Kidney active fusion proteins and methods of treatment using the same

Abstract

Described herein are fusion proteins which include factor H functional domains and may include VHH domains and integrin binding domains, and the use of such fusion proteins in methods of treatment of focal segmental glomerulosclerosis.

Description

[0001] PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0002] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0003] KIDNEY ACTIVE FUSION PROTEINS AND METHODS OF TREATMENT USING THE SAME

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically in XML

[0006] 5 format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 28, 2024, is named 50694-108001_Sequence_Listing_3_28_24 and is 88,294 bytes in size.

[0007] BACKGROUND

[0008] The complement system plays a central role in the clearance of immune complexes and in immune responses to infectious agents, foreign antigens, virus-infected cells, and tumor cells. Complement activation occurs primarily by three pathways: the classical pathway, the lectin pathway, and the alternative pathway. The alternative pathway of complement is in a constant state of low-level activation. Uncontrolled activation or insufficient regulation of the complement alternative pathway (CAP) can lead to inflammation, cellular injury, and tissue damage. Local alternative pathway activation within

[0009] 15 the kidney is a contributor to renal pathology and loss of function. Thus, the complement alternative pathway has been implicated in the pathogenesis of a number of renal diseases. Inhibition or modulation of complement alternative pathway activity, in the absence of initiation of the lectin and classical pathway, has been recognized as a promising therapeutic strategy. For example, the alternative pathway plays a role in amplifying complement activation initiated from all three pathways. The number of treatment

[0010] 20 options available for diseases associated with the complement alternative pathway are limited. Thus, developing innovative strategies to treat diseases associated with complement alternative pathway activation or dysregulation, such as kidney diseases, which affect an estimated 37 million people in the U.S. alone, is a significant unmet need.

[0011] 25 SUMMARY OF THE DISCLOSURE

[0012] Described herein are fusion polypeptides that include a Factor H functional domain. The fusion proteins may be used to treat patients with diseases associated with complement alternative pathway activation or dysregulation, such as kidney diseases.

[0013] In an aspect, the disclosure provides a method of treating focal segmental glomerulosclerosis (FSGS) comprising administering an effective amount of a pharmaceutical composition comprising a fusion protein having an amino acid sequence with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1 to a subject in need thereof. In some embodiments, the fusion protein has an amino acid sequence of SEQ ID NO: 1.

[0014] 35 In some embodiments, the FSGS is primary FSGS. In some embodiments, the subject has been diagnosed with FSGS within 3 years (e.g., within 1 week, 1 month, 3 months, 6 months, 9 months, 1 year,

[0015] 1

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[0017] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0018] 18 months, 24 months, 30 months, or 3 years) prior to being administered the pharmaceutical composition.

[0019] In some embodiments, the pharmaceutical composition comprises phosphate buffer. In some embodiments, the phosphate buffer has a concentration of between 5 mM and 15 mM (e.g., between 5

[0020] 5 mM and 12 mM, 5 mM and 10 mM, 5 mM and 8 mM, 5 mM and 6 mM, 6 mM and 15 mM, 8 mM and 15 mM, 10 mM and 15 mM, 12 mM and 15 mM, 8 mM and 12 mM, or 9 mM and 11 mM). In some embodiments, the phosphate buffer has a concentration of about 10 mM. In some embodiments, the pharmaceutical composition comprises one or more of L-arginine, arginine HCI, sucrose, or polysorbate. In some embodiments, the pharmaceutical composition comprises between 0.5 mg / mL and 3 mg / mL (e.g., between 0.5 mg / mL and 2.5 mg / mL, 0.5 mg / mL and 2 mg / mL, 0.5 mg / mL and 1 .5 mg / mL, 0.5 mg / mL and 1 mg / mL, 1 mg / mL and 3 mg / mL, 1.5 mg / mL and 3 mg / mL, 2 mg / mL and 3 mg / mL, 2.5 mg / mL and 3 mg / mL, 1 mg / mL and 2 mg / mL, or 1 .2 mg / mL and 1 .6 mg / mL) L-arginine. In some embodiments, the pharmaceutical composition comprises about 1 .4 mg / mL L-arginine. In some embodiments, the pharmaceutical composition comprises between 20 mg / mL and 35 mg / mL (e.g.,

[0021] 15 between 20 mg / mL and 30 mg / mL, 20 mg / mL and 25 mg / mL, 25 mg / mL and 35 mg / mL, 30 mg / mL and 35 mg / mL, 25 mg / mL and 30 mg / mL, or 20 mg / mL and 30 mg / mL) arginine HCI. In some embodiments, the pharmaceutical composition comprises about 27.8 mg / mL arginine HCI. In some embodiments, the pharmaceutical composition comprises between 10 mg / mL and 20 mg / mL (e.g., between 10 mg / mL and 18 mg / mL, 10 mg / mL and 15 mg / mL, 10 mg / mL and 12 mg / mL, 12 mg / mL and 20 mg / mL, 15 mg / mL and

[0022] 20 20 mg / mL, 18 mg / mL and 20 mg / mL, 10 mg / mL and 18 mg / mL, 10 mg / mL and 15 mg / mL, 10 mg / mL and 12 mg / mL, 12 mg / mL and 15 mg / mL, or 11 mg / mL and 14 mg / mL) sucrose. In some embodiments, the pharmaceutical composition comprises about 13.7 mg / mL sucrose. In some embodiments, the pharmaceutical composition comprises polysorbate 80. In some embodiments, the pharmaceutical composition comprises between 0.1 mg / mL and 1.5 mg / mL (e.g., between 0.1 mg / mL and 1 mg / mL, 0.1

[0023] 25 mg / mL and 0.5 mg / mL, 0.5 mg / mL and 1 .5 mg / mL, 1 mg / mL and 1 .5 mg / mL, or 0.2 mg / mL and 0.8 mg / mL) and polysorbate. In some embodiments, the fusion protein is formulated with about 0.5 mg / mL polysorbate. In some embodiments, the pharmaceutical composition comprises between 25 mg / mL and 75 mg / mL (e.g., between 25 mg / mL and 60 mg / mL, 25 mg / mL and 50 mg / mL, 25 mg / mL and 40 mg / mL, 25 mg / mL and 35 mg / mL, 35 mg / mL and 75 mg / mL, 50 mg / mL and 75 mg / mL, 60 mg / mL and 75 mg / mL, 40 mg / mL and 60 mg / mL, 30 mg / mL and 70 mg / mL, or 45 mg / mL and 55 mg / mL) of the fusion protein. In some embodiments, the pharmaceutical composition comprises about 50 mg / mL of the fusion protein.

[0024] In some embodiments, the subject is 18 years or more in age. In some embodiments, the subject is 75 years or less in age. In some embodiments, the subject is from 18 to 75 year old. In some embodiments, the subject had acute onset of nephrotic syndrome at the time of FSGS diagnosis. In

[0025] 35 some embodiments, the subject had proteinuria corresponding to at least 3.5 g / day (e.g., at least 3.5 g / day, 3.7 g / day, 4 g / day, 4.2 g / day, 4.5 g / day, 4.7 g / day, or 5 g / day) of protein in urine at the time of diagnosis. In some embodiments, the subject had hypoalbuminemia corresponding to less than 3.5 g / dL

[0026] 2

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[0028] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0029] (e.g., less than 3.5 g / dL, 3.2 g / dL, 3 g / dL, 2.8 g / dL, 3 g / dL, or 3.3 g / dL) of albumin in serum at the time of diagnosis. In some embodiments, the subject had peripheral edema at the time of diagnosis. In some embodiments, the subject has a urine protein creatinine ratio of at least 1 .5 g / g (e.g., at least 1 .5 g / g, 1 .7 g / g, 2 g / g, 2.2 g / g, 2.5 g / g, 2.7 g / g, or 3 g / g) priorto administration of the pharmaceutical composition. In

[0030] 5 some embodiments, the subject does not have kidney disease other than FSGS.

[0031] In some embodiments, the subject shows a reduction in proteinuria relative to baseline after at least 24 weeks of administration of the pharmaceutical composition. In some embodiments, the subject shows a reduction in proteinuria of greater than 30% (e.g., greater than 35%, 40% 45%, 50%, 55%, 60%, 65%, 75%, 85%, or 95%) relative to baseline after at least 24 weeks of administration of the pharmaceutical composition. In some embodiments, the subject shows a reduction in proteinuria of greater than 50% (e.g., greaterthan 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) relative to baseline after at least 24 weeks of administration of the pharmaceutical composition. In some embodiments, the subject shows a change in estimated glomerular filtration rate (eGFR) relative to baseline after at least 24 weeks of administration of the pharmaceutical composition. In some

[0032] 15 embodiments, the subject shows a change in serum albumin relative to baseline after at least 24 weeks of administration of the pharmaceutical composition. In some embodiments, the subject shows partial remission after at least 24 weeks of administration of the pharmaceutical composition. In some embodiments, the subject shows complete remission after at least 24 weeks of administration of the pharmaceutical composition. In some embodiments, the subject shows improved kidney function relative

[0033] 20 to baseline during administration of the fusion protein after at least 24 weeks of administration of the pharmaceutical composition.

[0034] In some embodiments, the subject is administered at least 180 mg (e.g., at least 190 mg, 200 mg, 250 mg, 300 mg, or 350 mg, or any intervening dose) of the fusion protein weekly. In some embodiments, the subject is administered about 180 mg of the fusion protein weekly. In some

[0035] 25 embodiments, the subject is administered between 170 mg and 460 mg (including any intervening dose, as well as the endpoints; e.g., between 170 mg and 450 mg, 170 mg and 425 mg, 170 mg and 400 mg, 170 mg and 350 mg, 170 mg and 325 mg, 170 mg and 300 mg, 170 mg and 275 mg, 170 mg and 250 mg, 170 mg and 225 mg, 170 mg and 200 mg, 200 mg and 450 mg, 250 mg and 450 mg, 275 mg and

[0036] 450 mg, 300 mg and 450 mg, 325 mg and 450 mg, 350 mg and 450 mg, 375 mg and 450 mg, 300 mg and 450 mg, 425 mg and 450 mg, 180 mg and 450 mg, or 250 and 350 mg) of the fusion protein weekly.

[0037] In some embodiments, the subject is administered about 450 mg of the fusion protein weekly. In some embodiments, the subject does not experience a treatment emergent adverse event.

[0038] In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a

[0039] 35 human.

[0040] 3

[0041] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0042] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a schematic drawing of the components of Compound A.

[0045] FIG. 2 is a schematic representation of Compound A with the disulfide bonds.

[0046] FIG. 3 is a representation of the first-in-human Compound A study design.

[0047] 5 FIG. 4 is a graph showing the mean Serum Concentration of Compound A over time after single subcutaneous or intravenous dose for cohorts 1-5.

[0048] FIG. 5 is a graph showing the mean percent change in complement alternative pathway (CAP) Activity from baseline over time for cohorts 1 -5.

[0049] FIG. 6 is a graph showing the mean complement fH serum concentrations over time for each of the randomized treatment groups.

[0050] FIG. 7 shows the study design for the proof of concept study of Compound A in the treatment of adult patients with primary focal segmental glomerulosclerosis (FSGS).

[0051] FIG. 8A and FIG. 8B are graphs showing the simulated PK Profiles of Compound A in patients with FSGS who are administered 180 mg of Compound A per week (FIG. 8A) and 450 mg of Compound

[0052] 15 A per week (FIG. 8B).

[0053] Definitions

[0054] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0055] 20 As used herein, “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Fusion proteins may be administered by any method known to those skilled in the art. Suitable methods for administering the fusion protein may be, for example, orally, by injection (e.g., intravenously, intraperitoneally, intramuscularly, intravitreally, and subcutaneously), drop infusion preparations, inhalation, intranasally, and the like. In some embodiments, administration is

[0056] 25 via intravenous and / or subcutaneous infusions. Fusion proteins prepared, as described herein, may be administered in various forms depending on the disorderto be treated and the age, condition, and body weight of the subject, as is known in the art. A preparation can be administered prophylactically; that is, administered to decrease the likelihood of developing a disease or condition.

[0057] As used herein, the terms “binding affinity,” “specifically binds,” “affinity” refer to the strength of the total noncovalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a specific interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by standard methods known in the art, including those described herein. A low-affinity complex contains a

[0058] 35 molecule that generally tends to dissociate readily from its binding partner, whereas a high-affinity complex contains a molecule that generally tends to remain bound to its binding partner for a longer duration. “Specifically binds” refers to molecules and binding partner pairs that have a Kd of at least

[0059] 4

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[0062] 1x1 O'6M or lower (e.g., in the range of 1x1 O'6M to 1x1 O'12M, such as 1x10’7M, IxW^ M, 1x10'9M, 1x1 O'10M, 1x10-11M, and 1x1012M).

[0063] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically or substantially specifically binds to, or is immunologically reactive with, a particular antigen. The antibody

[0064] 5 can be, for example, a natural or artificial mono- or polyvalent antibody including, but not limited to, a polyclonal, monoclonal, multi-specific, human, humanized, or chimeric antibody. An antibody may be a genetically engineered or otherwise modified form of an antibody, including but not limited to, heteroconjugate antibodies (e.g., bi-, tri-, and tetra-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, single domain, VHH, Fab', F(ab')2, Fab, Fv, rlgG and scFv fragments.

[0065] As used herein, the term “complement alternative pathway” refers to one of three pathways of complement activation (the others being the classical pathway and the lectin pathway).

[0066] As used herein, the term “complement alternative pathway activation or dysregulation” refers to any aberration in the ability of the complement alternative pathway to provide host defense against

[0067] 15 pathogens and clear immune complexes and damaged cells for immunoregulation. Complement alternative pathway activation or dysregulation can occur in the fluid phase and at the cell surface. Complement alternative pathway activation or dysregulation can lead to excessive complement activation or insufficient regulation, both causing tissue injury.

[0068] As used herein, the term “disease” refers to an interruption, cessation, or disorder of body

[0069] 20 functions, systems, or organs. Disease(s) or disorders of interest include those that would benefit from treatment with a fusion protein or by a method described herein.

[0070] As used herein, "Factor H" refers to a protein component of the complement alternative pathway encoded by the complement factor H gene (“FH NM000186; GenelD:3075; UniProt ID P08603; Ripoche, J. et al., Biochem. J., 249:593-602, 1988). Factor H is translated as a 1 ,213 amino acid

[0071] 25 precursor polypeptide that is processed by removal of an 18 amino acid signal peptide, resulting in the mature factor H protein (amino acids 19-1231). Factor H consists of 20 short complement regulator (SCR) domains. Amino acids 1-18 comprise the signal peptide, residues 21-80 comprise SCR1 : EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQK (SEQ ID NO: 6), residues 85-141 comprise SCR 2: RPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEV (SEQ ID NO: 7), residues 146-205 comprise SCR3: VKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVE (SEQ ID NO: 8), residues 201-262 comprise SCR 4: ISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEE (SEQ ID NO: 9),

[0072] 35 residues 267-320 comprise SCR 5: KSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLK (SEQ ID NO: 10), residues 326-384 comprise SCR 6:

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[0076] PCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCL (SEQ ID NO: 11). Factor H regulates complement activation on self-cells by possessing both cofactor activity for the factor l-mediated C3b cleavage, and decay accelerating activity against the alternative pathway C3 convertase, C3bBb.

[0077] 5 Cleavage of C3 results initially in the generation and deposition of C3b on the activating cell surface. The C3b fragment is involved in the generation of enzymatic complexes that amplify the complement cascade. On a cell surface, C3b is rapidly converted to inactive iC3b, for example when deposited on a host surface containing regulators of complement activation (i.e., most host tissue). Even in the absence of membrane-bound complement regulators, substantial levels of iC3b are formed because of the action of serum factor H and serum factor I. iC3b is subsequently digested to the membrane-bound fragments C3dg and then C3d by factor I and other proteases and cofactors, but this process is relatively slow.

[0078] As used herein, the term “fragment” refers to less than 100% of the amino acid sequence of a full- length reference protein (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, of the full-length

[0079] 15 sequence etc.), but including, e.g., 5, 10, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, or more amino acids. A fragment can be of sufficient length such that a desirable function of the full-length protein is maintained. For example, the regulation of the complement alternative pathway in the fluid phase by fragments of, for example, factor H, is maintained. Such fragments are “biologically active fragments.” As used herein, a “functional fragment" or a “biologically active fragment” refers to a fragment, or

[0080] 20 portion, of a protein having some or all of the activities of the full-length protein. For example, a functional or biologically active fragment of factor H, refers to any fragment of a factor H protein having some or all of the activities of factor H, e.g., complement alternative pathway regulatory activity of the full-length factor H protein. Examples include, but are not limited to, factor H fragments, joined from N-terminus to C terminus, containing the following SCRs: [1-4], [1-5], [1-6], [1 -7], [1-20], [19-20], [1 -4 and 19-20], and [1-5]

[0081] 25 and [19-20], A functional fragment” or a “biologically active fragment” of FHRP5 protein is one having some or all of the activities of FHRP5, e.g., complement alternative pathway regulatory activity of the full-length FHRP5 protein. Examples include, but are not limited to, FHRP5 fragments, from N-terminus to C-terminus, containing the following SCRs: [7-8], As used herein, the term “fused” or “joined” refers to the combination or attachment of two or more elements, components, or protein domains, e.g., polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., disulfide bonds and amide bonds. For example, two single polypeptides can be joined to form one contiguous protein structure through recombinant expression, chemical conjugation, a chemical bond, a peptide linker, or any other means of covalent linkage.

[0082] As used herein, the term “fusion protein” refers to a composite polypeptide made up of two (or

[0083] 35 more) distinct, heterologous polypeptides. The heterologous polypeptides can either be full-length proteins or fragments of full-length proteins. Fusion proteins herein can be prepared by either synthetic or recombinant techniques known in the art.

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[0087] As used herein, the term “host cell" refers to any kind of cellular system that can be engineered to generate the fusion proteins described herein. Non-limiting examples of host cells include Expi CHO-S, Expi 293 F, HEK, HEK 293, HT-1080, CHO, Pichia pastoris, Saccharomyces cerevisiae, and transformable insect cells such as High Five, Sf9, and Sf21 cells.

[0088] 5 As used herein, the term “integrin recognition motif’ refers to a polypeptide oligomer of repeating arginylglycylaspartic acid moieties, e.g., (RGD)i-s, such as (RGD)I-4 (SEQ ID NO: 4). In some embodiments, the arginylglycylaspartic acid moieties may be cyclized.

[0089] As used herein, the term “intrarenal residence time” refers to a time period during which a compound, such as Compound A described herein, is present in extravascular compartments, for example along the kidney epithelium or within Bowman’s capsule within the kidney. The intrarenal residence time may be measured using longitudinal in vivo imaging. For example, in animal studies, the MS Spectrum Imaging System (PerkinElmer Inc., Waltham, MA) can be used for image acquisition. Fluorescent imaging analysis can be performed using Living Image 4.5.1 software (PerkinElmer Inc., Waltham, MA), with automatic 2D epi-illumination exposure settings, field of view (FOV) C, F / Stop 2,

[0090] 15 medium binning and 800nm emission / 750nm excitation filters, with subjects receiving, for example, 1 mg / kg of AlexaFluor 750-labeled test article via intravenous injection. In clinical settings, for example, longitudinal in vivo imaging may be accomplished using radiolabeled test article and PET or SPECT imaging.

[0091] As used herein, the terms “linker,” “L1 ,” and “L2” refer to a linkage between two elements, e.g.,

[0092] 20 polypeptides or protein domains. A linker can be a covalent bond. A linker can also be a molecule of any length that can be used to couple, for example, a factor H fragment and / or a VHH and / or an integrin recognition motif. A linker also refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1 -200 amino acid, 1-150 amino acid, 1-100, a 5-50 amino acid, or a 1-10 amino acid sequence, such as amino acids with smaller side chains and / or flexible amino acid sequences)

[0093] 25 occurring between two polypeptides or polypeptide domains to provide space and / or flexibility between the two polypeptides or polypeptide domains. An amino acid linker may be part of the primary sequence of a polypeptide (e.g., joined to the linked polypeptides or polypeptide domains via the polypeptide backbone). Non-limiting examples include (G4A)2G4S, G4A, (G4A)3, and (G4A)2GsAG4S (SEQ ID NOs: 12, 13, 14, and 15).

