Complement C3 antigen-binding protein

Abstract

An antigen-binding protein having specificity for complement C3 is provided. Antigen binding proteins having specificity for complement C3 and C3b are provided. Methods for treating complement C3-mediated diseases and disorders, methods for inhibiting the activity of the classical (CP), lectin (LP), and / or alternative (AP) complement pathways, and methods for inhibiting the activity of choroidally-localized complement C3 are also provided.

Description

[Technical field]

[0001] The present disclosure relates to antigen binding proteins that target complement C3 and methods of treating complement C3-mediated diseases. [Background technology]

[0002] A major challenge in the treatment of certain ocular diseases and disorders is the delivery of therapeutic molecules to the deep layers of the retina. Delivery is hindered by multiple factors, including multiple physical 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 the capillary endothelial cells (inner BRB) and the retinal pigment epithelial cells (see, e.g., Jiang et al. Int J Ophthalmol. 2018; 11(6): 1038-1044). Ocular diseases in which delivery to the retina is particularly important are complement-mediated diseases, such as geographic atrophy (GA). Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD) characterized by loss of retinal pigment epithelium and photoreceptors in the macula. When GA affects the fovea, irreversible vision loss occurs. Patients with early stages of GA typically experience visual impairment even before vision is affected. The pathophysiology underlying geographic atrophy is not fully understood, but dysregulation of complement activity is thought to be a contributing factor. Several complement activation products, including C3a, C5a, C5b-9, and complement factor H (CFH), have shown elevated levels in vitreous samples, Bruch's membrane, and other parts of the choroid of GA patients compared to controls. In addition, complement inhibitors such as CD59, a membrane-bound inhibitor of membrane attack complex (MAC) formation, and membrane cofactor protein (MCP), a membrane-bound complement regulator with cofactor activity for complement factor I (CFI), have been reported to have decreased levels in GA.

[0003] There are currently no approved treatments for GA. Several investigational approaches targeting the complement pathway are being investigated but are not yet 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). More recently, findings from a phase II clinical trial of APL-2 (clinical trial NCT0250332 "Study of APL-2 Therapy in Patients with Geographic Atrophy (FILLY)") further implicate the complement pathway in the pathogenesis of GA, showing a positive treatment effect in suppressing the progression of GA via complement inhibition. These results also suggest that central APL-2 inhibition of the complement cascade at C3 (the convergence of all complement pathways; see Figure 1) may have the potential to treat GA more effectively than is possible with inhibitors that result in partial inhibition of the complement pathway. Nevertheless, the reduction in lesion growth in GA obtained with APL-2 remains modest. APL-2 has characteristics that may limit its efficacy. APL-2, a pegylated derivative of the cyclic 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. APL-2 has an effective period of only 1 month, probably due to its low concentration of 3.5 mM. APL-2 is also a PEGylated molecule, which may increase its viscosity and make it difficult to inject into the eye. Therefore, there is a need to more efficiently reduce GA progression.

[0004] One of the major challenges in the treatment of GA is that the observed dysregulation of complement activity occurs in the deep layers of the retina. We hypothesize that to achieve greater reduction in lesion growth in GA, better penetration into disease-related retinal tissues (i.e., retinal pigment epithelium (RPE), Bruch's membrane, and other parts of the choroid) is necessary. For this reason, small antibody fragments have several advantages compared to other biologics and antibodies. Small antibody formats may allow 1) better intraocular penetration into relevant retinal tissues; and 2) more drug per mg or mL delivered by intravitreal injection. Summary of the Invention

[0005] The present disclosure provides antigen binding proteins having specificity for complement C3. In one aspect, the present disclosure provides an antigen binding protein or fragment thereof that binds to an epitope on complement C3 and is capable of inhibiting pathways of complement activation, including the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). In certain embodiments, the antigen binding protein or fragment thereof is capable of binding to complement C3 and C3b. In certain embodiments, the antigen binding protein or fragment thereof is capable of binding to an epitope on complement C3, such binding preventing the formation of C3 convertase.

[0006] In certain embodiments, the antigen binding protein or fragment thereof can compete with one or more antigen binding proteins including: M0122, M0123, M0124, M0228, and M0251. In certain embodiments, the antigen binding protein or fragment thereof comprises a single chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, a diabody, a small antibody mimetic or a single domain antibody, such as an sdAb, an sdFv, a nanobody, a V-Nar or a VHH. In a preferred embodiment, the antigen binding protein or fragment thereof comprises an scFv or a VHH. In certain embodiments, the antigen binding protein or fragment thereof comprises a CDR-H3 having at least 80% similarity to a sequence of 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. In certain embodiments, the antigen binding protein or fragment thereof comprises a CDR-H3 having at least 80% identity to a sequence of 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.

[0007] In certain embodiments, the antigen binding protein or 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 NOs: 1, 4, 7, 13, and 19, a CDR-H2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 14, and 20, and a CDR-H3 sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 15, and 21; and the VL comprises a CDR-L1 sequence selected from the group consisting of SEQ ID NOs: 10, 16, and 22, a CDR-L2 sequence selected from the group consisting of SEQ ID NOs: 11, 17, and 23, and a CDR-L3 sequence selected from the group consisting of SEQ ID NOs: 12, 18, and 24.

[0008] In certain embodiments, the VH has at least 80% similarity to a sequence in the group consisting of SEQ ID NOs: 25, 26, 27, 29, and 31, and / or the VL has at least 80% similarity to a sequence in the group consisting of SEQ ID NOs: 28, 30, and 32. In certain embodiments, the VH has at least 80% identity to a sequence in the group consisting of SEQ ID NOs: 25, 26, 27, 29, and 31, and / or the VL has at least 80% similarity to a sequence in the group consisting of SEQ ID NOs: 28, 30, and 32. In certain embodiments, the antigen binding protein or fragment thereof comprises a VH and a VL, wherein the VH comprises a CDR-H1 sequence of SEQ ID NO:7, a CDR-H2 sequence of SEQ ID NO:8, and a CDR-H3 sequence of SEQ ID NO:9; and the VL comprises a CDR-L1 sequence of SEQ ID NO:10, a CDR-L2 sequence of SEQ ID NO:11, and a CDR-L3 sequence of SEQ ID NO:12. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:27, and the VL comprises the amino acid sequence of SEQ ID NO:28.

[0009] In certain embodiments, the antigen binding protein or fragment thereof comprises a VH and a VL, wherein the VH comprises a CDR-H1 sequence of SEQ ID NO: 13, a CDR-H2 sequence of SEQ ID NO: 14, and a CDR-H3 sequence of SEQ ID NO: 15; and the VL comprises a CDR-L1 sequence of SEQ ID NO: 16, a CDR-L2 sequence of SEQ ID NO: 17, and a CDR-L3 sequence of SEQ ID NO: 18. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:29, and the VL comprises the amino acid sequence of SEQ ID NO:30. In certain embodiments, the antigen binding protein or fragment thereof comprises a VH and a VL, wherein the VH comprises a CDR-H1 sequence of SEQ ID NO: 19, a CDR-H2 sequence of SEQ ID NO: 20, and a CDR-H3 sequence of SEQ ID NO: 21; and the VL comprises a CDR-L1 sequence of SEQ ID NO: 22, a CDR-L2 sequence of SEQ ID NO: 23, and a CDR-L3 sequence of SEQ ID NO: 24.

[0010] In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:31, and the VL comprises the amino acid sequence of SEQ ID NO:32. In certain embodiments, the antigen binding protein or fragment thereof comprises a VHH domain, wherein the VHH domain comprises a CDR-H1 sequence of SEQ ID NO:1, a CDR-H2 sequence of SEQ ID NO:2, and a CDR-H3 sequence of SEQ ID NO:3. In certain embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO:25. In certain embodiments, the antigen binding protein or fragment thereof comprises a VHH domain, wherein the VHH domain comprises a CDR-H1 sequence of SEQ ID NO:4, a CDR-H2 sequence of SEQ ID NO:5, and a CDR-H3 sequence of SEQ ID NO:6. In certain embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO:26.

[0011] In certain embodiments, the antigen binding protein or fragment thereof has an affinity for at least about 10 -8In certain embodiments, the antigen binding protein or fragment thereof has a binding affinity of about 10 to C3 and C3b. -9 M~about 10 -14 In certain embodiments, the antigen binding protein or fragment thereof has a binding affinity of about 10 to C3 and C3b. -10 M~about 10 -12 In certain embodiments, the antigen binding protein or fragment thereof comprises approximately equal binding affinity for C3 and C3b. In certain embodiments, the binding affinity for C3 is within 10-fold of the binding affinity for C3b. In certain embodiments, the antigen binding protein or fragment thereof has an affinity of about 10 to C3a, iC3b, C4, C4b, C5, and / or C5b. -4 In certain embodiments, the antigen binding protein or fragment thereof comprises a binding affinity of M or weaker for C3a, iC3b, C4, C4b, C5, and / or C5b compared to its binding affinity for C3 and C3b. In certain embodiments, the antigen binding protein or fragment thereof does not comprise binding affinity for C3a, iC3b, C4, C4b, C5, and / or C5b.

[0012] In certain embodiments, the antigen binding protein or fragment thereof can inhibit 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%. In certain embodiments, the antigen binding protein or fragment thereof is capable of inhibiting the activity of the CP, LP, and AP complement pathways equally or nearly equally, hi certain embodiments, inhibition of the activity 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%. In certain embodiments, CP, LP, and AP complement pathway activity is determined by measuring the level of red blood cell hemolysis in the presence of the antigen binding protein or fragment thereof compared to the level of red blood cell hemolysis in the absence of the antigen binding protein or fragment thereof. In certain embodiments, CP, LP, and AP complement pathway activity is determined by measuring membrane attack complex (MAC) formation in the presence of an antigen binding protein or fragment thereof compared to MAC formation in the absence of the antigen binding protein or fragment thereof.

