Novel complement system inhibiting antibodies

Abstract

Novel anti-C3 antibodies and anti-C5 antibodies are provided that are capable of modulating complement activity by specifically binding to the human complement factors C3, C3a, C3b and / or C5. Also described herein are recombinant adeno-associated virus (AAV) (rAAV) comprising a variant adeno-associated virus (AAV) capsid and a transgene encoding an anti-C3 antibody and / or an anti-C5 antibody. Also provided are methods of delivering a transgene to the retina, as well as methods of treating dry age-related macular degeneration and map-like atrophy secondary to an age-related macular degeneration disorder by contacting retinal cells with rAAV.

Description

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 63 / 503,745 and 63 / 503,768, both filed on May 23, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence list submission via EFS-WEB A computer-readable XML file, created on May 21, 2024, with a file size of approximately 249,282 bytes and named “090400-5023-WOSequence Listing”, contains the sequence list of this application and is incorporated herein by reference in its entirety. Background of the Invention Many diseases are caused by complement dysregulation, leading to complement-mediated damage to autologous tissues. Complement dysregulation can originate from somatic or germline mutations in complement regulators or related genes, causing these regulators to malfunction. In particular, there are common and rare human diseases caused by excessive complement activation resulting from dysregulation of the complement activation cascade.

[0004] Current treatment approaches focus on the development of agents such as monoclonal antibodies (mAbs), peptides, or other small molecules that bind to and block specific alternative or terminal complement pathway components. A clinically validated example is eculizumab, a humanized mAb targeting complement C5, which has been approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Other described approaches include mAbs targeting factor B (fB), factor D (fD), or properin (fP), as well as cyclic peptides that bind to and inhibit C3. The limitation of these approaches is that they require repeated and inconvenient intravenous (IV) administration to patients. Furthermore, because these treatments block alternative or terminal pathways, they pose a risk of impairing host defenses. In fact, patients receiving eculizumab therapy must be vaccinated against bacterial strains that cause fatal meningitis, and these patients also receive prophylactic antibiotic therapy before treatment with the approved mAb.

[0005] Other approaches have tested recombinant regulatory proteins such as soluble DAF, CR1, CRIg, and proteins containing the minimal domains of the liquid-phase regulator fH (N-terminal short shared repeats [SCR] 1–5 and C-terminal SCR 19–20), or fusion proteins between fH and CR2 (TT30). However, large-scale heterologous expression of such proteins as therapeutic agents requires significant effort, and animal studies have shown rapid in vivo clearance after administration, making such therapeutic strategies cumbersome and impractical due to the need for multiple and frequent administrations of these protein drugs.

[0006] There remains a need in the art for compositions that can be used to treat complement-mediated diseases with greater and more lasting efficacy. Invention Overview This article describes peptides comprising antibodies, preferably peptides comprising a single variable domain (VHH or nanobody) on the heavy chain, which can modulate complement activity by specifically binding to human complement factors C3, C3a, C3b, C5, C5a and / or C5b. Preferably, the peptide comprises a VH or VHH domain of a camel heavy chain antibody, or a VH or VHH domain derived from a camel heavy chain antibody.

[0008] In some aspects, the VH or VHH domain includes one or more complementarity-determining regions (CDRs) as shown in Tables 1 and 2. In a preferred aspect, the VH or VHH domain includes CDRs (CDR1, CDR2, and CDR3) of one or more antibodies selected from SEQ ID No:1-14 and SEQ ID No:57-65. In other preferred aspects, CDR1, CDR2, and CDR3 are separated by frame regions FR1, FR2, FR3, and FR4 of the VH or VHH domain.

[0009] In some preferred aspects, the VH or VHH domain comprises or consists of any of the amino acid sequences shown in SEQ ID No:1-14 or any of the amino acid sequences shown in SEQ ID No:57-65.

[0010] In other preferred aspects, the VH or VHH domain is humanized and comprises or consists of any of the amino acid sequences shown in SEQ ID No: 94-136 or SEQ ID No: 137-163.

[0011] In other preferred aspects, the VH or VHH domain contains a tag, such as a Strep tag or a his6 tag. In other aspects, the VH or VHH domain is fused to another polypeptide.

[0012] In other embodiments, a polynucleotide (DNA or RNA) is provided, wherein the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising one or more CDRs containing a VH or VHH domain as described herein, or encoding a polypeptide containing a VH or VHH domain as described herein. In related embodiments, an expression vector comprising the polynucleotide is provided, wherein the nucleotide sequence is operatively linked to an expression control sequence (e.g., a promoter). In other embodiments, pharmaceutical compositions comprising a polypeptide or expression vector as described herein and a pharmaceutically acceptable vector are provided.

[0013] Also provided are recombinant AAV (rAAV) viral particles comprising a variant AAV capsid sequence encapsulating a heterologous nucleic acid encoding a nucleotide sequence of an anti-C3 antibody and / or an anti-C5 antibody as described herein. Preferably, the nucleotide sequence encoding the antibody is operatively linked to an expression control sequence.

[0014] The rAAV variant AAV capsid protein comprises, relative to the corresponding parental AAV capsid protein, a capsid protein containing, preferably in the GH ring of the capsid protein, a peptide insert (“heteropeptide” or “peptide insert”) of about 7 to about 20 amino acids in the surface exposed region of the GH ring, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO: 168). In some preferred aspects, the peptide insert comprises one to three spacer amino acids (Y1-Y3) at the amino terminus and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO: 168), wherein Y1-Y3 are each independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. In a particularly preferred embodiment, the peptide insert comprises, is substantially composed of, or is composed of the amino acid sequence LAISDQTKHA (SEQ ID NO: 169). In some preferred embodiments, the peptide is inserted after any amino acid at positions 584-591 in the VP1 of AAV2, or at the corresponding position in another AAV serotype (i.e., the insertion site is between amino acids 587 and 588 in the VP1 of AAV2, or between amino acids 588 and 589 in AAV2, between amino acids 584 and 585, between amino acids 585 and 586, between amino acids 586 and 587, between amino acids 590 and 591, or between amino acids 591 and 592, or at the corresponding position in the capsid protein of another AAV serotype). In some embodiments, the capsid protein further comprises one or more amino acid substitutions relative to the VP1 capsid of AAV2, or one or more corresponding substitutions in another AAV serotype, preferably wherein the capsid protein further comprises a P34A amino acid substitution relative to the VP1 capsid of AAV2, or a corresponding substitution in another AAV serotype.

[0015] In other embodiments, a method is provided for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding an antibody as described herein to a mammalian subject, the method comprising administering to the mammal an effective amount of rAAV as described herein or a pharmaceutical composition comprising thereas, preferably wherein said rAAV or pharmaceutical composition is administered via intravitreal injection. In some aspects, the heterologous nucleic acid is delivered to the subject's retinal cells, such as the subject's photoreceptor cells (e.g., rod cells; cone cells), retinal ganglion cells (RGCs), glial cells (e.g., Müller glial cells, microglia), bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelium (RPE) cells.

[0016] In some embodiments, after rAAV administration to a subject, detectable plasma levels of the antibody are present in the subject for at least one week, at least two weeks, at least three weeks, at least one month, at least two months, or at least six months. In a particularly preferred embodiment, rAAV is administered to the subject via intravitreal administration.

[0017] In other embodiments, pharmaceutical compositions are provided comprising rAAV as described herein and pharmaceutically acceptable excipients.

[0018] In other respects, methods are provided for treating complement-related conditions by delivering rAAV or pharmaceutical compositions containing rAAV as described herein to a subject. Complement-related conditions that can be treated include, but are not limited to, membranoproliferative glomerulonephritis, atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD), geographic atrophy secondary to AMD, microangiopathic hemolytic anemia, thrombocytopenia, acute renal failure, paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, ischemic stroke, and / or bacterial infections caused by recruitment of bacterial pathogens.

[0019] In a further aspect, a method is provided for treating dry age-related macular degeneration (AMD) (e.g., advanced dry AMD) in a subject by delivering to a subject an effective amount of rAAV as described herein or a pharmaceutical composition containing rAAV. In a related aspect, the rAAV or pharmaceutical composition is administered to the subject for the treatment of geographic atrophy secondary to AMD. Preferably, the rAAV or pharmaceutical composition is administered to the subject via intravitreal injection. Brief description of the attached diagram Figure 1A -C-induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE) were transduced with the C3-N10 rAAV transgene (encoding the anti-C3 antibody SEQ ID NO:14) at three multiples of infection (MOIs) of 5,000, 10,000, and 20,000. Changes in nanobody (VHH) expression were observed. Figure 1A ) and MAC formation ( Figure 1B The dose-response relationship was observed. Immunocytochemistry characterized the deposition of complement membrane attack complex (MAC) in iPSC-RPE following yeast polysaccharide (a potent activator of the alternative complement pathway) and the inhibition of MAC deposition following C3-N10 rAAV transduction. Figure 1C ).

[0021] Figure 2A-C represents complement inhibition via wild-type lead nanobodies in the alternative, lectin, and classical complement pathways. The complement inhibitory activities of all three wild-type nanobodies were compared using Wieslab's alternative, MBL / lectin, and classical complement pathway assay kits. Figure 2A (alternative) Figure 2B (lectin) and Figure 2C As shown in the (classical) diagram, while C5S2 exhibits similarly strong inhibitory potency against all three pathways, C3S3 shows activity only against the alternative pathway. C3N10 appears to have a stronger inhibitory effect against the alternative pathway and a weaker potency against the lectin and classical pathways.

[0022] Figure 3A -C wild-type and humanized nanobodies were evaluated for their IC50 potency against the alternative complement pathway. To assess the potency differences between the wild-type and humanized counterparts, each nanobody was serially diluted and tested using the Wieslab Alternative Complement Pathway Kit, and its IC50 was calculated using GraphPad Prism. (See also: Anti-C3 nanobodies...) Figure 3A and 3B ) and anti-5C nanobody ( Figure 3C As shown in the figure, all humanized forms retain IC50 activity similar to their original wild type.

[0023] Figure 4A -BC3N10 and C3N10.3 are for the classical approach ( Figure 4A ) and lectin pathway ( Figure 4B The IC50 efficacy of these nanobodies was compared. To evaluate the inhibitory activity of the wild-type and humanized forms of C3N10 within the classical complement pathway and the lectin complement pathway, these nanobodies were serially diluted, and the samples were tested using separate Wieslab assay kits. Both forms exhibited similar IC50 efficacy against these assay pathways.

[0024] Figure 5 Cross-reactivity of the wild-type nanobody lead compound with mouse serum was demonstrated. The mouse cross-inhibitory activity of the wild-type nanobody was examined using Hycult's C3 mouse ELISA kit and mouse anti-mouse C3 antibody BB5.1 as controls. None of the anti-human C3 or C5 nanobodies, C3N10, C3S3, and C5S2, showed inhibitory activity against the mouse serum-mediated alternative complement pathway.

[0025] Figure 6A-IC50 of wild-type and humanized nanobodies against the alternative complement pathway in cynomolgus monkey serum. Wild-type and humanized counterparts were serially diluted, and their inhibitory activity against the alternative complement pathway mediated by cynomolgus monkey serum was calculated using separate Wieslab assays. C3N10 WT and C3N10.3 ( Figure 6A Together with C3S3 WT and C3S3.2 Figure 6B The assay showed activity against the cynomolgus monkey alternative complement pathway, with similar IC50s across each variant. C5S2 and C5S2.2 did not show any inhibitory activity in this NHP serum-mediated assay (data not shown).

[0026] Figure 7 Functional potency of anti-C3 and C5 VHH-Fc dimers in the alternative complement pathway in human serum. Humanized anti-C3 and C5 nanobodies were reengineered into G2-hinge-G4-Fc fusion proteins, and their IC50 was measured by serial dilution in a Wieslab alternative complement pathway assay. Data were plotted and IC50 calculated using GraphPad Prism. (CS52.2-FcIC50) 50 MAC = 9.076 nM; C3S3-Fc 50 MAC = 23.80 nM; C3N10.3-Fc 50 MAC = 49.31 nM) Figure 8 Amino acid interactions between C3N10.3 and human C3 protein were investigated. Humanized C3N10.3 was allowed to bind to human C3 protein, and CovalX crosslinking mass spectrometry was performed to identify potential binding peptide epitopes. Three epitopes (amino acids 1548-1551, 1568-1573, and 1583-1591) within amino acid range 1540-600 of the human C3 protein were identified.

[0027] Figure 9 Amino acid interactions between the nanobody C3S3.2 and human C3 protein. Humanized C3S3.2 was allowed to bind to human C3 protein, and cross-linking mass spectrometry (CovalX) was performed to identify potential binding peptide epitopes. Epitopes were identified in the human C3 protein between amino acids 110-130 (amino acids 114-124), and another epitope was identified between amino acids 490-510 (amino acids 497-502 and 502-505).

[0028] Figure 10Potential binding domains of C3N10.3 and C3S3.2 to human C3 protein are shown. Based on cross-linking mass spectrometry data, the binding regions of C3N10.3 and C3S3.2 to human C3 protein are displayed, with the former binding within the C345C domain of the α chain, and the latter interacting across the MG1 and MG5 domains of the β chain, respectively.

[0029] Figure 11 This study demonstrates the cross-blocking of eculizumab and C5S2.2 binding to human C5 using a probe-anti-human Fc sandwich method. A Gator / BLI instrument was used to examine whether C5S2.2 cross-blocks eculizumab, a clinically approved anti-human C5 antibody targeting hIgG4. Anti-human Fc probes were pre-bound with eculizumab and then washed. Individual human C5 or incrementally concentrated C5 pre-bound with C5S2.2 was then allowed to bind to the antibody-loaded probes. Binding kinetics were observed only with respect to free C5 protein, indicating that the pre-bound C5S2.2 VHH blocked the C5-binding epitope of eculizumab. Time (seconds) is on the X-axis, and displacement (nm) is on the Y-axis.

[0030] Figure 12 This study demonstrates the cross-blocking effect of eculizumab and C5S2.2 on human C5 binding: a probe-anti-StrepII sandwich assay. A Gator / BLI instrument was used to examine whether C5S2.2 cross-blocked eculizumab, a clinically approved anti-human C5 antibody targeting hIgG4. The anti-Strep II probe was pre-bound with a Strep-tagged anti-human C5 C5S2.2 VHH. After washing, the probe loaded with the nanobody was immersed in binding buffer containing human C5, and binding kinetics were observed. When this pre-bound complex was then immersed in a solution containing eculizumab, no interaction was observed, suggesting that the latter's binding epitope was blocked. Time (seconds) is on the X-axis, and displacement (nm) is on the Y-axis.

[0031] Figure 13A -C shows the binding of C5S2.2 to human and cynomolgus C5 mutants. Eculizumab is resistant to two naturally occurring variants: the human R885H polymorphism and the human W917S divergence relative to cynomolgus. Human C5 R885H, human C5 W917S, and cynomolgus C5 S917W mutant proteins were recombinantly generated, and the affinity of C5S2.2 for them was compared with that of eculizumab using Gator. Figure 14A -C).

[0032] Figure 14A-C indicates the binding of eculizumab to human and cynomolgus C5 mutants. Eculizumab is resistant to two naturally occurring variants: the human R885H polymorphism and the human W917S divergence relative to cynomolgus monkeys. The human C5 R885H, human C5 W917S, and cynomolgus monkey C5 S917W mutant proteins were recombinantly generated, and Gator was used to reflect the affinity of eculizumab for them. Figure 15 This displays the percentage of anti-drug antibody (ADA) responses in normal human donors to wild-type and humanized C3 and C5 nanobodies (Cap = Capsulesizumab / Cablivi; Beovu = busizumab). Normal human donor serum was tested for ADA against the listed nanobodies and controls using a mesoscale detection immunoassay. Samples were diluted 1:100 and applied to wells passively coated with the respective antibodies. Binding human antibodies were detected using sulfo-TAG goat anti-human IgG and measured on a Meso Sector S 600.

[0033] Figure 16A -C shows the functional evaluation of expressed VHH after plasmid transfection with the breakdown products of complement cascade-related proteins. Figure 16A C3 VHH inhibitors, rather than C5 VHH inhibitors, prevent C3 cleavage, as detected by the absence of C3a degradation products. Figure 16B C3 VHH inhibitors and C5 VHH inhibitors prevent C5 cleavage, as can be detected by the absence of C5a decomposition products. Figure 16C C3 VHH inhibitors, not C5 VHH inhibitors, prevent Bb cleavage, as detected by the absence of factor B degradation products. v, form; h, humanized; kDa, kilodaltons; NT, untransfected.

[0034] Figure 17 This is a Western blot that confirms single products for each VHH variant examined after transduction with AAV carrying VHH. NT, untransduced; kDa, kilodaltons; MOI 20,000; 33 µg protein loaded per lane; seven days post-transduction.

[0035] Figure 18A -C shows the expression of secreted VHH (C3N10 and humanized C3N10) after AAV transduction. Figure 18A C3S3 and humanized C3S3 Figure 18B C5S2 and humanized C5S2 Figure 18C), as measured by ELISA, and the observed dose response for each VHH is shown (MOI at 1,000, 5,000, and 20,000). NT, untransduced; MOI, multiplicity of infection; error bars ± standard deviation; n = 3 wells / MOI.

[0036] Figure 19 The function of secreted VHHs (C3N10, C3N10 h, C353, C353 h, C5S2, C5S2 h) is shown, as measured by complement inhibition as a percentage. Each VHH exhibits a certain level of complement inhibition. NT, untransduced; h, humanized; MOI, multiplicity of infection; error bars ± standard deviation; n = 3 wells / MOI. Invention Details definition The term “antibody” is used herein in the broadest sense to refer to a polypeptide or protein having an immunoglobulin-like domain capable of recognizing and binding an antigen, and includes the full-size antibody, its individual chains, and all its portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). The antigen-binding site of an antibody preferably contains at least one complementarity-determining region (CDR). This disclosure primarily relates to a single variable domain on a heavy chain (VHH) antibody. As used herein, the terms “single-domain antibody,” “single-variable-domain antibody,” “VHH antibody,” and “nanobody” have the same meaning, referring to the variable region of an antibody heavy chain, and constructing a single-domain antibody consisting of only one heavy chain variable region. Thus, the antigen-binding site of a single variable domain is formed by no more than three CDRs. Generally, a naturally deficient antibody with a light chain and a heavy chain constant region 1 (CH1) is first obtained, and the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. VHH antibodies are preferably derived from llamas.

[0038] An antigen is a molecule that contains at least one epitope. Antigens can be, for example, polypeptides, nucleic acids, polysaccharides, proteins, lipoproteins, or glycoproteins.

[0039] The "complementarity-determining region" or "CDR" is a hypervariable region of the antibody's antigen-binding domain. CDRs are inserted between more conserved regions called frame regions (FRs). The antibody's antigen-binding domain can therefore contain one or more CDRs and FRs. Typically, in each variable domain, three CDRs and four FRs are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0040] An epitope is a determinant that can specifically bind to an antibody. Epitopes can be contained, for example, within a polypeptide or protein. Epitopes can be adjacent or non-adjacent, wherein non-adjacent epitopes are conformational epitopes on an antigen, which are formed by at least two separate regions in the primary sequence of a protein, nucleic acid, or polysaccharide.

[0041] The term "affinity" refers to the strength of binding between an antibody and its antigen. Antibody affinity can be defined according to the dissociation constant KD, which is an equilibrium constant that measures the tendency of a molecular complex to reversibly separate (dissociate) into molecules forming the complex. In one aspect, KD... D Defined as ratio k 0ff / k on , where k 0ff and k on It is the rate constant for the binding and dissociation of the molecular complex. Preferably, the affinity is calculated by taking the dissociation constant K based on the IC50 value. D Therefore, affinity is measured as apparent affinity.