[0094] As used herein, the term “patient in need thereof’ or “subject in need thereof,” refers to a subject in need of treatment, e.g., based on the presence of a disease or disorder (e.g., one or more symptoms of the disease or disorder). A subject can be identified as having a need for treatment of a disease or disorder (e.g., kidney disorders, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic

[0095] 35 syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia reperfusion injury, or rejection of a transplanted organ, such as a

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[0098] PATENT ATTORNEY DOCKET NO.: 50694-108001 kidney) prior to administration of a treatment. In some embodiments, the disease is FSGS, and the need for treatment is based upon an earlier diagnosis by a person of skill in the art (e.g., a physician). For example, a patient is a mammal, such as a human.

[0099] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein to refer to

[0100] 5 polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component, among others.

[0101] “Percent (%) sequence identity,” with respect to a reference polynucleotide or polypeptide sequence, is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, or Megalign software.

[0102] 15 Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can

[0103] 20 alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0104] 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence

[0105] 25 alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0106] By “pharmaceutical composition” is meant any composition that contains a therapeutically or biologically active agent (e.g., fusion protein) that is suitable for administration to a subject. Any of these formulations can be prepared by well-known and accepted methods in the art. See, for example, Remington: The Science and Practice of Pharmacy (21st ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is hereby incorporated by reference.

[0107] 35 As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms, which are suitable for contact with the tissues of a subject, such as a

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[0110] PATENT ATTORNEY DOCKET NO.: 50694-108001 mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio.

[0111] The terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, or analogs thereof. A

[0112] 5 polynucleotide may include modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure described herein that is a doublestranded polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0113] As used herein, the terms “short complement regulator”, or “SCR”, also known as “short consensus repeat”, “sushi domains,” or “complement control protein” or “CCP,” describe domains found in all regulators of complement activation (RCA) gene clusters that contribute to their ability to regulate

[0114] 15 complement activation in the blood or on the cell surface to which they specifically bind. SCRs typically are composed of about 60 amino acids, with four cysteine residues disulfide bonded in a 1-3, 2-4 arrangement and a hydrophobic core built around an almost invariant tryptophan residue. SCRs are found in proteins including, but not limited to, factor H and FHRP5.

[0115] As used herein, the terms “single domain antibody” and “VHH” define molecules formed by a

[0116] 20 single immunoglobulin domain. Single domain antibodies include antibodies whose complementary determining regions (“CDRs”) are part of a single domain polypeptide. Single domain antibodies often include an antibody or antigen binding fragment thereof that specifically binds a single antigen (e.g., the VHH antibody binds an antigen with a KD of 1X10-6M or lower, e.g., a KD in the range of 1x10-6M to 1x10-12M, such as a Ko of 1x10-7M, 1x10-8M, 1x109M, 1x10'1° M, 1x10-11M, and 1x10-12M). Generally, the

[0117] 25 antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. The single variable domain may, for example, include a light chain variable domain sequence (a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or VHH sequence), or a suitable fragment thereof. Such antibodies can be derived, for example, from antibodies raised in Camelidae species, for example, in a camel, dromedary, llama, alpaca, or guanaco. Additional antibodies include, for example, immunoglobulin new antigen receptor (IgNAR) of cartilaginous fishes (e.g., sharks, e.g., nurse sharks). Other species besides Camelidae and cartilaginous fishes may produce antibodies whose CDRs are part of a single polypeptide. Antibodies can be prepared by either synthetic or recombinant techniques known in the art.

[0118] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not

[0119] 35 limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0120] 9

[0121] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0122] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0123] By “therapeutically effective amount” is meant the amount of a composition administered to improve, inhibit, or ameliorate a condition of a subject, or a symptom of a disorder or disease in a clinically relevant manner. Any improvement in the subject is considered sufficient to achieve treatment. In some embodiments, an amount sufficient to treat is an amount that reduces, inhibits, or prevents the

[0124] 5 occurrence or one or more symptoms of the disease or disorder (e.g., a disease or disorder mediated by complement alternative pathway activation or dysregulation) or is an amount that reduces the severity of, or the length of time during which a subject suffers from one or more symptoms of the disease or disorder, for example, any disease or disorder mediated by CAP activation or dysregulation, (e.g., by at least about 10%, about 20%, or about 30%, such as by at least about 50%, about 60%, or about 70%, and for example by at least about 80%, about 90%, about 95%, about 99%, or more, relative to a control subject that is not treated with a composition described herein). An effective amount of the pharmaceutical composition used to practice the methods described herein (e.g., the treatment of kidney diseases) may vary depending upon the manner of administration and the age, body weight, and general health of the subject being treated. A physician or researcher can decide the appropriate amount and

[0125] 15 dosage regimen. Dosage can vary, and can be administered in one or more dose administrations daily, weekly, monthly, or yearly, for one or several days.

[0126] As used herein, the terms “treatment,” “treating,” or “treat” refer to therapeutic treatment, in which the object is to inhibit or lessen an undesired physiological change or disorder or to promote a beneficial phenotype in a patient. For example, “treatment,” “treating,” or “treat” refer to clinical intervention in an

[0127] 20 attempt to alter the natural course of an individual’s affliction, disease, or disorder. The terms include, for example, prophylaxis before or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration, or palliation of the disease state, and improved prognosis. In some

[0128] 25 embodiments, fusion proteins are used to control the cellular and clinical manifestations of kidney disorders, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia reperfusion injury, or rejection of a transplanted organ, such as a kidney. In some embodiments, the disease is FSGS.

[0129] A “variant” refers to a polynucleotide or a polypeptide that is substantially homologous to a native or reference polynucleotide or polypeptide. For example, a variant polynucleotide is substantially homologous to a native or reference polynucleotide but has a polynucleotide sequence different from that

[0130] 35 of the native or reference polynucleotide because of one or a plurality of deletions, insertions, and / or substitutions. In another example, a variant polypeptide is substantially homologous to a native or reference polypeptide but has an amino acid sequence different from that of the native or reference

[0131] 10

[0132] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0133] PATENT ATTORNEY DOCKET NO.: 50694-108001 polypeptide because of one or a plurality of deletions, insertions, and / or substitutions. Variant polypeptide sequences encoding polynucleotide sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference polynucleotide sequence, that encode a variant protein or fragment thereof that retains activity. A wide

[0134] 5 variety of mutagenesis approaches are known in the art and can be applied by a person of ordinary skill in the art. A variant polynucleotide or polypeptide sequence can be at least 80%, at least 85%, at least at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings).

[0135] A “vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral

[0136] 15 vectors, liposomes, and other gene delivery vehicles.

[0137] DETAILED DESCRIPTION

[0138] Described herein are methods of treating focal segmental glomerulosclerosis (FSGS) in a subject in need thereof using a fusion protein that includes Complement Factor H (FH) functional domain short

[0139] 20 consensus repeats (SCRs) 1-5 for providing Factor l-mediated cofactor activity and decay acceleration functions through C3b binding. The fusion proteins described herein for treating FSGS include a singledomain, variable heavy chain only (VHH) camelid antibody, to enable kidney epithelial cell deposition, improve expression, facilitate purification, and provide an exogenous probe for detection. Collectively, the use of targeting residues, such as an integrin recognition motif (e.g., RGD loop), coupled with the

[0140] 25 inherent clearance kinetics of low molecular weight proteins, provide selective localization of CAP inhibitor fusion proteins to renal epithelial cells.

[0141] Fusion Proteins

[0142] As described herein, fusion proteins of the disclosure include a fragment of factor H and may include an integrin recognition motif. The constructs can be used as therapeutic agents to treat diseases mediated by complement alternative pathway activation or dysregulation (e.g., FSGS).

[0143] In humans, several regulatory proteins are encoded by a cluster of genes located on the long arm of chromosome 1 . This region is called the regulator of complement activation (RCA) gene cluster. Although the proteins within the RCA family vary in size, they share primary amino acid structure

[0144] 35 similarities. The best studied members of the RCA family are factor H, FHL-1 , CR1 , DAF, MCP, and C4b-binding protein (C4BP). The members of this family are organized in tandem structural units, termed

[0145] 11

[0146] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0147] PATENT ATTORNEY DOCKET NO.: 50694-108001 short consensus repeats (SCRs), which are present in multiple copies in the protein. Each SCR consists of -60-70 highly conserved amino acids, including four cysteine residues.

[0148] In some embodiments, the portion of the fusion protein suitable for inhibiting activity of the complement alternative pathway is fused with a VHH, for increased duration of effect.

[0149] 5 In certain embodiments, the portion of the fusion protein suitable for inhibiting activity of the complement alternative pathway includes a fragment of factor H. The fragment of factor H may include at least the first four N-terminal SCR domains of factor H (e.g., SCRs 1 , 2, 3, and 4). In certain embodiments, the fragment of factor H includes at least the first five N-terminal SCR domains of factor H (e.g., SCRs 1 , 2, 3, 4, and 5); also known as the cofactor and decay accelerating domains. In certain embodiments, the fragment of factor H includes at least the first six N-terminal SCR domains of factor H (e.g., SCRs 1 , 2, 3, 4, 5, and 6).

[0150] In some embodiments, the fragment of factor H may include a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 2.

[0151] 15 The fusion protein may include, in addition to a fragment of factor H, an integrin binding domain. The fragment of factor H in the fusion protein may include at least the first four, five, or six N-terminal SCR domains of factor H and the integrin binding domain may include an arginylglycylaspartic acid (RGD) peptide motif. The arginylglycylaspartic acid peptide motif may include a cyclo(RGD)4 peptide (SEQ ID NO: 4). In some embodiments, the fusion protein may include an integrin binding domain which includes

[0152] 20 a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 4.

[0153] In certain embodiments, the fragment of factor H includes at least the first five N-terminal SCR domains of factor H (e.g., SCRs 1 , 2, 3, 4, and 5) and the integrin binding domain includes a cyclo(RGD)4 peptide. In certain embodiments, the fragment of factor H includes at least the six five N-terminal SCR

[0154] 25 domains of factor H (e.g., SCRs 1 , 2, 3, 4, 5 and 6), and the integrin binding domain includes a cyclo(RGD)4 peptide.

[0155] In some embodiments, the fragment of the factor H portion of the fusion protein is a functional fragment of wild-type factor H. In some embodiments, the factor H, or fragment thereof, portion of the fusion protein is derived from a substituted (e.g., conservatively substituted) factor H or an engineered factor H (e.g., a factor H engineered to increase stability, activity, and / or other desirable properties of the protein, as determined by a predictive model or assay known to one of skill in the art, such as described herein).

[0156] Amino acid substitutions can be introduced into the fusion proteins described herein to improve functionality. For example, amino acid substitutions can be introduced into the fragment of factor H, the

[0157] 35 integrin binding domain, or the fragment of FHRP5, wherein an amino acid substitution increases binding affinity of the fragment of factor H, the integrin binding domain, or the fragment of FHRP5 for its ligand(s).

[0158] 12

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[0160] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0161] Similarly, amino acid substitutions can be introduced into the fragment of factor H, or fragment thereof, to increase functionality and / or to improve the pharmacokinetics of the fusion protein.

[0162] In certain embodiments, the fusion proteins described herein can be fused with another compound, such as a compound to increase the half-life of the polypeptide and / or to reduce potential

[0163] 5 immunogenicity of the fusion protein (for example, polyethylene glycol (PEG)). PEG can be used to improve water solubility, reduce the rate of kidney clearance, and reduce immunogenicity of the fusion protein (see, e.g., U.S. Pat. No. 6,214,966, the disclosure of which is incorporated herein by reference). The fusion proteins described herein can be PEGylated by any means known to one skilled in the art.

[0164] The fragment of factor H may be prepared by a number of synthetic methods of peptide synthesis by fragment condensation of one or more amino acid residues, according to conventional peptide synthesis methods known in the art (Amblard, M. et a / ., Mol. Biotechnol., 33:239-54, 2006).

[0165] Alternatively, a fragment of factor H, integrin binding domain, and / or fragment of FHRP5 may be produced by expression in a suitable prokaryotic or eukaryotic system. In some embodiments, a DNA construct may be inserted into a plasmid vector adapted for expression in a suitable host cell (such as E.

[0166] 15 coli) or a yeast cell (such as S. cerevisiae or P. pastoris), or into a baculovirus vector for expression in an insect cell, or a viral vector for expression in a mammalian cell. Examples of suitable mammalian cells for recombinant expression include, e.g., a human embryonic kidney cell (HEK) (e.g., HEK 293), a Chinese Hamster Ovary (CHO) cell, L cell, C127 cell, 3T3 cell, BHK cell, or COS-7 cell. Suitable expression vectors include the regulatory elements necessary and sufficient for expression of the DNA in the host

[0167] 20 cell. In some embodiments, a leader or secretory sequence or a sequence that is employed for purification of the fusion protein (e.g., a histidine tag), can be included in the fusion protein. The fragment of factor H, integrin binding domain, and / or fragment of FHRP5 produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art (See, e.g., Structural Genomics Consortium, Nat. Methods, 5:135-46, 2008).

[0168] 25 In certain embodiments, the cyclized integrin binding domain and fragments of FHRP5 are also produced by the same methods described for the expression and purification of fragments of factor H.

[0169] In some embodiments, the fusion protein has the structure, from N-terminus to C-terminus, of Formula I:

[0170] D1-L1-D2-L2-D3

[0171] Formula I wherein:

[0172] D1 is a fragment of FH (e.g., a fragment of FH with the amino acid sequence of SEQ ID NO: 2 or a variant thereof with 85% sequence identity thereto or greater);

[0173] L1 is absent (e.g., L1 is a covalent bond between D1 and D2 or between D1 and D3);

[0174] 35 D2 is a VHH domain, such as a single domain antibody (e.g., camelid single-domain antibody VHH with the amino acid sequence of SEQ ID NO: 3 or a variant thereof with 85% sequence identity thereto or greater);

[0175] 13

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[0177] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0178] L2 is absent (e.g., L2 is a covalent bond between D2 and D3), or is a linker with an amino acid sequence of at least one amino acid (e.g., the linker may have the amino acid sequence of any one of SEQ ID NOs: 16-98 or a variant thereof with 85% sequence identity thereto or greater; in particular embodiments the linker may have the amino acid sequence of SEQ ID NO: 16) between D2 and D3; and

[0179] 5 D3 is an integrin recognition domain (e.g., an arginylglycylaspartic acid (RGD) peptide motif e.g., cyclo(RGD)4 with the amino acid sequence of SEQ ID NO: 4 or a variant thereof with 85% sequence identity thereto or greater).

[0180] In some embodiments, the FH SCR domains are selected from the group consisting of SCR [1-5] or a variant thereof with at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to:

[0181] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQ KRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVK CLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEI SCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIP NGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLK (SEQ ID NO: 2).

[0182] 15 L1 and L2 may be linkers of the same type and / or sequence or of a different type and / or sequence.

[0183] In some embodiments, the composition of Formula I includes the amino acid sequence of SEQ ID NO: 1 , as shown below, and variants thereof with at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto:

[0184] 20 EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQ

[0185] KRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEWK CLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEI SCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIP NGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKQVQLVESGGGLVKP

[0186] 25 GGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYADSVKGRFTISRDNAKN

[0187] SLYLQMNSLRAEDTAVYYCAADLGDGSWVDYVNAEPYEYDYWGQGTLVTVSSGGGGACRGD RGDRGDRGDC (SEQ ID NO: 1).

[0188] In some embodiments, the composition of Formula I is encoded by the nucleic acid sequence of SEQ ID NO: 5, as shown below, and variants thereof with at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto: gaggattgcaatgagctgcctcctcggagaaacaccgagatcctgacaggctcttggagcgaccagacataccctgagggcaccca ggccatctacaagtgcagacctggctacagatccctgggcaacgtgatcatggtctgcagaaaaggcgagtgggtcgccctgaatcct ctgagaaagtgccagaagaggccttgcggacaccctggcgataccccttttggcacattcacactgaccggcggcaacgtgttcgagt atggcgtgaaggccgtgtacacctgtaacgagggatatcagctgctgggcgagatcaactacagagagtgtgataccgacggctgga

[0189] 35 ccaacgacatccctatctgcgaggtggtcaagtgcctgcctgtgacagcccctgagaatggcaagatcgtgtccagcgccatggaacc cgacagagagtatcactttggccaggccgtcagattcgtgtgcaacagcggctataagatcgagggcgacgaggaaatgcactgcag cgacgacggcttctggtccaaagaaaagcctaagtgcgtggaaatcagctgcaagagccccgacgtgatcaacggcagccctatca

[0190] 14

[0191] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0192] PATENT ATTORNEY DOCKET NO.: 50694-108001 gccagaagatcatctacaaagagaacgagcggttccagtacaagtgtaacatgggctacgagtacagcgagaggggcgacgccgt gtgtacagaatctggatggcgacctctgcctagctgcgaggaaaagagctgcgacaacccttacatccccaacggcgactacagccct ctgcggattaagcacagaaccggcgacgagatcacctaccagtgcagaaatggcttctaccccgccaccagaggcaataccgccaa gtgtacaagcaccggctggatccctgctcctagatgcacactgaaacaggtgcagctggtggaatctggcggcggacttgtgaaacctg

[0193] 5 gcggctctctgagactgtcttgtgccgccagcggcagaacctttagcagctacgccatgggatggttcagacaggcccctggcaaaga gagagagttcgttagcgccatcagcggctctggcggcagcacatattacgccgatagcgtgaagggcagattcaccatcagccggga caacgccaagaacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccgctgatctcggagat ggcagctgggtcgactatgtgaatgccgagccttacgagtacgactactggggccagggcacactggttacagttagttctggcggagg cggagcctgtaggggagacagaggcgatagaggcgacaggggagattgc (SEQ ID NO: 5).

[0194] Immunoglobin Proteins and Domains

[0195] The fusion proteins described herein may contain a single chain VHH domain. Such antibodies exist naturally in camelids and sharks (Saerens et al., Curr. Opin. Pharmacol., 8:600-608, 2008). Camelid antibodies are described in, for example, U.S. Pat. Nos 5,759,808; 5,800,988; 5,840,526; 5,874,541 ;

[0196] 15 6,005,079; and 6,015,695, the entire contents of each are incorporated herein by reference.

[0197] Exemplary VHH domains include those having the sequence of:QVQLVESGGGLVKPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAADLGDGSWVDYVNAEPYEYDYWGQGTL VTVSS (SEQ ID NO: 3).

[0198] 20 In some embodiments, the fusion protein may include a VHH domain which includes a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 3.

[0199] The fusion protein may include, from N-terminus to C-terminus, D1-L1-D2-L2-D3, in which D1 includes a fragment of an FH protein, such as FH SCR 1-5 or FH SCR 1-6, L1 is absent or includes a

[0200] 25 linker, D2 includes a VHH domain, L2 is absent or includes a linker, and D3 includes an integrin recognition domain, such as cyclo(RGD)4.

[0201] In some embodiments, the factor H fusion protein including a VHH domain has an increased intrarenal residence time along the kidney epithelial surface relative to a fusion protein lacking the VHH domain. Without being bound to a particular theory, the size of the fusion proteins described herein (e.g., about < 60 kDa (such as less than 60 kDa)) is believed to enable the fusion proteins to gain entry to extravascular compartments within the kidney inaccessible by monoclonal antibodies and albumin-bond bispecifics, and the use of a VHH domain in the fusion proteins described herein is believed to enable the fusion proteins to deposit on the apical membrane of proximal tubule and parietal epithelial cells, where naturally low levels of membrane-associated surface regulators confer susceptibility to CAP products, and

[0202] 35 to exhibit extended residence along the kidney epithelium. In some embodiments, the intrarenal residence time is increased by at least 1 fold (e.g., 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold) relative to a fusion protein lacking the VHH domain. In some embodiments, the intrarenal

[0203] 15

[0204] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0205] PATENT ATTORNEY DOCKET NO.: 50694-108001 residence time is between 24 hours and 96 hours (e.g., between 36 hours and 96 hours, 48 hours and 96 hours, 60 hours and 96 hours, 72 hours and 96 hours, and 60 hours and 84 hours).

