Complement c3 antigen binding proteins

Abstract

Antigen-binding proteins with specificity to complement C3 and C3b are provided. Methods of treating complement C3-mediated diseases and disorders, methods of inhibiting the activity of the complement Classical pathway (CP), Lectin pathway (LP), and / or Alternative pathway (AP), and methods of inhibiting the activity of choroidal-localized complement C3 are also provided.

Description

[Technical Field]

[0001] This invention relates to antigen-binding proteins targeting complement C3 and methods for treating complement C3-mediated diseases. [Previous Technology]

[0002] A major challenge in treating certain ocular diseases and conditions is delivering therapeutic molecules to the deep retinal layers. Delivery is hampered by many factors, including the numerous solid boundaries within the eye. These boundaries include the corneal and conjunctival epithelium, the blood-aqueous barrier (BAB), and the blood-retinal barrier (BRB), such as capillary endothelial cells (internal BRB) and retinal pigment epithelial cells (RPE cells, external BRB) (see, for example, Jiang et al., Int J Ophthalmol. 2018; 11(6): 1038-1044). Delivery to the retina is particularly important for complement-mediated diseases such as geographic atrophy (GA).

[0003] Geographic atrophy (GA) is a late form of age-related macular degeneration (AMD), characterized by damage to the retinal pigment epithelium and photoreceptors in the macula. Once GA involves the fovea, irreversible vision loss occurs. Even before vision is affected, patients with early stages of GA often experience visual impairment.

[0004] The underlying pathophysiology of geographic atrophy is not fully understood; however, complement dysregulation is considered a contributing factor. Increased levels of several complement activation products (including C3a, C5a, C5b-9, and complement factor H (CFH)) have been observed in vitreous samples, Bruch's membrane, and other parts of the choroid from patients with geographic atrophy (GA) compared to controls. Furthermore, decreased levels of complement inhibitors (such as CD59, a membrane-bound inhibitor of the membrane attack complex (MAC) formation) and membrane cofactor proteins (MCPs, a membrane-bound complement regulator with cofactor activity against complement factor I (CFI)) have been reported in GA.

[0005] Currently, there are no approved treatments for GA. Several investigational approaches targeting the complement pathway have been studied, but none have been approved or proven effective. Some examples of such approaches include eculizumab / SOLIRIS (Alexion), LFG-316 (Novartis / MorphoSys), ARC-1905 (Ophthotech), POT-4 (AL-78898A; Alcon), and lampalizumab (FCFD45142).

[0006] Recently, the results of the Phase II clinical trial of APL-2 (Clinical Trial NCT02503332, "Study of APL-2 Therapy in Patients Geographic Atrophy (FILLY)") further suggest the complement pathway in the pathogenesis of GA and confirm the positive therapeutic effect of reducing GA progression through complement inhibition. These results also indicate that APL-2 inhibition of the complement cascade centered on C3 (which is the convergence point of all complement pathways; see Figure 1) has the potential to treat GA more effectively than inhibitors that cause partial inhibition of the complement pathway. However, the reduction in lesion growth in GA achieved by APL-2 is still small. APL-2 has characteristics that may limit its effectiveness. APL-2, a PEGylated derivative of the cyclic decacyclic tridecapeptide compstatin (an inhibitor of complement component C3), has a large molecular weight equivalent of 350 kDa and a hydrodynamic radius of approximately 7.8 nm, making it difficult to penetrate deeply into the retina. Possibly due to its low concentration of 3.5 mM, APL-2 has a shelf life of only one month. APL-2 is also a PEGylated molecule, which increases its viscosity and may make it difficult to inject into the eye. Therefore, there is a need for more effective methods to reduce GA progression.

[0007] A major challenge in the treatment of GA is the observed dysregulation of complement activity in deeper retinal layers. We hypothesize that better penetration into disease-associated retinal tissues (i.e., the retinal pigment epithelium (RPE), Bruch's membrane, and other parts of the choroid) may be required to achieve greater reduction in lesion growth in GA. For this purpose, small antibody fragments offer several advantages over other biologics and antibodies. Small antibody formulations allow for: 1) better intraocular penetration into the relevant retinal tissues; and 2) delivery of more drug per milligram or milliliter via intravitreal injection. [Summary of the Invention]

[0008] The present invention provides an antigen-binding protein that is specific to complement C3.

[0009] In one embodiment, the present invention provides an antigen-binding protein or fragment thereof that binds to an antigenic determinant on complement C3, wherein the antigen-binding protein or fragment thereof is capable of inhibiting complement activation pathways, including the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP).

[0010] In some embodiments, the antigen-binding protein or a fragment thereof is capable of binding complement C3 and C3b.

[0011] In some embodiments, the antigen-binding protein or a fragment thereof is capable of binding to the antigenic determinant on complement C3, wherein such binding prevents the formation of C3 convertase.

[0012] In some embodiments, the antigen-binding protein or a fragment thereof is able to compete with one or more antigen-binding proteins (including MO122, MO123, MO124, MO228 and MO251).

[0013] In some embodiments, the antigen-binding protein or a fragment thereof comprises a single-chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, a bifunctional antibody, a small antibody mimic, or a single-domain antibody, such as sdAb, sdFv, nanoantibody, V-Nar, or VHH. In a preferred embodiment, the antigen-binding protein or a fragment thereof comprises scFv or VHH.

[0014] In some embodiments, the antigen-binding protein or a fragment thereof comprises CDR-H3 having at least 80% similarity to the sequences in the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:21.

[0015] In some embodiments, the antigen-binding protein or a fragment thereof comprises a CDR-H3 having at least 80% identity with a sequence from the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:21.

[0016] In some embodiments, the antigen-binding protein or a fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a CDR-H1 sequence selected from the group consisting of SEQ ID NO: 1, 4, 7, 13 and 19; a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2, 5, 8, 14 and 20; a CDR-H3 sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 15 and 21; and wherein the VL comprises a CDR-L1 sequence selected from the group consisting of SEQ ID NO: 10, 16 and 22; a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 11, 17 and 23; and a CDR-L3 sequence selected from the group consisting of SEQ ID NO: 12, 18 and 24.

[0017] In some embodiments, VH has at least 80% similarity to the sequence of the group consisting of SEQ ID NO:25, 26, 27, 29 and 31, and / or VL has at least 80% similarity to the sequence of the group consisting of SEQ ID NO:28, 30 and 32.

[0018] In some embodiments, VH has at least 80% identity with the sequence of the group consisting of SEQ ID NO:25, 26, 27, 29 and 31, and / or VL has at least 80% similarity with the sequence of the group consisting of SEQ ID NO:28, 30 and 32.

[0019] In some embodiments, the antigen-binding protein or a fragment thereof comprises VH and VL, wherein VH comprises the CDR-H1 sequence of SEQ ID NO:7, the CDR-H2 sequence of SEQ ID NO:8, and the CDR-H3 sequence of SEQ ID NO:9; and wherein VL comprises the CDR-L1 sequence of SEQ ID NO:10, the CDR-L2 sequence of SEQ ID NO:11, and the CDR-L3 sequence of SEQ ID NO:12.

[0020] In some embodiments, VH contains the amino acid sequence of SEQ ID NO:27 and VL contains the amino acid sequence of SEQ ID NO:28.

[0021] In some embodiments, the antigen-binding protein or a fragment thereof comprises VH and VL, wherein VH comprises the CDR-H1 sequence of SEQ ID NO:13, the CDR-H2 sequence of SEQ ID NO:14, and the CDR-H3 sequence of SEQ ID NO:15; and wherein VL comprises the CDR-L1 sequence of SEQ ID NO:16, the CDR-L2 sequence of SEQ ID NO:17, and the CDR-L3 sequence of SEQ ID NO:18.

[0022] In some embodiments, VH contains the amino acid sequence of SEQ ID NO:29 and VL contains the amino acid sequence of SEQ ID NO:30.

[0023] In some embodiments, the antigen-binding protein or a fragment thereof comprises VH and VL, wherein VH comprises the CDR-H1 sequence of SEQ ID NO:19, the CDR-H2 sequence of SEQ ID NO:20, and the CDR-H3 sequence of SEQ ID NO:21; and wherein VL comprises the CDR-L1 sequence of SEQ ID NO:22, the CDR-L2 sequence of SEQ ID NO:23, and the CDR-L3 sequence of SEQ ID NO:24.

[0024] In some embodiments, VH contains the amino acid sequence of SEQ ID NO:31 and VL contains the amino acid sequence of SEQ ID NO:32.

[0025] In some embodiments, the antigen-binding protein or a fragment thereof comprises a VHH domain, wherein the VHH domain comprises the CDR-H1 sequence of SEQ ID NO:1, the CDR-H2 sequence of SEQ ID NO:2, and the CDR-H3 sequence of SEQ ID NO:3.

[0026] In some embodiments, the VHH domain contains the amino acid sequence of SEQ ID NO:25.

[0027] In some embodiments, the antigen-binding protein or a fragment thereof comprises a VHH domain, wherein the VHH domain comprises the CDR-H1 sequence of SEQ ID NO:4, the CDR-H2 sequence of SEQ ID NO:5, and the CDR-H3 sequence of SEQ ID NO:6.

[0028] In some embodiments, the VHH domain contains the amino acid sequence of SEQ ID NO:26.

[0029] In some embodiments, the binding affinity of the antigen-binding protein or a fragment thereof for C3 and C3b is at least about 10⁻⁸ M. In some embodiments, the binding affinity of the antigen-binding protein or a fragment thereof for C3 and C3b is about 10⁻⁹ M to about 10⁻¹⁴ M. In some embodiments, the binding affinity of the antigen-binding protein or a fragment thereof for C3 and C3b is about 10⁻¹⁰ M to about 10⁻¹² M. In some embodiments, the antigen-binding protein or a fragment thereof has substantially equivalent binding affinity for C3 and C3b. In some embodiments, the binding affinity for C3 is less than one-tenth of the binding affinity for C3b.

[0030] In some embodiments, the antigen-binding protein or fragment thereof has a binding affinity of about 10⁻⁴ M or weaker for C3a, iC3b, C4, C4b, C5, and / or C5b. In some embodiments, the antigen-binding protein or fragment thereof has a weaker binding affinity for C3a, iC3b, C4, C4b, C5, and / or C5b compared to its binding affinity for C3 and C3b. In some embodiments, the antigen-binding protein or fragment thereof has no binding affinity for C3a, iC3b, C4, C4b, C5, and / or C5b.

[0031] In some embodiments, the antigen-binding protein or fragments thereof are capable of inhibiting the activity of the CP, LP and AP complement pathways by at least about 80%, at least about 85%, at least about 90% or at least about 95%.

[0032] In some embodiments, the antigen-binding protein or fragments thereof are capable of inhibiting the activity of the CP, LP, and AP complement pathways equivalently or substantially equivalently. In some embodiments, the activity of the CP, LP, and AP complement pathways is inhibited by at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0033] In some embodiments, the activity of the CP, LP and AP complement pathways is determined by measuring the level of hemolysis in the presence of antigen-binding proteins or fragments thereof, compared with the level of hemolysis in the absence of antigen-binding proteins or fragments thereof.

[0034] In some embodiments, the activity of the CP, LP and AP complement pathways is determined by measuring the formation of the membrane attack complex (MAC) in the presence of antigen-binding proteins or fragments thereof, compared with the formation of the membrane attack complex (MAC) in the absence of antigen-binding proteins or fragments thereof.

[0035] In some embodiments, the antigen-binding protein or a fragment thereof is capable of inhibiting the activity of C3 convertase by at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0036] In some embodiments, the antigen-binding protein or a fragment thereof is able to inhibit the C3 convertase amplification loop.

[0037] In some embodiments, the antigen-binding protein or a fragment thereof is capable of permeating the Bruch membrane.

[0038] In some embodiments, the antigen-binding protein or a fragment thereof can inhibit choroidal C3 activity.

[0039] In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 60 kDa or less. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 20 kDa to about 30 kDa. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 10 kDa to about 20 kDa. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 25 kDa. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 15 kDa.

[0040] In some embodiments, the antigen-binding protein or a fragment thereof is cross-reactive with cynomolgus monkey C3.

[0041] In one embodiment, the present invention provides a pharmaceutical composition comprising the antigen-binding protein or a fragment thereof described above and a pharmaceutically acceptable carrier. Thus, one embodiment is for use with the binding protein of the present invention in preparing a pharmaceutical composition for treating complement C3-mediated diseases or conditions in an individual.

[0042] In some embodiments, the pharmaceutical composition has low viscosity.

[0043] In some embodiments, the viscosity is between about 1 cP and about 50 cP. In some embodiments, the viscosity is less than or equal to about 20 cP.

[0044] In one embodiment, the present invention provides an isolated nucleic acid molecule encoding the antigen-binding protein or a fragment thereof described above.

[0045] In another embodiment, the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0046] In another embodiment, the present invention provides a host cell comprising the expression vector described above.

[0047] In another embodiment, a method for manufacturing an antigen-binding protein or a fragment thereof as described above is provided, comprising i) culturing host cells as described above under conditions that allow for the expression of the protein described herein; and ii) recovering the protein; and, where appropriate iii) further purifying and / or modifying and / or formulating the protein.

[0048] In one embodiment, the present invention provides a method for treating complement C3-mediated diseases or conditions in an individual, comprising administering to the individual in need an antigen-binding protein or a fragment thereof as described above. Therefore, the present invention also provides an antigen-binding protein or a fragment thereof as described above, which is used in a method for treating complement C3-mediated diseases or conditions. In some embodiments, the antigen-binding protein or a fragment thereof is administered topically, subconjunctivally, intravitreally, retroocularly, and / or intraanterior chamber.