[0013] In certain embodiments, the antigen binding protein or 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%. In certain embodiments, the antigen binding protein or fragment thereof is capable of inhibiting the C3 convertase amplification loop. In certain embodiments, the antigen binding protein or fragment thereof is capable of penetrating Bruch's membrane. In certain embodiments, the antigen binding protein or fragment thereof is capable of inhibiting choroidal C3 activity.

[0014] In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 60 kDa or less. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 20 kDa to about 30 kDa. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 10 kDa to about 20 kDa. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 25 kDa. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 15 kDa. In certain embodiments, the antigen binding protein or fragment thereof comprises cross-reactivity with cynomolgus C3. In one aspect, the present disclosure provides a pharmaceutical composition comprising the antigen binding protein or fragment thereof described above and a pharma- ceutically acceptable carrier.Accordingly, one aspect is the use of the binding protein of the present invention in the preparation of a pharmaceutical composition for treating a complement C3-mediated disease or disorder in a subject. In certain embodiments, the pharmaceutical composition comprises a low viscosity. In certain embodiments, the viscosity is from about 1 cP to about 50 cP, hi certain embodiments, the viscosity is less than or equal to about 20 cP.

[0015] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding the above-mentioned antigen binding protein or fragment thereof. In another aspect, the disclosure provides an expression vector comprising the above-described nucleic acid molecule. In yet another aspect, the disclosure provides a host cell comprising the above-described expression vector. In yet another aspect, there is provided a method for producing an antigen-binding protein or fragment thereof as described above, comprising the steps of: i) culturing said host cells under conditions allowing expression of the proteins described herein; ii) recovering the protein, and optionally iii) further purifying and / or modifying and / or formulating the protein; A method is provided that includes:

[0016] In one aspect, the present disclosure provides a method of treating a complement C3-mediated disease or disorder in a subject, comprising administering to a subject in need thereof an antigen binding protein or fragment thereof as described above. Accordingly, the present disclosure also provides an antigen binding protein or fragment thereof as described above for use in a method of treating a complement C3-mediated disease or disorder. In certain embodiments, the antigen binding protein or fragment thereof is administered via topical, subconjunctival, intravitreal, retrobulbar, and / or intracameral administration. In certain embodiments, the complement C3 mediated disease or disorder 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.

[0017] In one aspect, the present disclosure provides a method of inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative complement pathway (AP), comprising contacting complement C3 with an antigen binding protein or a fragment thereof that binds to an epitope on complement C3. Thus, the present disclosure provides an antigen binding protein or a fragment thereof as described herein for use in a method of treating a complement C3-mediated disease or disorder by inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative complement pathway (AP). The present disclosure also provides an antigen binding protein or a fragment thereof as described above for use in a method of treating a complement C3-mediated disease or disorder by inhibiting the activity of choroidally localized complement C3. In one aspect, the disclosure provides a method of inhibiting the activity of choroid-localized complement C3, comprising intraocular administration of an antigen binding protein or fragment thereof that binds to an epitope on complement C3.

[0018] In certain embodiments of the methods described herein, the antigen binding protein or fragment thereof is capable of binding to complement C3 and C3b. In certain embodiments, the antigen binding protein or fragment thereof is capable of binding to an epitope on complement C3, such binding preventing the formation of C3 convertase. In certain embodiments, the antigen binding protein or fragment thereof can compete with one or more antigen binding proteins including: M0122, M0123, M0124, M0228, and M0251. In certain embodiments, the antigen binding protein or fragment thereof comprises a single chain variable fragment (scFv), a Fab fragment, or a VHH. In certain embodiments, the antigen binding protein or fragment thereof comprises a CDR-H3 having at least 80% similarity to a sequence of 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. In certain embodiments, the antigen binding protein or fragment thereof comprises a CDR-H3 having at least 80% identity to a sequence of 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.

[0019] In certain embodiments, the antigen binding protein or 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 NOs: 1, 4, 7, 13, and 19, a CDR-H2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 14, and 20, and a CDR-H3 sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 15, and 21; a CDR-L1 sequence selected from the group consisting of SEQ ID NOs: 10, 16, and 22, a CDR-L2 sequence selected from the group consisting of SEQ ID NOs: 11, 17, and 23, and a CDR-L3 sequence selected from the group consisting of SEQ ID NOs: 12, 18, and 24. In certain embodiments, the VH has at least 80% similarity to a sequence in the group consisting of SEQ ID NOs: 25, 26, 27, 29, and 31, and / or the VL has at least 80% similarity to a sequence in the group consisting of SEQ ID NOs: 28, 30, and 32. In certain embodiments, the VH has at least 80% identity to a sequence in the group consisting of SEQ ID NOs: 25, 26, 27, 29, and 31, and / or the VL has at least 80% identity to a sequence in the group consisting of SEQ ID NOs: 28, 30, and 32.

[0020] In certain embodiments, the antigen binding protein or fragment thereof is capable of penetrating Bruch's membrane. In certain embodiments, the antigen binding protein or fragment thereof is capable of inhibiting choroidal C3 activity. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 60 kDa or less, e.g., 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, about 15 kDa or less. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 20 kDa to about 30 kDa. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 10 kDa to about 20 kDa. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 25 kDa. In certain embodiments, the antigen binding protein or fragment thereof comprises a molecular weight of about 15 kDa.

[0021] In one aspect, the disclosure provides a method for detecting one or both of C3 and C3b in a biological sample, comprising: (a) contacting a sample with at least one antigen binding protein or fragment thereof as described above; (b) allowing the formation of a complex between one or both of C3 and C3b in the sample and an antigen binding protein or fragment thereof; (c) detecting the antigen-binding protein or a fragment thereof; In a preferred embodiment, the antigen binding protein or fragment thereof is capable of binding to complement C3 and C3b. In one embodiment, the antigen binding protein or fragment thereof is detected by a detectable signal. In one embodiment, the antigen binding protein or fragment thereof is detected by ELISA, immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and / or flow cytometry.

[0022] The biological sample may be a tissue sample, for example retinal tissue of a human subject, such as a fixed tissue sample. The fixed tissue sample may be a formalin-fixed and paraffin-embedded tissue sample. In one aspect, a kit for detecting C3 is provided, comprising an antigen binding protein or fragment thereof as described above and instructions for use. The above and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings, in which: Copies of this patent or patent application publication and color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows three complement pathways that converge on C3: the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP). [Diagram 2] FIG. 1 shows the process of generating an anti-C3 antibody library. [Figure 3A-3B] Figure 3 shows ELISA assays confirming a good immune response to C3 in rabbits and llamas. Figure 3A shows an ELISA assay testing C3 protein isolated from human plasma. Figure 3B shows an ELISA assay testing the presence of anti-C3 antibodies in the serum of rabbits (top panel) and llamas (bottom panel) injected with the isolated human C3 shown in Figure 3A. [Figure 4] FIG. 4A shows a summary of the anti-C3 antibody library, and FIG. 4B shows the diversity of CDR-H3 amino acid lengths. [Diagram 5] FIG. 1 shows the screening process of anti-C3 antibodies. [Figure 6] Figure 1 shows the screening of candidate antibodies targeting C3 for their ability to inhibit all three complement pathways in human serum. Each antibody was used at a concentration of 2 μM. [Figure 7A-7D]Figure 7 shows that the four anti-C3 antibodies that inhibit all three complement pathways recognize three different epitopes on C3. Figure 7A shows a competition assay showing that there is no competition between M0122 and each of the other three anti-C3 antibodies. Figure 7B shows a competition assay showing that there is no competition between M0124 and each of the other three anti-C3 antibodies. Figure 7C shows a competition assay showing that there is competition between M0228 and M0251, but not between M0124 and M0122. Figure 7D shows a competition assay showing that there is competition between M0123 and M0251, and M0123 and M0228, but not between M0124 and M0122. [Figure 8A-8B] Figure 8A shows an ELISA assay demonstrating that M0122, M0124 and M0228 bind directly to both C3 and C3b. Figure 8B shows an ELISA assay demonstrating that M0122, M0124 and M0228 bind directly to C3b. [Figure 9A-9B] Figure 9 shows that M0122, M0124 and M0228 potently inhibit the classical and alternative pathways. Figure 9A shows that M0122, M0124 and M0228 potently inhibit the classical pathway. Figure 9B shows that M0122, M0124 and M0228 potently inhibit the alternative pathway. [Figure 10] FIG. 1 shows affinity parameters of M0122, M0124 and M0228. [Figure 11] Schematic diagram of the anatomical structure of the retina and choroid, e.g., Bruch's membrane, is shown. The anti-C3 scFv antibody of the present disclosure is shown to be able to penetrate Bruch's membrane and enter deep into the choroid, while the comparative C3 binding therapeutic APL-2 is shown to be unable to penetrate Bruch's membrane. The same principle applies to other antigen binding protein formats of the present invention. [Figure 12] FIG. 1 shows the negative relationship between hydrodynamic radius and permeability and complement fixation therapy shown in comparison to scFvs of the present disclosure. [Figure 13]Figures 13A and 14B show a comparison of scFv and APL-2 surrogates for penetrating Bruch's membrane. Figure 14A shows barium iodide staining (PEG) and Figure 13B shows Coomassie staining (protein). The APL2-surrogate contains one APL-1 moiety on a 40 kDa linear PEG. SC - sample chamber, DC - diffusion chamber, LC - loading control (initial concentration of SC). [Figures 14A-14C] Figure 14 shows that M0122, M0124 and M0251 potently inhibit the classical pathway, the alternative pathway and the lectin pathway in cyanoserum. Figure 14A shows that M0122, M0124 and M0251 potently inhibit all three pathways. Each antibody was used at a concentration of 2 μM. Figure 14B shows that M0122, M0124 and M0251 potently inhibit the classical pathway. Figure 14C shows that M0122, M0124 and M0251 potently inhibit the alternative pathway. [Figure 15A-15B] Figure 15 shows that M0122, M0124, and M0251 bind to cyno C3. Figure 15A shows M0122 and M0124. Figure 15B shows M0251. [Figure 16] FIG. 1 shows that M0122, M0123 and M0124 potently inhibit the lectin pathway. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Antigen binding proteins having binding specificity for complement C3 and the complement C3 cleavage product, C3b, are provided. Methods of treating or preventing complement C3-mediated diseases and disorders are also provided. In certain aspects, the antigen binding proteins described herein can inhibit the classical complement pathway (CP), the lectin pathway (LP), and the alternative complement 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.

