Bispecific antibodies and methods for treating eye diseases

Abstract

The present disclosure relates to the treatment and / or prevention of age-related macular degeneration (AMD), including both neovascular AMD ("wet" AMD) and non-neovascular AMD ("dry" AMD), as well as advanced non-neovascular AMD (geographic atrophy or GA). In particular, the present disclosure provides novel therapeutic bispecific antibodies that target components of the alternative pathway of the complement activation system.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 336,766, filed April 29, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing The text of the computer readable sequence listing submitted herewith, entitled "40820_601_SequenceListing" (created on April 27, 2023, file size 1,823,343 bytes), is hereby incorporated by reference in its entirety.

[0003] Embodiments of the present disclosure relate to the treatment and / or prevention of age-related macular degeneration (AMD), including both neovascular AMD ("wet" AMD) and non-neovascular AMD ("dry" AMD), as well as advanced non-neovascular AMD (geographic atrophy or GA). In particular, the present disclosure provides novel therapeutic bispecific antibodies that target components of the alternative pathway of the complement activation system. [Background technology]

[0004] Age-related macular degeneration (AMD) is the leading cause of severe vision loss in adults over the age of 50. According to estimates from the Centers for Disease Control and Prevention, 1.8 million people have AMD and an additional 7.3 million are at substantial risk of vision loss due to AMD. This eye disease occurs when changes occur in the macula (a small portion of the retina located at the inner posterior layer of the eye). AMD is a loss of central vision and can occur in two forms: "dry" (atrophic or non-neovascular) and "wet" (exudative or neovascular). Most people with macular degeneration have the dry form. Geographic atrophy (GA) is considered a later stage of the dry form of AMD and is characterized by a focal and progressive loss of photoreceptors. GA is less common than neovascular AMD and is responsible for 10-20% of cases of legal blindness in this condition, affecting over 5 million people worldwide. There are currently no approved or effective treatments to prevent either the onset or progression of GA. However, in recent years, there have been significant advances in understanding the pathogenesis of GA, leading to several new potential therapies. Treatments for wet AMD are rapidly evolving, with several now targeting specific biochemical events such as angiogenesis. The use of drug therapies targeting vascular endothelial growth factor (VEGF) has been shown to slow vision loss and even lead to vision improvement in some patients with wet macular degeneration. However, the ability to treat both wet and dry AMD with a single biologic has not been established. Summary of the Invention

[0005] Embodiments of the present disclosure include bispecific antibodies, or antigen-binding fragments thereof, that specifically bind to human complement factor H-related 4 (CFHR) and human complement component 3 (C3). In some embodiments, the human CFHR4 is the CFHR4b variant (CFHR4b), which is optionally a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 580. In some embodiments, the human C3 is the b variant of C3 (C3b), which is optionally a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1645.

[0006] In some embodiments, the bispecific antibody or fragment thereof is monoclonal and optionally recombinant. In some embodiments, the bispecific antibody or fragment thereof is human, humanized, or chimeric. In some embodiments, the bispecific antibody or fragment thereof is a full-length antibody, a single-chain antibody, a single-chain variable fragment (scFv), a variable fragment (Fv), a fragment antigen-binding region (Fab), Fab-C, Fab'-SH, (Fab')2, a single-domain antibody (sdAb), a VHH antibody, a nanobody, a camelid-derived single-domain antibody, a shark IgNAR-derived single-domain antibody fragment (VNAR), a diabody, a triabody, an anticalin, or an aptamer, and optionally the antibody is a full-length antibody comprising an Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 region.

[0007] In some embodiments, the bispecific antibody or fragment thereof is optionally conjugated to at least one additional moiety selected from an antigen-binding moiety, such as an antibody or antigen-binding fragment thereof capable of specifically binding to a target other than human CFHR4 or human C3, preferably wherein the target is expressed in the human eye; a therapeutic or cytotoxic moiety; a detection moiety; a purification moiety; a half-life extending moiety; optionally a polypeptide at least 20 amino acids in length and comprising any combination of G, A, ST, E, and P residues, which is conjugated to the C-terminus or N-terminus of the antibody.

[0008] In some embodiments, the bispecific antibody or fragment thereof is a polypeptide comprising one, two or all three HCDRs of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 3, and one, two or all three corresponding LCDRs of said exemplary antibodies, and / or a VH sequence having at least 90% identity to the VH sequence of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 4, and optionally a VL sequence having at least 90% identity to the corresponding VL sequence of said exemplary antibody, preferably with no mutations allowed in the HCDRs or LCDRs, and / or all six CDRs of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 3, the VH and VL sequences of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 4, and / or the full-length heavy chain (VH+constant) sequence of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 5, and optionally the corresponding full-length light chain (VL+constant) sequence of said exemplary antibody.

[0009] In some embodiments, the bispecific antibody or fragment thereof is a polypeptide having one, two, or all three HCDRs of any one of the exemplary C3 antibodies whose sequence is shown in Table 6, and one, two, or all three of the corresponding LCDRs of the exemplary antibodies, and / or a VH sequence having at least 90% identity to the VH sequence of any one of the exemplary C3 antibodies whose sequence is shown in Table 7, and optionally at least 90% identity to the corresponding VL sequence of the exemplary antibody shown in Table 8. A polypeptide comprising a VL sequence, preferably no mutations in the HCDRs or LCDRs are tolerated, and / or all six CDRs of any one of the exemplary C3 antibodies whose sequences are shown in Table 6, the VH and VL sequences of any one of the exemplary C3 antibodies whose sequences are shown in Tables 7 and 8, and / or the full-length heavy chain (VH+constant) sequence of any one of the exemplary C3 antibodies whose sequences are shown in Table 9, and optionally the corresponding full-length light chain (VL+constant) sequence of the exemplary antibody shown in Table 10.

[0010] Embodiments of the present disclosure also include polynucleotides encoding bispecific antibodies or fragments thereof. In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, or 100% identity to the nucleic acid sequence of any one of the exemplary antibodies whose sequence is set forth in Tables 5, 9, and 10. Embodiments of the present disclosure also include an expression vector comprising the polynucleotide, which is optionally an adeno-associated virus (AAV) vector, a lentivirus (LV) vector, a herpes simplex virus (HSV) vector, or a retrovirus vector.

[0011]

[0023] Embodiments of the present disclosure also include pharmaceutical compositions comprising the bispecific antibody or fragment thereof, the corresponding polynucleotide, or a vector comprising the polynucleotide. In some embodiments, the composition comprises at least one pharma- ceutically acceptable carrier, diluent, or preservative, and / or at least one additional active ingredient.

[0012] In some embodiments, the pharmaceutical compositions are suitable for ocular administration to a subject, optionally by delivery using conjunctival inserts, contact lenses, gels, nanoparticles, mucoadhesive polymers, ointments, solutions, suspensions, eye drops, and / or implants, preferably by injection into the vitreous humor.

[0013] The embodiments of the present disclosure also include the bispecific antibody or fragment thereof, corresponding polynucleotide, vector comprising the polynucleotide, or pharmaceutical composition for use as a medicament, optionally for use in a method for treating an ocular disease in a subject. In some embodiments, the disease is characterized by increased activation of the complement system, particularly the alternative pathway, particularly in the subject's eye, for example in drusen or retinal pigment epithelial (RPE) cells of the subject. In some embodiments, the method includes ocular administration of the antibody, preferably by injection into the vitreous humor, which preferably relieves at least one symptom in the subject selected from visual distortion, reduced central vision, blurred vision, and / or difficulty adapting to low light. In some embodiments, the disease is age-related macular degeneration (AMD), including dry AMD, which may be in early, intermediate, or advanced stages (the latter otherwise known as geographic atrophy, GA).

[0014] In accordance with the above, embodiments of the present disclosure include a bispecific antibody comprising a first antigen-binding site for complement factor H-related 4 (CFHR4) and a second antigen-binding site for complement component 3 (C3). In some embodiments, the first anti-CFHR4 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406, and the second anti-C3 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3.

[0015] Embodiments of the present disclosure include bispecific antibodies comprising a first antigen-binding site for complement factor H-related 4 (CFHR4) and a second antigen-binding site for complement component 3 (C3). In some embodiments, the first anti-CFHR4 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the second anti-C3 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 1127-1200, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 1275-1348.

[0016] Embodiments of the present disclosure include bispecific antibodies comprising a first antigen-binding site for complement factor H-related 4 (CFHR4) and a second antigen-binding site for complement component 3 (C3). In some embodiments, the first anti-CFHR4 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380, and a second antigen-binding site for complement component 3 (C3) from at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406. the first anti-C3 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 1127 to 1200, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 1275 to 1348.

[0017] In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:2, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:9, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:15. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:3, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:10, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:4, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:11, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:5, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:12, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:6, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:13, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:19. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:7, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:14, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:22, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:33, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:23, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:34, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:24, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:35, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:45.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:25, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:36, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:46. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:26, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:37, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:27, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:38, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:48. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:28, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:39, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:49. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:29, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:40, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:50. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:30, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:41, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:31, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:42, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:54, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:66, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:77. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:55, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:67, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:78.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:56, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:68, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:79. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:57, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:69, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:80. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:58, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:70, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:81. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:59, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:71, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:82. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:60, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:72, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:83. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:61, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:73, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:84. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:62, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:74, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:63, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:75, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:64, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:76, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:87.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 89, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 103, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 90, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 104, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 117. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 91, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 105, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 92, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 106, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:93, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:107, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:120. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:94, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:108, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:121. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:95, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:109, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:122. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:96, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:110, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:123. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:97, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:111, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:124.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:98, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:112, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:125. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:99, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:113, and HCDR3 comprises the amino acid sequence of SEQ ID NO:126. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:100, HCDR2 comprises the amino acid sequence of SEQ ID NO:114, and HCDR3 comprises the amino acid sequence of SEQ ID NO:127. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:101, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:115, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:128.

[0018] In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 130, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 153, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 131, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 154, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 132, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 155, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 133, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 156, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 178. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 134, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 157, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 179. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 135, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 158, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 136, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 159, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 181. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 137, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 160, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:138, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:161, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:183.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 139, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 162, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 184. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 140, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 163, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 185. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 141, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 164, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 142, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 165, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 143, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 166, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 144, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 167, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 189. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 145, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 168, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 190. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 146, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 169, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 191. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:147, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:170, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:192.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 148, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 171, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 193. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 149, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 172, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 194. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 150, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 173, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 195. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 151, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 174, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 196. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 198, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 201, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 199, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 202, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 207, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 217, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 227. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 208, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 218, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 228. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:209, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:219, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:229.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:210, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:220, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:230. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:211, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:221, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:231. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:212, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:222, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:232. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:213, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:223, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:233. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:214, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:224, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:234. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:215, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:225, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:235. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:237, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:245, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:253. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:238, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:246, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:254. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:239, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:247, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:255.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:240, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:248, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:256. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:241, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:249, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:257. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:242, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:250, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:258. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:243, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:251, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:259.

[0019] In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:583, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:602, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:620. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:584, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:603, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:621. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:585, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:604, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:622. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:586, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:605, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:623. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:587, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:606, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:624. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:588, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:607, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:625. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:589, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:608, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:626. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:590, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:609, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:627. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 591, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 610, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 628. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 592, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 611, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 629.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:593, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:612, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:630. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:594, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:613, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:631. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:595, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:614, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:632. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:596, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:615, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:633. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:597, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:616, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:634. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:598, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:617, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:635. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:599, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:618, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:636. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:600, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:619, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:637. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 639, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 652, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 664. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 640, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 653, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 665.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 641, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 654, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 642, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 655, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 667. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 643, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 656, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 668. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 644, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 657, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 669. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 645, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 658, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 670. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 646, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 659, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 671. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 647, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 660, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 672. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 648, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 661, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 673. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 649, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 662, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 674. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 650, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 663, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 675.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 677, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 706, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 734. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 678, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 707, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 735. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 679, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 708, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 736. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 680, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 709, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 737. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 681, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 710, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 738. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 682, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 711, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 739. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 683, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 712, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 740. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 684, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 713, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 741. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 685, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 714, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 742. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 686, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 715, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 743.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 687, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 716, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 744. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 688, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 717, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 745. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 689, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 718, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 745. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 690, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 719, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 747. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 691, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 720, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 748. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 692, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 721, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 749. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 693, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 722, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 750. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 694, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 723, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 751. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 695, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 724, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 752. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 696, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 725, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 753. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 697, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 726, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 754. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:698, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:727, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:755.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 699, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 728, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 756. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 700, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 729, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 757. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 701, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 730, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 758. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 702, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 731, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 759. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 703, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 732, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 760. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 704, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 733, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 761. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 763, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 771, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 778. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 764, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 772, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 779. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 765, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 773, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 780. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 766, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 774, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 781.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 767, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 775, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 782. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 768, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 776, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 783. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 769, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 777, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 784. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 786, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 794, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 801. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 787, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 795, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 802. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 788, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 796, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 803. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 789, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 797, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 804. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 790, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 798, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 805. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 791, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 799, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 806. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 792, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 800, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 807.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 809, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 812, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 815. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 810, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 813, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 816.

[0020] In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 818, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 850, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 881. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 819, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 851, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 882. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 820, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 852, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 883. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 821, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 853, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 884. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 822, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 854, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 885. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 823, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 855, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 886. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 824, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 856, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 887. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 825, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 857, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 888. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 826, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 858, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 889. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 827, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 859, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 890.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 828, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 860, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 891. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 829, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 861, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 892. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 830, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 862, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 893. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 831, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 863, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 894. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 832, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 864, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 895. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 833, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 865, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 896. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 834, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 866, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 897. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 835, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 867, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 898. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 836, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 868, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 899. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 837, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 869, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 900.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 838, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 870, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 901. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 839, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 871, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 902. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 840, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 872, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 903. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 841, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 873, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 904. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 842, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 874, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 905. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 843, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 875, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 906. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 844, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 876, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 907. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 845, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 877, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 908. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 846, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 878, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 909. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 847, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 879, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 910.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 848, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 880, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 911. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 913, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 916, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 919. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 914, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 917, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 920. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 914, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 917, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 920. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:922, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:931, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:940. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:923, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:932, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:941. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:924, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:933, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:942. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:925, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:934, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:943. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 926, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 935, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 944. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 927, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 936, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 945.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:928, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:937, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:946. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:929, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:938, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:947. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:949, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:984, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1019. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:950, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:985, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1020. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:951, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:986, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1021. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:952, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:987, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1022. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:953, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:988, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1023. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:954, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:989, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1024. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 955, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 990, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 990. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 956, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 991, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1026. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 957, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 992, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1027. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 958, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 993, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1028. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 959, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 994, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1029. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:960, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:995, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1030. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:961, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:996, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1031. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:962, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:997, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1032. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:963, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:998, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1033. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:964, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:999, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1034.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:965, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1000, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1035. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:966, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1001, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1036. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:967, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1002, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1037. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:968, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1003, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1038. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:969, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1004, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1039. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:970, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1005, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1040. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:971, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1006, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1041. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:972, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1007, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1042. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:973, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1008, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1043.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:974, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1009, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1044. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:975, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1010, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1045. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:976, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1011, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1046. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:977, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1012, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1047. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:978, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1013, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1048. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:979, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1014, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1049. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:980, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1015, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1050. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:981, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1016, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1051. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:982, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1017, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1052.

[0021] In some embodiments, the anti-CFHR4 VH comprises an amino acid sequence having at least 90% sequence identity to at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380. In some embodiments, the anti-CFHR4 VL comprises an amino acid sequence having at least 90% sequence identity to at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406. In some embodiments, the anti-C3 VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to at least one of SEQ ID NOs: 1127-1200. In some embodiments, the anti-C3 VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to at least one of SEQ ID NOs:1275-1348.

[0022] In some embodiments, the bispecific antibody binds CFHR4 and / or C3 and reduces complement activation. In some embodiments, the bispecific antibody binds CFHR4b and / or C3b and reduces complement activation. In some embodiments, the bispecific antibody K D binds to human CFHR4 and / or C3 with approximately 100 nM or less.

[0023] In some embodiments, the bispecific antibody comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises a polypeptide that is at least 20 amino acids in length and comprises any combination of G, A, ST, E, and P residues. In some embodiments, the half-life extending polypeptide is attached to the C-terminus or N-terminus of the antibody.

[0024] Embodiments of the present disclosure also include pharmaceutical compositions comprising any of the bispecific antibodies described herein. In some embodiments, the compositions are suitable for ocular administration. In some embodiments, ocular administration comprises injection into the vitreous humor. In some embodiments, ocular administration comprises delivering the antibody using a conjunctival insert, a contact lens, a gel, a nanoparticle, a mucoadhesive polymer, an ointment, a solution, a suspension, an eye drop, and / or an implant.

