLESGGGLVQ PGGSLRLSCAASGRISSIIH MAWFRQAPGK ERELVSEISR VGTTVYADSVKGRFTISRDN SKNTLYLQMN SLKPEDTAVY YCNALQYEKH GGADYWGQGT LVTVSS (SEQ ID NO: 55).

[0062] In some embodiments, the anti-properdin binding portion of the fusion protein comprises SEQ ID NO:55.

[0063] The portion encoding the anti-albumin binding portion may be at least 90% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of:

[0064] QVQLVESGGG LVKPGGSLRL SCAASGRPVS NYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTIS RDNAKNSLYL QMNSLRAEDT AVYYCAAVFR VVAPKTQYDY DYWGQGTLVTVSS (SEQ ID NO: 56).

[0065] In some embodiments, the anti-properdin binding portion of the fusion protein comprises SEQ ID NO:56.

[0066] In some embodiments, the fusion protein is encoded by a nucleic acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to:

[0067] (SEQ ID NO:57).

[0068] In some embodiments, the fusion protein is encoded by a nucleic acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the fusion protein is encoded by the nucleic acid sequence of SEQ ID NO: 57.

[0069] In some embodiments, the fusion protein has an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following amino acid sequence:

[0070] QVQLVESGGG LVKPGGSLRL SCAASGRPVS NYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTIS RDNAKNSLYL QMNSLRAEDT AVYYCAAVFR VVAPKTQYDY DYWGQGTLVTVSSGGGGEGGGGEGGGGEVQ LLESGGGLVQ PGGSLRLSCAASGRISSIIH MAWFRQAPGK ERELVSEISRVGTTVYADSV KGRFTISRDN SKNTLYLQMN SLKPEDTAVY YCNALQYEKH GGADYWGQGT LVTVSS (SEQ ID NO: 1).

[0071] In some embodiments, the fusion protein has an amino acid sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1. In some embodiments, the fusion protein has an amino acid sequence of SEQ ID NO: 1.

[0072] In some embodiments, the C-terminal residue of the properdin binding domain of the fusion protein may be fused directly or via a linker to the N-terminal residue of the human serum albumin binding domain. In other embodiments, the C-terminal residue of the complement component human serum albumin binding domain of the fusion protein may be fused directly or via a peptide to the N-terminal residue of the properdin binding domain. The fusion proteins described herein may comprise one or more modified amino acid residues. For example, the amino acid sequence of SEQ ID NO: 1 may comprise one or more amino acid modifications. The amino acid modifications described herein include all amino acid modifications known in the art (see, e.g., Liu et al., The Journal of Biological Chemistry 286 (13: 11211-11217, 2011) and Manning et al., Pharmaceutical Research 27 (4): 544-575, 2010). In all contexts, known conversions of specific amino acids will be included, e.g., during processing or purification of the fusion polypeptide, e.g., conversion of an exposed N-terminal glutamine to pyroglutamate.

[0073] Connectors

[0074] As described herein, a linker is used to describe a bond or connection between a polypeptide or protein domain and / or an associated non-protein moiety. In some embodiments, a linker is a bond or connection between at least two polypeptide constructs, for example, such that two polypeptide constructs are joined to each other in a tandem series (e.g., a monovalent antibody connected to a second polypeptide or monovalent antibody). A linker can connect the N-terminus or C-terminus of an antibody construct to the N-terminus or C-terminus of a second polypeptide construct.

[0075] Fusion proteins comprising engineered proteins that specifically bind albumin and properdin are described herein, wherein the engineered proteins are fused directly or connected via one or more suitable linkers or spacers. For example, a peptide linker can be inserted or included at the transition between the engineered proteins of the fusion protein at the sequence level. The identity and sequence of the amino acid residues in the linker can vary depending on the desired secondary structure.

[0076] The linker can be a simple covalent bond (e.g., a peptide bond), a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer), or any type of bond produced by a chemical reaction (e.g., chemical conjugation). In the case where the linker is a peptide bond, the carboxylic acid group at the C-terminus of one protein domain can react with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In particular, the peptide bond can be formed by synthetic means by conventional organic chemical reactions well known in the art, or by natural production by a host cell, where a polynucleotide sequence encoding a DNA sequence of two proteins (e.g., two antibody constructs in a tandem series) can be directly transcribed and translated into a continuous polypeptide encoding the two proteins by the necessary molecular machinery in the host cell (e.g., DNA polymerase and ribosomes).

[0077] Where the linker is a synthetic polymer (eg, a PEG polymer), the polymer may be functionalized at each end with reactive chemical groups to react with the terminal amino acids of the two protein-linking ends.

[0078] In the case where the linker (other than the peptide bond described above) is made by chemical reaction, a chemical functional group, such as an amine, carboxylic acid, ester, azide or other functional groups commonly used in the art, can be synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. The two functional groups can then react to form a chemical bond by synthetic chemical means, thereby linking the two proteins together. Such chemical conjugation procedures are routine to those skilled in the art.

[0079] As described herein, the linker between two peptide constructs can be an amino acid linker comprising 1-200 (e.g., 1-4, 1-10, 1-20, 1-30, 1-40, 2-10, 2-12, 2-16, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200) amino acids. Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine.

[0080] Glycine, serine and alanine can be used for the joint with maximum flexibility.Any amino acid residue can be regarded as a joint combined with one or more other amino acid residues, which can be the same or different from the first amino acid residue, thereby depending on the desired properties, a larger peptide joint is constructed when necessary.In other embodiments, the joint is GGGGEGGGGEGGGGE (SEQ ID NO: 10).In other embodiments, the joint is GGGGSGGGGSGGGGS (SEQ ID NO: 11). Additional peptide linkers suitable for use in generating fusion proteins described herein include, for example, G4S (SEQ ID NO: 12), (G4S)2 (SEQ ID NO: 13), (G4S)3 (SEQ ID NO: 14), (G4S)4 (SEQ ID NO: 15), (G4S)5 (SEQ ID NO: 16), (G4S)6 (SEQ ID NO: 17), (EAAAK)3 (SEQ ID NO: 18), PAPAP (SEQ ID NO: 19), G4SPAPAP (SEQ ID NO: 20), PAPAPG4S (SEQ ID NO: 21), (GGGDS)2 (SEQ ID NO: 22), (GGGES)2 (SEQ ID NO: 23), GGGDSGGGGS (SEQ ID NO: 24), GGGASGGGGS (SEQ ID NO: 25), GGGESGGGGS (SEQ ID NO: 26), ASTKGP (SEQ ID NO: 27), ASTKGPSVFPLAP (SEQ ID NO: 28), ASTKGPSVFPLAP (SEQ ID NO: 29), ASTKGPSVFPLAP (SEQ ID NO: 30), ASTKGPSVFPLAP (SEQ ID NO: 31), ASTKGPSVFPLAP (SEQ ID NO: 32), ASTKGPSVFPLAP (SEQ ID NO: 33), ASTKGPSVFPLAP (SEQ ID NO: 34), ASTKGPSVFPLAP (SEQ ID NO: 35), ASTKGPSVFPLAP (SEQ ID NO: 36), ASTKGPSVFPLAP (SEQ ID NO: 37), ASTKGPSVFPLAP (SEQ ID NO: 38), ASTKGPSVFPLAP (SEQ ID NO: 39), ASTKGPSVFPLAP (SEQ ID NO: 40), ASTKGPSVFPLAP (SEQ ID NO: 41), ASTKGPSVFPLAP (SEQ ID NO: NO:28), G3P (SEQ ID NO:29), G7P (SEQ ID NO:30), PAPNLLGGP (SEQ ID NO:31), G6 (SEQ ID NO:32), G 12(SEQ ID NO:33), APELPGGP (SEQ ID NO:34), SEPQPQPG (SEQ ID NO:35), (G3S2)3 (SEQ ID NO:36), GGGGGGGGGSGGGS (SEQ ID NO:37), GGGGSGGGGGGGGGS (SEQ ID NO:38), (GGSSS)3 (SEQ ID NO:39), (GS4)3 (SEQ ID NO:40), G4A(G4S)2(SEQ ID NO:41), G4SG4AG4S(SEQ ID NO:42), G3AS(G4S)2(SEQ ID NO:43), G4SG3ASG4S(SEQ ID NO:44), G4SAG3SG4S(SEQ ID NO:45), (G4S)2AG3S(SEQ ID NO:46), G4SAG3SAG3S (SEQ ID NO:47), G4D (G4S)2 (SEQ ID NO:48), G4SG4DG4S (SEQ ID NO:49), (G4D)2G4S (SEQ ID NO:50), G4E(G4S)2 (SEQ ID NO:51), G4SG4EG4S (SEQ ID NO:52), (G4E)2G4S (SEQ ID NO:53), and GGGGAGGGGAGGGGS (SEQ ID NO:54). One skilled in the art can select the linker, for example, to reduce or eliminate post-translational modifications, such as glycosylation, such as xylosylation. In certain embodiments, the fusion protein comprises at least two sdAbs, Dabs, VHH antibodies, VHH antibody fragments, or a combination thereof, wherein at least one of the sdAbs, Dabs, VHH antibodies, or VHH antibody fragments is directed against albumin and one of the sdAbs, Dabs, VHH antibodies, or VHH antibody fragments is directed against properdin, such that the resulting fusion protein is multivalent or multispecific. The binding domain or portion may be directed to, for example, HSA, cynomolgus serum albumin, human properdin and / or cynomolgus properdin.

[0081] Treatments for sickle cell disease

[0082] Provided herein are methods for treating sickle cell disease (SCD) in a human patient, the method comprising administering to the patient a fusion polypeptide according to a specific clinical dosage regimen (i.e., at a specific dosage and according to a specific dosing regimen), the fusion polypeptide comprising an engineered polypeptide that specifically binds human properdin fused to a polypeptide that specifically binds human serum albumin via a peptide linker.

[0083] In one embodiment, the fusion polypeptide is administered to the patient at a dosage of 300 mg. In some embodiments, the fusion polypeptide is administered once a week. The fusion polypeptide may be administered to the patient between 1 week and 13 weeks (e.g., between 1 week and 12 weeks, 1 week to 10 weeks, 1 week to 8 weeks, 1 week to 6 weeks, 1 week to 4 weeks, 4 weeks to 13 weeks, 6 weeks to 13 weeks, 8 weeks to 13 weeks, 10 weeks to 13 weeks, or 12 weeks to 13 weeks).

[0084] In some embodiments, the fusion polypeptide is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or every day. In another embodiment, the fusion polypeptide is administered twice a day. In another embodiment, the fusion polypeptide is administered once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, or once every twelve weeks.

[0085] In another embodiment, the fusion polypeptide is administered to the patient every two weeks. (e.g., once every two weeks). The fusion polypeptide may be administered for up to 12 weeks. In some embodiments, the fusion polypeptide is administered up to six times (e.g., once, twice, three times, four times, five times, or six times). In some embodiments, the fusion polypeptide is administered up to four times (e.g., once, twice, three times, or four times).

[0086] In another embodiment, the fusion polypeptide is administered to the patient at a dose of 600 mg. In some embodiments, the fusion polypeptide is administered to the patient once every 4 weeks. The fusion polypeptide can be administered to the patient up to 4 times (e.g., once, twice, three times, or four times).

[0087] The patient to whom the fusion polypeptide is administered may have been clinically diagnosed by a clinician as having sickle cell disease. Sickle cell disease may include HbSS or HbSβ 0 -thalassemia. The age of the human patient may be between 18 and 65 years old. In some embodiments, the weight of the human patient is ≥40 kg. In some embodiments, hydroxyurea is further administered to the patient. In some embodiments, the patient has received a stable dose of hydroxyurea for at least 3 months prior to administration of the antibody or antigen-binding fragment thereof. In another embodiment, the patient has not been administered hydroxyurea for at least 30 days prior to administration of the antibody or antigen-binding fragment thereof.

[0088] In some embodiments, the fusion polypeptide is formulated for subcutaneous administration.For example, the fusion polypeptide can be formulated at a concentration of 150 mg / mL at pH 5.4 in an aqueous solution comprising 20 nM sodium acetate, 250 mM sucrose, and 0.05% polysorbate-80.

[0089] In some embodiments, the fusion polypeptide is administered using a prefilled syringe. In other embodiments, the fusion polypeptide is administered using an automatic injector device. For example, an automatic injector device may include a single vial system, such as a pen-type injector device for solution delivery. Such devices are commercially available from manufacturers such as BD Pens, BD and Genotronorm Humatro Roferon J-tip Needle-Free For example, Becton Dickinson (Franklin Lakes, NJ), Ypsomed (Burgdorf, Switzerland), www.ypsomed.com ), Bioject, Portland, OR., National Medical Products, Weston Medical (Peterborough, UK), Medi-Ject Corp (Minneapolis, MN), and Zogenix, Inc, Emeryville, CA. Recognized devices including dual-vial systems include those for reconstituting a lyophilized drug in a cartridge to deliver a reconstituted solution, such as pen-type injector systems. In one embodiment, the autoinjector is a YpsoMate 2.25 or YpsoMate 2.25 Pro (Medical Biotechnology) disposable injection device.