[0049] In some embodiments, complement C3-mediated diseases or conditions are selected from the group consisting of: age-related macular degeneration, geographic atrophy, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, retrolental fibrosis, autoimmune uveitis, chorioretinitis, retinitis, rheumatoid arthritis, psoriasis, and atherosclerosis.

[0050] In one embodiment, the present invention provides a method for inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP), the method comprising contacting complement C3 with an antigen-binding protein or fragment thereof that binds to an antigenic determinant on complement C3. Therefore, the present invention provides an antigen-binding protein or fragment thereof as described herein, which is used in a method for treating complement C3-mediated diseases or conditions by inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The present invention also provides an antigen-binding protein or fragment thereof as described above, which is used in a method for treating complement C3-mediated diseases or conditions by inhibiting the activity of complement C3 locally in the choroid.

[0051] In one embodiment, the present invention provides a method for inhibiting the activity of local complement C3 in the choroid, the method comprising intraocularly administering an antigen-binding protein or a fragment thereof that binds to an antigenic determinant on complement C3.

[0052] In some embodiments of the methods described herein, the antigen-binding protein or a fragment thereof is capable of binding complement C3 and C3b.

[0053] In some embodiments, the antigen-binding protein or a fragment thereof is capable of binding to the antigenic determinant on complement C3, wherein such binding prevents the formation of C3 convertase.

[0054] In some embodiments, the antigen-binding protein or a fragment thereof can compete with one or more antigen-binding proteins (including M0122, M0123, M0124, M0228 and M0251).

[0055] In some embodiments, the antigen-binding protein or a fragment thereof comprises a single-chain variable fragment (scFv), a Fab fragment, or a VHH.

[0056] In some embodiments, the antigen-binding protein or a fragment thereof comprises CDR-H3 having at least 80% similarity to sequences from the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:21.

[0057] In some embodiments, the antigen-binding protein or a fragment thereof comprises a CDR-H3 having at least 80% identity with a sequence from the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:21.

[0058] In some embodiments, the antigen-binding protein or a fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a CDR-H1 sequence selected from the group consisting of SEQ ID NO: 1, 4, 7, 13 and 19; a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2, 5, 8, 14 and 20; a CDR-H3 sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 15 and 21; and wherein the VL comprises a CDR-L1 sequence selected from the group consisting of SEQ ID NO: 10, 16 and 22; a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 11, 17 and 23; and a CDR-L3 sequence selected from the group consisting of SEQ ID NO: 12, 18 and 24.

[0059] In some embodiments, VH has at least 80% similarity to the sequence of the group consisting of SEQ ID NO:25, 26, 27, 29 and 31, and / or VL has at least 80% similarity to the sequence of the group consisting of SEQ ID NO:28, 30 and 32.

[0060] In some embodiments, VH has at least 80% identity with the sequence of the group consisting of SEQ ID NO:25, 26, 27, 29 and 31, and / or VL has at least 80% identity with the sequence of the group consisting of SEQ ID NO:28, 30 and 32.

[0061] In some embodiments, the antigen-binding protein or a fragment thereof is capable of permeating the Bruch membrane.

[0062] In some embodiments, the antigen-binding protein or a fragment thereof can inhibit choroidal C3 activity.

[0063] In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 60 kDa or less, such as about 50 kDa or less, about 40 kDa or less, about 35 kDa or less, about 30 kDa or less, about 25 kDa or less, about 20 kDa or less, or about 15 kDa or less. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 20 kDa to about 30 kDa. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 10 kDa to about 20 kDa. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 25 kDa. In some embodiments, the molecular weight of the antigen-binding protein or a fragment thereof is about 15 kDa.

[0064] In one embodiment, the present invention provides a method for detecting one or both of C3 and C3b in a biological sample, comprising (a) contacting the sample with at least one antigen-binding protein or fragment thereof as described above; (b) allowing one or both of C3 and C3b in the sample to form a complex with the antigen-binding protein or fragment thereof; and (c) detecting the antigen-binding protein or fragment thereof. In a preferred embodiment, the antigen-binding protein or fragment thereof is capable of binding complement C3 and C3b.

[0065] In one embodiment, an antigen-binding protein or a fragment thereof is detected by a detectable signal.

[0066] In one embodiment, antigen-binding proteins or fragments thereof are detected by ELISA, immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting, and / or flow cytometry.

[0067] Biological samples may be tissue samples, such as retinal tissue from a human individual, or fixed tissue samples. Fixed tissue samples may be formalin-fixed and paraffin-embedded tissue samples.

[0068] In one sample, a kit for detecting C3 is provided, which contains the antigen-binding protein or a fragment thereof as described above and instructions for use.

Implementation Method

[0086] Provides an antigen-binding protein with binding specificity to complement C3 and complement C3 cleavage product C3b. Also provides methods for treating or preventing complement C3-mediated diseases and symptoms.

[0087] In certain states, the antigen-binding proteins described herein can inhibit the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The antigen-binding proteins described herein can inhibit all three pathways simultaneously. The antigen-binding proteins described herein can inhibit all three pathways in the choroid of the eye.

[0088] Generally, the nomenclature used in conjunction with cell and tissue cultures, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein is well-known and commonly used in this art. Unless otherwise specified, the methods and techniques provided herein are generally performed according to well-known methods in this art and as described in various general and more specific literature cited and discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions as commonly implemented in this art or as described herein. The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry as described herein, as well as their laboratory procedures and techniques, is well-known and commonly used in this art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

[0089] Unless otherwise specified herein, the scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. In the event of any potential discrepancy, the definitions provided herein shall take precedence over any dictionary or foreign definition. Unless otherwise required, singular terms shall include plural terms and plural terms shall include singular terms. Unless otherwise stated, the use of "or" means "and / or". The use of the term "including" and other forms (such as "includes" and "included") is not restrictive.

[0090] To make the present invention easier to understand, some terms are first defined. Antigen-binding protein

[0091] As used herein, the term "antibody" or "antigen-binding protein" refers to an immunoglobulin molecule that specifically binds to or undergoes an immune response to an antigen or antigenic determinant, and includes polyclonal and monoclonal antibodies as well as functional antibody fragments, including (but not limited to) fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanoantibodies, VHH) fragments. The term "antibody" includes genetically engineered or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide bodies, chimeric antibodies, fully human antibodies, humanized antibodies, heterobinding antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem di-scFv, tandem tri-scFv) and analogues. Unless otherwise stated, the term "antibody" should be understood to encompass its functional antibody fragment. As used herein, the term "antibody fragment" includes artificial proteins designed to selectively bind to antigens, i.e., antibody mimics. Typically, one or more CDRs are grafted onto a non-Ig backbone to mimic the CDR conformation from a parent antibody. Non-limiting examples of such antibody mimics include fluctuation-regulated affinity proteins (FLAPs), monofunctional antibodies, and affinities. Antibody mimics may contain one, two, three, four, five, or six CDRs as described herein.

[0092] As used herein, "Fab fragment" is an antibody fragment comprising a light chain fragment containing a variable light chain (VL) domain and a constant domain of the light chain (CL), and a variable heavy chain (VH) domain and a first constant domain (CH1) of the heavy chain. Fab fragments typically have a molecular weight of about 50 kDa and a hydrodynamic radius of about 3.0 nm.

[0093] As used herein, a "single-chain variable fragment" (scFv) is an antigen-binding protein comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL). The VH and VL domains of the scFv are linked via any suitable linker recognized in this art. Such linkers include (but are not limited to) repeating GGGGS amino acid sequences or variations thereof. scFvs typically do not contain antibody constant domain regions, but the scFvs of the present invention may be linked to or attached to antibody constant domain regions (e.g., antibody Fc domains) to alter various properties of the scFv, including (but not limited to) increased serum or tissue half-life. scFvs typically have a molecular weight of about 25 kDa and a hydrodynamic radius of about 2.5 nm.

[0094] As used herein, "VHH", "nano antibody" or "heavy chain only antibody" refers to an antigen-binding protein containing a single heavy chain variable domain derived from species of the Camelidae family, including camels, llamas, and alpacas. The molecular weight of VHH is typically about 15 kDa.

[0095] As used herein, the term "complementarity-determining region" or "CDR" refers to a non-adjacent amino acid sequence in the variable region of an antibody that provides antigen specificity and binding affinity. Typically, each heavy chain variable region contains three CDRs (CDR-H1, CDR-H2, CDR-H3) and each light chain variable region contains three CDRs (CDR-L1, CDR-L2, CDR-L3). "Frame region" and "FR" are known in this art and refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, each heavy chain variable region contains four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) and each light chain variable region contains four FRs (FR-L1, FR-L2, FR-L3, and FR-L4). Regarding the VHH antibody, only three heavy chain CDRs are present, and no light chain CDRs are present.

[0096] The exact amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in the following literature: Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell "Receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol, January 2003; 27(1):55-77 ("IMGT" numbering scheme); and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, June 8, 2001; 309(3):657-70 ("Aho" numbering scheme).

[0097] The boundaries of a given CDR or FR can vary depending on the identification scheme used. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the largest common antibody region sequence, where insertions and deletions (e.g., "30a") occur in some antibodies. The two schemes place certain insertions and deletions at different locations ("insertions and deletions"), resulting in different numbering. The Contact scheme is based on the analysis of the complex's crystal structure and is similar to the Chothia numbering scheme in several ways.

[0098] Variants of the antibodies provided herein can be generated by introducing deletions, substitutions, additions, and / or modifications into the framework and / or CDR. The desired function of the antibody variant can then be tested using the methods described herein. Any combination of deletions, substitutions, additions, modifications, and insertions can be made to the antigen-binding protein or fragments thereof, provided that the resulting variant possesses the desired characteristics that can be screened using appropriate methods.

[0099] As used herein, "conservative substitution" refers to a modification that maintains the functional properties of the parent antibody. For example, conserved amino acid substitution includes substitution in which an amino acid residue is replaced by an amino acid residue having similar properties. Examples include: valine (V) replacing alanine (A); lysine (K) replacing arginine (R); glutamic acid (Q) replacing aspartic acid (N); glutamic acid (E) replacing aspartic acid (D); serine (S) replacing cysteine ​​(C); aspartic acid (D) replacing glutamic acid (E); alanine (A) replacing glycine (G); and arginine (R) or lysine (... K) replaces histidine (H); leucine (L) replaces isoleucine (I); leucine (L) replaces methionine (M); tyrosine (Y) replaces phenylalanine (F); threonine (T) replaces serine (S); tyrosine (Y) replaces tryptophan (W); tryptophan (W) replaces phenylalanine (F); and / or leucine (L) replaces valine (V), and vice versa.

[0100] Therefore, unless otherwise stated, the “CDR” or “complementarity-determining region” or individually designated CDR (e.g., CDR-H1, CDR-H2) of a particular antibody or its region (such as its variable region) should be understood to encompass the complementarity-determining region (or specific complementarity-determining region) as defined by any known protocol. Similarly, unless otherwise stated, the “FR” or “framework region” or individually designated FR (e.g., FR-H1, FR-H2) of a particular antibody or its region (such as its variable region) should be understood to encompass the framework region (or specific framework region) as defined by any known protocol. In some cases, a protocol for identifying a particular CDR or FR is specified, such as a CDR defined by the Kabat, Chothia, Contact, IMGT, or AHo methods. In other cases, the specific amino acid sequence of the CDR or FR is provided.The CDR and FR numbering is further described in Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol, January 2003; 27(1): 55-77 (IMGT numbering scheme); and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, June 8, 2001; 309(3):657-70, (AHo numbering scheme).

[0101] The terms "competition" or "cross-competition" are used interchangeably herein to refer to the ability of an antibody molecule to interfere with the binding of an antibody molecule (e.g., an antigen-binding protein described herein) to a target (e.g., human C3 and / or C3b). Interference with binding can be direct or indirect (e.g., via ectopic regulation of the antigen-binding molecule or the target). Competitive binding assays (e.g., FACS, ELISA, or BIACORE assays) can be used to determine the extent to which an antigen-binding molecule can interfere with the binding of another antigen-binding molecule to a target and therefore whether it can be described as competitive. In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, when the binding of the first antibody molecule to the target is reduced by 10% or more in a competitive binding assay (e.g., the competitive assay described herein), such reduction is described as 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, the first antigen-binding molecule is said to compete with the second antigen-binding molecule for binding to the target.

[0102] As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antibody and the antigenic determinant it binds to. As will be readily understood by those skilled in the art, the affinity of an antibody or antigen-binding protein can be reported as a dissociation constant (KD) in the form of mole concentration (M). The KD value of the antibodies of the present invention can be in the range of 10⁻⁵ to 10⁻¹² M. The KD value of high-affinity antibodies is 10⁻⁹ M (1 nanomole, nM) and lower. For example, the KD value of high-affinity antibodies can be in the range of about 1 nM to about 0.01 nM. The KD value of high-affinity antibodies can be about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM or about 0.1 nM. Extremely high affinity antibodies have a KD value of 10⁻¹² M (1 pmol, pM) or lower. Weak or low affinity antibodies have a KD value in the range of 10⁻¹ to 10⁻⁴ M. Low affinity antibodies have a KD value of 10⁻⁴ or higher, such as 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

[0103] In some embodiments, the binding affinity of the antigen-binding protein of the present invention for C3 and C3b is about 10⁻⁸ M to about 10⁻¹⁴ M. In some embodiments, the binding affinity of the antigen-binding protein of the present invention for C3 and C3b is about 10⁻¹⁰ M to about 10⁻¹² M. In some embodiments, the binding affinity of the antigen-binding protein of the present invention for C3 and C3b is at least about 10⁻⁸ M, at least about 10⁻⁹ M, at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, or at least about 10⁻¹² M.