[0025] Embodiments of the present disclosure also include a method of treating age-related macular degeneration (AMD), comprising administering a pharmaceutical composition comprising an effective amount of any of the bispecific antibodies described herein to a subject in need of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is administered to the eye to treat at least one AMD symptom. In some embodiments, the AMD comprises wet AMD. In some embodiments, the AMD comprises dry AMD. In some embodiments, the AMD symptoms comprise visual distortion, reduced central vision, blurred vision, and / or difficulty adapting to low light. In some embodiments, administration of the pharmaceutical composition reduces complement activation in the subject's eye. In some embodiments, the pharmaceutical composition is administered at a dose ranging from about 0.0001 mg / dose to about 100 mg / dose. In some embodiments, the pharmaceutical composition is administered at a dose ranging from about 0.0001 mg / ml to about 100 mg / ml. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a representative diagram of the molecular mechanisms underlying the treatment of advanced age-related macular degeneration (AMD), including geographic atrophy (GA), with the anti-CFHR4 antibodies of the present disclosure. [Diagram 2] Figure 1 shows the results (see Table 1) plotting odds ratios and confidence intervals of haplotype effects when the Y402 haplotype has a deletion (tagged by one of the two variants) or a pQTL variant. Associations were performed relative to the reference haplotype at OR=1. [Figure 3A]FIG. 1 shows representative ELISA results used to determine antigen-positive serum titers of mice immunized with CFHR4 according to different immunization protocols / cohorts (cohort 1). [Figure 3B] FIG. 1 shows representative ELISA results used to determine antigen-positive serum titers of mice immunized with CFHR4 according to different immunization protocols / cohorts (cohort 2). [Figure 3C] FIG. 1 shows representative ELISA results used to determine antigen-positive serum titers of mice immunized with CFHR4 according to different immunization protocols / cohorts (cohort 3). [Figure 4] Representative results of the thermal stability of a human CFHR4 monoclonal antibody of the present disclosure (see Table 2, "ATX" antibody). Thermal stability was assessed by differential scanning fluorimetry (DSF) using a protein thermal shift (PTS) assay. [Figure 5A] FIG. 1 shows representative results of a CFHR4 antibody cross-blocking experiment, including data from a representative heat map analyzing the ability of antibodies to block each other for binding to the antigen. [Figure 5B] 1 is a representative network plot that gradually groups antibodies with similar competitive profiles. [Figure 6] FIG. 1 shows representative results of CFHR4 antibody binding kinetics. [Figure 7] FIG. 1 shows representative results of CFHR4 antibody cross-reactivity with cynomolgus monkey CFHR4b protein (cCFHR4b). [Figure 8] Representative results of CFHR4 antibody cross-reactivity with human CFHR4a protein. [Figure 9] Representative results of CFHR4 antibody cross-reactivity with human CFHR3 protein. [Figure 10] FIG. 1 shows representative results of CFHR4 antibody cross-reactivity with human CFHL-1(Y402H) protein. [Figure 11A]FIG. 1 is a representative schematic of a C3 convertase assembly assay used to assess the ability of CFHR4 antibodies of the disclosure to affect C3 convertase formation. [Figure 11B] FIG. 1 includes representative dose-response curves for the top inhibitory CFHR4 antibodies. [Figure 11C] FIG. 11B includes the IC50 values ​​calculated for each antibody tested. [Figure 11D] FIG. 1 provides a representative summary of data showing at least three functional classes of anti-CFHR4 antibodies. [Figure 12] Figure 12 shows representative ELISA results used to determine antigen-positive serum titers of mice immunized with human C3 and cynomolgus C3 according to different immunization protocols / cohorts (cohort 1: Figure 12A (human C3) and Figure 12B (cynomolgus C3); cohort 2: Figure 12C (human C3); Figure 12D (cynomolgus C3)). [Figure 13] FIG. 1 shows representative ELISA results of polyclonal phage pools to confirm enrichment and examine cross-reactivity. [Figure 14] 1 is a representative table containing binding kinetics of the C3 antibody of the present disclosure. [Figure 15] 1 is a representative table containing results of C3 antibody cross-reactivity with human C3b protein, cynomolgus monkey C3 protein, and human C3a protein. [Figure 16A] 1 is a representative table containing binding kinetics of C3 antibody to human C3 protein. [Figure 16B] 1 is a representative table containing binding kinetics of C3 antibody to human C3b protein. [Figure 17A] FIG. 13 shows representative results of a C3 antibody epitope experiment, including a table with binning assignments. [Figure 17B] FIG. 1 shows data from a representative heat map analyzing the ability of antibodies to block each other for binding to the antigen. [Figure 17C]1 is a representative network plot that gradually groups antibodies with similar competitive profiles. [Figure 17D] 1 is a representative network plot that gradually groups antibodies with similar competitive profiles. [Figure 18A] Representative binning network plots based on recombinant human affinity and cynomolgus binding performed by C3b. [Figure 18B] FIG. 1. C3b affinity and ligand blocking. [Figure 18C] FIG. 11. Bin vs. community diagram for blocking CFHR4b. [Figure 18D] Bin vs. community for CFH blocking. [Figure 18E] FIG. 11. Diagram of material availability and common versus diverse light chains. [Figure 19] FIG. 13. Representative results of C3 antibody polyreactivity with baculovirus particles (BVP). [Figure 20] FIG. 11: Representative results of C3 antibody polyreactivity with various proteins as indicated. [Figure 21A] FIG. 11. Representative results of C3 antibody polyreactivity with CFHR4. [Figure 21B] FIG. 11. Representative results of C3 antibody polyreactivity with CFH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Embodiments of the present disclosure relate to the treatment and / or prevention of age-related macular degeneration (AMD), including both neovascular AMD ("wet" AMD) and non-neovascular AMD ("dry" AMD), as well as advanced non-neovascular AMD (geographic atrophy or GA). In particular, the present disclosure provides novel therapeutic bispecific antibodies that target components of the alternative pathway of the complement activation system.

[0028] The human complement system comprises a family of proteins essential for the human immune response to infection. Complement recognizes microorganisms or damaged host cells and then triggers an enzyme cascade that serves primarily to mark target cells for phagocytosis by immune cells, produce chemotactic factors, and directly kill target cells via pore formation. Unwanted complement activation on the body's own cells is a key pathological driver in a wide range of immune diseases, including autoimmune, inflammatory, and degenerative diseases. Knowledge of complement activation and how it can be regulated is crucial for the current and future development of therapeutic complement inhibitors.

[0029] Human complement factor H-related protein (CFHR)4 belongs to the factor H family of plasma glycoproteins consisting of short consensus repeat (SCR) domains. Although factor H is a well-known inhibitor of the alternative complement pathway, the function of the CFHR protein is less clear. For example, CFHR4 lacks an SCR that is homologous to the complement inhibitory domain of factor H. However, its complement regulatory activity is not fully understood. Previous studies have shown that CFHR4 binds C-reactive protein through its most N-terminal SCR, resulting in activation of the classical complement pathway. CFHR4 binds C3b through its C-terminus, but the importance of this interaction is less well understood. Recent reports have shown that CFHR4 may serve as a platform for the assembly of the alternative pathway C3 convertase by binding to C3b. This is based, at least in part, on the sustained ability of CFHR4-bound C3b to bind factor B and properdin, resulting in an active convertase that generates C3a and C3b from C3. CFHR4-C3bBb convertase is less susceptible to factor H-mediated degradation compared to C3bBb convertase. CFHR4 mutants containing exchanges of conserved residues within the C-terminal C3b-binding site showed significantly reduced C3b binding and alternative pathway complement activation. These initial data indicate that, in contrast to complement inhibitor factor H, CFHR4 acts as an enhancer of opsonization by promoting complement activation. CFHR4 is detected as two distinct glycoproteins in human plasma. The long 86 kDa isoform, designated CFHR4A, consists of nine SCRs. The shorter ∼45 kDa isoform, designated CFHR4B, consists of five SCRs, representing SCR1 and SCR6-9 of CFHR4A. In CFHR4A, SCR1-4 and SCR5-8 are highly related based on nucleotide and amino acid sequence identity and may be the result of intramolecular duplication. Like all CFHRs, both CFHR4 isoforms lack SCRs homologous to the N-terminal complement inhibitory domains SCR1-4 of FH and CFHL1. Consistent with this, no significant cofactor and convertase decay-accelerating activity has been reported for CFHR4B.The two most C-terminal domains of CFHR4A and CFHR4B are homologous to the C-terminal FH domain SCR19-20, which contains the C3b / C3d binding site. CFHR4B has been shown to bind to the C3d region of C3b via its C-terminus SCR4-5. However, no significant function has been associated with this C3b binding ability, except for a slight enhancement of the cofactor activity of FH in the presence of high CFHR4B concentrations. The CFHR4A isoform has not yet been characterized for its interaction with C3b and complement regulatory activity.

[0030] Convertases play a central role in the complement cascade because they cleave C3 and C5, which mediate nearly all complement effector functions. C3 convertase cleaves C3 into C3a, a chemotactic molecule, and C3b, which covalently binds to target surfaces and triggers phagocytosis. C5 convertase cleaves C5 into C5a, a potent mediator of leukocyte recruitment and inflammation, and C5b, an initiator of the membrane attack complex and cell lysis. The complement cascade begins with specific recognition of target cells in the classical (CP) and lectin (LP) pathways. In the CP, antibodies bind epitopes on target cells and subsequently recruit the C1 complex (C1qr2s2). Upon binding to the antibody platform, the C1q-related protease C1s converts C4 and C2 to generate C3 convertase (C4b2a) on the cell surface. Similarly, the lectin pathway also forms C4b2a through activation of mannose-binding lectin-associated serine proteases. The resulting CP / LP C4b2a convertase cleaves C3 into C3a and C3b. Following cleavage, reactive thioesters in C3b are exposed, allowing its covalent attachment to the target cell surface, resulting in recognition of the cell by phagocytes. Target cell labeling by C3b is amplified by the alternative pathway (AP), in which surface-bound C3b binds to factor B (FB). The proconvertase C3bB is then cleaved by factor D (FD) to form the active C3 convertase complex (C3bBb) consisting of C3b and the protease fragment Bb. As the resulting active AP C3 convertase (C3bBb) is composed of C3b itself, cleavage of the substrate generates additional convertases, further spreading C3b deposition. When the density of C3b molecules on the cell surface becomes high enough, the preexisting C3 convertases (C4b2a and C3bBb) acquire the ability to cleave C5, forming C5a and C5b.

[0031] Age-related macular degeneration (AMD) is a progressive retinal disease characterized in its early stages by a relatively small number of small drusen in the macula. As AMD progresses, the size and number of drusen increase, eventually leading to more advanced stages of AMD. Two forms of advanced AMD are distinguished. The first form, neovascular AMD, is characterized by the infiltration of abnormal blood vessels into the retina. These newly formed blood vessels are fragile, and when they break, lead to sudden vision loss due to leakage of blood components in the retina. The second form of advanced AMD, geographic atrophy (GA), is the result of a gradual degeneration of the RPE and photoreceptor cells. Although angiogenesis occurs in only 15-20% of AMD cases, it is responsible for the majority of vision loss resulting from AMD. Drugs targeting vascular endothelial growth factor (VEGF), one of the central molecules in angiogenesis, have proven highly successful in neovascular AMD. However, there is no available treatment for the majority of the remaining cases of early, intermediate, or geographic atrophy AMD, and there is no effective means of preventing the progression from early to advanced stages.Although AMD is known to be the result of a complex interaction of environmental and genetic risk factors, research into the molecular components of drusen suggests that AMD may have an immunological component.This suggestion was made after proteins involved in inflammatory and / or other immune-related responses, including components of the complement system, were found in drusen.In addition, there is currently no treatment that addresses both wet and dry forms of AMD in a single biomolecule.

[0032] With this in mind, embodiments of the present disclosure provide a therapeutic platform based on modulating different aspects of the complement activation pathway using bispecific antibodies targeting CFHR4 and C3. As further described herein, the bispecific antibodies of the present disclosure contain an antigen binding site for CFHR4 and an antigen binding site for C3. Such antigen binding sites are described herein and in International PCT Application No. PCT / US2023 / 020351, and U.S. Provisional Patent Application No. US63 / 462,638, both of which are incorporated by reference in their entirety for all purposes.

[0033] definition To aid in the understanding of the present technology, several terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0034] In the context of describing embodiments of the present disclosure (particularly in the context of the claims below), use of the terms "a," "an," and "the," as well as "at least one" and similar referents, should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. Use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, without limitation"), unless otherwise stated herein. The recitation of ranges of values ​​herein is intended to serve merely as a shorthand way of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of some and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate various embodiments of the disclosure, and does not limit the scope of the disclosure unless otherwise asserted. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of various embodiments of the disclosure.

[0035] As used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or" unless the context clearly indicates otherwise. The term "based on" is not exclusive and allows for based on additional unlisted factors unless the context clearly indicates otherwise. Additionally, throughout the specification, the meanings of "a," "an," and "the" include plural references. The meaning of "in" includes "in" and "on."

[0036] The transitional phrase "consisting essentially of," when used in the claims of this application, limits the scope of the claim to certain materials or steps "and which do not materially affect the basic and novel characteristic(s)" of the claimed invention, as stated in In re Herz, 537 F.2d 549, 551-52,190 USPQ 461,463 (CCPA 1976). For example, a composition "consisting essentially of" the recited elements may contain a level of an unrecited contaminant such that, although the contaminant is present, the contaminant does not alter the function of the recited composition as compared to the pure composition (i.e., a composition "consisting of" the recited components).

[0037] The term "one or more," as used herein, refers to a number greater than 1. For example, the term "one or more" encompasses any of the following: 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 50 or more, 100 or more, or even more.

[0038] The terms "one or more, but less than a larger number," "two or more, but less than a larger number," "three or more, but less than a larger number," "four or more, but less than a larger number," "five or more, but less than a larger number," "six or more, but less than a larger number," "seven or more, but less than a larger number," "eight or more, but less than a larger number," "nine or more, but less than a larger number," "ten or more, but less than a larger number," "eleven or more, but less than a larger number," "twelve or more, but less than a larger number," "thirteen or more, but less than a larger number," "fourteen or more, but less than a larger number," or "fifteen or more, but less than a larger number" are not limited to the larger number. For example, the larger number can be 10,000, 1,000, 100, 50, etc. For example, the larger number can be about 50 (e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2).

[0039] The term "immunoglobulin" or "antibody", as used herein, refers to a protein found in the blood or other body fluids of vertebrates and is used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Typically, an immunoglobulin or antibody is a protein that contains at least one complementarity determining region (CDR). The CDRs form the "hypervariable region" of the antibody and are involved in antigen binding (described further below). A whole antibody usually consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (V H ) region and three C-terminal constant (C H1 , C H2 , and C H3 ) region, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L) region. The light chains of antibodies can be assigned to one of two different types, either kappa (κ) or lambda (λ), based on the amino acid sequence of their constant domain. In a typical antibody, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. The variable region of the light chain is aligned with the variable region of the heavy chain, and the constant region of the light chain is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0040] The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H and V L The regions have the same general structure, with each region containing four framework (FW or FR) regions. The term "framework region" as used herein refers to the relatively conserved amino acid sequences in the variable region located between the CDRs. There are four framework regions in each variable domain, which are called FR1, FR2, FR3, and FR4. The framework regions form a β-sheet that provides the structural framework of the variable region (see, for example, CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)).

[0041] The framework regions are connected by three CDRs. As mentioned above, the three CDRs (known as CDR1, CDR2, and CDR3) form the "hypervariable region" of the antibody involved in antigen binding. The CDRs form loops that connect to, and sometimes include part of, the beta sheet structure formed by the framework regions. The constant regions of the light and heavy chains are not directly involved in the binding of the antibody to the antigen, although the constant regions may affect the orientation of the variable regions. The constant regions also exhibit various effector functions, such as participating in antibody-dependent complement-mediated lysis or antibody-dependent cellular cytotoxicity through interactions with effector molecules and cells.

[0042] As used herein, when an antibody or other entity (e.g., an antigen-binding domain) "specifically recognizes" or "specifically binds" an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with substantially higher affinity than other entities that do not display the antigen or epitope. In this context, "substantially higher affinity" means an affinity that is high enough to allow detection of the antigen or epitope as distinguished from the entity using the desired assay or measurement device. It is usually expressed as a binding constant (K a ) but at least 10 7 M -1 (For example, >10 7 M -1 , >10 8 M -1 , >10 9 M -1 , >10 10 M -1 , >10 11 M -1 , >10 12 M -1 , >10 13 M -1 etc. In certain such embodiments, an antibody can bind to different antigens, so long as the different antigens contain that particular epitope. In certain cases, for example, homologous proteins from different species may contain the same epitope.

[0043] The terms "antibody fragment," "antibody fragment," and "antigen-binding fragment" of an antibody are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Any antigen-binding fragment of an antibody described herein is within the scope of the present disclosure. An antibody fragment desirably contains, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include, without limitation, the following: (i) V L , VH , C L , and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide bridges at the hinge region; and (iii) a V fragment, which is a single arm of an antibody. L and V H (iv) Fab' fragments resulting from cleavage of the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) antibody single variable region domains (V H or V L ) Domain antibodies (dAbs) are polypeptides.

[0044] The term "monoclonal antibody" as used herein refers to an antibody produced by a single clone of B lymphocytes against a single epitope on an antigen. Monoclonal antibodies are usually produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991)), or produced from transgenic mice with a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181:69-97 (2008)). In contrast, "polyclonal" antibodies are antibodies secreted by different B cell lineages within an animal. Polyclonal antibodies are a population of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0045] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0046] The terms "nucleic acid", "polynucleotide", "nucleotide sequence", and "oligonucleotide" are used interchangeably herein and refer to polymers or oligomers of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). These terms encompass any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid components, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states. In some embodiments, the nucleic acid or nucleic acid sequence includes other types of nucleic acid structures, such as DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002) and U.S. Pat. No. 5,034,506), locked nucleic acids (LNAs; see, e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see, Wang, J. Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes. The terms "nucleic acid" and "nucleic acid sequence" can also encompass strands that include non-natural nucleotides, modified nucleotides, and / or non-nucleotide components that can exhibit the same function as natural nucleotides (e.g., "nucleotide analogs").

[0047] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial," in which only a portion of the bases of the nucleic acids match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between strands of nucleic acids affects the efficiency and strength of hybridization between strands of nucleic acids. This is particularly important in amplification reactions and in detection methods that depend on binding between nucleic acids.

[0048] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA or a polypeptide or a precursor thereof. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of a coding sequence, so long as the desired activity or functional property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. The term "portion," when used in reference to a gene, refers to a fragment of that gene. A fragment can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleotide sequence comprising at least a portion of a gene" can include a fragment of a gene or the entire gene.

[0049] The term "gene" also includes the coding region of a structural gene, including sequences located adjacent to the coding region at both the 5' and 3' ends, for example, at a distance of about 1 kb on either end, such that the gene corresponds to the length of the full-length mRNA (e.g., including coding, regulatory, structural, and other sequences). Sequences located 5' of the coding region and present on the mRNA are referred to as 5' untranslated or non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' untranslated or non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), genomic forms or clones of a gene contain the coding region interrupted by non-coding sequences referred to as "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA), and introns may contain regulatory elements such as enhancers. Introns are absent in the messenger RNA (mRNA) transcript because they are removed or "spliced ​​out" from the nuclear or primary transcript, which functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0050] In addition to containing introns, genomic forms of a gene may also include sequences located both 5' and 3' to the sequences present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage, and polyadenylation.

[0051] The term "wild type", when used in reference to a gene, refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term "wild type", when used in reference to a gene product, refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term "wild type", when used in reference to a protein, refers to a protein that has the characteristics of a naturally occurring protein. The term "naturally occurring", when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and is present in an organism (including viruses) that has not been intentionally modified by the hand of man in the laboratory, is naturally occurring. A wild type gene is often that gene or allele that is most frequently observed in a population and is therefore arbitrarily referred to as the "normal" or "wild type" form of the gene. In contrast, the terms "modified" or "mutant", when used in reference to a gene or gene product, refer to a gene or gene product, respectively, that exhibits modifications in sequence and / or functional properties (e.g., altered characteristics) when compared to the wild type gene or gene product. It should be noted that naturally occurring mutants can be isolated. These are identified by the fact that they have altered properties when compared to the wild-type gene or gene product.

[0052] The term "allele" refers to a genetic variation, including, without limitation, variants and mutations, polymorphic and single nucleotide polymorphic loci, frameshifts, and splice variants. Alleles may occur naturally within a population or may arise during the lifetime of any particular individual in a population.

[0053] Thus, the terms "variant" and "mutant," when used in reference to a nucleotide sequence, refer to a nucleic acid sequence that differs from another, normally associated, nucleotide sequence by one or more nucleotides. A "mutation" is a difference between two different nucleotide sequences, usually one of which is a reference sequence.

[0054] Nucleic acid or amino acid sequence "identity" as described herein can be determined by comparing a subject nucleic acid or amino acid sequence to a reference nucleic acid or amino acid sequence. Many mathematical algorithms for obtaining optimal alignment and calculating identity between two or more sequences are known and are incorporated into many available software programs. Examples of such programs include: CLUSTAL-W, T-Coffee, and ALIGN (for aligning nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST2.1, BL2SEQ, and later versions), and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed, for example, in: Altschul et al., J. Molecular Biol., 215(3):403~410(1990), Beigert et al., Proc. Natl. Acad. Acids,Cambridge University Press,Cambridge,UK(2009),Soding,Bioinformatics,21(7):951~960(2005),Altschul et al.,Nucleic Acids Res.,25(17):3389~3402(1997),and Gusfield,Algorithms on Strings,Trees and Sequences,Cambridge University Press,Cambridge UK (1997)).