[0090] In some embodiments, patients treated according to the methods described herein have been vaccinated against meningococcal infection within three years prior to or at the time of initiation of the study drug. In one embodiment, patients who initiate treatment less than two weeks after receiving a meningococcal vaccine receive appropriate prophylactic antibiotic therapy until two weeks after vaccination. In another embodiment, patients treated according to the methods described herein are vaccinated against meningococcal serotypes A, C, Y, W135, and / or B.

[0091] result

[0092] In some embodiments, administration of any of the fusion polypeptides described herein can result in a patient experiencing no adverse events after 12 weeks of treatment. In some embodiments, the patient experienced no serious adverse events after 12 weeks of treatment. In some embodiments, the patient experienced no adverse events after 12 weeks of treatment.

[0093] In some embodiments, changes in serum concentrations of the fusion polypeptide may be measured up to 30 weeks after initiation of treatment. For example, changes in serum concentrations of the fusion polypeptide may be measured up to 12 weeks after initiation of treatment. In some embodiments, changes in blood concentrations of anti-drug antibodies may be measured up to 30 weeks after initiation of treatment. For example, changes in blood concentrations of anti-drug antibodies may be measured up to 12 weeks after initiation of treatment. In some embodiments, changes in serum concentrations of total and free properdin relative to baseline may be measured up to 30 weeks after initiation of treatment. For example, changes in serum concentrations of total and free properdin relative to baseline may be measured up to 30 weeks after initiation of treatment.

[0094] In some embodiments, the patient experiences a change in serum concentration of complement component Ba (Ba), complement component C3a (C3a), or soluble complement component C5B-9 (sC5B9) relative to baseline after 12 weeks of treatment. In another embodiment, the patient may experience a change in blood or serum concentration of hemoglobin, nitric oxide, an inflammatory marker, or a cell adhesion marker relative to baseline after 12 weeks of treatment. The inflammatory marker may be interleukin-1. The cell adhesion marker may be soluble P-selectin.

[0095] In some embodiments, after 12 weeks of treatment, the patient may experience a change in hemoglobin level relative to baseline after 12 weeks of treatment. In another embodiment, the patient may experience a change in serum LDH level, indirect bilirubin, haptoglobin, or hemoglobin binding protein relative to baseline after 12 weeks. In another embodiment, the patient may experience a change in reticulocyte level relative to baseline after 12 weeks.

[0096] In some embodiments, the patient may experience a reduced rate of vaso-occlusive crises after 12 weeks of treatment compared to baseline. In some embodiments, the patient experiences an increased time to first vaso-occlusive crisis after 12 weeks of treatment compared to baseline. Vaso-occlusive crises may occur when sickled red blood cells block blood flow to the point where tissues become deprived of oxygen, which can lead to an inflammatory response as the body tries to correct the problem, and can cause symptoms including pain, which can affect any part of the body but most commonly occurs in the back, chest, or extremities.

[0097] Kits and Unit Dosage Forms

[0098] Also provided herein is a kit comprising a pharmaceutical composition comprising a therapeutically effective amount of a fusion polypeptide described herein (such as a fusion polypeptide having an amino acid sequence of SEQ ID NO: 1) suitable for use in the aforementioned methods and a pharmaceutically acceptable carrier. The kit may also optionally include instructions, such as instructions including an administration schedule, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein, thereby administering the composition to a patient suffering from MG. The kit may also include a syringe.

[0099] The kit may optionally include multiple packages of a single-dose pharmaceutical composition, each package containing an effective amount of a fusion polypeptide for a single administration according to the method provided above. The instruments or equipment required for administering (multiple) pharmaceutical compositions may also be included in the kit. The kit may provide one or more pre-filled syringes containing a certain amount of fusion polypeptide. The kit may include one or more automatic injectors containing a certain amount of fusion polypeptide.

[0100] The following examples are exemplary only and should not be interpreted as limiting the scope of this disclosure in any way, because after reading this disclosure, many variations and equivalents will be apparent to those skilled in the art. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are clearly incorporated herein by reference.

[0101] Examples

[0102] The following examples illustrate specific embodiments of the present invention and various uses thereof. They are set forth for illustrative purposes only, and they should not be construed as limiting the scope of the present invention in any way.

[0103] Example 1: A Phase 2a, Randomized, Open-Label Study to Evaluate a Multiple-Dosing Regimen of a Subcutaneous Anti-Properdin / Anti-Serum Albumin Bispecific Single Variable Domain Antibody in Adult Patients with Sickle Cell Disease

[0104] 1.1 Research Principles:

[0105] The fusion polypeptides described herein (anti-properdin / anti-serum albumin bispecific single variable domain on heavy chain [VHH] antibodies) are novel properdin blockers being developed for the treatment of diseases involving dysregulated complement activity. The fusion polypeptide molecules described herein are bispecific and comprise a VHH antibody domain that binds and blocks properdin, which is linked to a VHH domain that binds serum albumin via a linker, thereby conferring an extended circulating half-life on the molecule. The fusion polypeptide formulations described herein are designed for subcutaneous (SC) administration. The purpose of this study is to evaluate the safety, tolerability, efficacy, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of multiple doses and dosing regimens of the fusion polypeptides described herein SC in patients with sickle cell disease (SCD).

[0106] The study will include up to 3 cohorts. Data from this study are expected to aid in the design of future studies in patients with SCD and other complement-mediated diseases.

[0107] Goals and End Points

[0108] Table 1: Mapping of goals to endpoints for patients with sickle cell disease

[0109]

[0110] Table 1: Mapping of goals to endpoints for patients with sickle cell disease

[0111]

[0112] Abbreviations: ADA = antidrug antibodies; AP = alternative pathway; CAP = alternative pathway of complement; PD = pharmacodynamics; PK = pharmacokinetics; SAE = serious adverse event; SC = subcutaneous; SCD = sickle cell disease; TEAE = treatment-emergent adverse event; VOC = vaso-occlusive crisis.

[0113] 1.2 Overall Design

[0114] This is a study in SCD (HbSS and HbSβ 0 This is a Phase 2a study with up to 3 multiple dose cohorts of the fusion polypeptide described herein SC in adult patients with β-thalassemia (β-thalassemia) with open label.

[0115] The study is conducted in up to 30 adult patients with SCD enrolled in up to 3 open-label cohorts (Cohorts 1, 2, and 3 [optional]) to receive multiple SC doses of an open-label fusion polypeptide described herein. The fusion polypeptides described herein are administered as described in Table 2.

[0116] Table 2: Fusion peptide dosing cohorts

[0117]

[0118] Abbreviations: N = number of participants; QW = once a week; Q2W = every 2 weeks; Q4W = every 4 weeks; SC = subcutaneous; SCD = sickle cell disease.

[0119] The dose and dosing interval for Cohorts 1 and 2 were determined using cumulative safety data, interim PK / PD analysis of participants enrolled in the studies described herein, and data from a 6-month Good Laboratory Practice (GLP) toxicology study in monkeys. After evaluating the safety and PK / PD data from Cohorts 1 and 2, an optional Cohort 3 was initiated at the discretion of the personnel evaluating the study.

[0120] Cohort 1 and cohort 2 were run in parallel, and patients were randomly assigned to either cohort at 1:1 when eligibility was determined. The decision to start the optional cohort 3 was based on the discretion of the person conducting the study and was based on the analysis of PK / PD and safety after at least 8 patients (4 in each cohort) were enrolled from cohorts 1 and 2. In addition, the enrollment of the optional cohort 3 began after cohorts 1 and 2 were fully enrolled. Each cohort was stratified to ensure that SCD patients treated with a stable dose of hydroxyurea and SCD patients who were not currently treated with hydroxyurea were included. The treatment period for cohorts 1 and 2 was 12 weeks, and for cohort 3 was 6 weeks. For patients who were previously treated with hydroxyurea but are not currently using hydroxyurea, treatment must have been stopped for at least 30 days before providing informed consent.

[0121] At the investigator's discretion and in consultation with the Data Monitoring Committee (DMC), additional SCD participants could be enrolled as replacement participants if a participant discontinued during the dosing period for reasons other than drug-related adverse events (AEs).

[0122] Disclosure Statement

[0123] This was an open-label, parallel-group intervention study with up to 3 treatment arms.

[0124] Number of participants:

[0125] Up to 30 patients with SCD (HbSS and HbSβ 0 Adult patients with SCD-thalassemia (SCD-thalassemia) were enrolled in up to 3 cohorts and received multiple open-label SC doses of the fusion polypeptides described herein. The cohorts included SCD patients who were treated with a stable dose of hydroxyurea and SCD patients who were not currently treated with hydroxyurea. For patients who were previously treated with hydroxyurea but are not currently using it, treatment must be stopped for at least 30 days before providing informed consent.

[0126] Intervention Groups and Duration:

[0127] For Cohorts 1 and 2, the planned study duration is approximately 38 weeks: a screening period of up to 56 days (8 weeks), a treatment period of 84 days (12 weeks), and a follow-up period of 126 days (18 weeks). Patients will attend outpatient visits during treatment and follow-up and may choose to remain in an inpatient facility. The expected end of study (EOS) for each individual patient is Day 211 (Day 210) or, if later than Day 211, the time point at which complement activity has returned to the normal range or to 80% of baseline.

[0128] For the optional Cohort 3, the planned study duration is approximately 32 weeks: a screening period of up to 56 days (8 weeks), a treatment period of 42 days (6 weeks), and a follow-up period of 126 days (18 weeks). Patients will attend outpatient visits during treatment and follow-up and may choose to remain in an inpatient facility. The EOS for each individual patient is expected to be Day 169 (Day 168) or, if later than Day 169, the time point at which complement activity has returned to the normal range or to 80% of baseline. A schematic diagram of the study is presented in Figure 1 middle.

[0129] Data Monitoring Committee: An independent DMC was used to monitor safety and conduct planned interim analyses of the study.

[0130] Statistical analysis - all cohorts:

[0131] Populations used for analysis For the purpose of analysis, the following populations were defined:

[0132]

[0133] Abbreviations: ADA = anti-drug antibodies; CAP = alternative complement pathway; CCP = classical complement pathway; ICF = informed consent; PK = pharmacokinetics.

[0134] Security Analysis

[0135] All safety analyses were performed on the safety population and reported per cohort and treatment group.

[0136] Safety analyses included analysis of all treatment-emergent adverse events (TEAEs), electrocardiograms (ECGs), clinical laboratory data, physical examinations, and vital sign measurements using descriptive statistics. No inferential statistical analysis was planned for the safety parameters of this study. The prevalence of AEs and serious adverse events (SAEs) was summarized by relationship to the study drug for each cohort and treatment group and overall within each treatment group by system organ classification (SOC) and preferences. AEs were summarized by severity by cohort and overall. SAEs and AEs that led to withdrawal from the study were listed. Participants with multiple AEs within a category (e.g., overall, SOC, preferences) were counted once in that category. For severity tables, the most severe events for participants within a category were counted.

[0137] All concomitant medications were coded using the World Health Organization Drug Dictionary, and the frequency and percentage of concomitant medications were summarized.

[0138] Efficacy analysis

[0139] The absolute and percentage changes from baseline in complement biomarkers, hemoglobin, and hemolysis markers were evaluated at the end of the treatment period (12 weeks) for both Cohort 1 and Cohort 2. The duration of hemoglobin response (defined as an increase in hemoglobin level >1 g / dL from baseline) was evaluated at the end of the treatment period (12 weeks) for both Cohort 1 and Cohort 2.

[0140] Pharmacokinetic analysis

[0141] PK was characterized by a population PK analysis approach using individual serum concentration data from participants who received a SC dose of a fusion polypeptide described herein at actual sampling dates and times.

[0142] Pharmacodynamic analysis

[0143] The PD effects of all SC doses of the fusion polypeptides described herein administered were evaluated by assessing changes in serum total and free properdin concentrations and complement alternative pathway (CAP) activity using the Wieslab alternative pathway (AP) assay. In addition, other measures of complement classical pathway activity and properdin activity over time may be considered appropriate (Section 8.6).

[0144] Immunogenicity analysis

[0145] Immunogenicity, as measured by the incidence of anti-drug antibodies (ADA) directed against the fusion polypeptides described herein, is summarized.

[0146] Exploratory analysis

[0147] Additional exploratory analyses of biomarker assays and clinical efficacy endpoints may be performed. Details of these analyses are presented in the Statistical Analysis Plan (SAP).

[0148] Interim Analysis

[0149] An interim analysis was performed after at least 12 patients (6 from each cohort) from Cohorts 1 and 2 had been enrolled and completed the treatment period (12 weeks). Details of this analysis are presented in the SAP.