[0025] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclature used in connection with analytical chemistry, organic synthetic chemistry, and medicinal chemistry described herein, as well as the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and patient treatment.

[0026] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or foreign definitions. Unless otherwise required by context, singular words include plurals and plural words include the singular. The use of "or" means "and / or" unless otherwise specified. The use of the term "including" and other forms such as "includes" and "included" are non-limiting. In order that the present invention may be more readily understood, certain terms are first defined.

[0027] Antigen-binding proteins As used herein, the term "antibody" or "antigen-binding protein" refers to an immunoglobulin molecule that specifically binds or is immunologically reactive with an antigen or epitope, including both polyclonal and monoclonal antibodies, as well as functional antibody fragments, such as, 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, nanobodies, VHH) fragments. The term "antibody" includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv). Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term "antibody fragment" as used herein includes artificial proteins designed to selectively bind antigens, i.e., antibody mimics. Typically, one or more CDRs are grafted onto a non-Ig scaffold, thereby mimicking the CDR conformation from the parent antibody. Non-limiting examples of such antibody mimics include wobble-regulated affinity proteins (FLAPs), monobodies, and affimers. Antibody mimics may contain one, two, three, four, five, or six of the CDRs, as described herein.

[0028] As used herein, a "Fab fragment" is an antibody fragment that contains the variable light (VL) domain and the constant domain of the light chain (CL), and the light chain fragment that contains the variable heavy (VH) domain and the first constant domain (CH1) of the heavy chain. Fab fragments generally have a molecular weight of about 50 kDa and a hydrodynamic radius of about 3.0 nm. 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 art-recognized linker. Such linkers include, but are not limited to, repeating GGGGS amino acid sequences or variants thereof. Although scFvs generally do not contain antibody constant domain regions, the scFvs of the present disclosure can be linked or conjugated 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 generally have a molecular weight of about 25 kDa and a hydrodynamic radius of about 2.5 nm. As used herein, a "VHH", "nanobody" or "heavy chain only antibody" is an antigen binding protein containing a single heavy chain variable domain derived from a species of the Camelidae family, including camel, llama and alpaca. VHHs generally have a molecular weight of about 15 kDa.

[0029] As used herein, the term "complementarity determining region" or "CDR" refers to a non-contiguous sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" or "FR" is known in the art to refer to the non-CDR portion of the variable regions of heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each light chain variable region. For VHH antibodies, there are only three heavy chain CDRs and no light chain CDRs.

[0030] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined by any of several well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. 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, 2003 January; 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, 2001 June. 8; 309(3):657-70 (AHo numbering scheme).

[0031] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions accommodated by the insertion letter, e.g., "30a", and deletions that appear in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0032] Variants of the antibodies provided herein can be generated by introducing deletions, substitutions, additions, and / or modifications into the framework and / or CDRs. The antibody variants can then be tested for the desired functionality using the methods described herein. Any combination(s) of deletions, substitutions, additions, modifications, and insertions can be made to the antigen-binding protein or fragment thereof, provided that the generated variants have the desired characteristics, which can be screened using appropriate methods.

[0033] As used herein, "conservative substitution" refers to a modification that maintains the functional properties of a parent antibody. For example, conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having similar properties. For example, alanine (A) is replaced with valine (V); arginine (R) is replaced with lysine (K); asparagine (N) is replaced with glutamine (Q); aspartic acid (D) is replaced with glutamic acid (E); cysteine ​​(C) is replaced with serine (S); glutamic acid (E) is replaced with aspartic acid (D); glycine (G) is replaced with alanine (A); histidine (H) is replaced with arginine (R) or lysine (K); leucine (L) for isoleucine (I); leucine (L) for methionine (M); tyrosine (Y) for phenylalanine (F); threonine (T) for serine (S); tyrosine (Y) for tryptophan (W); tryptophan (W) for phenylalanine (F); and / or leucine (L) for valine (V), and vice versa.

[0034] Thus, unless otherwise specified, a given antibody or region thereof, such as a "CDR" or "complementary determining region" of its variable region, or each particular CDR (e.g., "CDR-H1, CDR-H2"), should be understood to encompass a (or particular) complementary determining region, as defined by any of the known schemes. Similarly, unless otherwise specified, a given antibody or region thereof, such as a "FR" or "framework region" of its variable region, or each particular FR (e.g., "FR-H1", "FR-H2"), should be understood to encompass a (or particular) framework region, as defined by any of the known schemes. In some cases, CDRs are identified as defined by a scheme for identifying specific CDRs or FRs, such as the Kabat, Chothia, Contact, IMGT, or AHo method. In other cases, the specific amino acid sequence of the CDR or FR is given.The numbering of CDRs and FRs is based on Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. 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, 2001 Jun. 8; 309(3):657-70 (AHo numbering scheme).

[0035] The terms "compete" or "cross-compete" are used interchangeably herein to mean the ability of an antibody molecule, such as an antigen-binding protein described herein, to interfere with the binding of the antibody molecule to a target, such as human C3 and / or C3b. The interference of binding can be direct or indirect (e.g., via allosteric regulation of the antigen-binding molecule or the target). 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 said to compete, can be determined using a competitive binding assay, such as a FACS assay, an ELISA, or a BIACORE assay. In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, a first antigen-binding molecule is said to compete with a second antigen-binding molecule for binding to a target if binding of the first antibody molecule to the target is reduced by 10% or more, e.g., 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 in a competitive binding assay (e.g., a competitive assay described herein).

[0036] As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antibody and the epitope it binds. As will be readily understood by those of skill in the art, antibody or antigen-binding protein affinity is measured as a function of the molar (M) dissociation constant (K D The antibodies of the present disclosure can be reported as 10 -5 ~10 -12 K in the M range D A high affinity antibody can have a value of 10 -9 K below M (1 nanomolar, nM) D For example, a high affinity antibody has a K value in the range of about 1 nM to about 0.01 nM. D High affinity antibodies can have a K value of 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. D Very high affinity antibodies can have a value of 10 -12K below M (1 picomole, pM) D Weak or low affinity antibodies have a value of 10 -1 ~10 -4 K in the M range D Low affinity antibodies can have a value of 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 10 such as M -4 K over M D It can have a value.

[0037] In certain embodiments, the antigen binding proteins of the present disclosure have an affinity for C3 and C3b of about 10 -8 M~about 10 -14 In certain embodiments, the antigen binding proteins of the present disclosure have a binding affinity of about 10 to C3 and C3b. -10 M~about 10 -12 In certain embodiments, the antigen binding proteins of the disclosure have a binding affinity of at least about 10 for C3 and C3b. -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 It has a binding affinity of M. In certain embodiments, the antigen binding protein or fragment thereof comprises approximately equal binding affinity for C3 and C3b. For example, but not limited to, the antigen binding protein or fragment thereof comprises approximately equal binding affinity for C3b. -10 The binding affinity of M for C3b is approximately 10 -10 In certain embodiments, the antigen binding protein or fragment thereof has a binding affinity of about 10 to C3. -11 The binding affinity of M for C3b is approximately 10 -11 In certain embodiments, the antigen binding protein or fragment thereof has a binding affinity of about 10 to C3. -12 The binding affinity of M for C3b is approximately 10 -12 The binding affinity of M In certain embodiments, the binding affinity for C3 is within 10-fold of the binding affinity for C3b. For example, and without limitation, the antigen binding protein or fragment thereof has a binding affinity for C3 of about 10 -10 The binding affinity of M for C3b is approximately 10 -11 In certain embodiments, the antigen binding protein or fragment thereof has a binding affinity of about 10 to C3. -11 Binding affinity of M and approximately 10 for C3b -12 The binding affinity of M

[0038] In certain embodiments, the antigen binding protein or fragment thereof comprises cross-reactivity with cynomolgus monkey C3. Cynomolgus monkey (Macaca fascicularis) C3 is 95.1% identical to human C3, and the cross-reactivity allows for preclinical and toxicity testing of the antigen binding proteins of the present disclosure in relevant animal models. For the avoidance of doubt, and unless otherwise indicated, C3 as used herein refers to the human form of complement component 3, 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 cleavage of C3. In certain embodiments, the antigen binding proteins of the disclosure are monovalent and have a K of about 200 nM or less as measured using biolayer interferometry (BLI). D In certain embodiments, K binds to human C3 and C3b. D is about 200 pM or less, for example, about 100 pM, about 10 pM, about 1 pM, or about 0.1 pM.