[0055] As one of skill in the art would know based on this disclosure, one or more amino acids of the bispecific antibodies described above, or antigenic fragments thereof, can be exchanged or substituted with different amino acids. An amino acid "exchange" or "substitution" refers to the replacement of an amino acid at a given position or residue with another amino acid at the same position or residue in a polypeptide sequence. Amino acids are broadly classified as "aromatic" or "aliphatic". Aromatic amino acids contain an aromatic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly classified as "aliphatic". Examples of "aliphatic" amino acids include: Glycine (G or Gly), Alanine (A or Ala), Valine (V or Val), Leucine (L or Leu), Isoleucine (I or Ile), Methionine (M or Met), Serine (S or Ser), Threonine (T or Thr), Cysteine ​​(C or Cys), Proline (P or Pro), Glutamic Acid (E or Glu), Aspartic Acid (A or Asp), Asparagine (N or Asn), Glutamine (Q or Gln), Lysine (K or Lys), and Arginine (R or Arg). The aliphatic amino acids can be further classified into four subgroups. The "major aliphatic non-polar subgroup" consists of valine, leucine, and isoleucine. The "aliphatic slightly polar subgroup" consists of methionine, serine, threonine, and cysteine. The "aliphatic polar / charged subgroup" consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine. The "small residue subgroup" consists of glycine and alanine. The group of charged / polar amino acids can be further divided into three subgroups: the "positively charged subgroup" consisting of lysine and arginine, the "negatively charged subgroup" consisting of glutamic acid and aspartic acid, and the "polar subgroup" consisting of asparagine and glutamine. The aromatic amino acids can be further divided into two subgroups.the "nitrogen ring subgroup" consisting of histidine and tryptophan, and the "phenyl subgroup" consisting of phenylalanine and tyrosine.

[0056] Amino acid exchanges or substitutions can be conservative, semi-conservative, or non-conservative. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that has common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, groups of amino acids can be defined as those that preferentially exchange with each other and are therefore most similar to each other in the effect they have on the overall protein structure. Examples of conservative amino acid substitutions include: Substitution of amino acids within the aforementioned subgroups, such as lysine for arginine and vice versa so that a positive charge can be maintained, glutamic acid for aspartic acid and vice versa so that a negative charge can be maintained, serine for threonine so that a free -OH can be maintained, and glutamine for asparagine so that a free -NH2 can be maintained, etc. "Semi-conservative mutations" include amino acid substitutions of amino acids within the same group but not within the same subgroup listed above. For example, substitutions of aspartic acid with asparagine, or asparagine with lysine, involve amino acids within the same group but in different subgroups. "Non-conservative mutations" include amino acid substitutions between different groups (e.g., lysine with tryptophan, or phenylalanine with serine, etc.).

[0057] Additionally, one or more amino acids can be inserted into the bispecific antibodies of the present disclosure (e.g., inserted into the heavy and / or light chain variable region amino acid sequence). Any number of suitable amino acids can be inserted into the amino acid sequence of the antibody or antigen-binding fragment thereof. In this regard, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids), can be inserted into the amino acid sequence of the antibody or antigen-binding fragment thereof. For example, 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may be inserted into the amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof. In this regard, the amino acid(s) can be inserted into the antibody or antigen-binding fragment thereof at any suitable position. Preferably, the amino acid(s) is inserted into the CDR (e.g., CDR1, CDR2, or CDR3) of the antibody or antigen-binding fragment thereof.

[0058] The terms "immunogen" and "antigen" are used interchangeably herein and refer to any molecule, compound, or substance that induces an immune response in an animal (e.g., a mammal). An "immune response" can involve, for example, antibody production and / or activation of immune effector cells. An antigen in the context of this disclosure can include any subunit, fragment, or epitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate or lipid) molecule that elicits an immune response in a mammal. The term "epitope" refers to the sequence of an antigen that is recognized by an antibody or an antigen receptor. Epitopes are also referred to in the art as "antigenic determinants." In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope can include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope can have specific three-dimensional structural characteristics (e.g., a "conformational" epitope) and / or specific charge characteristics. The antigen may be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which elicits an immune response in a mammal, preferably leading to protective immunity.

[0059] "Pharmaceutically acceptable carriers" as used herein generally refer to ingredients in a pharmaceutical formulation, other than the active ingredient, that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0060] The term "pharmaceutical formulation," as used herein, generally refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein (e.g., the bispecific antibodies of the present disclosure) to be effective, and that does not contain additional components that are unacceptably toxic to a subject to which the formulation will be administered.

[0061] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") generally refers to a clinical intervention that seeks to alter the natural course of the individual being treated, and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, without limitation, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or alleviation of disease symptoms, and remission or improved prognosis. In some embodiments, the bispecific antibodies of the present disclosure, or other compositions comprising the bispecific antibodies of the present disclosure (e.g., antibody conjugates, fusion proteins, or polymer formulations), are used to delay disease onset or slow disease progression.

[0062] The term "half-life," as used herein, generally refers to the time required for the concentration of a substance (e.g., a bispecific antibody, an antibody conjugate, a fusion protein (e.g., a Fab fusion protein), or a polymer formulation) to decrease by half in vivo (e.g., in the eye (e.g., the vitreous)) or in vitro.

[0063] An "effective amount" of an agent (eg, a pharmaceutical formulation), as used herein, generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0064] An "individual" or "subject" is a mammal. Mammals include, without limitation, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, an individual or subject is a human. A "subject" may also be a "patient."

[0065] Anti-CFHR4 / anti-C3 bispecific antibody As further described herein, embodiments of the present disclosure relate to the treatment and / or prevention of age-related macular degeneration (AMD), including both neovascular AMD ("wet" AMD) and non-neovascular AMD ("dry" AMD), as well as advanced non-neovascular AMD (geographic atrophy or GA). In particular, the present disclosure provides novel therapeutic bispecific antibodies that target components of the alternative pathway of the complement activation system to treat AMD. As further described herein, anti-CFHR4 antibodies and anti-C3 have been generated and characterized structurally and functionally. Based on these data, corresponding bispecific antibodies have been generated with one antigen-binding site against CFHR4 and the other antigen-binding site against C3.

[0066] According to these embodiments, the present disclosure provides bispecific antibodies, or antigen-binding fragments thereof, that specifically bind to human complement factor H-related 4 (CFHR) and / or human complement component 3 (C3). In some embodiments, the human CFHR4 is the CFHR4b variant (CFHR4b), which is optionally a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 580. Thus, the bispecific antibodies of the present disclosure can bind to CFHR4 and / or CFHR4B polypeptides and proteins. In some embodiments, the human C3 is the b variant of C3 (C3b), which is optionally a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1645. Thus, the bispecific antibodies of the present disclosure can bind to complement component 3 (C3), complement component 3a (C3a), and / or complement component 3b (C3b) polypeptides and proteins.

[0067] In some embodiments, the bispecific antibody or fragment thereof is monoclonal and optionally recombinant. In some embodiments, the bispecific antibody or fragment thereof is human, humanized, or chimeric. In some embodiments, the bispecific antibody or fragment thereof is a full-length antibody, a single-chain antibody, a single-chain variable fragment (scFv), a variable fragment (Fv), a fragment antigen-binding region (Fab), Fab-C, Fab'-SH, (Fab')2, a single-domain antibody (sdAb), a VHH antibody, a nanobody, a camelid-derived single-domain antibody, a shark IgNAR-derived single-domain antibody fragment (VNAR), a diabody, a triabody, an anticalin, or an aptamer, and optionally the antibody is a full-length antibody comprising an Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 region.

[0068] In some embodiments, the bispecific antibody or fragment thereof is optionally conjugated to at least one additional moiety selected from an antigen-binding moiety, such as an antibody or antigen-binding fragment thereof capable of specifically binding to a target other than human CFHR4 or human C3, preferably wherein the target is expressed in the human eye; a therapeutic or cytotoxic moiety; a detection moiety; a purification moiety; a half-life extending moiety; optionally a polypeptide at least 20 amino acids in length and comprising any combination of G, A, ST, E, and P residues, which is conjugated to the C-terminus or N-terminus of the antibody.

[0069] In some embodiments, the bispecific antibody or fragment thereof is a polypeptide comprising one, two or all three HCDRs of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 3, and one, two or all three corresponding LCDRs of said exemplary antibodies, and / or a VH sequence having at least 90% identity to the VH sequence of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 4, and optionally a VL sequence having at least 90% identity to the corresponding VL sequence of said exemplary antibody, preferably with no mutations allowed in the HCDRs or LCDRs, and / or all six CDRs of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 3, the VH and VL sequences of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 4, and / or the full-length heavy chain (VH+constant) sequence of any one of the exemplary CFHR4 antibodies whose sequence is shown in Table 5, and optionally the corresponding full-length light chain (VL+constant) sequence of said exemplary antibody.

[0070] In some embodiments, the bispecific antibody or fragment thereof is a polypeptide having one, two, or all three HCDRs of any one of the exemplary C3 antibodies whose sequence is shown in Table 6, and one, two, or all three of the corresponding LCDRs of the exemplary antibodies, and / or a VH sequence having at least 90% identity to the VH sequence of any one of the exemplary C3 antibodies whose sequence is shown in Table 7, and optionally at least 90% identity to the corresponding VL sequence of the exemplary antibody shown in Table 8. A polypeptide comprising a VL sequence, preferably no mutations in the HCDRs or LCDRs are tolerated, and / or all six CDRs of any one of the exemplary C3 antibodies whose sequences are shown in Table 6, the VH and VL sequences of any one of the exemplary C3 antibodies whose sequences are shown in Tables 7 and 8, and / or the full-length heavy chain (VH+constant) sequence of any one of the exemplary C3 antibodies whose sequences are shown in Table 9, and optionally the corresponding full-length light chain (VL+constant) sequence of the exemplary antibody shown in Table 10.

[0071] Embodiments of the present disclosure also include polynucleotides encoding bispecific antibodies or fragments thereof. In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, or 100% identity to the nucleic acid sequence of any one of the exemplary antibodies whose sequence is set forth in Tables 5, 9, and 10. Embodiments of the present disclosure also include an expression vector comprising the polynucleotide, which is optionally an adeno-associated virus (AAV) vector, a lentivirus (LV) vector, a herpes simplex virus (HSV) vector, or a retrovirus vector.

[0072] Embodiments of the present disclosure also include pharmaceutical compositions comprising the bispecific antibody or fragment thereof, corresponding polynucleotide, or vector comprising the polynucleotide. In some embodiments, the composition comprises at least one pharma- ceutically acceptable carrier, diluent, or preservative, and / or at least one additional active ingredient. In some embodiments, the pharmaceutical composition is suitable for ocular administration to a subject, optionally by delivery using conjunctival inserts, contact lenses, gels, nanoparticles, mucoadhesive polymers, ointments, solutions, suspensions, eye drops, and / or implants, preferably by injection into the vitreous humor.

[0073] The embodiments of the present disclosure also include the bispecific antibody or fragment thereof, corresponding polynucleotide, vector comprising the polynucleotide, or pharmaceutical composition for use as a medicament, optionally for use in a method for treating an ocular disease in a subject. In some embodiments, the disease is characterized by increased activation of the complement system, particularly the alternative pathway, particularly in the subject's eye, for example in drusen or retinal pigment epithelial (RPE) cells of the subject. In some embodiments, the method includes ocular administration of the antibody, preferably by injection into the vitreous humor, which preferably relieves at least one symptom in the subject selected from visual distortion, reduced central vision, blurred vision, and / or difficulty adapting to low light. In some embodiments, the disease is age-related macular degeneration (AMD), including dry AMD, which may be in early, intermediate, or advanced stages (the latter otherwise known as geographic atrophy, GA).

[0074] In accordance with the above, embodiments of the present disclosure include a bispecific antibody comprising a first antigen-binding site for complement factor H-related 4 (CFHR4) and a second antigen-binding site for complement component 3 (C3). In some embodiments, the first anti-CFHR4 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406, and the second anti-C3 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3.

[0075] Embodiments of the present disclosure include bispecific antibodies comprising a first antigen-binding site for complement factor H-related 4 (CFHR4) and a second antigen-binding site for complement component 3 (C3). In some embodiments, the first anti-CFHR4 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the second anti-C3 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 1127-1200, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 1275-1348.

[0076] Embodiments of the present disclosure include bispecific antibodies comprising a first antigen-binding site for complement factor H-related 4 (CFHR4) and a second antigen-binding site for complement component 3 (C3). In some embodiments, the first anti-CFHR4 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380, and a second antigen-binding site for complement component 3 (C3) from at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406. the first anti-C3 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 1127 to 1200, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 1275 to 1348.

[0077] In some embodiments, a bispecific antibody of the disclosure comprises an anti-CFHR4 VH comprising an amino acid sequence having at least 90% sequence identity to at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380. In some embodiments, a bispecific antibody of the disclosure comprises an anti-CFHR4 VL comprising an amino acid sequence having at least 90% sequence identity to at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406.

[0078] According to these embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:2, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:9, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:15. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:3, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:10, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:4, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:11, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:5, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:12, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:6, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:13, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:19. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:7, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:14, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:22, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:33, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:23, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:34, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:24, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:35, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:45.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:25, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:36, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:46. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:26, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:37, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:27, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:38, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:48. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:28, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:39, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:49. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:29, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:40, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:50. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:30, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:41, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:31, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:42, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:54, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:66, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:77. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:55, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:67, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:78.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:56, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:68, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:79. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:57, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:69, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:80. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:58, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:70, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:81. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:59, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:71, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:82. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:60, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:72, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:83. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:61, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:73, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:84. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:62, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:74, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:63, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:75, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:64, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:76, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:87.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 89, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 103, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 90, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 104, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 117. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 91, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 105, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO: 92, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO: 106, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:93, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:107, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:120. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:94, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:108, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:121. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:95, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:109, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:122. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:96, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:110, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:123. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:97, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:111, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:124.In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:98, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:112, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:125. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:99, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:113, and HCDR3 comprises the amino acid sequence of SEQ ID NO:126. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:100, HCDR2 comprises the amino acid sequence of SEQ ID NO:114, and HCDR3 comprises the amino acid sequence of SEQ ID NO:127. In some embodiments, anti-CFHR4 HCDR1 comprises the amino acid sequence of SEQ ID NO:101, anti-CFHR4 HCDR2 comprises the amino acid sequence of SEQ ID NO:115, and anti-CFHR4 HCDR3 comprises the amino acid sequence of SEQ ID NO:128.

[0079] In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 130, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 153, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 131, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 154, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 132, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 155, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 133, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 156, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 178. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 134, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 157, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 179. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 135, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 158, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 136, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 159, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 181. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 137, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 160, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:138, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:161, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:183.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 139, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 162, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 184. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 140, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 163, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 185. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 141, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 164, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 142, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 165, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 143, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 166, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 144, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 167, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 189. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 145, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 168, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 190. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 146, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 169, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 191. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:147, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:170, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:192.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 148, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 171, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 193. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 149, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 172, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 194. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 150, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 173, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 195. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 151, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 174, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 196. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 198, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 201, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 199, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 202, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 207, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 217, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 227. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO: 208, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO: 218, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO: 228. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:209, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:219, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:229.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:210, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:220, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:230. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:211, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:221, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:231. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:212, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:222, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:232. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:213, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:223, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:233. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:214, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:224, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:234. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:215, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:225, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:235. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:237, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:245, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:253. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:238, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:246, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:254. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:239, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:247, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:255.In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:240, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:248, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:256. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:241, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:249, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:257. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:242, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:250, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:258. In some embodiments, anti-CFHR4 LCDR1 comprises the amino acid sequence of SEQ ID NO:243, anti-CFHR4 LCDR2 comprises the amino acid sequence of SEQ ID NO:251, and anti-CFHR4 LCDR3 comprises the amino acid sequence of SEQ ID NO:259.

[0080] In some embodiments, a bispecific antibody of the present disclosure comprises an anti-C3 VH comprising an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to at least one of SEQ ID NOs: 1127-1200. In some embodiments, a bispecific antibody of the present disclosure comprises an anti-C3 VL comprising an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to at least one of SEQ ID NOs: 1275-1348.

[0081] According to these embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:583, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:602, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:620. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:584, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:603, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:621. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:585, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:604, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:622. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:586, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:605, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:623. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:587, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:606, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:624. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:588, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:607, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:625. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:589, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:608, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:626. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:590, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:609, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:627. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 591, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 610, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 628. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 592, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 611, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 629.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:593, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:612, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:630. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:594, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:613, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:631. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:595, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:614, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:632. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:596, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:615, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:633. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:597, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:616, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:634. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:598, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:617, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:635. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:599, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:618, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:636. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO:600, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO:619, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO:637. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 639, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 652, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 664. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 640, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 653, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 665.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 641, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 654, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 642, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 655, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 667. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 643, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 656, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 668. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 644, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 657, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 669. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 645, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 658, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 670. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 646, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 659, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 671. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 647, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 660, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 672. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 648, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 661, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 673. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 649, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 662, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 674. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 650, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 663, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 675.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 677, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 706, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 734. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 678, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 707, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 735. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 679, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 708, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 736. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 680, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 709, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 737. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 681, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 710, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 738. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 682, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 711, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 739. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 683, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 712, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 740. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 684, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 713, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 741. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 685, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 714, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 742. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 686, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 715, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 743.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 687, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 716, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 744. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 688, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 717, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 745. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 689, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 718, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 746. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 690, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 719, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 747. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 691, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 720, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 748. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 692, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 721, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 749. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 693, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 722, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 750. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 694, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 723, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 751. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 695, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 724, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 752. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 696, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 725, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 753. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 697, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 726, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 754. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 698, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 727, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 755.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 699, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 728, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 756. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 700, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 729, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 757. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 701, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 730, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 758. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 702, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 731, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 759. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 703, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 732, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 760. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 704, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 733, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 761. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 763, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 771, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 778. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 764, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 772, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 779. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 765, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 773, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 780. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 766, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 774, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 781.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 767, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 775, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 782. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 768, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 776, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 783. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 769, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 777, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 784. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 786, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 794, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 801. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 787, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 795, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 802. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 788, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 796, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 803. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 789, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 797, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 804. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 790, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 798, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 805. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 791, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 799, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 806. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 792, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 800, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 807.In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 809, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 812, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 815. In some embodiments, anti-C3 HCDR1 comprises the amino acid sequence of SEQ ID NO: 810, anti-C3 HCDR2 comprises the amino acid sequence of SEQ ID NO: 813, and anti-C3 HCDR3 comprises the amino acid sequence of SEQ ID NO: 816.