[0042] The term "isolated" refers to biological material (cells, nucleic acids, or proteins) that has been removed from its original environment (the environment in which it naturally exists). For example, a polynucleotide that exists naturally in a plant or animal is not isolated; however, the same polynucleotide isolated from a neighboring nucleic acid in which it naturally exists is considered "isolated."

[0043] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. While “stop codons” (TAG, TGA, or TAA) are not typically translated into amino acids, they can be considered part of a coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of a coding region. The boundaries of a coding region are typically defined by a start codon at the 5' end (encoding the amino terminus of the resulting polypeptide) and a translation stop codon at the 3' end (encoding the carboxyl terminus of the resulting polypeptide). Two or more coding regions can exist in a single polynucleotide construct, for example, on a single vector, or in separate polynucleotide constructs, for example, on separate (different) vectors. Therefore, a subsequent single vector may contain only a single coding region or contain two or more coding regions.

[0044] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequence), inside, or downstream (3' non-coding sequence) of a coding region that influences transcription, RNA processing, stability, or translation of the relevant coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences are typically located at the 3' end of the coding sequence.

[0045] As used in this article, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and refers to a polymer of nucleotides.

[0046] Polynucleotides encoding gene products, such as polypeptides, may include promoters and / or other transcriptional or translational control elements that are operatively bound to one or more coding regions. In operative binding, the coding region of a gene product, such as a polypeptide, binds to one or more regulatory regions in such a manner to place the expression of the gene product under the influence or control of the regulatory regions. For example, the coding region and the promoter are “operatively bound” if the induction of promoter function leads to the transcription of mRNA encoding the gene product encoded by the coding region, and if the binding nature between the promoter and the coding region does not interfere with the promoter’s ability to direct the expression of the gene product or the ability to transcribe the DNA template. In addition to promoters, other transcriptional control elements, such as enhancers, operons, repressors, and transcription termination signals, may also be operatively bound to coding regions to direct the expression of gene products.

[0047] "Transcriptional control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, etc., that provide for the expression of coding sequences in host cells. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (an immediate early promoter that binds to intron A), simian virus 40 (an early promoter), and retroviruses (e.g., Raoult's sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters induced by interferon or interleukin).

[0048] Similarly, various translation control elements are known to those skilled in the art. These elements include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from microRNAs (particularly internal ribosome entry sites, or IRES, also known as CITE sequences).

[0049] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or a polypeptide. It includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of mRNA into a polypeptide. Expression produces a “gene product.” As used herein, a gene product can be a nucleic acid, such as messenger RNA produced by gene transcription, or a polypeptide translated from a transcript. Gene products described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation or splicing), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, lipid addition, binding to other protein subunits, or proteolytic cleavage).

[0050] "Promoter" and "promoter sequence" are used interchangeably and refer to the DNA sequence that controls the expression of a coding sequence or functional RNA. Generally, the coding sequence is located at the 3' end of the promoter sequence. A promoter can be derived entirely from a natural gene, or composed of different elements derived from different promoters found in nature, or even contain synthetic DNA segments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. Promoters that induce gene expression in most cell types most of the time are generally called "constitutive promoters." Promoters that induce gene expression in specific cell types are generally called "cell-specific promoters" or "tissue-specific promoters." Promoters that induce gene expression at specific developmental stages or cell differentiation stages are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and induce gene expression after exposure or treatment of cells with promoter-inducing agents, biomolecules, chemicals, ligands, light, etc., are generally called "inducible promoters" or "regulatory promoters." It was further recognized that, since the exact boundaries of the regulatory sequence are not fully defined in most cases, DNA fragments of different lengths can have the same promoter activity.

[0051] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the cell's central metabolic processes and is typically in the form of a circular double-stranded DNA molecule. Such elements can be derived from any source and have single-stranded or double-stranded DNA or RNA, linear, circular, or supercoiled autonomously replicating sequences, genome-integrated sequences, bacteriophage sequences, or nucleotide sequences in which multiple nucleotide sequences have been linked or rearranged into a unique structure capable of introducing promoter fragments and the DNA sequence of selected gene products, along with appropriate 3' untranslated sequences, into the cell.

[0052] A polynucleotide or polypeptide sharing a certain percentage of "sequence identity" with another polynucleotide or polypeptide means the percentage of identical bases or amino acids when comparing two sequences during alignment. As described in various aspects of the invention, sequence identity is related to sequence homology. Homology comparisons can be performed visually or, more generally, by means of readily available sequence comparison programs. Sequence similarity or sequence homology can be determined in a variety of different ways. Commercially available computer programs can calculate the percentage (%) homology between two or more sequences, and can also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. Sequence homology can be generated by any of a variety of computer programs known in the art, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) software package from Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), edited by Doolittle, Academic Press, Inc. Of particular interest are alignment procedures that allow for gaps in the sequence. Smith-Waterman is one type of algorithm that allows for gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Additionally, the GAP procedure using the Needleman and Wunsch alignment methods can be used for sequence alignment. See J. Mol. Biol. 48: 443-453 (1970).

[0053] Homology can be calculated on adjacent sequences, that is, one sequence is aligned with another, and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called "vacancy-free" alignment. Typically, this type of vacancy-free alignment is performed only on a relatively short number of residues.

[0054] While this is a very simple and consistent approach, it fails to account for the fact that, for example, in otherwise identical sequence pairs, an insertion or deletion can lead to misalignment of subsequent amino acid residues, potentially resulting in a significant reduction in % homology when performing global alignment. Therefore, most sequence comparison methods are designed to produce optimal alignments that account for possible insertions and deletions without unduly penalizing overall homology or identity scores. This is achieved by inserting “gaps” in the sequence alignment in an attempt to maximize local homology or identity.

[0055] However, these more sophisticated methods assign a "vacancy penalty" to each vacancy that appears in the alignment, so that for the same number of identical amino acids, sequence alignments with the fewest possible vacancies—reflecting a higher correlation between the two compared sequences—can achieve higher scores than alignments with many vacancies. Typically, an "affinity vacancy cost" is used, which charges a relatively high cost for the presence of a vacancy and a small penalty for each subsequent residue within the vacancy. This is the most commonly used vacancy scoring system. Of course, a high vacancy penalty can produce an optimized alignment with fewer vacancies. Most alignment programs allow modification of the vacancy penalty. However, when using such software for sequence alignment, the default values ​​are preferred. For example, when using the GCG Wisconsin Bestfit package, the default vacancy penalty for amino acid sequences is -12 for vacancies and -4 for each extension.

[0056] The calculation of maximum % homology therefore first requires consideration of optimal alignment generation for gap penalties. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit software package (Devereux et al., 1984, Nuc. Acids Research 12, p. 387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST software package (see Ausubel et al., 1999, Short Protocols in Molecular Biology, 4th ed. – Chapter 18), FASTA (Altschul et al., 1990, J Mol. Biol. 403–410), and the GNEWORKS comparison tool suite. Both BLAST and FASTA can be used for both offline and online searches (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pp. 7–58–7–60). However, for some applications, the GCG Bestfit program is preferred. A new tool called BLAST 2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8, and the National Center for Biotechnology Information website at the National Institutes for Health).

[0057] Although the final % homology can be measured as identity, the alignment process itself is not typically based on all-or-nothing pair comparisons. Instead, a scaled similarity score matrix is ​​generally used, which assigns a score to each pair comparison based on chemical similarity or evolutionary distance. A common example of such a matrix is ​​the BLOSUM62 matrix—the default matrix for the BLAST program suite. The GCG Wisconsin program generally uses public defaults or a custom symbol comparison table (if provided) (see the user manual for further details). For some applications, it is preferred to use the public defaults for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62, is used.

[0058] Alternatively, based on an algorithm similar to CLUSTAL (Higgins D G & Sharp PM (1988), Gene 73(1), 237-244), percentage homology can be calculated using the multiple sequence alignment features in DNASIS™ (Hitachi Software). Once the software has generated the optimal alignment, calculating % homology and, ideally, % sequence identity becomes possible. The software typically includes this as part of the sequence comparison and generates numerical results.

[0059] Sequences can also have deletions, insertions, or substitutions of amino acid residues, producing silencing changes and resulting in functionally equivalent substances. Intentional amino acid substitutions can be made based on similarities in amino acid properties (e.g., polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of residues), and can therefore be used to group amino acids together into functional groups. Amino acids can also be grouped together based on the properties of their individual side chains. However, including mutation data is also more useful. Therefore, the derived amino acid sets are likely to be conserved for structural reasons. These sets can be described in the form of Venn diagrams (Livingstone CD and Barton GJ (1993) “Protein sequence alignments: astrategy for the hierarchical analysis of residue conservation” Comput. Appl. Biosci. 9: 745-756) (Taylor WR (1986) “The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conserved substitutions can be made, for example, according to the table below, which describes the generally accepted Venn diagram grouping of amino acids.

[0060] Embodiments of the present invention include sequences (polynucleotides or polypeptides) that may contain homologous substitutions (substitution and substitution are used herein to mean the exchange of an existing amino acid residue or nucleotide with a substitute residue or nucleotide), i.e., same-kind substitutions in the case of amino acids, such as basic substitution for basic, acidic substitution for acidic, polar substitution for polar, etc. Non-homologous substitutions may also occur, i.e., substitutions from one class of residues to another, or alternatively, may involve non-natural amino acids, such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), ortholeucine ornithine (hereinafter referred to as O), pyridylalanine, thiophenylalanine, naphthylalanine, and phenylglycine.

[0061] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues in the sequence, including alkyl groups such as methyl, ethyl, or propyl groups, plus amino acid spacers such as glycine or β-alanine residues. Further variant forms will be well understood by those skilled in the art, which involve the presence of one or more amino acid residues in a peptide-like form. For the avoidance of doubt, “peptide-like form” is used to refer to variant amino acid residues in which an α-carbon substituent is placed on the nitrogen atom of the residue instead of the α-carbon. Methods for preparing peptides in peptide-like forms are known in the art, for example, Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0062] The term "amino acid substitution" and its synonyms described above are intended to cover amino acid sequence modifications by replacing an amino acid with another substituted amino acid. Substitution can be conservative substitution. It can also be non-conservative substitution. The term "conservative" when referring to two amino acids is intended to mean that the amino acids share common properties recognized by those skilled in the art. For example, amino acids having hydrophobic non-acidic side chains, amino acids having hydrophobic acidic side chains, amino acids having hydrophilic non-acidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains. Common properties can also include amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids having polar neutral side chains, amino acids having charged side chains, amino acids having charged acidic side chains, and amino acids having charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and can be used as substituted amino acids in embodiments. Methods for replacing amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. The use of “one or more” in this document is intended to cover, for example, 1, 2, 3, 4, 5, 6 or more individual implementations.

[0063] As used herein, the terms “treatment,” “treating,” etc., refer to achieving the desired pharmacological and / or physiological effect. The effect may be preventative in the complete or partial prevention of the disease or its symptoms, and / or therapeutic in the partial or complete cure of the disease and / or adverse effects attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease (and / or symptoms caused by the disease) in a subject who may be susceptible to the disease or at risk of acquiring the disease but has not yet been diagnosed with it; (b) suppressing the disease (and / or symptoms caused by the disease), i.e., preventing its development; and (c) alleviating the disease (and / or symptoms caused by the disease), i.e., promoting the resolution of the disease (and / or symptoms caused by the disease), i.e., improving the disease and / or one or more symptoms of the disease.

[0064] As used herein, the term "treatment of complement factor H syndrome" can encompass the reduction, decrease, and / or improvement of symptoms, and / or prevention of the development of additional symptoms associated with complement factor H syndrome, which can manifest in several different phenotypes, including asymptomatic, recurrent bacterial infections, and renal failure. This is typically characterized by reduced serum levels of factor H, complement component C3, and other terminal complement components, indicating activation of the alternative complement pathway. Serum levels of complement component C5 may also be reduced. This syndrome is associated with a variety of renal diseases with variable clinical presentation and progression, including C3 glomerulonephropathy and atypical hemolytic uremic syndrome. This article also provides compositions and methods for treating one or more of the following: dry age-related macular degeneration (AMD), geographic atrophy secondary to AMD, atypical hemolytic uremic syndromes (including, for example, microangiopathic hemolytic anemia syndrome, thrombocytopenia, acute renal failure), paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, ischemic stroke, and / or prevention or treatment, especially of bacterial pathogens (e.g., Aspergillus species; Borrelia burgdorferi). Borrelia burgdorferi ); Borrelia d'Arc ( B. duttonii ); Regressive thermophobic spirochetes ( B. recurrentis ); Candida albicans ( Candida albicans ); Tulafrancsis ( Francisella tularensis ); Haemophilus influenzae ( Haemophilus influenzae ); Neisseria meningitidis ( Neisseria meningitidis ); Streptococcus pyogenes ( Streptococcus pyogenes Compositions and methods for recruiting bacteria that cause infection, such as borborygmus factor H binding proteins (CRASP-1, CRASP-2, CRASP-3, CRASP-4 or CRASP-5).

[0065] As used in this article, the term “treatment of complement-related disorders” includes alleviating, reducing and / or improving symptoms of both the complement factor H disorders identified above and other disorders associated with uncontrolled alternative pathway complement regulation.

[0066] "Complement-mediated symptoms" can encompass symptoms associated with complement dysregulation, which can manifest in several different phenotypes, including asymptomatic recurrent bacterial infections and various tissue damages, including but not limited to kidney disease. Unless otherwise stated, this definition includes both homozygous and heterozygous subjects. Complement dysregulation is typically caused by loss-of-function mutations in complement regulatory proteins (including but not limited to fH, factor I (fI), and membrane cofactor protein (MCP)) or by autoantibodies against complement regulatory proteins, or by gain-of-function mutations in other complement proteins (including but not limited to C3, C5, and factor B (fB)). Complement dysregulation is typically (though not always) characterized by reduced serum levels of factor H, complement component C3, fB, and other terminal complement components, indicating activation of alternative and / or terminal complement pathways. Complement-mediated pathological conditions treatable by the compositions and methods of the present invention include, but are not limited to, the following diseases with different clinical presentations and progressions: C3 glomerulonephropathy (formally known as membranoproliferative glomerulonephritis type II or MPGNII), in which two known forms exist – dense deposit disease (DDD) and C3 glomerulonephritis (C3GN); thrombotic microangiopathy (TMA), including but not limited to atypical hemolytic uremic syndrome (aHUS) and Shiga toxin-producing Escherichia coli HUS. (STEC-HUS) and thrombotic thrombocytopenic purpura (TTP); retinal degenerative eye diseases, including age-related macular degeneration (AMD), RPE degeneration, choroidal retinal degeneration, photoreceptor cell degeneration, paroxysmal nocturnal hemoglobinuria (PNH), ischemia-reperfusion injury of all organs and environments, rheumatoid arthritis, hemodialysis, diabetic nephropathy, diabetic vascular disease, asthma, systemic lupus erythematosus (SLE), ischemic stroke, abdominal aortic aneurysm (AAA), antineutrophil cytoplasmic antibody (ANCA)-mediated vasculitis (ANCA vasculitis), and ANCA-mediated hemorrhagic lung injury. And diseases, ANCA glomerulonephritis, graft-versus-host disease (GvHD), acute or delayed graft rejection in organ transplantation, Crohn's disease, psoriasis, multiple sclerosis, antiphospholipid syndrome, preeclampsia, atherosclerosis, neuromyelitis optica (NMO), autoimmune cutaneous bullous disease, bullous pemphigoid (BP), Alzheimer's disease (AD), and bacterial infections caused by the recruitment of bacterial pathogens (e.g., Aspergillus species; Borrelia burgdorferi; Borrelia davidiana; Borrelia relapsingis; Candida albicans; Tula Francisella; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pyogenes).

[0067] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably in this document and refer to mammals, including but not limited to primates (e.g., humans; non-human primates, including apes); mammalian locomotion animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0068] As used herein, the term "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. An effective amount may be administered in one or more doses. For the purposes of this disclosure, an effective amount of a compound (e.g., infectious rAAV viral particles) is an amount sufficient to mitigate, improve, stabilize, reverse, prevent, slow, or delay the progression of a particular disease state (e.g., a condition associated with complement dysfunction) (and / or associated symptoms). Therefore, an effective amount of infectious rAAV viral particles is an amount of infectious rAAV viral particles capable of effectively delivering heterologous nucleic acids to an individual's target cells (or multiple target cells). An effective amount can be clinically determined, for example, by detecting a gene product (RNA, protein) encoded by a heterologous nucleic acid sequence in cells or tissues using techniques well understood in the art, such as RT-PCR, Western blotting, ELISA, fluorescence, or other reporter gene readouts. An effective dose can be determined clinically, for example, by detecting changes in the onset or progression of disease using methods known in the art, such as the 6-minute walk test, left ventricular ejection fraction, handheld dynamometer, Vignos scale, etc., as described herein and as known in the art. Invention Details This article describes novel anti-C3 and anti-C5 antibodies and the nucleic acids encoding them. It also describes rAAV viral particles containing nucleic acids encoding anti-C3 antibodies and / or antibodies. These rAAV viral particles are characterized by persistent and stable expression of anti-C3 and anti-C5 antibodies in the retina, and increased efficacy in treating conditions related to C3 and C5, as well as other complement disorders.

[0070] These rAAV viral particles can be delivered to subjects in need via various routes, preferably via intravitreal administration. Methods for using these rAAV viral particles in treatment regimens for complement factor C3 and C5-related conditions, particularly dry AMD and geographic atrophy secondary to AMD, are also provided.

[0071] Antibody Naturally occurring human antibodies are heterotetramers. The antibodies described herein contain, in one respect, an antigen-binding site within a single polypeptide. The antibodies are therefore referred to herein as “monovariable domain” or “VHH” antibodies. Monovariable domain antibodies are also known as nanobodies. However, in some embodiments, the monovariable domain antibodies disclosed herein may be bispecific or multispecific monovariable domain antibodies, wherein the monovariable domain antibody is conjugated.

[0072] A monovariable domain antibody is an antibody fragment composed of a single monomeric variable antibody domain. Like a complete antibody, it can selectively bind to a specific antigen. Monovariable domain antibodies typically have a molecular weight in the range of 12-15 kDa, which is much lower than that of common antibodies, which typically range from 150 to 160 kDa. Monovariable domain antibodies are also smaller than the Fab fragment (~50 kDa) of heterotetrameric antibodies, which contain one light chain and half a heavy chain.

[0073] Single-domain antibodies can be derived from those found in nature, such as those found in camels (VHH) and cartilaginous fish (VNAR). The new shark or nurse shark antigen receptor (NAR) protein exists as a dimer of two heavy chains without a binding light chain. Each chain consists of one variable domain (V) and five constant domains. The NAR protein thus constitutes a single immunoglobulin-like domain. Single-heavy-chain antibodies have also been found in camels such as dromedary camels, Bactrian camels, llamas, and alpacas, where the heavy chain has lost one of its constant domains and undergone modifications in the variable domains, both of which are essential structural elements for light chain binding.

[0074] However, monovariable domain antibodies can also be modified using recombinant methods. One approach is to disassemble the dimeric variable domain of common human or mouse immunoglobulin G (IgG) into monomers. The single domain derived from the light chain also binds specifically to the target epitope. Therefore, monovariable domain antibodies can be derived from any suitable organism.

