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] This disclosure relates to an antigen-binding protein that targets complement C3, and to a method for treating complement C3-mediated diseases. [Background technology]

[0002] A major challenge in treating certain eye diseases and disorders is the delivery of therapeutic molecules to the deep layers of the retina. Delivery is hindered by multiple factors, including several physical boundaries within the eye. These boundaries include the corneal and conjunctival epithelium, the blood-water barrier (BAB), and the blood-retinal barrier (BRB), such as capillary endothelial cells (medial BRB) and retinal pigment epithelial cells (see, e.g., Jiang et al. Int J Ophthalmol. 2018; 11(6): 1038-1044). Eye diseases in which delivery to the retina is particularly important include complement-mediated diseases such as geographic atrophy (GA). Geographic atrophy (GA) is a progressive form of age-related macular degeneration (AMD) characterized by the loss of retinal pigment epithelium and photoreceptors in the macula. When GA affects the fovea, irreversible vision loss occurs. Patients in the early stages of GA typically experience visual impairment even before their vision is affected. Although the pathophysiology underlying geographic atrophy is not fully understood, dysregulation of complement activity is considered a contributing factor. Several complement activators, 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 from GA patients compared to controls. Furthermore, complement inhibitors such as CD59, a membrane-bound inhibitor of membrane invasion complex (MAC) formation, and membrane cofactor proteins (MCPs), membrane-bound complement regulators with cofactor activity of complement factor I (CFI), have been reported to be reduced in GA.

[0003] Currently, there are no approved treatments for GA. Several investigative approaches targeting the complement pathway have been investigated, but none have been approved and have not yet demonstrated efficacy. Some examples of such approaches include eculizumab / SOLIRIS (Alexion), LFG-316 (Novartis / MorphoSys), ARC-1905 (Ophthotech), POT-4 (AL-78898A; Alcon), and lamparizumab (FCFD45142). More recently, findings from a Phase II clinical trial of APL-2 (Clinical Trial NCT0250332 "A Study of APL-2 Therapy in Patients with Geographic Atrophy (FILLY)") have further implicated the complement pathway in the pathogenesis of GA and demonstrated a positive therapeutic effect in inhibiting GA progression via complement inhibition. These results also suggest that APL-2 inhibition, which is central to the complement cascade at C3 (the convergence of all complement pathways; see Figure 1), may have the potential to treat GA more effectively than inhibitors that result in partial inhibition of the complement pathway. Nevertheless, the reduction in lesion proliferation in GA achieved with APL-2 remains modest. APL-2 has characteristics that may limit its effectiveness. APL-2 (an inhibitor of complement component C3), a pegylated derivative of the cyclic tridecapeptide compstatin, 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 likely has a short effective period of only one month due to its low concentration of 3.5 mM. APL-2 is also a PEGylated molecule, which can increase its viscosity and make injection into the eye difficult. Therefore, it needs to reduce GA progression more efficiently.

[0004] One of the major challenges in treating GA is that the observed dysregulation of complement activity occurs in the deeper layers of the retina. We hypothesize that better penetration into disease-related retinal tissue (i.e., retinal pigment epithelium (RPE), Bruch's membrane, and other parts of the choroid) is necessary to achieve a greater reduction in lesion proliferation in GA. For this reason, small antibody fragments have several advantages compared to other biologics and antibodies. The small antibody format can enable 1) better intraocular penetration into the relevant retinal tissue; and 2) more drug per 1 mg or mL delivered by intravitreous injection. [Overview of the Initiative]

[0005] This disclosure provides an antigen-binding protein having specificity for complement C3. In one embodiment, the present disclosure provides an antigen-binding protein or fragment thereof that binds to an epitope on complement C3 and can inhibit a complement activation pathway, including the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). In certain embodiments, an antigen-binding protein or a fragment thereof can bind to complement C3 and C3b. In certain embodiments, an antigen-binding protein or a fragment thereof can bind to an epitope on complement C3, and such binding prevents the formation of C3 convertase.

[0006] In certain embodiments, an antigen-binding protein or fragment thereof may 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-strand 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 sdAb, sdFv, a nanobody, V-Nar, or VHH. In preferred embodiments, the antigen-binding protein or fragment thereof comprises scFv or VHH. In a particular embodiment, the antigen-binding protein or fragment thereof comprises a CDR-H3 having at least 80% similarity to the sequences 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 a particular embodiment, the antigen-binding protein or fragment thereof comprises a CDR-H3 having at least 80% identity with the sequences 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 a particular embodiment, 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 a particular embodiment, VH has at least 80% similarity to the sequence of the group consisting of SEQ ID NOs. 25, 26, 27, 29, and 31, and / or VL has at least 80% similarity to the sequence of the group consisting of SEQ ID NOs. 28, 30, and 32. In a particular embodiment, VH has at least 80% identity with the sequence of the group consisting of sequence numbers 25, 26, 27, 29, and 31, and / or VL has at least 80% similarity with the sequence of the group consisting of sequence numbers 28, 30, and 32. In a particular embodiment, the antigen-binding protein or fragment thereof comprises VH and VL, where VH comprises the CDR-H1 sequence of SEQ ID NO: 7, the CDR-H2 sequence of SEQ ID NO: 8, and the CDR-H3 sequence of SEQ ID NO: 9; and 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. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO: 27 and VL comprises the amino acid sequence of SEQ ID NO: 28.

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

[0010] In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO: 31 and 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, and the VHH domain comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 2, and the CDR-H3 sequence of SEQ ID NO: 3. 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, and the VHH domain comprises the CDR-H1 sequence of SEQ ID NO: 4, the CDR-H2 sequence of SEQ ID NO: 5, and the CDR-H3 sequence of SEQ ID NO: 6. 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 at least about 10 to C3 and C3b -8It includes binding affinity for M. In certain embodiments, the antigen-binding protein or fragment thereof has about 10% binding affinity for C3 and C3b. -9 M~about 10 -14 It includes binding affinity for M. In certain embodiments, the antigen-binding protein or fragment thereof has about 10% binding affinity for C3 and C3b. -10 M~about 10 -12 It includes binding affinity for M. In certain embodiments, the antigen-binding protein or fragment thereof has approximately equivalent binding affinity for C3 and C3b. In certain embodiments, the binding affinity for C3 is within 10 times the binding affinity for C3b. In a particular embodiment, the antigen-binding protein or fragment thereof is approximately 10 to C3a, iC3b, C4, C4b, C5 and / or C5b. -4 The antigen-binding protein or fragment thereof contains a binding affinity of M or weaker. In certain embodiments, the antigen-binding protein or fragment thereof contains a weak binding affinity to C3a, iC3b, C4, C4b, C5, and / or C5b compared to its binding affinity to C3 and C3b. In certain embodiments, the antigen-binding protein or fragment thereof does not contain a binding affinity to C3a, iC3b, C4, C4b, C5, and / or C5b.

[0012] In certain embodiments, antigen-binding proteins or fragments 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, antigen-binding proteins or fragments thereof can inhibit the activity of the CP, LP, and AP complement pathways to an equivalent or near-equal degree. 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%. In certain embodiments, the activity of the CP, LP, and AP complement pathways is determined by measuring the level of erythrolysis in the presence of antigen-binding proteins or fragments, compared to the level of erythrolysis in the absence of antigen-binding proteins or fragments thereof. In certain embodiments, the activity of the CP, LP, and AP complement pathways is determined by measuring membrane invasion complex (MAC) formation in the presence of antigen-binding proteins or fragments, compared to MAC formation in the absence of antigen-binding proteins or fragments thereof.

[0013] In certain embodiments, the antigen-binding protein or a fragment thereof can inhibit the activity of the C3 convertase by at least about 80%, at least about 85%, at least about 90%, or at least about 95%. In certain embodiments, antigen-binding proteins or fragments thereof can inhibit the C3 convertase amplification loop. In certain embodiments, antigen-binding proteins or fragments thereof can penetrate Bruch's membrane. In certain embodiments, antigen-binding proteins or fragments thereof can inhibit choroidal C3 activity.