[0206] Integrin Binding Domain

[0207] 5 The fusion protein may also have an integrin binding domain, which may act as a targeting motif to improve the pharmacokinetics of the fusion protein and mediate renal cell-specific targeting at sites of damage or remodeling. The integrin binding domain may be added as an additional domain to any one of the fusion proteins described herein.

[0208] Exemplary integrin binding domains include one or more cyclic arginylglycylaspartic acid (RGD) peptide motifs fused to either the N- or C-terminus of the fusion protein. RGD motifs engage the extracellular domains of integrin a- and p-subunits on the cell surface that can be upregulated in response to injury (e.g., including renal fibrosis mediated by TGF-p signaling). Without being bound to a particular theory, it is expected that the inclusion of a cyclic RGD motif may also limit pro-TGF-p ligand binding and prevent pro-fibrotic signaling. Different variants of integrin binding motifs can be constructed and

[0209] 15 attached to the fusion protein. In some embodiments, the fusion protein may include an integrin binding domain which includes an amino sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to CRGDRGDRGDRGDC (SEQ ID NO: 4).

[0210] The fusion protein may include, from N-terminus to C-terminus, D1-L1-D2-L2-D3, in which D1 includes a fragment of an FH protein, such as an FH with SCR [1-5] or an FH with SCR [1-6], The fusion

[0211] 20 protein may have the amino acid sequence of SEQ ID NO: 1 or variant thereof with at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to SEQ ID NO: 1 . The fusion protein may also have a fragment of a FH protein, such as an FH with SCR [1-5] or an FH with SCR [1 -6], Linkers for the Fusion Proteins

[0212] The L1 and L2 domains of the fusion proteins described herein are linkers. A linker is used to

[0213] 25 create a linkage or connection between, for example, polypeptides, or protein domains. For example, a fragment of factor H may be linked directly to a VHH domain (e.g., single-domain camelid VHH domain) by one or more suitable linkers. A linker can be a simple covalent bond, e.g., a peptide bond, a synthetic polymer, e.g., a PEG polymer, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. The peptide linker can be, for example, a linker of one or more amino acid residues inserted or included at the transition between the two domains (e.g., a fragment of the FH protein and a VHH domain). The identity and sequence of amino acid residues in the linker may vary depending on the desired secondary structure. For example, glycine, serine, and alanine are useful for linkers given their flexibility. Any amino acid residue can be considered as a linker in combination with one or more other amino acid residues, which may be the same as or different from the first amino acid residue, to construct

[0214] 35 larger peptide linkers as necessary depending on the desired length and / or properties.

[0215] A variety of linkers can be used to fuse two or more protein domains together (e.g., a fragment of factor H and a VHH domain). Linkers may be flexible, rigid, or cleavable. Linkers may be structured or

[0216] 16

[0217] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0218] PATENT ATTORNEY DOCKET NO.: 50694-108001 unstructured. The residues for the linker may be selected from naturally occurring amino acids, non-naturally occurring amino acids, and modified amino acids. The linker may include at least 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more amino acid residues. Peptide linkers can include, but are not limited to, glycine linkers, glycine-rich linkers, serine-glycine linkers, and the like.

[0219] 5 A glycine-rich linker includes at least about 50% glycine.

[0220] In some embodiments, the linker(s) used confers one or more other favorable properties or functionality to the polypeptide(s) described herein, and / or provides one or more sites for the formation of derivatives and / or for the attachment of functional groups. For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification, and / or purification. A skilled artisan will be able to determine the optimal linkers for use in a specific polypeptide.

[0221] When two or more linkers are used for a polypeptide, the linkers may be the same or different.

[0222] Linkers can contain motifs, e.g., multiple or repeating motifs. In one embodiment, the linker has the amino acid sequence GS, or repeats thereof (Huston, J. et al., Methods Enzymol., 203:46-88, 1991).

[0223] 15 In another embodiment, the linker includes the amino acid sequence EK, or repeats thereof (Whitlow, M. et al., Protein Eng., 6:989-95, 1993). In another embodiment, the linker includes the amino acid sequence GGS, or repeats thereof.

[0224] In another embodiment, the linker includes the amino acid sequence GGGGA (SEQ ID NO: 16) or repeats thereof. In certain embodiments, the linker contains more than one repeat of GGS or GGGGS

[0225] 20 (US Pat. No. 6,541 ,219, the entire contents of which are herein incorporated by reference). In one embodiment, the peptide linker may be rich in small or polar amino acids, such as G and S, but can contain additional amino acids, such as T and A, to maintain flexibility, as well as polar amino acids, such as K and E, to improve solubility.

[0226] Exemplary linkers include, but are not limited to: G4S (SEQ ID NO: 17), (G4A)2G4S (SEQ ID NO:

[0227] 25 18), (G4A)2G3AG4S (SEQ ID NO: 19), G4AG3AG4S (SEQ ID NO: 20), G4SDA (SEQ ID NO: 21), G4SDAA (SEQ ID NO: 22), G4S (SEQ ID NO: 17), (G4S)2(SEQ ID NO: 24), (G4S)3(SEQ ID NO: 25), (G4S)4(SEQ ID NO: 26), (G4S)5(SEQ ID NO: 27), (G4S)6(SEQ ID NO: 28), EAAAK (SEQ ID NO: 29), (EAAAK)3(SEQ ID NO: 30), PAPAP (SEQ ID NO: 31), G4SPAPAP (SEQ ID NO: 32), PAPAPG4S (SEQ ID NO: 33), GSTSGKSSEGKG (SEQ ID NO: 34), (GGGDS)2(SEQ ID NO: 35), (GGGES)2(SEQ ID NO: 36), GGGDSGGGGS (SEQ ID NO: 37), GGGASGGGGS (SEQ ID NO: 38), GGGESGGGGS (SEQ ID NO: 39), ASTKGP (SEQ ID NO: 40), ASTKGPSVFPLAP (SEQ ID NO: 41), G3P (SEQ ID NO: 42), G7P (SEQ ID NO: 43), PAPNLLGGP (SEQ ID NO: 44), G6(SEQ ID NO: 45), G12 (SEQ ID NO: 46), APELPGGP (SEQ ID NO: 47), SEPQPQPG (SEQ ID NO: 48), (G3S2)3(SEQ ID NO: 49), GGGGGGGGGSGGGS (SEQ ID NO: 50), GGGGSGGGGGGGGGS (SEQ ID NO: 51), (GGSSS)3(SEQ ID NO: 52), (GS4)3(SEQ

[0228] 35 ID NO: 53), G4A(G4S)2(SEQ ID NO: 54), G4SG4AG4S (SEQ ID NO: 55), G3AS(G4S)2 (SEQ ID NO: 56), G4SG3ASG4S (SEQ ID NO: 57), G4SAG3SG4S (SEQ ID NO: 58), (G4S)2AG3S (SEQ ID NO: 59), G4SAG3SAG3S (SEQ ID NO: 60), G4D(G4S)2(SEQ ID NO: 61), G4SG4DG4S (SEQ ID NO: 62), (G4D)2G4S

[0229] 17

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[0231] PATENT

[0232] ATTORNEY DOCKET NO.: 50694-108001

[0233] (SEQ ID NO: 63), G4E(G4S)2(SEQ ID NO: 64), G4SG4EG4S (SEQ ID NO: 65), and (G4E)2G4S (SEQ ID NO: 66), (GGGGS)n, wherein n can be any number, KESGSVSSEQLAQFRSLD (SEQ ID NO: 67), and EGKSSGSGSESKST (SEQ ID NO: 68), (Gly)a (SEQ ID NO: 69), GSAGSAAGSGEF(SEQ ID NO: 70), and (Gly)s (SEQ ID NO: 71). Exemplary rigid linkers include but are not limited to A(EAAAK)A (SEQ ID

[0234] 5 NO: 72), A(EAAAK)nA, wherein n can be any number, or (XP)nwherein n can be any number, with X designating any amino acid. Exemplary in vivo cleavable linkers include, for example, LEAGCKNFFPRSFTSCGSLE (SEQ ID NO: 73), GSST (SEQ ID NO: 23), and CRRRRRREAEAC (SEQ ID NO: 74). In some embodiments, a linker can contain 2 to 12 amino acids including motifs of GS, e.g., GS, GSGS (SEQ ID NO: 75), GSGSGS (SEQ ID NO: 76), GSGSGSGS (SEQ ID NO: 77), GSGSGSGSGS (SEQ ID NO: 78), or GSGSGSGSGSGS (SEQ ID NO: 79). In certain other embodiments, a linker can contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 80), GGSGGSGGS (SEQ ID NO: 81), and GGSGGSGGSGGS (SEQ ID NO: 82). In yet other embodiments, a linker can contain 4 to 12 amino acids including motifs of GGSG, e.g., GGSG (SEQ ID NO: 83), GGSGGGSG (SEQ ID NO: 84), or GGSGGGSGGGSG (SEQ ID NO: 85). In other

[0235] 15 embodiments, a linker can contain motifs of GGGGS (SEQ ID NO: 17). In other embodiments, a linker can also contain amino acids otherthan glycine and serine, e.g., GENLYFQSGG (SEQ ID NO: 86), SACYCELS (SEQ ID NO: 87), RSIAT (SEQ ID NO: 88), RPACKIPNDLKQKVMNH (SEQ ID NO: 89), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGT GSG (SEQ ID NO: 90), AAANSSIDLISVPVDSR (SEQ ID NO: 91), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 92),

[0236] 20 GGGGAGGGGAGGGGS (SEQ ID NO: 93), GGGGAGGGGAGGGGAGGGGS (SEQ ID NO: 94), DAAGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 95), GGGGAGGGGAGGGGA (SEQ ID NO: 96), GGGGAGGGGAGGGAGGGGS (SEQ ID NO: 97), or GGSSRSSSSGGGGAGGGG (SEQ ID NO: 98).

[0237] In one embodiment, the linker is a cleavable linker, such as an enzymatically cleavable linker.

[0238] 25 Inclusion of a cleavable linker can aid in detection of the fusion protein. An enzymatically cleavable linker can be cleavable, for example, by trypsin, Human Rhinovirus 3C Protease (3C), enterokinase (Ekt), Factor Xa (FXa), Tobacco Etch Virus protease (TEV), or thrombin (Thr). Cleavage sequences for each of these enzymes are well known in the art. For example, trypsin cleaves peptides on the C-terminal side of lysine and arginine amino acid residues. If a proline residue is on the carboxyl side of the cleavage site, the cleavage will not occur. If an acidic residue is on either side of the cleavage site, the rate of hydrolysis has been shown to be slower. The following linkers are examples of linkers that can be cleaved using trypsin: K(G4A)2G3AG4SK, R(G4A)2G3AG4SR, K(G4A)2G3AG4SR, R(G4A)2G3AG4SK, K(G4A)2G4SK, K(G4A)2G4SR, R(G4A)2G4SK, and R(G4A)2G4SR.

[0239] An example of a protease cleavage site that can be included in an enzymatically cleavable linker

[0240] 35 is a tobacco etch virus (TEV) protease cleavage site, e.g., ENLYTQS, where the protease cleaves between the glutamine and the serine. Another example of a protease cleavage site that can be included in an enzymatically cleavable linker is an enterokinase cleavage site, e.g., DDDDK, where cleavage

[0241] 18

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[0243] PATENT ATTORNEY DOCKET NO.: 50694-108001 occurs after the lysine residue. Another example of a protease cleavage site that can be included in an enzymatically cleavable linker is a thrombin cleavage site, e.g., LVPR. For Human Rhinovirus 3C Protease, the cleavage site is LEVLFQGP where cleavage occurs between the glutamine and glycine residues. A cleavage site for Factor Xa protease is IEDGR, where cleavage occurs between the glutamic

[0244] 5 acid and aspartic acid residues.

[0245] The inclusion of the cleavable linker is useful in that it has a sequence of amino acids that is unique from other peptides in the human proteome that are generated with the above-mentioned enzymes. As such this excised linker may serve as a unique identifying peptide of the fusion protein when administered as a pharmaceutical preparation to humans. In this way, the cleavable linker may be detected and quantitated by mass spectrometry and be used to monitor the pharmacokinetics of the fusion protein.

[0246] In another embodiment, the linker is a polymeric or oligomeric glycine linker, and can include a lysine at the N-terminus, the C-terminus, or both the N- and the C-termini.

[0247] With reference to Formulas I and III above, the C-terminus of D1 may be linked to the N-terminus

[0248] 15 of D2. In a certain embodiment, the C-terminus of the FH fragment is linked to the N-terminus of a VHH. In certain embodiments, the C-terminus of an integrin binding domain is linked to the N-terminus of a VHH. In a certain embodiment, the C-terminus of D2 may be linked to the N-terminus of D3. In a certain embodiment, the C-terminus of a VHH may be linked to the N-terminus of an integrin binding domain. In a certain embodiment, the C-terminus of the VHH may be linked to the N-terminus of the FH fragment. In

[0249] 20 another example, the C-terminus of D1 may be linked to the N-terminus of D3. In certain embodiments, the C-terminus of the FH fragment is linked to the N-terminus of the integrin binding domain. In a certain embodiment, the C-terminus of the integrin binding domain is linked to the N-terminus of the FH fragment. In another example, the C-terminus of D2 may be linked to the N-terminus of D3. In certain embodiments, the C-terminus of the VHH may be linked to the N-terminus of the integrin binding domain. In certain embodiments, the C-terminus of the VHH may be linked to the N-terminus of the FH fragment.

[0250] With reference to Formula II above, the C-terminus of D1 may be linked to the N-terminus of D2. In a certain embodiment, the C-terminus of the FH fragment is linked to the N-terminus of the FHRP5 fragment.

[0251] 30 Table 1 : Compound A with the sequence, from N-terminus to C-terminus, of D1-L1-D2-L2-D3 indicates the absence of a feature.

[0252] 19

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[0254] PATENT

[0255] ATTORNEY DOCKET NO.: 50694-108001

[0256] Production of Fusion Proteins

[0257] Described herein are methods for producing a fusion protein using nucleic acid molecules encoding the fusion proteins, such as the fusion protein shown in Table 1 (e.g., Compound A). The nucleic acid molecule can be operably linked to a suitable control sequence to form an expression unit

[0258] 5 encoding the protein. The expression unit can be used to transform a suitable host cell, and the transformed host cell can be cultured under conditions that allow the production of the recombinant protein. Optionally, the recombinant protein can be isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated. Additional residues may be included at the N- or C-terminus of the protein coding sequence to facilitate purification (e.g., a histidine tag) and, if desired, subsequently removed to form the final protein product.

[0259] The fusion protein can be expressed from a single polynucleotide that encodes the entire fusion protein or as multiple (e.g., two or more) polynucleotides that may be expressed by suitable expression systems or may be co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may

[0260] 15 associate through, e.g., disulfide bonds or other means to form a functional fusion protein. For example, the light chain portion of monoclonal antibody may be encoded by a separate polynucleotide from the heavy chain portion of a monoclonal antibody. When co-expressed in a host cell, the heavy chain polypeptides will associate with the light chain polypeptides to form the monoclonal antibody.

[0261] Typically, a nucleic acid encoding the desired fusion protein is generated using molecular cloning

[0262] 20 methods and is generally placed within a vector, such as a plasmid or virus. The vector is used to transform the nucleic acid into a host cell appropriate for the expression of the fusion polypeptide. Representative methods are disclosed, for example, in Maniatis et al. (Cold Springs Harbor Laboratory, 1989). Many cell types can be used as appropriate host cells, although mammalian cells are often selected because they are able to confer appropriate post-translational modifications. Host cells can

[0263] 25 include, e.g., a Human Embryonic Kidney (HEK) (e.g., HEK 293) cell, Chinese Hamster Ovary (CHO) cell, L cell, C127 cell, 3T3 cell, BHK cell, COS-7 cell, or any other suitable host cell known in the art.

[0264] In one embodiment, a nucleic acid or polynucleotide encoding the fusion protein is provided. In one embodiment, a vector including a nucleic acid or polynucleotide encoding the fusion protein is provided. In one embodiment, a host cell including one or more polynucleotides encoding the fusion protein is provided. In certain embodiments a host cell including one or more fusion expression vectors is provided. The fusion proteins can be produced by expression of a nucleotide sequence in any suitable expression system known in the art. Any expression system may be used, including yeast, bacterial, animal, plant, eukaryotic, and prokaryotic systems. In some embodiments, yeast systems that have been modified to reduce native yeast glycosylation, hyper-glycosylation or proteolytic activity may be used.

[0265] 35 Furthermore, any in vivo expression systems designed for high level expression of recombinant proteins within organisms known in the art can be used for producing the fusion proteins specified herein. In some embodiments, the factor H fusion protein, as described herein, is produced by culturing one or more host

[0266] 20

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[0268] PATENT ATTORNEY DOCKET NO.: 50694-108001 cells including one or more nucleic acid molecules capable of expressing the fusion protein under conditions suitable for expression of the fusion protein. In some embodiments, the factor H fusion protein is obtained from the cell culture or culture medium.

[0269] The fusion protein can also be produced using chemical methods to synthesize the desired amino

[0270] 5 acid sequence, in whole or in part. For example, polypeptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high-performance liquid chromatography (e.g., Creighton (1983) Proteins: Structures and Molecular Principles, WH Freeman and Co, New York N.Y.). The composition of the synthetic polypeptides can be confirmed by amino acid analysis or sequencing. Additionally, the amino acid sequence of a fusion protein or any part thereof, can be altered during direct synthesis and / or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant polypeptide.

[0271] Isolation / Purification of Fusion Proteins

[0272] Secreted, biologically active fusion proteins described herein, such as the fusion protein

[0273] 15 described in Table 1 (e.g., Compound A), may be purified by techniques, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, e.g., protein A affinity chromatography, size exclusion chromatography, and the like, known in the art. The conditions used to purify a particular protein depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., as would be apparent to a skilled artisan.

[0274] 20

[0275] Assays for Fusion Protein Activity

[0276] Hemolytic Assay

[0277] The fusion proteins described herein were assessed for activity using a complement pathway hemolysis assay, which measures complement-mediated lysis of rabbit erythrocytes secondary to

[0278] 25 activation of the alternative pathway on a cell surface. Rabbit erythrocytes generally activate complement-mediated lysis in mouse or human serum. As serum C3 is activated, C3 convertases, C3 activation fragments, and C5 convertases are deposited on rabbit RBCs. Serum complement alternative pathway activity in the presence of a fusion protein comprising a fragment of factor H and a VHH domain, a fragment of factor H and a fragment of FHRP5 or a fragment of factor H, a VHH, and an integrin binding domain (e.g., the fusion protein of Table 1 , e.g., Compound A), for example, were evaluated in a concentration-dependent manner in human or mouse serum supplemented with Mg++ and EGTA as Ca sequestrant, thus favoring the alternative pathway of complement activation. Incubation of rabbit erythrocytes in normal mouse or human serum causes cell lysis, while addition of nanomolar quantities of a fusion protein comprising a fragment of factor H and a VHH domain, or a fragment of factor H and a

[0279] 35 fragment of FHRP5, or a fragment of factor H, a VHH domain, and an integrin binding domain, for example, decreases the degree of lysis. The fusion protein of the disclosure may exhibit a half maximal inhibitory concentration (IC50) of between about 15 nM to about 250 nM (e.g., between about 15 nM to

[0280] 21

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[0282] PATENT ATTORNEY DOCKET NO.: 50694-108001 about 240 nM, between about 15 nM to about 220 nM, between about 200 nM to about 150 nM, between about 15 nM to about 100 nM, between about 15 nM to about 40 nM, or between about 15 nM to about 50 nM . In some embodiments, the fusion protein may exhibit an IC50 of between 19 nM and 240 nM (e.g., between about 19 nM and about 230 nM, about 50 nM and about 240 nM, about 100 nM and about

[0283] 5 240 nM, about 150 nM and about 240 nM, about 200 nM and about 240 nM, about 19 nM and 50 nM, about 19 nM and about 100 nM, about 19 nM and about 150 nM, about 19 nM and about 200 nM, and about 19 nM and about 230 nM).