[0082] In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 818, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 850, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 881. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 819, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 851, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 882. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 820, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 852, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 883. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 821, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 853, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 884. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 822, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 854, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 885. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 823, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 855, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 886. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 824, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 856, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 887. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 825, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 857, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 888. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 826, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 858, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 889. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 827, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 859, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 890.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 828, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 860, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 891. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 829, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 861, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 892. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 830, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 862, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 893. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 831, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 863, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 894. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 832, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 864, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 895. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 833, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 865, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 896. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 834, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 866, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 897. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 835, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 867, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 898. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 836, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 868, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 899. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 837, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 869, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 900.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 838, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 870, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 901. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 839, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 871, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 902. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 840, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 872, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 903. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 841, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 873, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 904. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 842, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 874, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 905. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 843, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 875, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 906. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 844, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 876, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 907. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 845, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 877, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 908. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 846, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 878, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 909. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 847, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 879, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 910.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 848, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 880, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 911. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 913, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 916, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 919. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 914, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 917, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 920. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 914, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 917, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 920. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:922, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:931, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:940. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:923, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:932, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:941. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:924, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:933, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:942. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:925, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:934, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:943. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 926, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 935, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 944. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 927, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 936, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 945.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:928, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:937, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:946. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:929, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:938, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:947. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:949, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:984, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1019. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:950, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:985, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1020. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 951, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 986, and anti-C3 LCDR3 comprises. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 952, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 987, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1022. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 953, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 988, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1023. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 954, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 989, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1024. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO: 955, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO: 990, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO: 1025. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:956, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:991, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1026. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:957, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:992, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1027. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:958, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:993, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1028. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:959, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:994, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1029. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:960, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:995, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1030.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:961, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:996, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1031. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:962, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:997, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1032. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:963, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:998, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1033. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:964, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:999, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1034. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:965, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1000, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1035. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:966, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1001, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1036. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:967, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1002, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1037. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:968, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1003, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1038. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:969, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1004, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1039.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:970, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1005, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1040. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:971, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1006, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1041. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:972, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1007, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1042. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:973, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1008, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1043. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:974, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1009, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1044. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:975, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1010, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1045. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:976, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1011, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1046. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:977, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1012, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1047. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:978, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1013, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1048.In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:979, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1014, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1049. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:980, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1015, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1050. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:981, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1016, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1051. In some embodiments, anti-C3 LCDR1 comprises the amino acid sequence of SEQ ID NO:982, anti-C3 LCDR2 comprises the amino acid sequence of SEQ ID NO:1017, and anti-C3 LCDR3 comprises the amino acid sequence of SEQ ID NO:1052.

[0083] In some embodiments, the bispecific antibody of the present disclosure comprises an anti-C3 antigen binding site comprising a VH and VL that are at least 90% identical to the VH and VL from any anti-C3 antibody. In some embodiments, the bispecific antibody of the present disclosure comprises an anti-CFHR4 antigen binding site, or an antigen-binding fragment thereof, and an anti-C3 antigen binding site, or an antigen-binding fragment thereof. Such bispecific antibodies can be used to target the complement activation pathway from two different mechanisms, thus providing additional therapeutic benefits. In some embodiments, the second anti-C3 antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from the NGM621 antibody (or an antigen-binding fragment thereof). NGM621 is a humanized IgG1 monoclonal antibody engineered to inhibit complement C3. In some embodiments, the second anti-C3 antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-C3 antibody disclosed in U.S. Patent No. 9,815,890 or PCT Application No. PCT / US2019 / 025123, or an antigen-binding fragment thereof, both of which are incorporated herein by reference.

[0084] In other embodiments, the anti-CFHR4 antigen binding site can be any of the anti-CFHR4 antibodies of the present disclosure, and the anti-C3 arm can be any C3 antagonist, such as, but not limited to, POT-4 (AL-78898A) and APL-2. POT-4 (AL-78898A) is a synthetic cyclic peptide conjugated to a PEG polymer (e.g., a compstatin analog) that specifically binds C3, and was the first complement inhibitor to be tested in the treatment of macular degeneration. APL-2 (pegcetacoplan) is a symmetric molecule composed of two identical pentadecapeptides covalently attached to the termini of a linear 40 kDa polyethylene glycol molecule, and binds both C3 and C3b.

[0085] In some embodiments, the second anti-C3 antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from any antibody that binds to complement component 3b (C3b). In some embodiments, the second anti-C3b antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from the S77 antibody (or an antigen-binding fragment thereof). S77 is an antibody that selectively recognizes C3b but not the non-activated molecule C3. In some embodiments, the second anti-C3 antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from the anti-C3 antibody, or an antigen-binding fragment thereof, disclosed in U.S. Pat. No. 8,012,473 or PCT Application No. PCT / US2008 / 065771. Both publications are incorporated herein by reference (see also Katschke, KJ et al., Journal of Biological Chemistry, 2009, vol. 284(16):10473-10479).

[0086] Anti-C3 / anti-VEGF bispecific antibody Embodiments of the present disclosure relate to the treatment and / or prevention of age-related macular degeneration (AMD), including both neovascular AMD ("wet" AMD) and non-neovascular AMD ("dry" AMD), as well as advanced non-neovascular AMD (geographic atrophy or GA). In some embodiments, the present disclosure provides novel therapeutic bispecific antibodies that target components of the alternative pathway of the complement activation system and the vascular endothelial growth factor (VEGF) signaling pathway to treat AMD. As further described herein, embodiments of the present disclosure also include bispecific antibodies that include an antigen-binding site for CFHR4 or C3 and an antigen-binding site for VEGF. According to these embodiments, the anti-CFHR4 antigen-binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from one or more of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from one or more of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406, and the second anti-VEGF antigen-binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-VEGF antibody. In some embodiments, the anti-C3 antigen binding site comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from one or more of SEQ ID NOs: 1127-1200, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from one or more of SEQ ID NOs: 1275-1348, and the second anti-VEGF antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-VEGF antibody.

[0087] In some embodiments, the second anti-VEGF antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from any anti-VEGF antibody. In some embodiments, the bispecific antibody of the present disclosure comprises an anti-CFHR4 antigen binding site, or an antigen-binding fragment thereof, and an anti-VEGF antigen binding site, or an antigen-binding fragment thereof. Such bispecific antibodies can be used to treat AMD through inhibition of the complement activation pathway and inhibition of the VEGF signaling pathway, thus providing additional therapeutic effects. The VEGF antigen binding site can be selected from any anti-VEGF antibody, such as, but not limited to, bevacizumab, ranibizumab, brolucizumab, faricimab, and vanucizumab, or antigen-binding fragments thereof.

[0088] In some embodiments, the second anti-VEGF antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from bevacizumab (or an antigen binding fragment thereof). Bevacizumab (Avastin) is an anti-VEGF human monoclonal antibody. In some embodiments, the second anti-VEGF antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-VEGF antibody disclosed in EP Patent Nos. EP0666868 and EP0817648, or PCT Application No. PCT / US1992 / 009218, or an antigen binding fragment thereof, all of which are incorporated herein by reference.

[0089] In some embodiments, the second anti-VEGF antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from ranibizumab (or an antigen-binding fragment thereof). Ranibizumab (Lucentis) is a Fab fragment derived from bevacizumab. In some embodiments, the second anti-VEGF antigen binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-VEGF antibody disclosed in U.S. Patent Application No. 11 / 840,279 or PCT Application No. PCT / EP2007 / 007276, or an antigen-binding fragment thereof, both of which are incorporated herein by reference.

[0090] In some embodiments, the second anti-VEGF antigen-binding site comprises a VH and VL that are at least 90% identical to the VH and VL from brolucizumab (or an antigen-binding fragment thereof). Brolucizumab (RTH258; Beovu) is a humanized scFv that is an inhibitor of VEGF-A. In some embodiments, the second anti-VEGF antigen-binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-VEGF antibody disclosed in PCT Application No. PCT / US2015 / 059575 or PCT Application No. PCT / US2015 / 059571, or an antigen-binding fragment thereof, both of which are incorporated herein by reference.

[0091] In some embodiments, the second anti-VEGF antigen-binding site comprises a VH and VL that are at least 90% identical to the VH and VL from faricimab (or an antigen-binding fragment thereof). Faricimab (RG7716) is a bispecific monoclonal antibody specifically designed for the eye that simultaneously binds to and neutralizes both angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A). In some embodiments, the second anti-VEGF antigen-binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-VEGF antibody disclosed in U.S. Pat. No. 10,072,075 or PCT Application No. PCT / US2016 / 053454, or an antigen-binding fragment thereof, both of which are incorporated herein by reference.

[0092] In some embodiments, the second anti-VEGF antigen-binding site comprises a VH and VL that are at least 90% identical to the VH and VL from vanucizumab (or an antigen-binding fragment thereof). Vanucizumab (RG7721) is a humanized bispecific monoclonal antibody designed for the treatment of cancer, consisting of two different heavy chains and two different light chains, one arm of which binds Ang2 and the other arm of which binds VEGF (bevacizumab). In some embodiments, the second anti-VEGF antigen-binding site comprises a VH and VL that are at least 90% identical to the VH and VL from an anti-VEGF antibody disclosed in U.S. Pat. No. 8,945,552 or PCT Application No. PCT / EP2011 / 054504, or an antigen-binding fragment thereof, both of which are incorporated herein by reference.

[0093] In other embodiments, the bispecific antibodies of the present disclosure comprise an anti-CFHR4 or anti-C3 antigen binding site (or antigen-binding fragment thereof) and an anti-VEGF antigen binding site (or antigen-binding fragment thereof) from any of the following VEGF antagonists, including, but not limited to, anti-VEGF antibodies (e.g., bevacizumab, sevacizumab, and ranibizumab), anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab, tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules (e.g., icrucumab, aflibercept (VEGF Trap-Eye, EYLEA®), and div-aflibercept (VEGF trap, ZALTRAP®)), the anti-VEGF arm of a VEGF bispecific antibody (e.g., MP-0250, vanucizumab (VEGF-ANG2)), such as the anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms.

[0094] multispecific antibody The amino acid sequence of the bispecific antibody of the present disclosure is not limited to the specific amino acid sequence described herein. Indeed, the anti-CFHR4 / anti-C3 bispecific antibody, anti-CFHR4 / anti-VEGF bispecific antibody, and anti-C3 / anti-VEGF bispecific antibody, or antigen-binding fragment thereof, can include any heavy or light chain polypeptide that competes with the various antigen-binding sites of these antibodies. Antibody competition can be assayed using conventional peptide competition assays (e.g., ELISA, Western blot, or immunohistochemistry, etc.) (see, e.g., U.S. Patent Nos. 4,828,981 and 8,568,992, and Braitbard et al., Proteome Sci., 4:12 (2006)).

[0095] The bispecific antibodies of the present disclosure may be whole antibodies or antigen-binding fragments of whole antibodies. As defined herein, antigen-binding antibody fragments encompassed by the present disclosure include, without limitation, F(ab')2, Fab', Fab, Fv, scFv, dsFv, dAb, and single-chain binding polypeptides. Antibody fragments and their therapeutic utility are further described, for example, in Nelson, AL, MAbs. 2010 Jan-Feb; 2(1): 77-83; Joosten et al., Microbial Cell Factories volume 2, Article number: 1 (2003); and Bates A, Power CA., Antibodies (Basel). 2019; 8(2): 28; doi: 10.3390 / antib8020028). In some embodiments, the anti-CFHR4, anti-C3, and / or anti-VEGF antigen-binding fragments are single-chain variable fragments (scFv), which are engineered antibodies produced by the fusion of immunoglobulin heavy (VH) and light (VL) chains via a short polypeptide linker. Single-chain variable domain (Fv) fragments (scFv) have been used in the art for a variety of clinical and therapeutic applications, primarily due to their improved pharmacokinetic properties compared to parent monoclonal antibodies and their relative ease of production in large quantities at low cost (Monnier et al., Antibodies 2013, 2(2), 193-208; doi.org / 10.3390 / antib2020193; Safdari et al., Mol Med. 2016; 22:258-270; and Lu, R., Hwang, Y., Liu, I. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1(2020). https: / / doi.org / 10.1186 / s12929-019-0592-z).

[0096] The bispecific antibodies of the present disclosure may be diabodies. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). The anti-CFHR4, anti-C3, or anti-VEGF antibodies of the present disclosure may be single-domain antibodies (also referred to as nanobodies). Single-domain antibodies are antibody fragments that include all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0097] In other embodiments, the bispecific antibodies of the present disclosure are whole antibodies. As defined herein, a whole antibody comprises two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (V H ) region and three C-terminal constant (C H1 , C H2 , C H3 ) region, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L) and the heavy chain C-terminal constant region contains the fragment crystallizable (Fc) domain, which determines the antibody class and is responsible for humoral and cellular effector functions. Antibodies are classified into five major classes (or "isotypes"), IgG, IgM, IgA, IgD, and IgE, which differ in their functions in the immune system. IgG is the most abundant immunoglobulin in blood, accounting for 60% of all serum antibodies in humans. IgG antibodies can be subdivided into IgG1, IgG2, IgG3, and IgG4. These are named in order of their abundance in serum, with IgG1 being the most abundant (Vidarsson et al., Frontiers in Immunology. 5:520 (2014)). The fully bispecific antibodies described herein (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies) may be of any suitable class and / or subclass. In some embodiments, the antibody is of the class IgG (e.g., IgG1, IgG2, IgG3, or IgG4). For example, the antibody may be an IgG1 antibody.

[0098] As mentioned above, the Fc domain mediates some effector functions of the antibody, such as binding to receptors on target cells and complement fixation (which triggers the effector function of eliminating antigens). In some embodiments, the Fc domain may be modified or engineered to alter its effector functions. For example, the Fc domain may be modified to improve antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), as well as to control serum half-life. In some embodiments, the Fc domain of an anti-CFHR4 antibody may be engineered to modulate its affinity for Fc receptors (e.g., Fcγ receptors (FcγRs) and neonatal Fc receptors (FcRn)). Indeed, optimization of the interaction between antibodies and FcγRs has emerged as a promising approach to enhance the activity of therapeutic antibodies for the treatment of various diseases. (Mimoto et al.,Curr.Pharm.Biotechnol.17,1298~1314(2016);Lazar et al.,Proc.Natl Acad.Sci.USA 103,4005~4010(2006);Richards et al.,Mol.Cancer Ther.7,2517~2527(2008);Nordstrom et al. al., Breast Cancer Res.13, R123 (2011); and Kang, TH, Jung, ST, Exp Mol Med 51, 1-9 (2019)). The Fc domain may also be modified to improve serum half-life, for example, by engineering IgG Fc for higher FcRn binding (Zalevsky et al., Nat. Biotechnol. 28, 157-159 (2010); and Dall'Acqua et al., J. Immunol. 169, 5171-5180 (2002)). In other embodiments, the Fc domain may be modified to create monovalency or bispecificity of the antibody to improve therapeutic efficacy. For example, an Fc domain can be generated that does not form homodimers but remains as a soluble monomeric mFc that exhibits high affinity for FcγRI but no detectable binding to FcγRIIIa.In other embodiments, heterodimeric Fc domains can be generated to obtain bispecific properties for antigen binding that avoid homodimer formation. Engineered Fc domains can be generated by inducing point mutations or by modifying the glycosylation of the Fc domain (Saunders, KO, Front Immunol. 2019; 10: 1296; Kelley, RF, Meng, YG, Liu et al., J Biol Chem. 2014; 289: 3571-90; Monnet et al., MAbs. 2014; 6: 422-36; Li et al., Proc Natl Acad Sci US A. 2017; 114: 3485-90; and Lin et al., Proc Natl Acad Sci US A. 2015; 112: 10611-6; Kang and Jung, supra).

[0099] As previously mentioned, the bispecific antibodies of the disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies) can be monoclonal, human, humanized, and / or chimeric antibodies. In some embodiments, the antibody is a fragment selected from the group consisting of Fab, Fab-C, Fab′-SH, Fv, scFv, and (Fab′)2 fragments. In some embodiments, the bispecific antibodies of the disclosure comprise two or more single domain antibodies forming a bivalent, trivalent, or tetravalent antibody that recognizes different epitopes on the same or different antigens.

[0100] In some embodiments, the bispecific antibodies of the present disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies) are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA.: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable domain derived from a mouse, rat, hamster, rabbit, or a non-human primate, e.g., a monkey) and a human constant domain. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0101] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0102] Humanized antibodies and methods for their production are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., below. Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al. al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0103] In accordance with the foregoing embodiments, the bispecific antibodies of the disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies) can be made in bivalent, trivalent, or tetravalent formats. For example, the bispecific antibodies of the disclosure can be bivalent bispecific antibodies having heteromeric heavy chains (e.g., triomabs, knobs and holes (KIH), duobodies, etc.). Bispecific antibodies of the present disclosure can be tetravalent multispecific antibodies composed of IgG with other binding domains fused to either the N-terminus or C-terminus of either the heavy or light chain (e.g., dual variable domains [DVDs], IgG-scFv fusions, mabtyrin (an IgG with a non-antibody binding scaffold "sentilin" fused to the C-terminus of the heavy chain). Bispecific antibodies of the present disclosure can be composed of IgG with additional antigen binding sites added into the structure (e.g., two-in-one antibodies, MAT "Modular Antibody Technology" platform from F-Star). Bispecific antibodies of the present disclosure can be engineered antibody fragments linked by short peptide linkers that can be made into bivalent, trivalent, or tetravalent formats addressing two to three targets (e.g., bispecific T cell engagers (BiTEs), nanobody platforms, dual affinity retargeting (DART) antibodies, "tandem antibody" structures (TandAbs)). Bispecific antibodies of the present disclosure can also be composed of chemically conjugated IgG.

[0104] Polypeptides and Expression Vectors Embodiments of the present disclosure also include polynucleotides encoding any of the bispecific antibodies of the present disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies). In accordance with these embodiments, the present disclosure includes an expression vector comprising any of the polynucleotides encoding the bispecific antibodies of the present disclosure. In some embodiments, the expression vector is suitable for producing a bispecific antibody of the present disclosure for delivery of the antibody to a subject. In certain embodiments, the nucleic acid sequence is in the form of a vector. The vector can be, for example, a plasmid, an episome, a cosmid, a viral vector (e.g., a retrovirus or an adenovirus), or a phage. Suitable vectors and methods for vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 4th edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0105] In addition to the nucleic acid encoding the anti-CFHR4 antibody or antigen-binding fragment thereof, the vector desirably contains expression control sequences (e.g., promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc.) that provide for the expression of the nucleic acid sequence encoding the antibody in a host cell. Exemplary expression control sequences are known in the art and described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0106] Vectors containing nucleic acid sequences encoding the bispecific antibodies of the present disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies), or antigen-binding fragments thereof, may be introduced into a host cell capable of expressing the encoded polypeptide, including any suitable prokaryotic or eukaryotic cell. Examples of suitable prokaryotic cells include, without limitation, cells from the genera Bacillus (e.g., Bacillus subtilis and Bacillus brevis), Escherichia (e.g., E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of Escherichia coli (e.g., K12, HB101 (ATCC No. 33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102). Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Hansenula, Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, and Schizosaccharomyces. Suitable insect cells include Sf-9 and HIS cells (Invitrogen, Carlsbad, Calif.), as described, for example, in Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67:4566-4579 (1993). Examples of suitable mammalian cells include, but are not limited to, the following: Chinese hamster ovary cells (CHO) (ATCC number CCL61), CHO DHFR cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC number CRL1573), and 3T3 cells (ATCC number CCL92).Other suitable mammalian cell lines are monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and CV-1 cell line (ATCC No. CCL70). Further typical mammalian host cells include primate and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro culture of primary tissues, and primary explants are also suitable. Other suitable mammalian cell lines include, without limitation, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from ATCC. Methods for selecting suitable mammalian host cells, as well as methods for transforming, culturing, amplifying, screening, and purifying such cells, are well known in the art (see, e.g., Ausubel et al., eds., Short Protocols in Molecular Biology, 5th ed., John Wiley & Sons, Inc., Hoboken, NJ (2002)). Preferably, the mammalian cells are human cells.

