Polypeptides for binding complement component c5 or serum albumin and fusion proteins thereof

Abstract

To provide a modulator of C5 activity having desirable pharmaceutical properties (for example, half-life).SOLUTION: Provided is a fusion protein comprising an engineered polypeptide that specifically binds to human complement component C5 and an engineered polypeptide that specifically binds to human serum albumin, wherein the engineered polypeptide that specifically binds to human complement component C5 is fused, directly or via a peptide linker, to the polypeptide that specifically binds to human serum albumin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 531,215, filed July 11, 2017, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 24, 2018, has the file name AXJ-251PC_SL.txt, and is 376,575 bytes in size. [Background technology]

[0003] Complement component 5 (C5), the fifth component of complement, plays a key role in inflammation and cell killing processes. The activation peptide C5a, an anaphylatoxin with potent spasmogenic and chemotactic activities, is formed from the α polypeptide by cleavage with C5 convertase. The C5b macromolecular cleavage product can form a complex with the C6 complement component, which is the basis for the formation of the membrane attack complex (MAC), which contains additional complement components. Summary of the Invention [Problem to be solved by the invention]

[0004] Inadequate regulation of C5 can lead to immune-compromised patients or disorders characterized by excessive cell breakdown, such as hemolytic disorders caused by C5-mediated hemolysis.

[0005] Because misregulation of C5 can lead to severe and devastating phenotypes, there is a need for modulators of C5 activity with favorable pharmaceutical properties (e.g., half-life). [Means for solving the problem]

[0006] The present disclosure provides modified polypeptides that specifically bind to complement component C5 or serum albumin, which may be sdAb or Ig variable domains. In some embodiments, the modified polypeptides do not significantly reduce or inhibit binding of serum albumin to FcRn or significantly reduce the half-life of serum albumin. The present disclosure also provides fusion proteins, which may be multivalent, multispecific fusion proteins, comprising such modified polypeptides. Furthermore, the present disclosure provides nucleic acid molecules encoding such modified polypeptides or fusion proteins and methods for making such modified polypeptides or fusion proteins. Furthermore, the present disclosure provides pharmaceutical compositions comprising such modified polypeptides or fusion proteins and therapeutic methods using such modified polypeptides or fusion proteins.

[0007] In one embodiment, the present disclosure relates to a fusion protein comprising an altered polypeptide that specifically binds human complement component C5 and an altered polypeptide that specifically binds human serum albumin, wherein the altered polypeptide that specifically binds human complement component C5 is fused to the polypeptide that specifically binds human serum albumin, either directly or via a peptide linker. In certain embodiments, the C-terminal residue of the polypeptide that specifically binds human serum albumin is fused to the N-terminal residue of the polypeptide that specifically binds human complement component C5, either directly or via a linker. In certain embodiments, the C-terminal residue of the polypeptide that specifically binds human complement component C5 is fused to the N-terminal residue of the polypeptide that specifically binds human serum albumin, either directly or via a linker. In certain embodiments, the polypeptide that specifically binds human complement component C5 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and fragments thereof, and the polypeptide that specifically binds human serum albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34 and fragments thereof. In certain embodiments, the polypeptide that specifically binds to human complement component C5 comprises the amino acid sequence of SEQ ID NO: 11, and the polypeptide that specifically binds to human serum albumin has the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the fusion protein described herein further comprises a peptide linker having the amino acid sequence of SEQ ID NO: 102 or 103. In certain embodiments, the fusion protein has a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 96-101. In certain embodiments, the fusion protein consists of a sequence selected from the group consisting of SEQ ID NOs: 96-101. In certain embodiments, the fusion protein consists of the polypeptide sequence of SEQ ID NO: 96. In certain embodiments, the polypeptide that specifically binds to human complement component C5 comprises three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises any one of the amino acid sequences of SEQ ID NOs: 13-17, CDR2 comprises the amino acid sequence of SEQ ID NO: 18 or 19, and CDR3 has the amino acid sequence of SEQ ID NO: 20 or 21.In certain embodiments, a polypeptide that specifically binds to human serum albumin comprises three complementarity determining regions, CDR1, CDR2, and CDR3, where CDR1 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 35-43, CDR2 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 44-51, and CDR3 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 52-63. In some embodiments, the antigen-binding domains described herein can be modified or further engineered to bind to an antigen in a pH-dependent manner, e.g., with a higher affinity for the antigen at a higher pH and a lower affinity for antigen binding at a lower pH, or vice versa.

[0008] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein described herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition can include an agent that degrades or inactivates hyaluronan, such as hyaluronidase or recombinant hyaluronidase.

[0009] In one embodiment, the present disclosure relates to an isolated nucleic acid molecule having a nucleotide sequence encoding a fusion protein described herein. The nucleic acid molecule may be, for example, an expression vector. The present disclosure relates to host cells (e.g., Chinese hamster ovary (CHO) cells, HEK293 cells, Pichia pastoris cells, mammalian cells, yeast cells, plant cells, etc.) and expression systems that include or utilize nucleic acids encoding the fusion proteins described herein.

[0010] In one embodiment, the present disclosure relates to a modified polypeptide that binds to human complement component C5, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and fragments thereof. In certain embodiments, the modified polypeptide has an amino acid sequence at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a sequence selected from the group consisting of SEQ ID NOs: 1-12. For example, in one embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 1 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 2. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 3 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 3. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 4 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 4. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:5 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:5. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:6 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:6. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:7 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:7. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:8 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:8. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:9 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:9. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:10 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:10. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:11 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:11.In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:12 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:12.

[0011] In another embodiment, a modified polypeptide that binds to human complement component C5 is provided, the modified polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and fragments thereof. For example, in one embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 5. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 11. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 12.

[0012] In one embodiment, the present disclosure relates to modified polypeptides that specifically bind to human serum albumin, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34 and fragments thereof. In certain embodiments, the modified polypeptide has an amino acid sequence at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any one of the amino acid sequences of SEQ ID NOs: 22-34. For example, in one embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 22 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 22. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 23 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 23. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 24 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 24. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 25 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 25. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 26 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 26. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 27 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 27. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 28 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 28. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 29 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 29. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 30 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 30. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 31 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 31.In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 32, or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 32. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 33, or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 33. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 34, or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 34.

[0013] In another embodiment, the modified polypeptide that specifically binds to human serum albumin consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34 and fragments thereof. For example, in one embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 22. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 23. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 24. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 25. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 26. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 27. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 28. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 29. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 30. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 31. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 32. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 33. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 34.

[0014] In certain embodiments, the polypeptide that specifically binds to human serum albumin comprises three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 35 to 43, CDR2 comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 44 to 51, and CDR3 comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 52 to 63. In certain embodiments, the polypeptide specifically binds to the same epitope on human serum albumin as Alb1.

[0015] In one embodiment, the present disclosure relates to a method for producing a fusion protein described herein, comprising expressing in a host cell at least one nucleic acid molecule having a nucleotide sequence encoding a fusion protein described herein.

[0016] In one embodiment, the disclosure relates to a therapeutic kit comprising (a) a container containing a label; and (b) a composition comprising a fusion protein described herein, where the label indicates that the composition is to be administered to a patient having or suspected of having a complement-mediated disorder. The kit can optionally include an agent that degrades or inactivates hyaluronan, such as hyaluronidase or recombinant hyaluronidase.

[0017] In one embodiment, the disclosure relates to a method for treating a patient having a complement-mediated disorder, comprising administering to the patient a therapeutically effective amount of a fusion protein described herein. In certain embodiments, the complement-mediated disorder is selected from the group consisting of rheumatoid arthritis, lupus nephritis, asthma, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, paroxysmal nocturnal hemoglobinuria, macular degeneration, HELLP syndrome, Guillain-Barré syndrome, CHAPLE syndrome, myasthenia gravis, neuromyelitis optica, thrombotic microangiopathy after hematopoietic stem cell transplantation (post-HSCT TMA), post-bone marrow transplant TMA (post-BMT TMA), Degos disease, Gaucher disease, glomerulonephritis, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, oligoimmune vasculitis, epidermolysis bullosa, recurrent abortion, multiple sclerosis (MS), traumatic brain injury, and injury resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis. [Brief explanation of the drawings]

[0018] [Figure 1A] FIG. 1A shows the results of a classical complement pathway (CCP) hemolysis assay of the anti-C5 VHH domain. [Figure 1B] FIG. 1B shows the results of a classical complement pathway (CCP) hemolysis assay of the anti-C5 VHH domain. [Figure 2] FIG. 2 shows the results of a C5a release assay of anti-C5 VHH domains. [Figure 3A] FIG. 3A shows the results of a CCP hemolytic assay of the bispecific fusion protein. [Figure 3B] FIG. 3B shows the results of a CCP hemolytic assay of the bispecific fusion proteins. [Figure 3C] FIG. 3C shows the results of a CCP hemolytic assay of the bispecific fusion proteins. [Figure 3D] FIG. 3D shows the results of a CCP hemolytic assay of the bispecific fusion proteins. [Figure 4] FIG. 4 shows the results of a Wieslab CCP assay of the bispecific fusion proteins. [Figure 5] FIG. 5 shows the results of a C5a release assay of the bispecific fusion proteins. [Figure 6A] FIG. 6A shows the results of an LC-MS-based quantitative assay demonstrating the pharmacokinetics of the bispecific fusion protein. [Figure 6B] FIG. 6B shows the results of an LC-MS-based quantitative assay demonstrating the pharmacokinetics of the bispecific fusion protein. [Figure 7] Figure 7A shows a Biacore sensorgram showing FcRn binding to HSA without a VHH domain (control) in HBS-EP buffer at pH 6.0. Figure 7B shows a Biacore sensorgram showing FcRn binding to MSA21 in HBS-EP buffer at pH 6.0. Figure 7C shows a Biacore sensorgram showing FcRn binding to HAS040 in HBS-EP buffer at pH 6.0. Figure 7D shows a Biacore sensorgram showing FcRn binding to HAS041 in HBS-EP buffer at pH 6.0. [Figure 8] Figure 8A shows a Biacore sensorgram showing albumin binding by the VHH domain HAS040 competing with Alb1 VHH. Figure 8B shows a Biacore sensorgram showing albumin binding by the VHH domain HAS041 competing with Alb1 VHH. Figure 8C shows a Biacore sensorgram showing albumin binding by the VHH domain HAS020 competing with Alb1 VHH. Figure 8D shows a Biacore sensorgram showing albumin binding by the VHH domain HAS044 competing with Alb1 VHH. [Figure 9A] FIG. 9A shows the ability of various bispecific fusion proteins to inhibit hemolysis. [Figure 9B] FIG. 9B shows the ability of various bispecific fusion proteins to inhibit hemolysis. [Figure 10] Figure 10 shows that CRL0952 (SEQ ID NO: 96) is functionally very similar to CRL0500 in preventing hemolysis, which is a bispecific C5-albumin binding fusion protein linked with a (G4S)3 (SEQ ID NO: 106) linker. [Figure 11A] FIG. 11A shows the pH-dependent binding of histidine-substituted fusion proteins. [Figure 11B] FIG. 11B shows the pH-dependent binding of histidine-substituted fusion proteins. [Figure 11C] FIG. 11C shows the pH-dependent binding of histidine-substituted fusion proteins. [Figure 11D] FIG. 11D shows the pH-dependent binding of histidine-substituted fusion proteins. [Figure 12A] FIG. 12A shows the pH-dependent binding of histidine-substituted fusion proteins. [Figure 12B] FIG. 12B shows the pH-dependent binding of histidine-substituted fusion proteins. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present disclosure provides modified polypeptides that specifically bind to serum albumin or complement component C5, and may be, for example, single-domain antibodies (sdAbs) or immunoglobulin (IgG) variable domains. In some embodiments, the modified polypeptides do not significantly reduce or inhibit serum albumin binding to FcRn or significantly shorten the half-life of serum albumin. The present disclosure also provides fusion proteins comprising the modified polypeptides, which may be, for example, multivalent, multispecific fusion proteins. Furthermore, the present disclosure provides nucleic acid molecules encoding the modified polypeptides or fusion proteins and methods for making such modified polypeptides or fusion proteins. Furthermore, the present disclosure provides pharmaceutical compositions comprising the modified polypeptides or fusion proteins and therapeutic methods using such modified polypeptides or fusion proteins.