[0150] 1.3 Schedule of Activities (SoA)

[0151] The SoAs for once-weekly (QW) dosing for multiple-dose Cohort 1 from screening to Day 85 are presented in Table 3.

[0152] The SoAs for once-every-four-week (Q4W) dosing for multiple-dose Cohort 2 from screening to Day 85 are presented in Table 4.

[0153] Table 5 presents the SoA for intensive PK / PD sampling for a subset of participants in Cohorts 1 and 2. Table 6 presents the SoA for Cohorts 1 and 2 from Day 99 to the end of the follow-up period.

[0154] The SoAs for the optional multiple-dose Cohort 3 once-daily (Q2W) dosing for screening through Day 43 are presented in Table 7. Table 8 presents the SoAs for Cohort 3 from Day 57 to the end of the follow-up period.

[0155]

[0156]

[0157]

[0158]

[0159] f Obtain non-fasting blood samples.

[0160] g All female participants of childbearing potential (i.e., who have reached menarche) require a serum pregnancy test to confirm that the female participant is not pregnant prior to the first dose. A urine pregnancy test may be performed if the Screening Visit is within 3 weeks of the Day 1 Visit. A urine (or serum, if required by local site policy) pregnancy test is performed at all other required time points.

[0161] h At Screening, supine and standing (upright) blood pressure measurements are taken.

[0162] iA pre-dose 12-lead ECG in triplicate was performed on Day 1 only and 3 times approximately 2, 1, and 0.5 hours before the start of dosing (if this schedule is not feasible, ECGs performed at least 20 minutes apart are acceptable). If the planned dosing is delayed, a pre-dose ECG performed within 4 hours of dosing does not need to be repeated. A post-dose ECG was performed within 30 minutes before PK / PD sample collection.

[0163] j PK and PD samples were collected before and 2 to 4 hours after dosing.

[0164] k PD samples were collected to assess serum total and free properdin and CAP activity.

[0165] l Evaluations for injection site reactions were performed on dosing days and, if a reaction occurred, continued until the reaction had completely resolved.

[0166] m Blood samples for biomarkers were collected twice before the first dose (once during screening and then again on day 1 before dosing, with at least 2 weeks between collections). Blood (for serum, citrate and K2-EDTA, or P100 plasma) and urine were collected.

[0167] n In any case of suspected hypersensitivity or anaphylaxis, additional samples for evaluation of ADA may be collected at or near the time of the event.

[0168] o If VOCs occur, samples are collected as unscheduled visits.

[0169] p Patients recorded VOC symptoms in a paper diary throughout the study. The investigator or a qualified designee reviewed the patient diary at each visit.

[0170] q The Investigator or qualified designee met with participants at each visit to ensure that they were carrying their Clinical Research Participant Safety Card at all times and to review the potential meningococcal infection risk of the fusion polypeptides described herein and to address any safety concerns of the participant.

[0171]

[0172]

[0173]

[0174] bEligibility was assessed at Screening and Day 1.

[0175] c Randomization was performed after all eligibility requirements were confirmed.

[0176] d To reduce the risk of meningococcal infection (Neisseria meningitidis), all patients should be vaccinated with MCV4 and serogroup B meningococcal vaccine at least 14 days before the first dose if they have not been vaccinated within 3 years before the first dose (or according to national / local guidelines). Participants who initiate study intervention less than 14 days after receiving a meningococcal vaccine must receive appropriate prophylactic antibiotic therapy until at least 2 weeks after vaccination. Every effort should be made to start the meningococcal vaccination series at least 14 days before randomization.

[0177] e Height and BMI at screening only.

[0178] f Obtain non-fasting blood samples.

[0179] g All female participants of childbearing potential (i.e., who have reached menarche) require a serum pregnancy test to confirm that the female participant is not pregnant prior to the first dose. A urine pregnancy test may be performed if the Screening Visit is within 3 weeks of the Day 1 Visit. Perform a urine (or serum, if required by site policy) local pregnancy test at all other required time points.

[0180] h At Screening, supine and standing (upright) blood pressure measurements are taken.

[0181] i A pre-dose 12-lead ECG in triplicate was performed on Day 1 only and 3 times approximately 2, 1, and 0.5 hours before the start of dosing (if this schedule is not feasible, ECGs performed at least 20 minutes apart are acceptable). If the planned dosing is delayed, a pre-dose ECG performed within 4 hours of dosing does not need to be repeated. A post-dose ECG was performed within 30 minutes before PK / PD sample collection.

[0182] j PK and PD samples were collected before and 2 to 4 hours after dosing.

[0183] k PD samples were collected to assess serum total and free properdin and CAP activity.

[0184] l Evaluations for injection site reactions were performed on dosing days and, if a reaction occurred, continued until the reaction had completely resolved.

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] g All female participants of childbearing potential (i.e., who have reached menarche) require a serum pregnancy test to confirm that the female participant is not pregnant prior to the first dose. A urine pregnancy test may be performed if the Screening Visit is within 3 weeks of the Day 1 Visit. Perform a urine (or serum, if required by site policy) local pregnancy test at all other required time points.

[0193] h At Screening, supine and standing (upright) blood pressure measurements are taken.

[0194] i A pre-dose 12-lead ECG in triplicate was performed on Day 1 only and 3 times approximately 2, 1, and 0.5 hours before the start of dosing (if this schedule is not feasible, ECGs performed at least 20 minutes apart are acceptable). If the planned dosing is delayed, a pre-dose ECG performed within 4 hours of dosing does not need to be repeated. A post-dose ECG was performed within 30 minutes before PK / PD sample collection.

[0195] j PK and PD samples were collected before and 2 to 4 hours after dosing.

[0196] k PD samples were collected to assess serum total and free properdin and CAP activity.

[0197] l Evaluations for injection site reactions were performed on dosing days and, if a reaction occurred, continued until the reaction had completely resolved.

[0198] m Blood samples for biomarkers were collected twice before the first dose (once during screening and then again on day 1 before dosing, with at least 2 weeks between collections). Blood (for serum, citrate and K2-EDTA, or P100 plasma) and urine were collected.

[0199] n In any case of suspected hypersensitivity or anaphylaxis, additional samples for evaluation of ADA may be collected at or near the time of the event.

[0200] o If a VOC crisis occurred, samples were collected during an unscheduled visit.

[0201] p Patients recorded VOC symptoms in a paper diary throughout the study. The investigator or a qualified designee reviewed the patient diary at each visit.

[0202] q The Investigator or qualified designee met with participants at each visit to ensure that they were carrying their Clinical Research Participant Safety Card at all times and to review the potential meningococcal infection risk of the fusion polypeptides described herein and to address any safety concerns of the participant.

[0203] r After signing the ICF, adverse events and serious adverse events will be collected.

[0204]

[0205]

[0206] f Evaluation of VOCs must be performed by a healthcare provider.

[0207] g Patients recorded VOC symptoms in a paper diary throughout the study. The investigator or a qualified designee reviewed the patient diary at each visit.

[0208] h The Investigator or qualified designee met with participants at each visit to ensure that they carried their clinical trial participant safety card at all times and to review the potential meningococcal infection risk of the fusion polypeptides described herein and to address any safety concerns on the part of the participant.

[0209] i After signing the ICF, adverse events and serious adverse events will be collected.

[0210] Abbreviations: ADA = antidrug antibodies; CAP: alternative pathway of complement; ECG = electrocardiogram; ET = premature termination; ICF = informed consent; K2-EDTA = potassium ethylenediaminetetraacetate; PD = pharmacodynamics; PK = pharmacokinetics; VOC = vaso-occlusive crisis.

[0211] 2. Introduction

[0212] 2.1 Research Principle

[0213] The fusion polypeptide described herein (anti-properdin / anti-serum albumin bispecific VHH antibody) is a novel properdin blocker being developed for the treatment of diseases involving dysregulated CAP activity. The fusion polypeptide molecule described herein is bispecific and comprises a VHH antibody domain that binds and blocks properdin, which is connected to a VHH domain that binds serum albumin via a linker, thereby conferring a prolonged circulating half-life to the molecule.

[0214] The fusion polypeptides described herein bind to properdin with high affinity to prevent stabilization of the CAP complement component C3 (C3) and complement component C5 (C5) convertases, which cleave C3 and C5 into their activated products. Based on experience with properdin binding antibodies, quantitative blocking of properdin has been shown to be safe in humans (see Section 4.3). The fusion polypeptides described herein are currently being evaluated in an ongoing Phase 1 study in healthy adult participants, and the cumulative safety data and interim PK / PD analysis from participants enrolled in the ongoing Phase 1 study and data from 6-month GLP toxicology in monkeys are used to determine the dose and dosing regimen for participants in the study.

[0215] The purpose of this study in SCD patients is to evaluate the safety, tolerability, efficacy, PK, PD, and immunogenicity of multiple doses and dosing schedules of subcutaneous administration of the fusion polypeptides described herein.

[0216] The study includes up to 3 cohorts using multiple SC doses and dosing schedules of the open-label fusion polypeptides described herein in adult patients with SCD. Data from this study are expected to aid in the design of future studies for patients with SCD and other complement-mediated diseases.

[0217] 2.2 Background

[0218] A detailed description of the available chemical, pharmacological and toxicological data for the fusion polypeptides described herein is provided in the Investigator's Brochure (IB).

[0219] 2.2.1 Chemistry

[0220] The fusion polypeptide described herein is a recombinant humanized VHH bispecific antibody that binds to human properdin and serum albumin. The antibody consists of a single polypeptide chain of 256 amino acids, which consists of an anti-albumin domain fused to an anti-properdin domain at the N-terminus via a 15 amino acid linker to the C-terminus. The variable region domains that form the serum albumin and properdin binding sites consist of llama complementarity determining regions transplanted into a human germline framework. Within the framework region, llama residues at 11 positions remain unchanged to maintain antigen binding, water solubility and overall stability. There are 2 intrachain disulfide bonds, with 1 disulfide bond located in each VHH domain. The theoretical average molecular weight of the antibody is 27,350.2 Da. At pH 7.4, the fusion polypeptide described herein exhibits a binding dissociation constant (K) of 323 pM for human properdin. D ) and a K of 439 pM for human serum albumin D IC of the fusion polypeptide described herein (20% v / v final serum) on hemolysis of human CAP 50 The fusion polypeptide described herein blocks the deposition of C3 fragments, properdin and complement component C9 (C9) onto the myeloperoxidase substrate by human serum (20% v / v), IC 50 The values ​​were approximately 20 nM (C3), 15 nM (properdin), and 19 nM (C9).

[0221] 2.2.2 Nonclinical pharmacology

[0222] In studies of the efficacy of the fusion polypeptides described herein in blocking in vitro CAP hemolysis using sera from a range of species, significant species cross-reactivity was observed only when sera from cynomolgus monkeys and rhesus monkeys were used. CAP hemolytic activity in sera from mice, rats, guinea pigs, minipigs, beagles, and rabbits was not inhibited by the highest concentrations of the fusion polypeptides described herein (>100 μg / mL) tested. IC values ​​for CAP hemolysis blocking by the fusion polypeptides described herein (20% v / v final serum) were 0.1% and 0.2% respectively. 50 The fusion polypeptide described herein exhibits a binding K of 2.9 nM for cynomolgus monkey properdin. D and a K of 2.1 nM for cynomolgus monkey serum albumin D The fusion polypeptides described herein blocked the deposition of C3 fragments, properdin and C9 onto myeloperoxidase substrates by cynomolgus monkey serum (20% v / v). 50The values ​​are about 11 nM (C3), 9 nM (properdin) and 17 nM (C9). In addition to the above-mentioned primates, the lack of species cross-reactivity has prevented the testing of the biological activity of the fusion polypeptides described herein in traditional rodent models of diseases involving dysregulated complement activity. Therefore, in summary, non-clinical in vitro and in vivo studies are being conducted in cynomolgus monkeys to evaluate the pharmacological, PK, PD and toxicological properties of the fusion polypeptides described herein.

[0223] 2.2.3 Toxicology

[0224] The non-clinical safety profile of the fusion polypeptides described herein has been evaluated in in vitro GLP tissue cross-reactivity (TCR) studies and in non-GLP and GLP in vivo studies in cynomolgus monkeys. In GLP toxicology studies, the fusion polypeptides described herein were administered by intravenous (IV) administration (single dose of up to 100 mg / kg) and SC administration (26-week dose of up to 300 mg / kg / week). The fusion polypeptides described herein did not show any nonspecific binding to human tissues in TCR studies. Based on the absence of any adverse systemic or local toxicity in cynomolgus monkeys, 300 mg / kg / week was considered to be the no-observed-adverse-effect-level (NOAEL) for SC administration of the fusion polypeptides described herein, while 100 mg / kg was considered to be the NOAEL for IV administration. After the last dose at the NOAEL in the SC group, systemic exposure (maximum observed serum concentration [C max ] and the area under the concentration-time curve [AUC 0-168 ] 8,570 μg / mL and 1,160,000 μg·hr / mL, respectively) produced exposures that were approximately 30 times the expected exposure at the expected human dose of 300 mg QW. When observed in a very small number of monkeys, anti-drug antibodies did not have any effect on systemic exposure or toxicity profiles.