[0014] In certain embodiments, the antigen-binding protein or fragment thereof has a molecular weight of about 60 kDa or less. In certain embodiments, the antigen-binding protein or fragment thereof has a molecular weight of about 20 kDa to about 30 kDa. In certain embodiments, the antigen-binding protein or fragment thereof has a molecular weight of about 10 kDa to about 20 kDa. In certain embodiments, the antigen-binding protein or fragment thereof has a molecular weight of about 25 kDa. In certain embodiments, the antigen-binding protein or fragment thereof has a molecular weight of about 15 kDa. In certain embodiments, the antigen-binding protein or a fragment thereof is cross-reactive with cynomolgus monkey C3. In one embodiment, the present disclosure provides a pharmaceutical composition comprising the aforementioned antigen-binding protein or fragment thereof and a pharmaceutically acceptable carrier. Thus, one embodiment is the use of the binding protein of the present invention in the preparation of a pharmaceutical composition for treating complement C3-mediated diseases or disorders in a subject. In certain embodiments, the pharmaceutical composition includes low viscosity. In certain embodiments, the viscosity is approximately 1 cP to approximately 50 cP. In certain embodiments, the viscosity is less than or equal to approximately 20 cP.

[0015] In one embodiment, the present disclosure provides an isolated nucleic acid molecule encoding the antigen-binding protein or a fragment thereof as described above. In another embodiment, the present disclosure provides an expression vector comprising the nucleic acid molecule described above. In yet another aspect, the disclosure provides a host cell containing the expression vector described above. In yet another embodiment, a method for producing the antigen-binding protein or fragment thereof, i) A step of culturing the above-mentioned host cells under conditions that enable the expression of the proteins described herein, ii) A step to recover the protein, and optionally iii) A step of further purifying and / or modifying and / or formulating the protein. A method including this is provided.

[0016] In one embodiment, the Disclosure provides a method for treating a complement C3-mediated disorder or impairment in a subject, comprising administering the aforementioned antigen-binding protein or fragment thereof to a subject in need. Accordingly, the Disclosure also provides the aforementioned antigen-binding protein or fragment thereof for use in a method for treating a complement C3-mediated disorder or impairment. In certain embodiments, the antigen-binding protein or fragment thereof is administered via topical, subconjunctival, intravitreous, retrobulbar, and / or anterior chamber administration. In certain embodiments, complement C3-mediated diseases or disorders are selected from the group consisting of age-related macular degeneration, geographic atrophy, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, posterior lens fibrosis, autoimmune uveitis, chorioretinitis, retinitis, rheumatoid arthritis, psoriasis, and atherosclerosis.

[0017] In one embodiment, the Disclosure provides a method for inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP), comprising contacting complement C3 with an antigen-binding protein or fragment thereof that binds to an epitope on complement C3. Accordingly, the Disclosure provides antigen-binding proteins or fragments thereof described herein for use in a method for treating complement C3-mediated diseases or disorders by inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The Disclosure also provides the aforementioned antigen-binding proteins or fragments thereof for use in a method for treating complement C3-mediated diseases or disorders by inhibiting the activity of choroidally localized complement C3. In one embodiment, the present disclosure provides a method for inhibiting the activity of choroidally 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, antigen-binding proteins or fragments thereof can be bound to complement C3 and C3b. In certain embodiments, an antigen-binding protein or a fragment thereof can bind to an epitope on complement C3, and such binding prevents the formation of C3 convertase. In certain embodiments, an antigen-binding protein or fragment thereof may compete with one or more antigen-binding proteins, including M0122, M0123, M0124, M0228, and M0251. In certain embodiments, the antigen-binding protein or a fragment thereof comprises a single-stranded variable fragment (scFv), a Fab fragment, or a VHH. In a particular embodiment, the antigen-binding protein or fragment thereof comprises a CDR-H3 having at least 80% similarity to the sequences 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 a particular embodiment, the antigen-binding protein or fragment thereof comprises a CDR-H3 having at least 80% identity with the sequences 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 a particular embodiment, 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 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 a particular embodiment, VH has at least 80% similarity to the sequence of the group consisting of SEQ ID NOs. 25, 26, 27, 29, and 31, and / or VL has at least 80% similarity to the sequence of the group consisting of SEQ ID NOs. 28, 30, and 32. In a particular embodiment, VH has at least 80% identity with the sequence of the group consisting of sequence numbers 25, 26, 27, 29, and 31, and / or VL has at least 80% identity with the sequence of the group consisting of sequence numbers 28, 30, and 32.

[0020] In certain embodiments, antigen-binding proteins or fragments thereof can penetrate Bruch's membrane. In certain embodiments, antigen-binding proteins or fragments thereof can inhibit choroidal C3 activity. In certain embodiments, the antigen-binding protein or fragment thereof includes molecular weights of about 60 kDa or less, for example, 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, and about 15 kDa or less. In certain embodiments, the antigen-binding protein or fragment thereof includes molecular weights of about 20 kDa to about 30 kDa. In certain embodiments, the antigen-binding protein or fragment thereof includes molecular weights of about 10 kDa to about 20 kDa. In certain embodiments, the antigen-binding protein or fragment thereof includes molecular weights of about 25 kDa. In certain embodiments, the antigen-binding protein or fragment thereof includes molecular weights of about 15 kDa.

[0021] In one embodiment, the present disclosure relates to a method for detecting one or both of C3 and C3b in a biological sample, (a) A step of contacting the sample with at least one antigen-binding protein or fragment thereof as described above, (b) A step that enables the formation of a complex between one or both of C3 and C3b in the sample and an antigen-binding protein or a fragment thereof, (c) A step of detecting the antigen-binding protein or a fragment thereof, The present invention provides a method comprising the following: In a preferred embodiment, the antigen-binding protein or a fragment thereof can be bound to complement C3 and C3b. In one embodiment, the antigen-binding protein or a 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] Biological specimens may be tissue specimens, such as retinal tissue from human subjects, including fixed tissue specimens. Fixed tissue specimens may be formalin-fixed and paraffin-embedded tissue specimens. In one embodiment, a kit for detecting C3 is provided, comprising the aforementioned antigen-binding protein or a fragment thereof and instructions for use. The above-mentioned and other features and advantages of the present invention will be better understood from the following detailed description of exemplary embodiments taken in conjunction with the accompanying drawings. Copies of this patent or publication of the patent application and color drawings(s) will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0023] [Figure 1] This diagram shows the three complement pathways that converge at C3: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). [Figure 2] This diagram shows the process for creating an anti-C3 antibody library. [Figure 3A-3B] This figure shows ELISA assays that confirm a superior immune response to C3 in rabbits and llamas. Figure 3A shows an ELISA assay testing for C3 protein isolated from human plasma. Figure 3B shows an ELISA assay testing for the presence of anti-C3 antibodies in the serum of rabbits (upper panel) and llamas (lower panel) injected with the isolated human C3 shown in Figure 3A. [Figure 4] Figure 4A shows a summary of the anti-C3 antibody library, and Figure 4B shows the diversity of CDR-H3 amino acid lengths. [Figure 5] This diagram shows the screening process for anti-C3 antibodies. [Figure 6] This figure 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. [Figures 7A-7D]This figure shows that four anti-C3 antibodies, which inhibit all three complement pathways, recognize three different epitopes on C3. Figure 7A shows a competitive assay showing no competition between M0122 and each of the other three anti-C3 antibodies. Figure 7B shows a competitive assay showing no competition between M0124 and each of the other three anti-C3 antibodies. Figure 7C shows a competitive assay showing competition between M0228 and M0251, but no competition between M0124 and M0122. Figure 7D shows a competitive assay showing competition between M0123 and M0251, and between M0123 and M0228, but no competition between M0124 and M0122. [Figures 8A-8B] This figure shows that M0122, M0124, and M0228 directly bind to both C3 and C3b. Figure 8A shows an ELISA assay demonstrating that M0122, M0124, and M0228 directly bind to C3. Figure 8B shows an ELISA assay demonstrating that M0122, M0124, and M0228 directly bind to C3b. [Figure 9A-9B] This figure shows that M0122, M0124, and M0228 strongly inhibit both the classical and alternative pathways. Figure 9A shows that M0122, M0124, and M0228 strongly inhibit the classical pathway. Figure 9B shows that M0122, M0124, and M0228 strongly inhibit the alternative pathway. [Figure 10] This figure shows the affinity parameters for M0122, M0124, and M0228. [Figure 11] A schematic diagram of the anatomical structure of the retina and choroid, such as Bruch's membrane, is shown. The anti-C3 scFv antibody of this 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] This figure shows the negative relationship between hydrodynamic radius and permeability, and the complement-binding therapy shown in comparison to the scFv of this disclosure. [Figure 13]Figures 13A and 14B show a comparison of scFv and APL-2 substitutes for penetrating the Bruch membrane. Figure 14A shows barium iodide staining (PEG), and Figure 13B shows Coomassi staining (protein). The APL2 substitute contains one APL-1 moiety on a 40 kDa linear PEG. SC - sample chamber, DC - diffuser chamber, LC - loading control (initial concentration of SC). [Figure 14A-14C] This figure shows that M0122, M0124, and M0251 strongly inhibit the classical, alternative, and lectin pathways in cyanose serum. Figure 14A shows that M0122, M0124, and M0251 strongly inhibit all three pathways. Each antibody was used at a concentration of 2 μM. Figure 14B shows that M0122, M0124, and M0251 strongly inhibit the classical pathway. Figure 14C shows that M0122, M0124, and M0251 strongly inhibit the alternative pathway. [Figures 15A-15B] This figure shows that M0122, M0124, and M0251 bind to cyno C3. Figure 15A shows M0122 and M0124. Figure 15B shows M0251. [Figure 16] This figure shows that M0122, M0123, and M0124 strongly inhibit the lectin pathway. [Modes for carrying out the invention]