[0104] In some embodiments, the antigen-binding protein or a fragment thereof has substantially equivalent binding affinity for C3 and C3b. For example, but not limitingly, the binding affinity of the antigen-binding protein or a fragment thereof for C3 may be about 10⁻¹⁰ M and for C3b may be about 10⁻¹⁰ M. In some embodiments, the binding affinity of the antigen-binding protein or a fragment thereof for C3 may be about 10⁻¹¹ M and for C3b may be about 10⁻¹¹ M. In some embodiments, the binding affinity of the antigen-binding protein or a fragment thereof for C3 may be about 10⁻¹² M and for C3b may be about 10⁻¹² M.

[0105] In some embodiments, the binding affinity for C3 is less than one-tenth of the binding affinity for C3b. For example, but not limitingly, the binding affinity of the antigen-binding protein or a fragment thereof for C3 may be about 10⁻¹⁰ M and the binding affinity for C3b may be about 10⁻¹¹ M. In some embodiments, the binding affinity of the antigen-binding protein or a fragment thereof for C3 is about 10⁻¹¹ M and the binding affinity for C3b is about 10⁻¹² M.

[0106] In some embodiments, the antigen-binding protein or a fragment thereof is cross-reactive with cynomolgus monkey C3. The cynomolgus monkey (Macaca fascicularis) C3 has 95.1% similarity to human C3, and this cross-reactivity allows for preclinical and toxicological testing of the antigen-binding protein of the present invention in relevant animal models.

[0107] To avoid doubt and unless otherwise indicated, C3 as used herein refers to human complement component 3 of UniProt P01024 and the nucleic acid sequence encoding that protein. C3b is derived from native C3 and is the larger of the two elements formed by the cleavage of C3.

[0108] In some embodiments, the antigen-binding protein of the present invention is monovalent and binds to human C3 and C3b with a KD of about 200 nM or less, as measured by biological layer interferometry (BLI). In some embodiments, the KD is about 200 pM or less, such as about 100 pM, about 10 pM, about 1 pM, or about 0.1 pM.

[0109] The ability of an antigen-binding domain to bind to a specific antigen determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in this technique (e.g., surface plasma resonance (SPR) technique (analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Anticomplement C3 antigen-binding protein

[0110] In one embodiment, the present invention provides an antigen-binding protein that has binding specificity to complement C3 protein. In some embodiments, the anti-C3 antigen-binding protein is scFv, a Fab fragment, or VHH.

[0111] Exemplary anti-C3 antigen-binding proteins CDRs are described in Table 1 below. The variable heavy chain and variable light chain domains of exemplary anti-C3 antigen-binding proteins are described in Table 2 below. The exemplary anti-C3 antigen-binding proteins described below were generated by immunizing rabbits and llamas with human C3 protein isolated from human plasma. The VH and VL domains of exemplary M0122, M0123, and M0124 are derived from rabbits immunized with human C3 protein and are wild-type rabbit sequences. The exemplary VHH domains of M0228 and M0251 are derived from llamas immunized with human C3 protein and are wild-type llama sequences.

[0112] Table 1 - CDR sequence of anti-C3 antigen-binding protein SEQ ID NO: sequence Notes 1 DYTMG M0228_CDR-H1 2 AINWRGSSTYYADSVKG M0228_CDR-H2 3 QVSPYVELTATAAY M0228_CDR-H3 4 NWAMG M0251_CDR-H1 5 AIRWSVGTTNYRDSVKG M0251_CDR-H2 6 GTPFVLARINGYDY M0251_CDR-H3 7 NYAMN M0122_ CDR-H1 8 IINTDGNTNYASWAKG M0122_ CDR-H2 9 AVGYHHHALDP M0122_ CDR-H3 10 TLSSAHKTYTID M0122_ CDR-L1 11 LKSDGSYTKGT M0122_ CDR-L2 12 GTDYGGGYV M0122_ CDR-L3 13 SYHMS M0123_ CDR-H1 14 IIYTDGNTDYANWAKG M0123_ CDR-H2 15 RGYADYGYTFNL M0123_ CDR-H3 16 TADTLSRNYAS M0123_ CDR-L1 17 RDTSRPS M0123_ CDR-L2 18 ATGDGSGSSYQFV M0123_ CDR-L3 19 RYWMN M0124_ CDR-H1 20 YITTNDKTYYANWAKG M0124_ CDR-H2 21 RSSGAYDI M0124_ CDR-H3 22 TLSSAHKTYYIE M0124_ CDR-L1 23 LKSDGTYTKGT M0124_ CDR-L2 24 GVTGGNVYV M0124_ CDR-L3

[0113] Table 2 - VH / VL Sequences of Anti-C3 Antigen Binding Proteins SEQ ID NO: Sequence Annotation 25 EVQLVESGGGLVQAGGSLRLSCAASGRTINDYTMGWFRQAPGKDREFVSAINWRGSSTYYADSVKGRFTISRDNAKKTIYLQMNLLKPEDTAVYYCARQVSPYVELTATAAYWGQGTQVTVSS M0228_VHH 26 EVQLVESGGGLVQAGGSLRLSCVASGHTFGNWAMGWFRQAPGKEREFVGAIRWSVGTTNYRDSVKGRFAISRDNARNTVYLQMNRLKPEDTAVYYCAAGTPFVLARINGYDYWGQGTQVTVSS M0251_VHH 27 QSVKESGGRLVTPGTPLTLTCTVSGFSLYNYAMNWVRQAPGKGLEWIGIINTDGNTNYASWAKGRFTISTTSSTTVDLKITSPTTEDTATYFCPRAVGYHHHALDPWGPGTLVTVSS M0122_ VH 28 ELVLTQSPSVSAALGASAKLTCTLSSAHKTYTIDWYQQQQGEAPRYLMQLKSDGSYTKGTGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGTDYGGGYVFGGGTQLTVTG M0122_VL 29 QSVKESEGRLVTPGTPLTLTCTASGFTIGSYHMSWVRQAPGKGLEWIGIIYTDGNTDYANWAKGRFTISKTSTTMDLKMTSLTAADTATYFCARRGYADYGYTFNLWGQGTLVTISS M0123_ VH 30 ELVLTQPASVQVNLGQTVSLTCTADTLSRNYASWYQQKPGQAPVLLIYRDTSRPSGVPDRFSGSSSGNTATLTISGAQAGDEADYYCATGDGSGSSYQFVFGGGTQLTVTG M0123_VL 31 QSVKESGGRLVTPGTPLTLTCTVSGIDLSRYWMNWVRQAPGKGLEWIGYITTNDKTYYANWAKGRYTISKTSSTTVDLKMTSLTTEDTATYFCARRSSGAYDIWGPGTLVTISS M0124_VH 32 QPVLTQSPSASATLGASAKLTCTLSSAHKTYYIEWYQQQQGEAPRYLMQLKSDGTYTKGTGVPDRFSGSSSGADRYLIISSVQAEDEADYICGVTGGNVYVFGGGTQLTVTG M0124_VL

[0114] In some embodiments, the anti-C3 antigen binding protein of the present invention has at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence similarity or identity with any sequence in Table 1 or Table 2.

[0115] In some embodiments, the anti-C3 antigen-binding protein of the present invention is selected for its ability to inhibit one or more complement pathways (classical pathway, alternative pathway, and lectin pathway). In some embodiments, the anti-C3 antigen-binding protein of the present invention is selected for its ability to inhibit all three complement pathways (classical pathway, alternative pathway, and lectin pathway). In some embodiments, the anti-C3 antigen-binding protein of the present invention is capable of inhibiting all three complement pathways in the eye. In some embodiments, the anti-C3 antigen-binding protein of the present invention is capable of inhibiting all three complement pathways in the choroidal region of the eye. The choroidal region is a vascularized layer arranged on the back of the eye between the retina and the sclera. The choroidal region is divided into four layers: Haller's layer, Sattler's layer, choroidal capillary layer, and Bruch's membrane. Bruch's membrane, also known as the vitreous layer, is the innermost layer of the choroid and is adjacent to the retinal pigment epithelium (RPE). In some embodiments, the anti-C3 antigen-binding protein of the present invention can penetrate or diffuse across the Bruch membrane and enter other layers of the choroid, such as (but not limited to) the choroidal capillary layer.

[0116] The retina has a solid barrier that prevents large molecules (such as full-length immunoglobulins) from penetrating deeper layers, which can reduce the effectiveness of treatment (Jackson et al., Invest Ophthalmol Vis Sci. 2003;44(5): 2141-6). In contrast, smaller antibody derivatives can penetrate deeper into the retina. Exemplary antibody derivatives with a molecular weight of about 60 kDa or less are antibody fragments, including (but not limited to) Fab, Fab' fragments, scFab, scFv, Fv fragments, nanoantibodies, VHH, dAb, V-Nar, sdAb, sdFv, and bispecific and bivalent antibodies, such as single-chain bifunctional antibodies (scDb) or DART. In some embodiments, the molecular weight of the anti-C3 antigen-binding protein of the present invention is about 60 kDa or less, such as about 55 kDa, about 50 kDa, about 45 kDa, about 40 kDa, about 35 kDa, about 30 kDa, about 25 kDa, about 20 kDa, about 15 kDa or less.

[0117] In some embodiments, the anti-C3 antigen-binding protein of the present invention is able to penetrate or diffuse across a Bruch membrane, partly due to a sufficiently low size to facilitate penetration. In some embodiments, the size of the antigen-binding protein of the present invention is measured by molecular weight. In some embodiments, the molecular weight of the antigen-binding protein of the present invention is less than about 60 kDa. In some embodiments, the antigen-binding protein of the present invention is about 20 kDa to about 30 kDa or about 10 kDa to about 20 kDa. In some embodiments, the antigen-binding protein of the present invention is about 25 kDa. In some embodiments, the antigen-binding protein of the present invention is about 15 kDa. In some embodiments, the size of the antigen-binding protein of the present invention is measured by hydrodynamic radius. In some embodiments, the hydrodynamic radius of the antigen-binding protein of the present invention is less than or equal to about 3.0 nm. In some embodiments, the hydrodynamic radius of the antigen-binding protein of the present invention is less than or equal to about 2.5 nm. In some embodiments, the hydrodynamic radius of the antigen-binding protein of the present invention is less than or equal to about 2.0 nm.

[0118] In some embodiments, the anti-C3 antigen-binding protein of the present invention can compete with one or more antigen-binding proteins, including M0122, M0123, M0124, M0228, and M0251. Antibody competition can be measured by any analytical method known in this art. In some embodiments, in a C3 binding ELISA, an antibody can be labeled with a marker (such as biotin) and incubated with other anti-C3 antibodies. Typically, when an excess of competing antigen-binding proteins is present, it will reduce the specific binding of the antigen-binding protein or fragment thereof to C3 and / or C3b as described herein (i.e., its cross-blocking binding) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some embodiments, in the presence of competing antigen-binding proteins, the binding of the antigen-binding proteins or fragments thereof described herein is reduced by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0119] Complement C3 is a large protein composed of 13 different domains with a molecular size of 185 kilodaltons. During complement activation, C3 undergoes proteolytic cleavage and structural modifications at various sites. C3-derived fragments perform different effector functions and form convertases, which facilitate the amplification of the three complement pathways. In the classical and lectin pathways, C3 convertases C4bC2a cleave the full-length C3 into C3b and anaphylatoxin C3a. The alternative pathway also produces C3b and C3a, but utilizes the alternative pathway C3 convertase C3bBb. Furthermore, other C3 degradation products can be generated in the complement pathway. Complement factor I (CFI) is a plasma serine protease capable of permanently deactivating C3b to iC3b. Subsequently, iC3b is cleaved by CFI into other fragments (C3dg and C3c). Another C3 protein hydrolysate, C3d, binds to complement receptor 2 (CR2) and plays an important role in B cell cell cycle control. Besides C3-derived protein products, the complement pathway includes (but is not limited to) C1, C2, C4, C4b, C4a, C5, C5b, C5a, C6, C7, C8, C9, C1q, C1r, C1s, factor B, factor D, factor P, factor H, factor I, CD46 (MCP), CD55 (DAF), CD59 (MAC-IP), CR1 (CD35), CR2 (CD21), CR3, CR4, C3aR, C5aR1, C5aR2, CRIg, C4BPα chain, C4BPβ chain, ficolin-1, mannose-binding lectin (MBL), MBL-associated serine protease-1 (MASP-1), and MBL-associated serine protease-2 (MASP-2). The complement pathway and its various components are described in further detail in Noris et al., Semin Nephrol. 2013; 33(6): 479-492.

[0120] In some embodiments, the present invention provides an anti-C3 antigen-binding protein capable of binding to C3 and C3b. In some embodiments, the anti-C3 antigen-binding protein of the present invention has a weaker binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b than for C3 and C3b. In some embodiments, the anti-C3 antigen-binding protein of the present invention has a binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b of about 10⁻⁴ M or less. In some embodiments, the anti-C3 antigen-binding protein of the present invention has no binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b. As used herein, "no binding affinity" means that in one or more binding affinity assays known in the art (such as (but not limited to) ELISA assays), there is no detectable binding affinity relative to the background.

[0121] In some embodiments, the antigen-binding protein is capable of binding to the antigenic determinant on complement C3, wherein such binding prevents the formation of C3 convertase. In some embodiments, the antigen-binding protein of the present invention inhibits the activity of C3 convertase. In some embodiments, the antigen-binding protein of the present invention inhibits the C3 convertase amplification loop.