[0107] In some embodiments, the vector can include a means for attaching a detection moiety to the bispecific antibody of the disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibody, anti-CFHR4 / anti-VEGF bispecific antibody, and anti-C3 / anti-VEGF bispecific antibody). In some embodiments, the vector can include a means for attaching a purification moiety to the bispecific antibody of the disclosure. Exemplary detection and / or purification moieties / tags that can be attached to the bispecific antibodies of the disclosure include, without limitation, hemagglutinin (HA), c-Myc, V5, DYKDDDDK, His tag (e.g., 6x-HIS), glutathione S-transferase (GST), maltose binding protein (MBP), fluorophores (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), mCherry, chromophores, and / or luminescent peptides (e.g., luciferase).

[0108] In some embodiments, the expression vector is suitable for use in gene therapy (e.g., an expression vector for delivering a polynucleotide encoding a bispecific antibody of the present disclosure to a subject). In some embodiments, the expression vector is a herpes simplex virus (HSV) vector, or a retroviral vector. In some embodiments, the expression vector is an adeno-associated virus (AAV) vector or comprises an AAV backbone. For example, AAV vectors have been designed, produced, and used to mediate gene delivery in human subjects, including for therapeutic purposes. Typically, AAV vectors for use in gene transfer comprise a replication-deficient AAV genome that lacks functional Rep and Cap coding viral sequences. Such replication-deficient AAV vectors more preferably lack most or all of the Rep and Cap coding sequences and substantially retain one or two AAV ITR sequences and packaging sequences. The defective genome is packaged into a viral particle to form a defective recombinant AAV virus, also referred to as an "AAV vector." Methods for producing such AAV vectors have been disclosed in the literature, including the use of packaging cells, accessory viruses or plasmids, and / or baculovirus systems (Samulski et al., (1989) J. Virology 63, 3822; Xiao et al., (1998) J. Virology 72, 2224; Inoue et al., (1998) J. Virol. 72, 7024; WO98 / 22607; WO2005 / 072364). Methods for producing pseudotyped AAV vectors have also been reported (e.g., WO00 / 28004), as well as various modifications or formulations of AAV vectors to reduce immunogenicity upon in vivo administration (see, e.g., WO01 / 23001, WOOO / 73316, WO04 / 112727, W005 / 005610, WO99 / 06562). AAV vectors may be prepared or derived from various serotypes of AAV, which may further be mixed together or mixed with other types of viruses to produce chimeric (e.g., pseudotyped) AAV viruses.Examples of tAAV are human AAV4 vectors, human AAV7 vectors, human AAV9 vectors, human AAV10 vectors, or bovine AAV vectors. AAV vectors can be derived from a single AAV serotype, or can contain sequences or components derived from at least two distinct AAV serotypes (pseudotyped AAV vectors). For example, an AAV vector comprises an AAV genome derived from one AAV serotype (e.g., AAV9) and a capsid derived at least partially from a distinct AAV serotype. An AAV vector, as used herein, is a vector that comprises at least one component part derived from an adeno-associated virus. Preferably, the component part is involved in the biological mechanism by which the vector infects or transduces target cells and expresses the anti-CFHR4 antibody of the present disclosure (e.g., ocular delivery / expression).

[0109] In other embodiments, the expression vector is a lentiviral vector (LV) or comprises a LV backbone. Lentiviruses are part of the larger retrovirus group. A detailed list of lentiviruses can be found in Coffin (1997) "Retroviruses" Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). For example, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype "slow virus" Visna / Maedi virus (VMV), as well as the related Caprine Arthritis Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Maedi-Visna Virus (MVV) and Bovine Immunodeficiency Virus (BIV). In one embodiment, the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or Visna lentivirus. The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells (Lewis et al (1992) EM BO J 11(8):3053-3058 and Lewis and Emerman (1994) J Virol 68(1):510-516). In contrast, other retroviruses (e.g., MLV) are unable to infect non-dividing or slowly dividing cells (e.g., those that make up muscle, brain, lung and liver tissues). A lentiviral vector, as used herein, is a vector that includes at least one component part derived from a lentivirus. Preferably, that component part is involved in the biological mechanism by which the vector infects or transduces target cells and expresses the anti-CFHR4 antibodies of the disclosure (e.g., ocular delivery / expression).

[0110] Further compositions and methods for ocular gene therapy can be found, for example, in Bordet, T., and Behar-Cohen, F., "Ocular gene therapies in clinical practice: viral vectors and nonviral alternatives", Drug Discovery Today, Volume 24, Issue 8, August 2019, Pages 1685-1693. In some embodiments, gene therapy platforms, methods, and compositions that can be used to deliver (e.g., ocular delivery) bispecific antibodies of the present disclosure to a subject include those disclosed in US20220025396, US20220011308, US20210371877, US20210363192, US20190078099, US20190038724, and US10494646B2, which are incorporated herein by reference. In other embodiments, gene therapy platforms, methods, and compositions that can be used to deliver the bispecific antibodies of the present disclosure to a subject (e.g., ocular delivery) include platforms, methods, and compositions based on HMR59 (Hemera Biosciences). HMR59 blocks the membrane attack complex formed during the terminal steps in the complement cascade through its protein product, soluble CD59. HMR59 is designed to be administered as a single intraocular injection.

[0111] In accordance with these embodiments, the present disclosure also provides a method of administering ocular gene therapy to a subject in need thereof, comprising injecting a pharmaceutical composition comprising an effective amount of an expression vector described herein (e.g., an expression vector comprising a polynucleotide encoding a bispecific antibody of the present disclosure). As further described below, the present disclosure also provides a method of treating AMD (wet and / or dry AMD) and / or GA, comprising administering a pharmaceutical composition comprising an effective amount of an expression vector described herein (e.g., an expression vector comprising a polynucleotide encoding a bispecific antibody of the present disclosure). In some embodiments, administering the pharmaceutical composition treats at least one AMD symptom (wet and / or dry AMD symptom) and / or at least one GA symptom.

[0112] Methods for producing bispecific antibodies In general, the modular architecture of antibodies has led to the production of over 100 different bispecific antibody formats. These formats differ in many ways, including based on their molecular weight, the number of antigen binding sites, the spatial relationship between the different binding sites, the valency for each antigen, the ability to assist secondary immune functions, and pharmacokinetic half-life. Recombinant bispecific antibodies can be categorized into two classes: bispecific formats with Fc regions and bispecific formats with Fc regions. Bispecific antibodies with Fc regions retain Fc-mediated effector functions (e.g., CDC and ADCC). These formats broadly include "knobs and holes" IgG, crossmab, ortho-FabIgG, DVD-Ig, two-in-one IgG, IgG-scFv, and scFv2-Fc.

[0113] Fc-less bispecific antibodies lack Fc-mediated effector functions. However, the small size of such antibodies allows better tumor tissue penetration than IgG-like formats. In this format, the variable domains of each parent monoclonal antibody and a linker are cloned and linked to form a single-chain bispecific antibody. These bispecific antibodies represent many formats, such as tandem scFv, diabody formats, single-chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DART), dock-and-lock (DNL), and nanobodies.

[0114] Several strategies have been developed to generate bispecific antibodies. Hybrid hybridoma (also called quadroma) was the earliest technology used to produce bispecific antibodies. It is based on somatic cell fusion of two different hybridoma cell lines expressing mouse IgG of the desired specificity. However, the actual percentage of functional bispecific antibodies by quadroma cell lines is unpredictable and requires a laborious process of isolating bispecific antibodies from by-products. By using molecular cloning techniques, bispecific IgG antibodies can be assembled from two different heavy and light chains expressed in the same production line cells. The production of bispecific antibodies requires at least two plasmids for the heterodimerized heavy chains and one plasmid for the common light chain or two light chain plasmids if two different light chains are used. In particular, expressing the HC and LC on separate plasmids can be advantageous because the manipulation of the plasmid ratio is an easy and efficient approach to optimize protein assembly for the desired product. A laborious and time-consuming process is then usually required to select the most desirable clonal cell line from the heterogeneous stable transfectant pool for large-scale antibody production. Generally, methods for generating bispecific antibodies can be divided into three groups: chemical recombination, cell fusion, and genetic engineering (Reichert, JM, and Dhimolea, E., Drug Discov Today, 2012 Sep;17(17-18):954-63). Recent reviews on different strategies in constructing bispecific antibodies with emphasis on therapeutic applications include Wu, C., Drug News Perspect. 2009 Oct;22(8):453-8; Gu, J. and Ghayur, T., Methods Enzymol. 2012;502:25-41; and Kontermann, R., MAbs, Mar-Apr 2012;4(2):182-97.

[0115] The bispecific antibodies of the present disclosure can be made using any method known in the art. One approach for single-cell manufacturing of bispecific antibodies is to use an antibody based on the same light chain in combination with a minimal set of Fc mutations that drive heavy chain heterodimerization. The resulting bispecific antibody can be easily purified from the two undesired by-products. As used herein, the term "common light chain" refers to an immunoglobulin light chain that includes a variable domain, which can productively associate with multiple heavy chain variable domains to form a paratope, and each heavy chain variable region can specifically bind to the antibody-bound epitope that the heavy chain variable region originally encountered. The heavy or light chains, or portions thereof, of various antibodies are herein said to be "related" to the corresponding chains, or portions thereof, of the enumerated or specifically identified antibodies. This means that the chains, or portions thereof, have the same sequence as the corresponding chains, or portions thereof, of the enumerated or specifically identified antibodies, except for the specifically indicated modifications, such as amino acid substitutions, which may not be present. As further described herein, bispecific antibodies of the present disclosure can be generated using this "common light chain" approach, involving the use of one or more light chains of an anti-CHFR4 antibody.

[0116] Compositions and methods of treatment The bispecific antibodies of the present disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies) can be administered as part of a pharmaceutical composition in a therapeutically effective amount to treat ocular diseases (e.g., AMD or GA). In some embodiments, the composition is suitable for ocular administration. In some embodiments, ocular administration comprises injection into the vitreous humor. In some embodiments, ocular administration comprises delivering the antibody using a conjunctival insert, contact lens, gel, nanoparticles, mucoadhesive polymers, ointments, solutions, suspensions, eye drops, and / or implants (e.g., Susvimo™). Recent methods and formulations for ocular administration can be found, for example, in Souto, EB, et al. "Advanced Formulation Approaches for Ocular Drug Delivery: State-Of-The-Art and Recent Patents", Pharmaceutics, 2019 Sep;11(9):460).

[0117] According to these embodiments, the method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of a bispecific antibody of the present disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibody, anti-CFHR4 / anti-VEGF bispecific antibody, and anti-C3 / anti-VEGF bispecific antibody). In some embodiments, the pharmaceutical composition is administered to the eye to treat at least one AMD symptom. In some embodiments, the AMD comprises wet AMD. In some embodiments, the AMD comprises dry AMD (e.g., GA). In some embodiments, the at least one AMD symptom comprises visual distortion, reduced central vision, blurred vision, and / or difficulty adapting to low light. In some embodiments, administration of the pharmaceutical composition reduces complement activation in the subject's eye.

[0118] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. In some embodiments, the effect is therapeutic, i.e., the effect partially or completely treats the disease and / or adverse symptoms resulting from the disease. To this end, the methods of the present disclosure include administering a "therapeutically effective amount" of a bispecific antibody, or a composition comprising a bispecific antibody. A "therapeutically effective amount" refers to an amount effective for the dosage and duration necessary to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the monoclonal antibody to elicit a desired response in the individual. For example, a therapeutically effective amount of a bispecific antibody of the present disclosure is an amount that treats at least one AMD and / or GA symptom in a subject. In some embodiments, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the present disclosure include administering a "prophylactically effective amount" of a bispecific antibody, or a composition comprising a bispecific antibody. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (eg, prevention of the onset of AMD and / or GA).

[0119] Typical doses of a therapeutically effective amount of a bispecific antibody of the disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibody, anti-CFHR4 / anti-VEGF bispecific antibody, and anti-C3 / anti-VEGF bispecific antibody) can range, for example, from about 0.0001 mg / dose to about 100 mg / dose for each eye to be treated. In some embodiments, a therapeutically effective amount of a bispecific antibody of the disclosure can range from about 0.001 mg / dose to about 100 mg / dose, about 0.01 mg / dose to about 100 mg / dose, about 0.05 mg / dose to about 50 mg / dose, about 0.1 mg / dose to about 10 mg / dose, about 0.5 mg / dose to about 5 mg / dose, and about 1 mg / dose to about 10 mg / dose. In some embodiments, a therapeutically effective concentration of a bispecific antibody of the disclosure can be, for example, from about 0.0001 mg to about 100 mg of antibody per milliliter of solution. In some embodiments, a therapeutically effective concentration of a bispecific antibody of the present disclosure can range from about 0.001 mg / ml to about 100 mg / ml, from about 0.01 mg / ml to about 100 mg / ml, from about 0.1 mg / ml to about 100 mg / ml, from about 1.0 mg / ml to about 100 mg / ml, from about 0.001 mg / ml to about 50 mg / ml, from about 0.01 mg / ml to about 50 mg / ml, from about 0.1 mg / ml to about 50 mg / ml, from about 0.1 mg / ml to about 25 mg / ml, from about 0.1 mg / ml to about 10 mg / ml, and from about 1.0 mg / ml to about 10 mg / ml. In some embodiments, a therapeutically effective dose of a bispecific antibody of the disclosure can be exactly or approximately 0.1 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 2.0 mg, 3.0 mg, 4.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, or 25.0 mg, or can fall within a range bounded by any two of the foregoing values.For example, in certain embodiments, a sustained release formulation (e.g., an ocular implant) can contain exactly or approximately 0.1 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 2.0 mg, 3.0 mg, 4.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, or 25.0 mg of a bispecific antibody, or an amount contained in a range bounded by any two of the foregoing values.

[0120] The therapeutic or prophylactic effect can be monitored by regular evaluation of the treated patient. In the case of repeated administration over several days or more, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the present disclosure. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administration of the composition, or by continuous infusion administration of the composition. The composition comprising the bispecific antibody, or antigen-binding fragment thereof, can be administered to a mammal using standard administration techniques (e.g., ocular, oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration). The composition is preferably suitable for ocular administration.

[0121] In accordance with the compositions and methods of treatment described herein, embodiments of the present disclosure include bispecific antibodies with extended half-life (e.g., following ocular administration) such that the bispecific antibody may be administered less frequently to a subject. In some embodiments, the antibody comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises a polypeptide capable of binding to a bispecific antibody of the present disclosure by any means known in the art (e.g., generation of a fusion protein). In some embodiments, the polypeptide capable of binding to a bispecific antibody of the present disclosure is at least 20 amino acids in length and comprises any combination of G, A, ST, E, and P residues. In some embodiments, the half-life extending polypeptide is attached to the C-terminus or N-terminus of the antibody. In some embodiments, this is referred to as "XTEN-based" and is further described in US8933197, US7846445, US7855279, US8492530, US9938331, US8673860, US9371369, US9926351, US10961287, US10172953, and US10953073.

[0122] The present disclosure also provides compositions comprising any of the bispecific antibodies or antigen-binding fragments thereof described herein (e.g., anti-CFHR4 / anti-C3 bispecific antibodies, anti-CFHR4 / anti-VEGF bispecific antibodies, and anti-C3 / anti-VEGF bispecific antibodies). The compositions are desirably pharma- ceutically acceptable (e.g., physiologically acceptable) compositions comprising a carrier, preferably a pharma- ceutically acceptable (e.g., physiologically acceptable) carrier, and the bispecific antibody or antigen-binding fragment thereof. Any suitable carrier may be used within the context of the present disclosure, and such carriers are well known in the art. For example, the compositions may include a preservative (e.g., methylparaben, propylparaben, sodium benzoate, benzalkonium chloride, and the like). Mixtures of two or more preservatives may optionally be used. In addition, a buffering agent may be included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. Mixtures of two or more buffering agents may optionally be used. Methods for preparing compositions for pharmaceutical use are known to those skilled in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0123] Once administered to a mammal (e.g., a human), the biological activity of a bispecific antibody, or antigen-binding fragment thereof, can be measured by any suitable method known in the art. For example, biological activity can be assessed by determining the stability of the bispecific antibody. The biological activity of a bispecific antibody can also be assessed by determining its binding affinity to an antigen-containing peptide and / or by assessing its binding affinity to a peptide with which the bispecific antibody can cross-react. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is defined as the dissociation constant (K DThe affinity of a binder for a ligand, e.g., the affinity of an antibody for an epitope, can be, for example, from about 1 femtomolar (fM) to about 1 millimolar (mM) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM), or from about 1 nM to about 1 micromolar (μM)). In some embodiments, the affinity of an anti-CFHR4 antibody can be from about 1 nm to about 20 nm, desirably from about 5 nm to about 10 nm. Antibody affinity for an antigen or epitope of interest can be measured using any art-recognized assay. Such methods include, for example, fluorescence-activated cell sorting (FACS), separable beads (e.g., magnetic beads), antigen panning, and / or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY, 2001).

[0124] In some embodiments, the bispecific antibody or the composition comprising the bispecific antibody may be administered alone or in combination with other drugs. For example, the bispecific antibody of the present disclosure (e.g., anti-CFHR4 / anti-C3 bispecific antibody, anti-CFHR4 / anti-VEGF bispecific antibody, and anti-C3 / anti-VEGF bispecific antibody) may be administered in combination with other agents for the treatment or prevention of AMD and / or GA as disclosed herein. For example, the bispecific antibody of the present disclosure, or an antibody conjugate, fusion protein, or polymeric formulation thereof, may be used in therapy, either alone or in combination with other agents. For example, the bispecific antibody may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is another antibody, a chemotherapeutic agent, a cytotoxic drug, an angiogenesis inhibitor, an immunosuppressant, a prodrug, a cytokine, a cytokine antagonist, a cytotoxic radiation therapy, a corticosteroid, an antiemetic drug, a cancer vaccine, an analgesic drug, a growth inhibitory agent, or a combination thereof.

[0125] For example, in certain embodiments, any of the aforementioned methods further comprise administering one or more additional compounds. In certain embodiments, the bispecific antibody, antibody conjugate, fusion protein, or polymer formulation is administered simultaneously with the additional compound(s). In certain embodiments, the bispecific antibody, antibody conjugate, fusion protein, or polymer formulation is administered before or after the additional compound(s). In certain embodiments, the additional compound binds to a second biomolecule selected from the group consisting of: VEGF, IL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin / APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically linked to AMD risk, such as complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In some embodiments, the additional compound is an antibody or an antigen-binding fragment thereof. In some embodiments according to (or as applied to) any of the preceding embodiments, the ocular disorder is an intraocular neovascular disease selected from the group consisting of proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), geographic atrophy (GA), diabetic and other ischemia-related retinopathies, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (including CRVO and BRVO), corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (ROP).