[0284] Table 2: Complement alternative pathway (CAP) hemolytic assay half maximal inhibitory concentration (IC50) summary

[0285] Complement Activity Assay

[0286] The fusion proteins described herein (e.g., the fusion protein of Table 1 , e.g. Compound A) can

[0287] 15 be evaluated for complement alternative pathway activity in the fluid phase using a complement alternative pathway assay kit, for example, Complement system Alternative Pathway WIESLAB®, Lund, Sweden. This method combines principles of the hemolytic assay for complement activation with the use of labeled antibodies specific for a neoantigen produced as a result of complement activation. The amount of neoantigen generated is proportional to the functional activity of the alternative pathway. In the

[0288] 20 Complement system Alternative Pathway kit, wells of the plate are coated with specific activators of the alternative pathway. Serum is diluted in diluent containing specific blockers to ensure that only the alternative pathway is activated. Anti-properdin VHH, for example, can be spiked into the patient’s blood in a concentration-dependent manner. During the incubation of the diluted patient serum in the wells, complement is activated by the specific coating. The wells are then washed and C5b-9 is detected with a specific alkaline phosphatase-labelled antibody to the neoantigen as a result of complement activation. The amount of complement activation correlates with the color intensity and is measured in terms of absorbance (optical density (OD)) at 405 nm. The addition of nanomolar quantities of a factor H fusion protein according to the disclosure, for example, decreases the degree of activity. Additional exemplary assays for determining complement pathway activity include those described in Hebell et al., (Science

[0289] 30 (1991) 254(5028):102-105).

[0290] Pharmaceutical Compositions, Dosage, and Administration

[0291] The fusion protein described herein (e.g., Compound A; see Table 1) can be incorporated into a pharmaceutical composition for administration to a subject (e.g., a human subject). Pharmaceutical

[0292] 22

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[0294] PATENT ATTORNEY DOCKET NO.: 50694-108001 compositions including factor H fusion proteins described herein can be formulated for administration at individual doses ranging, e.g., from 0.01 mg / kg to 500 mg / kg or in doses of 10 mg to 1 ,000 mg (e.g., 180 mg or 450 mg). In some embodiments, the fusion protein is formulated for administration of about 180 mg of the fusion protein per week. In some embodiments, the fusion protein is formulated for

[0295] 5 administration of about 450 mg of the fusion protein per week. The pharmaceutical composition may contain, e.g., from 0.1 pg / 0.5 mL to 1 g / 5 mL of the fusion protein. In some embodiments, the pharmaceutical composition described herein contains about 1-200 mg / mL, such as about 30-100 mg / mL, for example, between 25 mg / mL and 75 mg / mL (e.g., between 25 mg / mL and 65 mg / mL, 25 mg / mL and 50 mg / mL, 25 mg / mL and 35 mg / mL, 35 mg / mL and 75 mg / mL, 50 mg / mL and 5 mg / mL, 60 mg / mL and 75 mg / mL, 40 mg / mL and 60 mg / mL, or 45 mg / mL and 55 mg / mL). In some embodiments, the pharmaceutical composition comprising Compound A has a concentration of about 50 mg / mL (e.g., 50 mg / mL) of Compound A.

[0296] Compositions including factor H fusion proteins can also be formulated for either a single or multiple dosage regimens. Doses can be formulated for administration, e.g., hourly, bihourly, daily,

[0297] 15 bidaily, twice a week, three times a week, four times a week, five times a week, six times a week, or weekly. In some embodiments, the pharmaceutical composition is formulated for administration to the subject weekly. In some embodiments, the pharmaceutical composition is formulated for administration to the subject biweekly.

[0298] The pharmaceutical compositions including factor H fusion proteins can be formulated according

[0299] 20 to standard methods. Pharmaceutical formulation is a well-established art, and is further described in, e.g., Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000) Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 3rd Edition (ISBN:

[0300] 25 091733096X).

[0301] The pharmaceutical composition can include the fusion protein and at least one pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The term “pharmaceutically acceptable carrier” excludes tissue culture medium including bovine or horse serum. Pharmaceutically acceptable carriers or adjuvants, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. Therefore, pharmaceutically acceptable carriers are inherently non-toxic and nontherapeutic, and are known to the person skilled in the art. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline,

[0302] 35 dextrose, glycerol, ethanol, and the like, as well as combinations thereof. Some embodiments will include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances include minor amounts of auxiliary substances

[0303] 23

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[0305] PATENT ATTORNEY DOCKET NO.: 50694-108001 such as wetting or emulsifying agents, preservatives, stabilizers, or buffers, which enhance the shelf life or effectiveness of the antibody. In some embodiments, the pharmaceutical composition comprises phosphate buffer. The phosphate buffer may have a concentration in the pharmaceutical composition oof between 5 mM and 15 mM (e.g., between 5 mM and 12 mM, 5 mM and 10 mM, 5 mM and 7 mM, 7 mM

[0306] 5 and 15 mM, 10 mM and 15 mM, 12 mM and 15 mM, or 7 mM and 12 mM). For example, the phosphate buffer may have a concentration of about 10 mM. The pharmaceutical composition may include one or more of L-arginine, arginine HCI, sucrose, or polysorbate.

[0307] In some embodiments, the pharmaceutical composition includes between 0.5 mg / mL and 3 mg / mL of L-arginine (e.g., between 0.5 mg / mL and 2.5 mg / mL, 0.5 mg / mL and 2 mg / mL, 0.5 mg / mL and 1 .5 mg / mL, 0.5 mg / mL and 1 mg / mL, 1 mg / mL and 3 mg / mL, 1 .5 mg / mL and 3 mg / mL, 2 mg / mL and 3 mg / mL, 2.5 mg / mL and 3 mg / mL, or 1 .2 mg / mL and 1 .8 mg / mL). For example, the pharmaceutical composition may include about 1 .4 mg / mL of L-arginine. In some embodiments, the pharmaceutical composition may include between 20 mg / mL and 35 mg / mL (e.g., between 20 mg / mL and 30 mg / mL, 20 mg / mL and 25 mg / mL, 25 mg / mL and 35 mg / mL, 30 mg / mL and 35 mg / mL, or 25 mg / mL and 30 mg / mL)

[0308] 15 arginine HCI. For example, the pharmaceutical composition may include about 27.8 mg / mL arginine HCI.

[0309] In some embodiments, the pharmaceutical composition includes between 10 mg / mL and 20 mg / mL (e.g., between 10 mg / mL and 18 mg / mL, 10 mg / mL and 15 mg / mL, 10 mg / mL and 12 mg / mL, 12 mg / mL and 20 mg / mL, 15 mg / mL and 20 mg / mL, 18 mg / mL and 20 mg / mL, or 12 mg / mL and 15 mg / mL) sucrose. For example, the pharmaceutical composition may include about 13.7 mg / mL sucrose.

[0310] 20 In some embodiments, the pharmaceutical composition includes polysorbate 80. In some embodiments, the pharmaceutical composition includes between 0.1 mg / mL and 1.5 mg / mL (e.g., between 0.1 mg / mL and 1.5 mg / mL, 0.1 mg / mL and 1 mg / mL, 0.1 mg / mL and 0.5 mg / mL, 0.5 mg / mL and 1 .5 mg / mL, 1 mg / mL and 1 .5 mg / mL, or 0.2 mg / mL and 0.8 mg / mL) polysorbate. For example, the fusion protein may be formulated with about 0.5 mg / mL polysorbate.

[0311] 25 The compositions described herein may be prepared in a variety of forms. These include, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions. Such formulations can be prepared by methods known in the art such as, e.g., the methods described in Epstein et al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in, e.g., U.S. Patent No. 5,013,556.

[0312] Pharmaceutical compositions including factor H fusion proteins can also be formulated with a carrier that will protect the composition (e.g., a factor H fusion protein) against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides,

[0313] 35 polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art. See, e.g., J.R. Robinson (1978) Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York.

[0314] 24

[0315] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0316] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0317] The final form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The composition(s) can be delivered by, for example, parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, or

[0318] 5 intramuscular injection) or by local administration (e.g., directly to the kidneys). For example, the pharmaceutical composition comprising Compound A may be formulated for subcutaneous administration.

[0319] The pharmaceutical compositions can be provided in a sterile form and stable under the conditions of manufacture and storage.

[0320] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the fusion protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. The form chosen depends, in part, on the intended mode of administration and therapeutic application. For

[0321] 15 example, compositions intended for systemic or local delivery can be in the form of injectable or infusible solutions. The composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage at 2-8°C (e.g., 4°C). A composition can also be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). A composition can further be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six

[0322] 20 months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1 % years, or 2 years) at 2-8°C (e.g., 4°C). Thus, the compositions described herein can be stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

[0323] The fusion proteins described herein can be administered by a variety of methods known in the art, although for many therapeutic applications, the chosen route / mode of administration is intravenous

[0324] 25 injection or infusion. The fusion proteins can also be administered by intramuscular or subcutaneous injection. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. For treating FSGS, the fusion protein (Compound A) can be administered by subcutaneous injection (e.g., once per week).

[0325] The pharmaceutical composition(s) may have a pH of about 6.5-8.0. For example, the pH may be about 6.2, 6.5, 6.75, 7.0, or 7.5, such as pH 7.0. The pharmaceutical compositions may be formulated for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, topical, intravitreal, or parenteral administration. Parenteral administration may include intradermal, subcutaneous (SC, s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (IV), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intravitreal (eye), intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and

[0326] 35 intrathecal (spinal fluids) injection or infusion. SC administration may include an SC infusion or an SC push. Any device suitable for parenteral injection or infusion of drug formulations may be used for such

[0327] 25

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[0329] PATENT ATTORNEY DOCKET NO.: 50694-108001 administration. For example, the pharmaceutical composition may be contained in a sterile pre-filled syringe.

[0330] Additional active compounds can also be incorporated into the composition. In certain embodiments, a fusion protein is co-formulated with and / or co-administered with one or more additional

[0331] 5 therapeutic agents. When compositions are to be used in combination with a second active agent, the compositions can be co-formulated with the second agent, or the compositions can be formulated separately from the second agent formulation. For example, the respective pharmaceutical compositions can be mixed, e.g., just prior to administration, and administered together or can be administered separately, e.g., at the same or different times. In some embodiments, a fusion protein can be co-formulated and / or co-administered with one or more additional antibodies that bind other targets (e.g., antibodies that bind regulators of the complement alternative pathway). Such combination therapies may utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies. Additionally, the compositions described herein can be co-formulated or co-administered with other therapeutic agents to ameliorate side effects of

[0332] 15 administering the compositions described herein (e.g., therapeutic agents that minimize risk of infection in an immunocompromised environment, for example, anti-bacterial agents, anti-fungal agents and anti-viral agents).

[0333] The pharmaceutical compositions can include a “therapeutically effective amount” or a “prophylactically effective amount” of a fusion protein. A “therapeutically effective amount” refers to an

[0334] 20 amount effective, at dosages, and for periods of time sufficient, to achieve the desired therapeutic result. A therapeutically effective amount of the fusion protein can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the fusion protein to elicit a desired response in the individual. A “prophylactically effective amount” refers to an amount effective, at dosages, and for periods of time sufficient, to achieve the desired prophylactic result. In some

[0335] 25 embodiments, a prophylactic dose is used in subjects prior to or at an earlier stage of disease where the prophylactically effective amount will be less than the therapeutically effective amount.

[0336] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. It is to be noted that dosage values can

[0337] 35 vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the administering clinician.

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[0340] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0341] The efficacy of treatment with a fusion protein as described herein can be assessed based on an improvement in one or more symptoms or indicators of the disease state or disorder being treated (e.g., an improvement in one or more symptoms of a complement alternative pathway (CAP)-mediated disease or disorder, such as a kidney disease or disorder mediated by dysregulation of the CAP). An

[0342] 5 improvement of at least 10% (increase or decrease, depending upon the indicator being measured) in one or more clinical indicators is considered “effective treatment,’’ although greater improvements are possible, such as 20%, 30%, 40%, 50%, 75%, 90%, or even 100%, or, depending upon the indicator being measured, more than 100% (e.g., two-fold, three-fold, ten-fold, etc., up to and including attainment of a disease-free state.

[0343] Methods of Treatment Using the Fusion Proteins

[0344] The complement factor H fusion proteins described herein (e.g., Compound A; see Table 1) can be used to treat a disease mediated by complement alternative pathway activation or dysregulation (e.g., FSGS) by inhibiting the complement alternative pathway activation in a mammal (e.g., a human). The

[0345] 15 fusion protein(s) described herein can be used to treat a variety of diseases or disorders mediated by complement alternative pathway activation or dysregulation. Such disorders include, without limitation, kidney disorders, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis,

[0346] 20 membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia reperfusion injury, or rejection of a transplanted organ, such as a kidney.

[0347] In particular embodiments, the Compound A having the amino acid sequence of SEQ ID NO: 1 or a variant thereof with at least 85% (e.g., at least 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

[0348] 25 97%, 98%, or 99%) sequence identity thereto) may be used to treat FSGS in subject in need thereof. The subject may be a mammal. In some embodiments, the subject is a human.

[0349] A therapeutically effective amount of a complement factor H fusion protein, as disclosed herein (e.g., a fusion protein having SEQ ID NO: 1 or a variant thereof with at least 85% (e.g., at least 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto) can be administered to a mammalian subject in need of such treatment. For example, the subject may be administered at least 180 mg of Compound A per week. In some embodiments, Compound A may be administered to the subject in an amount of between 170 mg and 460 mg (e.g., 180±20 mg, 200±20 mg, 220±20 mg, 240+20 mg, 260+20 mg, 280 +20 mg, 300+20 mg, 320±20 mg, 340+20 mg, 360+20 mg, 380±20 mg, 400±20 mg, 420±20 mg, 440±20 mg, or 460±20 mg) per week. In some embodiments, the

[0350] 35 subject is administered Compound A in an amount of 180 mg per week. In some embodiments, the subject is administered Compound A in an amount of 450 mg per week. In some embodiments, the subject is administered Compound A weekly. A physician or researcher can decide the appropriate

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[0353] PATENT ATTORNEY DOCKET NO.: 50694-108001 frequency of administration based on the concentration of the serum concentration of the fusion protein in the subject, assessment of the activity of the fusion protein using a complement pathway hemolysis assay, or evaluation of alternative pathway activity using, for example, a complement system alternative pathway Wl ESLAB® assay.

[0354] 5 In some embodiments, the subject is a human patient. The amount administered should be sufficient to inhibit complement activation and / or restore normal complement alternative pathway regulation. The route of administration may affect the recommended dose. Repeated systemic doses (e.g., by subcutaneous injection) are contemplated to maintain an effective level, e.g., to attenuate or inhibit complement activation in a patient's system, depending on the mode of administration adopted.

[0355] The compositions and methods described herein may be useful for treatment of a kidney disorder mediated by dysregulation of the CAP, such as FSGS. FSGS is characterized by obliteration of glomerular capillary tufts with increased matrix deposition and scarring (D'Agati et al., Am J Kidney Dis. 43(2):368-382, 2004). The incidence of FSGS has increased over the past decades and it is one of the leading causes of nephrotic syndrome in adults (Korbet, J Am Soc Nephrol. 23(11):1769-1776, 2012).

[0356] 15 Spontaneous remission is rare (<5%) and presence of persistent nephrotic syndrome indicates a poor prognosis with 50% of patients progressing to end-stage renal disease (ESRD) 6-8 years after initial diagnosis (Korbet, Nephrol Dial Transplant. 14 Suppl 3:68-73, 1999). Primary FSGS is responsible for 3.3% of all the cases of end-stage renal disease (ESRD) resulting from primary kidney disease in the United States. The complement system has been shown to be activated in patients with primary FSGS

[0357] 20 and elevated levels of plasma Ba, indicative of activation of the alternative pathway, correlates with disease severity. Patients with low serum C3 had a higher percentage of interstitial injury. Furthermore, renal survival was found to be higher in patients with normal serum C3 as compared to those with low serum C3. Low serum C3 is indicative of complement activation. Therefore, activation of the complement system may play a crucial role in the pathogenesis and outcome of FSGS (Liu et al.,

[0358] 25 Scientific Reports, 7: 4095, 2017). In humans, tubulointerstitial deposition of the complement membrane attack complex (C5b-9) is correlated with interstitial myofibroblast accumulation and proteinuria. Experimentally, in focal segmental glomerulosclerosis, the intratubular formation of C5b-9 was found to promote peritubular myofibroblast accumulation. Myofibroblasts may act as sentinel inflammatory cells and deposit extracellular matrix. These cells may also constrict kidney tubules leading to atubular glomeruli. By this mechanism, complement activation may contribute to tubulointerstitial injury and fibrosis in FSGS (Rangan et al., Kidney Int. 66:1838-1848, 2004). Factor B and factor D deficient mice have lower proteinuria than WT controls in the adriamycin-induced FSGS model, suggesting that activation of CAP has a pathogenic role (Lenderink et al., Am. J. Physiol. Renal Physio. 293:F555-F564, 2007). The alternative pathway of complement is activated in the glomeruli and tubulointerstitium of mice

[0359] 35 with adriamycin nephropathy (Turnberg et al., J Immunol. 177(6):4094-4102, 2006). Furthermore, complement factor H deficient mice display higher C3b glomerular deposition and more severe kidney damage than wild-type controls (Morigi et al., Sci Rep. 6:28445, 2016), which confirms a previously

[0360] 28

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[0362] PATENT ATTORNEY DOCKET NO.: 50694-108001 unrecognized role of C3a in proteinuric progressive nephropathy. Therefore, an inhibitor of the alternative pathway of complement activation can be used to achieve clinical utility in FSGS. The FSGS may be primary FSGS.

[0363] The subject may have been diagnosed with FSGS within 3 years prior to being administered

[0364] 5 Compound A (e.g., within 1 day, 1 week, 1 month, 3 months, 6 months, 9 months, 1 year, 18 months, 2 years, 30 months, or 36 months prior to being administered Compound A). The subject may not have any other kidney disease other than FSGS. At the time the subject is diagnosed with FSGS, the subject may also have acute onset of nephrotic syndrome. In some embodiments, at the time the subject is diagnosed with FSGS, the subject has proteinuria. The proteinuria may correspond to a protein concentration in the urine of at least 3.5 g / day. In some embodiments, the subject has hypoalbuminemia at the time of diagnosis with FSGS. The hypoalbuminemia may correspond to serum album levels of less than 3.5 g / d L. At the time of FSGS diagnosis, the subject may have peripheral edema at the time of diagnosis. The subject may also have a urine protein creatinine ratio of at least 1 .5 g / g prior to administration of Compound A.

[0365] 15 The disclosure further relates to a composition including a fusion protein of Compound A (e.g., a fusion protein having the amino acid sequence of SEQ ID NO: 1) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to SEQ ID NO: 1) for use in treatment of kidney disorders.

[0366] The disclosure further relates to a composition including a fusion protein of Compound A (e.g., a fusion protein having the amino acid sequence of SEQ ID NO: 1-) or a variant thereof (e.g., a fusion

[0367] 20 protein having at least 85% sequence identity to SEQ ID NO: 1) for use in treatment of FSGS.

[0368] In some embodiments, the disclosure relates to a pharmaceutical composition for treating kidney disorders, containing a fusion protein of Compound A (e.g., a fusion protein having the amino acid sequence of SEQ ID NO: 1) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to SEQ ID NO: 1) as an active ingredient.