[0122] In some embodiments, the anti-C3 antibody of the present invention is expected to have better efficacy and safety in treating GA or other ocular conditions, due to the following characteristics described below.

[0123] The anti-C3 antibody of the present invention may include (but is not limited to) scFv and VHH antibody fragments having a molecular weight of less than about 60 kDa. For example, but not limitingly, the molecular weight of the scFv of the present invention may be about 25 kDa and the molecular weight of the VHH of the present invention may be about 15 kDa, while other therapeutic agents may have larger molecular weights. Based on hydrodynamic radius estimation, the anti-C3 antibody of the present invention is expected to have better choroidal C3 inhibitory activity because it can penetrate the Bruch's membrane more effectively and enter the choroid of the eye more effectively.

[0124] The therapeutic duration of the anti-C3 antibody of the present invention can exceed one month, which is a longer duration compared with other therapeutic agents. The extended therapeutic duration can be attributed to the anti-C3 antibody of the present invention having a molar concentration of up to 7 mM.

[0125] Compared to other therapeutic agents, the anti-C3 antibody of the present invention can be easily injected into the eye. The anti-C3 antibody of the present invention does not contain PEG, thereby reducing its viscosity. Therefore, the viscosity of the anti-C3 antibody of the present invention is expected to be lower than that of other therapeutic agents. Solutions with reduced viscosity, such as solutions with a viscosity of less than or equal to 20 centipoise (cP), are easier to inject into the eye due to reduced back pressure. Performance of the antigen-binding peptide.

[0126] In one state, a polynucleotide encoding a binding polypeptide (e.g., an antigen-binding protein) disclosed herein is provided. A method for preparing the binding polypeptide, comprising expressing such polynucleotides, is also provided.

[0127] Typically, a polynucleotide encoding a binding polypeptide disclosed herein is inserted into an expression vector for introduction into host cells, which can be used to produce the desired amount of the claimed antibody or fragment thereof. Thus, in some embodiments, the present invention provides an expression vector comprising the polynucleotide disclosed herein and a host cell comprising such a vector and the polynucleotide.

[0128] The terms "vector" or "expression vector" are used herein to mean a vector used as a medium according to the present invention for introducing a desired gene into a cell and expressing the desired gene in the cell. As will be known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, bacteriophages, viruses, and retroviruses. Typically, vectors compatible with the present invention will contain selection markers, appropriate restriction sites for promoting the selective colonization of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0129] Numerous expression vector systems can be used to achieve the objectives of this invention. For example, one vector utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (e.g., RSV, MMTV, MOMLV or analogs thereof), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome binding sites. Furthermore, cells that integrate DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The markers can provide auxotrophic hosts with protonutrients, biocidal resistance (e.g., antibiotics), or resistance to heavy metals (such as copper). Optional marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Optimal mRNA synthesis may also require other elements. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and termination signals. In some embodiments, the selected variable region gene is inserted into the expression vector along with the heavy and light chain constant region genes (e.g., human constant region genes) synthesized as described above.

[0130] In other embodiments, polycistronic constructs can be used to express binding peptides. In such expression systems, a variety of gene products of interest, such as the heavy and light chains of antibodies, can be generated from a single polycistronic construct. These systems preferably utilize internal ribosome entry sites (IRES) to provide relatively high levels of peptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will appreciate that such expression systems can be used to efficiently generate all the peptides disclosed in this application.

[0131] More generally, after preparing a vector or DNA sequence encoding an antibody or a fragment thereof, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. The introduction of plasmids into host cells can be achieved using various techniques well known to those skilled in the art. These techniques include (but are not limited to) transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact viruses. See Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, Vectors, eds. Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Plastmids can be introduced into the host via electroporation. The transformed cells are grown under conditions suitable for the production of light and / or heavy chains, and the synthesis of heavy and / or light chain proteins is analyzed. Exemplary analytical techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting assay (FACS), immunohistochemistry, and similar techniques.

[0132] As used herein, the term "transformation" should be used in a broad sense and refers to the introduction of foreign DNA into recipient host cells, thereby altering the genotype and thus causing changes in the recipient cells. Genetically modified recipient cells may contain foreign sequences through temporary or stable transformation. For example, foreign sequences may be stably integrated into the genome sequence of the recipient cell at a target site or a random site. Cells modified by gene editing methods (e.g., using homologous recombination, transposon-mediated systems, the loxP-Cre system, CRISPR / Cas9, or TALEN methods) are within the scope of this invention. In some embodiments, stable cell lines are generated for producing antigen-binding proteins or fragments thereof. This advantageously results in the stable production of antigen-binding proteins or fragments thereof with uniform quality and yield.

[0133] Thus, "host cell" refers to a cell transformed by a vector, which is constructed using recombinant DNA technology and encoding at least one heterologous gene. In the description of the process of isolating polypeptides from the recombinant host, unless otherwise explicitly specified, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody. In other words, recovering polypeptides from "cells" can mean recovering them from rapidly centrifuged complete cells or cell cultures containing culture medium and suspended cells.

[0134] In one embodiment, the host cell line used for antibody expression is mammalian in origin. Those skilled in the art can determine the specific host cell line most suitable for expressing the desired gene product. Exemplary host cell lines include (but are not limited to) DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-deficient), HELA (human cervical cancer), CV-1 (monkey kidney cell line), COS (a derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney), and analogues thereof. In one embodiment, the cell line produces glycosylations altered by the antibodies it expresses, such as defucosylation (e.g., PER.C6® (Crucell) or FUT8 knockout CHO cell lines (Potelligent® cells) (Biowa, Princeton, NJ)). The host cell line is typically available from commercial services, such as the American Tissue Culture Collection, or from publicly available literature.

[0135] In vitro preparation allows for scale-up to obtain large quantities of the desired peptide. Techniques for culturing mammalian cells under tissue culture conditions are known in this art and include homogeneous suspension cultures, such as in airlift reactors or continuous stirred reactors, or immobilized or embedded cell cultures, such as in hollow fibers, microcapsules, agarose microbeads, or ceramic filter cartridges. Depending on requirements and / or needs, the peptide solution can be purified by conventional chromatographic methods (e.g., gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immunoaffinity) chromatography).

[0136] The gene encoding the antigen-binding protein provided in this invention can also be expressed in non-mammalian cells (such as bacterial, yeast, insect, or plant cells). In this regard, it should be understood that various single-celled non-mammalian microorganisms (such as bacteria), i.e., microorganisms capable of growing or fermenting in cultures, can also be transformed. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It should be further understood that when expressed in bacteria, the protein can become part of an inclusion body. The protein must be isolated, purified, and then assembled into a functional molecule.

[0137] Eukaryotic microorganisms can also be used in addition to prokaryotes. Among eukaryotic microorganisms, Saccharomyces cerevisiae or commonly used roasting yeast strains are most commonly used, but many other strains can also be used. For expression in yeast (Saccharomyces), plastid YRp7 is commonly used, for example (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). This plastid already contains the TRP1 gene, which provides a selection marker for mutant yeast strains that do not have the ability to grow in tryptophan (e.g., ATCC number 44076 or PEP4-1) (Jones, Genetics 85:12 (1977)). Therefore, the presence of trp1 lesions, which are characteristic of the yeast host cell genome, can provide an effective environment for detecting transformation by growth in the absence of tryptophan.

[0138] Therefore, in one embodiment, a method for manufacturing an antigen-binding protein or a fragment thereof as described above is provided, comprising the steps of: i) culturing host cells under conditions allowing for the expression of the protein described herein; and ii) recovering the protein; and, where appropriate, iii) further purifying and / or modifying and / or formulating the protein. Method for administering antigen-binding protein

[0139] Those skilled in the art will know or readily determine the methods for preparing and administering antigen-binding proteins (e.g., the antigen-binding proteins disclosed herein) to individuals. The routes of administration of the antigen-binding proteins of the present invention may be oral, parenteral, inhalation, topical, or intraocular. As used herein, parenteral administration includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Intraocular administration, as used herein, includes (but is not limited to) subconjunctival, intravitreal, retrobulbar, or anterior chamber administration. Topical administration, as used herein, includes (but is not limited to) administration via liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.

[0140] In some embodiments, the antigen-binding protein of the present invention is administered intraocularly. Delivering therapeutic compounds to different structures of the eye, such as the retina, is challenging. These challenges include, but are not limited to, several restrictive ocular barriers, tearing mechanisms (including removal of the delivered compound by blinking and tearing), limited local injection volume, limited local bioavailability, and low tolerance for impurities and contaminants (see, for example, Patel et al., World J Pharmacol. 2013; 2(2): 47-64; Morrison et al., Ther. Deliv. 2014; 5(12): 1297-1315). The antigen-binding protein of the present invention overcomes these challenges. The antigen-binding protein of the present invention has a molecular weight of about 60 kDa or less. Examples of antigen-binding proteins of about 60 kDa or less include, but are not limited to, scFv, VHH, and Fab fragments. The smaller size of the antigen-binding protein of the present invention relative to full-length antibodies allows for the delivery of more therapeutic antibodies per injection. This enables the delivery of high concentrations of antibodies to the eye. The smaller size of the antigen-binding protein of this invention also improves its penetration into disease-related tissues, namely, the choroidal region of the eye. The antigen-binding protein can penetrate one or more layers of the choroidal region, including the Haller's layer, the Sattler's layer, the choroidal capillary layer, and the Bruch's membrane, thereby targeting complement C3 and C3b within these layers of the choroidal region.

[0141] In some embodiments, intraocular drug delivery is performed using a drug delivery device, such as a choroidal drug delivery device or a subretinal drug delivery device. The choroidal drug delivery procedure involves delivering a drug into the suprachoroidal space of the eye and is typically performed using a choroidal drug delivery device, such as a microinjector with microneedles (see, for example, Hariprasad, Retinal Physician; 2016; 13: 20-23; Goldstein, 2014, Retina Today 9(5): 82-87; each of which is incorporated herein by reference in its entirety). Choroidal drug delivery devices that can be used to deposit the antigen-binding protein of the present invention in the suprachoroidal space include (but are not limited to) choroidal drug delivery devices manufactured by Clearside® Biomedical, Inc. (see, for example, Hariprasad, 2016, above). Subretinal drug delivery devices that can be used to deposit the antigen-binding protein of the present invention in the subretinal space via the suprachoroidal space include (but are not limited to) subretinal drug delivery devices manufactured by Janssen Pharmaceuticals, Inc. (see, for example, International Patent Application Publication No. WO 2016 / 040635).

[0142] In some embodiments, intravitreal drug delivery is achieved via an intravitreal route. Intravitreal drug delivery is typically performed using a syringe and a gauge 27 to 30 needle (see, for example, Jiang et al., above).

[0143] One form of administration is a solution for injection, particularly for intravitreal injection, but all such forms of administration are clearly within the scope of this invention. Typically, pharmaceutical compositions suitable for injection may contain buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbate), and stabilizers (e.g., human albumin), as appropriate. However, in other methods compatible with the teachings herein, modified antibodies may be delivered directly to sites of harmful cell populations, thereby increasing the exposure of diseased tissue to the therapeutic agent.

[0144] In some embodiments, the antigen-binding protein of the present invention is formulated in a solution with low viscosity. The viscosity of the solution is measured in centipoise (cP). High-viscosity antibody solutions may pose a challenge for administering the antigen-binding protein of the present invention to the eye. For example, solutions with a viscosity greater than 50 cP may be difficult to administer with a fine needle due to high back pressure. Therefore, it is necessary to formulate the antigen-binding protein of the present invention in a low-viscosity solution. In some embodiments, the viscosity of the antigen-binding protein of the present invention and its pharmaceutical composition is from about 1 cP to about 50 cP. In some embodiments, the viscosity of the antigen-binding protein of the present invention and its pharmaceutical composition is less than or equal to about 20 cP, about 15 cP, about 10 cP, about 5 cP, about 4 cP, about 3 cP, about 2 cP, or about 1 cP. Further details regarding antibody viscosity are described in Tomar et al., MAbs. 2016; 8(2): 216-228 and Fennell et al., MAbs. 2013; 5(6): 882-895.

[0145] Formulations used for drug delivery include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. In the compositions and methods of the present invention, pharmaceutically acceptable carriers include (but are not limited to) 0.01-0.1 M or 0.05 M phosphate buffer, or 0.8% physiological saline. Other commonly used parenteral mediators include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's dextrose, non-volatile oils, and the like. Intravenous mediators include (but are not limited to) fluids and nutritional supplements, electrolyte supplements (such as Ringer's dextrose-based supplements), and the like. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases and the like, may also be present. In some embodiments, pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (wherein which water is soluble) or dispersions and sterile powders for ready-to-use formulations of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and its flowability should be sufficient to allow for injection. It should be stable under manufacturing and storage conditions and should also be protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0146] Microbial activity can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. The composition may also include isotonic agents, such as sugars, polyols, or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including a delayed absorption agent (e.g., aluminum monostearate and gelatin) in the composition.

[0147] In any case, a sterile injectable solution can be prepared by incorporating the desired amount of an active compound (e.g., an antigen-binding protein or fragment thereof) and (if necessary) one or a combination of the components listed herein in a suitable solvent, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing an alkaline dispersion medium and other desired components listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation methods typically include vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired components of its previously sterile filtered solution. Preparations for injection are processed according to methods known in this art, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under sterile conditions.

[0148] The effective dosage of the composition of the present invention for treating the above-mentioned conditions varies depending on many different factors, including the method of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. Conventional methods known to those skilled in the art can be used to titrate the therapeutic dose to optimize safety and efficacy.