[0024] Antigen-binding proteins having binding specificity to complement C3 and C3b, a complement C3 cleavage product, are provided. Methods for treating or preventing complement C3-mediated diseases and disorders are also provided. In certain embodiments, the antigen-binding proteins described herein can inhibit the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The antigen-binding proteins described herein can inhibit all three pathways simultaneously. The antigen-binding proteins described herein can inhibit all three pathways in the choroid of the eye.

[0025] In general, the nomenclature used in relation to 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 carried out in accordance with conventional methods well known in the art, and, unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in the art or as described herein. The nomenclature used in relation to analytical chemistry, organic synthesis chemistry, and medicinal chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, preparation, formulation, delivery, and patient treatment of pharmaceuticals.

[0026] Unless otherwise defined herein, scientific and technical terms used herein have meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over dictionary or foreign definitions. Unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. The use of “or” shall mean “and / or” unless otherwise specified. The use of the term “including,” as well as other forms such as “includes” and “included,” is non-restrictive. To facilitate understanding of this invention, we will first define certain terms.

[0027] Antigen-binding protein As used herein, the terms “antibody” or “antigen-binding protein” refer to immunoglobulin molecules that specifically bind to an antigen or epitope, or are immunologically reactive, and include 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-strand variable fragments (scFv), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody, VHH). The term “antibody” also includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabody, peptide body, chimeric antibody, fully human antibody, humanized antibody, and heteroconjugate antibody (e.g., bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv). Unless otherwise stated, the term “antibody” should be understood to encompass its functional antibody fragments. As used herein, the term “antibody fragment” 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 a parent antibody. Non-limiting examples of such antibody mimics include fluctuation-regulating affinity proteins (FLAPs), monobodies, and affimers. An antibody mimic may comprise one, two, three, four, five, or six CDRs, as described herein.

[0028] As used herein, "Fab fragment" is an antibody fragment comprising a variable light chain (VL) domain and a constant domain of the light chain (CL), as well as a light chain fragment containing a variable heavy chain (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, “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 a linker recognized in any suitable art. Such linkers include, but are not limited to, repeating GGGGS amino acid sequences or variants thereof. While scFv generally do not contain an antibody constant domain region, the scFv of this disclosure can be linked to or conjugated to an antibody constant domain region (e.g., an antibody Fc domain) to alter various properties of the scFv, including, but not limited to, an increased serum or tissue half-life. scFv generally have a molecular weight of about 25 kDa and a hydrodynamic radius of about 2.5 nm. As used herein, “VHH,” “nanobody,” or “heavy-chain-only antibody” are antigen-binding proteins containing a single heavy-chain variable domain derived from camelid species, including camels, llamas, and alpacas. 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-adjacent sequence of amino acids within the antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region contains three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region contains three CDRs (CDR-L1, CDR-L2, CDR-L3). In the art, “framework region” or “FR” is known to refer to the non-CDR portion of the heavy and light chain variable regions. Generally, each heavy chain variable region contains four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each light chain variable region contains four FRs (FR-L1, FR-L2, FR-L3, and FR-L4). For VHH antibodies, only three heavy chain CDRs are present, and no light chain CDRs are present.

[0030] The precise amino acid sequence boundaries of a given CDR or FR can be easily determined by one of several well-known schemes, including: 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); and Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp This includes the schemes described in 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 Jun. 8; 309(3):657-70 (AHo numbering scheme).

[0031] The boundaries of a given CDR or FR can 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. Numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertions accommodated by inserting letters, 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] Antibody variants provided herein can be generated by introducing deletions, substitutions, additions, and / or modifications to the framework and / or CDR. The antibody variants can then be tested for desired functions using the methods described herein. Any combination(s) of deletions, substitutions, additions, modifications, and insertions can be applied to antigen-binding proteins or fragments thereof, provided that the generated variants possess the desired features that can be screened using appropriate methods.

[0033] As used herein, “conservative substitution” refers to modifications that maintain the functional properties of the parent antibody. For example, a conservative amino acid substitution includes substitutions 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. Substitute with n(K); substitute isoleucine(I) with leucine(L); substitute methionine(M) with leucine(L); substitute phenylalanine(F) with tyrosine(Y); substitute serine(S) with threonine(T); substitute tryptophan(W) with tyrosine(Y); substitute phenylalanine(F) with tryptophan(W); and / or substitute valine(V) with leucine(L), and vice versa.

[0034] Therefore, unless otherwise specified, a given antibody or region thereof, for example, its variable region's "CDR" or "complementary determination region," or individual specific CDRs (e.g., "CDR-H1, CDR-H2"), should be understood to encompass a certain (or specific) complementary determination region as defined by any known scheme. Similarly, unless otherwise specified, a given antibody or region thereof, for example, its variable region's "FR" or "framework region," or individual specific FRs (e.g., "FR-H1," "FR-H2"), should be understood to encompass a certain (or specific) framework region as defined by any known scheme. In some cases, a scheme for identifying a specific CDR or FR, such as Kabat, Chothia, Contact, IMGT, or AHo method, is used to identify the CDR. In other cases, a specific amino acid sequence of the CDR or FR is given.The CDR and FR numbering schemes are 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); and 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. Further details are provided in 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 "competing" or "cross-competing" 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 an antibody molecule to a target, such as human C3 and / or C3b. The interference with binding can be direct or indirect (e.g., via allosteric modulation of the antigen-binding molecule or target). The degree to which an antigen-binding molecule can interfere with the binding of another antigen-binding molecule to a target, and thus whether it can be said to compete, can be determined using a competitive binding assay, such as a FACS assay, ELISA or BIACORE assay. In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, a first antigen-binding molecule competes for binding to a target with a second antigen-binding molecule if the binding of the first antibody molecule to the target is reduced by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more 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 to which it binds. As will be readily understood by those skilled in the art, antibody or antigen-binding protein affinity can be reported as the dissociation constant (K D ) in molar concentration (M). The antibodies of the present disclosure can have K -5 values in the range of 10 -12 to 10 D M. High-affinity antibodies have K -9 values of 10 D M (1 nanomolar, nM) or less. For example, a high-affinity antibody can have a K D value in the range of about 1 nM to about 0.01 nM. A high-affinity antibody can have a K D 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. Very high-affinity antibodies have 10 -12K below M (1 picomole, pM) D It has a value. Antibodies with weak or low affinity are 10 -1 ~10 -4 K in the range of M D It can have a value. Low affinity antibodies are 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 M etc. -4 M or higher K D It can have a value.

[0037] In certain embodiments, the antigen-binding protein of this disclosure is approximately 10 to C3 and C3b. -8 M ~ about 10 -14 It has binding affinity for M. In certain embodiments, the antigen-binding protein of this disclosure has about 10% binding affinity for C3 and C3b. -10 M ~ about 10 -12 It has binding affinity for M. In certain embodiments, the antigen-binding protein of this disclosure has at least about 10 to 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 binding affinity for M. In certain embodiments, the antigen-binding protein or fragment thereof has approximately equivalent binding affinities to C3 and C3b. For example, but not limited to, the antigen-binding protein or fragment thereof has approximately 10% binding affinities to C3. -10 The binding affinity of M, and approximately 10 for C3b. -10 It may include binding affinity for M. In certain embodiments, the antigen-binding protein or a fragment thereof has about 10 to C3. -11 The binding affinity of M and approximately 10 for C3b -11 It includes binding affinity for M. In certain embodiments, the antigen-binding protein or fragment thereof has about 10 to C3. -12 The binding affinity of M and approximately 10 for C3b -12 Includes binding affinity of M. In certain embodiments, the binding affinity to C3 is within 10 times the binding affinity to C3b. For example, but not limited to, the antigen-binding protein or fragment thereof has a binding affinity of about 10 times that of C3b. -10 The binding affinity of M and approximately 10 for C3b -11 It may include binding affinity for M. In certain embodiments, the antigen-binding protein or a fragment thereof has about 10 to C3. -11 M's binding innocence and approximately 10% of C3b's affinity -12 Includes binding affinity of M.