[0126] In some cases, the bispecific antibodies, or antibody conjugates, fusion proteins, and / or polymeric formulations thereof of the present disclosure may be administered in combination with at least one additional therapeutic agent for the treatment of an ocular disorder, e.g., an ocular disorder described herein (e.g., AMD (e.g., wet AMD or dry AMD), GA, DME, DR, or RVO). Exemplary additional therapeutic agents for combination therapy for the treatment of ocular disorders include, without limitation, the following:Angiogenesis inhibitors, for example, VEGF antagonists, such as anti-VEGF antibodies (e.g., anti-VEGF FabLUCENTIS® (ranibizumab)), soluble receptor fusion proteins (e.g., recombinant soluble receptor fusion protein EYLEA® (aflibercept, also known as VEGF trap eye; Regeneron / Aventis)), aptamers (e.g., anti-VEGF pegylated aptamer MACUGEN® (pegaptanib sodium; NeXstar Pharmaceuticals / OSI Pharmaceuticals), and VEGFR tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)); tryptophanyl-tRNA synthetase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)); azeta (TrpRS); squalamine; RETAANE® (anecortave acetate for depot suspension; Alcon, Inc.); combretastatin A4 prodrug (CA4P); MIFEPREX® (mifepristone-ru486); subtenon triamcinolone acetonide; intravitreal crystalline triamcinolone acetonide; matrix metalloproteinase inhibitors (e.g., prinomastat (AG3340; Pfizer)); fluocinolone acetonide (including fluocinolone intraocular implant; Bausch & Lomb / Control Delivery Systems); linomide; inhibitors of integrin β3 function; angiostatin, and combinations thereof.

[0127] Further examples of additional therapeutic agents that can be used in combination with the bispecific antibodies, or antibody conjugates, fusion proteins, and / or polymeric formulations thereof of the present disclosure for the treatment of ocular disorders (e.g., AMD, GA, DME, DR, or RVO) include, without limitation, the following: VISUDYNE® (verteporfin; a photoactivated drug typically used in conjunction with photodynamic therapy using non-thermal lasers), PKC412, Endovion (NS 3728; NeuroSearch A / S), neurotrophic factors (e.g., glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, PHOTOTROP®, 9-cis-retinal, ophthalmic drugs (e.g., phosphoinositide, echothiophate, or carbonic anhydrase inhibitors), beovastat (AE-941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; Sirna Therapeutics, Inc.), neurotrophins (including, by way of example only, NT-4 / 5, Genentech), Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (including those manufactured by Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (e.g., EntreMed, Inc.(used by Allergan / Oculex), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan / Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (LynkeusBiotech), microalgae compounds (Aquasearch / Albany, Mera Pharmaceuticals), D-9120 (Celitech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme / Celtrix), tyrosine kinase inhibitors (e.g., those from Allergan, SUGEN, or Pfizer), NX-278-L (NeXstar Pharmaceuticals / Gilead Sciences), Opt-24 (OPTIS France SA), retinal ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporine A, therapeutic agents used in photodynamic therapy (e.g., VISUDYNE®; receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfimer sodium for injection with PDT; verteporfin, QLT Inc.; rostaporfin with PDT, Miravent Medical Technologies; talaporfin sodium with PDT, Nippon Petroleum; and motexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (including, by way of example, products tested by Novagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), and combinations thereof.

[0128] The bispecific antibodies of the present disclosure, or antibody conjugates, fusion proteins, and / or polymeric formulations thereof, may be administered in combination with therapeutic or surgical procedures for the treatment of ocular disorders (e.g., AMD, GADME, DR, or RVO). For example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser processing, macular hole surgery, macular relocation surgery, implantable miniature telescopes, phi motion angiography (also known as microlaser therapy and vascular endothelial procedures), proton beam therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckling, submacular surgery, transpupillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal rheopheresis (also known as membrane fractional filtration and rheotherapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy for hypoxia response element, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell grafts, Cell Genesys, Inc.), acupuncture, and combinations thereof.

[0129] In some embodiments, the bispecific antibodies, or antibody conjugates, fusion proteins, and / or polymeric formulations thereof of the present disclosure can be administered in combination with an angiogenesis inhibitor for the treatment of ocular disorders (e.g., AMD, GA, DME, DR, or RVO). Any suitable angiogenesis inhibitor can be used in combination with the antibodies of the present disclosure, including, without limitation, those listed by Carmeliet et al. Nature 407:249-257, 2000. In some embodiments, the angiogenesis inhibitor is a VEGF antagonist, including, without limitation, the following: Anti-VEGF antibodies (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single chain antibody fragment; Novartis), or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiopoietin 2 bispecific antibodies, e.g., RG-7716; Roche)), soluble recombinant receptor fusion proteins (e.g., EYLEA® (aflibercept)), VEGF variants, soluble VEGFR fragments, aptamers capable of blocking VEGF (e.g., pegaptanib) or aptamers capable of blocking VEGFR, neutralizing anti-VEGFR antibodies, small molecule inhibitors of VEGFR tyrosine kinases, anti-VEGF DARPins® (e.g., abicipar pegol), small interfering RNAs that inhibit the expression of VEGF or VEGFR, VEGFR tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)), and combinations thereof.

[0130] Other suitable angiogenesis inhibitors that may be administered in combination with the bispecific antibodies, or antibody conjugates, fusion proteins, and / or polymeric formulations thereof of the present disclosure for the treatment of ocular disorders (e.g., AMD, GA, DME, DR, or RVO) include the following: Corticosteroids, angiostatic steroids, anecortave acetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors, insulin-like growth factor binding protein 3 (IGFBP3), stromal derived factor (SDF-1) antagonists (e.g., anti-SDF-1 antibodies), pigment epithelium derived factor (PEDF), gamma-secretase, delta-like ligand 4, integrin antagonists, hypoxia inducible factor (HIF)-1α antagonists, protein kinase CK2 antagonists, agents that inhibit stem cells (e.g., endothelial progenitor cells) homing to sites of angiogenesis (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and / or anti-SDF-1 antibodies), and combinations thereof.

[0131] In some embodiments, the bispecific antibodies, or antibody conjugates, fusion proteins, and / or polymeric formulations thereof of the present disclosure can be administered in combination with agents active against angiogenesis for the treatment of ocular disorders (e.g., AMD, GA, DME, DR, or RVO), such as anti-inflammatory drugs, mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin, AFINITOR® (everolimus), and TORISEL® (temsirolimus)), cyclosporine, tumor necrosis factor (TNF) antagonists (e.g., anti-TNFα antibodies or antigen-binding fragments thereof (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab) or soluble receptor fusion proteins (e.g., etanercept)), anti-complement agents, nonsteroidal anti-inflammatory drugs (NSAIDs), or combinations thereof.

[0132] Any suitable AMD therapeutic can be administered as an additional therapeutic agent in combination with the bispecific antibodies, or antibody conjugates, fusion proteins, and / or polymeric formulations thereof of the present disclosure for the treatment of ocular disorders (e.g., AMD, GA, DME, DR, or RVO). For example, without limitation, VEGF antagonists, such as anti-VEGF antibodies (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single chain antibody fragment; Novartis), or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiopoietin 2 bispecific antibodies, e.g., RG-7716; Roche)), soluble VEGF receptor fusion proteins (e.g., EYLEA® (aflibercept)), anti-VEGF DRPins (e.g., abicipar pegol; Molecular Partners), and the like. AG / Allergan), or anti-VEGF aptamers (e.g., MACUGEN® (pegaptanib sodium)); platelet derived growth factor (PDGF) antagonists, such as anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB pegylated aptamers (e.g., FOVISTA®; Ophthotech / Novartis), soluble PDGFR receptor fusion proteins, or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / , anti-VEGF antibodies anti-inflammatory drugs); VISUDYNE® (verteporfin) in combination with photodynamic therapy; antioxidants; complement system antagonists, e.g., complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Opthotech) or anti-C5 antibodies (e.g., LFG-316; Novartis), properdin antagonists (e.g., anti-properdin antibodies, e.g., CLG-561; Alcon), or complement factor D antagonists (e.g., anti-complement factor D antibodies, e.g., lampalizumab; Roche)); visual cycle regulators (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102;Ohr Pharmaceutical; vitamin and mineral supplements (e.g., those described in Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and those described in Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin, and / or omega-3 fatty acids)); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (Janssen), OpRegen (Cell Cure Neurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics); tissue factor antagonists (e.g., hl-con1; Iconic Therapeutics); alpha-adrenergic receptor agonists (e.g., brimonidine tartrate); peptide vaccines (e.g., S-646240; Shionogi); amyloid beta antagonists (e.g., anti-beta amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP™; Lpath Inc); ROBO4 antagonists (e.g., anti-ROBO4 antibodies, e.g., DS-7080a; Daiichi Sankyo); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof. In some cases, the AMD therapeutics (including any of the aforementioned AMD therapeutics) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept (EYLEAR®). In some cases, such co-formulations can be administered in combination with an antibody of the present disclosure. In some cases, the ocular disorder is AMD (e.g., wet AMD);

[0133] In addition to therapeutic applications, the bispecific antibodies or antigen-binding fragments described herein can be used for diagnostic or research applications. Research applications include, for example, methods utilizing the bispecific antibodies and labels to detect the corresponding antigen(s) in a sample, for example, in a human body fluid, or in a cell or tissue extract. The bispecific antibodies or antigen-binding fragments thereof may be used in any suitable assay for measuring the corresponding antigen(s) in a sample for diagnostic and / or research purposes. Such assays include, but are not limited to, sandwich immunoassays, enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), lateral flow assays, competitive inhibition immunoassays (e.g., forward and reverse), competitive binding assays, Forster resonance energy transfer (FRET), one-step antibody detection assays, single molecule detection assays, radioimmunoassays (RIA), and FACS. Such methods are disclosed, for example, in the following documents: U.S. Patent Nos. 6,143,576, 6,113,855, 6,019,944, 5,985,579, 5,947,124, 5,939,272, 5,922,615 No. 5,885,527, No. 5,851,776, No. 5,824,799, No. 5,679,526, No. 5,525,524, and No. 5,480,792, and Adamczyk. et al., Anal. Chim. Acta, 579(1):61~67 (2006).

[0134] The bispecific antibody or antigen-binding fragment thereof can be provided in a kit, e.g., a packaged combination of predetermined amounts of reagents and instructions for carrying out an assay using the antibody (e.g., an assay to detect the corresponding antigen(s)). Thus, the present disclosure provides a kit including an antibody or antigen-binding fragment described herein and instructions for its use. The instructions can be in paper format or in computer readable format (e.g., disk, CD, DVD, etc.). Alternatively or additionally, the kit can include calibrators or controls, and / or at least one container (e.g., tube, microtiter plate, or strip) for carrying out the assay, and / or buffer (e.g., assay buffer or wash buffer). Ideally, the kit includes all components (i.e., reagents, standards, buffers, diluents, etc.) necessary to carry out the assay. Other additives may be included in the kit, e.g., stabilizers, buffers (e.g., blocking buffer or lysis buffer), etc. The relative amounts of the various reagents can be varied to provide concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. Reagents may be provided as dry powders (usually lyophilized) that contain excipients that, upon dissolution, provide a reagent solution of the appropriate concentration.

[0135] The following examples further illustrate various embodiments of the present disclosure but should not be construed as in any way limiting its scope. EXAMPLES

[0136] As will be readily apparent to those skilled in the art, other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and recognizable, and may be made using suitable equivalents without departing from the scope of the disclosure or aspects and embodiments disclosed herein. Having described the disclosure in detail, the disclosure will be more clearly understood by reference to the following examples, which are intended merely to illustrate some aspects and embodiments of the disclosure, and should not be considered as limiting the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referenced herein are incorporated herein by reference in their entirety.

[0137] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0138] Example 1 Genetic analysis of CFHR4 in geographic atrophy. This example describes an analysis performed to determine the impact of CFHR1 / 4 deletion on age-related macular degeneration (AMD) at FinnGen. There are three general haplotypes consisting of a variant in CFH (rs1061170 Y402H) and a variant that is an eQTL and pQTL for CFH and CFHR4 (rs10922109), which are associated with three different levels of risk. The high AMD risk haplotype (H402+high CFHR4 expression, rs106117_C(ref) / rs10922109_C(ref)) was set as the "reference" haplotype to allow comparison with other haplotypes (frequency in cases: 0.56, frequency in controls: 0.36). Haplotypes carrying Y402 (alternative allele at rs106117) with high CFHR4 / low CFH expression (reference allele at rs10922109) are associated with a "medium" AMD risk (frequency in cases: 0.196, frequency in controls: 0.21). "Low" risk haplotypes carrying Y402 (alternative allele at rs106117) with low CFHR4 / high CFH expression (alternative allele at rs10922109) with frequencies in cases: 0.22, frequency in controls: 0.43). It was hypothesized that haplotypes carrying CFHR1 / 4 deletions have a different risk of AMD in a medium risk background.

[0139] Genetic variation at this locus provides a natural experiment to determine the impact of the CFHR1 / 4 deletion on AMD. The CFHR1 / 4 deletion has not been directly genotyped in FinnGen, but there are variants (rs528922402 and rs188297593) that incompletely tag (r2=0.36, D'=1 in Finland) a non-overlapping subset of the deletion in the Finnish population in the 1000 Genomes dataset (HapMap Phase 3). The variant rs188297593 has a D'<1.0 in HapMap Phase 3 samples of European ancestry (one of four carriers does not tag the CFHR1 / 4 deletion). These two variants capture a subset of the deletion, but not all carriers.

[0140] To determine the effect of CFH variants (rs1061170), CFHR4 / CFHeQTL / pQTL (rs10922109), and CFHR1 / 4 deletions (tagged by rs528922402 and rs188297593), AMD risk was examined for individuals from Finn GenR6. Haplotypes consisting of the individual variants were generated and logistic regression was performed in R to predict the effect of the haplotype on AMD risk, adjusting for sex, age, and PC'. The reference haplotype was used as a reference so that the effects of all other haplotypes were relative to it.

[0141] Consistent with the hypothesis that CFHR4 directly contributes to AMD risk, the “moderate” risk haplotype with a CFHR4 deletion conferred increased protection from AMD compared with the “moderate” risk haplotype with an intact CFHR4.

[0142] [Table 1]

[0143] Example 2 Generation of recombinant CFHR4 proteins. Recombinant protein preparations were undertaken to produce specific proteins that could be used as immunogens / antigens, screening reagents, and / or control reagents. All efforts were directed toward the goal of generating a panel of anti-CFHR4b antibodies with desired properties.

[0144] Proteins with sequences corresponding to human CFHR4b, human CFHL-1 wild type, human CFHL-1 with tyrosine 402 mutated to histidine, and cynomolgus CFHR4b were successfully produced using a variety of protein tags. Upon termination, all protein preparations were greater than 90% pure by analytical methods and had <1 endotoxin unit / milliliter. Examples of final specifications for individual preparations are shown below.

[0145] Starting with in silico analysis, appropriate wild-type amino acid sequences of human and cynomolgus CFHR4b and human CFHL-1 were identified and extracted from publicly available databases. The sequences were then further analyzed and manually modified using Geneious Prime software according to the project plan. The sequences were examined for trends and modified to add additional amino acid sequences that code for "tags" to facilitate purification, reduce immunogenicity, or simplify analytical screening. Examples of protein tags used in this study include the HIS tag, the proprietary mouse IgG Fc tag, and the Avi tag. The tags were separated from each other and from the CFHR4b or CFHL-1 sequences by short linker sequences. All tags were added to the C-terminus of the sequences. Additionally, the native signal peptide was removed and replaced with a signal peptide that facilitates recombinant expression.

[0146] After all modifications were completed, the amino acid sequences were reverse translated into DNA sequences and optimized for the codon bias found in the human genome. These optimized DNA sequences were sent to Integrated DNA Technologies and produced as DNA fragment(s) with DNA overhangs added to the 5' and 3' ends. Using DNA overhangs and Gibson cloning methods, these DNA fragments were assembled into the expected sequence and cloned into a mammalian expression plasmid driven by a CMV promoter. The plasmids were propagated in E. coli with appropriate antibiotic selection and prepared at a scale useful for recombinant expression using a commercial preparation kit purchased from Qiagen. The sequences of the plasmids and expressed genes were then confirmed using Sanger sequencing.

[0147] The sequence verified plasmid was transfected using polyethyleneimine into human embryonic kidney cells adapted for recombinant expression. One day after transfection, the cells were supplemented with chemicals and nutrients designed to increase recombinant protein expression. These supplements included sodium propionate, valproic acid, glucose, glutamine, and various yeast lysates. Five days after transfection, the expressing cell cultures were harvested. As the recombinant protein was secreted into the growth medium, cells, cell debris, and cellular debris were removed by centrifugation and filtration through membranes with pores of 0.22 microns or less. The clarified culture medium conditioned by the recombinant protein was then ready for purification.

[0148] Proteins of interest were purified from cell culture media using FPLC (fast pressure liquid chromatography) and appropriate commercially available pre-packed affinity chromatography columns for the C-terminal tag(s) and immobilized on the chromatography column. For HIS-tagged proteins, a Ni-NTA agarose column was used. For Fc-tagged proteins, a proA agarose column was used. Multiple column washes followed (each specific for the column / tag / chromatography type). Proteins of interest were eluted from the column with 300 mM imidazole for HIS-tagged proteins and 100 mM citrate pH 3.5 for Fc-tagged proteins. After elution, Fc-tagged proteins were adjusted to neutral pH using 1 M Hepes pH 9. For HIS-tagged proteins, neutralization was not necessary as the elution buffer was at neutral pH.

[0149] Protein quality and quantity were assessed using a combination of SDS-PAGE gel electrophoresis, spectrophotometry, and analytical SEC (size exclusion chromatography). Most proteins for this project required further purification. FPLC and a second chromatography column were used to further purify the proteins. Size separation achieved with the size exclusion column allowed protein purity to be increased up to >90%. SEC also allowed buffer exchange from the affinity chromatography elution buffer into the final buffer of choice (PBS). Protein samples were then transferred to quality control. At any point during purification, if the protein concentration needed to be increased, it was concentrated by separating it from the buffer using an Amicon Ultra molecular weight cutoff (MWCO) filtration unit. The MWCO was selected to ensure compatibility with the size of the protein of interest. All final samples were concentrated to >1 milligram / milliliter before moving to final quality control.

[0150] Endotoxin contamination in the final protein samples was assayed using Charles River's Endosafe PTS system. Final protein concentration was determined by spectrophotometry. Three micrograms of the final sample was injected onto an analytical SEC column (YMC Diol300) to determine its final purity. Additionally, in some cases, SDS-PAGE electrophoresis was performed to determine final quality. Once all final metrics were passed, the protein was sterilized using a sterile 0.22 micron filter in a biosafety cabinet. This was followed by sterile aliquots and flash freezing in liquid nitrogen before storage at -80°C.

[0151] Example 3 Recovery of CFHR4 antibody sequences from immunized mice. CFHR4 Immunization: Three cohorts of Alloy Therapeutic transgenic humanized mice (ATX-GK) were immunized with human CFHR4b using the following 5-week protocol (Figures 3A-3C).

[0152] Cohort 1: Five ATX-GK mice immunized with human CFHR4B (ATX-P-57) using a standard 5-week RIMMS protocol (10ug subcutaneous administration of antigen emulsified in complete Freund's adjuvant followed by 5 weeks of subcutaneous administration of antigen emulsified in incomplete Freund's adjuvant). Cohort 2: Five ATX-GK mice immunized with human CFHR4B (ATX-P-57) using a 5-week RIMMS protocol (10ug subcutaneous administration of antigen emulsified in incomplete Freund's adjuvant every week). Cohort 3: Five ATX-GK mice immunized with human CFHR4B (ATX-P-57) using a 5-week RIBI IP protocol (10ug intraperitoneal administration of antigen emulsified in RIBI adjuvant every week).