[0149] As previously discussed, pharmaceutically effective amounts of the antigen-binding protein of the present invention, its immunoreactive fragments, or recombinants can be administered for in vivo treatment of mammalian diseases. In this regard, it should be understood that the disclosed antigen-binding protein will be formulated to facilitate drug administration and enhance the stability of the active agent.

[0150] The pharmaceutical compositions of the present invention generally comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffer solution, preservative, and the like. In this application, a pharmaceutically effective amount of the modified antigen-binding protein, its immunoreactive fragment, or recombinant (with or without binding to a therapeutic agent) must always mean an amount sufficient to achieve effective binding to the antigen and to provide benefit (e.g., improvement of symptoms of a disease or condition, or detection of substances or cells). In the case of tumor cells, the modified binding peptide will generally be able to interact with selected immunoreactive antigens on proliferative or immunoreactive cells and cause an increase in the death of such cells. Of course, the pharmaceutical compositions of the present invention can be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding peptide.

[0151] Consistent with the scope of this invention, the antigen-binding protein of this invention can be administered to humans or other animals in an amount sufficient to produce a therapeutic or preventative effect, according to the aforementioned treatment methods. The antigen-binding protein of this invention can be administered to such humans or other animals in known dosage forms prepared according to known techniques by combining the antibody of this invention with a known pharmaceutically acceptable carrier or diluent. Those skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent are indicated by the amount of the active ingredient it is combined with, the route of administration, and other well-known variables. Those skilled in the art will also understand that mixtures comprising one or more of the binding polypeptides described in this invention can prove particularly effective.

[0152] The bioactivity of the pharmaceutical compositions defined herein can be determined, for example, by complement inhibition assays, such as, but not limited to, enzyme immunoassays for determining the activity of functional classical, lectin, and alternative complement pathways in human serum. In some embodiments, the complement system screening Wieslab® (Euro Diagnostica AB, Malmö, Sweden) can be used to assess the inhibitory activity of the pharmaceutical compositions defined herein.

[0153] The purified complement components can be used for functional analysis to study the ability of the antibody of the present invention to inhibit the complement pathway, wherein the components are regenerated from the surface of red blood cells or artificial matrix, as described in Okroj et al., PLoS One.; 2012; 7(10): e47245.

[0154] The standard 50% hemolytic complement (CH50) assay is also a commonly used method to assess the ability of compounds to inhibit the functional activity of the classical complement pathway, as described by Jaskowski et al., Clinical and Diagnostic Laboratory Immunology; 1999; 6(1):137-9.

[0155] In some embodiments, the activity of the CP, LP, and AP complement pathways can be determined by measuring the level of hemolysis in the presence of the antigen-binding protein of the present invention, compared to the level of hemolysis in the absence of the antigen-binding protein of the present invention. In some embodiments, antibody-sensitized sheep erythrocytes can be used to measure complement-dependent hemolysis mediated by the classical pathway. In some embodiments, antibody-sensitized rabbit erythrocytes can be used to measure complement-dependent hemolysis mediated by alternative pathways, as described in Tomlinson et al., J Immunol. 1997; 159 (11): 5606-5609.

[0156] In some embodiments, the activity of the CP, LP, and AP complement pathways can be determined by measuring MAC formation in the presence of the antigen-binding protein of the present invention, compared to MAC formation in the absence of the antigen-binding protein of the present invention. MAC assays for activation of the classical complement pathway mediated by IgM in human serum cause MAC deposition on IgM-coated ELISA discs. MAC formation can be detected by using an alkaline phosphate-labeled antibody against C5b-9. In the presence of the antigen-binding protein of the present invention, the ELISA signal decreases in a dose-dependent manner. For testing alternative pathways, MAC deposition on LPS-coated ELISA discs can be performed using MAC assays for activation of the alternative complement pathway mediated by LPS in human serum. Suitable MAC assays include (but are not limited to) the Pacific Biomarkers Complement Membrane Attack Complex (SC5b-9) ELISA assay.

[0157] As used herein, "efficacy" or "in vivo efficacy" refers to the response to a therapy using the pharmaceutical composition of the present invention, using, for example, standardized response criteria, such as standard ophthalmic response criteria. The efficacy or in vivo efficacy of a therapy using the pharmaceutical composition of the present invention refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to produce its desired effect (i.e., to inhibit the complement pathway in the eye). In vivo efficacy can be monitored using established standard methods for various ocular diseases. Monitoring methods include (but are not limited to) the Amsler grid test, opthtalmoscopy, ocular fundus microscopy, ocular computed tomography, and optical coherence tomography. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used. Antibody engineering and optimization.

[0158] The antigen-binding protein of the present invention can be engineered or optimized. As used herein, "optimized" refers to changes made to improve one or more functional properties of the antigen-binding protein. Changes include (but are not limited to) the deletion, substitution, addition, and / or modification of one or more amino acids within the antigen-binding protein.

[0159] As used herein, the term "functional property" refers to a characteristic of an antigen-binding protein, the modification of which (e.g., relative to conventional antigen-binding proteins) is desirable and / or advantageous to those skilled in the art, for example, to improve the manufacturing properties or therapeutic efficacy of the antigen-binding protein. In one embodiment, the functional property is stability (e.g., thermal stability). In another embodiment, the functional property is solubility (e.g., under cellular conditions). In another embodiment, the functional property is an aggregated state. In another embodiment, the functional property is protein expression (e.g., in prokaryotic cells). In another embodiment, the functional property is the refolded state after the inclusion bodies dissolve during manufacturing. In some embodiments, the functional property is not an improvement in antigen-binding affinity. In another embodiment, the modification of one or more functional properties does not have a substantial effect on the binding affinity of the antigen-binding protein.

[0160] In some embodiments, the antigen-binding protein of the present invention is an scFv and is optimized by identifying preferred substituted, deleted, and / or added amino acid residues at the sites of interest in the antigen-binding protein (e.g., by comparing a database of scFv sequences having at least one desired characteristic (e.g., selected by quality control (QC) analysis) with a database of mature antibody sequences (e.g., the Kabat database). Therefore, the present invention also provides an "enrichment / exclusion" method for selecting specific amino acid residues. Furthermore, the present invention provides a method for engineering antigen-binding proteins (e.g., scFv) by mutating specific structural amino acid sites identified using the "functional consensus" method described herein. In some embodiments, structural amino acid sites are mutated by replacing existing amino acid residues with residues identified as "enriched" using the "enrichment / exclusion" analysis method described herein. In one embodiment, the present invention provides a method for identifying amino acid positions for mutation in a single-chain antibody (scFv) having VH and VL amino acid sequences, the method comprising: a) inputting the scFv VH, VL, or VH and VL amino acid sequences into a database containing a large number of antibody VH, VL, or VH and VL amino acid sequences, such that the scFv VH, VL, or VH and VL amino acid sequences are compared with antibody VH, VL, or VH and VL amino acid sequences in the database; b) comparing the amino acid positions within the scFv VH or VL amino acid sequences with the corresponding positions within the antibody VH or VL amino acid sequences in the database; c) measuring the scFv Whether the amino acid position within the VH or VL amino acid sequence is occupied by a conserved amino acid residue at the corresponding position within the antibody VH or VL amino acid sequence in the database; and d) when the amino acid position is not occupied by a conserved amino acid residue at the corresponding position within the antibody VH or VL amino acid sequence in the database, identifying the amino acid position within the scFv VH or VL amino acid sequence as the amino acid position for mutation. ScFV optimization is further described in detail in WO2008110348, WO2009000099, WO2009000098 and WO2009155725, all of which are incorporated herein by reference. Humanization:

[0161] In some embodiments, the antigen-binding protein of the present invention may be humanized. As used herein, the term "humanization" refers to a non-human donor antibody modified to increase its similarity to naturally occurring antibodies in humans. As used herein, the term "humanization" refers to the process of humanizing a non-human donor antibody. Humanization can be achieved by transplanting a CDR of a non-human donor antibody (e.g., a rabbit or llama antibody CDR) onto a human or humanized antibody receptor framework region (such as a soluble and stable light chain and / or heavy chain human antibody framework region). A common method for transplanting a CDR into a human receptor framework has been disclosed by Winter in U.S. Patent No. 5,225,539 and by Queen et al. in WO199007861, which are incorporated herein by reference. A suitable receptor framework region can exhibit superior functional properties, such as improved solubility and stability. In some embodiments, the antigen-binding protein of the present invention is a rabbit antibody. The CDRs of these rabbit antibodies can be transplanted into universal receptor architecture regions, such as those described in WO2009155726, which are incorporated herein by reference.

[0162] In some embodiments, the humanization / stabilization of non-human antibodies or the stabilization of human antibodies using human frameworks involves replacing the κ-binding segment in the κ variable light chain domain with a λ-binding segment, resulting in a κ-λ chimeric variable light chain domain with improved protein stability and reduced aggregation tendency. It also involves a mutation of the κ common residue at position AHo101 and a substitution by the λ common residue to support the filling of the λ-binding segment in the κ-λ chimeric variable light chain domain, further improving protein stability and further reducing aggregation tendency. Further details regarding these human framework regions are described in WO2014206561 and WO2019057787, which are incorporated herein by reference. Methods for treating complement C3-mediated diseases and symptoms.

[0163] Provides a method for treating complement C3-mediated diseases or conditions in individuals with complement C3-mediated diseases or conditions using the antigen-binding protein described herein.

[0164] In some embodiments, complement C3-mediated diseases or conditions are selected from the group consisting of: age-related macular degeneration (AMD), geographic atrophy (GA), neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, posterior fibrosis, autoimmune uveitis, chorioretinitis, retinitis, rheumatoid arthritis, psoriasis, and atherosclerosis. In some embodiments, C3-mediated diseases are a form of AMD. AMD is generally classified into two main categories: dry AMD and wet AMD. Dry AMD (also known as non-exudative AMD) is characterized by the presence of drusen (yellow deposits) in the macular region. Wet AMD (also known as exudative AMD or neovascular AMD) is characterized by abnormal vascular growth from the choroid beneath the macula. This process is also known as choroidal neovascularization, and the new blood vessels can allow fluids (such as blood) to leak into and around the retina. Geographic atrophy (also known as atrophic AMD or late dry AMD) is a late form of AMD that can cause progressive and irreversible loss of retinal cells.

[0165] Treating ocular conditions (such as AMD as described above) is particularly challenging. As previously mentioned, the delivery of therapeutic agents to the eye is limited by several barriers, including (but not limited to) the blood-retinal barrier, such as the RPE. The ability to penetrate the RPE and enter the choroid of the eye would enhance the therapeutic potential of the drug. In some embodiments, the antigen-binding protein of the present invention is able to penetrate the RPE and Bruch's membrane in the choroidal region of the eye, thereby targeting complement C3 in the choroidal region. The ability of the antigen-binding protein of the present invention to penetrate the RPE and Bruch's membrane improves its therapeutic potential in treating complement C3-mediated diseases or conditions. The ability of the antigen-binding protein of the present invention to penetrate the RPE and Bruch's membrane is partly due to its sufficiently small size to facilitate penetration. In some embodiments, the size of the antigen-binding protein of the present invention is measured by molecular weight. In some embodiments, the molecular weight of the antigen-binding protein of the present invention is less than about 60 kDa. In some embodiments, the antigen-binding protein of the present invention is about 20 kDa to about 30 kDa or about 10 kDa to about 20 kDa. In some embodiments, the antigen-binding protein of the present invention has a size of about 25 kDa. In some embodiments, the antigen-binding protein of the present invention has a size of about 15 kDa. In some embodiments, the size of the antigen-binding protein of the present invention is measured by hydrodynamic radius. In some embodiments, the hydrodynamic radius of the antigen-binding protein of the present invention is less than or equal to about 3.0 nm. In some embodiments, the hydrodynamic radius of the antigen-binding protein of the present invention is less than or equal to about 2.5 nm. In some embodiments, the hydrodynamic radius of the antigen-binding protein of the present invention is less than or equal to about 2.0 nm.

[0166] In one embodiment, the present invention provides a method for inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP), the method comprising contacting complement C3 with an antigen-binding protein or fragment thereof that binds to an antigenic determinant on complement C3. The ability of the antigen-binding protein of the present invention to inhibit all three complement pathways further improves its therapeutic potential in treating complement C3-mediated diseases or conditions. It is not desired to be bound by theory; by compensating for the disease-promoting effects of blocking one active pathway with the other inactive pathways, inhibiting all three complement pathways can improve the therapeutic potential of the antigen-binding protein of the present invention.

[0167] In some embodiments, the antigen-binding protein or a fragment thereof is capable of substantially equivalently inhibiting the activity of the CP, LP, and AP complement pathways. For example, but not limitingly, the antigen-binding protein or a fragment thereof is capable of inhibiting the activity of the CP pathway by at least 80%, inhibiting the activity of the LP pathway by at least 80%, and inhibiting the activity of the AP pathway by at least 80%. In some embodiments, the activity of the CP, LP, and AP complement pathways is inhibited by at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0168] In another embodiment, the present invention provides a method for inhibiting the activity of complement C3 in the choroidal region, which is carried out by intraocular administration of an antigen-binding protein or a fragment thereof that binds to the antigenic determinant of complement C3. Activated complement pathways in the choroidal region of the eye can promote complement C3-mediated diseases or conditions. Therefore, one object of the present invention is to provide an antigen-binding protein capable of penetrating or diffusing into the choroidal region and targeting complement C3 and C3b. In some embodiments, the antigen-binding protein of the present invention inhibits the activity of C3 convertase in the choroidal region of the eye. In some embodiments, the antigen-binding protein of the present invention inhibits the C3 convertase amplification loop in the choroidal region of the eye. Medical Use

[0169] This invention also relates to methods for treating complement C3-mediated diseases or conditions in an individual using antigen-binding proteins or fragments thereof as disclosed herein. All technical features described herein regarding antigen-binding proteins or fragments thereof are applicable. Kit

[0170] This invention also covers kits comprising at least one antigen-binding protein or fragment thereof as described herein. In one embodiment, the kit comprises a composition containing an effective amount of the antigen-binding protein or fragment thereof in unit dosage form. Such kits may comprise a sterile container containing the composition; non-limiting examples of such containers include (but are not limited to) vials, ampoules, bottles, tubes, syringes, and blister packs. In some embodiments, the composition is a pharmaceutical composition and the container is made of a material suitable for preserving the pharmaceutical preparation. In one embodiment, the kit may comprise a lyophilized antigen-binding protein or fragment thereof in a first container and a diluent (e.g., sterile water) in a second container for reconstitution or dilution of the antigen-binding protein or fragment thereof. In some embodiments, the diluent is a pharmaceutically acceptable diluent.