[0369] 25 In some embodiments, the disclosure relates to a pharmaceutical composition for treating FSGS, containing a fusion protein of Compound A (e.g., a fusion protein having the amino acid sequence of SEQ ID NO: 1) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to SEQ ID NO: 1) as an active ingredient.

[0370] In some embodiments, the disclosure relates to use of a composition comprising a fusion protein of Compound A (e.g., a fusion protein having the amino acid sequence of SEQ ID NO: 1) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to SEQ ID NO :1) for the manufacture of a medicament for kidney disorders.

[0371] In some embodiments, the disclosure relates to use of a composition comprising a fusion protein of Compound A (e.g., a fusion protein having the amino acid sequence of SEQ ID NO: 1) or a variant

[0372] 35 thereof (e.g., a fusion protein having at least 85% sequence identity to SEQ ID NO: 1) for the manufacture of a medicament for FSGS.

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[0375] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0376] To treat FSGS in a subject, Compound A may be administered to a subject in need thereof for at least 24 weeks. In some embodiments, Compound A is administered to a subject in need thereof for at least 52 weeks.

[0377] Upon administration of Compound A to the subject, the subject may experience a reduction in

[0378] 5 proteinuria relative to baseline. For example, the subject may have a reduction in proteinuria of greater than 30% relative to baseline. In some embodiments, the subject shows a reduction in proteinuria of greater than 50% relative to baseline after. The subject may show the reduction in proteinuria after 24 weeks of administration of the pharmaceutical composition comprising Compound A. The subject may show the reduction in proteinuria after 52 weeks of administration of the pharmaceutical composition comprising Compound A.

[0379] The subject may show a change in estimated glomerular filtration rate (eGFR) relative to baseline after administration of the pharmaceutical composition. For example, the subject may demonstrate a change in eGFR from baseline after administration of Compound A for 24 weeks. In some embodiments, the subject experiences a change in eGFR from baseline after administration of Compound A for 52

[0380] 15 weeks.

[0381] The subject may show a change in serum albumin relative to baseline after administration of the pharmaceutical composition comprising Compound A. The subject may experience partial remission after administration of the pharmaceutical composition Compound A. The subject may experience complete remission after administration of the pharmaceutical composition comprising Compound A. The subject

[0382] 20 may experience improved kidney function relative to baseline during administration of the fusion protein after administration of the pharmaceutical composition comprising Compound A. The subject may experience a change in serum albumin relative to baseline, partial remission, complete remission, or improved kidney function relative to baseline after 24 weeks of administration of Compound A. The subject may experience a change in serum albumin relative to baseline, partial remission, complete

[0383] 25 remission, or improved kidney function relative to baseline after 52 weeks of administration of Compound A. In some embodiments, the subject who is administered Compound A does not experience a treatment emergent adverse event.

[0384] The dose may be increased to increase serum albumin concentration in a subject relative to baseline by up to 50% until the baseline metric result relative to a healthy subject (or a prior measurement from the subject) is restored or stabilized. In some embodiments, the dose is increased to increase kidney function in a subject relative to baseline by up to 50% until the baseline metric result relative to a healthy subject (or a prior measurement from the subject) is restored or stabilized.

[0385] EXAMPLES

[0386] 35 The following examples are put forth so as to provide those of ordinary skill in the art with a disclosure and description of how the methods and compounds claimed herein are performed, made. They are intended to be purely exemplary and are not intended to limit the scope of the disclosure.

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[0389] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0390] Example 1. A Phase 2, Randomized, Double-blind, Placebo-controlled Study to Evaluate the Efficacy and Safety of Compound A in Adult Participants with Primary FSGS

[0391] Compound A, a novel fusion protein designed to provide intrarenal CAP regulation, is being

[0392] 5 developed for treatment of patients with primary FSGS.

[0393] FSGS is a morphologic pattern of glomerular injury, rather than a specific disease entity, that is commonly found to underlie the nephrotic syndrome in adults and children. The glomerular injury is primarily directed at the glomerular visceral epithelial cell (the podocyte) and defined by the presence of sclerosis in parts (segmental) of some (focal) glomeruli by LM of a kidney biopsy specimen. IF commonly reveals IgM and complement (C3, C3d, and variably C1) in sclerotic lesions), with C3d deposition associated with worse clinical outcomes).

[0394] Primary FSGS is associated with diffuse (> 80%) podocyte foot process effacement on EM examination of the kidney biopsy and is presumed to be caused by a circulating permeability factor that is toxic to the podocyte and causes generalized podocyte dysfunction. The identity of the circulating

[0395] 15 factor(s) has not yet been clearly established, although several candidates have been proposed. Significant literature implicates the CAP as a disease driver in FSGS with C3 deposition occurring along parietal, proximal tubule epithelial cells. Published urine biomarker data (levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine) suggest intra-renal CAP activation is greater in FSGS than other renal diseases with equivalent proteinuria levels. Complement-dependent podocyte

[0396] 20 injury is a key event in the pathogenesis of FSGS resulting in nephrotic syndrome and leading to irreversible scarring in glomeruli in both children and adults.

[0397] There is currently no approved therapy for FSGS and current treatment options include glucocorticoids and CNI which present numerous AEs. Compound A is designed to bind and irreversibly inactivate intrarenal C3b and clear CAP C3 / C5 convertases. Negligible inhibition of systemic CAP activity

[0398] 25 was observed in toxicology studies in cynomolgus monkeys after a single SC dose at 10 to 200 mg / kg and in healthy adult participants after a single SC dose at 50 to 900 mg. This feature provides a substantial therapeutic advantage over nontargeted complement inhibitors by preserving the homeostatic defense benefits of all 3 pathways of the complement system and abrogating infection risks. Compound A’s mechanism of action is anticipated to be useful in the treatment of primary FSGS.

[0399] The safety, tolerability, PK, PD, and immunogenicity of Compound A were evaluated in a FIH Phase 1 study in healthy adult participants (Study NCT05751642), which assessed SAD of SC and IV administered Compound A in 48 healthy adult participants.

[0400] A Phase 2, double-blind, randomized, placebo-controlled, multicenter study will evaluate the efficacy and safety of Compound A in addition to SoC therapy in approximately 105 adult participants (>

[0401] 35 18 to < 75 years) with primary FSGS.

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[0404] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0405] 1.1. Product Name / Active Ingredient

[0406] Compound A is a fusion protein composed of a fH functional domain, a humanized camelid VHH domain and a cyclic RGD polypeptide of SEQ ID NO: 1.

[0407] 5 1.2. Chemical name, Established Name and / or Structure.

[0408] Compound A is a 453 amino-acid, single-chain protein. The N-terminal region consists of the first 5 SCR domains of human fH linked to a VHH fused via a short linker peptide (4 glycine and 1 alanine) to a cyclic integrin-binding motif, c(RGD)4, at the C-terminus. The fH domain has 1 endogenous glycosylation site (NGSP) with only trace levels of glycosylation because of the neighboring proline. The 5 SCRs bind to C3b to inhibit C3 convertase formation in the CAP. The c(RGD)4 motif binds integrin to enable transmembrane receptor binding to facilitate surface adhesion. A diagram of the Compound A molecular construct is illustrated in FIG. 1. The complete, confirmed amino acid sequence of Compound A is shown in SEQ ID NO: 1 and a schematic of the protein and disulfide bonding pattern is diagramed in FIG. 2.

[0409] 15 1.3. Proposed Indication or Context of Product Development

[0410] Compound A is proposed for the treatment of patients with primary FSGS.

[0411] 1.4. Dosage Form, Route of Administration, Dosing Regimen and Presentation(s)

[0412] Dosage form: solution for infusion

[0413] 20 Compound A is provided as a 50-mg / mL, 4-mL (200 mg / vial) solution for SC infusion use. The formulation is phosphate buffered to a pH of 7.0.

[0414] Route of Administration: SC

[0415] Dosage Regimen (Including frequency and duration):

[0416] • 450 mg weekly

[0417] • 180 mg weekly

[0418] Presentation:

[0419] Compound A is supplied in a 4R type I glass vial with a 13-mm bromobutyl stopper and an aluminum overseal. Each vial nominally contains 200 mg of Compound A in a volume of 4 ml_. The vials are filled with a target volume of 4.3 mL to allow for the labeled volume to be delivered.

[0420] 30

[0421] 1.5. Pharmacologic Class

[0422] Compound A is a complement inhibitor. Background Information

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[0425] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0426] 2. BACKGROUND INFORMATION ON FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS)

[0427] 2.1 .1 . Etiology and Pathophysiology

[0428] Etiology

[0429] 5 FSGS is a morphologic pattern of glomerular injury, rather than a specific disease entity, that is commonly found to underlie the nephrotic syndrome in adults and children. The glomerular injury is primarily directed at the glomerular visceral epithelial cell (the podocyte) and defined by the presence of sclerosis in parts (segmental) of some (focal) glomeruli by LM of a kidney biopsy specimen.

[0430] FSGS can be classified into the following forms, based upon clinical presentation and pathologic findings on kidney biopsy:

[0431] • Primary FSGS, which most often presents with the acute onset of nephrotic syndrome.

[0432] • Secondary FSGS, which most often presents with non-nephrotic proteinuria and, commonly, some degree of kidney function impairment. This category most commonly refers to FSGS that develops as a maladaptive response to glomerular hypertrophy or hyperfiltration. This includes

[0433] 15 disorders associated with a reduced kidney mass and / or renal vasodilation, such as unilateral renal agenesis. Other secondary causes of FSGS include drugs and toxins (including heroin, interferon, and pamidronate) and viral infections (such as COVID-19 and HIV).

[0434] • Genetic FSGS, which may present early in childhood with massive proteinuria and nephrotic syndrome or in adolescence or adulthood with less severe proteinuria.

[0435] 20 • FSGS of undetermined cause, in which clinical presentation and EM findings on biopsy are similar to those in patients with secondary FSGS, but a clear etiology cannot be determined despite extensive evaluation, including a comprehensive genetic analysis.

[0436] Pathology

[0437] • On LM, FSGS lesions are characterized by segmental consolidation of capillary loops with

[0438] 25 obliteration of the capillary lumen. FSGS lesions are often accompanied by hyalinosis, macrophage infiltration of the sclerosed tufts, and adhesion of the sclerosed loops to the Bowman capsule. EM of the FSGS lesions shows collapsed capillary loops often with hyaline entrapment; electron dense deposits are not present. Podocyte foot process effacement is a characteristic feature. Podocyte damage and loss are key events in the pathogenesis of FSGS, resulting in nephrotic syndrome and leading to irreversible scarring in glomeruli, declining kidney function and kidney failure in both children and adults.

[0439] • IF commonly reveals IgM and complement (C3, C3d, and variably C1) in sclerotic lesions, with C3d deposition associated with worse clinical outcomes. C3 deposition is also observed.

[0440] • The restricted presence of endogenous fH and other circulating regulators beyond the selectively

[0441] 35 permeable glomerular capillary wall, coupled with the FSGS-associated decrease of the

[0442] 33

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[0444] PATENT ATTORNEY DOCKET NO.: 50694-108001 complement surface regulator decay accelerating factor (DAF) confers the inability to control intrinsic CAP activation within the nephron.

[0445] Role of complement in FSGS

[0446] Mechanistic studies highlight the role of the complement axis in FSGS:

[0447] 5 • In a rodent model of primary FSGS, adriamycin exposure led to the deposition of the complement terminal components (C5b-9) in the lumen and apical surfaces of proximal tubular epithelial cells and the peritubular region of the tubulointerstitium. C6-deficient animals in this study were protected against peritubular fibrosis, and tubulointerstitial injury and inflammation.

[0448] • In another study using the same model, factor B- and factor D-deficient mice were protected against tubulointerstitial injury and proteinuria.

[0449] • In a mechanistic study using transgenic mice, podocyte-specific deficiency of DAF, a negative regulator of the complement pathway, resulted in complement dysregulation and complement- mediated kidney injury, with consequent proteinuria.

[0450] • Recently, the same group showed that inhibiting DAF release from the podocytes has a

[0451] 15 protective effect against FSGS.

[0452] • Most importantly, and relevant to the proposed study, fH deficiency predisposes mice to more severe podocyte depletion and glomerulosclerosis in an FSGS protein overload model.

[0453] Human studies support the critical role of complement in patients with primary FSGS. Complement activation (evaluated by measuring complement fragments in urine and plasma) showed strong

[0454] 20 associations with kidney injury and proteinuria, and distinguished primary FSGS from MCD. Furthermore, C3 deposition in the kidney, an irrefutable indicator of complement dysfunction, predicted the progression to ESRD and remained significant as a risk factor for treatment refractoriness in a multivariable analysis on primary FSGS patients. These studies are reviewed in more detail in Section 2.3.10.

[0455] 2.1.2. Clinical Manifestations

[0456] Patients with presumed primary FSGS most commonly present with the acute onset of nephrotic syndrome and the associated features of high-grade (> 3.5 g / day) proteinuria, hypoalbuminemia, and peripheral edema. Hematuria is common (approximately 50% of patients), and hypertension is present in approximately 20% of cases. An elevated serum creatinine may be seen in 25% to 50% of patients at

[0457] 30 presentation but may not necessarily reflect chronic kidney damage, since acute tubular necrosis can occur in patients with severe proteinuria.

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[0460] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0461] 2.1.3. Diagnosis

[0462] The identification of a primary FSGS lesion in a kidney biopsy of a patient with proteinuria does not establish a specific diagnosis; rather, it should prompt the clinician to pursue a thorough evaluation to determine the underlying cause of the FSGS lesion. A systematic approach is used that considers clinical

[0463] 5 and pathologic features of the patient to differentiate between primary, secondary, and genetic forms of FSGS. Most patients with primary or secondary FSGS can be distinguished by the presence or absence of nephrotic syndrome (urine protein excretion > 3.5 g / day and hypoalbuminemia), the presence of identifiable risk factors for secondary FSGS (e.g., drugs, toxins, and viruses), and the degree of podocyte foot process effacement visualized by EM examination of the kidney biopsy:

[0464] • If the patient has diffused (> 80%) foot process effacement on EM examination, diagnosis is most likely primary FSGS. However, genetic forms of FSGS lesions cannot be excluded with confidence. Thus, in such patients, distinguishing between primary and genetic FSGS is based upon the patient's response to initial immunosuppressive therapy with glucocorticoids or a CNI.

[0465] 15 o In patients who respond to initial immunosuppressive therapy with glucocorticoids or a CNI, a diagnosis of primary FSGS can be established since patients with genetic forms of FSGS rarely respond to treatment with immunosuppressive agents. In such patients, genetic testing is not performed for FSGS. o In patients who do not respond to initial immunosuppressive therapy with glucocorticoids

[0466] 20 or a CNI, a diagnosis of primary FSGS or genetic FSGS may be possible. In such patients, genetic testing is suggested, if available, to screen for monogenic forms of FSGS.

[0467] • If the patient has segmental (< 80%) foot process effacement on EM examination, a secondary or genetic cause of FSGS should be suspected. In patients with identifiable risk

[0468] 25 factors for secondary FSGS (eg, obesity, reflux nephropathy, reduced kidney mass, drugs, viruses), a diagnosis of secondary FSGS can be established and genetic testing for FSGS is not performed. In patients with no identifiable risk factors for secondary FSGS, a genetic form of FSGS may be possible; hence, genetic testing is suggested if available, to screen for monogenic forms of FSGS.

[0469] 2.1.4. Current Treatment Options

[0470] Currently, there are no approved therapies available for primary FSGS. Patient management consists of treatment with glucocorticoids as the current first-line therapy for primary FSGS. Patients often require long periods of therapy with significant risk of toxicity, relapse, and development of steroid resistance or. CNIs are administered alternatively to steroids in patients who have a high risk of steroid

[0471] 35 induced toxicity, with or without low dose steroids as an initial therapy. Since CNIs cause potential nephrotoxicity, they are not used in patients with reduced kidney function as a first line therapy. When these treatments fail, a third option will include MMF, rituximab, and cyclophosphamide.

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[0475] 2.1.5. Current Unmet Medical Need

[0476] FSGS is a leading cause of kidney failure worldwide, yet no treatment is approved for this condition. An estimated 50% to 70% of patients with primary FSGS progress to ESRD within 10 years of diagnosis. Podocyte foot process effacement leading to kidney failure is a characteristic feature. In the

[0477] 5 adult population, recurrence of FSGS after renal transplant ranges from 4% to 66%.

[0478] 2.2. Rationale for Development of Compound A for Treatment of FSGS

[0479] 2.2.1 . Mechanism of Action

[0480] Compound A is a novel fusion protein composed of a human fH functional domain, a humanized camelid VHH domain and a cyclic RGD polypeptide. Compound A is designed to provide intrarenal CAP regulation in complement-mediated kidney diseases. The molecule leverages the regulatory capabilities present in the first 5 SCRs of human fH, the major soluble regulator of the CAP. The human fH domain enables Compound A to bind to and irreversibly inactivate C3b and clear CAP C3 / C5 convertases. The molecule’s low molecular weight results in clearance via glomerular filtration, allowing access to the

[0481] 15 kidney lumen, where it accumulates in the ultrafiltrate within the Bowman’s capsule and deposits along the apical surface of the kidney epithelial cells via a novel mechanism provided by the Compound A VHH domain. The cyclic RGD motif on the C-terminus of Compound A is essential for the molecule’s MoA in the adriamycin nephropathy mouse model of FSGS and enables selective binding to aVp3 and aVf>6 integrins present at sites of damage. Localization to the kidney epithelium thereby enables Compound A

[0482] 20 to direct its protective effects to sites where naturally low levels of membrane-associated surface regulators of complement confer susceptibility to complement dysregulation. Negligible inhibition of systemic CAP activity was observed in healthy adult participants at single SC doses of 50 to 900 mg. Avoiding systemic CAP inhibition would provide a substantial therapeutic advantage over nontargeted complement inhibitors by preserving the homeostatic defense benefits of all 3 pathways of the

[0483] 25 complement system and abrogating infection risks.

[0484] By specifically targeting dysregulated CAP activity in disease-relevant renal tissues, Compound A has the potential to improve renal function through reducing proteinuria and attenuating progressive tubular damage associated with renal diseases in which dysregulated CAP activity has been implicated such as primary FSGS. The selection of primary FSGS as a lead indication for Compound A is based on the following:

[0485] • Demonstrated preclinical efficacy data using the adriamycin nephropathy mouse model with Compound A links MoA with primary endpoint of proteinuria (see Section 2.2.2)

[0486] • Significant literature implicating the CAP as a disease driver in FSGS with C3 deposition occurring along parietal, proximal tubule epithelial cells (see Section 2.1 .1)

[0487] 35 • Published urine biomarker data (levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine) suggest intra-renal CAP activation is greater in FSGS than other renal diseases with equivalent proteinuria levels

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[0491] • Complement-dependent podocyte injury is a key event in the pathogenesis of FSGS resulting in nephrotic syndrome and leading to irreversible scarring in glomeruli in both children and adults (see Section 2.1.1)

[0492] • Compound A was designed for indications with kidney-exclusive pathology, targeting parietal,

[0493] 5 proximal tubule epithelial cells

[0494] • Delivery of FH1-5 to parietal epithelial cells surrounding Bowman’s capsule directs Compound A’s protective CAP inhibitory MoA to the specific cells participating in sclerotic lesion formation. Emerging data suggest that parietal epithelial cells also provide a source for podocyte repopulation

[0495] • Lack of access by endogenous fH and other circulating regulators beyond the glomerular capillary wall, coupled with the FSGS-associated decrease of the complement surface regulator DAF confers an inability to control intrinsic CAP activation within the nephron (see Section 2.1.1)

[0496] 2.2.2. Nonclinical Proof of Concept

[0497] Nonclinical efficacy studies with Compound A were performed using an adriamycin nephropathy

[0498] 15 mouse model, a widely used and highly translatable model of FSGS characterized by podocyte injury, glomerulosclerosis and tubulointerstitial damage. Studies included a single-dose level proof-of-concept study and a minimum efficacious dose study that investigated effects across multiple-dose levels. In the model, male BALB / c OLA mice (Envigo, Loughborough, UK) were administered a single IV injection of adriamycin on Day 0 to induce podocyte damage and progressive renal disease mirroring clinical FSGS.