[0225] 2.3 Benefit / risk assessment

[0226] There may be potential benefits for patients with SCD (see section 2.3.2).

[0227] Identified and potential risks are described below. More detailed information about the known and anticipated benefits and risks of the fusion polypeptides described herein and the reasonably anticipated AEs can be found in the IB. Potential risk mitigation strategies are described in Table 9.

[0228] 2.3.1 Risk Assessment

[0229] In addition to the first human (FIH) Phase 1 study in healthy participants, this study is the second exposure of humans to the fusion polypeptides described herein and the first exposure to SCD patients. Due to limited clinical experience to date, potential risks are based on molecular classes and their mechanisms of action. Repeated dose toxicity studies were conducted in cynomolgus monkeys with the fusion polypeptides described herein after 26 weeks of SC dosing of up to 300 mg / kg / week or after a single IV dose of up to 100 mg / kg, and no potential risks were identified, and no safety issues were identified in healthy participants as of March 11, 2022 after 5 weeks of SC dosing of up to 150 mg or after a single SC dose of 1200 mg.

[0230] 2.3.1.1 Neisseria meningitidis infection

[0231] Increased susceptibility to Neisseria meningitidis infection is a known risk associated with properdin deficiency and has been well described in properdin-deficient patients (Figueroa, 1991). Similar to properdin deficiency, the main risk associated with the use of the fusion polypeptides described herein (properdin inhibitors) is expected to be the risk of meningococcal infection. Specific risk mitigation measures are in place to address this risk.

[0232] Clinically, the risk of N. meningitidis in properdin-deficient patients has been mitigated by vaccinating all patients with quadrivalent meningococcal conjugate vaccine (MCV4) and serogroup B vaccine against N. meningitidis prior to administration.

[0233] Patients should be vaccinated with MCV4 and serogroup B meningococcal vaccines at least 14 days prior to the first dose if they have not been vaccinated within 3 years prior to the first dose (or as per national / local guidelines). Participants who initiate the study intervention less than 14 days after receiving a meningococcal vaccine must receive appropriate prophylactic antibiotic therapy for at least 2 weeks after vaccination. Every effort should be made to start the meningococcal vaccination series at least 14 days prior to randomization.

[0234] In addition, participants could be treated with prophylactic antibiotics at the investigator's discretion.

[0235] 2.3.1.2 Immunogenicity and hypersensitivity reactions

[0236] The fusion polypeptides described herein have the potential for immunogenicity and may be associated with hypersensitivity reactions. It is also known that some healthy participants have pre-existing antibodies to VHH antibodies. Antibodies to the fusion polypeptides described herein have been observed in 14 of 100 healthy participant serum samples tested in an in vitro screening assay.

[0237] The immunogenicity of the fusion polypeptides described herein was monitored for use in this study as specified in the SoA (Section 1.3).

[0238] Table 9: Potential risks and mitigation strategies

[0239]

[0240]

[0241] 2.3.1.3 Coronavirus disease 2019

[0242] At the time of this protocol revision, the SARS-COV-2 disease (coronavirus disease 2019 [COVID-19]) global pandemic is active in many countries. Given this unique situation, specific consideration has been given to the risks and benefits of the research as they may be related to COVID-19 and the global and local changes that exist as a result of this pandemic.

[0243] 2.3.2. Benefit assessment

[0244] The potential benefit of the fusion polypeptides described herein for the treatment of SCD is measured by assessing anemia and hemolysis. An increase in hemoglobin of ≥1 g / dL relative to baseline is considered clinically significant. In addition, a reduction in hemolysis will also show a positive treatment effect, including improvements in serum levels of hemolytic markers (i.e., lactate dehydrogenase [LDH], indirect bilirubin, and haptoglobin). Other exploratory endpoints (vaso-occlusive crisis [VOC], etc.) may be assessed.

[0245] 2.3.2 Overall Benefit: Risk Conclusion

[0246] The fusion polypeptides described herein have been and are being evaluated in an ongoing Phase 1 study. The study described herein is the second exposure of humans to the fusion polypeptides described herein.

[0247] This study was conducted in patients with SCD, and dosing was initiated based on a review of safety, tolerability, and PK / PD data from Phase 1 studies. The doses administered in this study are expected to produce exposures below the highest exposures tested in previous studies, and the expected exposures for patients are below the NOAEL exposures established in 6-week and 6-month GLP monkey toxicology studies. Strict inclusion / exclusion criteria and a robust safety monitoring and risk mitigation plan were established. The DMC evaluated the available study data on the safety of participants at pre-specified time points and made recommendations on dose modifications or termination of the study. The doses selected are intended to deliver complete inhibition of properdin, thereby providing SCD patients with the potential benefits of the fusion polypeptides described herein, with a positive benefit / risk ratio.

[0248] The data obtained from this study are expected to provide a basis for future clinical research in SCD patients.

[0249] 3. Goals and End Points

[0250] The study objectives and corresponding endpoints are presented in Table 10.

[0251] Table 10: Mapping of goals to endpoints for patients with sickle cell disease

[0252]

[0253]

[0254] Abbreviations: ADA = antidrug antibodies; AP = alternative pathway; CAP = alternative pathway of complement; PD = pharmacodynamics; PK = pharmacokinetics; SAE = serious adverse event; SC = subcutaneous; SCD = sickle cell disease; TEAE = treatment-emergent adverse event; VOC = vaso-occlusive crisis.

[0255] 4. Study Design

[0256] 4.1 Overall Design

[0257] This is a study in SCD (HbSS and HbSβ 0 A Phase 2a study of up to 3 multiple-dose cohorts of a fusion polypeptide described herein with open label SC in adult patients with β-thalassemia.

[0258] The study is conducted in up to 30 adult patients with SCD enrolled in up to 3 open-label cohorts (Cohorts 1, 2, and 3 [optional]) to receive multiple SC doses of an open-label fusion polypeptide described herein. The fusion polypeptides described herein are administered as described in Table 11.

[0259] Table 11: Fusion peptide dosing cohort

[0260]

[0261]

[0262] Abbreviations: N = number of participants; QW = once a week; Q2W = once every 2 weeks; Q4W = once every 4 weeks; SC = subcutaneous.

[0263] The dose and dosing interval for Cohorts 1 and 2 were determined using cumulative safety data, interim PK / PD analysis from participants enrolled in the Phase 1 study, and data from a 6-month GLP toxicology study in monkeys. An optional Cohort 3 was initiated after evaluation of safety and PK / PD data from Cohorts 1 and 2.

[0264] Cohorts 1 and 2 were run in parallel, and patients were randomly assigned to either cohort at 1:1 when eligibility was determined. The decision to start the optional cohort 3 was made at the investigator's discretion and was based on the analysis of PK / PD and safety after at least 8 patients (4 in each cohort) were enrolled from cohorts 1 and 2. In addition, enrollment in the optional cohort 3 began after cohorts 1 and 2 were fully enrolled. Each cohort was stratified to ensure that patients with SCD who were treated with a stable dose of hydroxyurea and those who were not currently treated with hydroxyurea were included. For patients who were previously treated with hydroxyurea but were not currently using hydroxyurea, treatment must be stopped for at least 30 days before providing informed consent.

[0265] An independent DMC was used to monitor safety and conduct planned interim analyses of the study. At the investigator's discretion and in consultation with the DMC, additional SCD participants could be enrolled as replacement participants if a participant discontinued during dosing for reasons other than drug-related AEs.

[0266] 4.2 Scientific Principles of Research Design

[0267] The initial indication of the fusion polypeptide described herein is SCD. SCD affects approximately 20 to 25 million people worldwide (Aliyu, 2008) and in the US, approximately 100,000 people are affected (Hassell, 2010). The prevalence of SCD newborns and SCD carriers in the EU is approximately 1 to 5 in 10,000 and 1 in 150, respectively (Engert, 2016). In the US or EU, the few available life expectancy estimates for SCD patients vary widely between 45 and 65 years, which is about 20 years shorter than the general population (Gardner, 2016; Lubeck, 2019; Payne, 2020; Platt, 1994).

[0268] SCD is a group of inherited disorders. Mutations in the beta-hemoglobin gene are responsible for the synthesis of sickle hemoglobin (HbS). The most common genotypes in SCD are HbSS, HbSC, and HbSβ + Thalassemia. The most common clinical manifestations of SCD are chronic hemolysis and VOC (Kato, 2018; Pecker, 2021).

[0269] HbS has abnormal physicochemical properties and is prone to polymerization at low oxygen concentrations, resulting in characteristic sickle-shaped red blood cells (RBC) deformation. Sickling has many adverse consequences for RBC and multiple organs. Sickle red blood cells have a limited lifespan due to hemolysis. Hemolysis is believed to occur mainly through extravascular phagocytosis (about 2 / 3) and intravascular (about 1 / 3) hemolysis, which leads to anemia. Intravascular hemolysis of sickle red blood cells leads to the release of free hemoglobin, which in turn activates CAP (through free heme) and depletes nitric oxide, leading to endothelial damage. Hemolysis and sickling of RBC lead to endothelial cell activation, accompanied by increased expression of adhesion molecules and activation of neutrophils, monocytes and platelets (Kato, 2018). VOC, as a result of these processes, leads to blood flow obstruction to important organs such as kidneys, liver, lungs and heart, thereby promoting ischemia, acute pain attacks and necrosis. Subsequent ischemia / reperfusion injury leads to the generation of reactive oxygen species. This in turn leads to a chronic inflammatory state (Kato, 2018; Piel, 2017). Due to functional or actual asphyxia, patients have increased vulnerability to infection, particularly from encapsulated bacteria. Together, these mechanisms contribute to the development of chronic organ damage, including sickle nephropathy, pulmonary hypertension, avascular necrosis of bone, chronic lung disease, and reduced life expectancy (Kato, 2018).

[0270] Universal neonatal screening was established in the US to enable early diagnosis and treatment of SCD infants, thereby reducing morbidity and mortality. 0 -Infants with thalassemia receive penicillin prophylaxis and 23-valent pneumococcal polysaccharide vaccine to prevent invasive pneumococcal disease (Kato, 2018; Pecker, 2021).

[0271] Hydroxyurea, RBC transfusions, and opioids are commonly used to manage symptoms of SCD. Several new drugs have been approved in recent years to treat complications of sickle cell disease: L-glutamine Voxelotor and crizanlizumab However, none of these treatments address both anemia (hemolysis) and VOC. The only curative treatment option for SCD is hematopoietic stem cell transplantation. However, this is reserved for severely ill patients due to the risk of life-threatening complications (Pecker, 2021).

[0272] Recently, the role of the innate immune system in SCD has received much attention, especially the role of complement activation in the pathophysiology of SCD (Tampaki, 2021; Varelas, 2021). In investigator-initiated studies, eculizumab has demonstrated clinical efficacy in SCD patients with delayed hemolytic transfusion reactions, VOCs, and drug-induced immune hemolytic anemia (Chonat, 2020). CAP inhibitors have potential advantages over C5 inhibitors in the treatment of SCD. An increasing number of publications support the hypothesis that sickled RBCs are the focus of CAP activation, which triggers C3 opsonization on the cell surface and complement-mediated RBC hemolysis. Intravascular hemolysis is one of the main causes of anemia and also causes further amplification of CAP activation by releasing free heme from RBCs. C3 opsonization of sickled RBCs also contributes to anemia via extravascular hemolysis through the reticuloendothelial system. In addition, C3 opsonization has been shown to be a key contributor to VOCs. It has also been demonstrated that C3 opsonization can be promoted by exposure of phosphatidylserine on sickled RBCs and lead to VOCs by enhancing its interaction with adhesion molecules such as P-selectin and complement receptor 3 on activated endothelial cells (Lombardi, 2019). Thus, the SCD nonclinical literature collectively emphasizes the role of CAP activation in the pathophysiology of SCD.

[0273] The fusion polypeptides described herein bind with high affinity to human properdin, a component of CAP, thereby preventing it from stabilizing the CAP C3 and C5 convertases that cleave C3 and C5 into their activated products. By binding properdin, the fusion polypeptides described herein prevent activation of the alternative complement system and thus have the potential to treat SCD. In support of this, studies conducted in a mouse model of SCD demonstrated that pretreatment of animals with mouse anti-properdin antibodies significantly improved signs of hemolysis and vascular occlusion, two of the main clinical features of SCD.

[0274] The current nonclinical data and data from the first-in-human study support further investigation of the potential of the fusion polypeptides described herein as a treatment for patients with SCD. This study is designed to allow preliminary evaluation of changes in SCD disease-associated biomarkers and guide the design of further clinical studies in patients with SCD.