[0171] Typically, the kit will further include a separate sheet, manual, or card with instructions for use, provided in or with the container. If the kit is intended for medical use, it may further include one or more of the following: information on administration of the composition to individuals with complement C3-mediated diseases or conditions and the timing of administration; instructions on the therapeutic agent; precautions; warnings; indications; contraindications; information on overdose and / or adverse reactions. Diagnostic applications and / or detection.

[0172] The antigen-binding protein or fragment thereof of the present invention can be used for in vivo and / or in vitro detection or diagnostic purposes. For example, those skilled in the art know many immunoassays involving antigen-binding proteins for detecting the expression of specific cells or tissues. For such applications, the antigen-binding protein or fragment thereof disclosed herein may be labeled or unlabeled. For example, but not limitingly, unlabeled antigen-binding proteins may be used and detected by a secondary antibody that recognizes the antigenic determinant on the antigen-binding protein described herein. In another embodiment, the antigen-binding protein or fragment thereof binds to one or more substances that can be recognized by a detection agent substance, for example, the antigen-binding protein or fragment thereof binds to biotin that can be detected by streptoacidin. In some embodiments, the antigen-binding protein or fragment thereof is suitable for detecting the presence of C3 and / or C3b in a sample. In some embodiments, the sample is a biological sample. As used herein, the term "detection" encompasses quantitative and / or qualitative detection. In some embodiments, the biological sample comprises cells or tissues from a human patient, such as retinal tissue.

[0173] In some embodiments, the method includes contacting a biological sample with at least one antigen-binding protein or fragment thereof of the present invention; allowing C3 (if present) in the sample to form a complex with the antigen-binding protein or fragment thereof; and then detecting the antigen-binding protein or fragment thereof. In a preferred embodiment, the antigen-binding protein or fragment thereof is capable of binding complement C3 and C3b.

[0174] In one embodiment, antigen-binding proteins or fragments thereof are detected by a detectable signal. In another embodiment, antigen-binding proteins or fragments thereof are detected by ELISA, immunocytochemistry (ICC), immunohistochemistry (IHC), Western ink dot assay, and / or flow cytometry.

[0175] Biological samples may be tissue samples, such as retinal tissue. Tissue samples may be fixed tissue samples, such as formalin-fixed and paraffin-embedded tissue samples.

[0176] In one embodiment, such a method is used to select patients, that is, to determine whether an individual is eligible for a therapy using an antigen-binding protein or a fragment thereof as described herein.

[0177] Those skilled in the art will readily recognize that other suitable modifications and alterations to the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Certain embodiments have been described in detail, and these embodiments will be more clearly understood with reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. Example 1 - Generation and Characterization of Anti-C3 Antibody Library

[0178] In order to generate antibodies that can inhibit the complement cascade more effectively than partial inhibitors of complement, it is hypothesized that a large collection of anti-C3 antibodies with different antigenic determinant recognition can increase the probability of isolating antibodies with the desired function. To this end, a large antibody phage library is constructed using genomic information that encodes antibody variable domains derived from B cells of animals immunized with C3.

[0179] To generate a large number of antibodies capable of recognizing different antigenic determinants on C3, three New Zealand white rabbits and two llamas were immunized with native human C3 protein purified from serum (Figure 2). Each animal received four injections of C3 protein and either complete or incomplete Freund's adjuvant at different time points (Figure 3A). The immune response of each animal was tested by ELISA to quantify the presence of anti-C3 antibodies in serum samples from the immunized animals. The antibody titer in the serum indicated an excellent immune response (Figure 3B).

[0180] A cDNA library of scFv antibodies was constructed from RNA extracted from isolated rabbit PBMCs and spleen lymphocytes via PCR amplification. The coding sequences of the variable light and variable heavy domains were amplified separately and ligated through a series of overlapping polymerase chain reaction (PCR) steps to obtain the final scFv product.

[0181] For llamas, a large amount of blood was drawn, RNA was isolated from the blood, and reverse transcriptase was used to transduce it into cDNA. The cDNA was washed, and primers were used to attach it to the leader sequence region and the CH2 region to amplify the heavy chain fragment.

[0182] The amplified DNA sequences encoding rabbit scFv and llama VHH were digested using appropriate restriction enzymes and then conjugated into phage vectors. The phage vectors were then transformed into TG1 electrically competent cells well-suited for antibody phage presentation library production. These processes yielded four antibody libraries with a size greater than 10⁸ pure lines and approximately 100% insertion percentage (Figs. 4A and 4B). Example 2 - Screening for anti-C3 antibodies inhibiting all three complement pathways.

[0183] C3 is a large protein composed of 13 distinct domains with a molecular size of 185 kilodaltons. During complement activation, C3 undergoes proteolytic cleavage and structural modifications at various sites. C3-derived fragments perform different effector functions and form invertases, which contribute to the amplification loop of the complement pathway. The enzyme C3 invertase has the ability to cleave multiple C3 molecules into C3b in an effective amplification loop to produce more C3 invertase, thereby causing complete activation of the complement system. Antibodies that bind to different antigenic determinants on C3 and C3b and effectively block all three complement activation pathways (classical, lectin, and alternative) are identified using the screening methods described herein.

[0184] To screen for high-affinity anti-C3 antibodies, scFv and VHH antibodies presented on phages were prepared and subjected to several rounds of biopanning (selection) against native human C3 purified from serum. The selection rigor was increased in each round by reducing the concentration of C3 protein used in the biopanning or by increasing the stringency of the washes. Approximately 380 single phage strains were selected and screened for their C3 binding ability using ELISA analysis (Figure 5).

[0185] Based on ELISA data and DNA fingerprinting, 41 phage pure lines were selected for sequencing and prepared into antibody proteins in a recombinant manner. Their ability to bind to human C3 and C3b was evaluated and further characterized (Figure 5).

[0186] To identify antibodies that block all three complement pathways, screening was performed using the Wieslab® complement system (Svar Life Science AB, Malmö, Sweden). Enzyme immunoassay was used for the qualitative determination of functional classical, lectin, and alternative complement pathways in human serum. The amount of C5b-C9 neoantigen produced was proportional to the functional activity of the complement pathway. As shown in Figure 6, five antibodies, namely M0251, M0228, M0122, M0123, and M0124, were able to inhibit all three complement pathways in human serum (Quidel) by at least 90% at a fixed concentration of 2 µM. Example 3 - Characterization of anti-C3 antibodies: M0251, M0228, M0122, M0123, and M0124

[0187] M0251, M0228, M0122, M0123, and M0124 were tested in a paired combination to identify antibodies targeting the same region (antigenic determinant) on C3. In short, an antibody was labeled with biotin and cultured together with other antibody lines in a C3 binding ELISA. It was assumed that anti-C3 antibodies competing for the same binding region shared similar antigenic determinants and therefore had similar functions. This information allowed for a reduction in the number of potential antibody candidates while maintaining antigenic determinant diversity. Among the five antibodies inhibiting all three complement pathways, M0251, M0228, and M0123 were considered to share the same antigenic determinant on C3 (Figure 7D). It was hypothesized that inhibiting leads bind to three different antigenic determinants on C3 (Figures 7A-7D).

[0188] The ability of antibodies capable of inhibiting all three complement pathways to bind to cynomolgus monkey C3 was evaluated in ELISA. Briefly, 96-well ELISA pans were coated with a multi-strain goat antiserum believed to cross-react with cynomolgus monkey C3, followed by secondary binding with a custom-made cynomolgus monkey serum formulation (BioIVT, NB-151558). Serial dilutions of antibody molecules were added to the ELISA pans, and antibodies bound to cynomolgus monkey C3 were detected using rabbit anti-human κHRP antibody (Abcam, ab202549) or mouse anti-His Tag HRP antibody (R&D Systems, MAB050H). Guide molecules M0122, M0124, and M0251 demonstrated dose-responsive binding to cynomolgus monkey C3. Interestingly, although M0251, M0228, and M0123 compete for the same antigenic determinant on human C3, only M0251 exhibited binding activity against cynomolgus monkey C3 (Figures 15A and 15B).

[0189] The ability of anti-C3 antibodies to inhibit all complement activation pathways in cynomolgus monkey serum was evaluated using the Wieslab complement system screening. Anti-C3 antibodies were added to a custom-made cynomolgus monkey serum formulation. Figure 14A shows that M0122, M0124, and M0251 effectively inhibited all three complement pathways at a fixed concentration of 2 μM, indicating that M0122, M0124, and M0251 are potent inhibitors of complement-mediated MAC formation in cynomolgus monkey serum. M0228 did not show inhibitory activity against complement pathways in cynomolgus monkey serum, confirming that this antibody did not have observed binding activity against cynomolgus monkey C3 (Figure 14B). The dose-dependent inhibition of classical and alternative pathways in cynomolgus monkey serum by M0122, M0124, and M0251 was further evaluated using the corresponding Wieslab complement system kits (Figures 14B and 14C).

[0190] The binding ability of M0122, M0124, and M0228 to human C3 and C3b was evaluated in a direct binding ELISA assay (Figures 8A and 8B). Briefly, purified native human C3 or C3b (Complement Technology, A113 and A114) was coated onto 96-well ELISA discs. Serial dilutions of the antibody molecules were added to the discs and detected by rabbit anti-human κHRP antibody (Abcam, ab202549) or rabbit anti-His Tag HRP antibody (Abcam, ab1187). M0122, M0124, and M0228 exhibited high affinity binding to human C3 and C3b. Further analysis of the binding kinetics of M0122, M0124, and M0228 to human C3 using biolayer interferometry revealed low affinity in the pimozne range (Figure 10).

[0191] The dose-dependent inhibition of the alternative and classical pathways in human serum by M0122, M0124, and M0228 was evaluated using the corresponding Wieslab complement system kit. Anti-C3 antibodies M0122, M0124, and M0228 demonstrated potent inhibition of both the alternative and classical pathways in human serum (Figures 9A and 9B). The ability of anti-C3 antibodies M0122, M0123, and M0124 to inhibit the lectin pathway in a dose-responsive manner was further evaluated. Figure 16 shows the effective inhibition of the lectin pathway in human serum. In conclusion, these results further support the effectiveness of the antibodies of the present invention in inhibiting all three complement activation pathways. Example 4 - Anti-C3 antibodies are more likely to penetrate Bruch membranes compared to APL-2.

[0192] The complement system is known to play a role in the pathogenesis of geographic atrophy (GA). However, the extent to which complement activity is distributed in the eye and whether the efficacy of GA therapy depends on delivering the therapeutic agent to the correct anatomical acupoints within the eye are not fully understood. We hypothesize that better penetration into the disease-associated retinal tissues (i.e., the RPE, Bruch's membrane, and choroid) may be necessary to achieve greater reduction in lesion growth in GA. The inner portion of the choroid is called the choroidal capillary layer, which contains capillaries separated by an extracellular membrane called the Bruch's membrane (BrM) (Figure 11).

[0193] Bruch membranes are selectively permeable by antibodies and biologics. As reported by Clark et al. (Front. Immunol. 2017. 8:1-10), complement pathway proteins, except for FHL-1, factor D, and C5a, cannot cross Bruch membranes. In general, antibodies and biologics with large hydrodynamic radii are unlikely to cross Bruch membranes. As shown in Table 3 below, molecules with a hydrodynamic radius greater than 3.00, except for APL-2 and CDR2 (the anti-C3 scFv of this invention), cannot cross Bruch membranes; on the other hand, all molecules with a hydrodynamic radius greater than 3.00, except for C3a, can cross Bruch membranes. Table 3: Size Factors of Biologics List protein Hydrodynamic radius (nm) MW (kDa) Diffusion (BM > 70 years old) APL-2* Approximately 7-8 43 (Linear PEG) Nk IgG Approximately 6 150 no Factor H 5.56 155 no C3 4.84 180 no FHL-1 4.40 49 yes Factor B 3.22 83 no Factor I 3.07 65 no Fab fragment 2.91 50 yes CDR2* 2.5 26 Nk Factor D 2.08 twenty four yes C3a 1.56 9 no C5a 1.63 8.3 yes

[0194] In addition, Pitkänen et al. (Invest Ophthalmol Vis Sci. 2005; 46(2):641-6) studied the permeability of carboxyfluorescein and FITC-labeled polydextrose (molecular mass 4 to 80 kDa) to fresh RPE-choroidal membrane samples from bovine eyes. We plotted the permeability by molecular size (Fig. 12, black spots). We also used the study conducted by Hirvonen et al. (Pharm Res. 2016;33(8):2025-32) to derive the permeability values ​​of scFv, lucentis, eylea, and APL2 based on the hydrodynamic radius, and plotted the permeability against molecular weight in the same figure (Fig. 12, colored dots). The trend shows that the larger the molecular weight, the worse the permeability to the Bruch membrane.