[0499] 20 Natural history data demonstrated that elevated proteinuria levels and kidney pathology were detectable by Day 7, suggesting Day 7 as a clinically meaningful time point for therapeutic intervention with Compound A. A cohort of 4 adriamycin-treated animals (Group B) was terminated at Day 7 in this study with significant increases in tubular protein and tubular degeneration / dilation observed, confirming the presence of histopathological changes and a therapeutic intervention paradigm for the remaining groups.

[0500] 25 This study demonstrated that Compound A significantly reduced 16-hour protein and albumin excretion caused by adriamycin-induced renal disease. Exploratory analyses further showed that Compound A was effective in reducing C3 and properdin protein deposition in the kidneys as a result of induced renal disease. Additional histological analyses showed that Compound A treatment resulted in modest but significant reductions in tubular protein and tubular degeneration / dilation in the kidneys. In the study, significant reductions were observed in the tubule-specific kidney injury marker urine KIM-1 following therapeutic intervention with Compound A at both 10 and 3 mg / kg. Significant reductions were also observed in kidney weight (a surrogate readout for inflammation) across all Compound A treated groups.

[0501] The lack of effects observed with human FH1-5 or other molecular variants tested in the study

[0502] 35 indicated that both the VHH and RGD domains present in Compound A promoted therapeutic benefit.

[0503] 37

[0504] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0505] PATENT ATTORNEY DOCKET NO.: 50694-108001

[0506] 2.3. Status of Nonclinical Development Program

[0507] Nonclinical pharmacology, PK, and toxicology studies to evaluate the safety and efficacy of Compound A were conducted in mice and monkeys and in in-vitro studies. The therapeutic MoA Compound A involves the inactivation and clearance of the CAP activation product C3b, a component of

[0508] 5 CAP C3 and C5 convertases and immunostimulatory opsonin which binds to cell surfaces during CAP activation. This MoA was validated in 2 human in vitro systems - the CAP hemolysis assay and the CAP Wieslab assay.

[0509] Species cross-reactivity for blockade of CAP hemolytic activity (using sera from cynomolgus monkeys and mice) confirmed pharmacological activity in both species, supporting the relevance of each

[0510] 10 for GLP toxicology studies.

[0511] Nonclinical in-vivo pharmacology studies with Compound A were performed using the adriamycin nephropathy mouse model of FSGS. Non-GLP secondary pharmacology studies examined the off-target binding selectivity of Compound A. Collectively, the data from these studies, along with those generated in a standard GLP human tissue cross-reactivity study, revealed no findings that would preclude clinical development.

[0512] Compound A has been evaluated in a full panel of completed toxicology studies performed in C57BL / 6 mice (twice weekly up to 6 weeks) and cynomolgus monkeys (twice weekly up to 26 weeks) based on ICH S6 (R1). A special tissue cross-reactivity GLP study was also conducted using normal human tissues. According to ICH S6 (R1), additional toxicity studies, including carcinogenicity studies and

[0513] 20 genotoxicity studies, are not considered appropriate for humanized proteins such as Compound A. According to ICH S5 (R3), a non-GLP dose range finding EFD study in pregnant C57BL / 6 mice has been conducted, and an GLP EFD study in pregnant C57BL / 6 mice is underway.

[0514] A list of nonclinical studies completed to date with Compound A is provided in Table 3.

[0515] 38

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[0517] PATENT

[0518] ATTORNEY DOCKET NO: 50694-108001

[0519] Table 3: Completed Compound A Nonclinical Studies

[0520] 39

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[0522] PATENT

[0523] ATTORNEY DOCKET NO: 50694-108001

[0524] Table 3: Completed Compound A Nonclinical Studies

[0525] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0526] PATENT

[0527] ATTORNEY DOCKET NO: 50694-108001

[0528] Table 3: Completed Compound A Nonclinical Studies

[0529] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0530] PATENT

[0531] ATTORNEY DOCKET NO: 50694-108001

[0532] Table 3: Completed Compound A Nonclinical Studies

[0533] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0534] PATENT ATTORNEY DOCKET NO: 50694-108001

[0535] 2.3.1. Safety Pharmacology

[0536] Standalone safety pharmacology studies were not performed for Compound A. However, cardiovascular, neurobehavioral, respiratory, and indirect blood pressure parameters were assessed as part of the 6-week GLP toxicity study in cynomolgus monkeys. Compound A when given SC or IV

[0537] 5 twice weekly for up to 6 weeks did not produce any changes in cardiovascular parameters (heart rate; RR, PR, or QT intervals; QTc; or QRS duration), respiratory rate (count), indirect blood pressure measurements, or neurobehavioral parameters FOB at SC dose levels up to 203.6 mg / kg or IV dose level of 50 mg / kg.

[0538] 2.3.2. Non-GLP Toxicology Studies

[0539] In study another study (C57BL6 / J male mice), the parameters or endpoints evaluated included mortality, clinical signs, cytokine evaluation, PK and PD (CAP) parameters, ADA analysis, gross necropsy, and microscopic pathology examination (kidneys and lungs) and Compound A was well tolerated with no mortality or adverse findings at a single IV dose of 30 mg / kg or up to 5 SC doses of 100 mg / kg. In Study 1727-270 (female cynomolgus monkey), the parameters or endpoints

[0540] 15 evaluated included mortality, clinical signs, body weights, body weight gains, clinical pathology parameters (hematology, coagulation, and clinical chemistry), cytokine evaluation, PK and PD parameters (CAP and endogenous fH), ADA analysis, and macroscopic and microscopic pathology examinations. The NOAEL was considered to be the highest dose tested (150 mg / kg for IV and 50 mg / kg for SC).

[0541] 20 2.3.3. GLP 6-week Toxicology Studies in C57BL / 6 mice

[0542] In another study, the parameters / endpoints evaluated included mortality, clinical observations, body weights, body weight gains, dermal scoring, ophthalmology, clinical pathology parameters (hematology and clinical chemistry), TK parameters, ADA analysis, cytokine analysis, organ weights, and macroscopic and microscopic examinations. A potential hypersensitivity reaction arising from

[0543] 25 immunogenicity against Compound A was considered a possible cause for 19 unscheduled deaths in Compound A-treated SC animals. Clinical signs of poor tolerability combined with acute mortality after multiple doses without noteworthy postmortem findings in this study were consistent with type I hypersensitivity reaction associated with biopharmaceuticals administration in nonclinical species. A NOAEL of Compound A was not able to be established for the SC route. However, the mortality noted

[0544] 30 in the SC-treated groups could be related to immunogenicity directed towards Compound A. Therefore, after excluding potential immunogenicity effects, the NOAEL after 6 consecutive weeks of twice-weekly dosing was considered to be 200 mg / kg SC.

[0545] Compound A was tolerated in the 100 mg / kg IV group. Based on the survival data, the NOAEL after 6 consecutive weeks of twice-weekly dosing was considered to be 100 mg / kg IV.

[0546] 2.3.4. GLP 6-week Toxicology Studies in Cynomolgus Monkey

[0547] In another study, the parameters and endpoints evaluated included mortality, clinical signs, body weight, body weight gain, ophthalmology, electrocardiogram, FOB, evaluation of skin reaction, cardiovascular evaluations (jacketed telemetry), visual assessment of respiratory rate, indirect blood

[0548] 40 pressure, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), 43

[0549] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0550] PATENT ATTORNEY DOCKET NO: 50694-108001 cytokine analysis, PK and PD parameters, ADA, organ weights, macroscopic and microscopic examinations, and IHC pathology evaluations. Euthanasia was necessary for 1 male dosed at 203.6 mg / kg SC due to clinical signs of dehydration and abdominal distention likely secondary to vascular / perivascular inflammation throughout the gastrointestinal tract. The NOAEL after up to 12

[0551] 5 doses was 203.6 (female) and 10 (male) mg / kg SC and 50 mg / kg IV. Early euthanasia of 1 male at 203.6 mg / kg SC and, vascular / perivascular inflammation, renal tubular degeneration, and glomerular inflammation with erythrocytic cast formation at > 50 mg / kg SC in male monkeys were considered adverse due to greater severity and greater incidence. Observations at 10 mg / kg SC in males were not considered adverse after administration of Compound A for up to 6 weeks SC, owing to the absence of systemic observations and considering minimal severity of renal findings in the single male monkey.

[0552] 2.3.5. Rationale for Conducting Chronic toxicology study in cynomolgus monkey

[0553] Longer-term general toxicity studies in one species are usually considered sufficient as long

[0554] 15 as the toxicity findings from short-term toxicity studies in rodents and nonrodents are similar or the findings are understood from the mechanism of action of the test article. Since NHP is considered the pharmacologically relevant species to conduct toxicity studies for biotherapeutics due to higher genome sequence homology to humans and a similarity in physiological systems, such as the immune system and considering the high mortality that was observed due to potential immunogenicity

[0555] 20 in the GLP 6-week toxicology study conducted in C57BL / 6 mice, cynomolgus monkey was selected to conduct a GLP 6-month toxicity study.

[0556] 2.3.6. 26-weeks study in cynomolgus monkey

[0557] In another study, (cynomolgus monkey) the parameters and endpoints evaluated in this study

[0558] 25 included mortality, clinical signs, skin reactions, body weights, body weight gains, ophthalmology, physical examinations, electrocardiographic examinations, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), cytokines, ADA, IHC, TK parameters, PD parameters (CAP and endogenous fH), organ weights, macroscopic and microscopic examinations. The IV dosing groups were discontinued after dosing Day 64. In addition, the IV route is not the intended route of

[0559] 30 chronic administration in clinics.

[0560] The early euthanized 50 mg / kg SC animals with tissue-specific immune complex deposition showed clinical signs such as decreased activity and reactivity, pale mucous membranes, eyes closed, recumbency, elevated heart rate, edema of the abdomen and I or face, and / or skin thickening and scabbing on dorsal aspect such that no further sites were available for SC dosing. Clinical signs such as decreased activity, salivation, cold to touch, suspected dehydration, hunched posture, and a swollen face were observed in 1 early euthanized male at 50 mg / kg IV. These clinical signs in early euthanized SC and IV group animals were likely related to positive ADA in the animals.

[0561] Compound A -related generally dose-responsive clinical signs in SC animals that survived to their scheduled necropsy during the dosing phase (with their status at the end of the recovery period

[0562] 40 indicated) included abdominal distension at > 10 mg / kg (resolved), decreased activity at > 10 mg / kg 44

[0563] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0564] PATENT ATTORNEY DOCKET NO: 50694-108001 (resolved), skin thickening at abdominal, dorsal, and lumbar area at > 10 mg / kg (resolved), skin papule at dorsal area at > 10 mg / kg (resolved), swollen at dose sites and dorsal aspect at > 10 mg / kg (resolved), and generalized tremors > 10 mg / kg. Additionally, there were Compound A -related generally dose-responsive clinical signs of abnormal feces with red or purple colors and soft / liquid

[0565] 5 and / or mucoid material consistency at > 10 mg / kg that were resolved following recovery. These clinical signs were likely correlated to the positive ADA responses in these animals and supported by microscopic findings and IHC findings attributed to immune complex deposition. Excessive struggling was also observed in the Compound A -treated animals at > 50 mg / kg, and although the cause could not be definitively confirmed, were resolved following recovery. It should be noted that there were only recovery males at 50 mg / kg available for assessment due to females euthanized prior to recovery necropsy. Therefore, most of these clinical signs upon completion of the recovery period for the SC males at 50 mg / kg were considered to be recovered.

[0566] For the 50 mg / kg IV animals that survived to their scheduled necropsy, Compound A -related clinical signs observed during the dosing phase (with their status at the end of the recovery period

[0567] 15 indicated) included abdominal distension (persisted), decreased activity (resolved), loss of consciousness during dosing and postdose (resolved), and salivation (resolved), which were all likely correlated to positive ADA responses. Vocalization and excessive struggling were also observed in the Compound A -treated animals, and although the cause could not be definitively confirmed, they were resolved following recovery. Overall, the clinical signs correlated to the positive ADA responses

[0568] 20 in the Compound A treated SC and IV animals, as supported by the ADA titers and immune complex formation, in particular, changes at the injection site in all SC groups at < 200 mg / kg and kidney changes in SC and IV groups at 50 mg / kg.

[0569] Compound A -related decrease in mean body weight gain was noted at 200 mg / kg SC and in mean body weight at 50 mg / kg IV females at the end of the dosing phase when compared to the

[0570] 25 control group, which recovered after the recovery period. For animals that survived to their scheduled necropsy, there were minimal effects on serum proteins (reversible) in Compound A treated SC animals at > 50 mg / kg as characterized by minimal increases in mean globulins and / or minimal decreases in mean albumin: globulin on Days 86 and 122. These findings were reversible and likely related to the microscopic findings of minimal to marked vascular / perivascular mononuclear cell

[0571] 30 inflammation at the injection site. There were minimal inflammatory changes in Compound A treated IV animals that survived to their scheduled necropsy at 50 mg / kg as characterized by increases in white blood cells, neutrophils, and monocytes along with minimal increases in fibrinogen, globulins, and decrease in albumin: globulin ratio on Days 86 and 114. These findings correlated with the microscopic findings of minimal to mild mixed cell vascular / perivascular inflammation of the lung. Minimal diminished erythropoiesis was found in IV animals at 50 mg / kg that survived to their scheduled necropsy, as characterized by decreases in total red blood cell mass (RBC, hemoglobin, hematocrit) on Day 71 . This was also associated with a minimal increase in platelets suggesting a reactive process.

[0572] Minimal tubular degeneration / necrosis in the kidney (males at 50 mg / kg SC) and minimal to

[0573] 40 marked vascular / perivascular inflammation at the 2 SC injection sites (animals at < 200 mg / kg) 45

[0574] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0575] PATENT ATTORNEY DOCKET NO: 50694-108001 associated with fibroplasia (animals at < 50 mg / kg SC) were observed after 26 weeks of Compound A treatment by SC route. At recovery euthanasia (Day 225), there was no degeneration / necrosis of tubules noted in males at 50 mg / kg / dose SC, indicating full recovery of the kidney findings in males. Vascular / perivascular mononuclear cell inflammation of the SC injection sites persisted following

[0576] 5 recovery, but at a lower severity in males in all SC-treated dose groups (< 200 mg / kg) and in females at 10 and 200 mg / kg SC, indicating partial recovery. Fibroplasia of the SC injection sites persisted in recovery SC females at 10 mg / kg, however, at a decreased severity compared to terminal euthanasia animals. Fibroplasia of the SC injection sites was not observed in SC recovery males. These findings indicated full recovery of fibroplasia in males and partial recovery in females. It should be noted that there were no 50 mg / kg recovery females (SC administration) available for histopathology evaluation due to early deaths, and therefore evaluation of recovery at this dose level could not be determined for females. Minimal to mild mixed cell vascular / perivascular inflammation of the lung, minimal tubular degeneration / necrosis of the kidney, and minimal GN (animals at 50 mg / kg) were observed after 64 days of Compound A treatment by IV route. Microscopic findings in the kidney and lung were not

[0577] 15 observed at recovery (Day 114), indicating complete recovery from IV administration of Compound A. Microscopic findings in the kidney (tubular degeneration / necrosis) and injection sites (vascular / perivascular inflammation) associated with Compound A SC administration, and in the kidney (tubular degeneration / necrosis, GN) and lung (vascular / perivascular inflammation) associated with Compound A IV administration, were all considered adverse at 50 mg / kg. However, these

[0578] 20 findings were consistent with immune complex mediated tissue changes secondary to ADA formation against Compound A and were confirmed with positive immunohistochemical staining.

[0579] For the SC animals (males and females combined), the early euthanasia of the three 50 mg / kg SC females with a cause of death attributed to tissue-specific immune complex deposition was considered adverse while, at 10 mg / kg SC, all animals survived to their terminal necropsy, with

[0580] 25 clinical signs and histopathology findings generally of reduced incidence and / or severity when compared to those noted at 50 mg / kg SC after 26 weeks of Compound A treatment. Furthermore, clinical signs and histopathology findings noted at 10 mg / kg SC in terminal animals were mostly resolved following a 6-week recovery period. As a result, the NOAEL of Compound A in monkeys following twice-weekly SC dose administration for 26 weeks is determined to be 10 mg / kg. If the

[0581] 30 immunogenicity associated early euthanasia of the females and histopathology findings at 50 mg / kg SC are excluded in adversity determination, then the NOAEL of Compound A in monkeys following twice-weekly SC dose administration for 26 weeks would be determined to be the highest SC dose level administered at 200 mg / kg.

[0582] For the IV animals, the early euthanasia of the single Compound A treated male at 50 mg / kg with a cause of death attributed to tissue-specific immune complex deposition was considered adverse. Therefore, the NOAEL could not be determined for Compound A in monkeys following twice- weekly IV dose administration for up to 64 days at 50 mg / kg IV of Compound A (the only dose level administered). Positive ADA response against Compound A was noted in the majority of 50 mg / kg IV animals, with the cause of death in single male at 50 mg / kg IV attributed to tissue-specific immune

[0583] 40 complex deposition. Furthermore, some of the clinical signs and histopathology findings noted at 50 46

[0584] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0585] PATENT ATTORNEY DOCKET NO: 50694-108001 mg / kg IV in terminal animals were resolved following a 6-week recovery period. Therefore, if the early euthanized male due to immunogenicity and histopathology findings at 50 mg / kg IV were not included in adversity determination, the NOAEL of Compound A in monkeys following twice-weekly IV dose administration for up to 64 days would be determined to be the highest IV dose level administered at

[0586] 5 50 mg / kg.

[0587] 2.3.7. Conclusions of the Nonclinical Toxicology

[0588] Toxicology studies in mice and monkeys indicate no direct toxicity related to Compound A. Mortality (mouse and monkey), clinical signs (mouse and monkey), clinical pathology (monkey) and histopathology changes (mouse and monkey) observed can be collectively attributed to immune complex-mediated tissue changes secondary to ADA formation against Compound A. Positive IHC staining of IgG, IgM, C3, and sC5b-9 at / in close proximity of histopathology changes in selected tissues from monkeys was consistent with an immune complex deposition across all Compound A administered SC and IV groups. Overall observations for Compound A were consistent with similar

[0589] 15 observations reported in the literature for nonclinical species administered with other biologies intended for human administration).

[0590] Based on the weight of evidence, findings in multidose general toxicology studies (in mice and monkeys) were determined to be immune complex mediated and of limited value to human risk assessment and were consistent with similar observations in monkeys administered with biologies

[0591] 20 intended for human administration.

[0592] 2.3.8. DART

[0593] Studies are planned to be conducted in C57BL / 6 mice. The EFD studies are in progress. Because of the ethical consideration for the usage of monkeys in research, high-cost implications,

[0594] 25 and the duration of ePPND studies (1-3 years) as well as significant immunogenicity observed in the GLP 6-week toxicology study (1727-277) and the 26-week toxicology study (1727-294) conducted in cynomolgus monkey, the mouse was considered the suitable species to conduct DART studies for the Compound A program. A non-GLP dose range finding EFD study in pregnant C57BL / 6 mice (Study 2046-0827) was conducted at doses of 30, 100, and 200 mg / kg SC given qd for 10 days (Gestation

[0595] 30 Days 6 to 15). The parameters and endpoints evaluated in this study included mortality, clinical signs, injection site scoring, body weights, body weight gains, gravid uterine weights, TK parameters, ADA analysis, macroscopic examinations, intrauterine growth and survival, and external fetal morphology. One female at 30 mg / kg was euthanized in extremis on Gestation Day 9 while 1 female at 100 mg / kg was found dead on Gestation Day 14 in the TK groups. The cause of death was undetermined for both animals, however, the clinical findings noted for the female at 30 mg / kg group suggested a mechanical injury unrelated to Compound A administration.