[0038] In certain embodiments, the antigen-binding protein or fragment thereof involves cross-reactivity with cynomolgus monkey C3. Since cynomolgus monkey (Macaca fascicularis) C3 is 95.1% identical to human C3, the cross-reactivity allows for preclinical and toxicity testing of the antigen-binding proteins of this disclosure in relevant animal models. To avoid any ambiguity, and unless otherwise indicated, C3 as used herein refers to the human complement component 3 of UniProt P01024 and the nucleic acid sequence encoding its protein. C3b is derived from natural C3 and is the larger of the two elements formed by the cleavage of C3. In certain embodiments, the antigen-binding protein of this disclosure is monovalent and, when measured using biolayer interferometry (BLI), has a K content of approximately 200 nM or less. D It binds to human C3 and C3b. In a particular embodiment, K D This is approximately 200 pM or less, for example, approximately 100 pM, approximately 10 pM, approximately 1 pM, or approximately 0.1 pM.

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

[0040] Anti-complement C3 antigen-binding protein In one embodiment, the disclosure provides an antigen-binding protein having binding specificity to complement C3 protein. In certain embodiments, the anti-C3 antigen-binding protein is scFvs, Fab fragment, or VHH. Exemplary anti-C3 antigen-binding protein (CDR) sequences are listed in Table 1 below. Exemplary anti-C3 antigen-binding protein variable heavy chain domains and variable light chain 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 are derived from rabbits immunized with human C3 protein and are wild-type rabbit sequences. The exemplary VHH domains of M0228 and M0251 are derived from llamas immunized with human C3 protein and are wild-type llama sequences.

[0041] [Table 1]

[0042] [Table 2]

[0043] In certain embodiments, the anti-C3 antigen-binding proteins of this disclosure include at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence similarity or identity with any of the sequences in Table 1 or Table 2.

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

[0045] The retina has substantial physical barriers that can prevent large molecules, such as full-length immunoglobulins, from penetrating deeper, potentially leading to reduced therapeutic efficacy (Jackson et al. Invest Ophthalmol Vis Sci. 2003;44(5): 2141-6). Smaller antibody derivatives, in contrast, may penetrate deeper into the retina. Exemplary antibody derivatives with molecular weights of approximately 60 kDa or less include, but are not limited to, antibody fragments, such as Fab, Fab' fragments, scFab, scFv, Fv fragments, nanobodies, VHH, dAb, V-Nar, sdAb, sdFv, and bispecific and bivalent antibodies, such as single-stranded diabodies (scDb), or DART. In certain embodiments, the anti-C3 antigen-binding protein of the Disclosure has a molecular weight of about 60 kDa or less, for example, 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 Disclosure can penetrate or diffuse through the Bruch membrane, partly because their size is low enough to facilitate penetration. In certain embodiments, the size of the antigen-binding proteins of the Disclosure is measured by molecular weight. In certain embodiments, the antigen-binding proteins of the Disclosure have a molecular weight of less than about 60 kDa. In certain embodiments, the antigen-binding proteins of the 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 Disclosure are about 25 kDa. In certain embodiments, the antigen-binding proteins of the Disclosure are about 15 kDa. In certain embodiments, the size of the antigen-binding proteins of the Disclosure is measured by their hydrodynamic radius. In certain embodiments, the antigen-binding proteins of the Disclosure have a hydrodynamic radius of less than or equal to about 3.0 nm. In certain embodiments, the antigen-binding proteins of the Disclosure have a hydrodynamic radius of less than or equal to about 2.5 nm. In certain embodiments, the antigen-binding protein of this 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 this 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 there is an excess of competing antigen-binding proteins, it reduces the specific binding of the antigen-binding protein or its fragments to C3 and / or C3b, i.e., cross-blocks binding by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more, as described herein. In certain embodiments, the binding of the antigen-binding proteins or fragments thereof described herein is reduced by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more in the presence of competing antigen-binding proteins.

[0048] Complement C3 is a large protein with a molecular size of 185 kilodaltons, composed of 13 distinct domains. During complement activation, C3 undergoes proteolytic cleavage and structural modification at different sites. The C3-derived fragments exert different effector functions and form convertases that create amplification loops in the three complement pathways. The classical and lectin pathway C3 convertase, C4bC2a, cleaves full-length C3 into C3b and anaphylatoxin C3a. The alternative pathway also produces C3b and C3a, but utilizes the alternative pathway C3 convertase, C3bBb. Furthermore, the complement pathway may produce additional C3 degradation products. 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). Further C3 protein degradation products, such as C3d, bind to complement receptor 2 (CR2) and may play a crucial role in regulating the B cell cycle. 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, C4BPα chain, C4BPβ chain, phycolin-1, mannose-binding lectin (MBL), MBL-related serine protease-1 (MASP-1), and MBL-related serine protease-2 (MASP-2). The complement pathway and its various components are described in more detail in Noris et al. Semin Nephrol. 2013; 33(6): 479-492.

[0049] In certain embodiments, the Disclosure provides an anti-C3 antigen-binding protein that can bind to both C3 and C3b. In certain embodiments, the anti-C3 antigen-binding protein of the Disclosure has a binding affinity to C3a, iC3b, C4, C4b, C5, and / or C5b that is weaker than the binding affinity to C3 and C3b. In certain embodiments, the anti-C3 antigen-binding protein of the Disclosure has a binding affinity to C3a, iC3b, C4, C4b, C5, and / or C5b of about 10 -4 The binding affinity includes M or weaker. In certain embodiments, the anti-C3 antigen-binding proteins of this disclosure do not have binding affinity to C3a, iC3b, C4, C4b, C5, and / or C5b. As used herein, “no binding affinity” means no detectable binding affinity to background by one or more binding affinity assays known in the art, such as ELISA assays, but not limited to these.

[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 disclosure inhibits the activity of C3 convertase. In certain embodiments, the antigen-binding protein of the disclosure inhibits the C3 convertase amplification loop. In certain embodiments, the anti-C3 antibody of this disclosure is expected to have better efficacy and safety in treating GA or other eye disorders compared to other therapies, due to the following characteristics listed below.

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

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

[0053] Expression of antigen-binding polypeptides In one embodiment, polynucleotides encoding binding polypeptides (e.g., antigen-binding proteins) disclosed herein are provided. Methods for producing binding polypeptides, comprising expressing these polynucleotides, are also provided. The polynucleotides encoding the conjugated polypeptides disclosed herein are typically inserted into an expression vector for introduction into host cells that can be used to produce a desired amount of the requested antibody or fragment thereof. Thus, in certain embodiments, the present invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0054] The terms “vector” or “expression vector” are used herein to mean a vector used in accordance with the present invention as a vehicle for introducing and expressing a desired gene into a cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, a vector suitable for the present invention includes a selection marker, a suitable restriction site to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells. Numerous 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 papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (e.g., RSV, MMTV, MOMLV, etc.), or SV40 virus. Others involve the use of polycistrone systems having an internal ribosome binding site. Furthermore, cells into which the DNA has been incorporated into their chromosomes can be selected by introducing one or more markers that enable selection of the transfected host cell. The markers can provide prototrophicity to a trophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selection marker gene can be directly linked to the expressed DNA sequence or introduced into the same cell by co-transformation. Further elements may be required to optimize mRNA synthesis. These elements may include signal sequences, splice signals, and transcription promoters, enhancers, and termination signals. In some embodiments, the cloned variable region gene is inserted into an expression vector along with the synthesized heavy chain and light chain constant region genes (e.g., human constant region genes) as discussed above.

[0055] In other embodiments, the conjugated polypeptide may be expressed using a polycistronic construct. In such expression systems, multiple gene products of interest, such as the heavy and light chains of antibodies, can be produced from a single polycistronic construct. These systems advantageously utilize internal ribosome entry sites (IRESs) to provide relatively high levels of polypeptide in eukaryotic host cells. A compatible IRES sequence is 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 understand that such expression systems can be used to effectively generate the entire range of polypeptides disclosed in this 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. The introduction of plasmids into host cells can be achieved by various 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 envelope DNA, microinjection, and infection with intact viruses. See Ridgway, AAG “Mammalian Expression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introduction into the host is possible by electroporation. Transformed cells are grown under conditions suitable for light chain and / or heavy chain production and assayed for heavy chain and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescence-activated cell sorting (FACS), and immunohistochemistry.