[0075] Single-domain camel antibodies are equivalent to conventional antibodies in terms of specificity. Single-domain antibodies can be easily isolated, for example, using phage panning procedures. Their smaller size and single-domain architecture make these antibodies easier to express as proteins in bacterial cells for large-scale production, making them ideal for commercial use. The antibodies provided by this invention are therefore single variable-domain antibodies, preferably derived from camel antibodies, more preferably from llama antibodies, including their functional homologs, fragments, and fusion macromolecules containing VHHs covalently linked to glycans, nucleic acids, proteins, or chemical groups that are not macromolecules.

[0076] In some respects, the antibodies described herein are antibody fragments. In one respect, antibody fragments are Fab, Fab', Fab'-SH, or F(ab')2 fragments, particularly Fab fragments. Papain digestion of an intact antibody produces two identical antigen-binding fragments, referred to as "Fab" fragments, which contain variable domains (VH and VL, respectively) for each heavy and light chain, as well as a constant domain (CL) for the light chain and a first constant domain (CH1) for the heavy chain. The term "Fab fragment" therefore refers to an antibody fragment comprising a light chain containing the VL and CL domains, and a heavy chain containing the VH and CH1 domains. The difference between a "Fab' fragment" and a Fab fragment lies in the addition of residues at the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain carry a free thiol group. Pepsin treatment produces F(ab')2 fragments, which have two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0077] Antibody fragments can be prepared using various techniques, including but not limited to proteolytic digestion of intact antibodies and recombinant production via recombinant host cells (e.g., Escherichia coli, CHO).

[0078] In a preferred embodiment, the antibody provided herein is a Fab fragment. In one embodiment, the VH domain of the antibody provided herein comprises the human VH3 framework. In one embodiment, the VL domain of the antibody provided herein comprises the human Vκ1 framework. In one embodiment, the CL domain of the antibody provided herein has a κ isotype. In one embodiment, the CH1 domain of the antibody provided herein has a human IgG1 isotype.

[0079] In a preferred embodiment, the antibody provided herein is a Fab fragment comprising a CL domain having a κ isotype and a CH1 domain having a human IgG1 isotype.

[0080] In some respects, the antibodies described herein are multispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificity with respect to at least two distinct sites, namely, different epitopes on different antigens or different epitopes on the same antigen. In some respects, multispecific antibodies have three or more binding specificities.

[0081] Multispecific antibodies containing the antibodies provided herein, having three or more binding specificities, may be provided in an asymmetric form with domain exchanges in one or more binding arms that have the same antigen specificity, i.e., by exchanging the VH / VL domain (see, for example, WO 2009 / 080252 and WO 2015 / 150447), the CH1 / CL domain (see, for example, WO2009 / 080253), or the complete Fab arm (see, for example, WO 2009 / 080251, WO 2016 / 016299, also see Schaefer et al., PNAS, 108 (2011) 1187-1191 and Klein et al., MAbs 8 (2016) 1010-20). Various further molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

[0082] In some respects, amino acid sequence variants of the antibodies presented herein are considered. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions of residues within the amino acid sequence of the antibody, and / or insertions and / or substitutions within them. Any combination of deletions, insertions, and substitutions can be prepared to obtain the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0083] In some respects, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” below. More fundamental changes are provided under the heading “Exemplary Substitutions” below and are further described below as referring to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and desired activities can be screened in the product, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0084] Original exemplary preferred Residue substitution Ala (A) Val;Leu;Ile Val Arg (R) Lys;Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser;Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu (L) (Ile; Val; Met; Ala; Phe Ile) Lys (K) Arg;Gln;Asn Arg Met (M) Leu;Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val;Ser Ser Trp (W) Tyr;Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leucine Leu.

[0085] Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidity: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatics: Trp, Tyr, Phe.

[0086] Non-conservative substitution allows members of one of these categories to be exchanged for members of another category.

[0087] One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variants selected for further research are modified (e.g., improved) relative to the parent antibody in certain biological properties (e.g., increased affinity, decreased immunogenicity), and / or retain some biological properties substantially preserved by the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more CDR residues are mutated, and the variant antibody is displayed on a phage, and specific biological activities (e.g., binding affinity) are screened.

[0088] In some respects, substitutions, insertions, or deletions can occur within one or more CDRs, provided that such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity can be made in the CDR (e.g., conserved substitutions as described herein). Such changes can, for example, be located outside the antigen-contacting residues in the CDR. In some variant VH and VL sequences provided above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0089] A useful method for identifying target residues or regions in an antibody that can be used for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid locations that demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired properties.

[0090] Amino acid sequence insertions include N-terminal and / or C-terminal fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionine residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or a peptide that increases the serum half-life of the antibody.

[0091] Glycosylation variants In some respects, the antibodies presented herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites in antibodies can be conveniently achieved by altering the amino acid sequence, thereby creating or removing one or more glycosylation sites.

[0092] When an antibody contains an Fc region, the oligosaccharide to which it is attached can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are generally attached via an N-bond to the Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications to the oligosaccharides in the antibodies of the present invention can be prepared to produce antibody variants with certain improved properties.

[0093] In one aspect, antibody variants are provided that have non-fucosylated oligosaccharides, i.e., oligosaccharide structures lacking (directly or indirectly) fucose attached to the Fc region. Such non-fucosylated oligosaccharides (also referred to as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack the fucose residue of the first GlcNAc attached to the stem of the biantennary oligosaccharide structure. In another aspect, antibody variants are provided that have an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to natural or parental antibodies. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucose-containing oligosaccharides). For example, the percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucosylated residues relative to the sum of all oligosaccharides attached to Asn 297 (e.g., complex, mixed, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, as described in WO 2006 / 082515. Asn297 specifies an asparagine residue (EU number of Fc region residues) located near position 297 in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US2003 / 0157108; US 2004 / 0093621.

[0094] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells defective in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, particularly at Example 11), and knockout cell lines, such as α-1,6-fucosylation gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or absent activity of GDP-fucose synthesis or transport proteins (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

[0095] In a further aspect, antibody variants with dimeric oligosaccharides are provided, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are dimeric by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0096] Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO1999 / 22764.

[0097] Fc region variants In some respects, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0098] In some respects, the present invention considers antibody variants having some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity), but retains FcRn binding capacity. The primary cells used to mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox 96© non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402.To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929A1).

[0099] Antibodies with reduced effector function include those with substitutions of one or more residues from Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitutions at residues 265 and 297 to alanine (US Patent No. 7,332,581).

[0100] Certain antibody variants that exhibit improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604(2001).) In some respects, antibody variants contain one or more amino acid substitutions that improve ADCC, such as substituted Fc regions at positions 298, 333, and / or 334 of the Fc region (EU number of the residue).

[0101] In some aspects, the antibody variant includes one or more amino acid substitutions that reduce FcγR binding, for example, substituted Fc regions at positions 234 and 235 of the Fc region (EU number of the residues). In one aspect, the substitutions are L234A and L235A (LALA). In some aspects, the antibody variant further includes D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one aspect, the substitutions are L234A, L235A, and P329G in the Fc region derived from the human IgG1 Fc region (LALA-PG). (See, for example, WO 2012 / 130831). In another aspect, the substitutions are L234A, L235A, and D265A in the Fc region derived from the human IgG1 Fc region (LALA-DA).

[0102] In some respects, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0103] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934 (Hinton et al.), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies contain Fc regions with one or more substitutions that improve the binding of the Fc regions to FcRn. Such Fc variants include those with substitutions at one or more sites in the Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, the substitution of Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0104] Fc region residues key to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index number) are involved in the interaction (Medesan, C., et al. Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al. Int. Immunol. 13 (2001) 993; Kim, JK, et al. Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 have been found to be crucial for the interaction between human Fc and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined in Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671).

[0105] In some respects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 253, and / or 310, and / or 435 of the Fc region (EU number of the residue). In some respects, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one respect, the substitutions are I253A, H310A, and H435A derived from the Fc region of human IgG1. See, for example, Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0106] In some aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 310, and / or 433, and / or 436 of the Fc region (EU number of the residues). In some aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433, and 436. In one aspect, the substitutions are H310A, H433A, and Y436A derived from the Fc region of human IgG1. (See, for example, WO 2014 / 177460 A1).

[0107] In some aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, for example, substitutions at positions 252, and / or 254, and / or 256 of the Fc region (EU number of the residues). In some aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are M252Y, S254T, and T256E derived from the Fc region of human IgG1. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260; 5,624,821; and WO 94 / 29351, which relates to other examples of Fc region variants.

[0108] The C-terminus of the antibody heavy chain, as reported herein, can be a full C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain can also be a shortened C-terminus, wherein one or both of the C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect of all aspects reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbers of amino acid positions). In one aspect of all aspects reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, EU index number of amino acid position).

[0109] Cysteine-modified antibody variants In some respects, it may be desirable to generate cysteine-modified antibodies, such as THIOMAB™ antibodies, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain respects, the substituted residues appear at accessible sites on the antibody. By substituting these residues with cysteine, reactive thiol groups are thus positioned at accessible sites on the antibody and can be used to conjugate the antibody with other parts, such as pharmaceutical parts or linker-pharmaceutical parts, to produce immunoconjugates, as further described herein. Cysteine-modified antibodies can be generated as described, for example, in U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856.

[0110] Immunoconjugates The present invention also provides immunoconjugates comprising (chemically bonded) antibodies provided herein conjugated with one or more reagents; in one embodiment, said reagents are, for example, cytotoxic agents, chemotherapeutic agents, pharmaceuticals, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioisotopes.

[0111] In one embodiment, the present invention provides an immunoconjugation comprising an antibody conjugated herein with a polymer. The term “polymer” as used herein includes chemical polymers and protein polymers. In one embodiment, the immunoconjugation comprises an antibody conjugated herein with an extended recombinant polypeptide (XTEN). “Extended recombinant polypeptide” is known in the art and is disclosed, for example, in US20190083577. In one embodiment, the immunoconjugation comprises an XTEN that (a) comprises a sequence selected from GGSPAGSCTSP, GASASCAPSTG, TAEAAGCGTAEAA, and GPEPTCPAPSG, (b) has a length of 36 to 3000 L-amino acid residues, and / or (c) wherein the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) residues constitutes more than 90% of the total amino acid residues of the XTEN.

[0112] Recombination Method and Composition Antibodies can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0113] In one aspect, isolated nucleic acids encoding the antibodies of the present invention are provided. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody), or produced by recombinant methods or obtained by chemical synthesis.

[0114] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*.) After expression, the antibody can be separated from a soluble fraction in bacterial cell clumps and can be further purified. In one embodiment, the host cell is *E. coli* cells.

[0115] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth can be useful. Other examples of useful mammalian host cell lines include: monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293T cells, as described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); young hamster kidney cells (BHK); mouse Support cells (TM4 cells, as described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells (as described, for example, in Mather, JP et al., Annals NY Acad. Sci. 383 (1982)). (as described in 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR−CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0116] In one aspect, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In a preferred embodiment, the host cell is a CHO cell. The production of the antibody of the present invention in CHO cells can improve the injectability of the antibody.

[0117] anti-C3 antibody In some respects, anti-C3 antibodies, as described herein, are capable of specifically binding to epitopes of human complement factor C3 and / or proteolytic derivatives C3a and / or C3b. Therefore, anti-C3 antibodies are capable of specifically binding to epitopes in any region of a polypeptide sequence identified by GenBank accession number NP_000055.2, the entire contents of which are incorporated herein by reference.

[0118] The antibodies described herein contain one or more CDRs. In particular, CDRs can identify the specificity of the antibody, and accordingly preferably, the antigen binding site contains at least two, and more preferably three, CDRs.

[0119] Therefore, the antibodies provided herein are preferably derived from natural antibodies, such as camel antibodies. In some respects, the antibodies are selected from the following sequences or sequences having at least 75% identity with them (CDR sequences are underlined): C3S1: QVQLQESGGGLVQAGGSLRLSCAAS GNISAPSLM GWYRQAPGKEREFVA TITYGSITNY VDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA NTLGIYNTYGDHHY WGQGTQVTVSS (SEQ ID NO:1) C3S3: QVQLQESGGGLVQAGGSLRLSCAAS GTIFSRNYM GWYRQAPGKERELVA GIGYGSSTNY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA VPTYRPYYFY WGQGTQVTVSS (SEQ ID NO:2) C3S4: QVQLQESGGGLVQAGGSLRLSCAAS GTISGPGDMG WYRQAPGKERELVA GINYGAITYY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA DYETWAPSYEYFDY WGQGTQVTVSS (SEQ ID NO:3) C3S5: QVQLQESGGGLVQAGGSLRLSCAAS GTIFDDESMG WYRQAPGKEREFVA SINYGSTTYY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV GWSTNYDWTLSYYY WGQGTLVTVSS (SEQ ID NO:4) C3S6: QVQLQESGGGLVQAGGSLRLSCAAS GNIFVGHGMG WYRQAPGKEREFVA AIGYGAITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA SSYYPPRYWY WGQGTLVTVSS (SEQ ID NO:5) C3S7: QVQLQESGGGLVQAGGSLRLSCAAS GYIFTGVDMG WYRQAPGKEREFVA AISYGASTYY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA SLSNTYLLDLFFRY WGQGTQVTVSS (SEQ ID NO:6) C3S8: QVQLQESGGGLVQAGGSLRLSCAAS GTIFPTDRMG WYRQAPGKEREFVA TIDLGGTTNY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA AWEGPSRYGRHRYYSHRY WGQGTQVTVSS (SEQ ID NO:7) C3S9: QVQLQESGGGLVQAGGSLRLSCAAS GYIFVYSTMG WYRQAPGKEREFVA SINYGSITYY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV TDKSRYWLTY WGQGTQVTVSS (SEQ ID NO:8) C3S10: QVQLQESGGGLVQAGGSLRLSCAAS GYISGGSIMG WYRQAPGKEREFVA GINQGSNTYY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV GGRSYYYHHY WGQGTQVTVSS (SEQ ID NO:9) C3I3: DVQLVESGGGLVQPGGSLRLSCAAS GFTLDNYAIA WFRQAPGKEREGVS CISSSRGSTAY EDSVKGRFTIARDNAKNTVYLQMNSLKPEDTAVYYCAA RARGMCREFDYDY WGQGTQVTVSS (SEQ ID NO:10) C3I4: DVQLVESGGGLVQPGGSLRLSCATS GFTLDYYAIG WFRQAPGKEREGVS CITGPDGSTHY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA TAGLYGCGYGMDH WGKGTQVTVSS (SEQ ID NO:11) C3I8: DVQLVESGGGLVQPGGSLRLSCATS GFSFGVYDMS WVRQASGKGLEWVS GIDSGSNTAY ADSVKVRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAL GRETDYIPVTDF RTQGTQVTVSS (SEQ ID NO:12) C3I22: DVQLVESGGGLVQPGGSLRLSCTAS GFTFGVYAMS WVRQAPGKGLEWVS SIDSGVNTAY ADSVKGRFTISRDNAINTVFLQMNSLKPEDTAVYHCAL GRESDYEPVTGS WGQGTQVTVSS (SEQ ID NO:13) C3N10: DVQLVESGGGLVQAGGSLRLACAAS GLTFSPYAMG WFRQAPGKEREFVA GIRWISGNPYY ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA APLTRSYDY WGQGTQVTVSS (SEQ ID NO:14).

[0120] The CDRs of the above antibodies are listed in Table 1 below: Table 1 .

[0121] In some embodiments, the antibody comprises a CDR that differs from the CDRs shown in Table 1 by at least one, at least two, or at least three amino acids. In some embodiments, the antibody comprises a CDR1 having the sequence: G –F / L – T / S – F / L – S / G / D – P / V / Y / N – Y – A / D – M / I – G / S / A (where the first amino acid is G, the second amino acid is F or L, etc.), and / or comprises a CDR2 having the sequence: G / S / C – I – R / D / T / S / N –W / S / G / Q – I / G / P / S – S / V / D / R – G / N – N / T / S – P / A / T / Y – Y / H / A – Y / H, wherein the 11th amino acid is optional.

[0122] In the relevant implementation scheme, the antibody comprises CDR1, CDR2 and / or CDR3 selected from the CDR sequences shown in Table 1.

[0123] In some preferred embodiments, the antibody comprises a CDR1 selected from SEQ ID No: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, and 54, or comprises a CDR1 containing a sequence having at least 75% identity with any one of SEQ ID No: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, and 54; and / or comprises a CDR2 selected from SEQ ID No: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55, or comprises a CDR2 containing a sequence having at least 75% identity with any one of SEQ ID No: CDR2 comprising any one of SEQ ID Nos: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52 and 55 having at least 75% identity; and / or comprising CDR3 comprising any one of SEQ ID Nos: 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53 and 56, or comprising CDR1 comprising any one of SEQ ID Nos: 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53 and 56 having at least 75% identity. In one related embodiment, the antibody comprises CDR1, CDR2, and CDR3 selected from the following: SEQ ID No: 15-17, 18-20, 21-23, 24-26, 27-29, 30-32, 33-35, 36-38, 39-41, 42-44, 45-47, 48-50, 51-53, and 54-56, or comprises CDR1, CDR2, and CDR3 having at least 75% identity with SEQ ID No: 15-17, 18-20, 21-23, 24-26, 27-29, 30-32, 33-35, 36-38, 39-41, 42-44, 45-47, 48-50, 51-53, and 54-56.

[0124] In a particularly preferred embodiment, the antibody comprises CDR1, CDR2, and CDR3 having SEQ ID No: 54-56, respectively, or comprises CDRs having at least 75% (e.g., at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98%) identity with them.

[0125] The amino acid sequence of C3 in cynomolgus monkeys is as follows:

[0126] The amino acid sequence of human C3 is provided below:

[0127] In some respects, anti-C3 antibodies as described herein (e.g., C3N10.3) bind to an epitope within the C3α chain at the C345C domain. In some respects, anti-C3 antibodies as described herein bind to an epitope within amino acids 1540-1600 of human C3 or an amino acid sequence having at least 80% identity with it. In related respects, anti-C3 antibodies as described herein bind to an epitope containing amino acids 1548-1561 of the human C3 protein or an amino acid sequence having at least 80% identity with it, and / or bind to an epitope containing amino acids 1568-1573 of the human C3 protein or an amino acid sequence having at least 80% identity with it, and / or bind to an epitope containing amino acids 1583-1591 of the human C3 protein or an amino acid sequence having at least 80% identity with it.

[0128] In some aspects, anti-C3 antibodies as described herein (e.g., C3S3.2) bind to at least one non-near-neighbor epitope within the C3β chain at the MG1 and MG5 domains. In some aspects, anti-C3 antibodies as described herein bind to at least one non-near-neighbor epitope within amino acids 110-130 and 490-501 of human C3. In some aspects, anti-C3 antibodies as described herein bind to epitopes comprising amino acids 114-124 of human C3 or amino acid sequences having at least 80% identity with them, and / or bind to epitopes comprising amino acids 497-505 of human C3 or amino acid sequences having at least 80% identity with them.

[0129] In some preferred embodiments, the anti-C3 antibody comprises CDR1, CDR2 and / or CDR3 of any one of SEQ ID No: 2, 14, 98 and 135, or comprises CDR1, CDR2 and / or CDR3 having at least 70% identity with it (or comprising four or fewer amino acid substitutions relative to CDR1, CDR2, CDR3 having any one of SEQ ID No: 2, 14, 98 and 135).