[0596] Noteworthy findings in the main study animals at 200 mg / kg included a slightly higher mean percentage of post-implantation loss compared to the control group. This difference was attributed to 1 female at 200 mg / kg with an individual post-implantation loss of 60% compared to 0% to 25.0% for

[0597] 40 other females in control and 200 mg / kg groups. Lower, non-dose-responsive mean gravid uterine 47

[0598] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0599] PATENT ATTORNEY DOCKET NO: 50694-108001 weights were noted at 100 and 200 mg / kg and a higher mean percentage of post-implantation loss was noted at 200 mg / kg group which was attributed to a single animal. Overall, dose levels of 30, 100, and 200 mg / kg were well tolerated in the maternal mice. Also, based on the lack of any significant findings, dose levels of 30, 100, and 200 mg / kg / day were selected for a definitive

[0600] 5 embryo / fetal development study of Compound A administered via SC injection in female C57BL / 6 mice.

[0601] 2.3.9. Special Studies

[0602] In a GLP tissue cross-reactivity study using normal human tissues, Compound A stained extracellular material within the mammary glands (secretory material), thymus (keratinized material within Hassall’s corpuscles), and ureter (ureteral lumen). Staining within this study was considered expected.

[0603] 2.3.10. First-In-Human Study Compound A

[0604] 2.3.10.1. Overview

[0605] 15 The first in human Compound A study is a randomized, double-blind, placebo-controlled, FIH study to evaluate safety, tolerability, PK, PD, and immunogenicity of SADs of Compound A SC and Compound A IV. The study planned for 48 healthy adult participants, of which 36 participants will receive Compound A and 12 participants will receive placebo as described in FIG. 3.

[0606] For this FIH study, 8 participants were randomly assigned in a 3:1 (Compound A: placebo)

[0607] 20 ratio in each of the 6 treatment cohorts, with escalating doses of 50 mg SC (Cohort 1), 150 mg SC (Cohort 2), 450 mg SC (Cohort 3), and 900 mg SC (Cohort 4), followed by 450 mg IV (Cohort 5) and 900 mg SC in Japanese participants (Cohort 6).

[0608] Enrollment, dosing, and follow-up period of this study have been completed (data analysis and reporting are in progress). Preliminary data are available from Cohorts 1-5 and are summarized

[0609] 25 below. The data from Cohort 6 in Japanese participants were not available as of the data cutoff (08 Dec 2023).

[0610] 2.3.10.2. Summary of Data

[0611] Pharmacokinetics: Serum Compound A concentrations

[0612] Serum Compound A concentrations reached Cmax (0.33-5.26 pg / mL) at 6.0-18 hours and decreased with mean ti / 2 of 27-34 hours after SC dosing from 50 to 900 mg. Similar elimination rates were observed across all SC doses. Inter-subject variability in systemic exposure was low to moderate after SC dosing (Table 4). The Compound A Cmax after SC dosing increased in a dose proportional manner because the estimate of the slope was 0.967 and the corresponding 90% Cl

[0613] 35 included 1.0. The Compound A AUCo-inf after SC dosing increased in a slightly greater than dose proportional manner because the estimate of the slope was 1 .074 and the 90% Cl did not include 1 .0 (FIG. 4). The SC bioavailability was 61 .5% at the 450 mg dose.

[0614] 48

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[0616] PATENT

[0617] ATTORNEY DOCKET NO: 50694-108001

[0618] Table 4: Summary of PK Parameters Following Single SC or IV Dose (Cohorts 1-5)

[0619] Values are presented as geometric mean (geometric %CV) for all except median (range) for Tmax.

[0620] Source: Table 4.

[0621] 5

[0622] Pharmacodynamics: Systemic CAP activity

[0623] Following a single SC dose administration from 50 to 900 mg, no significant changes from Baseline overtime were observed in systemic CAP activity. The mean systemic CAP activity decreased by approximately 30% from Baseline and activity returned to > 90% of Baseline by 72 hours postdose, thus suggesting that the changes in the mean systemic CAP activity were transient (FIG. 6).

[0624] Following a single IV dose administration of 450 mg, the mean systemic CAP activity decreased rapidly with the nadir at approximately 0.5 hours postdose. The mean systemic CAP activity decreased by approximately 40% from Baseline and returned to > 90% of Baseline by

[0625] 15 24 hours postdose (FIG. 6) .

[0626] Pharmacodynamics: Complement fH serum concentrations

[0627] Following a single SC dose administration from 50 to 900 mg and a single IV dose administration of 450 mg, no significant changes from Baseline overtime were observed in serum fH

[0628] 20 concentration (FIG. 5). The changes in the mean serum fH concentrations were within the normal range.

[0629] Immunogenicity

[0630] All protein therapeutics, including Compound A, have the potential for the development of ADA that may trigger symptoms resembling an allergic reaction (including anaphylaxis) and may impact the PK / PD profile of the product, leading to loss of efficacy. In the study, none of the 30 participants treated with Compound A showed pre-existing immunoreactivity and the majority of the study participants were ADA negative with only 3 of 30 exhibiting treatment emergent ADA responses. The observed 3 ADA positive responses were in the SC-treated arm, one in each of 50

[0631] 30 mg, 450 mg, and 900 mg dosing cohorts. In the same study, no treatment emergent ADA response was observed in the IV cohort. The ADA titers in the 3 treatment emergent ADA responses were low (range 30 to 120) and were not associated with any hypersensitivity and anaphylaxis reactions, or any other immune mediated adverse event across the cohorts. No difference in the PK was observed between ADA positive and ADA negative study participants. The ADA response was assessed on day

[0632] 49

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[0634] PATENT

[0635] ATTORNEY DOCKET NO: 50694-108001

[0636] 29 after dosing and the 27 to 34 hr half-life of Compound A in serum suggests that the elicited ADA may not have interacted with the drug before its elimination, hence showing no impact on PK.

[0637] Safety

[0638] 5 No potential safety concerns have been identified, and Compound A was well tolerated at all dose levels. In total, 24 of 30 participants exposed to Compound A reported 66 AEs and these are summarized in Table 5 and Table 6.

[0639] Overall, most AEs were non-serious, mild to moderate in severity and had resolved. There were no deaths. There was 1 SAE reported (PT: Campylobacter colitis infection; assessed as not

[0640] 10 related to Compound A by the investigator). There were no clinically significant abnormalities observed in vital signs, ECG, or laboratory values.

[0641] Table 5: Summary of Adverse Event Data from Study as of Cutoff Date 08 Dec 2023

[0642] Table 6: Frequency of Treatment-emergent Adverse Events by System Organ Class and Preferred Term

[0643] 50

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[0645] PATENT

[0646] ATTORNEY DOCKET NO: 50694-108001

[0647] Table 6: Frequency of Treatment-emergent Adverse Events by System Organ Class and Preferred Term

[0648] 51

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[0650] PATENT

[0651] ATTORNEY DOCKET NO: 50694-108001

[0652] Table 6: Frequency of Treatment-emergent Adverse Events by System Organ Class and Preferred Term

[0653] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0654] PATENT ATTORNEY DOCKET NO: 50694-108001

[0655] 2.3.11. Proposed Phase 2 Study of Compound A

[0656] Compound A is being evaluated in adult participants with primary FSGS in a Phase 2, randomized, double-blinded, placebo-controlled study. This study is designed to evaluate the efficacy

[0657] 5 and safety of Compound A in addition to SoC in adult participants with primary FSGS administered SC compared to placebo to demonstrate proof-of-concept of localized internal CAP inhibition in this disease.

[0658] The study will randomize participants 1 :1 :1 to receive Compound A 450 mg (N=35), Compound A 180 mg (N=35), or placebo (N=35) for a total of 105 participants. Randomization will be stratified by baseline proteinuria based on proteinuria level > 3 g / g and 1 .5 — 3 g / g.

[0659] A > 20% to 30% relative treatment effect over SOC in change in proteinuria is considered clinically relevant. To target at least a 25% relative treatment effect, the GMR of 24-week to baseline in proteinuria is assumed to be 0.65 (i.e., a 35% reduction in proteinuria) for the combined Compound A treatment groups, and 0.85 (i.e., a 15% reduction in proteinuria) for the placebo group. Sample size

[0660] 15 calculations are based on a one-sided two-sample t-test of log-transformed proteinuria values. The log change from baseline in proteinuria is calculated as log(0.65) and log(0.85) for the Compound A (combined) and placebo treatment groups. Assuming a common standard deviation of 0.7 of the log transformed percentage change from baseline and a 10% participant loss, this sample size will provide approximately 70% power to detect a relative treatment effect of 25% comparing the combined

[0661] 20 Compound A treated participants to placebo with a 1 -sided significance level of 0.1 .

[0662] The study consists of an up to 4-week Screening period and a 24-week Treatment Period. After the Treatment Period, participants will have the option to enter an Extended Treatment Period for up to 52 weeks. During the Extended Treatment Period, Compound A treated participants will continue with their assigned dose during their Treatment Period. Participants who were in the placebo

[0663] 25 group will be randomized (1 :1) to receive either 450 mg or 180 mg. A 6-week post-treatment Safety Follow-up Period will be required of all patients whether or not they participate in the Extended Treatment Period. Thus, the total treatment duration is up to 76 weeks and the total study duration is up to 86 weeks (FIG. 7).

[0664] The purpose of the 4-week screening period is to confirm and optimize the stabilization of concomitant therapy and establish persistent baseline proteinuria. In the post-treatment Safety Follow-up Period, all participants will continue to receive SoC and will be monitored for safety, clinical events of interest, and kidney function.

[0665] A randomized, double-blind, placebo-controlled study design is selected to provide the most robust evidence of efficacy and safety of Compound A. Randomization minimizes the effect of

[0666] 35 confounding factors on the study endpoints. The use of placebo comparator allows for the true treatment effect of Compound A by minimizing the risk of selection bias. In the FSGS-CT study (NCT00135811), patients were treated with CSA vs steroids + MMF for 1 year. The earliest urine protein reduction was observed between 4 to 6 months. In an extensive analysis of the NEPTUNE cohort on the FSGS subset, most patients would have partial or complete remission between 4 to 8

[0667] 53

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[0669] PATENT ATTORNEY DOCKET NO: 50694-108001 months. Given the uncertainty around the PD of Compound A and the timing of an early signal for urine protein reduction, a 24-week treatment period is proposed.

[0670] An interim analysis may be conducted when 100% of participants have been randomly assigned to study treatment or placebo and have had the opportunity to complete 12-weeks of the

[0671] 5 Treatment Period. This interim analysis, if performed, will be conducted by a separate unblinded team and will be for Phase 3 planning purposes only with no impact on the progression of the study.

[0672] 2.3.11.1. Dose Rationale for Compound A

[0673] This study proposes 2 dose levels of Compound A, 180 mg SC qw and 450 mg SC qw to fully characterize the PK, PD, dose-response, biomarker, efficacy, and safety data in participants with primary FSGS.

[0674] The dose selection is based on the integrated analysis of the preliminary safety, PK, and PD data from the FIH study in healthy adult participants (Cohort 1 to Cohort 5), in vitro and in vivo preclinical pharmacology studies, and modeling and simulation.

[0675] 15 A nonclinical in vivo pharmacology study with therapeutic administration of Compound A in the adriamycin nephropathy mouse model of FSGS demonstrated 50% reductions in proteinuria / albuminuria at 10 mg / kg dosed twice a week. This dose translates to a human equivalent dose of 178 mg SC per week based on allometric scaling. A second study using the same model did not replicate the proteinuria / albuminuria reductions observed in the first study, but significant dose¬

[0676] 20 dependent effects were observed in biomarkers assessed in the study, including urine KIM-1 and kidney weight (a surrogate indicator of inflammation). The urine KIM-1 and kidney weight data from the previous study appeared to reach a plateau at > 3 mg / kg. In addition, 2 in vivo pharmacology studies investigating prophylactic administration of Compound A in the PHN rat model of MN demonstrated prevention of proteinuria onset at 10 mg / kg (Study RTR-0073) which translates to a

[0677] 25 human equivalent dose of 322 mg SC per week based on allometric scaling. Therefore, 180 mg SC qw is selected as the minimum therapeutic dosage to be evaluated in this Phase 2 study.

[0678] In a single-dose PK study in adriamycin nephropathy mouse model of FSGS, the PK profiles of Compound A in proteinuric FSGS mice were comparable to those in healthy mice, suggesting that the PK properties of Compound A in healthy participants may be comparable to those in patients with

[0679] 30 FSGS.

[0680] In the FIH study, SAD of 50 mg, 150 mg, 450 mg and 900 mg SC were safe and well tolerated in healthy adult participants. After a single SC dose, Compound A was absorbed slowly with peak concentrations at 6 to 18 hours, and the serum Compound A concentrations declined in a biphasic manner with a half-life of 27 to 34 hours. The exposure to Compound A increased with increasing doses in a dose-proportional manner.

[0681] A population PK (PopPK) model (based on the data from Cohorts 1 to 5) was developed and PK profiles for multiple dosing in participants with primary FSGS were simulated. Based on the PopPK model, Compound A PK was adequately described by 2-compartment model with first-order absorption with a lag time. The estimated population PK parameters were CL (1 .98 L / h), Vc (24 L),

[0682] 40 Q (0.135 L / h), VP(0.029 L), Ka(0.024 1 / h), and Tiag(0.449 h). Preliminary covariate analysis (sex, 54

[0683] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0684] PATENT ATTORNEY DOCKET NO: 50694-108001 race, CrCL) showed no impact on Compound A PK. Simulations were performed using body weight (range 21 .1 kg - 154.0 kg) in the FSGS patient population for 180 and 450 mg SC to predict concentration-time profiles of Compound A following weekly SC doses. The simulated concentrationtime profiles and exposure indicated no accumulation following repeated qw doses (FIG. 8A and FIG.

[0685] 5 8B).

[0686] 55

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[0688] PATENT

[0689] ATTORNEY DOCKET NO: 50694-108001

[0690] Table 7: Predicted PK Exposure and Safety Margins in Patients with FSGS

[0691] Values are presented as geometric mean (range). aNOAEL: 200 mg / kg biw SC (26-week GLP NHP toxicology study; Study 1727-294).

[0692] 5 Note: AUCo-i68h is used for human subjects and AUCo-72h is used for NHPs.

[0693] To fully characterize exposure-response in FSGS participants and due to the novel selective, unmonitorable tissue (kidney) targeting mechanism of Compound A, the inclusion of a higher dose level of 450 mg SC qw is needed to inform the Phase 3 dose selection. The proposed high dose of 450 mg SC is ~3x the anticipated therapeutic dose, safe and well tolerated in the FIH study and has a 39-fold safety margin based on 1) the exposure at the NOAEL dose from the 26-week GLP toxicology study in cynomolgus monkeys, and 2) the simulated systemic exposure in participants with FSGS after multiple doses of 450 mg SC qw.

[0694] 15 2.3.11.2. Main Eligibility Criteria

[0695] The study population will include adult primary FSGS participants (> 18 and < 75 of years) with concomitant therapy consistent with SoC. The inclusion and exclusion criteria have been carefully selected to focus on patients with primary FSGS, and are listed below:

[0696] 1 . Diagnosis of primary FSGS by biopsy including both LM and EM within 3 years of screening:

[0697] 20 • At time of diagnosis, participants must have had acute onset of nephrotic syndrome and its associated features of high grade (3.5 g / day) proteinuria, hypoalbuminemia (< 3.5 g / dL), and peripheral edema.

[0698] • Participants must have received at least 16 weeks of therapy with at least 1 immunosuppressant, including corticosteroids except participants with contraindication to corticosteroids.

[0699] 2. Persistent UPCR > 1 .5 g / g based on 2 complete and valid 24-hour urine collection during the Screening Period

[0700] 3. Stable SoC (ACE / ARB / SGLT2i / immunosuppressant) dose for 16 weeks prior to randomization and do not have plans to alter the regimen during the study except to maintain

[0701] 30 therapeutic immunosuppression or in the event of AEs.

[0702] 4. Participants who have taken rituximab or cyclophosphamide will be eligible provided they have not taken these medications for 6 months prior to randomization.

[0703] 5. eGFR > 45 mL / min / 1 .73 m2during Screening

[0704] 56

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[0706] PATENT ATTORNEY DOCKET NO: 50694-108001

[0707] The proposal to enroll adult participants with primary FSGS who have received at least 16 weeks of therapy with at least 1 immunosuppressant at a stable dose is intended to allow for stabilization of participants with SoC before enrolling into the study and minimizes residual anti-proteinuria effect of the immunosuppressant. To enroll participants with persistent proteinuria despite SoC and to

[0708] 5 demonstrate reduction in proteinuria, the proposed inclusion criteria require persistent UPCR > 1.5 g / g during the Screening period. The inclusion of participants with eGFR > 45 mL / min / 1 ,73m2allows enrollment of participants with adequate kidney function and to demonstrate improvement in other kidney functions.

[0709] The study population is reflective of the characteristics of primary FSGS patients with persistent proteinuria despite SoC who may benefit from Compound A.

[0710] 2.3.11.3. Endpoint Selection

[0711] The primary endpoint for the study is percentage change in proteinuria from Baseline to Week 24 based on 24-hour urine collection at each timepoint. Change in proteinuria is a valid

[0712] 15 surrogate endpoint for kidney survival in glomerular diseases. Change in proteinuria is also an acceptable endpoint for clinical studies as concluded during a scientific workshop facilitated by the National Kidney Foundation in collaboration with FDA and EMA in 2018. A > 20% to 30% relative treatment effect over SoC in change in proteinuria is considered clinically relevant. Changes in proteinuria become evident before traditional markers of kidney survival (eg, ESRD, dialysis or transplant), thus especially useful for detecting signals of efficacy in a proof-of-concept study. Accordingly, the effect of Compound A on the percentage change reduction of proteinuria at Week 24 compared to the Baseline was selected as the primary endpoint for this study.

[0713] For the secondary endpoint, change in eGFR is considered as a suitable surrogate marker for renal survival in clinical studies of glomerular disease. There is strong evidence that nephrotic

[0714] 25 patients who experience a complete remission have a favorable prognosis. A partial remission in proteinuria and its maintenance are important therapeutic targets in FSGS, with implications for both slowing progression rate and improving renal survival. As such, partial and complete remission are other secondary endpoints.

[0715] 57

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[0717] PATENT

[0718] ATTORNEY DOCKET NO: 50694-108001

[0719] Table 8: Primary and Secondary Endpoints

[0720] 58

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[0722] PATENT

[0723] ATTORNEY DOCKET NO: 50694-108001

[0724] For the primary analysis at Week 24, the percentage change from baseline in proteinuria will be analyzed using all available data up to Week 24 with an MMRM model. To reduce skewness, the natural logarithm will be used to transform proteinuria values before the analysis is conducted. The

[0725] 5 model will include change from baseline in log-transformed proteinuria as the response variable and fixed, categorical effects of treatment group (combined Compound A treated at 450 mg / 180 mg: placebo), randomization stratification factor, visit, and treatment group by visit interaction as well as a fixed, continuous effect of baseline log proteinuria as a covariate. The treatment effect will be evaluated for the combined Compound A 450 mg / 180 mg and placebo treatment groups using a contrast for treatment group- by- vis it term at Week 24. The point estimate and 2-sided 90% Cl for the mean difference of log-transformed proteinuria will be back-transformed (via exponentiation) to obtain the GMR and corresponding 2-sided 90% Cl. The values will then be expressed as percentage change in adjusted geometric mean of proteinuria at Week 24 relative to baseline.

[0726] For the secondary analysis, binary endpoints, including participants who achieve > 30% and > 50%

[0727] 15 reduction in proteinuria at Week 24 compared to Baseline, and participants who meet the criteria for partial remission (defined as 40% proteinuria reduction and proteinuria < 1 .5 g / g) and complete remission (defined as reduction of UPCR < 300 mg / g, stable serum creatinine and albumin > 3.5 g / dL) at Week 24, will use a CMH test to assess the difference between treatment (combined Compound A 450 mg / 180 mg vs. placebo) in proportions of participants achieving the reduction in proteinuria at

[0728] 20 Week 24 compared to baseline, controlling for the randomization stratification factor.