[0275] 4.3 Rationale for Dosage

[0276] The dose and dosing frequency are based on all available data, including overall safety, tolerability, PK / PD modeling from ongoing cohorts in the Phase 1 study; available nonclinical data include PK, PD, and efficacy in the SCD mouse model, and toxicology data from the GLP 6-week and 6-month studies in cynomolgus monkeys.

[0277] A preliminary PK / PD model has been developed based on data from a Phase 1 study in healthy participants. This semi-mechanistic model assumes that the monovalent fusion polypeptides described herein bind to trimeric properdin at three binding sites. The relationship between free properdin and CAP activity is determined by a sigmoidal E max Model Characterization. The model provided a good fit to the observed data (fusion polypeptides described herein, total and free properdin, and CAP activity) as judged by the model diagnostics. To estimate the therapeutic dose in SCD patients, the following assumptions have been made:

[0278] Based on data from the mouse SCD model (described in detail in the fusion polypeptide IB described herein), complete inhibition of CAP activity (<1% of baseline activity) is required for clinical efficacy

[0279] Sickle cell patients have baseline properdin concentrations that are approximately 20% higher than healthy subjects (Strauss, 1977)

[0280] In addition, the clearance of the fusion polypeptides described herein in sickle cell patients was set to be similar to or 40% higher than that in healthy participants (seen in other antibody treatments for SCD (Lizanlizumab, 2019)) to evaluate the effect of increased clearance on exposure and CAP inhibition. Based on these assumptions and analyses, a dose of 300 mg QW is expected to inhibit CAP activity to <1% of baseline values ​​(upper limit of the 90% prediction interval) and maintain this effect during treatment. Doses of 600 mg Q4W or 300 mg Q2W also showed inhibition of CAP activity by <1% during most dosing intervals. After terminating dosing of the fusion polypeptides described herein, CAP activity slowly recovered and returned to baseline. Based on the NOAEL exposure established in a 6-month (or 26-week) monkey toxicology study, the safety margin at 300 mg QW is approximately 30-fold. Based on the NOAEL exposure, the safety margin at 600 mg Q4W or 300 mg Q2W is approximately 60-fold.

[0281] 4.4 Definition of study end

[0282] A participant is considered to have completed the study if he / she has completed all phases of the study, including the final planned procedure as indicated in the SoA (Section 1.3).

[0283] End of Study (EOS) was defined as the date when the last participant completed the last visit, as indicated in the SoA (Section 1.3).

[0284] 5. Study population

[0285] Prospective approval of protocol deviations (also known as protocol waivers or exemptions) from recruitment and inclusion criteria is not permitted.

[0286] 5.1 Inclusion criteria

[0287] age

[0288] 1. Participants must be between 18 and 65 years old (inclusive) when signing the informed consent form.

[0289] Participant Types and Disease Characteristics

[0290] 2. Confirmed diagnosis of SCD (HbSS or HbSβ 0 -thalassemia).

[0291] weight

[0292] 3. Body weight ≥40kg (inclusive) at the time of screening.

[0293] gender

[0294] 4. Male or female contraceptive measures should comply with local regulations on contraceptive methods for participation in clinical studies. Female participants of childbearing potential and male participants with female partners of childbearing potential must be willing to follow the contraceptive instructions specified in the protocol during treatment and for at least 6 months after the last dose of study drug.

[0295] Other inclusion criteria

[0296] 5. Hemoglobin between 5.5 and 10 g / dL at screening.

[0297] 6. There have been 1 to 10 VOCs in the past 12 months.

[0298] 7. Patients receiving hydroxyurea must have been taking a stable dose for ≥ 3 months before providing informed consent, and it is expected that no dose adjustment will be required during the study. For patients who have previously used hydroxyurea but are not currently receiving hydroxyurea treatment (due to non-responsiveness, intolerance, or unwillingness to take hydroxyurea), hydroxyurea treatment must be discontinued for at least 30 days before providing informed consent.

[0299] 8. Vaccinate these patients with MCV4 and serogroup B meningococcal vaccines at least 14 days prior to dosing if they have not been vaccinated within 3 years prior to the first dose (or as per national / local guidelines). Participants initiating study intervention treatment less than 14 days after receiving meningococcal vaccine must receive appropriate prophylactic antibiotic therapy until at least 2 weeks after vaccination.

[0300] 9. Vaccinations against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae are up to date according to current national / local vaccination guidelines for individuals with SCD.

[0301] 10. Must be willing to comply with all research requirements and restrictions.

[0302] Informed consent

[0303] 11. Ability to sign informed consent (or assent, as applicable), including compliance with the requirements and restrictions outlined in the ICF and this protocol.

[0304] 5.2 Exclusion criteria

[0305] Participants were excluded from the study if they met any of the following criteria:

[0306] 1. Planned initiation, discontinuation, or dose change of hydroxyurea during the study.

[0307] 2. Received OXBRYTA or ADAKVEO within 60 days of providing informed consent.

[0308] 3. Receiving treatment with recombinant human erythropoietin (e.g., epoetin alfa).

[0309] 4. Treatment with complement inhibitors within 6 months prior to the first dose.

[0310] 5. Patients who have received long-term blood transfusion or blood transfusion within 60 days of the first administration.

[0311] 6. Any significant illness or disorder that the investigator believes may put the participant at risk.

[0312] 7. History of complement deficiency.

[0313] 8. History of infection with Neisseria meningitidis, Streptococcus pneumoniae or Haemophilus influenzae.

[0314] 9. History of malignancy, except for non-melanoma skin cancer or cervical carcinoma in situ that has been treated and has no signs of recurrence within 5 years.

[0315] 10. Evidence of hepatitis B (positive hepatitis surface antigen [HBsAg] or positive core antibody (anti-HBc) with negative surface antibody [anti-HBs]) or hepatitis C virus infection (positive hepatitis C virus [HCV] antibody, except for patients with documented successful treatment and documented sustained virologic response) at screening.

[0316] 11. Active systemic bacterial, viral or fungal infection within 14 days before administration.

[0317] Previous / contemporaneous clinical research experience

[0318] 12. Participation in a clinical study within 90 days or 5 half-lives of the study drug (whichever is longer) prior to initiation of dosing on Day 1 (i.e., the last protocol-required study visit).

[0319] 13. Participation in more than 1 clinical study of a monoclonal antibody (mAb) within 6 months or 5 half-lives of the mAb (whichever is longer) prior to screening, or participation in a clinical study of a mAb (i.e., the last protocol-required study visit) during which the participant was exposed to active study drug.

[0320] Diagnostic evaluation

[0321] 14. Severe renal impairment (estimated glomerular filtration rate [eGFR] < 30 mL / min / 1.73 m 2 ) or long-term dialysis.

[0322] Other Exclusions

[0323] 15. Female participants who are pregnant or breastfeeding.

[0324] 16. History of allergy or hypersensitivity to excipients of the fusion polypeptides described herein (eg, polysorbate 80).

[0325] 5.3 Lifestyle considerations

[0326] Not applicable, the study described in this article did not include any specific lifestyle considerations.

[0327] 5.4 Screening Failure

[0328] Screen failures are defined as participants who consented to participate in a clinical study but were subsequently not assigned study drug due to failure to meet eligibility criteria. A minimum set of screen failure information is required to ensure transparency in reporting of participants with screen failures to meet Consolidated Standards of Reporting Trials publication requirements and respond to inquiries from regulatory agencies. Minimum information includes demographics, screen failure details (e.g., failure to pass eligibility criteria), and any AEs that occurred during the screening period, including any SAEs and any relevant concomitant medications.

[0329] Participants who do not meet the criteria for participation in this study for reasons that are expected to resolve or have resolved (i.e., screening failure) may be rescreened based on discussion and consensus between the Investigator and the Medical Monitor. Any abnormal laboratory parameter results that are outside the reference range at the time of screening may be repeated for the purpose of further determining eligibility at the discretion of the Investigator.

[0330] 6. Study Intervention

[0331] A study intervention is defined as any one or more study interventions, one or more marketed products, placebo, or one or more medical devices intended to be administered to study participants according to the study protocol. For the present study described herein, the study intervention is the fusion polypeptide described herein and is also referred to as the study drug throughout the present study protocol.

[0332] 6.1 Study intervention implemented

[0333] The investigational pharmaceutical compositions and doses to be administered (open label, SC) for the fusion polypeptides described herein in this study are presented in Table 12.

[0334] Table 12: Dose reference chart for the study

[0335]

[0336] Abbreviations: SC = subcutaneous.

[0337] In the event of a missed dose, unscheduled dosing may be necessary to ensure therapeutic coverage. Unscheduled dosing is determined on an individual basis by the study clinical pharmacologist.

[0338] 6.2 Preparation / Handling / Storage / Accountability

[0339] Detailed information regarding the preparation, handling, storage, accountability, and administration of study medications is discussed below. Additional guidance is provided in the pharmacy manual.

[0340] 1. The fusion polypeptides described herein will be stored at 2°C-8°C. The investigator or designee must confirm that all study drugs received maintain appropriate temperature conditions during transportation and report and resolve any discrepancies before use of the study drugs.

[0341] 2. Only participants enrolled in the study can receive study drugs, and only authorized research center staff can provide or administer study drugs. All study drugs must be stored in a safe, environmentally controlled and monitored (manual or automatic) area according to the labeled storage conditions, and only accessible to researchers and authorized research center staff.

[0342] 3. The investigator's site's pharmacy staff is responsible for study medication accountability, reconciliation, and record maintenance (i.e., receipt, reconciliation, and final disposition records). The site's pharmacy assembles the supplies into individually labeled participant-dose syringes and certifies the assembled products as qualified personnel. Further guidance on preparation, handling, storage, and accountability, as well as information on final disposition of unused study interventions, is provided in the pharmacy manual.

[0343] 6.3 Measures to reduce bias: randomization and blinding

[0344] All eligible participants in Cohorts 1, 2, and optionally Cohort 3 who meet all inclusion criteria and no exclusion criteria receive an open-label fusion polypeptide as described herein.

[0345] 6.4 Compliance with study interventions

[0346] Administration of the study intervention to participants was supervised by the Investigator or his / her designee to ensure that participants received appropriate doses at appropriate points during the study.

[0347] The date and time of each dose administered in the clinic was recorded on the source document Case Report Form (CRF).

[0348] The dose of study intervention and the identity of study participants were confirmed at the time of dosing by study site personnel, not the person administering the study drug.

[0349] Additional information regarding compliance and administration of study interventions is provided in the pharmacy brochure.

[0350] 6.5 Concomitant therapy

[0351] Any medications (including over-the-counter or prescription medications, vitamins and / or herbal supplements), vaccines, or other specific categories of interest that the participant is taking at the time of enrollment or receives during the study must be recorded along with the following:

[0352] Reasons for use

[0353] Date of application, including start and end dates

[0354] Dosage information, including dose and frequency

[0355] The Medical Monitor should be contacted if there are any questions regarding concomitant or prior therapy.

[0356] 6.5.1 Permitted drugs and therapies

[0357] During the study, multivitamins, birth control pills, and paracetamol (i.e., acetaminophen at a dose of ≤2 g / day) were allowed at the investigator's discretion.Topical skin products should not be applied to the study drug injection site from 24 hours before until 24 hours after study drug administration.

[0358] See section 8.2.8 for detailed information on the administration and duration of prophylactic antibiotics as concomitant medication to mitigate the risk of N. meningitidis.

[0359] If necessary, the investigator may consider other concomitant medications on a case-by-case basis in consultation with the Medical Monitor. Concomitant procedures are not permitted unless medically indicated.

[0360] Drugs and therapies are permitted for SCD, except those presented in Section 6.5.2.

[0361] 6.5.2 Unacceptable drugs and therapies

[0362] During the screening and treatment period, hydroxyurea should not be initiated if the patient is not currently treated with hydroxyurea. If the patient is taking a stable dose of hydroxyurea, the dose of hydroxyurea should not be changed or discontinued. Vorseloto, rizumab, erythropoietin, other complement inhibitors, and blood transfusions are not permitted. If any of the above medications / therapies are used, discontinue the patient as per section 7.1. The above medications / therapies are permitted during safety follow-up.

[0363] 6.6 Dosage Modifications

[0364] The investigator and / or DMC make decisions to continue or modify dosing after reviewing safety data. The DMC may also make recommendations regarding safety issues, study conduct, or study suspension.

[0365] 6.7 Intervention after the Study

[0366] No follow-up intervention was planned.

[0367] 7. Discontinuation of Study Intervention and Participant Discontinuation / Withdrawal

[0368] 7.1 Discontinuation of Study Intervention

[0369] In rare cases, participants may need to permanently discontinue (terminal withdrawal) the study intervention. If the study intervention is definitively discontinued, participants should remain in the study for safety follow-up assessments. See the SoA (section 1.3) for data to be collected at study intervention discontinuation and follow-up, and any further evaluations to be completed.