[0130] anti-C5 antibody In some respects, anti-C5 antibodies (e.g., nanobodies) as described herein are capable of specifically binding to epitopes of human complement factor C5 and / or proteolytic derivatives C5a and / or C5b. Therefore, anti-C5 antibodies are capable of specifically binding to epitopes in any region of a polypeptide sequence identified by GenBank accession number NP_001304092.1, the entire contents of which are incorporated herein by reference.

[0131] The antibodies described herein contain one or more CDRs. In particular, CDRs can identify the specificity of the antibody, and accordingly preferably, the antigen binding site contains at least two, and more preferably three, CDRs.

[0132] Therefore, the antibodies provided herein are preferably derived from natural antibodies, such as camel antibodies. In some respects, the antibodies are selected from the following sequences or sequences having at least 75% identity with them (CDR sequences are underlined): C5S1: QVQLQESGGGLVQAGGSLRLSCAAS GYISYDDDMG WYRQAPGKEREFVA AIDVGGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV RDGYYWDHDY WGQGTQVTVSS (SEQ ID NO:57) C5S2: QVQLQESGGGLVQAGGSLRLSCAAS GNIFHQSDMG WYRQAPGKEREFVA AIDYGTNTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA RDTSYWDHVY WGQGTQVTVSS (SEQ ID NO:58) C5S3: QVQLQESGGGLVQAGGSLRLSCAAS GSISFDTDMG WYRQAPGKEREFVA TIDGGTSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA EAGYFSLLGPYFRY WGQGTQVTVSS (SEQ ID NO:59) C5S4: QVQLQESGGGLVQAGGSLRLSCAAS GYIFAATDMG WYRQAPGKEREFVA TIDYGANTN YADSVKGRFTIGRDNAKNTVYLQMNSLKPEDTAVYYCAA RDNNYYDLVY WGQGTQVTVSS (SEQ ID NO:60) C5S5: QVQLQESGGGLVQAGGSLRLSCAAS GTISPYEGMG WYRQAPGKERELVA AIDGGGITY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA GIWTLSYYDY WGQGTQVTVSS (SEQ ID NO:61) C5S6: QVQLQESGGGLVQAGGSLRLSCAAS GTIFYSYKMG WYRQAPGKEREFVA GITYGGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA RAYYVSVYDTALYY WGQGTQVTVSS (SEQ ID NO:62) C5S7: QVQLQESGGGLVQAGGSLRLSCAAS GYIFYDDDMG WYRQAPGKEREFVA TIDVGGNTN YADSVKGRFTIGRDNAKNTVYLQMNSLKPEDTAVYYCAV RDNNYYDHVY WGQGTQVTVSS (SEQ ID NO:63) C5S8: QVQLQESGGGLVQAGGSLRLSCAAS GNIFDEWDMG WYRQAPGKEREFVA AIDDGASTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA RQTYAWDYRY WGQGTQVTVSS (SEQ ID NO:64) C5S12: QVQLQESGGGLVQAGGSLRLSCAAS GYIFPSDDMG WYRQAPGKEREFVA AINDGASTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV QLNYYWDYVY WGQGTQVTVSS (SEQ ID NO:65).

[0133] The CDRs of the above antibodies are listed in Table 2 below: Table 2 .

[0134] In some embodiments, the antibody comprises a CDR that differs from the CDRs shown in Table 2 by at least one, at least two, or at least three amino acids. In some embodiments, the antibody comprises a CDR1 having the sequence: G – Y / N / S / T – I – S / F – Y / H / F / A / P / D – D / Q / A / Y / S / E – D / S / T / E / Y / W – D / K / G – M – G (where the first amino acid is G, the second amino acid is Y, N, S, or T, etc.), and / or comprises a CDR2 having the sequence: A / T / G – I – D / T / N – V / Y / G / D – G – G / T / A – S / N / I – T – Y / N. In relevant embodiments, the antibody comprises CDR1 having the following sequence: G – Y / N / S / T – I – F – Y / H / F / A / P / D – D / Q / A / Y / S / E – D / S / T / E / Y / W – D – M – G, and / or comprises CDR2 having the following sequence: – A / T – I – D – V / Y / G / D – G – A – S / N – T – Y.

[0135] In the relevant implementation scheme, the antibody comprises CDR1, CDR2 and / or CDR3 selected from the CDR sequences shown in Table 2.

[0136] In a preferred embodiment, the antibody comprises CDR1 selected from SEQ ID Nos: 66, 69, 72, 75, 78, 81, 84, 87, and 90, or comprises CDR1 containing a sequence having at least 75% identity with any one of SEQ ID Nos: 66, 69, 72, 75, 78, 81, 84, 87, and 90; and / or comprises CDR2 selected from SEQ ID Nos: 67, 70, 73, 76, 79, 82, 85, 88, and 91, or comprises CDR2 containing a sequence having at least 75% identity with any one of SEQ ID Nos: 67, 70, 73, 76, 79, 82, 85, 88, and 91; and / or comprises CDR3 selected from SEQ ID Nos: 68, 71, 74, 77, 80, 83, 86, 89, and 92, or comprises CDR3 containing a sequence having at least 75% identity with any one of SEQ ID Nos: 66, 69, 72, 75, 78, 81, 84, 87, and 90; and / or comprises CDR3 containing a sequence having at least 75% identity with any one of SEQ ID Nos: 66, 69, 72, 75, 78, 81, 84, 87, and 92. CDR1 having at least 75% identity with any one of SEQ ID Nos: 68, 71, 74, 77, 80, 83, 86, 89, and 92. In one related embodiment, the antibody comprises CDR1, CDR2, and CDR3 selected from the following: SEQ ID Nos: 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86, 87-89, and 90-92, or comprises CDR1, CDR2, and CDR3 having at least 75% identity with SEQ ID Nos: 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86, 87-89, and 90-92.

[0137] The amino acid sequence of C5 in cynomolgus monkeys is provided below:

[0138] The amino acid sequence of human C5 is provided below:

[0139] In some implementations, anti-C5 antibodies (e.g., C5S2.2) as described herein competitively inhibit the binding of eculizumab to human C5.

[0140] In some preferred embodiments, the anti-C5 antibody comprises CDR1, CDR2 and / or CDR3 of any of the anti-C5 antibodies in SEQ ID No: 58 and 141, or comprises CDR1, CDR2 and / or CDR3 having at least 70% identity with them (or comprising four or fewer amino acid substitutions relative to CDR1, CDR2, CDR3 having any of SEQ ID No: 58 and 141).

[0141] The antibodies provided herein also include their functional variants. The term "functional variant" is intended to include variants that retain some or substantially all of the ability of an antibody to selectively bind its antigen or ligand (such as any ligand mentioned below herein). A functional variant includes any variant that has at least 75% identity with the antibodies provided herein, such as those identified by SEQ ID NO: 1-14 and 57-65, having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, such as 90, 91, 92, 93, 94, 95, 96, such as 97, 98, 99, or at least 99.5% identity. In a preferred embodiment, the antibody comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with it.

[0142] Functional variants include any variant antibody containing one or more CDRs, said one or more CDRs having at least 75% identity with the CDRs of the antibodies provided herein, for example having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, such as 90, 91, 92, 93, 94, 95, 96, such as 97, such as 98, such as 99, such as at least 99.5% identity with said CDRs. The CDRs of the antibodies identified by SEQ ID NO: 1-14 and 57-65 are indicated above as SEQ ID No: 15-56 and 66-92, respectively. Therefore, an antibody comprising one or more regions is provided, said one or more regions having at least 75%, such as at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, such as 90, 91, 92, 93, 94, 95, 96, such as 97, 98, 99, such as 99, such as at least 99.5%, identity with one or more regions identified by any one of SEQ ID No: 15-56 and 66-92.

[0143] Amino acid substitution includes conserved amino acid substitution, which refers to the substitution of one amino acid by another amino acid residue having a side chain with similar properties. For example, a group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains are serine and threonine; a group of amino acids with amide-containing side chains are asparagine and glutamine; a group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains are lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. Preferred groups of conserved amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Within the meaning of the term "conserved amino acid substitution" as used herein, one amino acid may substitute for another amino acid within the group of amino acids indicated below: i) Amino acids with polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys) ii) Amino acids with nonpolar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met) iii) Amino acids with aliphatic side chains (Gly, Ala, Val, Leu, lie) iv) Amino acids with cyclic side chains (Phe, Tyr, Trp, His, Pro) v) Amino acids with aromatic side chains (Phe, Tyr, Trp) vi) Amino acids with acidic side chains (Asp, Glu) vii) Amino acids with basic side chains (Lys, Arg, His) viii) Amino acids with amide side chains (Asn, Gin) ix) Amino acids with hydroxyl side chains (Ser, Thr) x) Amino acids with sulfur-containing side chains (Cys, Met) xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr) xii) Hydrophilic and acidic amino acids (Gln, Asn, Glu, Asp), and xiii) Hydrophobic amino acids (Leu, Lie, Val).

[0144] In a preferred embodiment, the functional variant of the antibody can be a fragment of the antibody, preferably an antigen-binding fragment or a variable region. Examples of antibody fragments available for use in this invention include fragments of VHH and VNAR.

[0145] Antibodies can therefore be obtained through the immunity of any suitable organism, particularly camels, sharks, etc. However, antibodies can also be generated from synthetic libraries with randomized or designed CDRs. In one embodiment, antibodies are provided that specifically bind to epitopes of human complement factors C3, C3a, and / or C3b. In another embodiment, antibodies are provided that specifically bind to epitopes of human complement factors C5, C5a, and / or C5b.

[0146] In one embodiment, the antibody is conjugated to a histidine tag, such as a his6 tag, at the N-terminus or C-terminus of the polypeptide. In a preferred embodiment, the antibody is conjugated to a non-natural Strep or his6 tag at the C-terminus (e.g., WSHPQFEKHHHHHH (SEQ ID NO: 93)). In another embodiment, the antibody is conjugated to an Fc fragment at the N-terminus. N-terminal addition is particularly preferred, and in a preferred embodiment, the antibody provided herein includes an additional N-terminal region. Depending on the intended application of the antibody and the desired functionality of the final antibody product, the additional N-terminal region may be selected from any other relevant portion. Albumin may be added to increase cycle time and protect the product from degradation. Other antigen-binding fragments, antibodies, or fragments thereof may be added to introduce a second affinity / binding specificity into the antibody product.

[0147] In some embodiments, the anti-C3 antibody, as described herein, forms the first domain of the fusion protein. In some aspects, the fusion protein is a heterodimeric fusion protein comprising a first domain and a second domain, the first domain comprising a polypeptide containing the antibody as described herein, and the second domain comprising an immunoglobulin Fc domain. In some aspects, the first and second domains are linked by a linker. In a preferred embodiment, the fusion protein is a heterodimeric fusion protein comprising an amino acid sequence of any one of SEQ ID NO: 178-180 or an amino acid sequence having at least 70% identity with it. In other aspects, the fusion protein is a homodimeric fusion protein comprising a first domain and a second domain, the first domain comprising a polypeptide containing the antibody as described herein, and the second domain being identical to the first domain. In some aspects, the first and second domains are linked by a linker, such as a polyglycine-serine (G4S) linker, preferably a (G4S)2 linker. In a preferred embodiment, the fusion protein is a homodimeric fusion protein comprising the amino acid sequence of SEQ ID NO: 181 or 182 or an amino acid sequence having at least 70% identity with it.

[0148] In some embodiments, bispecific / multispecific antibodies are provided, for example, a single peptide chain comprising two antigen-binding regions, which can be separated by a linker sequence, wherein one of the two antigen-binding regions comprises a VHH domain as described herein. In some aspects, a VHH domain as described herein is coupled to another VHH domain to obtain a bispecific antibody comprising or including: (i) a VHH domain as described herein, and (ii) a VHH domain capable of specifically binding to an epitope of a second target.

[0149] In a preferred embodiment, the antibodies disclosed herein may include modifications that improve the antibody's function. For example, it is not always desirable to use non-human antibodies for human therapies, and accordingly, the antibodies provided herein may be humanized antibodies.