[0195] It is highly likely that, compared to APL-2 (two anti-C3 cyclic APL-1 peptides linked to a 40 kDa linear PEG, totaling 43 kDa) with a hydrodynamic radius of at least 7 nm, the anti-C3 antibodies of the present invention, in the form of antibody fragments (such as (but not limited to) scFv or VHH types) and with a hydrodynamic radius of about 2.5 nm and smaller, can better permeate Bruch membranes.

[0196] To test this hypothesis, the ability of anti-C3 molecules to cross the BrM was assessed using enriched porcine BrM mounted in a using chamber. In short, enriched BrM membranes were isolated from the porcine eye and mounted in a using diffusion chamber (Multi Channel Systems MCS GmbH, catalog number 660026). After mounting, the 5 mm diameter BrM membrane served as the sole barrier between the two identical compartments. Both sides of the BrM membrane were washed with 1 ml PBS for at least 5 minutes at room temperature. For a leakage test, 1 ml PBS was added to the sample chamber and leakage to the second compartment was monitored for 5 minutes. If no leakage was detected (leakage indicates impaired membrane integrity), antibody protein was added to the sample chamber at 100 µg / ml in 1 ml PBS, and 1 ml PBS was added to the second compartment (diffuser chamber). The entire using chamber was incubated at room temperature for 24 hours with gentle shaking to avoid creating a gradient of diffusing proteins. Samples (15 µl) from each compartment were analyzed by gel electrophoresis. A pre-prepared 4–12% NuPAGE Bis Tris SDS gel (Thermo Fisher Scientific) was operated at 200 V for 40 minutes under reducing conditions. The gel was stained with Instant Blue (Expedeon) for 60 minutes at room temperature for antibody protein detection, or stained with barium iodide solution for PEG detection (the gel was fixed with 0.1 M perchloric acid, which was replaced after 15 minutes with a premix of 20 ml 5% BaCl2 and 8 ml 0.1 M iodine solution, and then repeatedly replaced with deionized water every 10 minutes for 1 hour). To calculate the protein percentage in the sample or diffusion compartment, the band density in the Instant Blue-stained or BaI2-stained SDS gels was measured using ImageJ software. The average intensity of these bands was compared with the density of a control band representing 100% protein loading (i.e., 15 µl, 100 µg / ml). The calculated protein percentage ± SD was then plotted. The ability of a scFv derivative of M0123 (26 kDa) and an APL-2 substitute (a C3-linked cyclic APL-1 peptide linked to a 40 kDa linear PEG, totaling 42 kDa) to cross porcine BrM was compared between the two scFv derivatives of M0123 cultured simultaneously on four different BrM formulations from porcine eyes. The amount of scFv crossing BrM in all four membrane formulations was significantly higher than that of the APL-2 substitute (Figures 13A and 13B). [Simplified Explanation of the Diagram]

[0069] The foregoing descriptions and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings. Upon application and payment of the necessary fees, the Patent Office will provide a copy of the patent application or patent application publication containing color drawings.

[0070] Figure 1 depicts three complement pathways: the classical (CP), lectin (LP), and alternative (AP) pathways, which converge at C3.

[0071] Figure 2 depicts a method for generating an anti-C3 antibody library.

[0072] Figures 3A-3B depict an ELISA assay that confirms an excellent immune response against C3 in rabbits and llamas. Figure 3A depicts an ELISA assay used to test for C3 protein isolated from human plasma. Figure 3B depicts an ELISA assay used to test for the presence of anti-C3 antibodies in the serum of rabbits (top inset) and llamas (bottom inset) injected with isolated human C3 as shown in Figure 3A.

[0073] Figure 4A depicts an overview of the anti-C3 antibody library and Figure 4B shows the diversity of CDR-H3 amino acid lengths.

[0074] Figure 5 depicts a method for screening anti-C3 antibodies.

[0075] Figure 6 depicts the screening of candidate antibodies targeting C3 for their ability to inhibit all three complement pathways in human serum. Each antibody system was used at a concentration of 2 µM.

[0076] Figures 7A-7D illustrate the recognition of three different antigenic determinants on C3 by four anti-C3 antibodies that inhibit all three complement pathways. Figure 7A depicts a competitive analysis, which confirms that M0122 does not compete with any of the other three anti-C3 antibodies. Figure 7B depicts a competitive analysis, which confirms that M0124 does not compete with any of the other three anti-C3 antibodies. Figure 7C depicts a competitive analysis, which confirms that M0228 competes with M0251, but not with M0124 and M0122. Figure 7D depicts a competitive analysis, which confirms that M0123 competes with M0251 and with M0228, but not with M0124 and M0122.

[0077] Figures 8A and 8B depict M0122, M0124, and M0228 directly binding to both C3 and C3b. Figure 8A depicts the ELISA analysis method, demonstrating that M0122, M0124, and M0228 directly bind to C3. Figure 8B depicts the ELISA analysis method, demonstrating that M0122, M0124, and M0228 directly bind to C3b.

[0078] Figures 9A and 9B depict the effective inhibition of classical and alternative pathways by M0122, M0124, and M0228. Figure 9A depicts the effective inhibition of classical pathways by M0122, M0124, and M0228. Figure 9B depicts the effective inhibition of alternative pathways by M0122, M0124, and M0228.

[0079] Figure 10 depicts the affinity parameters of M0122, M0124 and M0228.

[0080] Figure 11 is a schematic diagram depicting the anatomical structure of the retina and choroid (including Bruch's membrane). The anti-C3 scFv antibody of the present invention is depicted as capable of penetrating Bruch's membrane and entering the choroid more deeply, while the comparative C3-binding therapeutic agent APL-2 cannot penetrate Bruch's membrane. The same principle applies to other antigen-binding protein types of the present invention.

[0081] Figure 12 illustrates the negative correlation between the hydrodynamic radius and permeability of the complement binding therapy compared to the scFv of the present invention.

[0082] Figures 13A and 13B depict a comparison of scFv and the APL-2 substitute in terms of permeation through the Bruch membrane. Figure 13A shows barium iodide staining (PEG), and Figure 13B shows Coomassie staining (protein). The APL2-substitute contains one APL-1 moiety on a 40 kDa linear PEG. SC - Sample chamber, DC - Diffusion chamber, LC - Internal reference (initial concentration in SC).

[0083] Figures 14A-14C illustrate the effective inhibition of classical, alternative, and lectin pathways in cynomolgus monkey serum by M0122, M0124, and M0251. Figure 14A illustrates the effective inhibition of all three pathways by M0122, M0124, and M0251. Each anti-molecular system was used at a concentration of 2 µM. Figure 14B illustrates the effective inhibition of the classical pathway by M0122, M0124, and M0251. Figure 14C illustrates the effective inhibition of the alternative pathway by M0122, M0124, and M0251.

[0084] Figures 15A-15B depict M0122, M0124 and M0251 in combination with the cynomolgus monkey C3. Figure 15A depicts M0122 and M0124. Figure 15B depicts M0251.

[0085] Figure 16 illustrates how M0122, M0123 and M0124 effectively inhibit the lectin pathway. [Sequence List]

[0197] <![CDATA[ <110> CDR-LIFE AG (Germany) and CDR Life International GmbH (Switzerland) <![CDATA[ <120> complement C3 antigen-binding protein <![CDATA[ <130> 02-0558]]> <![CDATA[ <140> TW 111105011]]> <![CDATA[ <141> 2022-02-11 <![CDATA[ <150> PCT / EP2021 / 053526]]> <![CDATA[ <151> 2021-02-12 <![CDATA[ <160> 32]]> <![CDATA[ <170> BiSSAP 1.3.6 <![CDATA[ <210> 1]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 1]]> Asp Tyr Thr Met Gly 1 5 <![CDATA[ <210> 2]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 2]]> Ala Ile Asn Trp Arg Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <![CDATA[ <210> 3]]> <![CDATA[ <211> 14]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 3]]> Gln Val Ser Pro Tyr Val Glu Leu Thr Ala Thr Ala Ala Tyr 1 5 10 <![CDATA[ <210> 4]]> <![CDATA[ <211> 5]]><![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 4]]> Asn Trp Ala Met Gly 1 5 <![CDATA[ <210> 5]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 5]]> Ala Ile Arg Trp Ser Val Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys 1 5 10 15 Gly <![CDATA[ <210> 6]]> <![CDATA[ <211> 14]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 6]]> Gly Thr Pro Phe Val Leu Ala Arg Ile Asn Gly Tyr Asp Tyr 1 5 10 <![CDATA[ <210> 7]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 7]]> Asn Tyr Ala Met Asn 1 5 <![CDATA[ <210> 8]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 8]]> Ile Ile Asn Thr Asp Gly Asn Thr Asn Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <![CDATA[ <210> 9]]> <![CDATA[<211 >11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 9]]> Ala Val Gly Tyr His His His Ala Leu Asp Pro 1 5 10 <![CDATA[ <210> 10]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 10]]> Thr Leu Ser Ser Ala His Lys Thr Tyr Thr Ile Asp 1 5 10 <![CDATA[ <210> 11]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 11]]> Leu Lys Ser Asp Gly Ser Tyr Thr Lys Gly Thr 1 5 10 <![CDATA[ <210> 12]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 12]]> Gly Thr Asp Tyr Gly Gly Gly Tyr Val 1 5 <![CDATA[ <210> 13]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 13]]> Ser Tyr His Met Ser 1 5 <![CDATA[ <210> 14]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]><![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 14]]> Ile Ile Tyr Thr Asp Gly Asn Thr Asp Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <![CDATA[ <210> 15]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 15]]> Arg Gly Tyr Ala Asp Tyr Gly Tyr Thr Phe Asn Leu 1 5 10 <![CDATA[ <210> 16]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 16]]> Thr Ala Asp Thr Leu Ser Arg Asn Tyr Ala Ser 1 5 10 <![CDATA[ <210> 17]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 17]]> Arg Asp Thr Ser Arg Pro Ser 1 5 <![CDATA[ <210> 18]]> <![CDATA[ <211> 13]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 18]]> Ala Thr Gly Asp Gly Ser Gly Ser Ser Ser Tyr Gln Phe Val 1 5 10 <![CDATA[ <210> 19]]> <![CDATA[ <211> 5]] ><![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 19]]> Arg Tyr Trp Met Asn 1 5 <![CDATA[ <210> 20]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 20]]> Tyr Ile Thr Thr Asn Asp Lys Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <![CDATA[ <210> 21]]> <![CDATA[<21]]> 1> 8]]>br / > <![CDATA[ <212> PRT]]>br / > <![CDATA[ <213> Artificial sequence]]>br / >br / >br / > <![CDATA[ <220> ]]>br / > <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 21]]>br / > <![CDATA[Arg Ser Ser Gly Ala Tyr Asp Ile 1 5 <![CDATA[ <210> 22]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 22]]> Thr Leu Ser Ser Ala His Lys Thr Tyr Tyr Ile Glu 1 5 10 <![CDATA[<21]]> 0> 23]]>br / > <![CDATA[ <211> 11]]>br / > <![CDATA[ <212> PRT]]>br / > <![CDATA[ <213> Artificial sequence]]>br / >br / >br / > <![CDATA[ <220> ]]>br / > <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 23]]>br / > <![CDATA[Leu Lys Ser AspGly Thr Tyr Thr Lys Gly Thr 1 5 10 <![CDATA[ <210> 24]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 24]]> Gly Val Thr Gly Gly Asn Val Tyr Val 1 5 <![CDATA[ <210> 25]]> <![CDATA[ <211> 123]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 2]]>5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ile Asn Asp Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Arg Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Ile Tyr 65 70 75 80 Leu Gln Met Asn Leu Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Val Ser Pro Tyr Val Glu Leu Thr Ala Thr Ala Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <![CDATA[ <210> 26]]> <![CDATA[ <211> 123]]><![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic polypeptide]]> <![CDATA[<400> 26]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly His Thr Phe Gly Asn Trp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Gly Ala Ile Arg Trp Ser Val Gly Thr Thr Asn Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Thr Pro Phe Val Leu Ala Arg Ile Asn Gly Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <![CDATA[<210> 27]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic polypeptide]]> <![CDATA[<400> 27]]> Gln Ser Val Lys Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Tyr Asn Tyr Ala 20 25 30 Met Asn Trp Val Arg GlnAla Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Asn Thr Asp Gly Asn Thr Asn Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Thr Thr Ser Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Pro Arg Ala 85 90 95 Val Gly Tyr His His His Ala Leu Asp Pro Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[<210> 28]]> <![CDATA[<211> 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic polypeptide]]> <![CDATA[<400> 28]]> Glu Leu Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ala 1 5 10 15 Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Ala His Lys Thr Tyr Thr 20 25 30 Ile Asp Trp Tyr Gln Gln Gln Gln Gly Glu Ala Pro Arg Tyr Leu Met 35 40 45 Gln Leu Lys Ser Asp Gly Ser Tyr Thr Lys Gly Thr Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro 65 70 75 80 Ser Val Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Asp Tyr 85 90 95 Gly Gly Gly Tyr Val Phe Gly Gly GlyThr Gln Leu Thr Val Thr Gly 100 105 110 <![CDATA[ <210> 29]]> <![CDATA[ <211> 117]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 29]]> Gln Ser Val Lys Glu Ser Glu Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Thr Ile Gly Ser Tyr His 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Thr Asp Gly Asn Thr Asp Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Met Asp Leu Lys Met Thr 65 70 75 80 Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Arg Gly 85 90 95 Tyr Ala Asp Tyr Gly Tyr Thr Phe Asn Leu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Ile Ser Ser 115 <![CDATA[ <210> 30]]> <![CDATA[ <211> 111]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <400> 30]]> Glu Leu Val Leu Thr Gln Pro Ala Ser Val Gln Val Asn Leu Gly Gln 1 5 10 15 Thr Val Ser Leu Thr Cys Thr Ala Asp ThrLeu Ser Arg Asn Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Leu Ile Tyr 35 40 45 Arg Asp Thr Ser Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Gly Asp Gly Ser Gly Ser Ser 85 90 95 Tyr Gln Phe Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly 100 105 110 <![CDATA[<210> 31]]> <![CDATA[<211> 114]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic polypeptide]]> <![CDATA[<400> 31]]> Gln Ser Val Lys Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Tyr Trp 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Thr Thr Asn Asp Lys Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Tyr Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Arg 85 90 95 Ser SerGly Ala Tyr Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 100 105 110 Ser Ser <![CDATA[<210> 32]]> <![CDATA[<211> 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic polypeptide]]> <![CDATA[<400> 32]]> Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Thr Leu Gly Ala 1 5 10 15 Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Ala His Lys Thr Tyr Tyr 20 25 30 Ile Glu Trp Tyr Gln Gln Gln Gln Gly Glu Ala Pro Arg Tyr Leu Met 35 40 45 Gln Leu Lys Ser Asp Gly Thr Tyr Thr Lys Gly Thr Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Ser 65 70 75 80 Ser Val Gln Ala Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Thr Gly 85 90 95 Gly Asn Val Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly 100 105 110

Claims

1. An antigen-binding protein or a fragment thereof that binds to an antigenic determinant on complement C3, wherein the antigen-binding protein or fragment thereof can inhibit complement activation pathways, including the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP).