[0370] 7.2 Stopping criteria

[0371] 7.2.1 Individual stopping rules

[0372] Participants should be considered to have discontinued the intervention if any of the following occurred during the study:

[0373] Severe hypersensitivity reactions;

[0374] Grade 3 or higher injection site reactions;

[0375] Severe uncontrolled infection;

[0376] Severe infection with Neisseria meningitidis, Streptococcus pneumoniae, or Haemophilus influenzae;

[0377] · use of impermissible drugs as defined in Section 6.5.2;

[0378] are pregnant or planning to become pregnant; or

[0379] Any AE, laboratory abnormality, or intercurrent illness that, in the investigator's judgment, presents a clinically significant risk to the patient of continuing study drug administration.

[0380] 7.2.2 Study stopping rules

[0381] The study may be terminated based on the DMC's recommendation if the following circumstances occur and are considered to be related to the study drug:

[0382] Two or more meningococcal infections;

[0383] Two or more severe (≥Grade 3) pneumococcal infections;

[0384] Two or more severe (≥Grade 3) Haemophilus influenzae infections;

[0385] A meningococcal, pneumococcal, or Haemophilus influenzae infection with fatal outcome.

[0386] 7.3 Participant Discontinuation / Withdrawal from the Study

[0387] Every effort should be made to ensure that participants are willing to comply with study participation prior to conducting screening procedures. Study staff should notify the investigator and their site monitor of all study withdrawals as soon as possible. The reason for participant discontinuation must be documented in the source documents and the electronic case report form (eCRF).

[0388] A participant may withdraw from the study at any time at his / her own request or at the investigator's discretion for safety, compliance, or administrative reasons. This is expected to be uncommon.

[0389] In the event of study discontinuation, an early discontinuation visit should be conducted, if possible, as indicated in the SoA (Section 1.3). Refer to the SoA for data to be collected at study discontinuation and follow-up, and any further evaluations that need to be completed.

[0390] Participants will then permanently discontinue both the study drug and the study.

[0391] If a participant withdraws consent for disclosure of future information, the researcher may retain and continue to use any data collected prior to the withdrawal of consent.

[0392] If a participant withdraws from the study, he or she may request that any samples collected and not tested be destroyed, and the investigator must record this in the site study records.

[0393] Participants who discontinued during the screening or dosing period for reasons other than drug-related AEs could be replaced.

[0394] 7.4 Loss to follow-up

[0395] If a participant failed to return for scheduled visits multiple times and could not be contacted by the study center, he or she was considered lost to follow-up.

[0396] If a participant fails to return to the clinic for a required study visit, the following actions must be taken:

[0397] The site must attempt to contact participants and reschedule missed visits as quickly as possible, counsel participants on the importance of maintaining the assigned visit schedule, and determine whether the participant wishes and / or should continue in the study.

[0398] Before a participant is considered lost to follow-up, the investigator or designee must make every effort to re-contact the participant (email, 3 phone calls where possible, and, if necessary, certified mail to the participant's last known mailing address or local equivalent). These attempts at contact should be documented in the participant's medical record.

[0399] · If the participant continued to be unreachable, he / she was considered lost to follow-up.

[0400] 8. Research Evaluation and Procedures

[0401] The study procedures and their timing are summarized in the SoA (Section 1.3). No waivers or exemptions of the protocol will be permitted.

[0402] Immediate safety concerns should be discussed with the investigator promptly upon occurrence or awareness to determine whether the participant should continue or discontinue the fusion polypeptides described herein.

[0403] Adherence to study design requirements, including those specified in the SoA, is essential for the conduct of the study.

[0404] All screening assessments must be completed and reviewed to confirm that potential participants meet all eligibility criteria. The investigator maintains a screening log to record details of all screened participants and confirm eligibility or record reasons for screening failure, if applicable.

[0405] Procedures performed as part of the participant's routine clinical management (e.g., blood counts) and obtained prior to signing the ICF may be used for screening or baseline purposes, provided that the procedures meet protocol-specified criteria and, if consistent with site standard operating procedures, are performed within the timeframe defined in the SoA.

[0406] 8.1 Efficacy evaluation

[0407] The timing of assessment collections for Cohort 1 is described in detail in Section 1.3, Table 3, and Table 6. The timing of intensive collection of PK and PD samples for Cohorts 1 and 2 is described in detail in Section 1.3, Table 5. The timing of assessment collections for Cohort 2 is described in detail in Section 1.3, Table 4, and Table 6. The timing of assessment collections for the optional Cohort 3, if performed, is described in detail in Tables 7 and 8.

[0408] 8.1.1 Changes in complement biomarkers

[0409] The following complement markers were measured during the study (absolute change and percentage change from baseline):

[0410] Complement component Ba (Ba)

[0411] Complement component C3a (C3a)

[0412] Soluble complement component C5B-9 (sC5B9)

[0413] If available, other complement markers can be evaluated.

[0414] 8.1.2 Changes in VOC-related biomarkers

[0415] The following VOC markers were measured during the study period (absolute change and percentage change from baseline):

[0416] Hemoglobin

[0417] Nitric oxide

[0418] Inflammatory markers (eg, interleukin-1)

[0419] Cell adhesion markers (eg, soluble P-selectin)

[0420] If feasible, other markers can be evaluated.

[0421] 8.1.3 Changes in hemoglobin

[0422] Blood samples were collected to assess changes in hemoglobin from baseline.

[0423] 8.1.4 Hemolysis markers

[0424] The following hemolytic markers were measured during the study:

[0425] Serum LDH level

[0426] Absolute reticulocyte count

[0427] Serum indirect bilirubin

[0428] Serum haptoglobin and hemoglobin

[0429] 8.1.5 VOC Assessment

[0430] Sickle cell disease-related painful crises (VOCs) were collected throughout the study and are detailed in the SoA (Section 1.3).

[0431] After signing the informed consent, patients were issued a paper diary to record VOC symptoms. The diary was collected by the investigator or designee at each visit to the study center, and a new diary was provided.

[0432] 8.2 Safety Assessment

[0433] The planned time points for all safety assessments for all cohorts are provided in the SoA (Section 1.3).

[0434] 8.2.1 Physical Examination

[0435] A complete physical examination includes, at a minimum, an assessment of the general appearance, skin, head, ears, eyes, nose and throat, neck, lymph nodes, chest, heart, abdomen, extremities, central nervous system, and musculoskeletal system.

[0436] A brief physical examination includes, at a minimum, an assessment of the skin, lungs, cardiovascular system, and abdomen (liver and spleen).

[0437] Record height, weight and BMI according to Section 1.3 SoA.

[0438] Investigators should pay special attention to clinical signs associated with pre-existing serious illness.

[0439] 8.2.2 Vital signs

[0440] Vital sign measurements, including temperature (tympanic or oral), respiratory rate, supine BP, and pulse, were taken after the participant had rested in a supine or semi-supine position for at least 5 minutes. Ideally, the same arm should be used for BP and pulse measurements for each participant. Orthostatic (standing) BP was measured only at screening.

[0441] The timing of vital sign measurements is described in the SoA (Section 1.3).

[0442] Out-of-range blood pressure or pulse measurements were repeated at the investigator's discretion. Clinically significant vital sign measurements were recorded as AEs.

[0443] 8.2.3 Electrocardiogram

[0444] Record triplicate 12-lead ECGs at the time points described in SoA (Section 1.3) to obtain heart rate, PR, QRS, and QT intervals. Perform 12-lead ECG recordings after the participant has rested in a supine position for at least 10 min.

[0445] At each time point when a triple ECG is required, 3 separate ECG tracings should be obtained as nearly consecutively as possible but no more than 2 minutes apart.

[0446] 8.2.3.1 Safety review of 12-lead electrocardiogram

[0447] All recorded ECGs were reviewed by the investigator or qualified designee. If a participant presented with an abnormal ECG, additional safety documentation was performed and the abnormality was followed until resolved.

[0448] 8.2.4 Clinical laboratory evaluation

[0449] See Table 13 for a list of clinical laboratory tests to be performed, and the timing and frequency are given in the SoA (Section 1.3). Clinical laboratory assessments were performed by a central laboratory unless otherwise specified.

[0450] The investigator must review the laboratory reports, document the review, and record any clinically relevant changes that occur during the study in the AE section of the eCRF. Laboratory reports must be archived with the source documents. Clinically significant abnormal laboratory findings are those that are not related to an underlying medical condition unless, in the investigator's judgment, are more severe than expected for the participant.

[0451] All laboratory tests with values ​​considered clinically significant abnormal should be repeated during study participation until such values ​​return to normal or baseline or are no longer considered clinically significant by the Investigator or Medical Monitor.

[0452] If these values ​​do not return to normal / baseline within a time deemed reasonable by the investigator, the cause should be identified and the investigator notified.

[0453] ·All protocol-required laboratory evaluations must be performed in accordance with the laboratory manual and SoA.

[0454] If laboratory values ​​from non-protocol-specified laboratory assessments performed at the institution's local laboratory require a change in participant management or are considered clinically significant by the investigator (e.g., SAE or AE or dose modification), the results must be documented in the eCRF.

[0455] 8.2.5 Clinical safety laboratory evaluation

[0456] 8.2.5.1 Viral serology

[0457] Blood samples collected at screening were analyzed for HIV-1, HIV-2, HBsAg, HBc antibody (anti-HBc IgG + IgM if IgG positive), and HCV antibody titers.

[0458] 8.2.6 Injection site evaluation

[0459] Subcutaneous injection site evaluations were performed at the time points specified in the SoA (Section 1.3). Injection site reactions were recorded as AEs unless considered clinically significant.

[0460] 8.2.7 Injection-related reactions

[0461] Injection-related reactions were defined as systemic AEs occurring during or within 24 hours after the start of SC infusion (e.g., fever, chills, flushing, changes in heart rate and blood pressure, dyspnea, nausea, vomiting, diarrhea, and systemic rash) that were assessed by the investigator to be related to study drug.

[0462] 8.2.8 Vaccine and antibiotic prevention

[0463] To mitigate the risk of N. meningitidis infection associated with complement inhibition, participants were administered the following treatments:

[0464] 1. Vaccinate patients with MCV4 and serogroup B meningococcal vaccines (if available) at least 14 days before the first dose if they have not been vaccinated within 3 years before the first dose (or as per national / local guidelines).

[0465] 2. Patients were treated with prophylactic antibiotics at the investigator's discretion.

[0466] Patients must be vaccinated against other pathogens (e.g., Haemophilus influenzae, Streptococcus pneumoniae) according to current national / local guidelines.

[0467] 8.3 Adverse Events and Serious Adverse Events

[0468] All AEs were reported by the participant (or, where appropriate, by the caregiver, surrogate, or legally authorized representative of the participant) to the investigator or qualified designee.

[0469] The Investigator and any qualified designees are responsible for detecting, recording, and documenting events that meet the definition of an AE or SAE and for continuing follow-up of events that are serious, considered to be related to the study intervention or study procedures, or that lead to discontinuation of the participant from the study intervention (see Section 7).

[0470] 8.3.1 Time period and frequency of collecting AE and SAE information

[0471] All AEs and SAEs were collected from the signing of the ICF until the last follow-up visit. All SAEs were recorded and reported to the investigator or designee immediately and in no case more than 24 hours later. The investigator submitted any updated SAE data to the investigator within 24 hours of the date the site became aware of the event.

[0472] The Investigator is under no obligation to actively seek AE or SAE data after study participation ends. However, if the Investigator learns of any SAE, including death, at any time after a participant has withdrawn from the study and he / she believes that the event is reasonably related to the study intervention or study participation, the Investigator must promptly notify the Investigator.

[0473] 8.3.2 Methods for detecting AE and SAE

[0474] Care should be taken not to introduce bias when detecting AEs and / or SAEs. Open-ended and non-leading verbal questioning of participants is the preferred method for inquiring about the occurrence of AEs.

[0475] 8.3.3 Follow-up of AEs and SAEs

[0476] The investigator was required to proactively follow up with each participant at subsequent visits / contacts following the initial AE / SAE report. All SAEs were followed up until resolved, stabilized, the event was otherwise explained, or the participant was lost to follow-up (as defined in Section 7.4).

[0477] 8.3.4 SAE Regulatory Reporting Requirements

[0478] Prompt notification of SAEs to investigators is essential in order to fulfill legal obligations and ethical responsibilities for the safety of participants and the safety of the investigational intervention in clinical research.

[0479] Investigators have a legal responsibility to inform local regulatory authorities and other supervisory authorities about the safety of investigational interventions in clinical studies. Investigators comply with country-specific regulatory requirements related to reporting safety to regulatory authorities, Institutional Review Boards (IRBs) / IECs, and investigators.

[0480] Suspected unexpected serious adverse reactions must be reported according to local regulatory requirements and forwarded to the investigator when necessary.