[0150] In some embodiments, the humanized antibody is a humanized form of the above-described llama nanobody and is selected from the following amino acid sequences or amino acid sequences having at least 75% identity with them (the amino acids in bold are the humanized amino acids that have been modified from the corresponding llama nanobody amino acids): C3S1.1: EVQLVESGGGLVQPGGSLRLSCAAS GNISAPSLM GWYRQAPGKEREFVA TITYGSITNY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA NTLGIYNTYGDHHY WGQGTLVTVSS (SEQ ID NO:94) C3S1.2: EVQLVESGGGLVQAGGSLRLSCAAS GNISAPSLM GWYRQAPGKEREFVA TITYGSITNY VDSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA NTLGIYNTYGDHHY WGQGTLVTVSS (SEQ ID NO:95) C3S1.3: QVQLVESGGGLVQAGGSLRLSCAAS GNISAPSLM GWYRQAPGKEREFVA TITYGSITNY VDSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA NTLGIYNTYGDHHY WGQGTLVTVSS (SEQ ID NO:96) C3S3.1: EVQLVESGGGLVQPGGSLRLSCAAS GTIFSRNYM GWYRQAPGKERELVA GIGYGSSTNY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA VPTYRPYYFY WGQGTLVTVSS (SEQ ID NO:97) C3S3.2: EVQLVESGGGLVQAGGSLRLSCAAS GTIFSRNYM GWYRQAPGKERELVA GIGYGSSTNY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA VPTYRPYYFY WGQGTLVTVSS (SEQ ID NO:98) C3S3.3: QVQLVESGGGLVQAGGSLRLSCAAS GTIFSRNYM GWYRQAPGKERELVA GIGYGSSTNY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA VPTYRPYYFY WGQGTLVTVSS (SEQ ID NO:99) C3S4.1: EVQLVESGGGLVQPGGSLRLSCAAS GTISGPGDMG WYRQAPGKERELVA GINYGAITYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DYETWAPSYEYFDY WGQGTLVTVSS (SEQ ID NO:100) C3S4.2: EVQLVESGGGLVQAGGSLRLSCAAS GTISGPGDMG WYRQAPGKERELVA GINYGAITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA DYETWAPSYEYFDY WGQGTLVTVSS (SEQ ID NO:101) C3S4.3: QVQLVESGGGLVQAGGSLRLSCAAS GTISGPGDMG WYRQAPGKERELVA GINYGAITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA DYETWAPSYEYFDY WGQGTLVTVSS (SEQ ID NO:102) C3S5.1: EVQLVESGGGLVQPGGSLRLSCAAS GTIFDDESMG WYRQAPGKEREFVA SINYGSTTYY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAV GWSTNYDWTLSYYY WGQGTLVTVSS (SEQ ID NO:103) C3S5.2: EVQLVESGGGLVQAGGSLRLSCAAS GTIFDDESMG WYRQAPGKEREFVA SINYGSTTYY ADSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCAV GWSTNYDWTLSYYY WGQGTLVTVSS (SEQ ID NO:104) C3S5.3: QVQLVESGGGLVQAGGSLRLSCAAS GTIFDDESMG WYRQAPGKEREFVA SINYGSTTYY ADSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCAV GWSTNYDWTLSYYY WGQGTLVTVSS (SEQ ID NO:105) C3S6.1: EVQLVESGGGLVQPGGSLRLSCAAS GNIFVGHGMG WYRQAPGKEREFVA AIGYGAITNY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA SSYYPPRYWY WGQGTLVTVSS (SEQ ID NO:106) C3S6.2: EVQLVESGGGLVQAGGSLRLSCAAS GNIFVGHGMG WYRQAPGKEREFVA AIGYGAITNY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA SSYYPPRYWY WGQGTLVTVSS (SEQ ID NO:107) C3S6.3: QVQLVESGGGLVQAGGSLRLSCAAS GNIFVGHGMG WYRQAPGKEREFVA AIGYGAITNY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA SSYYPPRYWY WGQGTLVTVSS (SEQ ID NO:108) C3S7.1: EVQLVESGGGLVQPGGSLRLSCAAS GYIFTGVDMG WYRQAPGKEREFVA AISYGASTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA SLSNTYLLDLFFRY WGQGTLVTVSS (SEQ ID NO:109) C3S7.2: EVQLVESGGGLVQAGGSLRLSCAAS GYIFTGVDMG WYRQAPGKEREFVA AISYGASTYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA SLSNTYLLDLFFRY WGQGTLVTVSS (SEQ ID NO:110) C3S7.3: QVQLVESGGGLVQAGGSLRLSCAAS GYIFTGVDMG WYRQAPGKEREFVA AISYGASTYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA SLSNTYLLDLFFRY WGQGTLVTVSS (SEQ ID NO:111) C3S8.1: EVQLVESGGGLVQPGGSLRLSCAAS GTIFPTDRMG WYRQAPGKEREFVA TIDLGGTTNY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA AWEGPSRYGRHRYYSHRY WGQGTLVTVSS (SEQ ID NO:112) C3S8.2: EVQLVESGGGLVQAGGSLRLSCAAS GTIFPTDRMG WYRQAPGKEREFVA TIDLGGTTNY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA AWEGPSRYGRHRYYSHRY WGQGTLVTVSS (SEQ ID NO:113) C3S8.3: QVQLVESGGGLVQAGGSLRLSCAAS GTIFPTDRMG WYRQAPGKEREFVA TIDLGGTTNY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA AWEGPSRYGRHRYYSHRY WGQGTLVTVSS (SEQ ID NO:114) C3S9.1: EVQLVESGGGLVQPGGSLRLSCAAS GYIFVYSTMG WYRQAPGKEREFVA SINYGSITYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAV TDKSRYWLTY WGQGTLVTVSS (SEQ ID NO:115) C3S9.2: EVQLVESGGGLVQAGGSLRLSCAAS GYIFVYSTMG WYRQAPGKEREFVA SINYGSITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAV TDKSRYWLTY WGQGTLVTVSS (SEQ ID NO:116) C3S9.3: QVQLVESGGGLVQAGGSLRLSCAAS GYIFVYSTMG WYRQAPGKEREFVA SINYGSITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAV TDKSRYWLTY WGQGTLVTVSS (SEQ ID NO:117) C3S10.1: EVQLVESGGGLVQPGGSLRLSCAAS GYISGGSIMG WYRQAPGKEREFVA GINQGSNTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAV GGRSYYYHHY WGQGTLVTVSS (SEQ ID NO:118) C3S10.2: EVQLVESGGGLVQAGGSLRLSCAAS GYISGGSIMG WYRQAPGKEREFVA GINQGSNTYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAV GGRSYYYHHY WGQGTLVTVSS (SEQ ID NO:119) C3S10.3: QVQLVESGGGLVQAGGSLRLSCAAS GYISGGSIMG WYRQAPGKEREFVA GINQGSNTYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAV GGRSYYYHHY WGQGTLVTVSS (SEQ ID NO:120) C3I3.1: EVQLLESGGGLVQPGGSLRLSCAAS GFTLDNYAIA WFRQAPGKEREGVS CISSSRGSTAY EDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA RARGMCREFDYDY WGQGTLVTVSS (SEQ ID NO:121) C3I3.2: EVQLLESGGGLVQPGGSLRLSCAAS GFTLDNYAIA WFRQAPGKEREGVS CISSSRGSTAY EDSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA RARGMCREFDYDY WGQGTLVTVSS (SEQ ID NO:122) C3I3.3: EVQLLESGGGLVQPGGSLRLSCAAS GFTLDNYAIA WFRQAPGKEREGVS CISSSRGSTAY EDSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAA RARGMCREFDYDY WGQGTLVTVSS (SEQ ID NO:123) C3I4.1: EVQLLESGGGLVQPGGSLRLSCATS GFTLDYYAIG WFRQAPGKEREGVS CITGPDGSTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA TAGLYGCGYGMDH WGKGTLVTVSS (SEQ ID NO:124) C3I4.2: EVQLLESGGGLVQPGGSLRLSCATS GFTLDYYAIG WFRQAPGKEREGVS CITGPDGSTHY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA TAGLYGCGYGMDH WGKGTLVTVSS (SEQ ID NO:125) C3I4.3: EVQLLESGGGLVQPGGSLRLSCATS GFTLDYYAIG WFRQAPGKEREGVS CITGPDGSTHY ADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAA TAGLYGCGYGMDH WGKGTLVTVSS (SEQ ID NO:126) C3I8.1: EVQLLESGGGLVQPGGSLRLSCATS GFSFGVYDMS WVRQASGKGLEWVS GIDSGSNTAY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAL GRETDYIPVTDF RTQGTLVTVSS (SEQ ID NO:127) C3I8.2: EVQLLESGGGLVQPGGSLRLSCATS GFSFGVYDMS WVRQASGKGLEWVS GIDSGSNTAY ADSVKGRFTISRDNSKNTLYLQMNNLRPEDTAVYYCAL GRETDYIPVTDF RTQGTLVTVSS (SEQ ID NO:128) C3I8.3: EVQLLESGGGLVQPGGSLRLSCATS GFSFGVYDMS WVRQASGKGLEWVS GIDSGSNTAY ADSVKVRFTISRDNSKNTLYLQMNNLKPEDTAVYYCAL GRETDYIPVTDF RTQGTLVTVSS (SEQ ID NO:129) C3I22.1: EVQLLESGGGLVQPGGSLRLSCTAS GFTFGVYAMS WVRQAPGKGLEWVS SIDSGVNTAY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCAL GRESDYEPVTGS WGQGTLVTVSS (SEQ ID NO:130) C3I22.2: EVQLLESGGGLVQPGGSLRLSCTAS GFTFGVYAMS WVRQAPGKGLEWVS SIDSGVNTAY ADSVKGRFTISRDNSINTLYLQMNSLRPEDTAVYHCAL GRESDYEPVTGS WGQGTLVTVSS (SEQ ID NO:131) C3I22.3: EVQLLESGGGLVQPGGSLRLSCTAS GFTFGVYAMS WVRQAPGKGLEWVS SIDSGVNTAY ADSVKGRFTISRDNSINTLFLQMNSLKPEDTAVYHCAL GRESDYEPVTGS WGQGTLVTVSS (SEQ ID NO:132) C3N10.1: QVQLVESGGGLVQPGGSLRLACAAS GLTFSPYAMG WFRQAPGKEREFVA GIRWISGNPY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCAA APLTRSYDY WGQGTQVTVSS (SEQ ID NO:133) C3N10.2: EVQLVESGGGLVQPGGSLRLACAAS GLTFSPYAMG WFRQAPGKEREFVA GIRWISGNPY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCAA APLTRSYDY WGQGTLVTVSS (SEQ ID NO:134) C3N10.3: EVQLVESGGGLVQAGGSLRLACAAS GLTFSPYAMG WFRQAPGKEREFVA GIRWISGNPY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYSCAA APLTRSYDY WGQGTLVTVSS (SEQ ID NO:135) C3N10.4: EVQLVESGGGLVQAGGSLRLACAAS GLTFSPYAMG WFRQAPGKEREFVA GIRWISGNPY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYSCAA APLTRSYDY WGQGTLVTVSS (SEQ ID NO:136) C5S1.1: EVQLVESGGGLVQPGGSLRLSCAAS GYISYDDDMG WYRQAPGKEREFVA AIDVGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAV RDGYYWDHDY WGQGTLVTVSS (SEQ ID NO:137) C5S1.2: EVQLVESGGGLVQAGGSLRLSCAAS GYISYDDDMG WYRQAPGKEREFVA AIDVGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAV RDGYYWDHDY WGQGTLVTVSS (SEQ ID NO:138) C5S1.3: QVQLVESGGGLVQAGGSLRLSCAAS GYISYDDDMG WYRQAPGKEREFVA AIDVGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAV RDGYYWDHDY WGQGTLVTVSS (SEQ ID NO:139) C5S2.1: EVQLVESGGGLVQPGGSLRLSCAAS GNIFHQSDMG WYRQAPGKEREFVA AIDYGTNTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA RDTSYWDHVY WGQGTLVTVSS (SEQ ID NO:140) C5S2.2: EVQLVESGGGLVQAGGSLRLSCAAS GNIFHQSDMG WYRQAPGKEREFVA AIDYGTNTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA RDTSYWDHVY WGQGTLVTVSS (SEQ ID NO:141) C5S2.3: QVQLVESGGGLVQAGGSLRLSCAAS GNIFHQSDMG WYRQAPGKEREFVA AIDYGTNTY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAA RDTSYWDHVY WGQGTLVTVSS (SEQ ID NO:142) C5S3.1: EVQLVESGGGLVQPGGSLRLSCAAS GSISFDTDMG WYRQAPGKEREFVA TIDGGTSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA EAGYFSLLGPYFRY WGQGTLVTVSS (SEQ ID NO:143) C5S3.2: EVQLVESGGGLVQAGGSLRLSCAAS GSISFDTDMG WYRQAPGKEREFVA TIDGGTSTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA EAGYFSLLGPYFRY WGQGTLVTVSS (SEQ ID NO:144) C5S3.3: QVQLVESGGGLVQAGGSLRLSCAAS GSISFDTDMG WYRQAPGKEREFVA TIDGGTSTY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAA EAGYFSLLGPYFRY WGQGTLVTVSS (SEQ ID NO:145) C5S4.1: EVQLVESGGGLVQPGGSLRLSCAAS GYIFAATDMG WYRQAPGKEREFVA TIDYGANTN YADSVKGRFTIGRDNSKNTLYLQMNSLRAEDTAVYYCAA RDNNYYDLVY WGQGTLVTVSS (SEQ ID NO:146) C5S4.2: EVQLVESGGGLVQAGGSLRLSCAAS GYIFAATDMG WYRQAPGKEREFVA TIDYGANTN YADSVKGRFTIGRDNSKNTLYLQMNSLRPEDTAVYYCAA RDNNYYDLVY WGQGTLVTVSS (SEQ ID NO:147) C5S4.3: QVQLVESGGGLVQAGGSLRLSCAAS GYIFAATDMG WYRQAPGKEREFVA TIDYGANTN YADSVKGRFTIGRDNSKNTLYLQMNSLKPEDTAVYYCAA RDNNYYDLVY WGQGTLVTVSS (SEQ ID NO:148) C5S5.1: EVQLVESGGGLVQPGGSLRLSCAAS GTISPYEGMG WYRQAPGKERELVA AIDGGGITY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA GIWTLSYYDY WGQGTLVTVSS (SEQ ID NO:149) C5S5.2: EVQLVESGGGLVQAGGSLRLSCAAS GTISPYEGMG WYRQAPGKERELVA AIDGGGITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA GIWTLSYYDY WGQGTLVTVSS (SEQ ID NO:150) C5S5.3: QVQLVESGGGLVQAGGSLRLSCAAS GTISPYEGMG WYRQAPGKERELVA AIDGGGITY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAA GIWTLSYYDY WGQGTLVTVSS (SEQ ID NO:151) C5S6.1: EVQLVESGGGLVQPGGSLRLSCAAS GTIFYSYKMG WYRQAPGKEREFVA GITYGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA RAYYVSVYDTALYY WGQGTLVTVSS (SEQ ID NO:152) C5S6.2: EVQLVESGGGLVQAGGSLRLSCAAS GTIFYSYKMG WYRQAPGKEREFVA GITYGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA RAYYVSVYDTALYY WGQGTLVTVSS (SEQ ID NO:153) C5S6.3: QVQLVESGGGLVQAGGSLRLSCAAS GTIFYSYKMG WYRQAPGKEREFVA GITYGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAA RAYYVSVYDTALYY WGQGTLVTVSS (SEQ ID NO:154) C5S7.1: EVQLVESGGGLVQPGGSLRLSCAAS GYIFYDDDMG WYRQAPGKEREFVA TIDVGGNTN YADSVKGRFTIGRDNSKNTLYLQMNSLRAEDTAVYYCAV RDNNYYDHVY WGQGTLVTVSS (SEQ ID NO:155) C5S7.2: EVQLVESGGGLVQAGGSLRLSCAAS GYIFYDDDMG WYRQAPGKEREFVA TIDVGGNTN YADSVKGRFTIGRDNSKNTLYLQMNSLRPEDTAVYYCAV RDNNYYDHVYWGQGTLVTVSS (SEQ ID NO:156) C5S7.3: QVQLVESGGGLVQAGGSLRLSCAAS GYIFYDDDMG WYRQAPGKEREFVA TIDVGGNTN YADSVKGRFTIGRDNSKNTLYLQMNSLKPEDTAVYYCAV RDNNYYDHVY WGQGTLVTVSS (SEQ ID NO:157) C5S8.1: EVQLVESGGGLVQPGGSLRLSCAAS GNIFDEWDMG WYRQAPGKEREFVA AIDDGASTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA RQTYAWDYRY WGQGTLVTVSS (SEQ ID NO:158) C5S8.2: EVQLVESGGGLVQAGGSLRLSCAAS GNIFDEWDMG WYRQAPGKEREFVA AIDDGASTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA RQTYAWDYRY WGQGTLVTVSS (SEQ ID NO:159) C5S8.3: QVQLVESGGGLVQAGGSLRLSCAAS GNIFDEWDMG WYRQAPGKEREFVA AIDDGASTY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAA RQTYAWDYRY WGQGTLVTVSS (SEQ ID NO:160) C5S12.1: EVQLVESGGGLVQPGGSLRLSCAAS GYIFPSDDMG WYRQAPGKEREFVA AINDGASTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAV QLNYYWDYVY WGQGTLVTVSS (SEQ ID NO:161) C5S12.2: EVQLVESGGGLVQAGGSLRLSCAAS GYIFPSDDMG WYRQAPGKEREFVA AINDGASTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAV QLNYYWDYVY WGQGTLVTVSS (SEQ ID NO:162) C5S12.3: QVQLVESGGGLVQAGGSLRLSCAAS GYIFPSDDMG WYRQAPGKEREFVA AINDGASTY YADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAV QLNYYWDYVY WGQGTLVTVSS (SEQ ID NO:163).

[0151] In some preferred embodiments, the antibody comprises a sequence of any one of SEQ ID No: 94-163, or comprises an amino acid sequence having at least 75%, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 99.5% identity with it, and preferably comprises crude amino acids.

[0152] In addition to the antibodies provided herein, nucleic acids comprising nucleotide sequences encoding antibodies as described herein are also provided. In relevant embodiments, the nucleic acid encoding an antibody as described herein constitutes part of an expression vector, wherein the nucleotide sequence encoding the antibody is operatively linked to a suitable promoter.

[0153] In one implementation, the RNA and / or cDNA coding sequences are designed for optimal expression in human cells. Codon-optimized coding regions can be designed using a variety of different methods. This optimization can be performed using methods available online, publicly available methods, or by companies that provide codon optimization services. One codon optimization method is described, for example, in WO2015 / 012924 A2, which is incorporated herein by reference. Briefly, the nucleic acid sequence encoding the product is modified with synonymous codon sequences. Appropriately, the entire length of the open reading frame (ORF) of the product is modified. However, in some implementations, only a fragment of the ORF can be modified. By using one of these methods, frequencies can be applied to any given polypeptide sequence, and nucleic acid fragments encoding codon-optimized coding regions of the polypeptide are produced.

[0154] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a nucleic acid comprising a nucleotide sequence encoding an antibody as described herein, said nucleotide sequence being operatively linked to a promoter. In other embodiments, a pharmaceutical composition is provided comprising an antibody as described herein and a pharmaceutically acceptable carrier.

[0155] Recombinant AAV viral particles encoding anti-C3 and / or anti-C5 antibodies The novel rAAV viral particles described herein comprise (i) a capsid as described herein, and (ii) a heterologous nucleic acid encoding an anti-C3 antibody and / or an anti-C5 antibody as described herein, preferably wherein the nucleotide sequence encoding the anti-C3 antibody and / or the anti-C5 antibody is operatively linked to an expression control sequence. Typically, the heterologous nucleic acid comprises an AAV genome, wherein the rep and cap genes are deleted and / or replaced with antibody sequences and their associated expression control sequences. The antibody sequence is typically inserted adjacent to one or both (i.e., flanked by) AAV TR or TR elements sufficient for viral replication (Xiao et al., 1997, J. Virol. 71(2): 941-948), in place of the nucleic acid encoding the viral rep and cap proteins. Additional regulatory sequences suitable for promoting tissue-specific expression of the antibody gene sequence in target cells (e.g., retinal cells) may also be included.

[0156] In some respects, anti-C3 antibodies encoded by the heteronucleotides of rAAV can prevent C3 from binding to C3 convertases in both the alternative and classical pathways, which in turn inhibit C3 cleavage.

[0157] heterologous nucleic acid components of rAAV In some respects, the rAAV viral particle contains a heterologous nucleic acid comprising (a) an AAV2 terminal repeat, (b) a transcriptional regulatory sequence, (c) a nucleotide sequence encoding an anti-C3 antibody as described herein, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat.

[0158] In other respects, the rAAV viral particle contains a heterologous nucleic acid comprising (a) an AAV2 terminal repeat, (b) a transcriptional regulatory sequence, (c) a nucleotide sequence encoding an anti-C5 antibody as described herein, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat.

[0159] The preferred inverted terminal repeat (ITR) for use in rAAV viral particles is an AAV sequence, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. The ITR can be a synthetic sequence that serves as an AAV inverted terminal repeat, such as the “double-D sequence” described in U.S. Patent No. 5,478,745 to Samulski et al., the entire contents of which are incorporated herein by reference. Typically, but not necessarily, the TRs originate from the same parvovirus; for example, both ITR sequences may be derived from AAV2.

[0160] In some respects, the heterologous nucleic acid encapsulated by rAAV viral particles contains a 5' ITR with the following sequence: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 164).

[0161] In related aspects, the heterologous nucleic acid encapsulated by rAAV viral particles contains a 3' ITR with the following sequence: AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO: 165).

[0162] The antibody gene encoded by rAAV is preferably operatively linked to at least one transcriptional control sequence, preferably a transcriptional control sequence heterologous to the nucleic acid. In some aspects, the transcriptional control sequence comprises a cell- or tissue-specific promoter that results in cell-specific expression of the nucleic acid, such as in photoreceptor cells, for example, the human rod photoreceptor-specific human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter or the human photoreceptor inter-retinol-binding protein (IRBP) promoter. In other aspects, the transcriptional control sequence comprises a constitutive promoter that results in similar expression levels of the nucleic acid in multiple cell types. Suitable constitutive promoters include the CAG promoter, which comprises (C) cytomegalovirus (CMV) immediate early enhancer element, (A) chicken β-actin The first exon and first intron of the gene, and (G) rabbit β-globinGene splice acceptors (see Miyazaki et al. (1989)) Gene 79(2): 269-277), Cytomegalovirus promoter (CMV) (Stinski et al., (1985) Journal of Virology 55(2): 431-441), Human extension factor 1α promoter (EF1α) (Kim et al. (1990) Gene 91(2): 217-223), Human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984)) Gene 32(3): 409-417), Mitochondrial heavy chain promoter (Loderio et al. (2012) PNAS 109(17): 6513-6518), and ubiquitous promoters (Wulff et al. (1990)). FEBS Letters 261: 101-105).

[0163] In a preferred aspect, the antibody gene encoded by rAAV is operatively linked to the CAG promoter. In a particularly preferred embodiment, the CAG promoter comprises the sequence of SEQ ID NO:166, or comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0164] In some respects, the heterologous nucleic acid encapsulated by rAAV viral particles contains SV40 polyadenylated sequences of the following: GGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCA (SEQ ID NO:167).

[0165] The capsid components of rAAV The rAAV variant AAV capsid—encapsulating a heterologous nucleic acid encoding an antibody or a functional variant thereof—contains a variant AAV capsid protein containing an insert of about 7 to about 20 amino acids (“heteropeptide” or “peptide insert”) within the GH ring of the parental AAV capsid protein, wherein said peptide contains the amino acid sequence ISDQTKH (SEQ ID NO: 168). Preferably, when present in AAV viral particles, the variant capsid protein confers increased infectivity to retinal cells compared to infectivity via AAV viral particles containing the corresponding parental capsid protein.

[0166] The “GH ring” or ring IV of an AAV capsid protein refers to the solvent-accessible portion, and is referred to in the art as the GH ring or ring IV of the AAV capsid protein. For more information on the GH ring / ring IV of the AAV capsid, see, for example, van Vliet et al. (2006). Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol.Ther. 15:1955. Therefore, for example, the insertion site could be located within approximately amino acid 570-611 of AAV2 VP1.

[0167] In some embodiments, the peptide insert has 1 to 3 spacer amino acids (Y1-Y3) at the N-terminus and / or C-terminus of the amino acid sequence ISDQTKH (SEQ ID NO: 168). Exemplary spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In some embodiments, the peptide insert comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insert comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus. In a preferred embodiment, the peptide insert comprises or is composed of the amino acid sequence LAISDQTKHA (SEQ ID NO: 169).

[0168] In some aspects, the variant AAV capsid protein includes a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO: 168), and further comprises one or more amino acid substitutions relative to the corresponding parental AAV capsid protein. Representative examples of amino acid substitutions can be found, for example, in column 26, lines 40-65 of U.S. Patent No. 11,576,983, the entire contents of which are incorporated herein by reference.

[0169] In some preferred embodiments, the variant AAV capsid protein includes a peptide insert containing the amino acid sequence ISDQTKH (SEQ ID NO:168), and further includes a P34A amino acid substitution relative to the VP1 capsid of AAV2, or a corresponding substitution in another AAV serotype.

[0170] In other respects, the variant capsid protein may include one or more features disclosed in U.S. Patent No. 11,576,983, particularly one or more features disclosed in columns 26, lines 66 through 29, and line 50 of U.S. Patent No. 11,576,983.

[0171] In a particularly preferred embodiment, the variant capsid protein comprises the following amino acid sequence, or comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPPKAAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:170).

[0172] The variant AAV capsid protein of SEQ ID NO:170 contains the following modifications relative to the native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34, the position being located inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids at amino acid position 588 (leucine-alanine-isoleucine-serine-aspartic acid-glutamine-threonine-lysine-histidine-alanine / LAISDQTKHA (SEQ ID NO:169)), the position being present in VP1, VP2, and VP3. In some embodiments, the capsid comprises a variant capsid protein containing a sequence having at least 90%, at least 95%, at least 98%, and at least 99% identity with SEQ ID NO:170, and containing a P34A substitution at amino acid position 588 and a LAISDQTKHA (SEQ ID NO:169) peptide insertion fragment.

[0173] This document also provides packaging cells, which are encompassed by “host cells”, which can be cultured to produce the packaging viral vectors of the present invention. The packaging cells of the present invention generally comprise cells having heterologous (1) viral vector function, (2) packaging function, and (3) helper function. These component functions are each discussed in subsequent sections.

[0174] Initially, the vector can be prepared by several methods known to those skilled in the art (see, for example, WO 2013 / 063379). A preferred method is described in Grieger, et al. 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. In short, efficient transfection of HEK293 cells serves as a starting point, in which adherent HEK293 cell lines from qualified clinical master cell banks are used for growth in shake flasks and WAVE bioreactors under suspension conditions without animal components, allowing for rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), at harvest time 48 hours post-transfection, suspension HEK293 cell lines yielded greater than 10 5 One particle (vg) containing the vector genome per cell or more than 10 14 Cell cultures in vg / L. More specifically, triple transfection refers to the fact that packaging cells are transfected with three plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various auxiliary functions (e.g., adenovirus or HSV proteins, such as E1a, E1b, E2a, E4, and VA RNA), and a third plasmid encodes the transgene and its various control elements (e.g., modified GLA genes and CAG promoters).