[0729] For continuous endpoints of change from Baseline in eGFR and the change from baseline in serum albumin at Week 24, the treatment comparisons will be based on an MMRM model similar to the primary analysis.

[0730] 25 Exploratory endpoints: complement as biomarker

[0731] This study will generate a unique setting to enrich the understanding of the role and the functional significance of complement in the pathogenesis of primary FSGS.

[0732] Currently, no gold standard prognostic or predictive biomarker exists for primary FSGS. Mechanistic studies strongly suggest that complement activation plays a critical role in the pathogenesis of FSGS (see Section 2.1.1). Human studies have shown the potential of complement as a biomarker of disease progression and response to therapy. Compound A ameliorates intrarenal CAP activation through targeted delivery of fH. fH is a regulatory factor that binds and irreversibly inactivates C3b, thereby inhibiting the progression of the complement cascade and deposition of MAC. Measurement of complement activation can therefore be used as an index of PD drug efficacy

[0733] 35 for Compound A.

[0734] Several lines of evidence point to the validity of complement measurements as an indicator of disease activity and progression in primary FSGS. In a 70-patient cohort of primary FSGS, plasma and urinary C5b-9 levels correlated with the severity of proteinuria, renal dysfunction, and interstitial 59

[0735] INCORPORATED BY REFERENCE (RULE 20.6) PCT / US25 / 21821 02 June 2025 (02.06.2025)

[0736] PATENT ATTORNEY DOCKET NO: 50694-108001 fibrosis. Additionally, plasma C5a correlated with the proportion of segmental sclerotic glomeruli, and urinary levels of Bb correlated with renal dysfunction and interstitial fibrosis. Furthermore, urinary Bb levels had predictive value for the occurrence of remission (HR = 3.348, 95% Cl: 1.264-8.870, p = 0.015) and ESRD (HR = 2.323, 95% Cl: 1 .222 - 4.418,p = 0.010). The deposition of complement

[0737] 5 in the kidney has also had predictive and prognostic significance in this disease. In a multivariable analysis on 58 patients with primary FSGS, the presence and intensity of C3 deposits in the patients who had IgM deposits in their kidney biopsies were independent risk factors for refractory disease (odds ratio, 11 .32; 95% Cl: 2.26 - 56.65; p = 0.003) and progression to renal dysfunction (HR = 5.67; 95% Cl: 1.34 - 23.84; p = 0.02).

[0738] Complement activation seems to even delineate a specific pathophysiological pathway in disease with glomerular proteinuria. In a comparison between 41 patients with primary FSGS and 15 patients with MCD that had similar levels of proteinuria, the levels of urinary sC5b-9 and C5a were noted to be significantly higher in the primary FSGS patients (8.7 [1 .7-52.3] and 1 .26 [0.45-1 .84] ) pg / mmol of creatinine, respectively in primary FSGS vs 0.8 (0.0-1 .5) and 0.06

[0739] 15 (0.01-0.15) pg / mmol of creatinine, respectively in MCD, p < 0.001],

[0740] Complement detection assays that were previously validated and utilized in clinical trials with ravulizumab in aHUS (approved in 2019) will be leveraged. To evaluate the state of complement system activation in kidney tissue, the deposition of C3 and C4d in the kidney will be assessed, and urinary Ba, Bb, C3a, C3b (for Alternative Pathway), C5a and C5b-9 (for Terminal Pathway), and C1q

[0741] 20 and C4d (for Classical Pathway) will be measured. In addition, the plasma levels of Ba, C3a, C5a, and C5b-9 will be assessed to evaluate the possible effects of Compound A on systemic complement activity.

[0742] In this study, changes in complement component levels will also be evaluated as a PD biomarker. The delivery Compound A (fH regulatory domain) to the kidney destabilizes C3 convertase

[0743] 25 (C3bBb) by displacing Bb and acting as a cofactor for factor l-mediated cleavage of C3b. Therefore, urinary Bb and C3b levels are direct indicators of Compound A action at the disease site (PD biomarker). Based on the above-mentioned observations in patients, complement activation directly affects proteinuria; therefore, urine protein excretion will be used as an additional PD biomarker.

[0744] 30 2.3.11.4. Immunogenicity Assessment

[0745] In the study, participants will be monitored for presence of ADAs using a validated assay throughout the duration of the study. Serum samples for ADA analysis will be collected at the timepoints according to the schedule of activities described in the study protocol. Samples that are confirmed ADA positive will be further characterized for ADA titers. The incidence of ADA response categories will be summarized as an absolute occurrence (n) and a percentage of all participants. Analysis will be provided for overall and by treatment group. An assessment of ADA impact on PK, PD, safety, and efficacy will be performed.

[0746] For the future Phase 3 study, confirmed ADA positive samples will be further characterized for ADA titers, domain specificity, endogenous cross-reactivity.

[0747] 40

[0748] 60

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[0750] PATENT ATTORNEY DOCKET NO: 50694-108001

[0751] 3. Compound A Drug Product Manufacturing

[0752] Chemistry, Manufacturing and Control Data

[0753] Compound A Drug Substance Manufacturing

[0754] Compound A is expressed in CHO cells and manufactured using a cell culture process free of animal

[0755] 5 derived components. MCB were prepared in animal component free media and tested in compliance with cGMP. No evidence of contamination was identified in the MCB.

[0756] The Compound A drug substance manufacturing process utilizes a series of inoculum expansion steps leading up to the production bioreactor together with a multi-step orthogonal purification process using Protein A affinity, cation and anion exchange chromatography, virus reduction steps (low pH viral inactivation and virus filtration) and ultrafiltration / diafiltration step.

[0757] The drug substance does not contain any animal derived products.

[0758] The product is concentrated and formulated at a concentration of 50 mg / mL in 10 mM phosphate buffer, 1.4 mg / mL L-arginine, 27.8 mg / mL L-arginine HCI, 13.7 mg / mL sucrose, 0.5 mg / mL polysorbate 80, pH 7.0. The formulated Compound A bulk drug substance is stored in 4 or 6 L FFT

[0759] 15 bioprocess bags sterilized using ionizing radiation and stored at 15°C to 30°C protected from light. The Compound A drug substance will be tested and released based on release specifications for attributes that confirm identity, purity, activity, and microbiological attributes.

[0760] The Compound A drug product is a sterile, preservative-free formulated liquid solution contained in a single use 4 mL vial. The drug product contains a concentration of 50 mg / mL in 10 mM

[0761] 20 phosphate buffer, 1.4 mg / mL L-arginine, 27.8 mg / mL L-arginine HCI, 13.7 mg / mL sucrose, 0.5 mg / mL polysorbate 80, pH 7.0. Compound A drug product does not contain any novel excipient or excipient of animal or human origin.

[0762] The drug product manufacturing process does not include any compounding prior to the drug product fill. The formulated Compound A drug substance is pooled, mixed and then sterile filtered and

[0763] 25 filled into vials utilizing a process commonly used for biologies. Compound A drug product is filled into 4R borosilicate Type 1 clear glass vials and closed with elastomeric bromobutyl stoppers.

[0764] The Compound A drug product will be tested and released based on release specifications for attributes that confirm identity, purity, activity, and microbiological attributes. Testing includes appearance, pH, osmolality, protein concentration, SEC-HPLC, CE-SDS (reduced and non-reduced), specific activity, imaged capillary isoelectric focusing, peptide mapping, subvisible particulates, endotoxin, sterility, and volume in container.

[0765] Stability studies have been initiated for Compound A drug product and include evaluations at the long-term storage condition of 2°C-8°C, accelerated condition of 25°C ± 2°C protected from light. Each vial nominally contains 200 mg of Compound A in a volume of 4 mL. The vials are filled with a

[0766] 35 target volume of 4.3 mL to allow for the labeled volume to be delivered. The full composition of the drug product is listed in Table 9.

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[0769] PATENT

[0770] ATTORNEY DOCKET NO: 50694-108001

[0771] Table 9: Composition of Compound A, 50 mg / mL (200 mg / Vial) The fill volume of 4.33 mL is intended to contain NLT 4.0 mL of drug product.

[0772] The Compound A drug product is stored at 2°C to 8°C (36°F to 46°F), protected from light.

[0773] The same manufacturing process, methods fortesting, and quality from the Phase 1 study will be

[0774] 5 used for proposed Phase 2 study.

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[0777] PATENT

[0778] ATTORNEY DOCKET NO: 50694-108001

[0779] LIST OF ABBREVIATIONS

[0780] 63

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[0782] PATENT

[0783] ATTORNEY DOCKET NO: 50694-108001

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[0786] PATENT

[0787] ATTORNEY DOCKET NO: 50694-108001

[0788] Example 2. Treatment of FSGS in a subject with 180 mg per week of Compound A

[0789] A subject having FSGS is administered 180 mg of Compound A per week . Compound A is administered to the subject in a pharmaceutical composition comprising 50 mg / mL of Compound A in

[0790] 5 10 mM phosphate buffer, 1.4 mg / mL L-arginine, 27.8 mg / mL L-arginine HCI, 13.7 mg / mL sucrose,

[0791] 0.5 mg / mL polysorbate 80, pH 7.0. The pharmaceutical composition may be administered to the subject at least once per week but up to daily. The pharmaceutical composition is administered to the subject having FSGS subcutaneously.

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[0794] PATENT ATTORNEY DOCKET NO: 50694-108001

[0795] The pharmaceutical composition may be administered to the subject for 24 weeks or up to 52 weeks or for a longer period of time. Upon administration of Compound A to the subject, the subject may experience a reduction in proteinuria relative to baseline. For example, the subject may have a reduction in proteinuria of greater than 30% relative to baseline. The subject may experience

[0796] 5 reduction in proteinuria of greater than 50% relative to baseline after administration of Compound A. The subject may show the reduction in proteinuria after 24 weeks of administration of the pharmaceutical composition comprising Compound A. The subject may show the reduction in proteinuria after 52 weeks of administration of the pharmaceutical composition comprising Compound A.

[0797] The subject may show a change in estimated glomerular filtration rate (eGFR) relative to baseline after administration of the pharmaceutical composition. For example, the subject may demonstrate a change in eGFR from baseline after administration of Compound A for 24 weeks. In some embodiments, the subject experiences a change in eGFR from baseline after administration of Compound A for 52 weeks. The change in eGFR may be about 10% or greater than 10% relative to

[0798] 15 baseline.

[0799] The subject may show a change in serum albumin relative to baseline after administration of the pharmaceutical composition comprising Compound A. The change in serum albumin relative to baseline may be about a 10% change from baseline. The change in serum albumin relative to baseline may be greater than 10%; for example, the change from baseline may be greater than 30%.

[0800] 20 The subject may experience partial remission after administration of the pharmaceutical composition Compound A. The subject may experience complete remission after administration of the pharmaceutical composition comprising Compound A. The subject may experience improved kidney function relative to baseline during administration of the fusion protein after administration of the pharmaceutical composition comprising Compound A. The subject may experience a change in

[0801] 25 serum albumin relative to baseline, partial remission, complete remission, or improved kidney function relative to baseline after 24 weeks of administration of Compound A. The subject may experience a change in serum albumin relative to baseline, partial remission, complete remission, or improved kidney function relative to baseline after 52 weeks of administration of Compound A.

[0802] Example 3. Treatment of FSGS in a subject with 450 mg per week of Compound A

[0803] A subject having FSGS is administered 450 mg of Compound A per week . Compound A is administered to the subject in a pharmaceutical composition comprising 50 mg / mL of Compound A in 10 mM phosphate buffer, 1.4 mg / mL L-arginine, 27.8 mg / mL L-arginine HCI, 13.7 mg / mL sucrose, 0.5 mg / mL polysorbate 80, pH 7.0. The pharmaceutical composition may be administered to the subject at least once per week but up to daily. The pharmaceutical composition is administered to the

[0804] 35 subject having FSGS subcutaneously.

[0805] The pharmaceutical composition may be administered to the subject for 24 weeks or up to 52 weeks or for a longer period of time. Upon administration of Compound A to the subject, the subject may experience a reduction in proteinuria relative to baseline. For example, the subject may have a reduction in proteinuria of greater than 30% relative to baseline. The subject may experience

[0806] 66

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[0808] PATENT ATTORNEY DOCKET NO: 50694-108001 reduction in proteinuria of greater than 50% relative to baseline after administration of Compound A. The subject may show the reduction in proteinuria after 24 weeks of administration of the pharmaceutical composition comprising Compound A. The subject may show the reduction in proteinuria after 52 weeks of administration of the pharmaceutical composition comprising Compound

[0809] 5 A.

[0810] The subject may show a change in estimated glomerular filtration rate (eGFR) relative to baseline after administration of the pharmaceutical composition. For example, the subject may demonstrate a change in eGFR from baseline after administration of Compound A for 24 weeks. In some embodiments, the subject experiences a change in eGFR from baseline after administration of Compound A for 52 weeks. The change in eGFR may be about 10% or greater than 10% relative to baseline.

[0811] The subject may show a change in serum albumin relative to baseline after administration of the pharmaceutical composition comprising Compound A. The change in serum albumin relative to baseline may be about a 10% change from baseline. The change in serum albumin relative to

[0812] 15 baseline may be greater than 10%; for example, the change from baseline may be greater than 30%. The subject may experience partial remission after administration of the pharmaceutical composition Compound A. The subject may experience complete remission after administration of the pharmaceutical composition comprising Compound A. The subject may experience improved kidney function relative to baseline during administration of the fusion protein after administration of the

[0813] 20 pharmaceutical composition comprising Compound A. The subject may experience a change in serum albumin relative to baseline, partial remission, complete remission, or improved kidney function relative to baseline after 24 weeks of administration of Compound A. The subject may experience a change in serum albumin relative to baseline, partial remission, complete remission, or improved kidney function relative to baseline after 52 weeks of administration of Compound A.

[0814] Other Embodiments

[0815] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. While particular embodiments are herein described, one of skill in the art will appreciate that further modifications and

[0816] 30 embodiments are encompassed including variations, uses or adaptations generally following the principles described herein and including such departures from the present disclosure that come within known or customary practice within the art and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

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Claims

PCT / US25 / 21821 02 June 2025 (02.06.2025)PATENT ATTORNEY DOCKET NO: 50694-108001CLAIMS1 . A method of treating focal segmental glomerulosclerosis (FSGS) comprising administering an effective amount of a pharmaceutical composition comprising a fusion protein having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1 to a subject in need thereof.

2. The method of claim 1 , wherein the fusion protein has an amino acid sequence of SEQ ID NO: 1 .

3. The method of claim 1 or claim 2, wherein the FSGS is primary FSGS.

4. The method of any one of claims 1-3, wherein the subject has been diagnosed with FSGS within 3 years of being administered the pharmaceutical composition.

5. The method of any one of claims 1-4, wherein the pharmaceutical composition comprises phosphate buffer.

6. The method of claim 5, wherein the phosphate buffer has a concentration of between 5 mM and 15 mM, optionally wherein the phosphate buffer has a concentration of about 10 mM.

7. The method of any one of claims 1-6, wherein the pharmaceutical composition comprises one or more of L-arginine, arginine HCI, sucrose, or polysorbate.

8. The method of claim 7, wherein the pharmaceutical composition comprises between 0.5 mg / mL and 3 mg / mL L-arginine.

9. The method of claim 8, wherein the pharmaceutical composition comprises about 1 .4 mg / mL L- arginine.

10. The method of any one of claims 7-9, wherein the pharmaceutical composition comprises between 20 mg / mL and 35 mg / mL arginine HCI.11 . The method of claim 10, wherein the pharmaceutical composition comprises about 27.8 mg / mL arginine HCI.

12. The method of any one of claims 7-11 , wherein the pharmaceutical composition comprises between 10 mg / mL and 20 mg / mL sucrose.68INCORPORATED BY REFERENCE (RULE 20.6)PCT / US25 / 21821 02 June 2025 (02.06.2025)PATENT ATTORNEY DOCKET NO: 50694-10800113. The method of claim 12, wherein the pharmaceutical composition comprises about 13.7 mg / mL sucrose.

14. The method of any one of claims 7-13, wherein the pharmaceutical composition comprises polysorbate 80.

15. The method of any one of claims 7-14, wherein the pharmaceutical composition comprises between 0.1 mg / mL and 1 .5 mg / mL polysorbate.

16. The method of claim 15, wherein the fusion protein is formulated with about 0.5 mg / mL polysorbate.

17. The method of any one of claims 1-16, wherein the pharmaceutical composition comprises between25 mg / mL and 75 mg / mL of the fusion protein.

18. The method of claim 17, wherein the pharmaceutical composition comprises about 50 mg / mL of the fusion protein.

19. The method of any one of claims 1-18, wherein the subject is 18 years or more in age.

20. The method of any one of claims 1-18, wherein the subject is 75 years or less in age.

21. The method of any one of claims 1-20, wherein subject had acute onset of nephrotic syndrome at the time of FSGS diagnosis.

22. The method of any one of claims 1-21 , wherein the subject had proteinuria corresponding to at least 3.5 g / day of protein in urine at the time of diagnosis.

23. The method of any one of claims 1-22, wherein the subject had hypoalbuminemia corresponding to less than 3.5 g / d L of albumin in serum at the time of diagnosis.

24. The method of any one of claims 1-23, wherein the subject had peripheral edema at the time of diagnosis.

25. The method of any one of claims 1-24, wherein the subject has a urine protein creatinine ratio of at least 1.5 g / g prior to administration of the pharmaceutical composition.69INCORPORATED BY REFERENCE (RULE 20.6)PCT / US25 / 21821 02 June 2025 (02.06.2025)PATENT ATTORNEY DOCKET NO: 50694-10800126. The method of any one of claims 1-25, wherein the subject does not have kidney disease other than FSGS.

27. The method of any one of claims 1-26, wherein the subject shows a reduction in proteinuria relative to baseline after at least 24 weeks of administration of the pharmaceutical composition.

28. The method of any one of claims 1-27, wherein the subject shows a reduction in proteinuria of greater than 30% relative to baseline after at least 24 weeks of administration of the pharmaceutical composition.

29. The method of any one of claims 1-28, wherein the subject shows a reduction in proteinuria of greater than 50% relative to baseline after at least 24 weeks of administration of the pharmaceutical composition.

30. The method of any one of claims 1-29, wherein the subject shows a change in estimated glomerular filtration rate (eGFR) relative to baseline after at least 24 weeks of administration of the pharmaceutical composition.

31. The method of any one of claims 1-30, wherein the subject shows a change in serum albumin relative to baseline after at least 24 weeks of administration of the pharmaceutical composition.

32. The method of any one of claims 1-31 , wherein the subject shows partial remission after at least 24 weeks of administration of the pharmaceutical composition.

33. The method of any one of claims 1-31 , wherein the subject shows complete remission after at least 24 weeks of administration of the pharmaceutical composition.

34. The method of any one of claims 1-33, wherein the subject shows improved kidney function relative to baseline during administration of the fusion protein after at least 24 weeks of administration of the pharmaceutical composition.

35. The method of any one of claims 1-34, wherein the subject is administered at least 180 mg of the fusion protein weekly.

36. The method of claim 35, wherein the subject is administered about 180 mg of the fusion protein weekly.70INCORPORATED BY REFERENCE (RULE 20.6)PCT / US25 / 21821 02 June 2025 (02.06.2025)PATENTATTORNEY DOCKET NO: 50694-10800137. The method of any one of claims 1-34, wherein the subject is administered between 170 mg and 460 mg of the fusion protein weekly.

38. The method of claim 37, wherein the subject is administered about 450 mg of the fusion protein weekly.

39. The method of any one of claims 1-38, wherein the subject does not experience a treatment emergent adverse event.

40. The method of any one of claims 1-39, wherein the pharmaceutical composition is formulated for subcutaneous administration.

41. The method of any one of claims 1-40, wherein the subject is a mammal.

42. The method of claim 41 , wherein the mammal is a human.71INCORPORATED BY REFERENCE (RULE 20.6)

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