[0057] As used herein, the term “transformation” should be used in a broad sense to refer to the introduction of exogenous DNA into recipient host cells that alters the genotype and consequently brings about a change in the recipient cells. Genetically modified recipient cells may contain exogenous sequences by transient or stable transformation. For example, exogenous sequences may be stably incorporated into the recipient cell’s genomic sequence, at a target site, or at a random site. Cells modified by gene editing methods (e.g., homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9, or TALEN methods) are within the scope of this disclosure. In certain embodiments, stable cell lines are generated for the production of antigen-binding proteins or fragments thereof. This advantageously results in consistent production of uniform quality and yield of antigen-binding proteins or fragments thereof. Along with these same strains, “host cells” refers to cells constructed using recombinant DNA technology and transformed with a vector encoding at least one heterologous gene. In describing the process of isolating polypeptides from recombinant hosts, the terms “cells” and “cell culture” are used interchangeably to indicate the source of antibodies unless otherwise specified. In other words, the recovery of polypeptides from “cells” may mean from whole cells centrifuged or from cell cultures containing both culture medium and suspended cells.

[0058] In one embodiment, the host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine the specific host cell line best suited to the desired gene product expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR-negative), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with SV40T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). In one embodiment, the cell line provides a modified glycosylation, e.g., non-fucosylation, of the antibody expressed therefrom (e.g., PER.C6® (Crucell) or FUT8 knockout CHO cell line (Potelligent® cells) (Biowa, Princeton, NJ)). Host cell lines are typically available from commercial services, e.g., U.S. tissue culture collections, or from published literature.

[0059] In vitro production allows for scale-up to provide large quantities 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 an airlift reactor or a continuous stirring reactor, or cell culture immobilized or incorporated in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the polypeptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno)affinity chromatography.

[0060] The genes encoding the antigen-binding proteins characterized in this invention may also be expressed in non-mammalian cells such as bacteria, yeast, insects, 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 that are easily transformed include Enterobacteriaceae such as Escherichia coli or Salmonella; Bacillus species such as Bacillus subtilis; Streptococcus pneumoniae; and Haemophilus influenzae. Furthermore, it is understood that when expressed in bacteria, the proteins may become part of inclusion bodies. The proteins must be isolated, purified, and then assembled into functional molecules.

[0061] In addition to prokaryotes, eukaryotic microorganisms can also be used. Budding yeast (Saccharomyces cerevisiae), or common baker's yeast, is the most commonly used of the eukaryotic microorganisms, but several other strains are also readily available. For expression in Saccharomyces, 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 and provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, e.g., ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Subsequently, the presence of trp1 damage as a feature of the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan. Therefore, in one embodiment, a method for producing the above-mentioned antigen-binding protein or fragment thereof, i) A step of culturing host cells under conditions that enable the expression of the proteins described herein, ii) A step to recover the protein, and optionally iii) A step of further purifying and / or modifying and / or formulating the protein. A method including this is provided.

[0062] Method of administering antigen-binding proteins Methods for preparing and administering antigen-binding proteins (e.g., the antigen-binding proteins disclosed herein) to a subject are well known to those skilled in the art or readily determined by those skilled in the art. The routes of administration of the antigen-binding proteins disclosed herein may be oral, parenteral, inhaled, topical, or intraocular. As used herein, the term parenteral includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. As used herein, the term intraocular includes, but is not limited to, subconjunctival, intravitreous, retrobulbar, or anterior chamber administration. As used herein, the term topical includes, but is not limited to, administration in the form of liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.

[0063] In certain embodiments, the antigen-binding proteins of this disclosure are administered intraocularly. Delivery of therapeutic compounds to different structures of the eye, such as the retina, is challenging. Challenges include, but are not limited to, several restrictive ocular barriers, the tear mechanism including blinking and washing away 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 this disclosure can overcome these challenges. The antigen-binding proteins of this 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 the size of the antigen-binding protein of this disclosure compared to the full-length antibody, the more therapeutic antibody can be delivered per injection. This allows for higher concentrations of antibody to the eye. The smaller the size of the antigen-binding protein of this disclosure, the better their penetration into disease-associated tissue, namely the choroidal region of the eye. The antigen-binding protein can penetrate one or more layers of the choroidal region, including the Hallerian layer, Sattlerian layer, choroidal capillaries, and Bruch's membrane, thereby targeting complement C3 and C3b within those layers of the choroidal region.

[0064] In certain embodiments, intraocular administration is achieved using a drug delivery device, such as a choroidal drug delivery device or a subretinal drug delivery device. The choroidal administration procedure involves the administration of a drug into the suprachoroidal space of the eye and is typically performed using a choroidal drug delivery device, such as a microinjector having a microneedle (see, e.g., Hariprasad, Retinal Physician; 2016; 13: 20-23; Goldstein, 2014, Retina Today 9(5): 82-87; each is incorporated herein by reference in its entirety). Choroidal drug delivery devices that can be used to deposit the antigen-binding proteins of this disclosure in the suprachoroidal space include, but are not limited to, choroidal drug delivery devices manufactured by Clearside® Biomedical, Inc. (see, e.g., Hariprasad, 2016, cited above). Subretinal drug delivery devices that can be used to deposit the antigen-binding protein of this disclosure into the subretinal space via the epichoroidal space include, but are not limited to, subretinal drug delivery devices manufactured by Janssen Pharmaceuticals, Inc. (see, for example, International Patent Application Publication No. 2016 / 040635).

[0065] In certain embodiments, intraocular administration is achieved via an intravitreous route. Intravitreous administration is often performed using a syringe and a 27-gauge to 30-gauge needle (see, for example, Jiang et al., cited above). All of these dosage forms are clearly considered to be within the scope of this disclosure, but the dosage form is a solution for injection, particularly a solution for intravitreal injection. Typically, a pharmaceutical composition suitable for injection may include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizer (e.g., human albumin). However, in other ways compatible with the teachings herein, modified antibodies can be delivered directly to the site of a harmful cell population, thereby increasing the exposure of diseased tissue to the therapeutic agent.

[0066] In certain embodiments, the antigen-binding proteins of the Disclosure are formulated in low-viscosity solutions. The viscosity of the solutions is measured in centipoise (cP) units. High-viscosity antibody solutions may present challenges for administering the antigen-binding proteins of the Disclosure to the eyes. For example, solutions with a viscosity exceeding 50 cP may be difficult to administer with a fine needle due to high back pressure. Therefore, it is desirable to formulate the antigen-binding proteins of the Disclosure in low-viscosity solutions. In certain embodiments, the antigen-binding proteins and their pharmaceutical compositions of the Disclosure have a viscosity of about 1 cP to about 50 cP. In certain embodiments, the antigen-binding proteins and their pharmaceutical compositions of the Disclosure have a viscosity of about 20 cP, about 15 cP, about 10 cP, about 5 cP, about 4 cP, about 3 cP, about 2 cP, or less than or equal to 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] Preparations 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, alcohol / aqueous solutions, emulsions, or suspensions, and include physiological saline and buffering media. In the compositions and methods of this disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer, or 0.8% physiological saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's glucose, dextrose and sodium chloride, Ringer's lactate, and fixative oils. Intravenous vehicles include, but are not limited to, fluids and nutritional supplements, electrolyte supplements such as those based on Ringer's glucose. Preservatives such as antimicrobial agents, antioxidants, chelating agents, and inert gases, and other additives may also be present. In certain embodiments, a pharmaceutical composition suitable for injectable use comprises a sterile aqueous solution (if water-soluble) or dispersion, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. In such cases, the composition must be sterile and fluid enough to be readily injectable. It should be stable under manufacturing and storage conditions and should also be resistant to contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of a coating agent such as lecithin, maintaining the required particle size in the case of a dispersant, and the use of a surfactant.

[0068] Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, such as sugars, polyalcohols, or sodium chloride, may also be included in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0069] In any case, a sterile injectable solution can be prepared by incorporating the required amount of the active compound (e.g., an antigen-binding protein or a fragment thereof) into a suitable solvent, along with one or a combination of the components listed herein as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method typically involves vacuum drying and lyophilization, yielding a powder of the active ingredient and any additional desired components from its 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 sterile conditions according to methods known in the art.