[0020] Standard recombinant DNA methodologies are used to construct polynucleotides encoding the modified polypeptides or fusion proteins of the present disclosure, incorporate such polynucleotides into recombinant expression vectors, and introduce such vectors into host cells to produce the modified polypeptides or fusion proteins of the present disclosure. See, e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Unless specifically defined, the nomenclature, laboratory procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those known and commonly used in the art. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0021] definition As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: Unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0022] As used herein, the term "binding domain" refers to a portion of a protein or antibody that contains amino acid residues that interact with an antigen. Binding domains include, but are not limited to, antibodies (e.g., full-length antibodies) and antigen-binding portions thereof. A binding domain confers its specificity and affinity for an antigen to a binding agent. The term also encompasses proteins having binding domains that are homologous or largely homologous to immunoglobulin binding domains.

[0023] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single-chain versions thereof. In a preferred embodiment, an "antibody" refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (heavy chain variable region, herein referred to as V H The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (V L The light chain constant region is composed of one domain, CL. H Area and V L The region can be further divided into regions of hypervariability called complementarity-determining regions (CDRs). These CDRs are separated by more conserved regions called framework regions (FRs). H and V L Each antibody consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including the first component of the classical complement system (Clq) and various cells of the immune system (e.g., effector cells).

[0024] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. Such "fragments" are, for example, about 8 to about 1500 amino acids in length, suitably about 8 to about 745 amino acids in length, suitably about 8 to about 300, e.g., about 8 to about 200 amino acids or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, i.e., a V L , V H (ii) a monovalent fragment consisting of the CL and CH1 domains; (iii) a F(ab')2 fragment, i.e., a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region; and (iv) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H (vi) an isolated complementarity-determining region (CDR); or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Additionally, the two domains of the Fv fragment, V, are also included. L and V H are encoded by separate genes, but using recombinant methods, V L and V H can be joined by a synthetic linker that can form a single protein chain. L Area and V HThe domains are paired to form a monovalent molecule (known as a single-chain Fv (sFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0025] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells, such as transfectomas, transformed to express the antibody, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable regions contain antigen-binding domains, which are encoded by different genes that rearrange to form antibodies specific for foreign antigens. Not only rearrangement, but also variable regions The constant regions may be further modified by multiple single amino acid changes (called somatic mutation or hypermutation), increasing the affinity of the antibody for the foreign antigen. The constant regions will further change in response to antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules that encode light and heavy chain immunoglobulin polypeptides in response to antigen may not share sequence identity with the original nucleic acid molecule, but will be substantially identical or similar (i.e., have at least 80% identity).

[0026] As used herein, the term "human antibody" refers to an immunoglobulin (Ig) used by the immune system, for example, to bind and neutralize pathogens. This term includes antibodies having variable and constant regions that substantially correspond to human germline Ig sequences. In some embodiments, human antibodies are produced in non-human mammals, including, but not limited to, rodents such as mice and rats and lagomorphs such as rabbits. In other embodiments, human antibodies are produced in hybridoma cells. In still other embodiments, human antibodies are recombinantly produced. As used herein, human antibodies include all or portions of antibodies, including, for example, heavy and light chains, variable regions, constant regions, proteolytic fragments, complementarity-determining regions (CDRs), and other functional fragments.

[0027] As used herein, a "biologically active fragment" refers to a portion of a molecule, such as a gene, coding sequence, mRNA, polypeptide, or protein, having a desired length or biological function. A biologically active fragment of a protein can be, for example, a fragment of the full-length protein that retains one or more biological activities of the protein. A biologically active fragment of an mRNA can be, for example, a fragment that, when translated, expresses a biologically active protein fragment. Additionally, a biologically active mRNA fragment can include truncated versions of non-coding sequences, such as regulatory sequences, UTRs, etc. In general, a fragment of an enzyme or signaling molecule can be, for example, that portion of the molecule that retains its signaling or enzymatic activity. A fragment of a gene or coding sequence can be, for example, that portion of the gene or coding sequence that produces an expression product fragment. A fragment is not necessarily functionally defined, as it can refer to a portion of a molecule, but not the entire molecule, but has some desired property or length (e.g., restriction fragments, proteolytic fragments of a protein, amplified fragments, etc.).

[0028] A normal or conventional mammalian antibody is generally a tetramer composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (generally having a molecular weight of about 25 kDa) and one full-length "heavy" chain (generally having a molecular weight of about 50-70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to an Ig polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The N-terminal portion of each light and heavy chain generally contains a variable domain of about 100-110 amino acids in length or more that is generally responsible for antigen recognition. The C-terminal portion of each chain generally defines a constant domain responsible for effector function. Thus, in naturally occurring antibodies, a full-length heavy chain Ig polypeptide contains a variable domain (V H or VH) and three constant domains (C H1 or CH1, C H2 or CH2, CH3 or CH3), V H The domains are the N-terminus, C-terminus, and C-terminus of the polypeptide. H3 The domain is located at the C-terminus, and the full-length light chain Ig polypeptide contains the variable domain (V L or VL) and constant domains (C L or CL), V L The domains are the N-terminus, C-terminus, and C-terminus of the polypeptide. L The domain is at the C-terminus.

[0029] Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with heavy chains also containing a "D" region of about 10 or more amino acids. The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions called CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, enabling binding to a specific epitope. From the N-terminus to the C-terminus, the variable domains of both light and heavy chains typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0030] As used herein, the terms "substantially pure" or "substantially purified" refer to a compound or species that is the predominant species present in a composition (i.e., more abundant, on a molar basis, than any other species in the composition). For example, a substantially purified fraction can be a composition in which the predominant species comprises at least about 50% (on a molar basis) of all macromolecular species present. For example, a substantially pure composition can include a predominant species that represents more than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, when a composition consists essentially of a single macromolecular species, the predominant species can be purified to substantial homogeneity (contaminating species in the composition cannot be detected by conventional detection methods).

[0031] As used herein, the term "antigen" or "antigen target" refers to a molecule or portion of a molecule that can be bound by an antibody to one or more Ig binding domains or other immunological binding moieties, including, for example, modified polypeptides or fusion proteins disclosed herein. An antigen can be used in an animal to produce antibodies that can bind to an epitope of that antigen. An antigen may have one or more epitopes.

[0032] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from either contiguous or non-contiguous amino acid sequences, provided they are joined by tertiary folding of the protein. Epitopes formed from contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Epitopes generally contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitope a given antibody binds to (i.e., epitope mapping) are well known in the art and include, for example, immunoblot and immunoprecipitation assays, in which overlapping or contiguous peptides from an antigen are tested for reactivity with a given antibody. Methods of determining spatial conformation of epitopes include techniques in the art and described herein, such as x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0033] The terms "activity," "biological activity," or "biological property" as used in reference to a modified polypeptide or fusion protein of the present disclosure include, but are not limited to, epitope affinity and specificity, the ability to antagonize the activity of an antigenic target, the in vivo stability of a modified polypeptide or fusion protein of the present disclosure, and the immunogenic properties of a modified polypeptide or fusion protein of the present disclosure. Other distinguishing biological properties include, for example, cross-reactivity (e.g., with non-human homologs of the antigenic target, or with other antigenic targets or tissues in general) and the ability to retain high expression levels of the protein in mammalian cells.

[0034] An antibody, immunoglobulin, or immunologically functional immunoglobulin fragment, or modified polypeptide or fusion protein disclosed herein is said to "specifically" bind to an antigen when the molecule preferentially recognizes its antigen target in a complex mixture of proteins and / or macromolecules. As used herein, the phrase "specifically binds" refers to the ability of an antibody, immunoglobulin, or immunologically functional immunoglobulin fragment, or modified polypeptide or fusion protein of the disclosure to bind to a specific antigen. D is at least about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 It refers to the ability to bind to an antigen containing an epitope of M or greater and / or the ability to bind to an epitope with an affinity at least two-fold greater than the affinity for a nonspecific antigen.

[0035] As used herein, "K D The term "dissociation constant" refers to the dissociation constant of the interaction between an antibody, immunoglobulin, or immunologically functional immunoglobulin fragment, or modified polypeptide or fusion protein disclosed herein, and an antigen target. When a modified polypeptide or fusion protein of the present disclosure has a monovalent Ig sequence, the monovalent Ig sequence preferably has a molecular weight of, for example, 10 -5~10 -12 M or less or 10 -7 ~10 -12 M or less or 10 -3 ~10 -12 K of M D and / or at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 or at least 10 12 M -1 It binds to the desired antigen with a binding affinity of 10 -4 K is bigger than M D Values are generally considered to represent non-specific binding. In some embodiments, the monovalent Ig sequence of a modified polypeptide or fusion protein of the present disclosure binds to a desired antigen with an affinity of less than 500 mM, less than 200 nM, less than 10 nM, or less than 500 pM.

[0036] K D can be determined by methods known in the art, including, for example, surface plasmon resonance (SPR). Generally, SPR analysis measures real-time binding interactions between a ligand (target antigen on a biosensor matrix) and an analyte, using, for example, a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). SPR analysis can also be performed by immobilizing the analyte and presenting the ligand. Specific binding of a modified polypeptide or fusion protein of the present disclosure to an antigen or antigenic determinant can also be determined by any suitable method known in the art, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays, enzyme immunoassays (EIAs), and sandwich competition assays.

[0037] The term "bispecific" refers to a fusion protein of the present disclosure that is capable of binding to two antigens. The term "multivalent fusion protein" refers to a fusion protein that contains two or more antigen-binding sites.

[0038] The term "multispecific fusion protein" refers to a fusion protein of the present disclosure that is capable of binding two or more related or unrelated targets.

[0039] As used herein, the term "fused to" refers to a polypeptide that is made by joining two or more sequences, typically by cloning one sequence, such as a coding sequence, into an expression vector in frame with one or more second coding sequences so that the two (or more) coding sequences are transcribed and translated into a single, contiguous polypeptide. In addition to being made by recombinant techniques, portions of a polypeptide can be "fused" to one another by chemical reaction or other means known in the art for making custom polypeptides.

[0040] As used herein, the term "vector" refers to a molecule (e.g., a nucleic acid, a plasmid, or a virus) used to transfer coding information into an expression system (e.g., a host cell or an in vitro expression system). One type of vector is a "plasmid," which refers to a circular double-stranded DNA (dsDNA) molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be inserted into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Additionally, some vectors are capable of expressing coding sequences to which they are transcriptionally linked. Such vectors are referred to herein as "expression vectors."

[0041] As used herein, the phrase "controllably linked" refers to the arrangement of flanking sequences so constructed or organized that they perform a desired function. Thus, a flanking sequence controllably linked to a coding sequence may be capable of influencing the replication, transcription, and / or translation of the coding sequence. For example, the coding sequence is controllably linked to a promoter that can effect transcription of the coding sequence. A flanking sequence need not be contiguous with the coding sequence to be considered controllably linked, as long as it functions properly.

[0042] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, as some modifications may occur in subsequent generations due to mutations or environmental influences, but such cells are still within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the modified polypeptides or fusion proteins of the present disclosure, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems).

[0043] As used herein, the term "naturally occurring" as applied to a particular molecule refers to a molecule that is found in nature and has not been manipulated by man. Similarly, as used herein, the term "non-naturally occurring" refers to a molecule that is not found in nature or that has been modified or artificially synthesized.

[0044] As used herein, the term "modified" applies to certain molecules, e.g., polypeptides, that have been modified or engineered by mutation, truncation, deletion, substitution, addition, conjugation, or otherwise altering the primary sequence, chemical or three-dimensional structure, chemical characteristics, folding behavior, glycosylation state, or other attributes of the molecule so that the molecule differs from its corresponding naturally occurring molecule.

[0045] As used herein, the term "patient" includes human and animal subjects.

[0046] A "disorder" is any condition that would benefit from treatment using a modified polypeptide or fusion protein of the present disclosure. "Disorder" and "condition" are used interchangeably herein.

[0047] As used herein, "complement-mediated disorder" refers to a disorder caused directly or indirectly by misregulation of the complement pathway, e.g., activation or inhibition of the complement pathway, or a disorder mediated directly or indirectly by one or more components of the complement pathway or products produced by the complement pathway. The term also refers to a disorder exacerbated by one or more components of the complement pathway or products produced by the complement pathway.

[0048] As used herein, the terms "treatment" or "treating" refer to both therapeutic measures and prophylactic or preventative measures. Those in need of treatment include those with the disorder as well as those at risk of having the disorder or those in whom the disorder is to be prevented.

[0049] As used herein, a "therapeutically effective" amount, e.g., of a fusion protein or modified polypeptide described herein, is an amount that, when administered, results in a decrease in the severity of disease symptoms (e.g., a reduction in symptoms of a disorder associated with complement-mediated damage, a longer and more frequent period free of disease symptoms, or prevention of functional impairment or disability due to disease affliction. In some embodiments, a therapeutically effective amount of a therapeutic agent described herein can include an amount (or varying amounts, in the case of multiple administrations) that reduces hemolysis or ameliorates symptoms of complement-mediated damage.