[0039] The ability of an antigen-binding domain to bind to a specific antigenic determinant can be measured via either enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0040] Anti-complement C3 antigen-binding protein In one aspect, the present disclosure provides antigen binding proteins having binding specificity for complement C3 protein. In certain embodiments, the anti-C3 antigen binding proteins are scFvs, Fab fragments, or VHHs. Exemplary anti-C3 antigen binding protein CDRs are listed in Table 1 below. Exemplary anti-C3 antigen binding protein variable heavy and light domains are listed in Table 2 below. The exemplary anti-C3 antigen binding proteins listed below were generated by immunizing rabbits and llamas with human C3 protein isolated from human plasma. The exemplary VH and VL domains of M0122, M0123, and M0124 were derived from rabbits immunized with human C3 protein and are wild-type rabbit sequences. The exemplary VHH domains of M0228 and M0251 were derived from llamas immunized with human C3 protein and are wild-type llama sequences.

[0041] [Table 1]

[0042] [Table 2]

[0043] In certain embodiments, an anti-C3 antigen binding protein of the disclosure comprises 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 to any of the sequences in Table 1 or Table 2.

[0044] In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure are selected for their ability to inhibit one or more of the complement pathways: classical, alternative, and lectin. In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure are selected for their ability to inhibit all three complement pathways: classical, alternative, and lectin. In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure can inhibit all three complement pathways in the eye. In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure can inhibit all three complement pathways in the choroid region of the eye. The choroid region is a layer containing blood vessels that lines the back of the eye and is located between the retina and the sclera. The choroid region is divided into four layers: Haller's layer, Sattler's layer, choriocapillaris, and Bruch's membrane. Bruch's membrane, also known as the vitreous plate, is the innermost layer of the choroid and is adjacent to the retinal pigment epithelium (RPE). In certain embodiments, anti-C3 antigen binding proteins of the present disclosure can penetrate or diffuse through Bruch's membrane and enter other layers of the choroid, including, but not limited to, the choriocapillaris.

[0045] The retina has substantial physical barriers that can prevent large molecules, such as full-length immunoglobulins, from penetrating deeper layers, which can result in reduced therapeutic efficacy (Jackson et al. Invest Ophthalmol Vis Sci. 2003;44(5):2141-6). Smaller antibody derivatives, in contrast, can penetrate deeper into the retina. Exemplary antibody derivatives having a molecular weight of about 60 kDa or lower are antibody fragments, including, but not limited to, Fab, Fab' fragments, scFab, scFv, Fv fragments, nanobodies, VHH, dAb, V-Nar, sdAb, sdFv, and bispecific and bivalent antibodies, such as single chain diabodies (scDbs), or DARTs. In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure have a molecular weight of about 60 kDa or less, e.g., 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.

[0046] In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure are able to penetrate or diffuse through Bruch's membrane, in part because their size is low enough to facilitate penetration. In certain embodiments, the size of the antigen binding proteins of the present disclosure is measured by molecular weight. In certain embodiments, the antigen binding proteins of the present disclosure have a molecular weight of less than about 60 kDa. In certain embodiments, the antigen binding proteins of the present disclosure are between about 20 kDa and about 30 kDa or between about 10 kDa and about 20 kDa. In certain embodiments, the antigen binding proteins of the present disclosure are about 25 kDa. In certain embodiments, the antigen binding proteins of the present disclosure are about 15 kDa. In certain embodiments, the size of the antigen binding proteins of the present disclosure are measured by their hydrodynamic radius. In certain embodiments, the antigen binding proteins of the present disclosure have a hydrodynamic radius of less than or equal to about 3.0 nm. In certain embodiments, the antigen binding proteins of the present disclosure have a hydrodynamic radius of less than or equal to about 2.5 nm. In certain embodiments, an antigen binding protein of the disclosure has a hydrodynamic radius of less than or equal to about 2.0 nm.

[0047] In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure can compete with one or more antigen binding proteins, including M0122, M0123, M0124, M0228, and M0251. Antibody competition can be measured by any assay known in the art. In certain embodiments, one antibody can be labeled with a marker, such as biotin, and incubated with other anti-C3 antibodies in a C3-binding ELISA. Typically, when a competing antigen binding protein is present in excess, it reduces the specific binding of the antigen binding protein or fragment thereof to C3 and / or C3b, as described herein, i.e., cross-blocks the binding by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In certain embodiments, binding of an antigen binding protein or fragment thereof described herein in the presence of a competing antigen binding protein is reduced by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0048] Complement C3 is a large protein composed of 13 distinct domains and with a molecular size of 185 kilodaltons. During complement activation, C3 undergoes proteolytic cleavage and structural modifications at different sites. C3-derived fragments form convertases that exert different effector functions and enter amplification loops into the three complement pathways. The classical and lectin pathway C3 convertase, C4bC2a, cleaves full-length C3 into C3b and the anaphylatoxin C3a. The alternative pathway also generates C3b and C3a, but utilizes the alternative pathway C3 convertase, C3bBb. In addition, further C3 degradation products can be generated in the complement pathways. Complement factor I (CFI) is a plasma serine protease that can permanently inactivate C3b to iC3b. iC3b is then cleaved by CFI into further fragments (C3dg and C3c). A further C3 proteolysis product, C3d, binds to complement receptor 2 (CR2) and may play an important role in B cell cell cycle control. Along with 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, C4BP alpha chain, C4BP beta 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 various complement pathway components are described in further detail in Noris et al. Semin Nephrol. 2013; 33(6): 479-492.

[0049] In certain embodiments, the present disclosure provides anti-C3 antigen binding proteins capable of binding to both C3 and C3b. In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure comprise binding affinities for C3a, iC3b, C4, C4b, C5, and / or C5b that are weaker than their binding affinities for ... -4 In certain embodiments, the anti-C3 antigen binding proteins of the present disclosure have no binding affinity for C3a, iC3b, C4, C4b, C5, and / or C5b. As used herein, "no binding affinity" means no detectable binding affinity over background by one or more binding affinity assays known in the art, such as, but not limited to, ELISA assays.

[0050] In certain embodiments, the antigen binding protein can bind to an epitope on complement C3, and such binding prevents the formation of C3 convertase.In certain embodiments, the antigen binding protein of the present disclosure inhibits the activity of C3 convertase.In certain embodiments, the antigen binding protein of the present disclosure inhibits the C3 convertase amplification loop. In certain embodiments, the anti-C3 antibodies of the present disclosure are expected to have better efficacy and safety in treating GA or other ocular disorders compared to other therapies due to the following properties listed below:

[0051] The anti-C3 antibodies of the present disclosure can include, but are not limited to, scFv and VHH antibody fragments having a molecular weight of less than about 60 kDa. For example, but not limited to, the scFv of the present disclosure can have a molecular weight of about 25 kDa, the VHH of the present disclosure can have a molecular weight of about 15 kDa, and other therapeutic agents can have a larger molecular weight. Based on hydrodynamic radius estimation, the anti-C3 antibodies of the present disclosure are expected to have better inhibition of choroidal C3 because they can more efficiently penetrate Bruch's membrane and enter the choroid of the eye more efficiently.

[0052] The anti-C3 antibodies of the present disclosure may have a therapeutic effective period of more than one month, which may be longer than other therapeutic agents. The increased therapeutic effective period may be due to the molar concentration of the anti-C3 antibodies of the present disclosure, which may reach as high as 7 mM. The anti-C3 antibodies of the present disclosure can be easily injected into the eye compared to other therapeutic agents. The anti-C3 antibodies of the present disclosure do not contain PEG, thereby reducing their viscosity. Therefore, the viscosity of the anti-C3 antibodies of the present disclosure is expected to be lower than that of other therapeutic agents. A solution with reduced viscosity, such as a solution with less than or equal to 20 centipoise (cP), is more easily injected into the eye due to reduced back pressure.

[0053] Expression of antigen-binding polypeptides In one aspect, polynucleotides are provided that encode the binding polypeptides (e.g., antigen binding proteins) disclosed herein. Methods of making the binding polypeptides comprising expressing these polynucleotides are also provided. Polynucleotides encoding the binding polypeptides disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce desired amounts of the claimed antibodies, or fragments thereof. Thus, in certain aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0054] The term "vector" or "expression vector" is used herein to mean a vector used according to the present invention as a vehicle for introducing and expressing a desired gene in a cell. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present invention contain a selection marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells. A number of expression vector systems can be used for the purposes of the present invention. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (e.g., RSV, MMTV, MOMLV, etc.), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by cotransformation. Additional elements may be required to optimize the synthesis of mRNA. These elements can include signal sequences, splice signals, as well as transcription promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (eg, human constant region genes) synthesized as discussed above.

[0055] In other embodiments, the binding polypeptides may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as heavy and light chains of an antibody, can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides 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 effectively produce the full range of polypeptides disclosed in the present application.