[0153] Samples were bled on week 4 and tested by ELISA for antigen positive and purification tag negative serum titers. ELISA plates were coated with either 1ug / ml of CFHR4 immunogen or an irrelevant protein (P114) with the same purification tag as the immunogen. Antigen-coated plates were incubated with seven 10-fold serial dilutions of serum starting at 1:300. Antigen-bound antibodies were detected by anti-mouse IgG HRP secondary antibody and one-step TMB solution. Absorbance signal at 450nm was measured by an ELISA microplate reader.

[0154] Hybridomas: Immune tissues from high titer mice were harvested and stored for antibody discovery. Hybridoma cell lines producing CFHR4 antibodies were generated by fusion of single B cells from the spleen and lymph nodes of titer positive mice with myeloma cells. Twenty 96-well plates of hybridoma fusions were generated and expanded. Hybridomas expressing CFHR4 specific antibodies were detected by antigen binding by ELISA. The affinity of the antibodies in the hybridoma supernatants was measured by SPR using an Octet instrument. CFHR4 antibodies in the hybridoma supernatants were loaded onto a biosensor. The response was measured as a nm shift in the interference pattern and was proportional to the number of antibodies bound to the biosensor surface. The binding interaction of CFHR4 to the immobilized antibodies was measured as association (kon). After analyte association, the biosensor was immersed in PBS without CFHR4 to dissociate the bound antigen from the antibodies (kdis). KD(M) (or affinity of the antibody for CFHR4) was measured as kdis / kon. Heavy and light chains from confirmed hybridomas were sequenced. RNA was isolated from CFHR4 antibody secreting hybridomas and heavy and light chain variable regions were cloned by reverse transcription using gene specific primers followed by PCR amplification with variable chain gene specific primers. PCR products were sequenced by standard Sanger sequencing methods.

[0155] Phage display: Variable heavy and light chains were amplified from the spleens of high-titer immunized mice by reverse transcription using gene-specific primers followed by PCR amplification with variable chain gene-specific primers. The variable regions were cloned into a phage display vector designed to express Fabs on the phage g3p protein. A library of phages expressing unique Fabs was amplified and purified. Phages were bound to biotinylated CFHR4 antigen captured on streptavidin magnetic beads. Phages that remained bound to the antigen beads after several stringent washes were eluted using a basic triethylamine solution and neutralized by Tris buffer pH 8.0. The eluted phages were reinfected into TG1 bacterial cells, amplified by co-infection with M13 helper phage, and purified by PEG precipitation. Purified phages expressing Fabs were selected for antigen binding as described. Phages from the second round were diluted and infected into TG1 cells. Polyclonal pools of phage output from two rounds of panning were tested by ELISA to confirm that the pools contained CFHR4-specific phage. Variable heavy and light chain regions were sequenced from single infected bacterial colonies using rolling circle amplification and standard Sanger sequencing.

[0156] Antibody Sequencing: Unique variable heavy and light chain pairs from the hybridoma and phage display campaigns were cloned into vectors designed to express full-length antibodies as IgG in HEK293 cells under the control of the CMV promoter. The antibody expression vectors were complexed with polyethylenimine and transfected into HEK293 cultures. After 5 days of shaking at 37°C in 293 cell culture medium, the antibodies were captured on an agarose-based protein A resin. After several stringent washes, the antibodies were eluted in a glycine solution (pH 3), neutralized with Hepes (pH 9), and buffer exchanged into PBS.

[0157] Example 4 Human CFHR4 monoclonal antibody differential scanning fluorimetry (DSF). The development of an effective monoclonal antibody depends not only on its biological activity but also on its physicochemical properties such as homogeneity and stability. mAb stability can be affected by its formulation. Among the many techniques used to examine mAb stability, differential scanning fluorimetry (DSF) offers both excellent throughput and minimal material consumption. DSF measures the temperature of protein unfolding transition (Tm) based on the change in fluorescence intensity of an environmentally sensitive dye.

[0158] Experiments were performed to evaluate the thermal stability of the human CFHR4 monoclonal antibody of the present disclosure (the "ATX" antibody) by determining the melting temperature. Thermal stability was evaluated by (DSF) differential scanning fluorimetry using the Protein Thermal Shift (PTS) assay from Applied Biosystems. The assay was performed according to the manufacturer's instructions. Briefly, the antibody to be evaluated was prepared in triplicate by mixing with Protein Thermal Shift dye and buffer. Real-time melting experiments 25°C-95°C were performed on a QuantStudio 3. Data was analyzed by using Protein Thermal Shift Software, and melting temperatures (Tm) were calculated from the melting curves (Figure 4).

[0159] [Table 2]

[0160] Example 5 CFHR4 antibody cross-blocking. High-throughput epitope binning experiments were performed on a real-time label-free biosensor (Carterra LSA) to sort a large panel of mAbs into bins based on their ability to block each other from binding to the antigen. In pairwise epitope binning analysis, antigen and antibody 2 (analyte antibody) are sequentially applied to a sensor chip (HC200M) that is covalently preloaded with antibody 1 (ligand antibody). An increase in response upon exposure to the analyte antibody indicates noncompetition between the two antibodies, while no change in signal indicates competition. Antibodies with the same blocking profile relative to others in the test set are grouped into one bin. A community network plot is used to examine the clustering of mAbs that share similar, but not necessarily identical, competition profiles. Rather than strictly relying on sandwich / blocking assignments in the heatmap (Figure 5A), as the bin network plot does, hierarchical clustering is applied to the sorted heatmap to create a network plot (Figure 5B) that gradually groups the mAbs together.

[0161] Example 6 CFHR4 antibody binding kinetics. Kinetics experiments were performed on a Carterra LSA with a running buffer of PBS pH 7.40, 1% BSA, 0.05% Tween 20. Antibodies were covalently printed onto HC30M chips. The chips were activated with 33 mM s-NHS and 133 mM EDC in 100 mM MES pH 5.5 for 7 min. Antibodies at 10 mg / ml in acetate buffer pH 4.5 were used for printing for 10 min. The printed chips were then quenched with 1 M ethanolamine pH 8.5 for 7 min. For kinetic analysis, purified recombinant his-tagged proteins ATX-P-57, human CDFR4B-His, at concentrations from 0.076 nM to 1500 nM (serial 3-fold dilutions) were injected sequentially. For each concentration, 5 min of association was performed followed by 15 min of dissociation. Results were processed and analyzed in Carterra LSA Kinetics Software. Kinetic data were referenced by interstitial reference spots, double-referenced to buffer cycles, and then globally fitted to a 1:1 binding model to determine their apparent association and dissociation kinetic rate constants (ka and kd values). The ratio kd / ka was used to derive the KD value for each antigen / mAb interaction, i.e., KD=kd / ka (Figure 6).

[0162] Example 7 CFHR4 antibody cross-reactivity. Experiments were performed to determine the reactivity of the disclosed CFHR4 antibody (developed using the CFHR4b antigen, see Example 3) with various other CFHR4 proteins. Experiments were performed to determine the reactivity of the CFHR4 antibody with the cynomolgus monkey CFHR4b protein (cCFHR4b), as shown in FIG. 7. Binding experiments were performed on a Carterra LSA with a running buffer of PBS pH 7.40, 1% BSA, 0.05% Tween 20. Antibodies were covalently printed onto HC30M chips. The chips were activated with 33 mM s-NHS and 133 mM EDC in 100 mM MES pH 5.5 for 7 minutes. 10 mg / ml of antibody in acetate buffer pH 4.5 was used for printing for 10 minutes. The printed chips were quenched with 1 M ethanolamine pH 8.5 for 7 minutes. In the reactivity assay, ATX-P-141, cynomolgus CFHR4B-mFc at 160 nM was tested by 5 minutes of association followed by 15 minutes of dissociation. Results were processed and analyzed in Carterra LSA Kinetics Software. Data was referenced by a stromal reference spot, double referenced to a buffer cycle, and then the response (nm) after association was reported. An isotype control was used to determine the cutoff response for positive binding.

[0163] Experiments were also performed to determine the reactivity of CFHR4 antibodies with human CFHR4a protein (Figure 8). Binding experiments were performed on a Carterra LSA with a running buffer of PBS pH 7.40, 1% BSA, 0.05% Tween 20. Antibodies were covalently printed onto HC30M chips. The chips were activated with 33 mM s-NHS and 133 mM EDC in 100 mM MES pH 5.5 for 7 minutes. 10 mg / ml of antibody in acetate buffer pH 4.5 was used for printing for 10 minutes. The printed chips were quenched with 1 M ethanolamine pH 8.5 for 7 minutes. In the reactivity assay, ATX-P-56, human CFHL4A-His at 540 nM was tested by association for 5 minutes followed by dissociation for 15 minutes. Results were processed and analyzed in Carterra LSA Kinetics Software. Data was referenced to a stromal reference spot, double referenced to buffer cycles, and then reported as post-association responses (nm). Isotype controls were used to determine the cutoff response for positive binding.

[0164] Experiments were also performed to determine the reactivity of CFHR4 antibodies with human CFHR3 protein (Figure 9). Binding experiments were performed on a Carterra LSA with a running buffer of PBS pH 7.40, 1% BSA, 0.05% Tween 20. Antibodies were covalently printed onto HC30M chips. The chips were activated with 33 mM s-NHS and 133 mM EDC in 100 mM MES pH 5.5 for 7 minutes. 10 mg / ml of antibody in acetate buffer pH 4.5 was used for printing for 10 minutes. The printed chips were quenched with 1 M ethanolamine pH 8.5 for 7 minutes. In the reactivity assay, ATX-P-58 at 550 nM, human CFHL3-His was tested by association for 5 minutes followed by dissociation for 15 minutes. Results were processed and analyzed in Carterra LSA Kinetics Software. Data was referenced to a stromal reference spot, double referenced to buffer cycles, and then reported as post-association responses (nm). Isotype controls were used to determine the cutoff response for positive binding.

[0165] Experiments were also performed to determine the reactivity of CFHR4 antibodies with human CFHL-1 (Y402H) protein (Figure 10). Binding experiments were performed on a Carterra LSA with a running buffer of PBS pH 7.40, 1% BSA, 0.05% Tween 20. Antibodies were covalently printed onto HC30M chips. The chips were activated with 33 mM s-NHS and 133 mM EDC in 100 mM MES pH 5.5 for 7 minutes. 10 mg / ml of antibody in acetate buffer pH 4.5 was used for printing for 10 minutes. The printed chips were quenched with 1 M ethanolamine pH 8.5 for 7 minutes. In the reactivity assay, ATX-P-421, human CFHL1-Y402H-His at 380 nM was tested by association for 5 minutes followed by dissociation for 15 minutes. Results were processed and analyzed in Carterra LSA Kinetics Software. Data was referenced to a stromal reference spot, double referenced to a buffer cycle, and then reported as post-association responses (nm). Isotype controls were used to determine the cutoff response for positive binding.

[0166] Example 8 C3 Convertase Assembly Assay. A screening assay was developed to assess the ability of the disclosed CFHR4 antibodies to affect C3 convertase formation (FIG. 11A). C3bBb is the C3 convertase (EC 3.4.21.43) of the alternative pathway of complement activation. The C3 enzyme is involved in the amplification of pathway activation and deposition of C3b and the membrane attack complex on target cells. It is regulated by serum proteins factor H, factor I (EC 3.4.21.45), and properdin. Formation of C3 convertase requires C3b, factor B, factor D (EC 3.4.21.46), and Mg2+. After formation of the C3bB(Mg) reversible bimolecular complex, factor D cleaves factor B to release the activation fragment Ba and generate the active enzyme C3bBb. The C3 enzyme is a serine protease whose catalytic site resides within the Bb subunit. Furthermore, CFHR4 has been reported to directly promote C3 convertase formation by binding to C3b, has a greater ability to assemble C3bBb on CFHR4, and the C3bBb-CFHR4 complex is more resistant to CFH cleavage compared with C3bBb.

[0167] Thus, an assay is designed to test the ability of CHFR4 antibodies to disrupt the formation of the C3 convertase complex by measuring the amount of factor B present using an anti-FB monoclonal antibody conjugated to a reporter moiety. As shown in Figure 11A, in the presence of CFHR4 (in the absence of an effective CFHR4 antibody), the C3 convertase complex is formed, the anti-FB antibody binds to the FB, and a signal is detected (i.e., complement activation). However, in the presence of an effective anti-CFHR4 antibody, the C3 convertase complex is disrupted and the antibody-bound FB is washed away, resulting in a reduced signal (i.e., no complement activation).

[0168] An example of an assay protocol is provided below.

[0169] Preparation of FHR-4 coated plates - (1) Coat plates with 5 μg / ml FHR-4 in PBS and incubate overnight (O / N) at room temperature (RT) in a plastic bag with moist paper. (2) Wash 3x300 μl with Wieslab wash buffer. (3) Block wells with 150 μl 3% BSA in PBS. (4) Incubate at least 1 h at RT. (5) Aspirate wells and wash with 1x300 μl superblock. (6) Dry plates at 37°C, 16% RH for at least 1 h. (7) Store in plate bag in refrigerator.

[0170] Assay protocol - (1) Bring diluent, plate, substrate, and stop solution to RT. (2) Dilute anti-OVA antibody to desired concentration in AP diluent. (3) Add 50 μl of anti-OVA, AP diluent (blank), and diluent control to plate. (4) Cover and incubate for 1 h at room temperature. (5) Wash with 3x300 μl Wieslab wash buffer. (6) Add 50 μl of 5 μg / ml C3b or AP diluent to plate. (7) Incubate plate for 30 min at 37°C. (8) Wash with 3x300 μl Wieslab wash buffer. (9) Add 50 μl of protein mix (FB, FD, properdin) to plate. (10) Incubate plate for 30 min at 37°C. (11) Wash with 3x300 μl Wieslab wash buffer. (12) 50 μl of anti-factor B antibody diluted 1:2000 in AP diluent was added to the entire plate. (13) Plate was incubated at room temperature for 1 h. (14) Washed with 3x300 μl of Wieslab wash buffer. (15) 50 μl of rabbit anti-goat IgG-HRP antibody diluted 1:5000 in Stabilzyme was added to the entire plate. (16) Plate was incubated at room temperature for 1 h. (17) Washed with 3x300 μl of Wieslab wash buffer. (18) 50 μl of substrate was added to the entire plate. (19) Plate was incubated at room temperature for 20 min. (20) 50 μl of stop solution was added to the entire plate. (21) Plate was read at 450-620 nm.

[0171] As shown in Figure 11B, dose-response curves were generated for the top eight inhibitory antibodies screened in the C3 convertase assembly assay. Percent C3 convertase activity is plotted relative to the negative control (diluent only). Figure 11C includes IC50 values ​​calculated for each antibody tested in Figure 11B. Figure 11D also provides a representative summary of the data showing at least three functional classes of anti-CFHR4 antibodies.

[0172] Example 9 Recovery of C3 antibody sequences from immunized mice. C3 antibody sequences were recovered from immunized mice and subsequently subjected to phage display with either fixed or diverse light chains. Selected leads were produced as fully aglyco-IgG1 and extensively characterized.

[0173] C3 recombinant protein. C3 protein was sourced from Complement Technologies (Tyler, TX). The protein was purified directly from relevant species (human or cynomolgus monkey) serum. The recombinant protein was qualified by analytical SEC and binding tests to established antibodies.

[0174] C3 Immunization. Two cohorts of transgenic humanized mice (ATX-GK Mix) from Alloy Therapeutics were immunized with either human C3 or cynomolgus C3 using the following 5 week protocol: Cohort 1: Five ATX-GX Mix mice were immunized with human C3 (REA23) using the standard 5 week RIMMS protocol with 10ug of antigen administered subcutaneously. The first dose was emulsified in complete Freund's adjuvant, followed by four weekly doses of 10ug emulsified in incomplete Freund's adjuvant. Cohort 2: As cohort 1 but with cynomolgus C3 (REA20).

[0175] Samples were bled at week 4 and tested for antigen positive titers and cross-reactivity titers (human for cynomolgus cohorts or cynomolgus for human cohorts). ELISA plates were coated overnight with 1ug / ml of the relevant recombinant protein. The coated plates were incubated with a 5x10-fold descending range of sera starting at 1:100 and ending at a 1:10E7 dilution. Antibodies bound to the coated antigen were detected by anti-mouse IgG HRP secondary antibody and quantified using a one-step TMB solution and OD determined at 450nM using a microplate reader. Note that the positive control antibody used in the ELISA experiments is not cross-reactive to cynomolgus C3 protein (Figures 12A-12D).

[0176] Antibody sequence recovery - phage display. Spleen tissue from high titer mice was stored at the time of collection for sequence recovery via phage display. RNA was extracted from the tissue. Two sequence recovery strategies were used in this study: fixed light chain strategy and diverse light chain strategy. In both strategies, variable heavy chains were amplified by reverse transcription from RNA prepared from spleen tissue. After reverse transcription, DNA fragments were generated using viable gene-specific primers and PCR. This could be cloned into a phage display vector designed to express Fab on the phage g3p protein. In the diverse light chain strategy, the same approach was used to amplify the natural diversity of light chains from spleen RNA and clone them into a Fab vector. In the fixed light chain strategy, the relevant part of the Fab vector was fixed to two specific sequences using commercially synthesized constructs that match the LC of ATX-P-569 and ATX-P-592.

[0177] Upon completion, libraries of phage expressing unique Fabs or Fabs with immobilized light chains were amplified, purified, and depleted on non-specific binders. Phages were bound to biotinylated C3 antigen (human and / or cynomolgus monkey) captured on streptavidin magnetic beads. Phages that remained bound to the antigen beads after several stringent washes were eluted using a basic triethylamine solution and neutralized with Tris buffer pH 8.0. Eluted phage were reinfected into TG1 bacterial cells, amplified by co-infection with M12 helper phage, and purified by PEG precipitation. A small sample was saved for polyclonal analysis. Purified phages expressing Fabs were subsequently selected for antigen binding as described above in a second round. Phages from the output of the second round were diluted and infected into TG1 cells. Polyclonal pools of output phages from both rounds were tested by ELISA to confirm enrichment and to check for cross-reactivity (Figure 13).

[0178] Subsequent monoclonal ELISAs on individual colonies were performed and from this second ELISA clones were identified for rolling circle amplification and Sanger sequencing of the variable heavy and light chain regions of the Fab, if necessary (only for the diverse library, only HC sequencing was required for the fixed LC library). From sequence analysis, a group of clones was advanced for reformatting into full-length antibodies. The selected group broadly represented the diversity of clonotypes found in both the fixed / cLC and diverse libraries. In total, 113 pairs of variable heavy and light chain sequences were generated across the fixed and diverse libraries.

[0179] Antibody production. Unique variable heavy and light chain pairs from the phage display campaign were cloned into vectors designed to express full-length antibodies as aglyco-IgG1 in HEK293 cells under the control of the CMV promoter. The antibody expression vectors were complexed with polyethylenimine and transfected into HEK293 cultures. After 5 days of shaking at 37° C. in 293 cell culture medium, the antibodies were captured on an agarose-based protein A resin. After several stringent washes, the antibodies were eluted in a citrate solution (pH 3), neutralized with Hepes (pH 9), and buffer-exchanged into PBS.

[0180] Example 10 C3 Antibody Binding Kinetics. Experiments were performed to determine the binding kinetics, species cross-reactivity, and epitope cross-blocking of anti-C3 antibodies.