[0050] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of eliciting a desired therapeutic effect when administered to a patient.

[0051] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing delivery of a modified polypeptide or fusion protein of the present disclosure.

[0052] The term "therapeutically effective amount," as used with respect to pharmaceutical compositions comprising one or more modified polypeptides or fusion proteins of the present disclosure, refers to an amount or dosage sufficient to produce a desired therapeutic result. Specifically, a therapeutically effective amount is an amount of one or more modified polypeptides or fusion proteins of the present disclosure sufficient to inhibit, over a period of time, one or more clinically defined pathological processes associated with the condition being treated, such as a complement-mediated disorder. The therapeutically effective amount may vary depending on the particular modified polypeptide or fusion protein used and is dependent on a variety of factors related to the patient and the severity of the disorder and the condition being treated.

[0053] complement system The complement system, in conjunction with the rest of the body's immune system, defends against invasion by cellular and viral pathogens. There are at least 25 complement proteins, a complex collection of plasma proteins and membrane cofactors. Plasma proteins account for approximately 10% of the globulins in vertebrate serum. Complement components exert their immune defense functions by interacting in a complex yet precise series of enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and lytic functions.

[0054] The complement cascade can proceed through the classical pathway (CP), lectin pathway, or alternative pathway (AP). The lectin pathway is generally initiated by the binding of mannose-binding lectin (MBL) to high-mannose substrates. The AP can be initiated by specific molecules on the surface of pathogens, independent of antibodies. The CP is generally initiated by antibody recognition and binding of antigenic sites on target cells. These pathways converge at C3 convertase, where complement component C3 is cleaved by active proteases to generate C3a and C3b.

[0055] Spontaneous hydrolysis of complement component C3, abundant in the plasma fraction of blood, can also trigger AP C3 convertase initiation. This process, known as "autoactivation," occurs when the thioester bond of C3 spontaneously cleaves to form C3i or C3(H20). Autoactivation is facilitated by the presence of surfaces that support the binding of activated C3 and / or possess neutral or positively charged properties (e.g., bacterial cell surfaces). Formation of C3(H20) allows the binding of the plasma protein factor B, which then allows factor D to cleave factor B into Ba and Bb. The Bb fragment remains bound to C3, forming a complex containing C3(H20)Bb, i.e., the "fluid-phase" or "initiation" C3 convertase. Although produced in small amounts, fluid-phase C3 convertase can cleave multiple C3 proteins into C3a and C3b, resulting in the generation of C3b and its subsequent covalent attachment to surfaces (e.g., bacterial surfaces). Factor B bound to surface-bound C3b is cleaved by factor D to form a surface-bound AP C3 convertase complex containing C3b and Bb.

[0056] AP C5 convertase ((C3b)2,Bb) is formed when a second C3b monomer is added to AP C3 convertase. The role of the second C3b molecule is to bind C5 and present it for cleavage by Bb. AP C3 and C5 convertases are stabilized by the addition of properdin, a trimeric protein. However, properdin binding is not required to form functional alternative pathway C3 or C5 convertases.

[0057] CP C3 convertase is formed upon interaction of complement component C1, a complex of C1q, C1r, and C1s, with an antibody bound to a target antigen (e.g., a microbial antigen). The binding of the C1q portion of C1 to the antibody-antigen complex induces a conformational change in C1 that activates C1r. Active C1r then cleaves the associated C1s, generating an active serine protease. Active C1s cleaves complement component C4 to C4b and C4a. Like C3b, the newly generated C4b fragment contains a highly reactive thiol that readily forms amide or ester bonds with appropriate molecules on target surfaces (e.g., microbial cell surfaces). C1s also cleaves complement component C2 to C2b and C2a. The complex formed by C4b and C2a is the CP C3 convertase, which can process C3 to C3a and C3b. CP C5 convertase (C4b, C2a, C3b) is formed when a C3b monomer is added to the CP C3 convertase.

[0058] In addition to its role in the C3 and C5 convertases, C3b also functions as an opsonin through its interaction with complement receptors present on the surface of antigen-presenting cells such as macrophages and dendritic cells. The opsonic function of C3b is generally considered one of the most important anti-infective functions of the complement system. Patients with genetic disorders that inhibit C3b function are susceptible to infections by a wide variety of pathogenic organisms, whereas patients with defects later in the complement cascade sequence, i.e., those with disorders that inhibit C5 function, appear to be only slightly susceptible to Neisseria infection.

[0059] AP and CP C5 convertases cleave C5 into C5a and C5b. C5 cleavage releases C5a, a potent anaphylatoxin, and C5b, a chemotactic factor, allowing the formation of the lytic terminal complement complex C5b-9. C5b binds with C6, C7, and C8 to form the C5b-8 complex on the surface of target cells. Binding of several C9 molecules results in the formation of the membrane attack complex (MAC, C5b-9, terminal complement complex (“TCC”)). When sufficient numbers of MACs insert into the target cell membrane, the opening created by the MAC (MAC pore) triggers rapid osmotic lysis of the target cell.

[0060] A properly functioning complement system provides robust defense against infectious microorganisms, but improper regulation or activation of the complement pathway can lead to a variety of conditions, including rheumatoid arthritis (RA), lupus nephritis, asthma, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis type II (DDD), paroxysmal nocturnal hemoglobinuria (PNH), macular degeneration (e.g., age-related macular degeneration (AMD)), HELLP syndrome, Guillain-Barré syndrome (GBS), and protein-losing enteropathies (e.g., CHAPLE syndrome). It has been implicated in the pathogenesis of a variety of disorders, including myasthenia gravis (MG), neuromyelitis optica (NMO), thrombotic microangiopathy after hematopoietic stem cell transplantation (HSCT TMA), post-bone marrow transplant TMA (BMT TMA), Degos disease, Gaucher disease, glomerulonephritis, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, oligoimmune vasculitis, epidermolysis bullosa, recurrent abortion, multiple sclerosis (MS), traumatic brain injury, and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis (Holers, V., Immunol. Rev., 223:300-16, 2008). Downregulation of complement activation has been shown to be effective in treating several disease indications in various animal models (Rother, R. et al., Nat. Biotechnol., 25:1256-64, 2007; Wang, Y. et al., Proc. Natl. Acad. Sci. USA, 93:8563-8, 1996; Wang, Y. et al., Proc. Natl. Acad. Sci. USA, 92:8955-9, 1995; Rinder, C. et al., J. Clin. Invest., 96:1564-72, 1995) Kroshus, T. et al., Transplantation, 60:1194-202, 1995; Homeister, J. et al., J. Immunol., 150:1055-64, 1993; Weisman, H. et al., Science, 249:146-51, 1990; Amsterdam, E. et al., Am. J. Physiol., 268:H448-57, 1995; and Rabinovici, R. et al., J. Immunol., 149:1744-50, 1992).

[0061] Human serum albumin and neonatal Fc receptors Polypeptides capable of binding to human serum albumin (HSA) and extending the half-life of therapeutically relevant proteins have been described (International Publication Nos. WO 91 / 01743, WO 01 / 45746, and WO 02 / 076489). However, the peptide moieties described are of bacterial or synthetic origin and are not suitable for use in human therapeutics. International Publication No. WO 04 / 041865 describes single domain antibodies (sdAbs or Nanobodies®) against serum albumin (particularly HSA) that can bind to other proteins (such as one or more other single domain antibodies against desired targets) and extend their half-life.

[0062] The neonatal Fc receptor (FcRn), also known as the "Brambell receptor," is involved in extending the lifespan of albumin in the circulation (Chaudhury, C. et al., J. Exp. Med., 3:315-22, 2003). FcRn is an integral membrane glycoprotein composed of a soluble light chain consisting of β-microglobulin (β2m) noncovalently bound to a 43-kDa α chain with three extracellular domains, a transmembrane region, and a cytoplasmic tail approximately 50 amino acids long. The cytoplasmic tail contains an endocytosis signal, a dinucleotide motif, responsible for receptor internalization. The α chain is a member of the nonclassical MHC I family of proteins. The association of the α chain with β2m is important for the correct folding of FcRn and its exit from the endoplasmic reticulum to endosomes and the cell surface.

[0063] The overall structure of FcRn resembles that of class I molecules. The α-1 and α-2 regions resemble a platform composed of eight antiparallel strands forming a single β-sheet covered by two antiparallel α-helices, very similar to the peptide cleft of MHC I molecules. The bending of the C-terminal part of the α-2 helix and the overall rearrangement of the α-1 helix due to the helix cleavage introduced by the presence of Pro162 bring the FcRn helices into close proximity, blocking peptide binding. The side chain of Arg164 of FcRn also blocks potential interactions between the N-terminus of peptides and the MHC pocket. Furthermore, hydrophobic interactions and salt bridges between the α-1 and α-2 helices may also contribute to the closure of the cleft. Therefore, FcRn is not involved in antigen presentation, and the peptide cleft is empty.

[0064] FcRn binds IgG, transports it from the maternal circulation through placental syncytioblasts to the fetal circulation, and protects it from degradation in adults. In addition to homeostasis, FcRn regulates IgG transcytosis within tissues. FcRn is localized on epithelial cells, endothelial cells, and hepatocytes.

[0065] HSA binds to FcRn and forms a trimolecular complex with IgG. Albumin and IgG both bind non-cooperatively to distinct sites on FcRn. Human FcRn binding to Sepharose-HSA and Sepharose-hIgG is pH-dependent, maximal at pH 5 and undetectable at pH 7 to 8. The observation that FcRn binds to albumin in a pH-dependent manner similar to that of IgG suggests that the mechanism by which albumin interacts with FcRn to protect it from degradation is identical to that for IgG and is similarly pH-sensitive. Surface plasmon resonance measurements of the ability of individual HSA domains to bind immobilized soluble hFcRn, FcRn, and albumin revealed that the interaction occurs via the albumin DIII domain in a pH-dependent manner at a site distinct from the IgG-binding site (Chaudhury, C. et al., Biochemistry, 45:4983-90, 2006).

[0066] The modified polypeptide specifically binds to complement C5 or serum albumin Described herein are modified polypeptides having an Ig sequence, e.g., an Ig variable domain sequence, capable of binding to or otherwise associating with complement component C5 or serum albumin. The modified polypeptides described herein can specifically bind to serum albumin such that, when the modified polypeptide is bound to or otherwise associated with a serum albumin molecule, binding of the serum albumin molecule to FcRn is not significantly reduced or inhibited compared to binding of the serum albumin molecule to FcRn when the polypeptide is not bound. In this embodiment, "not significantly reduced or inhibited" means that the binding affinity of serum albumin to FcRn (as measured, for example, using an appropriate assay such as SPR) is not reduced by more than 50%, or more than 30%, or more than 10%, or more than 5%, or is not reduced at all. In this embodiment, "not significantly reduced or inhibited" also means that the half-life of the serum albumin molecule is not significantly shortened. In particular, the modified polypeptide can be an amino acid residue on serum albumin that is not involved in binding of serum albumin to FcRn. Specifically, a modified polypeptide, e.g., a modified polypeptide capable of binding to amino acid residues or sequences of serum albumin that form part of domain I and / or domain II, can bind to amino acid residues or sequences of serum albumin that do not form part of domain III of serum albumin.

[0067] In some embodiments, the modified polypeptide is an sdAb or is suitable for use as an sdAb, and may itself be a heavy chain variable domain sequence or a light chain variable domain sequence, and in some embodiments is a heavy chain variable domain sequence of a heavy chain antibody. When the modified polypeptide is a single domain or a heavy chain variable domain sequence from a heavy chain antibody, such a sequence may be used in combination with a VHH antibody or VHH antibody. H H antibody, VHH antibody fragment or V H H antibody fragment or VHH domain or V H It is sometimes called the H domain.

[0068] "Heavy chain antibodies" refer to antibodies that consist of two heavy chains and lack the two light chains found in conventional antibodies. Camelids (members of the biological family Camelidae, the only surviving family of camelids in the suborder Tylopoda; extant camelids include dromedaries, Bactrian camels, wild and re-wild camels, llamas, alpacas, vicunas, and guanacos) are the only mammals that have single-chain VHH antibodies. Approximately 50% of camelid antibodies are heavy chain antibodies, and the remaining 50% are of the usual or conventional mammalian heavy / light chain antibody type.

[0069] "VHH domain" refers to the variable domain in a naturally occurring heavy chain antibody, and is distinguished from the heavy chain variable domain (herein referred to as "VH domain") present in conventional four-chain antibodies and the light chain variable domain (herein referred to as "VL domain") present in conventional four-chain antibodies.