[0481] Investigators who receive an Investigator Safety Report describing a SAE or other specific safety information (eg, a SAE summary or listing) from the Investigator review the Safety Report and then file it with the IB and notify the IRB / IEC, if appropriate according to local requirements.

[0482] 8.4 Overdose Treatment

[0483] For this study, any dose of study intervention greater than that specified in the protocol was considered an overdose. There is no specific treatment or antidote for overdose.

[0484] Overdose is a medication error that is not considered an AE unless there is an adverse medical event resulting from the overdose.

[0485] In the event of overdose or suspected overdose, the investigator should:

[0486] 1. Contact the Medical Monitor immediately.

[0487] 2. Closely monitor participants for any AEs / SAEs.

[0488] 3. If requested by the Medical Monitor (determined on a case-by-case basis), obtain samples for PK / PD analysis.

[0489] 4. Record the number of overdoses and the duration of the overdose in the eCRF.

[0490] Decisions regarding dose interruptions or modifications were made by the investigator in consultation with the medical monitor based on the participant's clinical evaluation.

[0491] 8.5 Pharmacokinetics

[0492] As specified in the SoA (Section 1.3), whole blood samples are collected for measuring serum concentrations of the fusion polypeptides described herein. Additional samples may be collected at other time points during the study if necessary and agreed with the Investigator. The total blood volume does not exceed the volume limit for the participant according to national / local guidelines. During the course of the study, the sampling time may be changed based on new available data (e.g., to obtain data closer to the time of peak serum concentration) to ensure appropriate monitoring.

[0493] Instructions for biological sample collection and processing were provided by the investigator. The actual date and time (24-hour clock) of each sample was recorded.

[0494] • Samples for evaluation of PK of fusion polypeptides described herein. Samples collected for analysis of serum concentrations of fusion polypeptides described herein may also be used to evaluate aspects of safety or efficacy related to issues that arise during or after the study.

[0495] - Samples may be used in research to develop methods, assays, prognoses and / or companion diagnostics related to dysregulated complement activity.

[0496] 8.6 Pharmacodynamics

[0497] As specified in the SoA (Section 1.3), whole blood samples are collected for measurement of serum total and free properdin concentrations, CAP activity, and potential other measures of complement activation. Additional samples may be collected at other time points during the study if necessary and agreed with the Investigator, and upon receipt of consent from the study participant. The total blood volume does not exceed the blood volume limit for the participant according to national / local guidelines. During the course of the study, sampling times may be changed based on new available data (e.g., to obtain data closer to the time of peak plasma concentration) to ensure appropriate monitoring.

[0498] Instructions for biological sample collection and processing were provided by the investigator. The actual date and time (24-hour clock) of each sample was recorded.

[0499] The samples are used to evaluate the PD of the fusion polypeptides described herein. Samples collected for analysis of the concentration of the fusion polypeptides described herein may also be used to evaluate safety or efficacy aspects related to issues that arise during or after the study. Unused samples may be retained for up to 25 years for additional evaluation as necessary.

[0500] 8.7 Genetics

[0501] Genetics were not evaluated in this study.

[0502] 8.8 Biomarkers

[0503] Sample collection for biomarker studies (eg, exploratory) is also part of this study.

[0504] As specified in the SoA (Section 1.3), the following samples for biomarker studies are required and collected from all participants in this study:

[0505] ·blood

[0506] Urine

[0507] Samples are collected for testing, which may include, but are not limited to, markers of complement dysregulation, inflammation, and endothelial activation / injury.

[0508] 8.9 Immunogenicity Assessment

[0509] Antibodies against the fusion polypeptide (ADA) were evaluated in whole blood samples collected from all participants according to SoA (Section 1.3).

[0510] Screen serum samples for ADA. If the screening is positive, the sample is analyzed using a confirmatory ADA assay and the titer of the confirmed positive sample is reported. The detection and characterization of antibodies to the fusion polypeptides described herein are performed by the researcher or under the supervision of the researcher using a validated assay method. If deemed necessary, the sample can be further characterized to determine the titer and presence of neutralizing antibodies (as an exploratory analysis).

[0511] The actual date and time (24-hour format) of each sample is recorded. Samples can be stored for a period of up to 25 years to allow for additional safety assessments if necessary.

[0512] Detailed instructions for the collection, handling, storage, and transportation of serum samples for immunogenicity analysis are provided in the laboratory manual.

[0513] 9. Statistical Considerations

[0514] 9.1 Statistical Assumptions

[0515] not applicable.

[0516] 9.2 Sample size determination

[0517] Twelve participants were enrolled in each of cohorts 1 and 2. In patients with stable SCD, hemoglobin levels are unlikely to change. The sample size was determined based on the change in target hemoglobin relative to baseline to exclude 0g / dL with a lower 2-sided 95% confidence limit. Assuming a standard deviation of 1g / dL, a sample size of 12 participants provides 88% power to detect a change of 1g / dL relative to baseline, with a 2-sided significance level of 0.05. In the optional cohort 3, 6 participants were enrolled to define the exposure / response relationship of the fusion polypeptide described herein by combining cohort 1, cohort 2, and cohort 3 data using a PK / PD modeling approach. The cohort 3 sample size was not determined for efficacy purposes.

[0518] 9.3 Population used for analysis

[0519] For the purpose of analysis, the following populations were defined:

[0520]

[0521]

[0522] Abbreviations: ADA = anti-drug antibodies; CAP = alternative complement pathway; CCP = classical complement pathway; ICF = informed consent; PK = pharmacokinetics.

[0523] 9.4 Statistical analysis

[0524] In general, descriptive statistics for continuous variables included the number of nonmissing values, arithmetic mean, standard deviation, median, minimum, and maximum. Descriptive statistics for PK parameters included the number of observations, arithmetic mean, standard deviation, arithmetic coefficient of variation (%CV), median, minimum, maximum, geometric mean, and geometric %CV. Categorical variables were summarized by cohort and time point using percentages and frequency counts.

[0525] The SAP was developed and completed prior to the first data cutoff / database lock and further described the participant population that would be included in the analysis and the procedures to be used to account for missing, unused, and spurious data, as appropriate. This section provides a high-level summary of the planned statistical analyses for the primary and secondary endpoints.

[0526] 9.4.1 Efficacy analysis

[0527] 9.4.1.1 Changes in complement biomarkers

[0528] The absolute and percent changes from baseline in complement biomarkers (eg, Ba, C3a, and sC5B9) were evaluated at the end of treatment (12 weeks) for Cohorts 1 and 2. Additional details are described in SAP.

[0529] 9.4.1.2 Changes in hemoglobin

[0530] The absolute and percentage changes from baseline in hemoglobin were evaluated at the end of treatment (12 weeks) for Cohorts 1 and 2. Additional details are described in SAP.

[0531] 9.4.1.3 Hematological Response Time

[0532] Hemoglobin response was defined as an increase in hemoglobin level >1 g / dL from baseline. Hemoglobin response was assessed at the end of treatment (12 weeks) for Cohorts 1 and 2. Additional details are described in SAP.

[0533] 9.4.1.4 Hemolysis markers

[0534] The absolute and percentage changes from baseline in hemolytic markers (serum LDH level, absolute reticulocyte count, serum indirect bilirubin, serum haptoglobin, and hemoglobin) were evaluated at the end of treatment (12 weeks) for Cohorts 1 and 2. Additional details are described in SAP.

[0535] 9.4.1.5 Exploratory Analysis of Changes in VOC-Related Biomarkers

[0536] After treatment completion (12 weeks for cohorts 1 and 2), VOC-related biomarkers can be assessed. Additional details are described in the SAP.

[0537] 9.4.2 Exploratory Evaluation of VOC

[0538] Sickle cell disease-related pain crises (VOCs) were defined as acute onset of pain with no medically determined cause other than VOC episodes that resulted in a healthcare facility visit and treatment with oral or parenteral narcotics or with parenteral nonsteroidal anti-inflammatory drugs. Uncomplicated VOCs were defined as the absence of any other SCD complications during the VOC episode. Complicated VOCs were defined as the presence of a diagnosis of other SCD complications during the VOC episode. Acute chest syndrome, liver sequestration, splenic sequestration, and priapism were considered VOC events in this study. Complicated VOCs were also reported as AEs.

[0539] If available, the following may also be assessed:

[0540] Number of VOCs resulting in medical visits

[0541] · Number of simple VOCs, acute chest syndrome, liver sequestration, spleen sequestration, and priapism

[0542] Time from first dose of study drug to first VOC

[0543] 9.4.3 Security Analysis

[0544] The primary endpoints of the study were safety and tolerability.

[0545] All safety analyses were performed on the safety population and reported per cohort.

[0546] Safety analysis includes analysis of all TEAEs, ECG, clinical laboratory data, physical examinations, and vital sign measurements using descriptive statistics. No inferential statistical analysis is planned for the safety parameters of this study. The prevalence of AEs and SAEs is summarized by SOC and preferences for each cohort and treatment group and overall within each treatment group, and by the relationship with the study drug. AEs are also summarized by cohort and treatment group and overall within each treatment group and by severity. SAEs and AEs that led to withdrawal from the study are listed. Participants with multiple AEs in a category (e.g., overall, SOC, preferences) are counted once in the category. For the severity table, the most serious events of participants in the category are counted.

[0547] Changes in vital sign measurements and laboratory assessments (e.g., clinical chemistry, blood cell counts with differences, and urinalysis) relative to baseline are summarized for each cohort and overall. Laboratory parameter values ​​are graded according to the Common Terminology Criteria for Adverse Events (CTCAE, v5.0, published on November 27, 2017). Shift tables by cohort and treatment group are generated for these laboratory parameters. These tables summarize the number of participants at each baseline grade relative to the reference range and the change in the worst highest grade assessed after dosing during the study.

[0548] All concomitant medications were coded using the World Health Organization Drug Dictionary, and the frequency and percentage of concomitant medications were summarized.

[0549] 9.4.3.1 ECG analysis

[0550] Cardiac assessments were performed in the safety population.

[0551] ECG parameters, including heart rate, PR, RR, QRS, QT, and QTcF intervals, were measured at defined time points. The mean of the triple ECG readings at the collected time points was calculated, and changes from pre-treatment baseline values ​​were assessed for each cohort and treatment group.

[0552] Outlier analyses were performed to summarize the absolute counts, frequencies, and percentages of participants who met any of the following outlier criteria at each visit by cohort and treatment group:

[0553] QT, QTcF interval > 450 msec

[0554] QT, QTcF interval > 480 msec

[0555] QT, QTcF interval > 500 msec

[0556] QT, QTcF interval increased by >30 msec from baseline

[0557] QT, QTcF interval increased by >60 msec from baseline

[0558] Analysis of drug-related QT / QTc interval changes relative to plasma PK concentrations can be performed for all dose regimens. The principle of this analysis follows the statistical method described by Garnett et al. (Garnett, 2018).

[0559] Detailed analysis is specified in the SAP or in a separate ECG analysis plan.

[0560] 9.4.4 Other analyses

[0561] 9.4.1.1 Pharmacokinetic Analysis

[0562] All PK analyses were performed on the PK population and reported by cohort.

[0563] Using individual serum concentration data from patients who received SC doses of the fusion polypeptides described herein at actual sampling dates and times, a population PK analysis approach was used to characterize PK. Details are provided in SAP.

[0564] 9.4.1.2 Pharmacodynamic Analysis

[0565] All PD analyses were performed on the PD population and reported by cohort.

[0566] All SC doses of the fusion polypeptides described herein administered were evaluated for PD effects by assessing changes in serum total and free properdin concentrations and CAP activity using the Wieslab AP assay. In addition, exploratory assessments of other measures of properdin activity over time may be considered appropriate.

[0567] 9.4.1.3 Immunogenicity Analysis

[0568] To assess immunogenicity, the incidence of confirmed positive ADA was summarized. In addition, after confirmation of ADA positivity, samples were assessed for ADA titer and the presence of neutralizing antibodies (if possible).

[0569] 9.4.1.4 Exploratory Analysis

[0570] Additional exploratory analyses of biomarker assays and clinical efficacy endpoints may be performed. Details of these analyses are presented in the SAP.

[0571] 9.5 Interim Analysis

[0572] After at least 12 patients from Cohorts 1 and 2 (6 from each cohort) have been enrolled and completed the treatment period, an interim analysis can be performed to provide the basis for later stages of the trial. The interim analysis includes safety, efficacy, PK / PD, and immunogenicity data. Details of this analysis are presented in the SAP.

[0573] 10. Laboratory Testing

[0574] Unless otherwise specified, the protocol-required clinical laboratory tests detailed in Table 13 were performed by a central laboratory.

[0575] Table 13: Laboratory evaluations required by the protocol

[0576]

[0577] Table 13: Laboratory evaluations required by the protocol

[0578]

[0579] Table 13: Laboratory evaluations required by the protocol

[0580]

[0581] a Unless local regulations or the IRB / IEC require serum testing, local urine testing is standard for this protocol.