[0070] The effective dose of the compositions disclosed herein varies depending on a number of different factors for treating the conditions described above, including the means of administration, the target site, the patient's physical condition, whether the patient is human or animal, other pharmaceuticals administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The treatment dose can be gradually increased using conventional methods known to those skilled in the art to optimize safety and efficacy. As previously discussed, the antigen-binding proteins, immunoreactive fragments, or recombinants thereof of this disclosure can be administered in pharmaceutically effective amounts for in vivo treatment of mammalian disorders. In this regard, it is understood that the disclosed antigen-binding proteins are formulated to facilitate the administration of activators and to promote stability.

[0071] The pharmaceutical compositions of this disclosure typically comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, a non-toxic buffer, or a preservative. For the purposes of this application, a pharmaceutically effective amount of a modified antigen-binding protein, its immunoreactive fragment, or recombinant, conjugated or unconjugated to a therapeutic agent, must be retained in an amount sufficient to achieve effective binding to an antigen and to achieve a benefit, for example, to improve the symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the modified binding polypeptide can typically interact with a selected immunoreactive antigen on neoplastic or immunoreactive cells, increasing the death of those cells. Naturally, the pharmaceutical compositions of this disclosure can be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0072] To the extent of this disclosure, the antigen-binding proteins of this disclosure may be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect, in accordance with the treatment methods described above. The antigen-binding proteins of this disclosure may be administered to such humans or other animals in a common dosage form prepared by combining the antibodies of this disclosure with conventional pharmaceutically acceptable carriers or diluents according to known techniques. It will be recognized by those skilled in the art that the form and characteristics of a pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient to be combined, the route of administration, and other well-known variables. Those skilled in the art will further understand that a cocktail containing one or more species of the binding polypeptides described in this disclosure may prove particularly effective.

[0073] The biological activity of the pharmaceutical compositions defined herein can be determined by complement inhibition assays, for example, by enzyme immunoassays for determining functional classical pathway, lectin pathway, and alternative complement pathway activity in human serum, though not limited to these assays. In certain embodiments, the inhibitory activity of the pharmaceutical compositions defined herein can be evaluated using the Wieslab® complement system screen (Euro Diagnostica AB, Malmo, Sweden). Functional assays to study the ability of the antibodies of this disclosure to inhibit the complement pathway can be performed using purified complement components from which the enzyme complex is reconstituted on the surface of erythrocytes or an artificial matrix, 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 for evaluating the ability of compounds 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 erythrocyte hemolysis in the presence of the antigen-binding proteins of this disclosure compared to the level of erythrocyte hemolysis in the absence of the antigen-binding proteins of this disclosure. In certain embodiments, complement-dependent hemolysis mediated by the classical pathway can be measured using antibody-sensitized sheep erythrocytes. In certain embodiments, complement-dependent hemolysis mediated by alternative pathways can be measured using antibody-sensitized rabbit erythrocytes, 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 MAC formation in the presence of the antigen-binding protein of this disclosure compared to MAC formation in the absence of the antigen-binding protein of this disclosure. A MAC assay for IgM-mediated activation of the classical complement pathway in human serum results in MAC deposition on an IgM-coated ELISA plate. MAC formation can be detected using an alkaline phosphatase-labeled antibody against C5b-9. In the presence of the antigen-binding protein of this disclosure, the ELISA signal is dose-dependently reduced. To test alternative pathways, a MAC assay for LPS-mediated activation of alternative complement pathways in human serum can be used for MAC deposition on an LPS-coated ELISA plate. Suitable MAC assays include, but are not limited to, the Pacific biomarker complement membrane invasion complex (SC5b-9) ELISA assay.

[0076] As used herein, “efficacy” or “in vivo efficacy” refers to the response to treatment with the pharmaceutical composition of this disclosure, for example, using standardized response criteria such as standard ophthalmic response criteria. The success of treatment or in vivo efficacy using the pharmaceutical composition of this disclosure refers to the effectiveness of the composition for its intended purpose, i.e., its desired effect, i.e., the ability of the composition to cause inhibition of the complement pathway in the eye. In vivo efficacy can be monitored by established standard methods for various eye diseases. Monitoring methods include, but are not limited to, the Amsler grid test, optomoscopy, fundus microscopy, computed tomography, and optical coherence tomography. Furthermore, various disease-specific clinical chemistry parameters and other established standard methods may be used.

[0077] Antibody Engineering and Optimization The antigen-binding proteins of this disclosure may be manipulated or optimized. As used herein, “optimized” or “optimized” means a modification of an antigen-binding protein to improve one or more functional properties. Modifications include, but are not limited to, the deletion, substitution, addition, and / or modification of one or more amino acids in the antigen-binding protein. As used herein, the term “functional property” means a property of an antigen-binding protein whose improvement (e.g., compared to a conventional antigen-binding protein) is desirable and / or advantageous to those skilled in the art for, for example, improving the manufacturing properties or therapeutic efficacy of the antigen-binding protein. In one embodiment, the functional property is stability (e.g., thermal stability). In another embodiment, the functional property is solubility (e.g., under cellular conditions). In yet another embodiment, the functional property is aggregation behavior. In yet another embodiment, the functional property is protein expression (e.g., in prokaryotic cells). In yet another embodiment, the functional property is refolding behavior after solubilization of the inclusion body in the manufacturing process. In a particular embodiment, the functional property is not an improvement in antigen-binding affinity. In another embodiment, an improvement in one or more functional properties has no substantial effect on the binding affinity of the antigen-binding protein.

[0078] In certain embodiments, the antigen-binding protein of the Disclosure is an scFv, and the antigen-binding protein is optimized by identifying preferred amino acid residues to be substituted, deleted, and / or added at a desired amino acid position (e.g., an amino acid position identified by comparing a database of scFv sequences having at least one desirable characteristic, such as those selected by a quality control (QC) assay, with a database of mature antibody sequences, e.g., the Kabat database). Accordingly, the Disclosure further provides a “concentration / exclusion” method for selecting specific amino acid residues. Furthermore, the Disclosure provides a method for manipulating an antigen-binding protein (e.g., an scFv) by mutating a specific framework amino acid position identified using the “functional consensus” approach described herein. In certain embodiments, the framework amino acid position is mutated by substituting an existing amino acid residue with a residue found to be a “concentrated” residue using the “concentration / exclusion” analytical method described herein. In one embodiment, the Disclosure provides a method for identifying amino acid sites for mutation in a single-chain antibody (scFv) having VH and VL amino acid sequences, the method comprising: a) inputting the scFv VH, VL, or VH and VL amino acid sequence into a database containing a large 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 in the database; b) comparing the amino acid sites in the scFv VH or VL amino acid sequence with the corresponding sites in the antibody VH or VL amino acid sequences in the database; c) determining whether the amino acid sites in the scFv VH or VL amino acid sequence are occupied by amino acid residues that are conserved at the corresponding sites in the antibody VH or VL amino acid sequences in the database; and d) if the amino acid sites are occupied by amino acid residues that are not conserved at the corresponding sites in the antibody VH or VL amino acid sequences in the database, identifying the amino acid sites in the scFv VH or VL amino acid sequence as amino acid sites for mutation.ScFV optimization is described in more detail in International Publication Nos. 2008110348, 2009000099, 2009000098, and 2009155725, all of which are incorporated herein by reference.

[0079] Humanization: In certain embodiments, the antigen-binding proteins of this disclosure may be humanized. As used herein, the term “humanized” refers to a non-human donor antibody modified to increase its 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 a CDR of a non-human donor antibody (e.g., a rabbit or llama antibody CDR) onto a human or humanized antibody receptor framework region, such as a soluble and stable light-chain and / or heavy-chain human antibody framework region. Common methods for grafting CDRs onto human receptor frameworks are disclosed by Winter in U.S. Patent No. 5,225,539 and Queen et al. in International Publication No. 199007861, and are incorporated herein by reference. Appropriate receptor framework regions can exhibit superior functional properties, such as improved solubility and stability. In certain embodiments, the antigen-binding proteins of this disclosure are rabbit antibodies. The CDR of the rabbit antibody may be grafted onto a universal receptor framework region, such as the framework region described in International Publication No. 2009155726, which is incorporated herein by reference.