[0070] VHH domains possess many unique structural and functional properties that make isolated VHH domains (as well as sdAbs, which are based on VHH domains and have the same structural and functional properties as naturally occurring VHH domains) and proteins containing VHH domains highly suitable for use as functional antigen-binding domains or proteins. For example, VHH domains and sdAbs that bind antigen without a VL can function as single, relatively small, functional antigen-binding structural units, domains, or proteins. The small size of these molecules distinguishes VHH domains from the VH and VL domains of conventional four-chain antibodies. The use of VHH domains and sdAbs as single antigen-binding proteins or as antigen-binding domains (e.g., as part of a larger protein or polypeptide) offers a number of significant advantages over conventional VH and VL domains and scFvs or conventional antibody fragments (e.g., Fab or F(ab')2 fragments). For example, only a single domain is required to bind antigen with high affinity and high selectivity; there is no need for two separate domains, nor is there a need to ensure that these two domains are present in a specific spatial conformation and organization (e.g., using a specific linker, as is the case with scFvs). VHH domains and sdAbs can also be expressed from a single gene and do not require post-translational folding or modification. VHH domains and sdAbs can be easily engineered into multivalent or multispecific formats. VHH domains and sdAbs are also highly soluble, do not tend to aggregate (Ward, E. et al., Nature, 341:544-6, 1989), and are highly stable to heat, pH, proteases, and other denaturing or denaturing conditions (Ewert, S. et al., Biochemistry, 41:3628-36, 2002). VHH domains and sdAbs can be prepared relatively easily and inexpensively, even at the scale required for production.For example, VHH domains, sdAbs, and polypeptides comprising VHH domains or sdAbs can be produced using microbial fermentation by methods known in the art, and do not require the use of mammalian expression systems, as are the case with, for example, conventional antibody fragments. VHH domains and sdAbs are relatively small (approximately 15 kDa, i.e., one-tenth the size of conventional IgG) compared to conventional four-chain antibodies and their antigen-binding fragments, and therefore have high tissue penetration potential (including, but not limited to, solid tumors and other dense tissues). VHH domains and sdAbs can exhibit so-called "cavity binding" properties (e.g., due to extended CDR3 loops), allowing them to access targets and epitopes inaccessible to conventional four-chain antibodies and their antigen-binding fragments. VHH domains and sdAbs have been shown, for example, to be capable of inhibiting enzymes (International Publication No. WO97 / 49805; Transue, T. et al., Proteins, 32:515-22, 1998; Lauwereys, M. et al., EMBO J., 17:3512-20, 1998).

[0071] As used herein, the term "single domain antibody" or "sdAb" refers to an antibody or fragment thereof consisting of a single monomeric variable antibody domain. It is not intended to be limited to a particular biological source or a particular method of preparation. sdAbs can be obtained, for example, by (1) isolating the VHH domain of a naturally occurring heavy chain antibody, (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain, (3) "humanizing" a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain, (4) "camelizing" a naturally occurring VH domain from any animal species, particularly a mammalian species such as human, or expressing a nucleic acid encoding such a camelized VH domain, (5) "camelizing" a "domain antibody" ("Dab") or expressing a nucleic acid encoding such a camelized VH domain, (6) using synthetic or semi-synthetic techniques to prepare engineered polypeptides or fusion proteins, (7) preparing nucleic acids encoding sdAbs using techniques for nucleic acid synthesis followed by expression of the resulting nucleic acid, and / or (8) any combination of the above.

[0072] The modified polypeptides or fusion proteins described herein may have the amino acid sequence of a naturally occurring VHH domain that has been "humanized", for example, by substituting one or more amino acid residues of the amino acid sequence of the naturally occurring VHH sequence with one or more amino acid residues that occur at the corresponding position in a VH domain of human origin.

[0073] The modified polypeptides or fusion proteins described herein can have the amino acid sequence of a naturally occurring VH domain that has been "camelized," for example, by substituting one or more amino acid residues of the naturally occurring VH sequence with, for example, one or more amino acid residues occurring at the corresponding positions in the VHH domain of a camelized antibody. This can be performed by methods known in the art. Such camelization may occur preferentially at amino acid positions present at the VH-VL interface and at so-called "Camelidae hallmark residues" (International Publication No. WO 94 / 04678). The VH domain or sequence used as the parent sequence or starting material for generating or designing a camelized sequence may be, for example, a VH sequence of mammalian origin, and in some embodiments, a human VH sequence. However, such camelized sequences can be obtained by any suitable method known in the art, and are not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring parent VH domain.

[0074] Both "humanized" and "camelized" refer to naturally occurring VHH domains or VHH domains, respectively. H After preparing a nucleotide sequence encoding the domain, this can be achieved by modifying one or more codons of the nucleotide sequence by methods known to those skilled in the art so that the new nucleotide sequence encodes a humanized or camelized sequence, respectively. Alternatively, a nucleotide sequence encoding a desired humanized or camelized sequence can be designed and synthesized de novo based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain using nucleic acid synthesis techniques known in the art, and the nucleotide sequence thus obtained can then be expressed by methods known in the art.

[0075] In some embodiments, the present disclosure provides modified polypeptides that specifically bind to the same epitope on human C5 as eculizumab or that bind to an epitope on C5 that prevents cleavage of C5 into C5a and C5b. In some embodiments, the present disclosure provides modified polypeptides that specifically bind to human complement component C5, comprising any one of the amino acid sequences set forth in SEQ ID NOs: 1-12 or fragments thereof. In other embodiments, the present disclosure provides modified polypeptides that specifically bind to human complement component C5, having an amino acid sequence that is at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-12. In other embodiments, the present disclosure provides modified polypeptides that specifically bind to human complement component C5, having an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-12. For example, in one embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 1 or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 1. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:2 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:2. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:3 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:3. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:4 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:4. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:5 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:5. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:6 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:6. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:7 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:7. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:8 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:8.In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 9 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 9. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 10 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 10. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 11 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 11. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 12 or a sequence at least 90% identical to said sequence.

[0076] In another embodiment, a modified polypeptide that binds to human complement component C5 is provided, wherein the modified polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and fragments thereof. For example, in one embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 5. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 11. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 12.

[0077] In another embodiment, the present disclosure provides a modified polypeptide that specifically binds to human complement component C5, comprising three complementarity determining regions, namely CDR1, CDR2, and CDR3, wherein CDR1 comprises any one of the amino acid sequences of SEQ ID NOs: 13-17 or comprises a sequence at least 90% identical to SEQ ID NOs: 13-17, CDR2 has the amino acid sequence of SEQ ID NO: 18 or 19 or comprises a sequence at least 90% identical to SEQ ID NO: 18 or 19, and CDR3 has the amino acid sequence of SEQ ID NO: 20 or 21 or has a sequence at least 90% identical to SEQ ID NO: 20 or 21.

[0078] In other embodiments, the present disclosure provides modified polypeptides that specifically bind to human serum albumin, comprising any one of the amino acid sequences set forth in SEQ ID NOs: 22-34 or fragments thereof. In other embodiments, the present disclosure provides modified polypeptides that specifically bind to human serum albumin, having an amino acid sequence at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 22-34. In other embodiments, the present disclosure provides modified polypeptides that specifically bind to human serum albumin, having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 22-34. For example, in one embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 22 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 22. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 23 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 23. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 24 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 24. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:25 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:25. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:26 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:26. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:27 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:27. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:28 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:28. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:29 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:29. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO:30 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO:30.In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 31 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 31. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 32 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 32. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 33 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 33. In another embodiment, the modified polypeptide has the amino acid sequence set forth in SEQ ID NO: 34 or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 34.

[0079] In another embodiment, the modified polypeptide that specifically binds to human serum albumin consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34 and fragments thereof. For example, in one embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 22. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 23. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 24. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 25. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 26. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 27. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 28. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 29. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 30. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 31. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 32. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 33. In another embodiment, the modified polypeptide consists of the amino acid sequence set forth in SEQ ID NO:34.

[0080] In another embodiment, the present disclosure provides a modified polypeptide that specifically binds to human serum albumin, comprising three complementarity determining regions, namely CDR1, CDR2, and CDR3, wherein CDR1 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 35-43 or a sequence at least 90% identical to SEQ ID NOs: 35-43, CDR2 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 44-51 or a sequence at least 90% identical to SEQ ID NOs: 44-51, and CDR3 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 52-63 or a sequence at least 90% identical to SEQ ID NOs: 52-63.

[0081] The modified polypeptides disclosed herein can specifically bind to the same epitope on human serum albumin as Alb1 (AVQLVESGGG LVQPGNSLRL SCAASGFTFR SFGMSWVRQA PGKEPEWVSS ISGSGSDTLY ADSVKGRFTI SRDNAKTTLY LQMNSLKPED TAVYYCTIGG SLSRSSQGTQ VTVSS; SEQ ID NO: 149), for example. In other embodiments, the modified polypeptide competitively inhibits the binding of Alb1 to human serum albumin.

[0082] Where the modified polypeptide comprises an Ig, a suitable fragment of an Ig, such as an Ig variable domain, can also be used in place of the entire Ig.

[0083] Methods for identifying CDRs within a given immunoglobulin variable domain are known in the art (Wu, T. & Kabat, E., J. Exp. Med., 132:211-50, 1970; Clothia, C. et al., Nature, 342:877-83, 1989; Al-Lazikani, B. et al., J. Mol. Biol., 273:927-48, 1997; and Ofran, Y. et al., J. Immunol., 181:6230-35, 2008).

[0084] A fusion protein that specifically binds to complement component C5 and serum albumin Described herein are fusion proteins comprising modified polypeptides that specifically bind to albumin and complement component C5, where the modified polypeptides are directly fused or linked via one or more suitable linkers or spacers. As used herein, the term "peptide linker" refers to one or more amino acid residues inserted or included between the modified polypeptides of a fusion protein. A peptide linker can be inserted or included, for example, at the sequence level, at the segment between the modified polypeptides of a fusion protein. The identity and sequence of amino acid residues in the linker vary depending on the desired secondary structure. For example, glycine, serine, and alanine are useful for linkers with maximum flexibility. Any amino acid residue can be considered a linker in combination with one or more other amino acid residues, which may be identical or different, to construct larger peptide linkers as needed depending on the desired properties. In another embodiment, the linker is GGGGAGGGGAGGGGS (SEQ ID NO: 102). In another embodiment, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 103). Additional peptide linkers suitable for generating the fusion proteins described herein include, for example, G4S (SEQ ID NO: 104), (G4S)2 (SEQ ID NO: 105), (G4S)3 (SEQ ID NO: 106), (G4S)4 (SEQ ID NO: 107), (G4S)5 (SEQ ID NO: 108), (G4S)6 (SEQ ID NO: 109), (EAAAK)3 (SEQ ID NO: 110), PAPAP (SEQ ID NO: 111), G4SPAPAP (SEQ ID NO: 112), PAPAPG4S (SEQ ID NO: 113), GSTSGK SSEGKG (SEQ ID NO: 114), (GGGDS)2 (SEQ ID NO: 115), (GGGES)2 (SEQ ID NO: 116), GGGDSGGGGS (SEQ ID NO: 117), GGGASGGGGS (SEQ ID NO: 118), GGGESGGGGS (SEQ ID NO: 119), ASTKGP (SEQ ID NO: 120), ASTKGPSVFPLAP (SEQ ID NO: 121), G3P (SEQ ID NO: 122), G7P (SEQ ID NO: 123), PAPNLLGGP (SEQ ID NO: 124), G6 (SEQ ID NO: 125), G 12(SEQ ID NO: 126), APELPGGP (SEQ ID NO: 127), SEPQPQPG (SEQ ID NO: 128), (G3S2)3 (SEQ ID NO: 129), GGGGGGGGGSGGGS (SEQ ID NO: 130), GGGGSGGGGGGGGGS (SEQ ID NO: 131), (GGSSS)3 (SEQ ID NO: 132), (GS4)3 (SEQ ID NO: 133), G4A(G4S)2 (SEQ ID NO: 134), G4SG4AG4S (SEQ ID NO: 135), G3AS(G4S)2 (SEQ ID NO: 136) , G4SG3ASG4S (SEQ ID NO: 137), G4SAG3SG4S (SEQ ID NO: 138), (G4S)2AG3S (SEQ ID NO: 139), G4SAG3SAG3S (SEQ ID NO: 140), G4D(G4S)2 (SEQ ID NO: 141), G4SG4DG4S (SEQ ID NO: 142), (G4D)2G4S (SEQ ID NO: 143), G4E(G4S)2 (SEQ ID NO: 144), G4SG4EG4S (SEQ ID NO: 145), and (G4E)2G4S (SEQ ID NO: 146). Those skilled in the art can select linkers, for example, to reduce or eliminate post-translational modifications, such as glycosylation, e.g., xylosylation. In some embodiments, the fusion protein comprises at least two sdAbs, Dabs, VHH antibodies, VHH antibody fragments, or a combination thereof, wherein at least one of the sdAbs, Dabs, VHH antibodies, or VHH antibody fragments is directed against albumin and one of the sdAbs, Dabs, VHH antibodies, or VHH antibody fragments is directed against complement component C5, thereby rendering the resulting fusion protein multivalent or multispecific. The binding domains or moieties can be, for example, directed against HSA, cynomolgus serum albumin, human C5, and / or cynomolgus C5.