2. The antigen-binding protein or fragment thereof, as requested in claim 1, is capable of binding complement C3 and C3b.

3. The antigen-binding protein or fragment thereof of claim 1, which is capable of binding to the antigenic determinant on complement C3, wherein this binding prevents the formation of C3 convertase.

4. The antigen-binding protein or fragment thereof of any of claims 1 to 3 is capable of penetrating Bruch's membrane.

5. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 is capable of competing with one or more antigen-binding proteins, including M0122, M0123, M0124, M0228 and M0251.

6. An antigen-binding protein or fragment thereof, as claimed in any of claims 1 to 3, comprising a single-chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, a bifunctional antibody (diabody), a small antibody mimic, or a single-domain antibody, such as sdAb, sdFv, a nanobody, V-Nar, or VHH.

7. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, comprising a CDR-H3 having at least 80% sequence identity with the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:

21.

8. An antigen-binding protein or fragment thereof of any one of claims 1 to 3, comprising a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a CDR-H1 sequence selected from the group consisting of SEQ ID NO: 1, 4, 7, 13 and 19; a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2, 5, 8, 14 and 20; a CDR-H3 sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 15 and 21; and wherein the VL comprises a CDR-L1 sequence selected from the group consisting of SEQ ID NO: 10, 16 and 22; a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 11, 17 and 23; and a CDR-L3 sequence selected from the group consisting of SEQ ID NO: 12, 18 and 24.

9. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, wherein the VH has at least 80% sequence identity with the group consisting of SEQ ID NO:25, 26, 27, 29 and 31, and / or the VL has at least 80% sequence identity with the group consisting of SEQ ID NO:28, 30 and 32.

10. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, comprising VH and VL, wherein the VH comprises the CDR-H1 sequence of SEQ ID NO:7, the CDR-H2 sequence of SEQ ID NO:8, and the CDR-H3 sequence of SEQ ID NO:9; and wherein the VL comprises the CDR-L1 sequence of SEQ ID NO:10, the CDR-L2 sequence of SEQ ID NO:11, and the CDR-L3 sequence of SEQ ID NO:

12.

11. The antigen-binding protein or a fragment thereof as claimed in claim 10, wherein the VH contains the amino acid sequence of SEQ ID NO:27 and the VL contains the amino acid sequence of SEQ ID NO:

28.

12. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, comprising VH and VL, wherein the VH comprises the CDR-H1 sequence of SEQ ID NO:13, the CDR-H2 sequence of SEQ ID NO:14, and the CDR-H3 sequence of SEQ ID NO:15; and wherein the VL comprises the CDR-L1 sequence of SEQ ID NO:16, the CDR-L2 sequence of SEQ ID NO:17, and the CDR-L3 sequence of SEQ ID NO:

18.

13. The antigen-binding protein or a fragment thereof as claimed in claim 12, wherein the VH contains the amino acid sequence of SEQ ID NO:29 and the VL contains the amino acid sequence of SEQ ID NO:

30.

14. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, comprising VH and VL, wherein the VH comprises the CDR-H1 sequence of SEQ ID NO:19, the CDR-H2 sequence of SEQ ID NO:20, and the CDR-H3 sequence of SEQ ID NO:21; and wherein the VL comprises the CDR-L1 sequence of SEQ ID NO:22, the CDR-L2 sequence of SEQ ID NO:23, and the CDR-L3 sequence of SEQ ID NO:

24.

15. The antigen-binding protein or a fragment thereof as claimed in claim 14, wherein the VH contains the amino acid sequence of SEQ ID NO:31 and the VL contains the amino acid sequence of SEQ ID NO:

32.

16. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, comprising a VHH domain, wherein the VHH domain comprises the CDR-H1 sequence of SEQ ID NO:1, the CDR-H2 sequence of SEQ ID NO:2, and the CDR-H3 sequence of SEQ ID NO:

3.

17. The antigen-binding protein or a fragment thereof as claimed in claim 16, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO:

25.

18. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, comprising a VHH domain, wherein the VHH domain comprises the CDR-H1 sequence of SEQ ID NO:4, the CDR-H2 sequence of SEQ ID NO:5, and the CDR-H3 sequence of SEQ ID NO:

6.

19. The antigen-binding protein or a fragment thereof as claimed in claim 18, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO:

26.

20. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 has a binding affinity for C3 and C3b of at least about 10⁻⁸ M.

21. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 has a binding affinity for C3 and C3b of about 10⁻⁹ M to about 10⁻¹⁴ M.

22. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 has a binding affinity for C3 and C3b of about 10⁻¹⁰ M to about 10⁻¹² M.

23. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 has substantially equivalent binding affinity for C3 and C3b.

24. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, wherein the binding affinity for C3 is less than one-tenth of the binding affinity for C3b.

25. The antigen-binding protein or fragment thereof of any of claims 1 to 3 has a binding affinity of about 10⁻⁴ M or less for C3a, iC3b, C4, C4b, C5 and / or C5b.

26. The antigen-binding protein or fragment thereof of any of claims 1 to 3 has a weaker binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b than for C3 and C3b.

27. The antigen-binding protein or fragment thereof of any of claims 1 to 3 has no binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b.

28. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 is capable of inhibiting the activity of any of the following groups: CP, LP and AP complement pathways.

29. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 is capable of inhibiting the activity of the CP, LP and / or AP complement pathway by at least about 80%, at least about 85%, at least about 90% or at least about 95%.

30. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 is capable of substantially equivalently inhibiting the activity of the CP, LP and AP complement pathways.

31. The antigen-binding protein or fragment thereof of claim 30, wherein the inhibition of the CP, LP and AP complement pathways is at least about 80%, at least about 85%, at least about 90% or at least about 95%.

32. The antigen-binding protein or fragment thereof, as requested in item 28, wherein the activity of the CP, LP and AP complement pathways is determined by measuring the level of hemolysis of erythrocytes in the presence of the antigen-binding protein or fragment thereof and the level of hemolysis of erythrocytes in the absence of the antigen-binding protein or fragment thereof.

33. The antigen-binding protein or fragment thereof as claimed in claim 28, wherein the activity of the CP, LP and AP complement pathways is determined by measuring the formation of the membrane attack complex (MAC) in the presence of the antigen-binding protein or fragment thereof compared with the formation of the MAC in the absence of the antigen-binding protein or fragment thereof.

34. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 is capable of inhibiting the activity of C3 convertase by at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

35. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 is capable of inhibiting the C3 convertase amplification loop.

36. The antigen-binding protein or fragment thereof of any one of claims 1 to 3 is capable of inhibiting choroidal C3 activity.

37. The antigen-binding protein or fragment thereof of any of claims 1 to 3, having a molecular weight of about 60 kDa or less.

38. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, having a molecular weight of about 20 kDa to about 30 kDa.

39. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, having a molecular weight of about 10 kDa to about 20 kDa.

40. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, having a molecular weight of about 25 kDa.

41. The antigen-binding protein or fragment thereof of any one of claims 1 to 3, having a molecular weight of approximately 15 kDa.

42. An antigen-binding protein or fragment thereof, as claimed in any of claims 1 to 3, wherein the antigen-binding protein or fragment thereof is cross-reactive with cynomolgus C3.

43. A pharmaceutical composition comprising an antigen-binding protein or a fragment thereof as claimed in any one of claims 1 to 42 and a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of claim 43, comprising low viscosity.

45. The pharmaceutical composition of claim 44, wherein the viscosity is between about 1 cP and about 50 cP.

46. ​​The pharmaceutical composition of claim 44, wherein the viscosity is less than or equal to about 20 cP.

47. An isolated nucleic acid molecule encoding an antigen-binding protein or a fragment thereof as described in any one of claims 1 to 42.

48. A representation vector comprising a nucleic acid molecule as claimed in claim 47.

49. A host cell comprising an expression vector as claimed in claim 48.

50. A method for manufacturing an antigen-binding protein or a fragment thereof as claimed in any one of claims 1 to 42, comprising the steps of: (i) culturing a host cell of claim 49 under conditions that allow the expression of a protein as claimed in any one of claims 1 to 42; (ii) recovering the protein; and, as appropriate (iii) further purifying and / or modifying and / or formulating the protein.

51. Use of an antigen-binding protein or a fragment thereof as claimed in any one of claims 1 to 42, for the preparation of a pharmaceutical composition for treating complement C3-mediated diseases or conditions in an individual.

52. As claimed in claim 51, wherein the antigen-binding protein or a fragment thereof is administered via topical, subconjunctival, intravitreal, retroocular, and / or intraanterior chamber administration.

53. As requested in paragraphs 51 or 52, wherein the complement C3-mediated disease or condition is selected from the group consisting of: age-related macular degeneration, geographic atrophy, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, retrolental fibrosis, autoimmune uveitis, chorioretinitis, retinitis, rheumatoid arthritis, psoriasis, and atherosclerosis.

54. Use of an antigen-binding protein or fragment thereof as claimed in any one of claims 1 to 42, for the preparation of a pharmaceutical composition for treating complement C3-mediated diseases or conditions in an individual by inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP) or by inhibiting the activity of complement C3 locally in the choroid.

55. Use of an antigen-binding protein or a fragment thereof as claimed in any one of claims 1 to 42, for the preparation of a pharmaceutical composition for inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP).

56. Use of an antigen-binding protein or a fragment thereof as claimed in any one of claims 1 to 42, for the preparation of a pharmaceutical composition for inhibiting the activity of local complement C3 in the choroid, wherein the pharmaceutical composition is for intraocular administration.

57. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof is capable of binding complement C3 and C3b.

58. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof is capable of binding to an antigenic determinant on complement C3, wherein such binding prevents the formation of C3 convertase.

59. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof is capable of competing with one or more antigen-binding proteins, including M0122, M0123, M0124, M0228 and M0251.

60. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a single-chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, a bifunctional antibody, a small antibody mimic, or a single-domain antibody such as sdAb, sdFv, a nano-antibody, V-Nar, or VHH.

61. As claimed in claim 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a CDR-H3 having at least 80% sequence identity with the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:

21.

62. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a CDR-H1 sequence selected from the group consisting of SEQ ID NO: 1, 4, 7, 13 and 19; a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2, 5, 8, 14 and 20; a CDR-H3 sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 15 and 21; and wherein the VL comprises a CDR-L1 sequence selected from the group consisting of SEQ ID NO: 10, 16 and 22; a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 11, 17 and 23; and a CDR-L3 sequence selected from the group consisting of SEQ ID NO: 12, 18 and 24.

63. As used in claim 62, wherein the VH has at least 80% identity with the sequence of the group consisting of SEQ ID NO:25, 26, 27, 29 and 31, and / or the VL has at least 80% identity with the sequence of the group consisting of SEQ ID NO:28, 30 and 32.

64. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof is capable of permeating a Bruch membrane.

65. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof is capable of inhibiting choroidal C3 activity.

66. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a molecular weight of about 60 kDa or less.

67. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a molecular weight of about 20 kDa to about 30 kDa.

68. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a molecular weight of about 10 kDa to about 20 kDa.

69. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a molecular weight of about 25 kDa.

70. As claimed in claims 55 or 56, wherein the antigen-binding protein or a fragment thereof comprises a molecular weight of about 15 kDa.

71. A method for detecting one or both of C3 and C3b in a biological sample, comprising the steps of: (a) contacting the sample with at least one antigen-binding protein or fragment thereof as claimed in any one of claims 1 to 42; (b) allowing one or both of C3 and C3b in the sample to form a complex with the antigen-binding protein or fragment thereof; and (c) detecting the antigen-binding protein or fragment thereof.

72. The method of claim 71, wherein the antigen-binding protein or a fragment thereof is capable of binding complement C3 and C3b.

73. The method of claim 71 or 72, wherein the antigen-binding protein or a fragment thereof is detected by a detectable signal.

74. The method of claim 71 or 72, wherein the biological sample is a tissue sample, such as retinal tissue.

75. A kit for detecting C3, comprising an antigen-binding protein or a fragment thereof as claimed in any one of claims 1 to 42 and instructions for use.

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