[0080] In certain embodiments, the human framework for humanizing / stabilizing non-human antibodies, or for stabilizing human antibodies, relates to the substitution of a κ-conjugation segment in a κ-λ chimeric variable light chain domain with a λ-conjugation segment, resulting in a κ-λ chimeric variable light chain domain with improved protein stability and reduced aggregation tendency. Furthermore, to further improve protein stability and further reduce aggregation tendency, the framework relates to mutations and substitutions of the κ consensus residue at position AHo101 with a λ consensus residue to assist in the packing of the λ-conjugation segment in the κ-λ chimeric variable light chain domain. Further details regarding these human framework regions are described in International Publication Nos. 2014206561 and 2019057787, which are incorporated herein by reference.

[0081] Methods for treating complement C3-mediated diseases and disorders A method for treating complement C3-mediated diseases and disorders in subjects suffering from such diseases is provided, using the antigen-binding proteins described herein. In certain embodiments, complement C3-mediated diseases or disorders are selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, posterior fibrosis of the lens, 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: atrophic AMD and exudative AMD. Atrophic AMD, also known as non-exudative AMD, is characterized by the presence of drusen (yellow deposits) in the macula. Exudative AMD, also known as exudative AMD or neovascular AMD, is characterized by abnormal proliferation of blood vessels from the submacula choroid. This process, also called choroidal neovascularization, can cause fluid, such as blood, to leak into and around the retina. Geographic atrophy, also known as atrophic AMD or progressive atrophic AMD, is a progressive form of AMD characterized by the progressive and irreversible loss of retinal cells.

[0082] Treating eye diseases such as AMD, as described above, is particularly challenging. As previously stated, the delivery of therapeutic agents to the eye is limited by several barriers, including, but not limited to, the blood-retinal barrier such as the RPE. The ability to penetrate the RPE and enter the choroid of the eye enhances the therapeutic potential of a drug. In certain embodiments, the antigen-binding proteins of this 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 this disclosure to penetrate the RPE and Bruch's membrane improves their therapeutic potential in the treatment of complement C3-mediated diseases or disorders. The antigen-binding proteins of this disclosure can penetrate the RPE and Bruch's membrane, partly because their size is low enough to facilitate penetration. In certain embodiments, the size of the antigen-binding proteins of this disclosure is measured by molecular weight. In certain embodiments, the antigen-binding proteins of this disclosure have a molecular weight of less than approximately 60 kDa. In certain embodiments, the antigen-binding proteins of the Disclosure are approximately 20 kDa to approximately 30 kDa or approximately 10 kDa to approximately 20 kDa. In certain embodiments, the antigen-binding proteins of the Disclosure are approximately 25 kDa. In certain embodiments, the antigen-binding proteins of the Disclosure are approximately 15 kDa. In certain embodiments, the size of the antigen-binding proteins of the Disclosure is measured by their hydrodynamic radius. In certain embodiments, the antigen-binding proteins of the Disclosure have a hydrodynamic radius of less than or equal to approximately 3.0 nm. In certain embodiments, the antigen-binding proteins of the Disclosure have a hydrodynamic radius of less than or equal to approximately 2.5 nm. In certain embodiments, the antigen-binding proteins of the Disclosure have a hydrodynamic radius of less than or equal to approximately 2.0 nm.

[0083] In one embodiment, the present disclosure provides a method for inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP), and the alternative pathway (AP), 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 protein of the present disclosure to inhibit all three complement pathways further improves their therapeutic potential in the treatment of complement C3-mediated diseases or disorders. While we do not wish to be constrained by theory, inhibiting all three complement pathways may improve the therapeutic potential of the antigen-binding protein of the present disclosure by preventing the disease-promoting effect of one active pathway from compensating for the other inactivating pathways.

[0084] In certain embodiments, antigen-binding proteins or fragments thereof can inhibit the activity of the CP, LP, and AP complement pathways to a substantially equal degree. For example, but not limited to, antigen-binding proteins or fragments thereof can inhibit the activity of the CP pathway by at least 80%, the activity of the LP pathway by at least 80%, and the activity of the AP pathway 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 embodiment, the Disclosure provides a method for inhibiting the activity of choroidally localized complement C3 by intraocular administration of an antigen-binding protein or fragment thereof that binds to an epitope on complement C3. Activated complement pathways in the choroidal region of the eye may contribute to complement C3-mediated diseases or disorders. Therefore, an object of the Disclosure is 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 Disclosure inhibits the activity of C3 convertase in the choroidal region of the eye. In certain embodiments, the antigen-binding protein of the Disclosure inhibits the C3 convertase amplification loop in the choroidal region of the eye.

[0086] medical use The present invention also relates to antigen-binding proteins or fragments thereof disclosed herein for use in methods for treating complement C3-mediated diseases or disorders in subjects. All technical features described herein relating to antigen-binding proteins or fragments thereof are applicable.

[0087] kit The present invention also comprises a kit comprising at least one antigen-binding protein or fragment thereof as described herein. In one embodiment, the kit comprises a composition comprising an effective amount of the antigen-binding protein or fragment thereof in a unit dose form. Such a kit may comprise a sterile container comprising the composition, and non-limiting examples of such containers include, but are not limited to, vials, ampoules, bottles, tubes, syringes, and blister packs. In some embodiments, the composition is a pharmaceutical composition, and the container is made of a material suitable for holding pharmaceuticals. In one embodiment, the kit may comprise a second container comprising a first container comprising an antigen-binding protein or fragment thereof in lyophilized form, and a diluent (e.g., sterile water) for reconstitution or dilution of the antigen-binding protein fragment. In some embodiments, the diluent is a pharmaceutically acceptable diluent. Typically, the kit further includes a separate sheet, pamphlet, or card supplied in or with the container along with instructions for use. If the kit is intended for pharmaceutical use, it may further include information for administering the composition to subjects with complement C3-mediated disorders or disabilities, and one or more of the following: a dosing schedule, a description of the therapeutic agent, prevention, warnings, indications, counterindications, overdose information, and / or side effects.

[0088] Diagnostic 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 applications, 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 an antigen-binding protein described herein. In another embodiment, the antigen-binding protein or fragment thereof is conjugated with one or more substances that can be recognized by a detection substance(s), for example, the antigen-binding protein or fragment thereof is conjugated with biotin that can be detected by streptavidin. In a particular embodiment, the antigen-binding protein or fragment thereof is useful for detecting the presence of C3 and / or C3b in a sample. In a particular embodiment, the sample is a biological sample. As used herein, the term “detect” includes quantitative and / or qualitative detection. In certain embodiments, the biological sample includes cells or tissues derived from a human patient, such as retinal tissue.

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

[0090] In one embodiment, such a method is used to select a patient, i.e., to determine the eligibility of a subject for treatment with an antigen-binding protein or a fragment thereof, as described herein. Those skilled in the art will readily see that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. While certain embodiments have been described in detail here, this specification will be better understood by reference to the following examples, which are included for illustrative purposes only and are not intended to limit it. [Examples]

[0091] (Example 1) Preparation and characterization of anti-C3 antibody libraries To generate antibodies that inhibit the complement cascade more efficiently than those possible with partial complement inhibitors, it was hypothesized that a broad collection of anti-C3 antibodies with diverse epitope recognition capabilities would increase the probability of isolating antibodies with desired function. For this purpose, a large antibody phage library was constructed using genomic information encoding antibody variable domains derived from B cells of C3-immunized animals. To generate a number of antibodies capable of recognizing different epitopes on C3, three New Zealand white rabbits and two llamas were immunized with naturally occurring human C3 protein purified from serum (Figure 2). Each animal received four injections of C3 protein at different time points using complete or incomplete Freund's adjuvant (Figure 3A). The immune response of each animal was tested using ELISA, and anti-C3 antibodies present in serum samples from the immunized animals were quantified. Antibody titers in serum indicated a superior immune response (Figure 3B).

[0092] An scFv antibody cDNA library was constructed from RNA extracted from rabbit PBMCs and splenic lymphocytes isolated by PCR amplification. The coding sequences of the variable light chain and heavy chain domains were amplified separately and ligated through a series of overlap polymerase chain reaction (PCR) steps to obtain the final scFv product. In the llama, massive bleeding was induced, and RNA was isolated from it and transcribed into cDNA using reverse transcriptase Kit. The cDNA was cleaned, and the heavy chain fragment was amplified using primer annealing in the leader sequence region and the CH2 region. Amplified DNA sequences encoding rabbit-derived scFv and llama-derived VHH were digested using appropriate restriction enzymes and then ligated to phagemid vectors. The phagemid vectors were transformed into TG1 electrocompetent cells, which are well-suited for antibody-phage display library construction. These processes were carried out in 10 8 This resulted in four antibody libraries with sizes larger than the individual clones, and the insert percentage was close to 100% (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 modification 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 and resulting in full activation of the complement system. Using the screening described herein, we identified antibodies that bind to different epitopes of 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 presented on phages were produced and subjected to several biopanning (selection) cycles against purified native human C3 from serum. The stringency of selection increased with each round by decreasing the concentration of C3 protein used in biopanning or increasing the stringency of washing. Approximately 380 monoclonal phages were selected and screened for their ability to bind to C3 in an ELISA assay (Figure 5). Based on ELISA data and DNA fingerprints, 41 phage clones were selected for sequencing, recombinantly produced as antibody proteins, and evaluated for their ability to bind to human C3 and C3b, as well as for further characterization (Figure 5).