[0582] Abbreviations: AP = alternative pathway; CAP = alternative pathway of complement; CCP = classical pathway of complement; HBc = hepatitis B core; IEC = independent ethics committee; IgG = immunoglobulin G; IgM = immunoglobulin M; IRB = institutional review board; LDH = lactate dehydrogenase; LP = lectin pathway; MCH = mean corpuscular hemoglobin; MCV = mean corpuscular volume; SAE = serious adverse event; VOC = vaso-occlusive crisis.

[0583] A list of abbreviations and their explanations is provided in Table 14.

[0584] Table 14: Abbreviations and technical terms

[0585] Abbreviation or term explain ADA Anti-drug antibodies AE Adverse Events AP Alternative Pathways

[0586] Table 14: Abbreviations and technical terms

[0587] Abbreviation or term explain <![CDATA[AUC 0-168 ]]> Area under the concentration-time curve from time 0 to 168 hours Ba Complement component Ba BMI body mass index C3 Complement component C3 C3a Complement component C3a C5 Complement component 5 C9 Complement component 9 CAP Alternative complement pathway CFR Federal regulations CIOMS Council for International Organizations of Medical Sciences <![CDATA[C max ]]> Maximum observed serum concentration COVID-19 Coronavirus Disease 2019 CTCAE Common Terminology Criteria for Adverse Events %CV Coefficient of variation DMC Data Monitoring Committee EC Ethics Committee ECG Electrocardiogram eCRF Electronic case report form EDC Electronic Data Capture EOS End of study FIH First human FSH Follicle-stimulating hormone GCP Good Clinical Practice GDS Global Drug Safety GDPR General Data Protection Regulation

[0588] Table 14: Abbreviations and technical terms

[0589] Abbreviation or term explain GLP Good Laboratory Practice HbC Hepatitis B Core HbS Sickle hemoglobin HBsAg Hepatitis B surface antigen HCV Hepatitis C virus Hi Haemophilus influenzae type b HIPAA Health Insurance Portability and Accountability Act HIV Human immunodeficiency virus HRT Hormone replacement therapy IB Investigator's Handbook ICF Informed consent ICH International Medical Regulation Council IEC Independent Ethics Committee IRB Institutional Review Board IV Intravenous <![CDATA[K D ]]> Dissociation constant LDH Lactate dehydrogenase mAbs Monoclonal antibodies MCV4 Quadrivalent meningococcal conjugate vaccine NOAEL No Observed Adverse Effect Level PD Pharmacodynamics PK Pharmacokinetics QTCE QT interval corrected using Fridericia's formula Q2W Every 2 weeks Q4W Every four weeks

[0590] Table 14: Abbreviations and technical terms

[0591] Abbreviation or term explain QW Once a week RBC Red blood cells SAE Serious adverse events SAP Statistical analysis plan SC Subcutaneous sC5 Soluble complement component C5b-9 SCD Sickle cell disease SoA Event Schedule SOC System Organ Class TCR Tissue cross-reactivity TEAE Treatment-emergent adverse events VHH Single variable domain on heavy chain antibody VOC Vaso-occlusive crisis WOCBP Women of childbearing potential

[0592] Example 2. Safety, Tolerability, Pharmacokinetics, Pharmacodynamics, and Immunogenicity of an Anti-Properdin / Anti-Serum Albumin Bispecific Single Variable Domain Antibody in Healthy Adults: Results of a Phase 1 Study

[0593] This randomized, double-blind, placebo-controlled study evaluated the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of fusion polypeptides having the amino acid sequence of SEQ ID NO: 1 or modifications thereof administered subcutaneously (SC) and intravenously (IV) in healthy adults.

[0594] In this Phase I first-in-human (FIH) study, healthy volunteers aged 18 to 65 years were randomized to the fusion peptide or placebo (3:1). The fusion peptide was administered SC in single and multiple ascending doses and as a single IV dose. Ten dosing cohorts were planned ( Figure 2 ). Two cohorts were not open for enrollment because the expected complete inhibition of complement AP would be >70 days. Because properdin inhibition may increase the risk of N. meningitidis infection, vaccination is required prior to receiving the fusion polypeptides described herein, and participants remain on antibiotics during treatment.

[0595] Sixty participants were randomized (fusion polypeptide, 45; placebo, 15). Cohorts 1, 3, 4, and 5 had 5 participants (each) treated with fusion polypeptide; cohorts 2, 6, and 9 had 6 participants treated with fusion polypeptide; and cohort 8 had 7 participants treated with fusion polypeptide. Demographic and baseline characteristics were similar for the fusion polypeptide and placebo groups. Four participants discontinued the study: 1 patient was lost to follow-up, 1 patient after taking contraindicated medications, and 2 patients withdrew consent (1 patient after an adverse event [AE] for placebo). There were no discontinuations due to AEs in participants exposed to fusion polypeptide. Most treatment-emergent AEs (TEAEs) were mild and assessed as unrelated to study treatment. There were no serious AEs, serious infectious events, or deaths. The incidence of TEAEs was similar across cohorts. Treatment-related TEAEs occurred more frequently in the fusion polypeptide group than in the placebo group (11 [24.4%] vs. 2 [13.3%]); the most common TEAEs related to fusion polypeptide treatment were nausea (n=2), headache (n=2), and infusion site erythema (n=2). No Neisseria meningitidis infections were reported.

[0596] Dose proportionality was observed in all single-dose cohorts except 50 mg. After multiple dosing, the geometric mean cumulative ratio R Cmax and R AUC The mean absolute bioavailability of the SC-administered fusion polypeptide was 94%. The complement alternative pathway (AP) activity decreased immediately after administration of the fusion polypeptide ( Figure 3 The duration of complete AP blockade (<1% of baseline) increased with increasing dose; in cohorts 4, 5, and 6, complete AP inhibition persisted for >30 days. In all single-dose cohorts, mean % AP hemolysis returned to baseline by the end of the observation period. In the multiple-dose cohorts, complete inhibition of AP activity was observed for 70 and 84 days (cohorts 8 and 9, respectively). There were no changes in classical pathway (CP) and lectin pathway (LP) activity.

[0597] Four participants had pre-existing immune reactivity at baseline, none of which was treatment-boosted. Treatment-emergent anti-drug antibodies (ADA) occurred in 20%, 40%, 100%, and 83.3% of participants in the 150 mg SC, 450 mg SC, 450 mg IV, and 1200 mg SC groups, respectively; most were of low titer and did not affect PK. In the multiple-dose cohorts (cohorts 8 and 9), 71.4% and 100% developed ADA, respectively, with most having low titers and short duration of response.

[0598] In this FIH study in healthy participants, the fusion polypeptides described herein showed no unexpected safety issues and were well tolerated. The inhibition of complement AP by the fusion polypeptides described herein was rapid and complete, and CP and LP were not affected.

[0599] Other embodiments

[0600] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. A method for treating a human patient with sickle cell disease, the method comprising administering to the patient a properdin binding antibody or antigen-binding fragment thereof, wherein the properdin binding antibody or antigen-binding fragment thereof comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 2, 3 and 4, respectively.

2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a human serum albumin binding sequence.

3. The method of claim 2, wherein the human serum albumin binding sequence is fused to the C-terminus of the properdin binding antibody or antigen-binding fragment thereof.

4. The method of claim 3, wherein the human serum albumin binding sequence is fused to the C-terminus of the properdin binding antibody or antigen-binding fragment thereof via a linker. The method of claim 4 , wherein the linker comprises the amino acid sequence of SEQ ID NO:

10.

6. The method of any one of claims 2-5, wherein the human serum albumin binding sequence comprises CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 5, 6 and 7.

7. The method of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 1 or a modification thereof.

8. The method of claim 7, wherein the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 into pyroglutamate.

9. The method of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof The patient was administered a dose of 300 mg.

10. The method of claim 9, wherein the antibody or antigen-binding fragment thereof is administered to the patient once a week.

11. The method of claim 9 or 10, wherein the antibody or antigen-binding fragment thereof is administered to the patient for up to 12 weeks.

12. The method of claim 9, wherein the antibody or antigen-binding fragment thereof is administered to the patient once every 2 weeks.

13. The method of claim 12, wherein the antibody or antigen-binding fragment thereof is administered up to 4 times.

14. The method of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof is administered to the patient at a dose of 600 mg.

15. The method of claim 14, wherein the antibody or antigen-binding fragment thereof is administered to the patient once every 4 weeks.

16. The method of claim 14 or 15, wherein the antibody or antigen-binding fragment thereof is administered to the patient up to 4 times.

17. A method for treating a human patient with sickle cell disease, the method comprising administering to the patient a properdin binding antibody or antigen-binding fragment thereof, wherein the properdin binding antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 1 or a modification thereof, and wherein the antibody or antigen-binding fragment thereof is administered to the patient at a dose of 300 mg once a week for up to 12 weeks.

18. A method for treating a human patient with sickle cell disease, the method comprising administering to the patient a properdin binding antibody or antigen-binding fragment thereof, wherein the properdin binding antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 1 or a modification thereof, and wherein the antibody or antigen-binding fragment thereof is administered to the patient at a dose of 300 mg once every 2 weeks for up to 4 times.

19. A method for treating a human patient with sickle cell disease, the method comprising administering to the patient a properdin binding antibody or antigen-binding fragment thereof, wherein the properdin binding antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 1 or a modification thereof, and wherein the antibody or antigen-binding fragment thereof is administered to the patient at a dose of 600 mg once every 4 weeks up to 4 times.

20. The method of any one of claims 17-19, wherein the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

21. The method of any one of claims 1-20, wherein the patient has been clinically diagnosed with sickle cell disease.

22. The method of claim 21, wherein the sickle cell disease is HbSS or HbSβ 0 - Thalassemia.

23. The method of any one of claims 1-22, wherein the patient is further administered hydroxyurea.

24. The method of claim 23, wherein the patient has been receiving a stable dose of hydroxyurea for at least 3 months prior to administration of the antibody or antigen-binding fragment thereof.

25. The method of any one of claims 1-22, wherein the patient has not been administered hydroxyurea for at least 30 days prior to administration of the antibody or antigen-binding fragment thereof.

26. The method of any one of claims 1-25, wherein the patient experiences no treatment-emergent adverse events after 12 weeks of treatment.

27. The method of any one of claims 1-26, wherein the patient experiences no serious adverse events after 12 weeks of treatment.

28. The method of any one of claims 1-26, wherein the patient experiences no adverse events after 12 weeks of treatment.

29. The method of any one of claims 1-28, further comprising measuring the change in serum concentration of the antibody or antigen-binding fragment thereof for up to 30 weeks after initiation of treatment.

30. The method of any one of claims 1-29, further comprising measuring changes in blood concentrations of anti-drug antibodies for up to 30 weeks after initiation of treatment.

31. The method of any one of claims 1-30, wherein the patient experiences a change from baseline in serum concentrations of total and free properdin after up to 30 weeks of treatment.

32. The method of any one of claims 1-31, wherein the patient experiences a change from baseline in serum concentration of complement component Ba (Ba), complement component C3a (C3a), or soluble complement component C5B-9 (sC5B9) after 12 weeks of treatment.

33. The method of any one of claims 1-32, wherein the patient experiences a change from baseline in blood or serum concentrations of hemoglobin, nitric oxide, an inflammatory marker, or a cell adhesion marker after 12 weeks of treatment.

34. The method of claim 33, wherein the inflammatory marker comprises interleukin-1.

35. The method of claim 33 or 34, wherein the cell adhesion marker comprises soluble P-selectin.

36. The method of any one of claims 1-30, wherein the patient experiences a change in hemoglobin level relative to baseline after 12 weeks of treatment.

37. The method of any one of claims 1-36, wherein the patient experiences a change from baseline in serum LDH levels, indirect bilirubin, haptoglobin, or hemopexin after 12 weeks.

38. The method of any one of claims 1-37, wherein the patient experiences a change in reticulocyte levels relative to baseline after 12 weeks.

39. The method of any one of claims 1-38, wherein the patient experiences a reduced rate of vaso-occlusive crises after 12 weeks of treatment compared to baseline.

40. The method of any one of claims 1-39, wherein the patient experiences an increased time to first vaso-occlusive crisis after 12 weeks of treatment compared to baseline.

41. The method of any one of claims 1-40, wherein the antibody or antigen-binding fragment thereof Formulated for subcutaneous administration.

42. The method of any one of claims 1-41, wherein the antibody or antigen-binding fragment thereof is formulated at a concentration of 150 mg / mL at pH 5.4 in an aqueous solution comprising 20 nM sodium acetate, 250 mM sucrose, and 0.05% polysorbate-80.

43. The method of any one of claims 1-42, wherein the human patient is between 18 and 65 years old.

44. The method of any one of claims 1-43, wherein the human patient has a body weight of ≥ 40 kg.

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