[0181] Kinetic experiments were performed on a Carterra LSA with a running buffer of PBS pH 7.40, 1% BSA, 0.05% Tween 20. Antibodies were captured on an anti-human Fc capture chip prepared with an HC30M chip. For kinetic analysis, C3a, C3b from Complement Technology, or C3-His from Acro Biosystems were injected sequentially at concentrations from 0.076 nM to 1500 nM (serial 3-fold dilutions). For each concentration, 5 minutes of association was performed followed by 15 minutes of dissociation. Results were processed and analyzed in Carterra LSA Kinetics Software. Kinetic data were referenced by an interstitial reference spot, double referenced to buffer cycles, and then globally fit to a 1:1 binding model to derive their apparent association and dissociation kinetic rate constants (K a and K D The ratio K D / K a Using the K D Value (K D =K D / K a ) was derived (Figure 14).

[0182] Cross-reactivity assay by SPRi. Binding experiments were performed on a Carterra LSA with a running buffer of PBS pH 7.40, 1% BSA, 0.05% Tween 20. Antibodies were covalently printed onto HC30M chips. The chips were activated with 33 mM s-NHS and 133 mM EDC in 100 mM MES pH 5.5 for 7 minutes. 10 mg / ml of antibody in acetate buffer pH 4.5 was used for printing for 10 minutes. The printed chips were quenched with 1 M ethanolamine pH 8.5 for 7 minutes. In the reactivity assay, human C3, C3a, C3b, and cynomolgus C3 from Complement Technology were tested by association for 5 minutes followed by dissociation for 15 minutes. Results were processed and analyzed in Carterra LSA Kinetics Software. Data was referenced to a stromal reference spot, double referenced to a buffer cycle, and then reported as response (nm) after association. Isotype controls were used to determine the cutoff response for positive binding (Figures 15 and 16).

[0183] Epitope binning. High-throughput epitope binning experiments were performed on a real-time label-free biosensor (Carterra LSA) to sort a large panel of mAbs into bins based on their ability to block each other from binding to the antigen. In pairwise epitope binning analysis, antigen and antibody 2 (analyte antibody) are sequentially applied to a sensor chip (HC200M) that is covalently preloaded with antibody 1 (ligand antibody). An increase in response upon exposure to the analyte antibody indicates noncompetition between the two antibodies, while no change in signal indicates competition. Antibodies with the same blocking profile relative to others in the test set are grouped into one bin. Community network plots are used to examine the clustering of mAbs that share similar, but not necessarily identical, competition profiles. Rather than strictly relying on sandwich / blocking assignments in the heatmap, as bin network plots do, hierarchical clustering is applied to the sorted heatmap to create a dendrogram and gradually group mAbs together (Figures 17-18).

[0184] Example 11 Experiments were performed to determine the reactivity of the disclosed C3 antibody with various other proteins using the C3 antibody polyreactivity ELISA and the Carterra LSA platform (Figures 19 and 20). The method used was similar to that reported by Hotzel et al. (2012). Briefly, baculovirus particles (BVP, Lake Pharma) were diluted 1:100 in 50 mM sodium bicarbonate (pH 9.3). After incubating 50 μL of BVP on an ELISA plate (3369, Corning) overnight at 4°C, unbound BVP was aspirated from the wells. All remaining steps were performed at room temperature. The plate was blocked with 100 μL of blocking buffer (PBS with 1% BSA) for 1 h before being washed three times with 100 μL of PBS. Next, 50 μL of 16 nM test antibody was added to the wells and incubated for 1 h, followed by washing with 100 μL of PBS. HRP-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch) at 1:1000 was used as the secondary antibody and incubated for 1 h, followed by washing as described above. Finally, 50 μL of TMB substrate (34021; Fisher Scientific) was added to each well and incubated for 10–15 min. The reaction was stopped by adding 50 μL of 2 M sulfuric acid to each well. BVP scores were determined by reading the absorbance at 450 nm and normalizing the absorbance by control wells that did not contain the test antibody.

[0185] Further experiments were performed to determine the blocking potential of the C3 antibody of the present disclosure against CFHR4 (Figure 21B) and CFH (Figure 21B).

[0186] array

[0187] The various amino acid and nucleic acid sequences referred to herein are provided below.

[0188] [Table 3-1]

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

[0189]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

Table 4-21

Table 4-22

Table 4-23

Table 4-24

Table 4-25

Table 4-26

Table 4-27

Table 4-28

[0190]

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Table 5-36

Table 5-37

Table 5-38

Table 5-39

Table 5-40

Table 5-41

Table 5-42

Table 5-43

Table 5-44

Table 5-45

Table 5-46

Table 5-47

Table 5-48

Table 5-49

Table 5-50

Table 5-51

Table 5-52

Table 5-53

Table 5-54

Table 5-55

Table 5-56

Table 5-57

Table 5-58

Table 5-59

Table 5-60

Table 5-61

Table 5-62

Table 5-63

Table 5-64

Table 5-65

Table 5-66

Table 5-67

Table 5-68

Table 5-69

Table 5-70

Table 5-71

Table 5-72

Table 5-73

Table 5-74

Table 5-75

Table 5-76

Table 5-77

Table 5-78

Table 5-79

Table 5-80

[0191] Human complement factor H related protein 4b (CFHR4b) (see, e.g., UniProt Accession No. Q92496): MLLLINVILTLWVSCANGQEVKPCDFPEIQHGGLYYKSLRRLYFPAAAGQSYSYYCDQNFVTPSGSYWDYIHCTQDGWSPTVPCLRTCSKSDIEIENGFISESSSIYILNKEIQYKCKPGYATADGNSSGSITCLQNGWSAQPICIKFCDMPVFENSRAKSNGMRFKLHDTLDYECYDGYEISYGNTTGSIVCGEDGWSHFPTCYNSSEKCGPPPPISNGDTTSFLLKVYVPQSRVEYQCQSYYELQGSNYVTCSNGEWSEPPRCIHPCIITEENMNKNNIQLKGKSDIKYYAKTGDTIEFMCKLGYNANTSVLSFQAVCREGIVEYPRCE (SEQ ID NO: 580).

[0192] Human CFHL1-Y402H-His(ATX-P-421): MYRMQLLSCIALSLALVTNSEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLT GGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKE NERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNHGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRVSFTLGGGGSGLNDIFEAQKIEWHEGGGGSHHHHHH (sequence number 581).

[0193]

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

[0194]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

Table 7-21

Table 7-22

Table 7-23

Table 7-24

Table 7-25

[0195]

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

Table 8-20

Table 8-21

Table 8-22

Table 8-23

[0196]

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

Table 9-19

Table 9-20

Table 9-21

Table 9-22

Table 9-23

Table 9-24

Table 9-25

Table 9-26

Table 9-27

Table 9-28

Table 9-29

Table 9-30

Table 9-31

Table 9-32

Table 9-33

Table 9-34

Table 9-35

Table 9-36

Table 9-37

Table 9-38

Table 9-39

Table 9-40

Table 9-41

Table 9-42

Table 9-43

Table 9-44

Table 9-45

Table 9-46

Table 9-47

Table 9-48

Table 9-49

Table 9-50

Table 9-51

Table 9-52

Table 9-53

Table 9-54

Table 9-55

Table 9-56

Table 9-57

Table 9-58

Table 9-59

Table 9-60

Table 9-61

Table 9-62

Table 9-63

Table 9-64

Table 9-65

Table 9-66

Table 9-67

Table 9-68

Table 9-69

Table 9-70

Table 9-71

Table 9-72

Table 9-73

Table 9-74

Table 9-75

Table 9-76

Table 9-77

Table 9-78

Table 9-79

Table 9-80

Table 9-81

Table 9-82

Table 9-83

Table 9-84

Table 9-85

Table 9-86

Table 9-87

Table 9-88

Table 9-89

Table 9-90

Table 9-91

Table 9-92

Table 9-93

Table 9-94

Table 9-95

Table 9-96

Table 9-97

Table 9-98

Table 9-99

[0197]

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-6

Table 10-7

Table 10-8

Table 10-9

Table 10-10

Table 10-11

Table 10-12

Table 10-13

Table 10-14

Table 10-15

Table 10-16

Table 10-17

Table 10-18

Table 10-19

Table 10-20

Table 10-21

Table 10-22

Table 10-23

Table 10-24

Table 10-25

Table 10-26

Table 10-27

Table 10-28

Table 10-29

Table 10-30

Table 10-31

Table 10-32

Table 10-33

Table 10-34

Table 10-35

Table 10-36

Table 10-37

Table 10-38

Table 10-39

Table 10-40

Table 10-41

Table 10-42

[0198] Human complement component 3 (C3), which includes complement component 3a (C3a) and complement component 3b (C3b), was used to generate the anti-C3 antibody of the disclosure (see, e.g., UniProt Accession No. M0QYC8): ITHRIHWESASLLRSEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETGIPSPIFLSSVFLEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLAFKVHQYFNVELIQPGAVKVYAYYNL (SEQ ID NO: 1645).

[0199] Various embodiments of the present disclosure are described herein. Variations of those embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will utilize such variations as necessary, and the inventors intend that the various embodiments of the present disclosure may be practiced other than as specifically described herein. Accordingly, the embodiments of the present disclosure include all modifications and equivalents of the subject matter recited in the appended claims to the extent permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations is encompassed in the various embodiments of the present disclosure unless otherwise indicated herein or clearly contradicted by context.

Claims

1. A bispecific antibody that specifically binds to human complement factor H-related 4 (CFHR4) and human complement component 3 (C3), Optionally, the human CFHR4 may be a CFHR4b variant (CFHR4b), which may contain the amino acid sequence of SEQ ID NO: 580 or be a polypeptide consisting of the amino acid sequence of SEQ ID NO:

580. Optionally, the human C3 is a b variant (C3b) of C3, which may be a polypeptide containing the amino acid sequence of SEQ ID NO: 1645 or consisting of the amino acid sequence of SEQ ID NO: 1645. The aforementioned bispecific antibody is One CFHR4 antibody selected from the group consisting of ATX-P-560, ATX-P-570, ATX-P-571, ATX-P-575, ATX-P-582, ATX-P-591, ATX-P-597, ATX-P-598, ATX-P-599, ATX-P-601, ATX-P-602, ATX-P-605, and ATX-P-607, with all six complementation-determining regions (CDRs) HCDR1, HCDR2, HCDR3 and their corresponding LCDR1, LCDR2, LCDR3 whose sequences are shown in Table 3, The sequences of any one of the C3 antibodies shown in Table 6, including all six complementation-determining regions (CDRs) HCDR1, HCDR2, HCDR3 and their corresponding LCDR1, LCDR2, LCDR3, and A bispecific antibody containing [specific antibody].

2. Any one of the CFHR4 antibodies defined in Claim 1, wherein the VH and VL amino acid sequences are shown in Table 4, and The VH and VL amino acid sequences of any one of the C3 antibodies whose sequences are shown in Tables 7 and 8. A bispecific antibody according to claim 1, which is a polypeptide comprising the above.

3. The bispecific antibody according to claim 1, which is monoclonal and optionally recombinant.

4. The bispecific antibody according to claim 1, which is human, humanized, or chimeric.

5. The bispecific antibody according to claim 1, which is a full-length antibody containing an Fc region such as the human IgG1, IgG2, IgG3, or IgG4 region.

6. At least one additional part, optionally, a. An antigen-binding portion, such as an antibody or its antigen-binding fragment, that can specifically bind to a target other than human CFHR4 or human C3, wherein the target is expressed in the human eye, and the antigen-binding portion, b. The treatment area or cytotoxic area, c. Detection part and, d. Purified portion and, e. The half-life extension portion is optionally a polypeptide having a length of at least 20 amino acids and comprising any combination of G, A, S, T, E, and P residues, which is conjugated to the C-terminus or N-terminus of the antibody. A bispecific antibody according to claim 1, conjugated to at least one additional portion selected from.

7. The bispecific antibody according to claim 1, wherein the sequence is a polypeptide comprising one full-length heavy chain (VH+ constant) sequence from any one of the CFHR4 antibodies shown in Table 5 and the corresponding full-length light chain (VL+ constant) sequence.

8. The bispecific antibody according to claim 1, wherein the sequence is a polypeptide comprising one full-length heavy chain (VH+ constant) sequence from any one of the C3 antibodies shown in Tables 9 and 10, and the corresponding full-length light chain (VL+ constant) sequence.

9. A polynucleotide encoding the antibody according to claim 1.

10. An expression vector comprising a polynucleotide as described in claim 9, wherein the expression vector is optionally an adeno-associated virus (AAV) vector, a lentivirus (LV) vector, a herpes simplex virus (HSV) vector, or a retroviral vector.

11. A comprising an antibody, polynucleotide, or vector according to any one of claims 1 to 10, and optionally, a. At least one pharmaceutically acceptable carrier, diluent, or preservative, and / or b. At least one further active ingredient, A pharmaceutical composition containing the following:

12. The pharmaceutical composition according to claim 11, which is suitable for ocular administration to a subject, and may also be suitable for administration by delivery using conjunctival implants, contact lenses, gels, nanoparticles, mucosal adhesive polymers, ointments, solutions, suspensions, eye drops, and / or implants, and is preferably suitable for administration by injection into the vitreous fluid.

13. The pharmaceutical composition according to claim 11, for use as a drug, and optionally for use in a method of treating an eye disease in a subject.

14. The pharmaceutical composition for use according to claim 13, wherein the disease is characterized by increased activation of the complement system, particularly of alternative pathways, especially increased activation within the eye of the subject, for example, within the drusen or retinal pigment epithelial (RPE) cells of the subject.

15. A pharmaceutical composition for use according to claim 13, formulated for ocular administration, preferably for injection into the vitreous fluid, preferably the administration alleviates at least one symptom in the subject selected from visual distortion, central vision impairment, blurred vision, and / or difficulty adapting to dim light, and / or the disease is age-related macular degeneration (AMD), optionally the AMD being dry AMD, and being in the early, middle, or advanced stage (the latter otherwise known as geographical atrophy, GA).

16. A bispecific antibody comprising a first antigen-binding site for human complement factor H-related 4 (CFHR4) and a second antigen-binding site for human complement component 3 (C3).

17. The bispecific antibody according to claim 16, wherein the first antigen-binding site for human complement factor H-related 4 (CFHR4) comprises a heavy chain variable region (VH) including complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380, and a light chain variable region (VL) including complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406.

18. The bispecific antibody according to claim 16, wherein the second antigen-binding site for human complement component 3 (C3) comprises a heavy chain variable region (VH) including complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 1127 to 1200, and a light chain variable region (VL) including complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 1275 to 1348.

19. i. The first antigen-binding site for human complement factor H-related 4 (CFHR4) comprises a heavy chain variable region (VH) including complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 260-265, 284-293, 324-334, and 368-380, and a light chain variable region (VL) including complementarity-determining regions (CDRs) LCDR1, LCDR2, and HCDR3 from at least one of SEQ ID NOs: 272-277, 304-313, 346-356, and 394-406, ii. The second antigen-binding site for human complement component 3 (C3) includes a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3 from at least one of SEQ ID NOs: 1127-1200, and a light chain variable region (VL) containing complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3 from at least one of SEQ ID NOs: 1275-1348. The bispecific antibody according to claim 16.

20. The bispecific antibody according to claim 16, wherein the first antigen-binding site comprises anti-CFHR4 HCDR1 containing the amino acid sequence of SEQ ID NO: 2, anti-CFHR4 HCDR2 containing the amino acid sequence of SEQ ID NO: 9, and anti-CFHR4 HCDR3 containing the amino acid sequence of SEQ ID NO:

15.

21. The bispecific antibody according to claim 16, wherein the first antigen-binding site comprises anti-CFHR4 HCDR1 containing the amino acid sequence of SEQ ID NO: 5, anti-CFHR4 HCDR2 containing the amino acid sequence of SEQ ID NO: 12, and anti-CFHR4 HCDR3 containing the amino acid sequence of SEQ ID NO:

18.

22. The bispecific antibody according to claim 16, wherein the second antigen-binding site comprises anti-C3 HCDR1 containing the amino acid sequence of SEQ ID NO: 583, anti-C3 HCDR2 containing the amino acid sequence of SEQ ID NO: 602, and anti-C3 HCDR3 containing the amino acid sequence of SEQ ID NO:

620.

23. The bispecific antibody according to claim 16, wherein the second antigen-binding site comprises anti-C3 HCDR1 containing the amino acid sequence of SEQ ID NO: 584, anti-C3 HCDR2 containing the amino acid sequence of SEQ ID NO: 603, and anti-C3 HCDR3 containing the amino acid sequence of SEQ ID NO:

621.

24. The bispecific antibody according to claim 16, wherein the second antigen-binding site comprises anti-C3 LCDR1 containing the amino acid sequence of SEQ ID NO: 840, anti-C3 LCDR2 containing the amino acid sequence of SEQ ID NO: 872, and anti-C3 LCDR3 containing the amino acid sequence of SEQ ID NO:

903.

25. The bispecific antibody according to claim 16, wherein the first antigen-binding site or the second antigen-binding site is human, humanized, or chimeric.

26. The bispecific antibody according to claim 16, wherein the bispecific antibody, the first antigen-binding site, or the second antigen-binding site is a full-length antibody, a single-chain antibody, a single-chain variable fragment (scFv), a variable fragment (Fv), a fragment antigen-binding region (Fab), Fab-C, Fab'-SH, (Fab')2, a single-domain antibody (sdAb), a VHH antibody, a nanobody, a camel-derived single-domain antibody, a shark IgNAR-derived single-domain antibody fragment (VNAR), a diabody, a triabody, an antikalin, or an aptamer, and optionally, the antibody is a full-length antibody containing an Fc region such as a human IgG1, IgG2, IgG3, or IgG4 region.

27. ​​One or more parts, a. An antigen-binding portion, such as an antibody or its antigen-binding fragment, that can specifically bind to a target other than human CFHR4 or human C3, wherein the target is expressed in the human eye, and the antigen-binding portion b. The area to be treated or the cytotoxic area, c. Detection section, d. Purified portion, and / or e. The half-life extension portion optionally comprises a polypeptide having a length of at least 20 amino acids and containing any combination of G, A, S, T, E, and P residues, which is conjugated to the C-terminus or N-terminus of the antibody. The bispecific antibody according to claim 16, which is conjugated to one or more portions independently selected from.

28. A pharmaceutical composition comprising a bispecific antibody or a pharmaceutically acceptable salt thereof according to any one of claims 16 to 27, and optionally a pharmaceutically acceptable carrier.

29. The pharmaceutical composition according to claim 28, which is suitable for ocular administration.

30. A pharmaceutical composition according to claim 28 for treating age-related macular degeneration (AMD).

31. The pharmaceutical composition according to claim 30, wherein the pharmaceutical composition is administered to the eye and treats at least one symptom of AMD.

32. The pharmaceutical composition according to claim 30, wherein the AMD includes a wet-type AMD or a dry-type AMD.

33. The pharmaceutical composition according to claim 31, wherein the at least one AMD symptom includes visual distortion, decreased central vision, blurred vision, and / or difficulty adapting to dim light.

34. The pharmaceutical composition according to claim 30, wherein administration of the pharmaceutical composition reduces complement activation in the target eye.

35. The pharmaceutical composition according to claim 30, wherein the pharmaceutical composition is administered in a dose in the range of about 0.0001 mg / dose to about 100 mg / dose, or in a dose in the range of about 0.0001 mg / ml to about 100 mg / ml.