[0175] To achieve the desired yield, multiple variables were optimized, such as selecting a serum-free suspension medium compatible with both growth and transfection, and choosing transfection reagents, transfection conditions, and cell density. A universal purification strategy based on ion-exchange chromatography was also developed, resulting in high-purity carrier formulations of AAV serotypes 1–6, 8, and 9, as well as various chimeric capsids. This user-friendly process can be completed within one week, resulting in a high complete particle / empty particle ratio (>90% complete particles), providing purified yields (>1 x 10^13 vg / L) and purity suitable for clinical applications, and is universal for all serotypes and chimeric particles. This scalable manufacturing technology has been used to manufacture GMP Phase I clinical AAV carriers for retinal neovascularization (AAV2), hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered to patients. In addition, by implementing the perfusion method, a minimum 5-fold increase is achieved in overall vector production, which allows for the harvesting of rAAV from the culture medium at multiple time points after transfection.

[0176] Packaging cells include viral vector functions, along with packaging and vector functions. Viral vector functions typically include a portion of a parvovirus genome, such as an AAV genome, where the rep and cap are deleted and replaced with modified GLA sequences and their associated expression control sequences. Viral vector functions include sufficient expression control sequences to result in replication of the viral vector used for packaging. Typically, viral vectors include a portion of a parvovirus genome, such as an AAV genome, where the rep and cap are deleted and replaced with transgenes and their associated expression control sequences. Transgenes are typically flanked by two AAV TRs, replacing the deleted viral rep and cap ORFs. Appropriate expression control sequences are included, such as tissue-specific promoters and other regulatory sequences suitable for promoting tissue-specific expression of the transgene in target cells. Transgenes are typically nucleic acid sequences that can be expressed to produce therapeutic peptides or marker peptides.

[0177] The terminal repeats (TRs) (distinguished and indistinguishable) used in the viral vector are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. Distinguished AAV TRs do not need to have a wild-type TR sequence (e.g., wild-type sequences can be altered by insertion, deletion, truncation, or missense mutations), as long as the TR mediates the desired function, such as viral packaging, integration, and / or provirus rescue. TRs can be synthetic sequences that act as inverted terminal repeats of AAVs, such as the “double-D sequence” described in U.S. Patent No. 5,478,745 to Samulski et al., the entire contents of which are incorporated herein by reference. Typically, but not necessarily, TRs originate from the same parvovirus; for example, both TR sequences may be from AAV2.

[0178] The packaging features include the variant coating components described above.

[0179] The packaged viral vector, comprising an anti-C3 antibody transgene and an expression control sequence flanked by TR elements, referred herein as a “transgene” or “transgene expression cassette,” is sufficient to result in the packaging of the vector DNA and subsequent expression of the gene sequence in transduced cells. The viral vector function can be provided to the cell, for example, as a component of a plasmid or amplicon. The viral vector function can exist extrachromosomally within the cell line and / or can integrate into the cell’s chromosomal DNA.

[0180] Any method can be used to introduce a nucleotide sequence carrying viral vector function into the cell host for replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments where viral vector function is provided by transfection with a viral vector, standard methods for producing viral infection can be used.

[0181] Packaging functions include genes for viral vector replication and packaging. Therefore, for example, packaging functions may include, as needed, functions required for viral gene expression, viral vector replication, rescue of the viral vector from its integrated state, viral gene expression, and packaging of the viral vector into viral particles. Packaging functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicones, baculoviruses, or HSV helper constructs. Packaging functions may exist extrachromosomally within packaging cells, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.

[0182] Accessory functions include helper viral elements required to establish active infection of packaging cells, which are essential for initiating viral vector packaging. Examples include functions derived from adenoviruses, baculoviruses, and / or herpesviruses sufficient to cause viral vector packaging. For example, adenoviral accessory functions typically include adenoviral components E1a, E1b, E2a, E4, and VA RNA. Packaging functions can be provided by infecting packaging cells with the desired virus. Packaging functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicon. See, for example, the pXR helper plasmid described in Rabinowitz et al., 2002, J. Virol. 76:791, and the pDG plasmid described in Grimm et al., 1998, Human Gene Therapy 9:2745-2760. Packaging functions can exist extrachromosomally within packaging cells, but are preferably integrated into the cell's chromosomal DNA (e.g., E1 or E3 in HEK 293 cells).

[0183] Any suitable helper virus function can be employed. For example, baculoviruses can act as helper viruses when the packaging cells are insect cells. Herpesviruses can also be used as helper viruses in AAV packaging methods. Hybridized herpesviruses encoding the AAV Rep protein can advantageously facilitate more scalable AAV vector production protocols.

[0184] Any method can be used to introduce a nucleotide sequence carrying an auxiliary function into the cell host for replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments where the auxiliary function is provided by transfection with a viral vector or infection with a helper virus, standard methods for producing viral infections can be used.

[0185] Any suitable permissive or packaging cells known in the art can be used for the production of packaged viral vectors. Mammalian or insect cells are preferred. Examples of cells that can be used for the production of packaging cells in the practice of this invention include, for example, human cell lines such as VERO, WI38, MRC5, A549, HEK 293 cells (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines. In one aspect, the packaging cells are capable of growth in suspension culture, more preferably, the cells are capable of growth in serum-free medium. In one embodiment, the packaging cells are HEK293 cells grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells described in U.S. Patent No. 9,441,206 and deposited as ATCC number PTA 13274. Numerous rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO 2002 / 46359. In another respect, packaging cells are cultured in the form of cell stacks (e.g., a 10-layer cell stack seeded with HEK293 cells).

[0186] Cell lines used as packaging cells include insect cell lines. Any insect cell line that allows AAV replication and can be maintained in culture can be used according to the present invention. Examples include fall armyworm (Spodoptera frugiperda) cell lines such as Sf9 or Sf21, Drosophila species cell lines, or mosquito cell lines such as those derived from Aedes albopictus. A preferred cell line is the fall armyworm Sf9 cell line. The following references, concerning their teachings on the use of insect cells for the expression of heterologous peptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture, are incorporated into this article: Methods in Molecular Biology, edited by Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., 1989, J. Virol. 63:3822-3828; Kajigaya et al., 1991, Proc. Nat'l. Acad. Sci. USA 88: 4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer et al., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; and Samulski et al., U.S. Patent No. 6,204,059.

[0187] The viral capsid according to the invention can be produced using any method known in the art, for example, by expression from baculoviruses (Brown et al., (1994) Virology 198:477-488). As a further alternative, the viral vector of the invention can be produced in insect cells using baculovirus vectors to deliver the rep / cap gene and rAAV template, as described, for example, by Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

[0188] In another aspect, the present invention provides a method for rAAV production in insect cells, wherein a baculovirus packaging system or vector carrying AAV Rep and Cap coding regions can be constructed by: modifying these genes into the polyhedral protein coding region of the baculovirus vector; and generating a viral recombinant by transfection into a host cell. Notably, when using baculoviruses for AAV production, the AAV DNA vector product is preferably a self-complementary AAV-like molecule without the need for mutations regarding the AAV ITR. This appears to be a byproduct of ineffective AAV Rep cleavage in insect cells, resulting in a self-complementary DNA molecule due to the lack of functional Rep enzyme activity. The host cell is a baculovirus-infected cell, or a cell in which additional nucleic acids encoding baculovirus helper functions have been introduced, or in which these baculovirus helper functions are included. These baculoviruses can express AAV components and subsequently promote capsid production.

[0189] During production, packaging cells typically include one or more viral vector functions, along with auxiliary and packaging functions sufficient to lead to viral vector replication and packaging. These various functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicones, and they can exist extrachromosomally within the cell line or be integrated into the cell's chromosome.

[0190] Cells can provide any one or more of the aforementioned functions that have been incorporated, such as cell lines with one or more carrier functions that are incorporated into or integrated into the chromosomal DNA of the cell, cell lines with one or more packaging functions that are incorporated into or integrated into the chromosomal DNA of the cell, or cell lines with auxiliary functions that are incorporated into or integrated into the chromosomal DNA of the cell.

[0191] The rAAV vector can be purified using methods standard in the art, such as column chromatography or a cesium chloride gradient. Methods for purifying the rAAV vector are known in the art and include those described in Clark et al., 1999, Human Gene Therapy 10(6):1031-1039; Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Patent Nos. 6,566,118 and WO 98 / 09657.

[0192] Methods for delivering nucleic acids encoding anti-C3 antibodies to the retina In several embodiments, methods are provided for delivering heterologous nucleotide sequences encoding anti-C3 and / or anti-C5 antibodies to the retina using rAAV as described herein. rAAV can be used to deliver nucleotide sequences encoding antibodies to retinal cells in vitro, for example, to produce antibody peptides or nucleic acids in vitro for ex vivo gene therapy. rAAV can also be used in methods for delivering nucleotide sequences to subjects in need, for example, to express antibodies in subjects in need, such as those with dry AMD or geographic atrophy. In this way, antibodies can thus be generated in the subject to restore complement regulation.

[0193] Therefore, in one aspect, a method is provided for delivering nucleic acids encoding anti-C3 antibodies and / or anti-C5 antibodies or antigen-binding fragments thereof to retinal cells, the method comprising contacting the retinal cells with rAAV viral particles as described herein.

[0194] In another aspect, a method is provided for delivering nucleic acids encoding anti-C3 antibodies and / or anti-C5 antibodies or antigen-binding variants thereof to retinal cells in a mammalian subject, the method comprising administering an effective amount of rAAV viral particles as described herein or a pharmaceutical preparation containing such particles to the mammalian subject.

[0195] rAAV can be administered to the subject's retina via any suitable route. In a preferred embodiment, rAAV is preferably administered intraocularly via subretinal, suprachoroidal, and / or intravitreal injection. In some particularly preferred embodiments, rAAV is administered via intravitreal injection, more preferably via a single intravitreal injection.

[0196] Treatment In some embodiments, a method is provided for treating dry AMD in a subject requiring such treatment, the method comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (i) a variant AAV capsid protein comprising a heterologous peptide insert of 7 to 20 amino acids covalently inserted into the GH ring of the AAV capsid protein, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO: 105), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding an anti-C3 antibody and / or an anti-C5 antibody or an antigen-binding variant thereof as described herein, the nucleotide sequence being operatively linked to a promoter, or administering to the subject a pharmaceutical composition comprising the rAAV and a pharmaceutically acceptable carrier, preferably wherein the rAAV or the pharmaceutical composition is administered to the subject via intravitreal injection. Use of the rAAV or pharmaceutical compositions comprising therein for treating dry AMD is also provided. Use of the rAAV in the manufacture of an agent for treating dry AMD is also provided.

[0197] In related embodiments, a method for treating geographic atrophy in subjects requiring such treatment is provided, the method comprising administering to the subject recombinant adeno-associated virus (rAAV) viral particles comprising: (i) a variant AAV capsid protein comprising a heterologous peptide insert of 7 to 20 amino acids covalently inserted into the GH ring of the AAV capsid protein, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO: 168), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding an anti-C3 antibody and / or an anti-C5 antibody or an antigen-binding variant thereof as described herein, the nucleotide sequence being operatively linked to a promoter; or administering to the subject a pharmaceutical composition comprising the rAAV viral particles and a pharmaceutically acceptable carrier, preferably wherein the rAAV or the pharmaceutical composition is administered to the subject via intravitreal injection. Use of the rAAV or pharmaceutical compositions comprising therein for treating geographic atrophy is also provided. Use of the rAAV in the manufacture of medicaments for treating geographic atrophy is also provided.

[0198] In some respects, the variant AAV capsid protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:170, and comprises a P34A substitution at amino acid position 588 and a LAISDQTKHA (SEQ ID NO:169) peptide insertion fragment.

[0199] In some preferred aspects, the heterologous nucleic acid encapsulated by the variant AAV capsid protein comprises a nucleotide sequence encoding an antibody comprising one or more CDRs, said one or more CDRs comprising an amino acid sequence according to Table 1 or Table 2, or comprising an amino acid sequence having at least 75% identity with it. In one preferred embodiment, the antibody comprises at least one, more preferably at least two, and most preferably three CDRs, each of said CDRs comprising an amino acid sequence selected as shown in SEQ ID No: 54-56, or comprising an amino acid sequence having at least 75% identity with an amino acid sequence selected as shown in SEQ ID No: 54-56. In a related aspect, the nucleotide sequence encapsulates an antibody comprising an amino acid sequence selected as shown in SEQ ID No: 14 and 133-136, or comprising an amino acid sequence having at least 75% identity (e.g., at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%) with an amino acid sequence selected as shown in SEQ ID No: 14 and 133-136. In other relevant aspects, the nucleotide sequence encoding the nanobody comprises a nucleotide sequence as shown in any one of SEQ ID Nos: 133-136. In another relevant aspect, the nucleotide sequence encodes an antibody comprising an amino acid sequence selected from those shown in SEQ ID Nos: 137-163, or comprising an amino acid sequence having at least 75% identity (e.g., at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity) with an amino acid sequence selected from those shown in SEQ ID Nos: 137-163.

[0200] In some respects, rAAV is administered via periocular, intravitreal, suprachoroidal, and / or subretinal injection, preferably via intravitreal injection, at a dose of approximately 1 × 10⁻⁶ for subjects with dry AMD and / or geographic atrophy. 8 One vector genome (vg) / eye to approximately 1×10 13 vg / eye, approximately 1×10 9 vg / eye to approximately 1×10 12 vg / eye, approximately 1×10 9 vg / eye to approximately 1×10 11 vg / eye, or approximately 6×10 9 vg / eye to approximately 6×10 10 vg / eye.

[0201] In some embodiments, a method is provided for treating dry AMD and / or geographic atrophy in subjects with this need, comprising administering to the subject an effective amount of rAAV viral particles comprising: (i) a capsid comprising a capsid protein comprising an amino acid sequence as shown in SEQ ID NO: 170; and (ii) a heterologous nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat; (b) a CAG or CMV promoter; (c) a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID No: 14 and 133-136; (d) a polyadenylated sequence; and (e) an AAV2 terminal repeat.

[0202] In relevant embodiments, a method is provided for treating dry AMD and / or geographic atrophy in subjects with this need, comprising administering a pharmaceutical composition to the subject via periocular, intravitreal, suprachoroidal, and / or subretinal injection, comprising a pharmaceutically acceptable carrier and rAAV viral particles comprising: (i) a capsid protein comprising the amino acid sequence shown in SEQ ID NO: 170, and (ii) a heterologous nucleic acid from 5' to 3' comprising: (a) an AAV2 terminal repeat, (b) a CAG or CMV promoter, (c) a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID No: 14 and 133-136, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat. In some aspects, the pharmaceutical composition comprises about 1 × 10 8 vg to approximately 1×10 13 vg, approximately 1×10 9 vg to approximately 1×10 12 vg, approximately 1×10 9 vg to approximately 1×10 11 vg, or approximately 6×10 9 vg to approximately 6×10 10 vg.

[0203] In some embodiments, a method is provided for treating dry AMD and / or geographic atrophy in subjects with this need, comprising administering to the subject an effective amount of rAAV viral particles comprising: (i) a capsid comprising a capsid protein comprising an amino acid sequence as shown in SEQ ID NO:170; and (ii) a heterologous nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat; (b) a CAG or CMV promoter; (c) a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID No:137-163; (d) a polyadenylated sequence; and (e) an AAV2 terminal repeat.

[0204] In relevant embodiments, a method is provided for treating dry AMD and / or geographic atrophy in subjects with this need, comprising administering a pharmaceutical composition to the subject via periocular, intravitreal, suprachoroidal, and / or subretinal injection, comprising a pharmaceutically acceptable carrier and rAAV viral particles comprising: (i) a capsid protein comprising the amino acid sequence shown in SEQ ID NO:170, and (ii) a heterologous nucleic acid from 5' to 3' comprising: (a) an AAV2 terminal repeat, (b) a CAG or CMV promoter, (c) a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID No:137-163, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat. In some aspects, the pharmaceutical composition comprises about 1 × 10 8 vg to approximately 1×10 13 vg, approximately 1×10 9 vg to approximately 1×10 12 vg, approximately 1×10 9 vg to approximately 1×10 11 vg, or approximately 6×10 9 vg to approximately 6×10 10 vg.

[0205] Pharmaceutical compositions comprising rAAV as described herein are provided. In some embodiments, the pharmaceutical composition comprises about 1 x 10 8 To approximately 1 x 10 14 One vector particle or vector genome, approximately 1 x 10 8 To approximately 1 x 10 13 One vector particle or vector genome, approximately 1 x 10 9 To approximately 1 x 10 12 One vector particle or vector genome, or approximately 1 x 10^6 9 Approximately 2 x 10 93 x 10 9 Approximately 4 x 10 9 Approximately 5 x 10 9 Approximately 6 x 10 9 Approximately 7 x 10 9 Approximately 8 x 10 9 Approximately 9 x 10 9 Approximately 1 x 10 10 Approximately 2 x 10 10 Approximately 3 x 10 10 Approximately 4 x 10 10 Approximately 5 x 10 10 Approximately 6 x 10 10 Approximately 7 x 10 10 Approximately 8 x 10 10 Approximately 9 x 10 10 Approximately 1 x 10 11 Approximately 2 x 10 11 Approximately 3 x 10 11 Approximately 4 x 10 11 Approximately 5 x 10 11 Approximately 6 x 10 11 Approximately 7 x 10 11 Approximately 8 x 10 11 Approximately 9 x 10 11 Or approximately 1 x 10 12 One carrier particle or carrier genome. In some aspects, the drug composition contains about 1 × 102 9 To approximately 1×10 11 vg, and preferably contains about 6×10 9 vg to approximately 6×10 10 vg. In some preferred embodiments, the pharmaceutical composition is administered via intravitreal injection to a person suffering from dry AMD and / or geographic atrophy. Example

[0206] The following examples illustrate preferred embodiments of the invention and are not intended to limit the scope of the invention in any way. While the invention has been described with respect to its preferred embodiments, various modifications thereto will be apparent to those skilled in the art upon reading this application.

[0207] Example 1 Data from patients with both dry and wet age-related macular degeneration (AMD) support the hypothesis that persistent 'tickover' of the alternative complement pathway leads to chronic deposition of the complement membrane attack complex (MAC) in the choroidal capillaries and retinal pigment epithelium (RPE). Sub-dissolution levels of MAC result in the production of cytokines and inflammatory molecules. Prevention of MAC deposition thus provides an effective treatment for both dry and geographic age-related macular degeneration.

[0208] Nanobodies targeting human complement factor C3 (SEQ ID No: 1-14) were isolated by phage display panning targeting the human C3 protein, and their ability to specifically bind to C3 and inhibit complement was tested. Recombinant AAVs containing a capsid of SEQ ID NO: 107 were generated, encapsulating heterologous nucleic acids encoding representative anti-C3 nanobodies, and complement inhibition was evaluated.

[0209] Materials and Methods Recombinant protein production For each nanobody identified, recombinant proteins were produced in HEK293 cells using NeoClone and GeneScript.

[0210] Cell culture HEK293T cells were grown on tissue culture plastics under 5% CO2, normoxic conditions, in DMEM, 10% fetal bovine serum (FBS), and with 1% penicillin / streptomycin.