[0056] More generally, after preparing a vector or DNA sequence encoding an antibody or fragment thereof, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These 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 virus. See Ridgway, AAG "Mammalian Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Introduction of the plasmid into the host can be by electroporation. The transformed cells are grown under conditions suitable for the production of light and / or heavy chains and assayed for heavy and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0057] As used herein, the term "transformation" shall be used in a broad sense to refer to the introduction of exogenous DNA into a recipient host cell, which changes the genotype and results in a change in the recipient cell. A genetically modified recipient cell may contain exogenous sequences by transient or stable transformation. For example, the exogenous sequences may be stably integrated into the recipient cell's genomic sequence, at a target site, or at a random site. Cells modified by gene editing methods (e.g., using homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9, or TALEN) are within the scope of the present disclosure. In certain embodiments, stable cell lines are generated for the production of antigen binding proteins or fragments thereof. This advantageously results in a uniform quality and yield of consistently produced antigen binding proteins or fragments thereof. Along with these same lines, "host cells" refer to cells that have been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing the process of isolating a polypeptide from a recombinant host, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the antibody, unless otherwise clearly specified. In other words, recovery of the polypeptide from the "cells" can mean either from centrifuged whole cells or from the cell culture containing both the medium and the suspended cells.

[0058] In one embodiment, the host cell line used for antibody expression is of mammalian origin. One skilled in the art can determine the particular host cell line that is most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney), and the like. In one embodiment, the cell line provides altered glycosylation, e.g., non-fucosylation, of an antibody expressed therefrom (e.g., PER.C6® (Crucell) or a FUT8 knockout CHO cell line (Potelligent® cells) (Biowa, Princeton, NJ)). Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from published literature.

[0059] In vitro production allows for scale-up to give large amounts of the desired polypeptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in airlift reactors or continuous stirred reactors, or immobilized or entrapped cell cultures, for example, in hollow fibers, microcapsules, agarose microbeads or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, for example gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose and / or (immuno) affinity chromatography.

[0060] Genes encoding the antigen-binding proteins featured in the present invention can also be expressed in non-mammalian cells, such as bacteria or yeast or insect or plant cells. In this regard, it is understood that various unicellular non-mammalian microorganisms, such as bacteria, can also be transformed, i.e., grown in culture or fermentation. Bacteria susceptible to transformation include bacteria of the Enterobacteriaceae family, such as Escherichia coli or Salmonella; Bacillus spp., such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus spp.; and Haemophilus influenzae. Furthermore, it is understood that when expressed in bacteria, the protein can become part of inclusion bodies. The protein must be isolated, purified, and then assembled into a functional molecule.

[0061] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although several other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, e.g. (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)), is commonly used. This plasmid already contains the TRP1 gene, providing a selection marker for mutant strains of yeast lacking the ability to grow in tryptophan, e.g., ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trp1 lesion as a feature of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Thus, in one aspect, there is provided a method for producing an antigen binding protein or fragment thereof as described above, comprising the steps of: i) culturing a host cell under conditions that allow expression of a protein as described herein; ii) recovering the protein; and optionally iii) further purifying and / or modifying and / or formulating the protein; A method is provided that includes:

[0062] Methods of Administering Antigen Binding Proteins Methods of preparing and administering antigen binding proteins (e.g., antigen binding proteins disclosed herein) to a subject are well known to or readily determined by those of skill in the art. Routes of administration of antigen binding proteins of the present disclosure may be oral, parenteral, inhalation, topical, or intraocular. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. The term intraocular as used herein includes, but is not limited to, subconjunctival, intravitreal, retrobulbar, or anterior chamber. The term topical as used herein includes, but is not limited to, administration by liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.

[0063] In certain embodiments, the antigen binding proteins of the present disclosure are administered intraocularly. The delivery of therapeutic compounds to different structures of the eye, such as the retina, is difficult. Challenges include, but are not limited to, several restrictive ocular barriers, tear mechanisms including blinking and washing away of delivered compounds, limited local injection volume, limited local bioavailability, and low tolerance to impurities and contaminants (see, e.g., Patel et al. World J Pharmacol. 2013; 2(2): 47-64; Morrison et al. Ther. Deliv. 2014; 5(12): 1297-1315). The antigen binding proteins of the present disclosure can overcome these challenges. The antigen binding proteins of the present disclosure have 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 proteins of the present disclosure compared to full-length antibodies allows for the delivery of more therapeutic antibodies per injection. This allows for a higher concentration of antibodies to the eye. The smaller size of the antigen-binding proteins of the present disclosure can also improve their penetration into disease-related tissues, i.e., the choroidal region of the eye. The antigen-binding proteins can penetrate one or more layers of the choroidal region, including the Haller layer, the Sattler layer, the choriocapillaris, and the Bruch's membrane, thereby targeting complement C3 and C3b in those layers of the choroidal region.

[0064] In certain embodiments, intraocular administration is accomplished using a drug delivery device, such as a suprachoroidal drug delivery device or a subretinal drug delivery device. Suprachoroidal administration procedures involve administration of a drug to the suprachoroidal space of the eye, and are typically performed using a suprachoroidal drug delivery device, such as a microinjector with a microneedle (see, e.g., 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). Suprachoroidal drug delivery devices that can be used to deposit the antigen binding proteins of the present disclosure in the suprachoroidal space include, but are not limited to, suprachoroidal drug delivery devices manufactured by Clearside® Biomedical, Inc. (see, e.g., Hariprasad, 2016, supra). Subretinal drug delivery devices that can be used to deposit antigen binding proteins of the present disclosure into the subretinal space via the suprachoroidal space include, but are not limited to, subretinal drug delivery devices manufactured by Janssen Pharmaceuticals, Inc. (see, e.g., International Patent Application Publication No. WO 2016 / 040635).

[0065] In certain embodiments, intraocular administration is accomplished via the intravitreal route, which is often accomplished using a syringe and a 27- to 30-gauge needle (see, e.g., Jiang et al., supra). Although all of these dosage forms are clearly contemplated to be within the scope of the present disclosure, the dosage form is a solution for injection, particularly a solution for intravitreal injection. Typically, pharmaceutical compositions suitable for injection may include buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin). However, in other methods consistent with the teachings herein, modified antibodies may be delivered directly to the site of harmful cell populations, thereby increasing the exposure of diseased tissues to therapeutic agents.

[0066] In certain embodiments, the antigen binding proteins of the present disclosure are formulated into a low viscosity solution. The viscosity of the solution is measured in centipoise (cP). High viscosity antibody solutions can pose challenges for administration of the antigen binding proteins of the present disclosure to the eye. For example, a solution having a viscosity of more than 50 cP can be difficult to administer with a thin needle due to high back pressure. Therefore, it is desirable to formulate the antigen binding proteins of the present disclosure in a low viscosity solution. In certain embodiments, the antigen binding proteins of the present disclosure and pharmaceutical compositions thereof have a viscosity of about 1 cP to about 50 cP. In certain embodiments, the antigen binding proteins of the present disclosure and pharmaceutical compositions thereof have a viscosity of 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.

[0067] Formulations for administration 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 saline and buffered media. In the compositions and methods of the present disclosure, pharma- ceutically acceptable carriers include, but are not limited to, 0.01-0.1M or 0.05M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, fixed oils, and the like. Intravenous vehicles include, but are not limited to, fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like. In certain embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0068] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Isotonic agents, for example, sugars, polyalcohols, or sodium chloride, can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0069] In any case, a sterile injectable solution can be prepared by incorporating the required amount of active compound (e.g., antigen-binding protein or fragment thereof) in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method typically includes vacuum drying and freeze-drying to produce a powder of the active ingredient and any additional desired ingredients from the previously sterile-filtered solution. The preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art.

[0070] The effective dose of the composition of the present disclosure for the treatment of the above-mentioned conditions varies depending on many different factors, such as the means of administration, the target site, the physical condition of the patient, whether the patient is human or animal, other medicaments administered, and whether the treatment is preventive or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Treatment doses can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy. As discussed above, the antigen binding proteins, immunoreactive fragments, or recombinants thereof of the present disclosure can be administered in a pharma- ceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it is understood that the disclosed antigen binding proteins are formulated to facilitate administration and promote stability of the active agent.

[0071] Pharmaceutical compositions according to the present disclosure typically include pharma- ceutical acceptable, non-toxic, sterile carriers, such as saline, non-toxic buffers, preservatives, etc. For the purposes of this application, a pharma- ceutical effective amount of the modified antigen-binding protein, its immunoreactive fragment or recombinant, conjugated or unconjugated to a therapeutic agent, must be held to mean an amount sufficient to achieve effective binding to the antigen and achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the modified binding polypeptides are typically capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells, increasing the death of those cells. Of course, the pharmaceutical compositions of the present disclosure can be administered in single or multiple doses to provide a pharma- ceutical effective amount of the modified binding polypeptide.

[0072] In accordance with the scope of the present disclosure, the antigen-binding proteins of the present disclosure can be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect, according to the treatment methods described above. The antigen-binding proteins of the present disclosure can be administered to such humans or other animals in a conventional dosage form prepared by combining the antibodies of the present disclosure with a conventional pharma-ceutically acceptable carrier or diluent, according to known techniques. It will be recognized by those skilled in the art that the form and characteristics of the pharma-ceutically acceptable carrier or diluent will be determined by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. Those skilled in the art will further appreciate that cocktails containing one or more species of the binding polypeptides described in the present disclosure may prove to be particularly effective.

[0073] The biological activity of the pharmaceutical compositions defined herein can be determined by complement inhibition assays, such as, but not limited to, enzyme immunoassays to determine functional classical, lectin, and alternative complement pathway activity in human serum. In certain embodiments, the inhibitory activity of the pharmaceutical compositions defined herein can be assessed using the Complement System Screen Wieslab® (Euro Diagnostica AB, Malmo, Sweden). Functional assays to study the ability of the antibodies of the present disclosure to inhibit the complement pathway can be performed using purified complement components, from which enzyme complexes are reconstituted on the surface of red blood cells or artificial matrices, as described in Okroj et al. PLoS One.; 2012; 7(10): e47245. The standard 50% hemolytic complement (CH50) assay is also a commonly used method to assess the ability of a compound to inhibit the functional activity of the classical complement pathway, as described in Jaskowski et al. Clinical and Diagnostic Laboratory Immunology; 1999; 6(1):137-9.