[0094] To identify antibodies that block all three complement pathways, the Wieslab® complement system screen (Svar Life Science AB, Malmo, Sweden) was used to screen antibodies using enzyme immunoassays for qualitative determination of the functional classical, lectin, and surrogate complement pathways in human serum. 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% in human serum (Quidel) at a fixed concentration of 2 μM.

[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 manner 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 thought to share similar epitopes and therefore have similar functions. This information allows for a reduction in the number of potential antibody candidates while maintaining epitope diversity. Of the five antibodies inhibiting all three complement pathways, M0251, M0228, and M0123 were thought to share the same epitope on C3 (Figure 7D). The inhibitory reads were thought 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 to cynomolgus monkey C3 in ELISA. Briefly, 96-well ELISA plates were coated with polyclonal goat antiserum, which is thought to be cross-reactive to cyno C3, and subsequently secondarily associated with a custom preparation of cynomolgus monkey serum (BioIVT, NB-151558). Serial dilutions of antibody molecules were added to the ELISA plates, and antibodies binding 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-response binding to cynomolgus monkey C3. Interestingly, while M0251, M0228, and M0123 compete for the same epitope on human C3, only M0251 showed binding activity to cyno C3 (Figures 15A and 15B).

[0097] The ability of anti-C3 antibodies to inhibit all pathways of complement activation in cynomolgus monkey serum was evaluated using the Wieslab complement system screen. Anti-C3 antibodies were added to custom preparations of cyno serum. Figure 14A shows potent 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 inhibitory activity of complement pathways 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 monkey 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 direct binding ELISA assays (Figures 8A and 8B). Briefly, 96-well ELISA plates were coated with purified natural human C3 or C3b (Complement Technology, A113 and A114). Serial dilutions of antibody molecules were added to the plates and detected with rabbit anti-human kappa HRP antibody (Abcam, ab202549) or rabbit anti-His Tag HRP antibody (Abcam, ab1187). M0122, M0124, and M0228 showed high affinity binding to both human C3 and C3b. The binding kinetics of M0122, M0124, and M0228 to human C3 were further analyzed by biolayer interferometry, which exhibits low picomolar affinity (Figure 10).

[0098] Dose-dependent inhibition of the surrogate and classical pathways in human serum was evaluated for M0122, M0124, and M0228 using the corresponding Wieslab complement system kits. The anti-C3 antibodies M0122, M0124, and M0228 exhibit potent inhibition of the surrogate and classical pathways in human serum (Figures 9A and 9B). The 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 the 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, how complement activity is compartmentalized in the eye, and whether the efficacy of GA therapy depends on delivering the drug to the correct anatomical site within the eye, is not yet fully understood. We hypothesized that better penetration into disease-related retinal tissue (i.e., RPE, Bruch's membrane, and choroid) may be necessary to achieve a significant reduction in lesion growth in GA. The inner part of the choroid is called the choroidal capillary, and it contains capillaries separated by a sheet of extracellular membrane called Bruch's membrane (BrM) (Figure 11).

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

[0101] [Table 3]

[0102] Furthermore, Pitkanen et al. (Invest Ophthalmol Vis Sci. 2005; 46(2):641-6) studied the permeability of carboxyfluorescein and fluorescein isothiocyanate (FITC)-labeled dextran with molecular weights of 4-80 kDa to fresh RPE-choroidal samples from bovine eyes. The inventors plotted permeability against molecular size (Figure 12, black dots). In addition, using the research conducted by Hirvonen et al. (Pharm Res. 2016;33(8):2025-32), the inventors derived permeability values ​​for scFv, lucentis, eylea, and APL2 based on hydrodynamic radius and plotted permeability against molecular weight on the same graph (Figure 12, colored dots). This trend indicates that the permeability of Bruch's membrane decreases as the molecular weight increases.

[0103] The inventors predicted that the anti-C3 antibodies of this disclosure, as antibody fragments such as scFv or VHH format, with a hydrodynamic radius of approximately 2.5 nm or less, are more likely 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, the ability of anti-C3 molecules to pass through BrM was evaluated using concentrated porcine BrM placed in a Ussing chamber. Briefly, concentrated Bruch membranes were isolated from porcine eyes and placed in a Ussing diffusion chamber (Multi Channel Systems MCS GmbH, catalog no. 660026). Once placed, a 5 mm diameter Bruch membrane was the only barrier between two identical compartments. Both sides of the Bruch membrane were washed with 1 ml of PBS at room temperature for at least 5 minutes. For leak testing, 1 ml of PBS was added to the sample chamber and leakage into the second compartment was tracked for 5 minutes. If no leakage indicating compromise of membrane integrity was detected, the antibody protein was added to the sample chamber with 1 ml of 100 μg / ml PBS, and 1 ml of PBS was added to the second compartment (diffused material chamber). The entire Ussing chamber was incubated at room temperature for 24 hours with gentle shaking to avoid creating a diffusion protein gradient. 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 minutes under reducing conditions. The gels were stained at room temperature for 60 minutes using Instant Blue staining (Expedeon) to detect antibody proteins, or stained with barium iodide solution to detect PEG (the gel was fixed with 0.1 M perchloric acid, replaced after 15 minutes with a preliminary mixture of 20 ml of 5% BaCl2 and 8 ml of 0.1 M iodine solution, and then replaced every 10 minutes for 1 hour with deionized water). To calculate the percentage of protein in the sample chamber or diffuse chamber, the band density in the Instant Blue-stained or BaCl2-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). Next, the calculated percentages of protein were plotted on ±SD.The ability to pass through porcine BrM was compared for the scFv derivative M0123 (26 kDa) and an APL-2 surrogate (one anti-C3 cyclic APL-1 peptide linked to a 40 kDa linear PEG, totaling 42 kDa), and they were simultaneously incubated on BrM preparations from four different pig eyes. Compared to the APL-2 surrogate, significantly higher amounts of scFv passed through BrM in all four membrane preparations (Figures 13A and 13B).

Claims

1. An antigen-binding protein or fragment thereof that binds to an epitope on complement C3, Including VH and VL, VH includes 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. VL is an antigen-binding protein or fragment thereof, comprising 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. The antigen-binding protein or fragment thereof according to 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. An antigen-binding protein or fragment thereof according to claim 1 or 2, comprising a single-stranded variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, or a diabody.

4. A pharmaceutical composition comprising an antigen-binding protein or a fragment thereof as described in any one of claims 1 to 3, and which may also comprise a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4 for treating complement C3-mediated diseases or disorders in a subject.

6. The pharmaceutical composition according to claim 5, wherein the antigen-binding protein or a fragment thereof is administered by topical, subconjunctival, intravitreous, retrobulbar, and / or intrachorally.

7. The pharmaceutical composition according to 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, postlentic fibrosis, autoimmune uveitis, chorioretinitis, retinitis, rheumatoid arthritis, psoriasis, and atherosclerosis.

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

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

10. A host cell comprising the expression vector described in claim 9.

11. A method for producing an antigen-binding protein or a fragment thereof according to any one of claims 1 to 3, (i) A step of culturing the host cells according to claim 10 under conditions that enable the expression of the protein according to any one of claims 1 to 3, (ii) The process of recovering the protein, A method that includes this.

12. A method for detecting one or both of C3 and C3b in a biological sample, (a) A step of contacting the sample with at least one antigen-binding protein or fragment thereof as described in any one of claims 1 to 3, (b) A step that enables the formation of a complex between one or both of C3 and C3b in the sample and an antigen-binding protein or a fragment thereof, (c) A step of detecting the antigen-binding protein or a fragment thereof A method that includes this.

13. A kit for detecting C3, comprising an antigen-binding protein or a fragment thereof as described in any one of claims 1 to 3, and instructions for use.

Images