[0211] iPSC-RPE was grown on Matrigel-coated plates and matured for 30 days in XVIVO-10 medium under normoxic conditions with 5% CO2.

[0212] Transduction iPSC-RPE cells were transduced ≥30 days after seeding at multiples of infection (MOI, vg / cell). Samples were collected 7 days post-infection for analysis.

[0213] complement inhibition assay The Wieslab® Complement System Alternative Pathway Kit (Eagle Biosciences Inc., Catalog No. COMPLAP 330) is an enzyme immunoassay for the qualitative determination of functional alternative complement pathways in human serum, performed according to the manufacturer's instructions. The Wieslab complement assay combines pathway-specific activation with the use of a labeled antibody specific to a novel epitope of the terminal complement complex C5b-9, which is generated as a result of complement activation (the amount of C5b-9 generated is proportional to the functional activity of the complement system). Briefly, for the Wieslab complement inhibition assay performed in Table 2, normal human serum or cynomolgus monkey serum (Complement Tech) was diluted 1:20, along with 3.75 μg of purified nanobody. Control and test sera were diluted 100 μL. μL / well was added to the provided plate. After incubation at 37°C for 1 hour, the plate was washed three times with washing buffer. An antibody-conjugate for C5b-9 labeled with alkaline phosphatase was added, and the plate was incubated at room temperature for 30 minutes. After washing, substrate solution was added to each well, and absorbance was read at 405 nm.

[0214] For the Wieslab complement inhibition assay performed in Figure 1, the supernatant after transfection or the recombinant purified nanobody (normalized to 1 mg / ml) used for dose-response mapping was diluted with the dilution buffer provided in the assay kit. 5% non-human primate (NHP) serum was prepared in the dilution buffer according to the protocol, and 100 μl of 5% NHP serum was transferred to a microtiter plate. The nanobody dilution was applied to the plate in triplicate and mixed on a plate mixer for 5 minutes. After mixing, the serum with the added nanobody dilution was transferred to the assay plate and incubated at 37°C for 60 minutes. After incubation, the plate was washed three times with the wash buffer provided in the kit and incubated with the conjugate at room temperature for 30 minutes. After incubation, the plate was washed three times with the wash buffer provided. 100 μl of the substrate solution provided in the kit was added to the microtiter plate, and the absorbance was read at 405 nm every 5 minutes for 60 minutes. For data analysis, dose-response curves were plotted at OD 405nm over 60 minutes based on concentration.

[0215] Immunocytochemistry (ICC) iPSC-RPE cells were exposed to 1% human serum or 1% human serum plus 5 μg / ml yeast polysaccharide to induce the alternative complement pathway. Twenty-four hours post-stimulation, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.01% Triton X-100. Primary antibody (C5b-9) was added at 4°C for 18 hours, followed by secondary antibody and DRAQ5 nuclear counterstaining at room temperature for 1 hour.

[0216] rAAV manufacturing For in vitro studies, rAAV with the capsid amino acid sequence listed in SEQ ID NO:107 was produced in HEK293 cells via PEI-mediated triple transfection. Cells were cultured in FBS-supplemented DMEM and maintained at 37°C in a 5% CO2 environment.

[0217] In short, cells were seeded into CellSTACK dishes and transfected using pHelper, the associated Rep-Cap plasmid, and the CAG-nanobody payload plasmid. Three to four days post-transfection, cells and supernatant were collected and lysed, treated with endonucleases, clarified via sterile filtration, and frozen. The thawed harvest was clarified, loaded onto the associated affinity resin, eluted at low pH, and immediately neutralized. Purified batches were prepared in a phosphate- or Tris-based buffer containing NaCl and 0.005% Pluronic F-68. Purified batches were stored at –80°C until use.

[0218] ELISA binding assay The antigen protein was pre-coated into a standard ELISA plate at 500 ng / well and then blocked with 1% BSA. The nanobodies were diluted to 10 μg / mL, and 150 μL was added to each well. After incubation and washing, the remaining bound nanobodies were detected using HRP-conjugated monoclonal rabbit anti-cameloid VHH. Control wells without nanobodies were used as background, and the reading was subtracted from the test wells.

[0219] Octet / Gator binding affinity measurement Biomembrane interferometry using Gator or Octet instruments was used to determine the binding affinity of nanobodies to their respective antigens. Anti-His probes were first equilibrated in buffer (PBST + 5% BSA) in 96-well plates at 37°C and 1000 rpm. After equilibration, nanobodies tagged with 6XHis (loaded sample) were immobilized onto the sensor. Once the probe reached 60% capacity, the loaded sensor was washed in buffer to remove any non-specific binding. The nanobodies-loaded probes were then immersed in a series of dilutions of the untagged target antigen protein (analyte sample) for the binding step to obtain “Kon”. The sensor was then transferred to blank buffer to obtain the dissociation rate “Koff”, the dissociation rate constant (1 / s). The ratio of Koff / Kon is “KD”, which is a direct measurement of the equilibrium dissociation rate constant of the binding affinity of the binding pair.

[0220] result Nanobody target binding and complement system inhibition Table 3 NB = unbound; WkB = weakly bound.

[0221] Table 3 lists the nanobody clones isolated by phage display panning targeting the human C3 protein, along with their respective characteristics (corresponding amino acid sequences as described). Production yield was calculated by the amount of purified protein extracted from 60 mL of supernatant from transiently transfected HEK293 suspension cells. ELISA binding to the human or cynomolgus monkey C3 protein was defined as a signal exceeding the antibody-free background by a multiple. Wieslab complement substitution pathway assays were determined by whether each nanobody inhibited at least 50% of complement activation at a specified concentration. Finally, the KD value of VHH binding to the human or cynomolgus monkey C3 protein was determined using BLI.

[0222] All nanobodies measured in Table 3, except for C3S5 and C3S10, bound to human and / or cynomolgus C3 protein (see corresponding ELISA binding and KD values). Similarly, all nanobodies measured in Table 3, except for C3S4, inhibited MAC formation at 4 μM and / or 0.1 μM, as shown in their corresponding Wieslab inhibition column.

[0223] Transduction of rAAV containing nanobody transgenes inhibits MAC formation in iPSC-RPE. Induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE) were transduced with rAAV carrying the C3-N10 transgene (SEQ ID No:14) at three multiples of infection (MOIs) of 5,000, 10,000, and 20,000, the rAAV having the capsid amino acid sequence listed in SEQ ID No:107. Seven days post-transduction, the levels of nanobodies (VHH) protein in the supernatant were analyzed by ELISA, and a dose-response in expression was observed (Fig. 1a). Complement inhibition was evaluated using the supernatant to determine MAC formation. A dose-response in MAC formation was observed after transduction at MOIs of 5,000, 10,000, and 20,000, compared to untransduced cells, with higher MOIs resulting in less MAC formation (Fig. 1b). In addition, MAC deposition on cells following the alternative pathway stimulant (yeast polysaccharide) was observed to be reversed after transduction of cells with positive controls inhibited by C3-N10 rAAV or the alternative complement pathway (Fig. 1c).

[0224] Example 2 Data from patients with both dry and wet age-related macular degeneration (AMD) support the hypothesis that the persistent 'slow operation' of the alternative complement pathway leads to chronic deposition of the complement membrane attack complex (MAC) in the choroidal capillaries and retinal pigment epithelium (RPE). Sub-dissolution levels of MAC result in the production of cytokines and inflammatory molecules. Prevention of MAC deposition thus provides an effective treatment for both dry and geographic age-related macular degeneration.

[0225] Nanobodies targeting human complement factor C5 (SEQ ID No: 57-65) were isolated by phage display panning targeting human C protein, and their ability to specifically bind to C5 and inhibit complement was tested.

[0226] Materials and Methods complement inhibition assay The Wieslab® Complement System Alternative Pathway Kit (Eagle Biosciences Inc., Catalog No. COMPLAP 330) is an enzyme immunoassay for the qualitative determination of functional alternative complement pathways in human serum, performed according to the manufacturer's instructions. The Wieslab complement assay combines pathway-specific activation with the use of a labeled antibody specific to a novel epitope of the terminal complement complex C5b-9, which is generated as a result of complement activation (the amount of C5b-9 generated is proportional to the functional activity of the complement system). Briefly, for the Wieslab complement inhibition assay performed in Table 2, normal human serum or cynomolgus monkey serum (Complement Tech) was diluted 1:20, along with 3.75 μg of purified nanobody. Control and test sera were diluted 100 μL. μL / well was added to the provided plate. After incubation at 37°C for 1 hour, the plate was washed three times with washing buffer. An antibody-conjugate for C5b-9 labeled with alkaline phosphatase was added, and the plate was incubated at room temperature for 30 minutes. After washing, substrate solution was added to each well, and absorbance was read at 405 nm.

[0227] ELISA binding assay The antigen protein was pre-coated into a standard ELISA plate at 500 ng / well and then blocked with 1% BSA. The nanobodies were diluted to 10 μg / mL, and 150 μL was added to each well. After incubation and washing, the remaining bound nanobodies were detected using HRP-conjugated monoclonal rabbit anti-cameloid VHH. Control wells without nanobodies were used as background, and the reading was subtracted from the test wells.

[0228] Octet / Gator binding affinity measurement Biomembrane interferometry using Gator or Octet instruments was used to determine the binding affinity of nanobodies to their respective antigens. Anti-His probes were first equilibrated in buffer (PBST + 5% BSA) in 96-well plates at 37°C and 1000 rpm. After equilibration, nanobodies tagged with 6XHis (loaded sample) were immobilized onto the sensor. Once the probe reached 60% capacity, the loaded sensor was washed in buffer to remove any non-specific binding. The nanobodies-loaded probes were then immersed in a series of dilutions of the untagged target antigen protein (analyte sample) for the binding step to obtain “Kon”. The sensor was then transferred to blank buffer to obtain the dissociation rate “Koff”, the dissociation rate constant (1 / s). The ratio of Koff / Kon is “KD”, which is a direct measurement of the equilibrium dissociation rate constant of the binding affinity of the binding pair.

[0229] result Nanobody target binding and complement system inhibition Table 4 NB = unbound; WkB = weakly bound.

[0230] Table 4 lists the nanobody clones isolated by phage display panning targeting the human C5 protein, along with their respective characteristics. Production yield was calculated as the amount of purified protein extracted from 60 mL of supernatant from transiently transfected HEK293 suspension cells. ELISA binding to human C5 or cynomolgus monkey C5a protein was defined as a fold increase in signal strength above the antibody-free background for each clone. Wieslab complement substitution pathway assays were determined by whether each nanobody inhibited at least 25% of complement activation at a specified concentration. Finally, the KD value of VHH binding to human C5 protein was determined using BLI.

[0231] All nanobodies measured in Table 4, except for C5S12, bound to human C5 protein (see corresponding ELISA binding and KD values). Similarly, all nanobodies measured in Table 4, except for C5S3, C5S4, and C5S7, inhibited MAC formation at 4 μM and / or 0.1 μM, as shown in their corresponding Wieslab inhibition column.

[0232] Example 3 Methods and Materials Recombinant complement protein production For recombinant protein production, both wild-type and mutant complement sequences, along with a C-terminal TEV cleavage site and a 6X His tag, were cloned into a pcDNA plasmid containing a mouse IgG secretion sequence and transiently transfected into Expi293 cells. Cells were harvested by rotating at 250Xg for 30 min and collecting the supernatant. The recombinant protein was purified by a single-step purification process using immobilized metal affinity chromatography (IMAC). For purification, the clarified supernatant was passed through a 0.2 μm filter and then through a 1 mL His excel capture column pre-equilibrated with equilibration buffer (50 mM Tris, 150 mM NaCl, pH 8.0) at a flow rate of 1 mL / min. After the capture step, the column was washed with washing buffer (10 mM imidazole, 50 mM Tris, 150 nM NaCl, pH 8) and eluted with his tag elution buffer (250 nM imidazole, 50 mM Tris, 150 mM NaCl, pH 8). The his eluent was subjected to TEV protease for 6XHis tag cleavage. After cleavage, the 6XHis tag was removed by passing the eluent through IMAC resin. The flow-through from the IMAC column was subjected to size exclusion chromatography, exchanged with buffer in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaH2PO4, 1.4 mM KH2PO4), and passed through a 0.4 nm filter and stored in aliquots at -20°C.

[0233] Reorganization of VHH production For recombinant nanobody production, both wild-type and humanized nanobody sequences, along with C-terminal strep II and 6XHis tags, were cloned into a pcDNA plasmid containing a mouse IgG secretion sequence and transiently transfected into Expi293 cells. Cells were harvested by rotating at 250Xg for 30 min, and the supernatant was collected. The recombinant nanobodies were purified by a single-step purification process using IMAC affinity chromatography. For purification, the clarified supernatant was passed through a 0.2 μm filter and then through a 1 mL His excel capture column pre-equilibrated with equilibration buffer (50 mM Tris, 150 mM NaCl, pH 8.0) at a flow rate of 1 mL / min. After the capture step, the column was washed with washing buffer (10 mM imidazole, 50 mM Tris, 150 mM NaCl, pH 8) and eluted with his tag elution buffer (250 nM imidazole, 50 mM Tris, 150 mM NaCl, pH 8). The his eluent was exchanged into PBS with buffer and passed through a 0.4 nm filter and stored as aliquots at -20°C.

[0234] Recombinant VHH-Fc production For the recombinant production of Fc-fused nanobodies, VHH cDNA was cloned into pCDNA3 with a C-terminal human IgG2 hinge-human IgG4 Fc to express nanobodies as Fc dimers in Expi293 cells, with a mouse IgG signal peptide for secretion in the supernatant. After transient transfection, cells were harvested and centrifuged at 250 x g for 30 min to form cell clumps, and the supernatant was passed through a 0.45 μm membrane. The recombinant VHH-Fc dimer was passed through a Mabselect Sure column in 1xPBS buffer at pH 7 to capture the VHH-Fc fusion. After loading, the column was washed with washing buffer and eluted with low pH buffer, 0.1 M citrate buffer, and 150 mM NaCl at pH 3. The eluent was immediately neutralized with 1 M Tris at pH 8, then exchanged for buffer in 1xPBS at pH 7 and aliquoted.

[0235] Wieslab Complement Activation Assay The Wieslab Alternative Complement Pathway Assay System is used for the semi-quantitative assessment of the inhibitory activity of nanobodies. The Wieslab Alternative Pathway Kit is an enzyme immunoassay for the qualitative determination of functional alternative complement pathways in human serum. The assay combines the principles of a hemolytic assay for complement activation with the use of labeled antibodies specific to neoantigens generated due to complement activation. In the complement activation AP assay, the assay plate is coated with a specific activator of the alternative pathway, and the provided diluent solution contains a specific blocker to ensure activation of only the alternative pathway. To assess the inhibitory effect of nanobodies on complement AP, normal human serum is diluted 1:20 in the provided diluent and added to 96-well plates. Recombinant purified nanobodies, along with control and test sera, are added in duplicate to the wells and mixed at 400 rpm for 5 minutes on a plate shaker. After mixing, the contents of the 96-well plate are transferred to the assay plate using a multichannel pipette. After incubation at 37°C for 1 hour, the plate was washed three times with washing buffer, and an alkaline phosphatase-labeled antibody conjugate with C5b-9 was added and incubated at room temperature for 30 minutes. After washing, substrate solution was added to the wells and incubated at room temperature for 45 minutes. The amount of complement activation was correlated with color intensity and measured as absorbance / density at 405 nm.

[0236] Assay for inhibition of classical and lectin pathways In addition, the complement inhibition of nanobodies in the classical and lectin pathways was investigated using Wieslab's classical and mannan-bound lectin pathway kits. In the classical pathway kit, the microplate wells were coated with a CP activator such as the C1q / C1 complex, and the provided diluent contained a specific activator for the classical pathway. Similarly, in the case of the lectin pathway MBL kit, microplates coated with mannan-bound lectins, opsonins, or fibrin were used to specifically activate the lectin pathway in combination with a diluent buffer containing specific inhibitors for both AP and CP.

[0237] Serum cross-reactivity in primates Cross-reactivity with primate sera was also investigated using the Wieslab AP assay kit. In this case, nanobodies were incorporated into a 1:20 dilution of normal cynomolgus monkey serum, and then the manufacturer’s guidelines for implementation were followed.

[0238] IC50 Calculation The half-maximal inhibitory concentration (IC50) of the membrane-associated complex (MAC) was determined using an alternative Wieslab assay. A series of dilutions of the recombinant purified nanobodies were doped into 20x diluted human serum at various concentrations and applied to Wieslab assay plates. After 60 min incubation, the plates were washed and treated with alkaline phosphatase conjugates labeled with C5b-9, followed by incubation for 30 min. After conjugate incubation, the plates were washed and incubated with the substrate solution, with readings taken at 405 nm at the 45 min time point. IC50 MAC formation was evaluated using curve fitting software.

[0239] Mouse alternative complement pathway assay The mouse C3 / C5 ELISA assay was used to examine the cross-functional activity of C3 and C5 nanobodies for mouse complement proteins. This assay was performed using LPS-coated microplates for specific activation of the alternative pathway. For this assay, mouse serum was diluted 1:20 in the provided dilution buffer and transferred to 96-well plates, with serial dilutions of the nanobodies, along with a positive control mouse BB5.1 antibody, added triplicate to the serum. The 96-well plates were agitated at 400 rpm for 5 minutes on a plate shaker, and the contents were transferred to LPS-coated microplates and incubated at 37°C for 60 minutes. After incubation, the plates were washed four times with the provided wash buffer, and a biotinylated tracer antibody was added, followed by another 60 minutes at 37°C, and then four more washes. A streptavidin-biotin peroxidase conjugate was added, and the plates were incubated at 37°C for 60 minutes, followed by four more washes. Add TMB solution and incubate at room temperature for 30 minutes. Then add stop solution to terminate the reaction and measure OD at 450 nm.

[0240] Gator assay for binding affinity (KD) Antistrep II antibody-coated probes were used to determine binding affinity via Octet / Gator. The antistrep II antibody probes were immersed in buffer (PBST + 5% BSA) for equilibration in 96-well plates at 37°C and 1000 rpm. After equilibration, the probes were immersed in wells containing VHHs tagged with VHH-strep for loading. Once approximately 60% loaded, the antistrep II antibody probes were immersed in buffer wells for a washing step to remove any VHHs that were not specifically bound to the probe. After washing, the probes were then immersed in a series of dilutions of recombinant purified human or cynomolgus monkey C3 or C5 wild-type or C5 single and double mutant proteins (untagged) for a binding step to obtain “Kon”, representing the binding rate constant (1 / s). Following binding, a dissociation step was performed in buffer to obtain the dissociation rate “Koff”, representing the dissociation rate constant (1 / s). The ratio of Koff / Kon is denoted as "KD", which is the equilibrium dissociation rate constant, a direct measure of the binding affinity of the binding pair.

[0241] Cross-blocking of antibody against C5 protein was determined by Gator BLI sandwich assay. The Gator BLI platform was used to investigate the binding epitopes of the C5S2.2 nanobody. Like eculizumab, the C5S2.2 nanobody did not show cross-binding with the cynomolgus C5 protein, nor did it exhibit inhibitory activity against alternative complement in cynomolgus serum. Therefore, the binding epitopes were queried to determine whether eculizumab and C5S2.2 cross-blocked the human C5 protein. A sandwich method was used on the BLI, where an anti-human Fc binding probe was used to immobilize eculizumab for 1–2 minutes. After washing, the pre-loaded probe was then immersed in the recommended binding buffer in either untagged C5 cells alone or C5 cells pre-incubated with C5S2 for 5–6 minutes to allow binding to C5 or the C5S2-C5 complex.