[0074] In certain embodiments, the activity of the CP, LP, and AP complement pathways can be determined by measuring the level of red blood cell hemolysis in the presence of an antigen binding protein of the present disclosure compared to the level of red blood cell hemolysis in the absence of the antigen binding protein of the present disclosure. In certain embodiments, antibody-sensitized sheep red blood cells can be used to measure complement-dependent hemolysis mediated by the classical pathway. In certain embodiments, antibody-sensitized rabbit red blood cells can be used to measure complement-dependent hemolysis mediated by the alternative pathway as described in Tomlinson et al. J Immunol.1997; 159 (11): 5606-5609.

[0075] In certain embodiments, the activity of the CP, LP, and AP complement pathways can be determined by measuring membrane attack complex (MAC) formation in the presence of an antigen binding protein of the present disclosure compared to MAC formation in the absence of an antigen binding protein of the present disclosure. A MAC assay for IgM-mediated activation of the classical complement pathway in human serum results in deposition of MAC on an IgM-coated ELISA plate. MAC formation can be detected using an alkaline phosphatase-labeled antibody against C5b-9. In the presence of an antigen binding protein of the present disclosure, the ELISA signal is reduced in a dose-dependent manner. To test for the alternative pathway, a MAC assay for LPS-mediated activation of the alternative complement pathway in human serum can be used for deposition of MAC on an LPS-coated ELISA plate. Suitable MAC assays include, but are not limited to, the Pacific biomarker complement membrane attack complex (SC5b-9) ELISA assay.

[0076] "Efficacy" or "in vivo efficacy" as used herein refers to the response to treatment with the pharmaceutical composition of the present disclosure, using standardized response criteria, such as standard ophthalmological response criteria. The success of treatment using the pharmaceutical composition of the present disclosure or in vivo efficacy refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to cause its desired effect, i.e., inhibition of the complement pathway in the eye. In vivo efficacy can be monitored by standard methods established for various ocular diseases. Monitoring methods include, but are not limited to, Amsler grid test, opthalmoscopy, 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.

[0077] Antibody Engineering and Optimization The antigen binding proteins of the present disclosure can be engineered or optimized. As used herein, "optimized" or "optimization" refers to alteration of an antigen binding protein to improve one or more functional properties. Alterations include, but are not limited to, deletion, substitution, addition, and / or modification of one or more amino acids in the antigen binding protein. As used herein, the term "functional property" is a property of an antigen binding protein whose improvement (e.g., relative to a conventional antigen binding protein) is desirable and / or advantageous to one of skill 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., thermostability). In another embodiment, the functional property is solubility (e.g., under cellular conditions). In yet another embodiment, the functional property is aggregation behavior. In yet another embodiment, the functional property is protein expression (e.g., in a prokaryotic cell). In yet another embodiment, the functional property is refolding behavior after inclusion body solubilization in a manufacturing process. In certain embodiments, the functional property is not an improvement in antigen binding affinity. In another embodiment, the improvement in one or more functional properties does not have a substantial effect on the binding affinity of the antigen binding protein.

[0078] In certain embodiments, the antigen binding proteins of the present disclosure are scFvs and are optimized in the antigen binding protein by identifying preferred amino acid residues to be substituted, deleted, and / or added at amino acid positions of interest (e.g., amino acid positions identified by comparing a database of scFv sequences having at least one desirable property as selected by a quality control (QC) assay with a database of mature antibody sequences, e.g., the Kabat database). Thus, the present disclosure further provides "enrichment / exclusion" methods for selecting specific amino acid residues. Still further, the present disclosure provides methods of engineering antigen binding proteins (e.g., scFvs) by mutating specific framework amino acid positions identified using the "functional consensus" approach described herein. In certain embodiments, framework amino acid positions are mutated by replacing existing amino acid residues with residues found to be "enriched" residues using the "enrichment / exclusion" analysis method described herein. In one aspect, the disclosure provides a method of identifying amino acid positions for mutation in a single chain antibody (scFv) having a VH and VL amino acid sequence, the method comprising: a) entering the scFv VH, VL or VH and VL amino acid sequence into a database comprising a number of antibody VH, VL or VH and VL amino acid sequences, thereby aligning the scFv VH, VL or VH and VL amino acid sequence with the antibody VH, VL or VH and VL amino acid sequences of the database; b) comparing amino acid positions within the scFv VH or VL amino acid sequence to corresponding positions within the antibody VH or VL amino acid sequences of the database; c) determining whether the amino acid position within the scFv VH or VL amino acid sequence is occupied by an amino acid residue that is conserved at the corresponding position within the antibody VH or VL amino acid sequences of the database; and d) identifying the amino acid position within the scFv VH or VL amino acid sequence as an amino acid position for mutation if the amino acid position is occupied by an amino acid residue that is not conserved at the corresponding position within the antibody VH or VL amino acid sequences of the database.ScFV optimization is described in further detail in WO2008110348, WO2009000099, WO2009000098, and WO2009155725, all of which are incorporated herein by reference.

[0079] Humanization: In certain embodiments, the antigen-binding proteins of the present disclosure may be humanized. As used herein, the term "humanized" refers to non-human donor antibodies that have been modified to increase their similarity to antibodies naturally produced in humans. As used herein, the term "humanization" refers to the process of humanizing a non-human donor antibody. Humanization can be achieved by grafting the CDRs of a non-human donor antibody (e.g., rabbit or llama antibody CDRs) onto a human or humanized antibody acceptor framework region, such as a soluble and stable light and / or heavy chain human antibody framework region. General methods of grafting CDRs onto a human acceptor framework are disclosed by Winter in U.S. Pat. No. 5,225,539 and Queen et al. in WO199007861, which are incorporated herein by reference. Appropriate acceptor framework regions can exhibit superior functional properties, such as improved solubility and stability. In certain embodiments, the antigen-binding proteins of the present disclosure are rabbit antibodies. The CDRs of said rabbit antibody may be grafted into universal acceptor framework regions, such as those described in WO2009155726, which is incorporated herein by reference.

[0080] In certain embodiments, the human framework for humanization / stabilization of non-human antibodies or stabilization of human antibodies relates to the replacement of a κ-junction segment in a κ variable light domain with a λ-junction segment resulting in a κ-λ chimeric variable light domain with improved protein stability and reduced aggregation tendency. Furthermore, it relates to the mutation and replacement of the κ consensus residue at position AHo101 with a λ consensus residue to aid in packing of the λ-junction segment in the κ-λ chimeric variable light domain to further improve protein stability and further reduce aggregation tendency. Further details regarding these human framework regions are described in WO2014206561 and WO2019057787, which are incorporated herein by reference.

[0081] Methods for Treating Complement C3-Mediated Diseases and Disorders - Patent application Methods of treating complement C3-mediated diseases and disorders using the antigen binding proteins described herein in a subject suffering from a complement C3-mediated disease or disorder are provided. In certain embodiments, the complement C3-mediated disease or disorder is selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, retrolental fibrosis, autoimmune uveitis, chorioretinitis, retinitis, rheumatoid arthritis, psoriasis, and atherosclerosis. In certain embodiments, the C3-mediated disease is a form of AMD. AMD is generally divided into two main classes: dry AMD and wet AMD. Dry AMD, also known as non-exudative AMD, is characterized by the presence of drusen (yellow deposits) in the macula. Wet AMD, also known as exudative AMD or neovascular AMD, is characterized by the growth of abnormal blood vessels from the choroid under the macula. This process is also called choroidal neovascularization and can result in leakage of fluid, such as blood, into and around the retina. Geographic atrophy, also known as atrophic AMD or advanced dry AMD, is an advanced form of AMD that can result in progressive and irreversible loss of retinal cells.

[0082] It is particularly difficult to treat eye diseases such as AMD mentioned above. As previously mentioned, delivery of therapeutic agents to the eye is limited due to 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 enhances the therapeutic potential of the drug. In certain embodiments, the antigen binding proteins of the present disclosure can 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 proteins of the present disclosure to penetrate the RPE and Bruch's membrane improves their therapeutic potential in treating complement C3-mediated diseases or disorders. The antigen binding proteins of the present disclosure can penetrate the RPE and Bruch's membrane, in part because their size is low enough to facilitate penetration. In certain embodiments, the size of the antigen binding proteins of the present disclosure is measured by molecular weight. In certain embodiments, the antigen binding proteins of the present disclosure have a molecular weight of less than about 60 kDa. In certain embodiments, the antigen binding proteins of the present disclosure are about 20 kDa to about 30 kDa or about 10 kDa to about 20 kDa. In certain embodiments, the antigen binding proteins of the present disclosure are about 25 kDa. In certain embodiments, the antigen binding proteins of the present disclosure are about 15 kDa. In certain embodiments, the size of the antigen binding proteins of the present disclosure is measured by their hydrodynamic radius. In certain embodiments, the antigen binding proteins of the present disclosure have a hydrodynamic radius of less than or equal to about 3.0 nm. In certain embodiments, the antigen binding proteins of the present disclosure have a hydrodynamic radius of less than or equal to about 2.5 nm. In certain embodiments, the antigen binding proteins of the present disclosure have a hydrodynamic radius of less than or equal to about 2.0 nm.