[0085] In some embodiments, the C-terminal residue of the albumin-binding domain of the fusion protein can be fused directly or via a peptide to the N-terminal residue of the complement component C5-binding domain, while in other embodiments, the C-terminal residue of the complement component C5-binding domain of the fusion protein can be fused directly or via a peptide to the N-terminal residue of the albumin-binding domain.

[0086] In some embodiments, the fusion protein comprises a complement component C5-binding domain comprising the amino acid sequence of SEQ ID NO: 1-12 or a fragment thereof, and the polypeptide that specifically binds to human serum albumin comprises the amino acid sequence of SEQ ID NO: 22-34 or a fragment thereof. In some embodiments, the first polypeptide is derived from the amino acid sequence set forth in any of SEQ ID NO: 1-12, and the second polypeptide is derived from the amino acid sequence set forth in any of SEQ ID NO: 22-34. The human complement component C5-binding domain can have, for example, the amino acid sequence of SEQ ID NO: 5 or 11, and the albumin-binding domain can have, for example, the amino acid sequence of SEQ ID NO: 26. In another embodiment, the present disclosure provides a fusion protein having any one of the amino acid sequences of SEQ ID NO: 64-95. In another embodiment, the present disclosure provides a fusion protein having the amino acid sequence of SEQ ID NO: 93. In another embodiment, the present disclosure provides a fusion protein having the amino acid sequence of SEQ ID NO: 77. In another embodiment, the present disclosure provides a fusion protein having any one of the amino acid sequences of SEQ ID NO: 96-101.

[0087] The fusion proteins disclosed herein can be produced by expressing at least one nucleic acid molecule having a nucleotide sequence encoding the fusion protein in a host cell. The host cell can be of mammalian, plant, or microbial origin. In addition to known mammalian host cells, yeast host cells, such as Pichia pastoris, Saccharomyces cerevisiae, and / or plant host cells can be used.

[0088] Therapeutic compositions comprising polypeptides that specifically bind to complement C5 or serum albumin or fusion proteins thereof and their administration In another embodiment, the present disclosure provides modified polypeptides having or consisting of the amino acid sequences disclosed herein. In another embodiment, the present disclosure provides fusion proteins, multivalent, multispecific fusion proteins, comprising or consisting of at least one modified polypeptide of the present disclosure linked, optionally via one or more suitable linkers or spacers, to at least one therapeutic or targeting moiety.

[0089] The present disclosure also relates to therapeutic uses of the modified polypeptides of the present disclosure, or fusion proteins and multivalent, multispecific fusion proteins comprising or consisting of such modified polypeptides, or pharmaceutical compositions comprising such modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins.

[0090] In some embodiments, the therapeutic or targeting moiety may comprise, for example, at least one sdAb, Dab, VHH or fragment thereof. In some embodiments, the modified polypeptides of the present disclosure are multivalent and / or multispecific fusion proteins comprising at least two sdAbs, Dabs, VHH antibodies, VHH antibody fragments or combinations thereof.

[0091] In some embodiments, the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins exhibit a higher affinity for HSA than for mouse serum albumin. In some embodiments, the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins exhibit a higher affinity for cynomolgus monkey serum albumin than for mouse serum albumin. In other embodiments, the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins exhibit a higher affinity for HSA than for cynomolgus monkey serum albumin.

[0092] In some embodiments, the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins exhibit higher affinity for human C5 than for mouse C5. In some embodiments, the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins exhibit higher affinity for cynomolgus monkey C5 than for mouse C5. In other embodiments, the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins exhibit higher affinity for human C5 than for cynomolgus monkey C5.

[0093] The modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins described herein can exhibit improved therapeutic properties, such as increased efficacy, bioavailability, half-life, or other therapeutically desirable properties, when compared to, for example, antibody or other therapeutic agents. In one embodiment, a fusion protein of the present disclosure comprises at least one modified polypeptide disclosed herein and at least one therapeutic or targeting moiety. In such fusion proteins, the fusion protein can have a longer half-life, for example, compared to the therapeutic binding domain alone. Generally, such fusion proteins have a half-life that is at least 1.5-fold, at least 2-fold, or at least 5-fold, or at least 10-fold, or even 20-fold longer than the corresponding therapeutic or targeting moiety alone. In some embodiments, the fusion proteins of the present disclosure have a half-life that is longer by more than 1 hour, more than 2 hours, more than 6 hours, or more than 12 hours compared to the corresponding therapeutic or targeting moiety alone. In other embodiments, the half-life of the fusion protein is greater than 1 hour, greater than 2 hours, greater than 6 hours, greater than 12 hours, about 1 day, about 2 days, about 1 week, about 2 weeks, about 3 weeks, or less than 2 months.

[0094] As used herein, the term "half-life" refers to the time it takes for the serum concentration of a modified polypeptide, fusion protein, or multivalent, multispecific fusion protein to decline by 50% in vivo, for example, as a result of molecular degradation and / or clearance or segregation of the molecule by physiological mechanisms. Methods for pharmacokinetic analysis and determining half-life are known to those of skill in the art.

[0095] General descriptions of multivalent, multispecific fusion proteins comprising one or more VHH antibodies and their preparation are known (Els Conrath, K. et al., J. Biol. Chem., 276:7346-50, 2001; Muyldermans, S., J. Biotechnol., 74:277-302 2001; International Publication Nos. WO96 / 34103, WO99 / 23221, WO04 / 041865).

[0096] The modified polypeptides, fusion proteins, and multivalent, multispecific fusion proteins disclosed herein can be expressed from or assembled into constructs comprising one or more elements, such as, for example, expression vectors (International Application Publication No. WO 04 / 041862).

[0097] The modified polypeptides, fusion proteins, and multivalent, multispecific fusion proteins disclosed herein can be expressed, for example, in an isolated host cell comprising a nucleic acid molecule encoding the modified polypeptides, fusion proteins, and multivalent, multispecific fusion proteins disclosed herein. Suitable host cells include, but are not limited to, mammalian and yeast cells.

[0098] Therapeutic or pharmaceutical compositions disclosed herein can comprise one or more modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins disclosed herein in a therapeutically effective amount in admixture with pharmaceutically or physiologically acceptable formulation materials selected to suit the mode of administration. Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

[0099] Acceptable formulation materials can be used, for example, to alter, maintain or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.Acceptable formulation ingredients include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavorings, diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as betaine, hydroxybenzoates ... and / or a pharmaceutical adjuvant (such as benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), a solvent (such as glycerin, propylene glycol, or polyethylene glycol), a sugar alcohol (such as mannitol or sorbitol), a suspending agent, a surfactant, or a wetting agent (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 or polysorbate 80, triton, tromethamine, lecithin, cholesterol, or tyloxapal), a stability enhancer (such as sucrose or sorbitol), a tonicity enhancer (such as an alkali metal halide, preferably sodium chloride or potassium chloride, or mannitol sorbitol), a delivery vehicle, a diluent, an excipient, and / or a pharmaceutical adjuvant (e.g., see REMINGTON'S Pharmaceuticals, the contents of which are incorporated herein by reference). Examples of useful reference materials include, but are not limited to, PHARMACEUTICAL SCIENCES (18th Ed., AR Gennaro, ed., Mack Publishing Company 1990 and subsequent editions thereof).

[0100] One skilled in the art can develop pharmaceutical compositions containing the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins disclosed herein depending, for example, on the intended route of administration, delivery format, and desired dosage.

[0101] The modified polypeptides, fusion proteins, and multivalent, multispecific fusion proteins disclosed herein may, in some embodiments, be administered to allow circulation, for example, because they may have a longer half-life. As such, they can be administered intravenously, subcutaneously, by injection or infusion, or any other suitable method that allows the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins to enter the body's circulation. The preparation of such pharmaceutical compositions is within the knowledge of those skilled in the art.

[0102] Any of the modified polypeptides, fusion proteins, and multivalent, multispecific fusion proteins disclosed herein can be administered in combination with additional therapies, i.e., in combination with other agents. As used herein, the term "co-administration" includes any or all of simultaneous, separate, or sequential administration of the modified polypeptides, fusion proteins, and multivalent, multispecific fusion proteins described herein with adjuvants and other agents, including administration as part of a dosing regimen.

[0103] The pharmaceutical compositions described herein can include one or more agents, for example, to improve delivery of a therapeutic agent. The additional agent can be co-administered, for example, as a co-injectable. For example, an agent that degrades hyaluronic acid can be included in the pharmaceutical compositions described herein, or such an agent can be co-administered with the pharmaceutical compositions described herein, for example, to facilitate the dispersion and absorption of the therapeutic agent described herein upon administration. One example of such an agent is recombinant hyaluronidase.

[0104] The pharmaceutical composition may also be selected for parenteral delivery. Alternatively, the composition may be selected for delivery via the digestive tract, such as by inhalation or orally. The preparation of such pharmaceutical compositions is within the knowledge of one skilled in the art.

[0105] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations for sustained or controlled delivery. Techniques for preparing sustained or controlled delivery formulations using, for example, liposome carriers, bioerodible microparticles or porous beads, and depot injections are known to those skilled in the art.

[0106] The present disclosure also encompasses therapeutic kits that include the modified polypeptides, fusion proteins, and multivalent and multispecific fusion proteins disclosed herein.In some embodiments, the kit includes both a first container containing dry protein and a second container containing aqueous formulation.In other embodiments, the kit includes single- and multi-chamber pre-filled syringes (for example, liquid syringes and lyosyringes).

[0107] The disclosure also includes an article of manufacture comprising a container containing a label and a composition comprising a modified polypeptide, fusion protein, or multivalent, multispecific fusion protein disclosed herein, wherein the label indicates that the composition is for administration to a patient with or suspected of having a complement-mediated disorder.

[0108] In one embodiment, the present disclosure provides a method for preventing and / or treating at least one disease, condition, or disorder that can be prevented or treated using the modified polypeptides, fusion proteins, or multivalent, multispecific fusion proteins disclosed herein, comprising administering a therapeutically or pharmaceutically effective amount of a modified polypeptide, fusion protein, or multivalent, multispecific fusion protein disclosed herein to a patient in need thereof. In certain embodiments, the disorder is, for example, rheumatoid arthritis (RA), lupus nephritis, asthma, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis type II (DDD), paroxysmal nocturnal hemoglobinuria (PNH), macular degeneration (such as, for example, age-related macular degeneration (AMD)), HELLP syndrome, Guillain-Barré syndrome (GBS), CHAPLE syndrome, myasthenia gravis (MG), optic nerve spinal cord injury, or other conditions. Complement disorders include myelitis (NMO), thrombotic microangiopathy after hematopoietic stem cell transplantation (post-HSCT TMA), post-bone marrow transplant TMA (post-BMT TMA), Degos disease, Gaucher disease, glomerulonephritis, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, oligoimmune vasculitis, epidermolysis bullosa, recurrent abortion, multiple sclerosis (MS), traumatic brain injury, and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

[0109] The effective amount of the pharmaceutical compositions disclosed herein to be used therapeutically will depend, for example, on the nature and purpose of the treatment. Those skilled in the art will understand that appropriate dosage levels for treatment will vary, in part, depending on the molecule being delivered, the indication for which the composition is being used, the route of administration, the size (weight, body surface, or organ size) and condition (age and general health) of the patient. [Example]

[0110] The following examples are illustrative of specific embodiments of the present disclosure and various applications thereof, and are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0111] Example 1 Immunization of llamas and construction of an anti-C5 VHH phage library Llamas were immunized starting with a primary injection followed by a secondary boost. Briefly, primary immunization began with 500 μg of human complement protein C5, followed by boosts of 500 μg of human complement protein C5 antigen at weeks 2 (boost 1), 4 (boost 2), 8 (boost 3), and 12 (boost 4). Serum titers were measured by ELISA, revealing that the titer was highest after boost 3, 10-fold greater than the pre-bleed signal at a 1:1,000,000 dilution. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood sample after boost 3. Trypan blue staining revealed cell viability of 98%. Immediately after PBMC isolation, cells were lysed in RNA lysis buffer. Total RNA was isolated from PBMCs, and cDNA was synthesized using llama heavy chain-specific primers. VHH (heavy chain only) fragments were separated from VH (conventional heavy chain) fragments by gel electrophoresis. These VHH fragments were cloned into pADL-10b (Antibody Design Labs, San Diego, CA), and the DNA library was transformed into TG1 cells. Random sequencing of 114 colonies yielded 101 (89%) correct sequences. The library was scraped, suspended in 25% glycerol, and stored at -80°C.