[0083] In one aspect, the present disclosure provides a method of inhibiting the activity of the classical (CP), lectin (LP), and alternative (AP) complement pathways, comprising contacting complement C3 with an antigen binding protein or fragment thereof that binds to an epitope on complement C3. The ability of the antigen binding proteins of the present disclosure to inhibit all three complement pathways further improves their therapeutic potential in treating complement C3-mediated diseases or disorders. Without wishing to be bound by theory, inhibiting all three complement pathways may improve the therapeutic potential of the antigen binding proteins of the present disclosure by preventing the disease-promoting effects of one activated pathway from compensating for the inactivated pathways of the other.

[0084] In certain embodiments, the antigen binding protein or fragment thereof can inhibit the activity of the CP, LP, and AP complement pathways approximately equally. For example, but not limited to, the antigen binding protein or fragment thereof can inhibit the activity of the CP pathway by at least 80%, the activity of the LP by at least 80%, and the activity of the AP by at least 80%. In certain embodiments, the inhibition of the activity 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%.

[0085] In another aspect, the present disclosure provides a method for inhibiting the activity of choroidally localized complement C3 via intraocular administration of an antigen binding protein or fragment thereof that binds to an epitope on complement C3. An activated complement pathway in the choroidal region of the eye may contribute to complement C3-mediated diseases or disorders. Therefore, it is an object of the present disclosure to provide an antigen binding protein that can penetrate or diffuse into the choroidal region and target complement C3 and C3b. In certain embodiments, the antigen binding protein of the present disclosure inhibits the activity of C3 convertase in the choroidal region of the eye. In certain embodiments, the antigen binding protein of the present disclosure inhibits the C3 convertase amplification loop in the choroidal region of the eye.

[0086] medical use The present invention also relates to an antigen-binding protein or fragment thereof disclosed herein for use in a method of treating a complement C3-mediated disease or disorder in a subject. All technical features described in this disclosure relating to the antigen-binding protein or fragment thereof are applicable.

[0087] kit The present invention also encompasses kits comprising at least one antigen binding protein or fragment thereof described herein. In one embodiment, the kit comprises a composition containing an effective amount of said antigen binding protein or fragment thereof in unit dosage form. Such kits may comprise a sterile container comprising the composition, non-limiting examples of such containers include, but are not limited to, vials, ampoules, bottles, tubes, syringes, blister packs. In some embodiments, the composition is a pharmaceutical composition and the container is made of a material suitable for holding a pharmaceutical. In one embodiment, the kit may comprise in a first container the antigen binding protein or fragment thereof in lyophilized form and a second container comprising a diluent (e.g., sterile water) for reconstitution or dilution of the antigen binding protein of the fragment. In some embodiments, the diluent is a pharmaceutically acceptable diluent. Typically, the kit further comprises a separate sheet, pamphlet or card provided in or with the container with instructions for use. If the kit is intended for medical use, it may further comprise information for administering the composition to a subject having a complement C3-mediated disease or disorder, and one or more of dosing schedules, therapeutic descriptions, preventions, warnings, indications, counterindications, overdose information and / or side effects.

[0088] Diagnostic Uses and / or Detection The antigen binding proteins or fragments thereof of the present invention can be used for in vivo and / or in vitro detection or diagnostic purposes. For example, a wide range of immunoassays involving antigen binding proteins for detecting expression in specific cells or tissues are known to those skilled in the art. For such uses, the antigen binding proteins or fragments thereof disclosed herein may be labeled or unlabeled. For example, but not limited to, unlabeled antigen binding proteins can be used and detected by a secondary antibody that recognizes an epitope on the antigen binding proteins described herein. In another embodiment, the antigen binding proteins or fragments thereof are conjugated to one or more substances that can be recognized by a detection substance(s), for example, the antigen binding proteins or fragments thereof are conjugated to biotin that can be detected by streptavidin. In certain embodiments, the antigen binding proteins or fragments thereof are useful for detecting the presence of C3 and / or C3b in a sample. In certain embodiments, the sample is a biological sample. As used herein, the term "detect" encompasses quantitative and / or qualitative detection. In certain embodiments, the biological sample comprises cells or tissue from a human patient, such as retinal tissue.

[0089] In certain embodiments, the method comprises contacting a biological sample with at least one antigen binding protein or fragment thereof of the present invention; allowing the formation of a complex between C3 (if present) in the sample and the antigen binding protein or fragment thereof; and then detecting said antigen binding protein or fragment thereof. In a preferred embodiment, the antigen binding protein or fragment thereof is capable of binding to both complement C3 and C3b. In one embodiment, the antigen binding protein or fragment thereof is detected by a detectable signal, hi another embodiment, the antigen binding protein or fragment thereof is detected by ELISA, immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and / or flow cytometry. The biological sample may be a tissue sample, such as retinal tissue. The tissue sample may be a fixed tissue sample, such as a formalin-fixed and paraffin-embedded tissue sample.

[0090] In one embodiment, such methods are used to select patients, i.e., to determine a subject's eligibility for treatment with an antigen binding protein or fragment thereof, as described herein. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Although certain embodiments have been described in detail herein, the present specification will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0091] Example 1 Generation and characterization of anti-C3 antibody library To generate antibodies that inhibit the complement cascade more efficiently than is possible with partial complement inhibitors, it was hypothesized that a broad collection of anti-C3 antibodies with diverse epitope recognition would increase the probability of isolating an antibody with the desired function. To this end, a large antibody phage library was constructed using genomic information encoding antibody variable domains from B cells of animals immunized with C3. To generate multiple antibodies capable of recognizing different epitopes 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 at different time points with complete or incomplete Freund's adjuvant (Figure 3A). The immune response of each animal was tested using ELISA to quantify the anti-C3 antibodies present in serum samples of immunized animals. Antibody titers in the serum indicated an excellent immune response (Figure 3B).

[0092] An scFv antibody cDNA library was constructed from RNA extracted from PBMCs and splenic lymphocytes isolated from rabbits by PCR amplification. The coding sequences for the variable light and heavy domains were amplified separately and linked through a series of overlapping polymerase chain reaction (PCR) steps to obtain the final scFv product. For llamas, a large bleed was performed, from which RNA was isolated and transcribed into cDNA using a reverse transcriptase kit. The cDNA was cleaned and a heavy chain fragment was amplified using primers annealing in the leader sequence region and CH2 region. The amplified DNA sequences encoding the rabbit-derived scFv and llama-derived VHH were digested with appropriate restriction enzymes and then ligated into a phagemid vector. The phagemid vector was transformed into TG1 electrocompetent cells, which are well suited for antibody phage display library generation. These processes were carried out over 10 8 This resulted in four antibody libraries with clone sizes greater than 1 and close to 100% insert percentage (Figures 4A and 4B).

[0093] Example 2 Screening for anti-C3 antibodies that inhibit all three complement pathways 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 different sites. C3-derived fragments form convertases that exert different effector functions and fuel the amplification loop of the complement pathway. The enzyme C3 convertase has the ability to cleave multiple C3 molecules into C3b in a strong amplification loop, generating more C3 convertase, leading to full activation of the complement system. Using the screen described herein, we identified antibodies that bind to distinct epitopes on both C3 and C3b and effectively block all three pathways of complement activation (classical, lectin, and alternative). To screen for high affinity anti-C3 antibodies, scFvs and VHH antibodies displayed on phage were produced and subjected to several rounds of biopanning (selection) against native human C3 purified from serum. The stringency of selection was increased with each round by decreasing the concentration of C3 protein used in biopanning or increasing the stringency of the washes. Approximately 380 monoclonal phages were selected and screened for their ability to bind C3 in an ELISA assay (Figure 5). Based on the ELISA data and DNA fingerprints, 41 phage clones were selected for sequencing, recombinantly produced as antibody proteins, and evaluated for their ability to bind human C3 and C3b and for further characterization (Figure 5).

[0094] To identify antibodies that block all three complement pathways, antibodies were screened using an enzyme immunoassay for the qualitative determination of functional classical, lectin and alternative complement pathways in human serum using the Wieslab® Complement System Screen (Svar Life Science AB, Malmo, Sweden). The amount of C5b-C9 neoantigen produced is proportional to the functional activity of the complement pathway. As shown in Figure 6, five antibodies, M0251, M0228, M0122, M0123 and M0124, were able to inhibit all three complement pathways by at least 90% at a fixed concentration of 2 μM in human serum (Quidel).

[0095] Example 3 Characterization of anti-C3 antibodies: M0251, M0228, M0122, M0123, and M0124 M0251, M0228, M0122, M0123 and M0124 were tested in a pairwise combinatorial fashion to identify those targeting the same region (epitope) on C3. Briefly, one antibody was labeled by biotinylation and incubated with other antibody clones in a C3-binding ELISA. These anti-C3 antibodies competing for the same binding region were assumed to share similar epitopes and therefore have similar functions. This information allows reducing the number of potential antibody candidates while maintaining epitope diversity. Of the five antibodies that inhibit all three complement pathways, M0251, M0228 and M0123 were assumed to share the same epitope on C3 (Figure 7D). The inhibitory leads were assumed to bind to three different epitopes on C3 (Figures 7A-7D).