[0112] Example 2 Phage display panning and screening of anti-C5 VHH domains TG1 cells containing the anti-human complement protein C5 VHH domain library were cultured in 2xYT medium containing 100 μg / mL carbenicillin and 2% glucose at 37°C until they reached logarithmic phase (OD 600The cells were grown to a pH of 0.4-0.8. These cells were infected with M13K07 helper phage for 30 minutes at 37°C, with or without shaking. The infected cells were pelleted at 4000 × g for 10 minutes and resuspended in 2xYT medium containing 100 μg / mL carbenicillin, 50 μg / mL kanamycin, and 1 mM IPTG. The bacteriophage was propagated overnight at 30°C and 250 rpm. The overnight culture was centrifuged at 9000 × g for 10 minutes at 4°C, and the phage was precipitated with 1 / 5 volume of PEG-NaCl solution [20% polyethylene glycol 6000, 1.5 M NaCl] by incubation on ice for 1 hour. The phage particles were pelleted by centrifugation at 9000 × g for 15 minutes at 4°C, and the supernatant was discarded. Phage particles were resuspended in Superblock blocking buffer and centrifuged at 7500 × g for 10 minutes in a microcentrifuge tube to pellet cell debris. The supernatant containing the phage particles was transferred to a new tube, and the phage was reprecipitated as described above. The concentrated phage particles were then heated at 70°C for 1 hour. Phage titers before and after heating were determined by infection of log-phase TG1 cells and plating onto 2xYT agar containing 100 μg / mL carbenicillin, 50 μg / mL kanamycin, and 2% glucose.

[0113] To obtain affinity-matched anti-C5 VHH domains with reactivity to both human and cynomolgus monkey species, library selection criteria included selection with biotinylated cynomolgus monkey (cyno) complement protein C5 and competition with an equimolar amount of non-biotinylated human complement protein C5. The phage-displayed VHH library was deselected with Dynabeads® M-280 streptavidin for 1 hour at room temperature. The deselected phage particles were affinity-matched to human and cynomolgus monkey C5 by incubating with an equimolar solution of biotinylated cynomolgus monkey C5 and non-biotinylated human C5 with Dynabeads® M-280 streptavidin for 30 minutes at room temperature. After washing five times with PBST and PBS, the phages were eluted from the beads using 0.1 M glycine (pH 2.2) containing 1 mg / mL BSA. The eluted supernatant was neutralized with 1 M Tris, pH 8.0. Log-phase TG1 cells were infected with the neutralized phage and plated on 2YTCG medium to determine the output titer. The output and input titers were compared to calculate the enrichment ratio. A higher ratio indicated successful isolation of C5-specific clones.

[0114] Individual clones were picked and inoculated into 96-deep well plates in 2xYT medium containing 100 μg / mL carbenicillin and 2% glucose and grown to logarithmic phase. The cells were infected with M13K07 and incubated overnight at 30°C to generate phage particles displaying individual VHH domains in the culture supernatant. Phage ELISA screening in four 96-well plates containing human C5 captured on streptavidin-coated plates suggested approximately 60% positive clones. Based on sequence analysis of CDR H3, 72 unique clones were selected as representatives of the 76 clones. The sequences of these representative VHH clones are shown in Table 1. For cloning purposes, the N- and C-terminal amino acids were modified to match those of the human VH-3 germline.

[0115] Suitable amino acid sequences for use in the modified polypeptides of the present disclosure include the amino acid sequences disclosed in Table 1 or fragments thereof.

[0116] [Table 1] TIFF2025118590000001.tif82170TIFF2025118590000002.tif252170TIFF2025118590000003.tif253170 TIFF2025118590000004.tif253170TIFF2025118590000005.tif254170TIFF2025118590000006.tif192170

[0117] Example 3 Cloning and expression of anti-C5 VHH domains Representative anti-C5 VHH domains were subcloned into mammalian expression vectors and expressed as VHH-His tag fusions in Expi293F cells. Culture supernatants were harvested when cell viability dropped to 50-60%. The supernatants were analyzed by SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue. Expression levels were calculated using Biolayer Interferometry on an Octet (ForteBio Inc.) instrument. His-tagged VHH domains were purified from the culture supernatants by immobilized metal affinity chromatography (IMAC) on an AKTA (GE Healthcare).

[0118] Example 4 Binding and functional analysis of anti-C5 VHH domains Binding assay for complement component C5 Representative anti-C5 VHH domains were sequenced, characterized, and analyzed using biolayer interferometry on an Octet (ForteBio Inc.) instrument. Binding to human, cynomolgus monkey (cyno), and mouse C5 proteins was assessed. Cell culture supernatants of expressed VHH-His domains were normalized to a concentration of 20 μg / mL in 2x kinetics buffer and captured onto an anti-penta-HIS (HIS1K) biosensor chip (ForteBio Inc.) for 300 seconds to fully saturate the sensor chip. The saturated chips were then exposed to 50 nM soluble C5 (human, cynomolgus monkey, or mouse) in 2x kinetics buffer for 600 seconds in separate experiments, followed by dissociation in 2x kinetics buffer for 600 seconds. VHH domains that showed binding to human C5 (hC5) or cynomolgus monkey C5 (cC5) are indicated with a "+" in Table 1.

[0119] Hemolytic assay of C5 antagonism The hemolysis assay measures the release of hemoglobin from sensitized chicken erythrocytes, which are lysed upon exposure to serum activated by the classical complement pathway (CCP). His-tagged VHH domains were expressed in Expi293 cells. Preliminary assays were used to select functional anti-C5 VHH domains, which were purified by IMAC. Ten purified VHH domains were analyzed at various concentrations for their ability to inhibit CCP-mediated hemolysis of sensitized chicken erythrocytes.

[0120] In this assay, no antibody was used for complete hemolysis, and 20 mM EDTA was not used for the no-hemolysis control. Ten VHH domains at different concentrations (32 μg / mL to 0.5 μg / mL) and anti-C5 IgG controls (labeled h5G1.1, BNJ441, and Ec-CHO) were preincubated with 0.1 mL of gelatin-veronal-buffered saline (GVB++, catalog no. B100, Comptech) containing 20% normal human serum (NHS) for 30 minutes at room temperature. 400 μL of chicken red blood cells (Lampire Biologicals, catalog no. 7201403) were washed four times with 1 mL of GVB++ and incubated at 5 × 10 7Sensitized cRBCs were prepared by incubating 1.5 × 10 cells / mL with rabbit anti-chicken IgG (Cat. No. 203-4139, Rockland) diluted 1:500 (v / v) and incubated at 4°C for 15 minutes. Cells were washed twice with GVB++ and resuspended in a final volume of 3.6 mL of GVB++. Sensitized cRBCs (2.5 × 10 cells) were incubated at 4°C for 15 minutes. 6 Thirty microliters of the solution (30 μL) was added to the pre-incubated human serum and antibody and incubated at 37°C for 30 minutes. The cells were pelleted by centrifugation at 1700 × g for 3 minutes at 4°C, and the supernatant (85 μL) was transferred to a new flat-bottom 96-well plate. The absorbance was measured at 415 nm. The hemolysis rate was calculated for each VHH domain and control antibody as follows:

[0121] [Number 1] ((A 415試料 -A 415溶血なし ) / (A 415完全溶血 -A 415溶血なし ))×100 In the formula, A 415試料 is the absorbance of the sample antibody at 415 nm, and A 415溶血なし is the absorbance at 415 nm of the control without hemolysis (20 mM EDTA), and A 415完全溶血 is the absorbance at 415 nm of the complete hemolysis control. The results are shown in Figure 1.

[0122] Identification of VHH domains that inhibit C5a releaseA Meso Scale Discovery (MSD) immunoassay was used to measure the cleavage of human C5 protein (e.g., the release of C5a by alternative pathway C5 convertase bound to the CAP activator zymosan). Anti-C5 VHH domains were expressed and purified as described above and analyzed for their ability to block human C5 protein cleavage by measuring the amount of hC5a released. The optimal concentration of the sample VHH domains was determined in pilot experiments. Sample VHH domains and control antibodies (h5G1.1, N19 / 8, BNJ441, and Ec-CHO) were added to GVB++ buffer (containing human C5 protein (final concentration 25 nM) (CompTech Inc.), 1% gelatin, and 2.5 mM NiCl) for 30 minutes at 37°C and then stored at 4°C until use. Anti-C5a antibody (2 μg / mL in phosphate-buffered saline (ThermoFisher) at 800 Hz) was coated onto an MSD high-binding 96-well plate and incubated for 1 hour. To activate the alternative complement pathway, zymosan was added to NHS in an equal ratio. This zymosan and NHS mixture was added to the pre-incubated VHH-hC5 solution and incubated at 37°C. The reaction was stopped at different time points (0, 30, 60, and 90 minutes) by adding Fusane-EDTA. The plate was centrifuged at 3600 rpm for 2 minutes, and the supernatant was transferred to a new polypropylene plate. Blocker A was added at room temperature for 1 hour to block nonspecific binding to the coated MSD plate. The MSD plate was washed, and the supernatants of the above samples were added. The plate was then incubated at room temperature for 15 minutes. A mixture of 1 μg / mL of detection antibody biotin Ab2942 (Abcam) and 0.5 μg / mL of streptavidin-conjugated sulfotag was added to each well and incubated at room temperature for 30 minutes. MSD 2x read buffer was added to each well, and the electrochemiluminescence signal was measured. The raw data was analyzed using MSD Workbench software. The results of this experiment are shown in Figure 2.

[0123] LCP0115, LCP0146, LCP0295, LCP0296, LCP0297 and LCP0302 inhibited C5a release and were therefore used for further characterization.

[0124] Example 5 Affinity analysis of anti-C5 VHH domains by Biacore Anti-C5 VHH domains were prioritized based on cross-reactivity with cynomolgus C5, and eight purified anti-C5 VHH domains were subjected to affinity analysis by Biacore. The kinetic parameters of binding to human and cynomolgus C5 for the initial eight candidates are shown in Table 2. From the eight affinity analysis candidates, five anti-C5 domains (LCP0115, LCP0143, LCP0146, LCP0296, and LCP0302) were selected and prioritized for further analysis based on matched affinity to human and cynomolgus C5.

[0125] [Table 2] TIFF2025118590000007.tif138170

[0126] Example 6 Humanization of anti-C5 VHH domain Five prioritized anti-C5 VHH domains (LCP0115, LCP0143, LCP0146, LCP0296, and LCP0302) were humanized by CDR-grafting into human germline domains with sequence similarity to the llama sequence. CDRs were based on higher amino acid position identity within the IMGT and Kabat definitions. To maintain VHH domain stability, llama FR2 hallmark residues were backmutated. The humanized variants were expressed in Expi293 cells and tested for binding to human C5 using biolayer interferometry.

[0127] Selected framework mutations of some variants were further introduced back to the parental llama residues to improve affinity for human C5. Constructs were expressed in HEK293F cells and assessed for binding by biolayer interferometry. To further optimize affinity, some variants were further mutated, with the N-terminus humanized to EVQLV (where required, SEQ ID NO: 147) and the C-terminus humanized to WGQGTLVTVSS (where required, SEQ ID NO: 148). The resulting prioritized anti-C5 VHH candidates are shown in Table 3 below. The CDRs of these candidates are shown in Table 4.

[0128] [Table 3] JPEG2025118590000008.jpg251170TIFF2025118590000009.tif241163TIFF2025118590000010.tif241163TIFF2025118590000011.tif155163

[0129] [Table 4] TIFF2025118590000012.tif88170TIFF2025118590000013.tif147170

[0130] Backmutations to parental Llama residues were introduced into frameworks selected from the humanization evaluation to improve the affinity of selected variants. The sequences of the variants after backmutation are shown in Table 5. Constructs were expressed in HEK293F cells and evaluated for binding by Biolayer Interferometry.

[0131] [Table 5] JPEG2025118590000014.jpg245170TIFF2025118590000015.tif91170

[0132] Example 7 Isolation of VHH domains that bind to human serum albumin Albumin is an abundant protein in serum and has a molecular weight sufficient to prevent its removal by filtration through the glomerular filtration barrier. Removal of albumin from serum by intracellular degradation is inhibited by the interaction of FcRn with albumin, which occurs at low pH. This interaction transports the albumin-FcRn complex back to the plasma membrane, where it is released back into the blood upon exposure to the neutral pH of blood.

[0133] Overview of the process for generating anti-HSA VHHs For the anti-C5 VHH domain and the anti-HSA VHH domain, an immune-biased VHH anti-HSA phage display library was generated from B cells of an immunized llama. When the endpoint titer against HSA exceeded 1,000,000, PBMCs were harvested, RNA was isolated, and VHH regions were genetically isolated. These anti-HSA VHH sequences were cloned into pIII fusion phagemids and displayed at 6 x 10 8 A library of independent clones was obtained. Standard phage display panning techniques were used to select VHH domains reactive to HSA and CSA (cynomolgus monkey serum albumin). The output of three rounds of panning was analyzed by ELISA and Sanger sequencing. In parallel, next-generation sequencing (NGS) was used to interrogate the population of sequences in the original library or enriched by panning. A total of approximately 1,000 clones were isolated and analyzed using these methods.

[0134] Immunization of llamas and construction of a VHH phage libraryLlamas were immunized with HSA. The primary immunization consisted of 500 μg of antigen mixed with complete Freund's adjuvant. Booster immunizations of 500 μg of antigen in incomplete Freund's adjuvant were administered at weeks 2, 4, 8, and 12. Approximately 2 weeks after each boost, blood samples were used to monitor serum titers. Blood samples were analyzed by ELISA to determine the titer of the immune response. Anti-HSA serum titers were detected with a signal 20-fold higher than the 1:100,000 dilution of the pre-bleed, so 500 mL of the prepared blood was processed to obtain approximately 7 × 10 cells for RNA isolation and library construction. 8 PBMCs Total RNA from PBMCs was purified by phenol / chloroform extraction, then purified on a silica spin column, and total RNA was eluted with RNase-free water. OD 260 / 280 The ratio was determined and the quality of the RNA was assessed by agarose gel electrophoresis. cDNA was synthesized using a llama heavy chain-specific reverse primer. VHH (heavy chain only) fragments were separated from VH (conventional heavy chain) fragments by gel electrophoresis.

[0135] The VHH fragments were modified with the SfiI site and cloned into pADL-10b, and the DNA library was transformed into TG1 cells. A total of 6 × 10 8 10 independent clones were obtained. All clones were recovered and stored in 25% glycerol at -80°C until use. The quality of the library was verified by analysis of 105 clones for the presence of inserts with the correct reading frame, uniqueness, and the presence of primer sequences.

[0136] Phage display panning and screening 3.75×10 10 Aliquots of glycerol stocks of the anti-HSA VHH library containing cells were cultured in 2xYT medium containing 2% glucose and 100 μg / mL carbenicillin. 600Cells were grown at 37°C with shaking at approximately 250 rpm until the RI was approximately 0.6. Helper phage was added at a multiplicity of infection (MOI) of 20, and the culture was incubated without shaking for 30 minutes, followed by 30 minutes with shaking at 37°C. Cells were harvested and resuspended in 2xYT medium supplemented with 25 μg / mL carbenicillin, 50 μg / mL kanamycin, and 200 μM IPTG. The culture was shaken overnight at 30°C and 250 rpm. The medium was clarified by centrifugation, and the phage were precipitated by adding 1 / 4 volume of 10% PEG-8000 / 2.5M NaCl and incubating on ice for 30 minutes. Phage were pelleted by centrifugation at 7500 rpm for 15 minutes at 4500 rpm in an SLA3000 rotor. The pellet was resuspended in Superblock (Thermo Scientific, 37515).

[0137] An aliquot of phage was deselected with M280 streptavidin beads (Life Technologies, 11205D) for 30 minutes at room temperature, the beads were removed using a magnet, and the phage-containing supernatant was transferred to a new Eppendorf tube. 10 μg of biotinylated HSA was added to the phage and incubated with rotation at room temperature for 30 minutes, after which M280 streptavidin beads were added to immobilize the biotinylated HSA. The beads were washed 11 times with PBS / 0.05% Tween wash buffer and eluted with 0.1 M glycine, pH 2.7. The elution buffer was then neutralized with 1 M Tris, pH 9.0. Eluted phage were rescued onto logarithmic phage TG1 cells, and growth was harvested in 250 cm x 250 cm LB-carbenicillin, 2% glucose trays. Titers were determined by serial dilution of the rescued phage aliquot. A second round of panning was performed essentially as described above, using an aliquot of the first round of growth and 5 μg of biotinylated HSA for selection.

[0138] To screen clones for reactivity to HSA, individual clones were picked into 96-well plates and cultured overnight at 37°C in a volume of 250 μL of 2xYT containing 100 μg / mL carbenicillin and 2% glucose. Each well was subcultured by transferring 5 μL of high-density overnight culture to 250 μL of fresh medium. An aliquot was used for rolling circle amplification sequencing analysis to determine the encoded insert. Cells were cultured at OD 600 After growth to an MOI of approximately 0.6, M13 helper phage was added at an MOI of 20 for 1 hour. Cells were harvested by centrifugation, and the medium was replaced with 250 μL per well of 2xYT supplemented with 100 μg / mL carbenicillin and 50 μg / mL kanamycin. The plates were then incubated overnight at 30°C with shaking at 250 rpm. The medium was clarified by centrifugation, and phage supernatant was prepared for use in ELISA assays.

[0139] For ELISA analysis, streptavidin-coated, pre-blocked 96-well plates (Pierce, 15500) were incubated with 2 μg / mL biotin for 30 minutes at room temperature with shaking. After washing the plates, blocking was repeated for 1 hour at room temperature. The plates were washed again, and 50 μL of clarified supernatant was added for 30 minutes at room temperature. The plates were washed three times and then incubated with anti-M13 HRP antibody (GE Healthcare, catalog number 27-9421-01) in blocking buffer for 30 minutes at room temperature. After washing the plates four times, 1-Step Ultra TMB ELISA Reagent (Thermo Scientific, catalog number 34029) was added, color was developed, and the reaction was stopped using 2M sulfuric acid stop solution. OD was measured using a BioRad iMark plate reader. 450 The reading was measured.

[0140] Next-generation sequencing (NGS) was used to examine the population of sequences in the original library or the population of sequences enriched by panning. For NGS, phagemid DNA was isolated from the initial library, the first round of panning, and the second round of panning. The VHH cassettes were excised from the phagemids by restriction digestion, and the VHH-encoding bands were isolated by agarose gel electrophoresis. The DNA was purified using a DNA affinity column. This DNA was used for library generation and analysis on the MiSeq 2x300 platform.

[0141] Example 8 Expression and purification of HSA-binding VHH domains VHH sequences selected using the methodology described above were synthesized with an N-terminal signal peptide and a C-terminal 6xHis tag (SEQ ID NO: 324) and cloned into mammalian expression constructs. GeneBlocks (Integrated DNA Technologies) synthesis and insert cloning into standard mammalian expression vectors were used to prepare the published MSA21 VHH domain (International Publication No. WO 2004 / 062551 A2) and genetically modified versions (deglycosylated or humanized) of individual clones. These constructs were transfected into 293expi cells, and supernatants were harvested 96 hours posttransfection. The supernatants were dialyzed against PBS, and VHH-His proteins were purified using standard chromatography methods. Purified proteins were buffer-exchanged into PBS and quantified using OD and extinction coefficients.

[0142] Example 9 Characterization of immobilized VHH domains binding to soluble HSA, CSA and mouse serum albumin Mammalian expression vectors were constructed for proteins produced in the 293 expi expression system and 112 VHH sequences. First, VHH sequences were analyzed by SDS-PAGE and Coomassie staining to determine approximate concentrations relative to known standards. Supernatant concentrations were then normalized and biolayer interferometry was performed on an Octet HTX (Pall / ForteBio). Penta-His sensors were exposed to kinetics buffer for 60 seconds to establish a baseline measurement. The sensors were then loaded with VHH-His-containing supernatants for 300 seconds, followed by a second baseline for 120 seconds in kinetics buffer. The chips were then incubated with 100 nM HSA or CSA in kinetics buffer for 600 seconds, and dissociation was measured for another 600 seconds.

[0143] Of the 112 VHH domains analyzed, 12 domains showed binding to biotinylated HSA, and three clones (HAS040, HAS041, and HAS042) interacted with both biotinylated CSA and biotinylated HSA. The sequences of these 12 anti-HSA VHH domains, including one or more humanized versions thereof, are shown in Table 6, and the CDRs of these anti-HSA VHH domains are shown in Table 7.

[0144] [Table 6] TIFF2025118590000016.tif235170TIFF2025118590000017.tif68170

[0145] [Table 7] TIFF2025118590000018.tif137170

[0146] Example 10. Characterization of albumin binding kinetics by Biacore The binding kinetics of VHH domains HAS040 and HAS041 to HSA or CSA was determined using SPR on a Biacore 3000 instrument. Biotinylated albumin was captured on a CAP chip (obtained from GE Healthcare) saturated with biotin CAPture reagent containing deoxyribonucleotides. Concentrates of purified VHH domains were injected for 5 min at a flow rate of 50 μL / min. Three concentrations per VHH domain were evaluated. Bound analytes were allowed to dissociate for 600 s. After each concentration, the chip surface was regenerated by injecting 6 M guanidine hydrochloride / 0.25 M NaOH at 10 μL / min for 2 min. A 1:1 Langmuir model (local R max Reaction kinetics were determined at pH 7.4 and pH 6.0 in HBS-EP buffer using a constant RI and double reference subtraction (subtraction of the buffer concentration cycle from the sample concentration cycle and subtraction of a parallel reference flow cell). MSA21 VHH domain (International Publication No. WO 2004 / 062551 A2) (sequence: LEQVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGTQVTVSS (SEQ ID NO: 322) was prepared and used as a comparator in these assays.

[0147] The results of this assay are shown in Table 8. Binding affinities were observed in the range of 0.3–5 nM, indicating that the HAS040 and HAS041 domains have sufficient affinity to promote half-life extension at both pH 6 and pH 7.4. Furthermore, these VHH domains exhibited binding to CSA and HSA with very similar affinities, enhancing the predictive nature of half-life extension studies performed in primates.

[0148] [Table 8] TIFF2025118590000019.tif93170

[0149] Example 11 Demonstration of non-competitive albumin binding by VHH and FcRn Albumin recycling from endocytic vesicles is mediated by its interaction with FcRn. Therefore, it was important to determine whether VHHs interfere with the interaction between HSA and FcRn. To determine whether the HAS040 and HAS041 VHH domains bind to the same epitope as FcRn, FcRn binding to HSA saturated with anti-HSA VHH domains was analyzed on a Biacore 3000 instrument in HBS-EP buffer, pH 6.0. HSA was directly immobilized on a CM5 chip using amine coupling to reach a target density of 250 RU (resonance units). VHH domains were diluted to approximately 1–10 μg / mL and injected to saturation (50 μL / min for 3 min). A concentration of FcRn was injected over the HSA:VHH surface, and kinetics were obtained for 5 min at 50 μL / min. Dissociation was allowed for 180 s before regeneration. The chip surface was regenerated by injecting 20 μL of 25 mM NaOH at 100 μL / min. max and constant RI) and double reference subtraction (subtraction of the buffer concentration cycle from the sample concentration cycle and subtraction of a parallel reference flow cell) were used to determine reaction kinetics.

[0150] The results are shown in Figure 7. In Figure 7A, direct interaction of FcRn with the HSA-saturated surface resulted in a 30 RU response difference. Similar RUs were obtained when 400 nM FcRn was injected onto surfaces saturated with complexes of HSA and MSA21 (ADL021) (Figure 7B), HAS040 (Figure 7C), or HAS041 (Figure 7D). Based on these data, we predict that HAS040 and HAS041 do not interfere with FcRn binding and are recycled from endosomes via the interaction of albumin with FcRn.

[0151] Example 12: Generation of an anti-C5 and anti-albumin bispecific fusion protein An anti-C5 VHH domain was fused to an anti-albumin domain to generate a bispecific molecule. Four linker lengths (G4S)3 (SEQ ID NO: 106), (G4S)4 (SEQ ID NO: 107), (G4S)5 (SEQ ID NO: 108), and (G4S)6 (SEQ ID NO: 109) and two different orientations of the anti-albumin domain (N- or C-terminus) were evaluated. The constructs were expressed in HEK293F cells and purified using Protein A affinity chromatography. The purified fusion molecules were evaluated in Biacore experiments. Human C5 was biotinylated and immobilized on a Biacore chip. The purified bispecific molecules were injected to saturate the chip, followed by injection of three concentrations of human serum albumin to determine reaction kinetics. The observed affinity for human serum albumin was used as a proxy to compare different linker lengths. (G4S)3 (SEQ ID NO: 106) was selected as the optimal linker length for generating a bispecific fusion. N- or C-terminal anti-albumin fusions were also evaluated in the same experiment. Different orientations were found to be optimal for different anti-C5 VHH domains. In Table 9, the N-to-C-terminal orientation of the constructs is noted below the construct name, with (C5 / HSA) indicating that the anti-C5 domain is located N-terminal to the anti-HSA domain. Similarly, (HSA / C5) indicating that the anti-HSA domain is located N-terminal to the anti-C5 domain.