[0096] Antibodies identified as capable of inhibiting all three complement pathways were evaluated for their ability to bind cynomolgus C3 in an ELISA. Briefly, 96-well ELISA plates were coated with a polyclonal goat antiserum believed to be cross-reactive to cyno C3, followed by secondary association with a custom preparation of cynomolgus serum (BioIVT, NB-151558). Serial dilutions of antibody molecules were added to the ELISA plate, and antibodies bound to cyno C3 were detected using rabbit anti-human kappa HRP antibody (Abcam, ab202549) or mouse anti-His Tag HRP antibody (R&D Systems, MAB050H). Leads M0122, M0124, and M0251 show dose-responsive binding to cynomolgus C3. Interestingly, although M0251, M0228 and M0123 compete for the same epitope on human C3, only M0251 showed binding activity to cyno C3 (FIGS. 15A and 15B).

[0097] The ability of anti-C3 antibodies to inhibit all pathways of complement activation in cynomolgus serum was evaluated using the Wieslab complement system screen. Anti-C3 antibodies were added to a custom preparation of cyno serum. Figure 14A shows the strong inhibition of all three complement pathways by M0122, M0124 and M0251 at a fixed concentration of 2 μM, suggesting that M0122, M0124 and M0251 are potent inhibitors of complement-mediated MAC formation in cyno serum. M0228 showed no complement pathway inhibitory activity in cyno serum, confirming the lack of binding activity observed for this antibody against cynoC3 (Figure 14B). Dose-dependent inhibition of classical and alternative pathways in cynomolgus serum was further evaluated for M0122, M0124 and M0251 using the corresponding Wieslab complement system kits (Figures 14B and 14C). M0122, M0124 and M0228 were evaluated for their ability to bind to both human C3 and C3b in a direct binding ELISA assay (Figure 8A and Figure 8B). Briefly, 96-well ELISA plates were coated with purified native human C3 or C3b (Complement Technology, A113 and A114). Serial dilutions of antibody molecules were added to the plate and detected by rabbit anti-human kappa HRP antibody (Abcam, ab202549) or rabbit anti-His Tag HRP antibody (Abcam, ab1187). M0122, M0124 and M0228 show high affinity binding to both human C3 and C3b. The binding kinetics of M0122, M0124 and M0228 to human C3 was further analyzed by biolayer interferometry showing affinities in the low picomolar range (Figure 10).

[0098] Dose-dependent inhibition of alternative and classical pathways in human serum was evaluated for M0122, M0124 and M0228 using the corresponding Wieslab complement system kit. Anti-C3 antibodies M0122, M0124 and M0228 show strong inhibition of alternative and classical pathways in human serum (Figure 9A and Figure 9B). Anti-C3 antibodies M0122, M0123 and M0124 were further evaluated for their ability to inhibit the lectin pathway in a dose-response manner. Figure 16 shows efficient inhibition of the lectin pathway in human serum. Taken together, these results further support the efficient inhibition of all three pathways of complement activation by the antibodies of the present invention.

[0099] Example 4 Anti-C3 antibodies are more likely to penetrate Bruch's membrane than APL-2 It is now known that the complement system is involved in the pathogenesis of geographic atrophy. However, it is still not fully understood how complement activity is compartmentalized in the eye and whether the efficacy of GA therapy depends on delivering therapeutics to the correct anatomical site in the eye. The inventors hypothesized that to achieve a significant reduction in lesion growth in GA, better penetration into disease-related retinal tissues (i.e., RPE, Bruch's membrane and choroid) may be necessary. The inside of the choroid is called the choriocapillaris and contains capillaries separated by a sheet of extracellular membrane called Bruch's membrane (BrM) (Figure 11).

[0100] Bruch's membrane is selectively permeable to antibodies and biologics. As reported by Clark et al. (Front. Immunol. 2017. 8:1-10), complement pathway proteins cannot pass through Bruch's membrane except for FHL-1, factor D, and C5a. Overall, antibodies and biologics with large hydrodynamic radii are less likely to pass through Bruch's membrane. As shown in Table 3 below, except for APL-2 and CDR2 (anti-C3 scFv of the present disclosure), the molecules listed with hydrodynamic radii greater than 3.00 cannot pass through Bruch's membrane except for FHL-1; whereas, all molecules listed with hydrodynamic radii greater than 3.00 can pass through Bruch's membrane except for C3a.

[0101] [Table 3]

[0102] Furthermore, Pitkanen et al. (Invest Ophthalmol Vis Sci. 2005; 46(2):641-6) studied the permeability of fresh RPE-choroid specimens from bovine eyes for carboxyfluorescein, fluorescein isothiocyanate (FITC)-labeled dextrans with molecular weights between 4 and 80 kDa. We plotted permeability against molecular size (Figure 12, black dots). We also derived permeability values ​​for scFv, lucentis, eylea and APL2 based on hydrodynamic radius using the work done by Hirvonen et al. (Pharm Res. 2016;33(8):2025-32) and plotted permeability against molecular weight on the same graph (Figure 12, colored dots). This trend indicates that the higher the molecular weight, the lower the permeability of Bruch's membrane.

[0103] The inventors predicted that the anti-C3 antibodies of the present disclosure as antibody fragments, such as, but not limited to, scFv or VHH formats, with a hydrodynamic radius of about 2.5 nm or smaller, will likely be able to penetrate Bruch's membrane better than APL-2 (two anti-C3 cyclic APL-1 peptides linked to a 40 kDa linear PEG, totaling 43 kDa), which has a hydrodynamic radius of at least 7 nm.

[0104] To test this hypothesis, enriched porcine BrM placed in an Ussing chamber was used to assess the ability of anti-C3 molecules to cross the BrM. Briefly, enriched Bruch's membrane was isolated from porcine eyes and placed in an Ussing diffusion chamber (Multi Channel Systems MCS GmbH, Cat. No. 660026). Once placed, the 5 mm diameter Bruch's membrane was the only barrier between the two identical compartments. Both sides of the Bruch's membrane were washed with 1 ml of PBS at room temperature for at least 5 min. For leakage testing, 1 ml of PBS was added to the sample chamber and leakage into the second compartment was followed for 5 min. If no leakage was detected indicating a compromise of the membrane integrity, antibody protein was added in 1 ml of PBS at 100 μg / ml to the sample chamber and 1 ml of PBS was added to the second compartment (diffusion chamber). The entire Ussing chamber was incubated with gentle shaking for 24 h at room temperature to prevent the development of a gradient of diffusing protein. Samples (15 μl) from each chamber were analyzed by gel electrophoresis. Precast 4-12% NuPAGE Bis Tris SDS gels (Thermo Fisher Scientific) were run at 200 V for 40 min under reducing conditions. Gels were stained at room temperature for 60 min with Instant Blue stain (Expedeon) to detect antibody proteins or with barium iodide solution to detect PEG (gels were fixed with 0.1 M perchloric acid and replaced after 15 min with a premix of 20 ml of 5% BaCl2 and 8 ml of 0.1 M iodine solution, then repeatedly replaced with deionized water every 10 min for 1 h). To calculate the percentage of protein in the sample or diffusant chambers, the density of bands in Instant Blue-stained or BaI2-stained SDS gels was measured using ImageJ software. The average intensity of these bands was compared to the density of a control band representing 100% loaded protein (i.e., 15 μl of 100 μg / ml). The calculated percentage of protein was then plotted ±SD.The ability to cross porcine BrM was compared for scFv derivatives of M0123 (26 kDa) and APL-2 surrogates (one anti-C3 cyclic APL-1 peptide linked to a 40 kDa linear PEG, totaling 42 kDa) incubated simultaneously on BrM preparations from four different porcine eyes. Significantly higher amounts of scFv crossed the BrM in all four membrane preparations compared to the APL-2 surrogate (Figures 13A and 13B).

Claims

1. An antigen-binding protein or a fragment thereof that binds to an epitope on complement C3, VH and VL, 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; An antigen-binding protein or fragment thereof, 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.

2. 2. The antigen-binding protein or fragment thereof of claim 1, wherein VH comprises the amino acid sequence of SEQ ID NO:27 and VL comprises the amino acid sequence of SEQ ID NO:

28.

3. The antigen-binding protein or fragment thereof of claim 1 or 2, comprising a single chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, a diabody, a small antibody mimetic or a single domain antibody, such as an sdAb, sdFv, nanobody, V-Nar or VHH.

4. A pharmaceutical composition comprising the antigen-binding protein or fragment thereof according to any one of claims 1 to 3, optionally together with a pharma- ceutically acceptable carrier.

5. The pharmaceutical composition of claim 4 for treating a complement C3-mediated disease or disorder in a subject.

6. The pharmaceutical composition of claim 5, wherein the antigen-binding protein or fragment thereof is administered by topical, subconjunctival, intravitreal, retrobulbar, and / or intracameral administration.

7. The pharmaceutical composition described in claim 5 or 6, wherein the complement C3-mediated disease or disorder 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.

8. An isolated nucleic acid molecule encoding the antigen-binding protein or fragment thereof according to any one of claims 1 to 3.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the expression vector of claim 9.

11. A method for producing the antigen-binding protein or fragment thereof according to any one of claims 1 to 3, comprising the steps of: (i) culturing a host cell according to claim 10 under conditions allowing expression of a protein according to any one of claims 1 to 3; (ii) recovering the protein; The method includes:

12. 1. A method for detecting one or both of C3 and C3b in a biological sample, comprising: (a) contacting a sample with at least one antigen binding protein or fragment thereof according to any one of claims 1 to 3; (b) allowing the formation of a complex between one or both of C3 and C3b in the sample and an antigen binding protein or fragment thereof; (c) detecting the antigen-binding protein or fragment thereof; The method includes:

13. A kit for detecting C3, comprising the antigen-binding protein or fragment thereof according to any one of claims 1 to 3 and instructions for use.

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