[0152] After selecting the optimal linker length, a series of different bispecific fusion molecules were generated using a humanized anti-C5 VHH domain fused to two different anti-albumin domains (shown in Table 8). These constructs were expressed in Expi293 cells and purified using Protein A chromatography. The purified bispecific fusion proteins were tested in a hemolytic assay. The results are shown in Figures 3A and 3B.

[0153] [Table 9] TIFF2025118590000020.tif241170TIFF2025118590000021.tif236170TIFF2025118590000022.tif236170TIFF2025118590000023.tif253170

[0154] Based on binding and functional assays, four bispecific molecules were prioritized: CRL0483, CRL0484, CRL0499, and CRL0500. Biacore affinity measurements for binding of CRL0483, CRL0484, CRL0499, and CRL0500 to human C5 are shown in Table 10, and functional evaluations are shown in Figures 3, 4, and 5. These four bispecific molecules were evaluated in an in vivo pharmacokinetic study in cynomolgus monkeys.

[0155] [Table 10] TIFF2025118590000024.tif157170

[0156] Example 13 Pharmacokinetic Analysis of Bispecific Fusion Proteins Purified proteins were administered intravenously or subcutaneously to cynomolgus monkeys at 10 mg / kg. Three monkeys were used per dose group for each test article. The pharmacokinetic properties of the bispecific molecules were determined by LC-MS-based quantitation using signature peptides for each construct. The PK profiles are shown in Figure 6, and the parameters are listed in Table 11.

[0157] [Table 11] TIFF2025118590000025.tif85170

[0158] Variant linker sequences for bispecific fusion proteins were also generated, and the sequences with these variant linker sequences are shown in Table 12.

[0159] [Table 12] TIFF2025118590000026.tif185170

[0160] Example 14 Various peptide linker sequences Constructs were made using the HAS042 (SEQ ID NO: 26) albumin binding domain and CRL0305 (SEQ ID NO: 11) humanized anti-C5 VHH. The constructs evaluated are listed in Table 13.

[0161] [Table 13] TIFF2025118590000027.tif173170

[0162] The 26 constructs listed in Table 13 were expressed, and the fusion proteins were evaluated for binding to human C5 and albumin (Table 13 - Octet Binding), aggregate formation, hydrophobicity (HIC HPLC), and glycosylation (electrospray mass spectrometry). For Octet analysis, biotinylated human C5 was captured on a CAP chip, followed by injection of the bispecific molecule to be tested. Various concentrations of albumin were then injected. Reaction kinetics were determined at pH 7.4 (Biacore 3000). All bispecific molecules bound both C5 and albumin, each with similar affinity for albumin (5-6 nM).

[0163] The bispecific fusion proteins were tested for their ability to inhibit hemolysis in an in vitro hemolysis assay, and the data are shown in Figures 9A and 9B.

[0164] Table 14 shows the binding kinetics of CRL0500 and CRL0952 binding to human C5 (hC5) and cynomolgus monkey C5 (cC5).

[0165] [Table 14] TIFF2025118590000028.tif58170

[0166] Table 15 shows the binding kinetics of CRL0500 and CRL0952 binding to Plasbumin® and cynomolgus monkey albumin.

[0167] [Table 15] TIFF2025118590000029.tif62170

[0168] Example 15 pH-dependent binding of anti-C5 VHH domains Histidine scanning was performed on all CDRs of the anti-C5 VHH domains LCP0115, LCP0143, LCP0146, and LCP0302. A single histidine substitution was made at each position in the CDR (shown in bold underlined text). The variants were transfected into Expi293 cell cultures and pH-dependent binding was assessed at pH 7.4, 6.0, and 5.5. Several variants per antibody showed pH-dependent binding. These variants are listed in Table 16, and their pH-dependent binding responses are shown in Figures 11A-11D.

[0169] [Table 16] JPEG2025118590000030.jpg193170

[0170] The single histidine mutations identified for pH-dependent binding were combined to enhance pH sensitivity. The sequences of these variants are shown in Table 17. These variants were evaluated by biolayer interferometry for pH-dependent binding, and the results are shown in Figures 12A and 12B.

[0171] [Table 17] JPEG2025118590000031.jpg229170

[0172] Example 16: Generation of anti-C5 and anti-albumin bispecific fusions Bispecific molecules were generated by fusing an anti-C5 VHH domain to an anti-albumin domain. Four linker lengths (G4S)3 (SEQ ID NO: 106), (G4S)4 (SEQ ID NO: 107), (G4S)5 (SEQ ID NO: 108), and (G4S)6 (SEQ ID NO: 109) were evaluated, as were two different orientations of the anti-albumin domain (N-terminus or C-terminus). The sequences of the generated molecules are shown in Table 18. The constructs were expressed in HEK293F cells and purified using Protein A affinity chromatography. The purified fusion molecules were evaluated in Biacore experiments. Human C5 was biotinylated and immobilized on a Biacore chip. The purified bispecific molecules were injected to saturate the chip, followed by injection of three concentrations of human serum albumin to determine the reaction kinetics. The observed affinity for human serum albumin was used as a proxy to compare different linker lengths. (G4S)3 (SEQ ID NO: 106) was selected as the optimal linker length to generate a bispecific fusion. N- or C-terminal anti-albumin fusions were also evaluated in the same experiment. Different orientations were found to be optimal for different anti-C5 VHH domains.

[0173] [Table 8] JPEG2025118590000032.jpg167170TIFF2025118590000033.tif254170

[0174] A series of different bispecific fusion molecules were generated using humanized anti-C5 VHH domains with or without pH-dependent binding. The anti-C5 VHH domain was fused to two different anti-albumin domains to generate bispecific molecules (shown in Table 9). These constructs were expressed in HEK293F cells and purified using Protein A chromatography. The purified bispecific fusions were tested in a hemolytic assay. The results are shown in Figures 3A-3D.

[0175] Based on binding and functional assays, four bispecific molecules were prioritized: CRL0483, CRL0484, CRL0499, and CRL0500. Biacore affinity measurements for binding of CRL0483, CRL0484, CRL0499, and CRL0500 to human C5 are shown in Table 10, and functional evaluations are shown in Figures 5, 6, and 7. These four bispecific molecules were evaluated in an in vivo pharmacokinetic study in cynomolgus monkeys.

[0176] Example 17 Pharmacokinetic analysis of bispecific fusion molecules Cynomolgus monkeys were dosed with purified proteins at 10 mg / kg intravenously or subcutaneously. Three monkeys were used per dose group for each test article. The pharmacokinetics of the bispecific molecules were measured by an LC-MS-based quantitative assay using signature peptides specific for each construct. The PK profiles are shown in Figures 6A and 6B, and the parameters are listed in Table 20.

[0177] [Table 20] TIFF2025118590000034.tif49170

[0178] While various embodiments have been described in this disclosure, it is understood that variations and modifications will occur to those skilled in the art. It is therefore intended that the appended claims cover all such equivalent variations. In addition, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0179] The embodiments described herein may be combined with other embodiments unless expressly stated to the contrary. In particular, any feature or embodiment indicated as being preferred or advantageous may be combined with any other feature or embodiment indicated as being preferred or advantageous, unless expressly stated to the contrary. All references cited in this application are expressly incorporated herein by reference.

Claims

1. 1. A fusion protein comprising a modified polypeptide that specifically binds to human complement component C5 and a modified polypeptide that specifically binds to human serum albumin, A fusion protein in which the modified polypeptide that specifically binds human complement component C5 is fused, directly or via a peptide linker, to the polypeptide that specifically binds human serum albumin.

2. The fusion protein of claim 1, wherein the C-terminal residue of the polypeptide that specifically binds to human serum albumin is fused directly or via a linker to the N-terminal residue of the polypeptide that specifically binds to human complement component C5.

3. The fusion protein of claim 1, wherein the C-terminal residue of the polypeptide that specifically binds to human complement component C5 is fused, directly or via a linker, to the N-terminal residue of the polypeptide that specifically binds to human serum albumin.

4. 2. The fusion protein of claim 1, wherein the polypeptide that specifically binds to human complement component C5 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and fragments thereof, and the polypeptide that specifically binds to human serum albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34 and fragments thereof.

5. The fusion protein of claim 4, wherein the polypeptide that specifically binds to human complement component C5 comprises the amino acid sequence of SEQ ID NO: 11, and the polypeptide that specifically binds to human serum albumin has the amino acid sequence of SEQ ID NO:

26.

6. The fusion protein of claim 5, further comprising a peptide linker having the amino acid sequence of SEQ ID NO: 102 or 103.

7. The fusion protein of claim 6, wherein the peptide linker has the amino acid sequence of SEQ ID NO:

102.

8. 2. The fusion protein of claim 1, having a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 96-101.

9. 9. The fusion protein of claim 8, consisting of a sequence selected from the group consisting of SEQ ID NOs: 96-101.

10. 10. The fusion protein of claim 9, consisting of the polypeptide sequence of SEQ ID NO:

96.

11. The fusion protein of claim 1, wherein the polypeptide that specifically binds to human complement component C5 comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises any one of the amino acid sequences of SEQ ID NOs: 13 to 17, CDR2 comprises the amino acid sequence of SEQ ID NO: 18 or 19, and CDR3 has the amino acid sequence of SEQ ID NO: 20 or 21.

12. The fusion protein of claim 1, wherein the polypeptide that specifically binds to human serum albumin comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises any one of the amino acid sequences of SEQ ID NOs: 35 to 43, CDR2 comprises any one of the amino acid sequences of SEQ ID NOs: 44 to 51, and CDR3 comprises any one of the amino acid sequences of SEQ ID NOs: 52 to 63.

13. The fusion protein of claim 1 , wherein one or both of the polypeptides that bind to human complement component C5 or albumin bind in a pH-dependent manner.

14. A pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. 15. The pharmaceutical composition of claim 14, further comprising hyaluronidase.

16. A nucleic acid molecule having a nucleotide sequence encoding the fusion protein of any one of claims 1 to 13.

17. 17. An expression vector comprising the nucleic acid molecule of claim 16.

18. 17. An isolated host cell comprising the nucleic acid molecule of claim 16.

19. 18. An isolated host cell comprising the expression vector of claim 17.

20. 20. The isolated host cell of claim 19, which is a mammalian cell or a yeast cell.

21. A modified polypeptide that binds to human complement component C5, having an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-12.

22. 22. The modified polypeptide of claim 21 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and fragments thereof.

23. A modified polypeptide that specifically binds to human serum albumin, having an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 22-34.

24. 24. The modified polypeptide of claim 23, wherein the modified polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34 and fragments thereof.

25. 25. The modified polypeptide of claim 24, wherein the polypeptide comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-43, CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-51, and CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-63.

26. 23. The modified polypeptide of claim 22, wherein the polypeptide specifically binds to the same epitope on human serum albumin as Alb1.

27. A method for producing a fusion protein according to any one of claims 1 to 13, comprising expressing in a host cell at least one nucleic acid molecule having a nucleotide sequence encoding said fusion protein.

28. (a) a container containing a label; (b) a composition comprising the fusion protein of any one of claims 1 to 13, A therapeutic kit, wherein the label indicates that the composition is to be administered to a patient having or suspected of having a complement-mediated disorder.

29. 29. The kit of claim 28, further comprising hyaluronidase.

30. 14. A method for treating a patient having a complement-mediated disorder, comprising administering to said patient a therapeutically effective amount of the fusion protein of any one of claims 1 to 13.

31. 31. The method of claim 30, wherein the complement-mediated disorder is selected from the group consisting of rheumatoid arthritis, lupus nephritis, asthma, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, paroxysmal nocturnal hemoglobinuria, macular degeneration, HELLP syndrome, Guillain-Barré syndrome, CHAPLE syndrome, myasthenia gravis, neuromyelitis optica, thrombotic microangiopathy after hematopoietic stem cell transplantation (post-HSCT TMA), post-bone marrow transplant TMA (post-BMT TMA), Degos disease, Gaucher disease, glomerulonephritis, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, oligoimmune vasculitis, epidermolysis bullosa, recurrent abortion, multiple sclerosis (MS), traumatic brain injury, and injury resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

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