[0242] Reverse sandwich measurement A reverse sandwich technique was performed by immobilizing a series of diluted C5S2 nanobodies onto anti-strep-II probes. After washing, the pre-loaded probes were immersed in a binding solution containing C5 for inspection, allowing the C5 protein to bind to the immobilized nanobodies for 2–3 minutes. Once the C5 protein was bound, the probes containing the VHH-C5 complex were immersed in wells containing eculizumab for binding.

[0243] Epitope mapping by cross-linking mass spectrometry (XL-MS) Epitope peptide mapping was performed using cross-linking mass spectrometry with humanized C3 nanobodies targeting the human C3 protein. Purified nanobodies and recombinant proteins were generated. Each nanobodies could bind to the target and then be chemically cross-linked using bis(succinimide) octanoate. The complex was then treated with a set of overlapping proteases, and the peptides were analyzed by mass spectrometry and subsequently compared with the native protein sequence.

[0244] QC checks for sample integrity As part of the initial QC check, high molecular weight MALDI MS analysis was performed on the recombinant proteins (two nanobodies, C3 and C5). Additionally, the same experiments were performed on the cross-linked recombinant proteins and compared to verify sample integrity and aggregation levels, which also served as controls for subsequent experiments.

[0245] Characterizing nanobody / C3 complex Once sample integrity was confirmed, C3N10 / C3 and C3S3 / C3 were characterized. For characterization, the complexes were prepared by incubation at two different concentrations, followed by measurements using an Autoflex MALDI / TOF mass spectrometer. For crosslinking experiments, the C3N10 / C3 and C3S3 / C3 complexes were treated with DDS for 180 min and measured on an Autoflex MALDI / TOF. Mass spectrometer readouts from the control and crosslinked samples were compared for any additional peaks.

[0246] Peptide quality fingerprinting For peptide mass fingerprinting, hC3 was cross-linked with DDS for 180 minutes and cleaved by five different proteases (trypsin, chymotrypsin, elastase, Asp-N, and thermophilic protease), with maximum sequence coverage measured. After cleavage, both the control and cross-linked samples underwent liquid chromatography for peptide separation, followed by mass spectrometry analysis on a Q-Exactive Plus mass spectrometer. The obtained peptides were analyzed using CovalX proprietary software, generating peptide fingerprints for both the cross-linked and control samples.

[0247] The Meso Scale Discovery immunoassay was used to measure the anti-drug antibody response against nanobodies in serum from healthy human donors. Purified nanobodies and controls were passively coated onto MSD LI5XA-3 plates at 1 μg / mL and then blocked with casein. Internal reservoirs of serum and plasma from normal human donors were diluted 1:100 and applied to each well, followed by incubation at room temperature with shaking for 2 hours to allow binding. After washing, bound antibodies were detected using Sulfo-TAG goat anti-human IgG. Purified human IgG was directly coated at multiple concentrations to track assay performance and serve as a positive control. After washing, diluted readout buffer was added to each well, and the plate was read out in a Meso Sector S 600.

[0248] Plasmid DNA cloning and isolation The AAV support vector containing the CAG promoter and other necessary AAV cartridge components was digested with restriction enzymes to remove unwanted payload. Custom gBlocks (GB) from Integrated DNA Technologies, containing the desired VHH, were cloned into the backbone.

[0249] Following cloning and transformation, Miniprep cultures were grown from the resulting colonies in Terrific Broth supplemented with 100 mg / mL kanamycin sulfate and incubated overnight at 30°C with shaking. DNA was isolated using the QIAprep Spin Miniprep Kit according to the manufacturer's protocol and sequenced using Primordium Lab sequencing technology to identify positive clones of sCFH and CFH.

[0250] Cell culture HEK293T cells were grown in DMEM medium containing 1% penicillin / streptomycin and 10% fetal bovine serum. Cells were passaged twice a week and used for no more than 20 passages.

[0251] iPSC-RPE was grown on Matrigel-coated plates and matured for 30 days in XVIVO-10 medium supplemented with 10 µM RHO / ROCK pathway inhibitor Y-27632 under normoxic conditions at 5% CO2.

[0252] AAV Manufacturing For in vitro studies, recombinant rAAV (containing the capsid of SEQ ID NO: 170) was produced in HEK293 cells via PEI-mediated triple transfection. Cells were cultured in DMEM supplemented with FBS and maintained at 37°C in a 5% CO2 environment.

[0253] In short, cells were seeded into CellSTACK dishes and transfected using pHelper, the associated Rep-Cap plasmid, and the CAG-nanobody payload plasmid. Three to four days post-transfection, cells and supernatant were collected and lysed, treated with endonucleases, clarified via sterile filtration, and frozen. The thawed harvest was clarified, loaded onto the associated affinity resin, eluted at low pH, and immediately neutralized. Purified batches were prepared in a phosphate- or Tris-based buffer containing NaCl and 0.005% Pluronic F-68. Purified batches were stored at –80°C until use.

[0254] VHH plasmid transfection HEK293T cells were used at 2×10 5 Cells / well were seeded into 12-well plates and transfected using various plasmid forms, including C3N10, C3S3, and C5S2. The supernatant was collected and transgene expression was tested using various assays.

[0255] Transduction iPSC-RPE cells were transduced at multiples of infection (MOI, vg / cell) of 1,000, 5,000, or 20,000 ≥30 days post-inoculation. Supernatant samples were collected 7 days post-inoculation for analysis.

[0256] VHH ELISA Anti-VHH capture antibody was diluted 1:500 in PBS and incubated overnight at 4 ºC in MaxiSorp microtiter plates. The plates were then washed 5x with PBST and incubated for 2 hours at room temperature in blocking solution of 2% BSA in PBS. Unless otherwise specified, plate washing was performed similarly between each subsequent step. Supernatant from transduced cells was diluted in PBS solution of 1% BSA; the dilution was consistent across all samples tested for each type of VHH. The diluted sample was applied to the microtiter plates and incubated for 2 hours at room temperature. The plates were then incubated for 1 hour at room temperature with HRP-conjugated anti-VHH detection antibody diluted 1:10,000 in PBST solution of 1% BSA. After incubation with the detection antibody, 100 µl of TMB substrate was applied to the plates and color development was monitored. 5–15 minutes after the addition of the TMB substrate, without a washing step, 100 µl of 450 nM TMB stop solution was applied to the plates. Within 15 minutes of applying the stop solution, the optical density (OD) was measured at 450 nm using a Cytation3 photometer.

[0257] Protein blot Following the manufacturer's instructions, the protein concentration in the cell supernatant was quantified using the Pierce Micro BCA Protein Assay. An appropriate volume of supernatant to reach 33 µg total protein was then diluted to 15 µl in PBS and mixed with 5.78 µl of 4x LDS buffer and 2.31 µl of 10x reducing agent, and incubated at 90 ºC for 10 min. Samples were loaded onto 15-well Bolt 12% Bis-Tris Plus polyacrylamide gels or Novex 10 to 20% Tricine polyacrylamide gels and run at 160 V for 45 min in 1× MOPS buffer. The isolated proteins were transferred to a PVDF membrane at 2.5 A for 10 min using a BioRad TransBlotTurbo device (catalog number 1704150, Bio-Rad). In the iBind Flex Solution with iBindFlex cards, membranes were probed with HRP-conjugated anti-VHH primary antibody, mouse anti-C3a, rabbit anti-C5a, or goat anti-human factor B accompanied by an appropriate HRP secondary antibody (if not conjugated). Proteins were visualized using SuperSignal West PicoPlus Chemiluminescent Substrate and imaged on a ChemiDoc MP.

[0258] Complement inhibition assay for evaluation of transduction samples The Wieslab® Complement System Alternative Pathway Kit is an enzyme immunoassay for the qualitative determination of functional alternative complement pathways in human serum, and should be performed according to the manufacturer's instructions. Normal human serum is diluted to 2% in the provided dilution buffer, and 200 µl of the 2% serum is transferred to a microtiter plate. A 20 µl supernatant sample from the transduced RPE culture is applied to the plate and mixed. After mixing, two aliquots of the diluted serum with the added supernatant sample are transferred to the assay plate, 100 µl per well, and incubated at 37°C for 60 min. After incubation, the plate is washed three times with the wash buffer provided in the kit and incubated with the conjugate at room temperature for 30 min. After incubation, the plate is washed three times with the wash buffer provided in the kit. 100 µL of the substrate solution provided in the kit is added to the microtiter plate, and the absorbance is read at 405 nm every 5 min for 90 min. The percentage complement inhibition for each sample is calculated according to the following formula and is considered semi-quantitative.

[0259] result The binding affinity of wild-type and humanized nanobodies for C3 and C5 targets.

[0260] Wild-type nanobodies targeting C3, C3N10, and C3S3, as well as C5 and C5S2, were humanized as C3N10.3, C3S3.2, and C5S2.2, respectively. All six were recombinantly expressed, purified, and further tested using transiently transfected human HEK293 suspension cells. Table 5 shows the binding affinity of wild-type and humanized nanobodies for human and cynomolgus monkey C3 and C5 complement protein targets. KD in nM was calculated using Gator via biomembrane interference (BLI). The wild-type nanobodies targeting C3 and C5 not only did not cross-bind with the other antigen, but none of them bound to other members of the α-2-macroglobulin family: human C4b, α-2-macroglobulin, and α-2-macroglobulin-like protein 1 (data not shown).

[0261] Table 5: Affinity of wild-type and humanized C3 and C5 nanobodies to human and cynomolgus monkey C3 and C5 proteins. (NB: No binding.) .

[0262] Mechanism of action: Inhibition of complement pathway activity The innate immune complement system is initiated via three separate pathways: the classical pathway, the lectin pathway, and the alternative pathway. The C3 protein acts as a convergence point for activation in each pathway, while C5 protein cleavage initiates the formation of the final component of complement activation: the membrane attack complex. To differentiate their mechanisms of action within the three pathways of the complement cascade, the functional potential of wild-type nanobodies was evaluated in human serum using a classical, alternative, and lectin pathway assay kit available from Wieslab. As shown in Figure 2, all three nanobodies exhibited inhibitory activity against the alternative pathway. However, while C5S2 showed strong inhibitory activity in all three pathways, C3S3 exhibited activity only against the alternative pathway. C3N10 showed the greatest inhibitory activity against the alternative pathway, and moderate potency against both the classical and lectin pathways.

[0263] IC50 efficacy of wild-type and humanized nanobodies against human serum-mediated alternative pathways.

[0264] To calculate IC50 and compare the potency between wild-type and humanized nanobodies, all were serially diluted 1:2 and retested using the same alternative assay. The humanized nanobodies exhibited similar potency to their original wild-type counterparts, suggesting minimal loss of inhibitory activity between variants. Figure 3A -C indicates their potency, and Table 6 summarizes these results. IC50 of C3N10 (wild type) 50 IC with a resolution of 133.8 nM and a C3N10.3 50 It is 69.47 nM ( Figure 3AC3S3 (wild type) IC 50 The IC is 34.51 nM; C3S3.2 50 42.86 nM ( Figure 3B C5S2 (wild type) IC 50 The IC has a resolution of 46.18 nM and is based on the C5S2.2 chip. 50 47.33 nM ( Figure 3C ).

[0265] IC50 of C3N10 WT and humanized C3N10 in the classical and lectin pathways Since C3N10 showed mild inhibition in both the classical and lectin pathways, the IC50 of wild-type and humanized C3N10 was calculated using more serial dilutions for both pathways, as shown in the figure. Figure 4A -B is shown and summarized in Table 6 below.

[0266] Table 6: Summary of IC50 inhibitory efficacy of wild-type and humanized nanobodies on the complement pathway. (NA: inactive; ND: incomplete) .

[0267] Activity of anti-human C3 and C5 wild-type nanobodies against mouse alternative pathways.

[0268] To examine whether human alternative complement pathway inhibitory nanobodies exhibit cross-functionality against mice, the wild-type form was tested using the Hycult mouse C3 / C5 ELISA kit (catalog # HK2002). This included the positive control mouse anti-mouse C5 inhibitory antibody BB5.1. Figure 5 As shown, C3N10, C3S3, and C5S2 showed no observable inhibitory activity in this mouse serum-mediated alternative complement pathway assay.

[0269] IC50 of anti-human C3 and C5 wild-type and humanized nanobodies against alternative complement activity in non-human primate serum Based on their binding affinity to their respective cynomolgus variants, the IC50 of wild-type and humanized nanobodies was evaluated in the same Wieslab alternative complement pathway assay using cynomolgus serum, such as... Figure 6A and 6B And as shown in Table 7 below. While both the wild-type and humanized nanobodies targeting C3 showed inhibitory activity, neither C5S2 nor its humanized form showed efficacy in the Wieslab cynomolgus serum-mediated alternative complement pathway (data not shown), reflecting their inability to bind to their cynomolgus C5 protein targets as shown in Table 5.

[0270] Table 7: IC50 of primate serum from alternative pathways for C3 and C5 nanobodies (NA: inactive) .

[0271] Inhibition of binding rate and alternative complement pathway of humanized nanobody-human Fc fusion protein Three humanized nanobodies, C3N10.3, C3S3.2, and C5S2.2, were engineered and expressed as homodimeric fusion proteins (also known as C3N10.3-Fc, C3S3.2-Fc, and C5S2.2-Fc) with human G2 hinge-G4 Fc domains to mimic the bivalent structure and function of native immunoglobulins. Their target binding rates and alternative pathway inhibitory potency were calculated using biomembrane interferometry and Wieslab assays and compared with the original monomers in Tables 8 and 9, respectively. Affinity binding was observed with C3S3.2-Fc and C5S2.2-Fc, while synergistic alternative pathway inhibitory activity (greater than two-fold) was observed with C5S2-Fc. Figure 7 ).

[0272] Table 8: Binding of humanized monomers and Fc fusion proteins to their targets (KD) .

[0273] Table 9: Inhibitory efficacy (IC50) against the alternative complement pathway .

[0274] Epitope peptide diagrams of C3N10.3 and C3S3.2 for human C3 protein To further differentiate the mechanisms of action of the two VHHs targeting C3, cross-linking mass spectrometry (XL-MS, CovalX) was used to identify the binding epitopes of C3N10.3 and C3S3.2. Both humanized nanobodies and purified human C3 protein are provided. The cross-linked peptides between the nanobodies and the antigens, along with the amino acid interactions shown in Table 10, are presented for C3N10.3 and 10, respectively. Figure 8 In, and for C3S3.2 it is shown in Table 11 and Figure 9Based on their interacting residues, the binding epitope of CN10.3 is located in the C345C domain within the α chain of C3 (1540-1600: GVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKK (SEQ ID NO:171)), while C3S3.2 appears to bind to two nonlinear epitopes spanning the MG1 (110-130: ATFGTQVVEKVVLVSLQSGYL (SEQ ID NO:172)) and MG5 (490-510: YTYLIMNKGRLLKAGRQVREP (SEQ ID NO:173) domains in the β chain. Figure 10 .

[0275] Epitope lookup for C5S2.2: Cross-blocking effect of eculizumab on human C5 protein Clinically approved eculizumab, like C5S2, binds to the human C5 protein, inhibits all three complement pathways in human serum, and does not bind to cynomolgus C5, nor inhibit complement pathways in cynomolgus serum (data not shown). Gator was used to investigate whether eculizumab and C5S2 also cross-block each other's binding to human C5.

[0276] exist Figure 11 In this process, anti-human Fc binding tips were first loaded with eculizumab and then washed. These pre-loaded tips were then immersed in either untagged C5 cells alone or C5 cells pre-incubated with C5S2 in the recommended binding buffer. As expected, the C5 protein alone bound rapidly to the tip pre-loaded with eculizumab, while the C5 cells pre-incubated with C5S2 did not, indicating that the latter pre-bound nanobodies blocked the epitopes of the former's full-size antibody.

[0277] exist Figure 12 In the alternative experiments, the C5S2 nanobody was first bound to the strep-II tip. After washing, the pre-loaded tip was immersed in a binding solution containing C5, and rapid binding was observed as expected. However, when the pre-bound C5S2-C5 complex tip was immersed in a binding solution containing eculizumab, the latter failed to bind, indicating that its binding site was blocked.

[0278] Epitope lookup of C5S2.2: Comparison with binding to mutant C5 protein of eculizumab. Eculizumab is resistant to two naturally occurring variants: the human R885H polymorphism and the human W917S variant relative to cynomolgus monkeys (Brachet G et al. 2016; Nishimura J et al. 2014). Human C5 R885H, human C5 W917S, and cynomolgus monkey C5 S917W mutant proteins were recombinantly generated, and the affinity of C5S2.2 and eculizumab for them was compared using Gator.

[0279] Figure 13A -C shows that while C5S2.2 has a high affinity for wild-type human C5 protein, it loses all binding activity for human C5 R885H and W917S mutants. On the other hand, although C5S2.2 cannot bind to wild-type cynomolgus monkey C5, it has a high affinity for the mutant cynomolgus monkey C5 S917W. Figure 14A The interaction pattern predicted in -C, which is reflected by eculizumab, indicates that C5S2.2 and eculizumab have similar binding epitopes on human C5.

[0280] Antidrug antibody response from serum of healthy donors against wild-type and humanized nanobodies To examine the relative potential immunogenicity of wild-type and humanized nanobodies, a sandwich immunoassay on an MSD with passively coated antigen and anti-human IgG detectors was used to measure the seropositivity rate of pre-existing antigen-specific antibodies in serum samples collected from individual healthy human donors. The percentage of donors with detectable pre-existing antibodies against each antigen is shown in Table 12 and graphically. Figure 15 The plots were drawn from both. When comparing different antigens, pre-existing antibodies against the monomeric C3 / 5 nanobody had a lower seropositivity rate in human serum samples than against the (G4S)2-linked homodimers C3N10Di and C3N10.3Di. The percentage of human samples with pre-existing antibodies against the clinically approved Beovu and Cablivi is shown as a comparative control. Beovu is a humanized monoclonal single-chain variable fragment (scFv) that binds to and inhibits vascular endothelial growth factor A, while Cablivi (capsulesizumab) is a trialanine-linked homodimer nanobody targeting the A1 domain of von Willebrand factor.

[0281] Table 12 .

[0282] AAV carrying VHH DNA leads to the production of functional proteins in human cells. In the preparation for AAV manufacturing, different forms of leader VHHs (C3N10, C3S3, and C5S2) are cloned into the AAV support framework. Prior to AAV manufacturing, the plasmids containing the VHHs are functionally evaluated by Western blotting. As depicted in Figure 16, after activation of the alternative complement cascade, C3 degrades into C3a and C3b. C3 degradation triggers activation of C5 degradation, resulting in C5a and C5b. The feedback loop of C3 activation can be initiated through this activation and leads to factor B degradation. C3 and C5 VHH inhibitors can be evaluated for their ability to stop these degradation products, as revealed by Western blotting.

[0283] The supernatant from transfected cells was subjected to Western blotting to examine degradation products that indicate activation of the complement cascade pathway. Figure 16A -C). All plasmids containing C3 VHH inhibitors blocked the formation of degradation product C3a, while inhibitors targeting C5, downstream of C3, did not, as expected. All VHH inhibitors targeting both C3 and C5 blocked the degradation of C5a. Next, the degradation products of factor B were evaluated, and all C3 VHH inhibitors blocked the degradation; however, inhibitors targeting the more downstream C5 VHH did not.

[0284] Following plasmid evaluation, VHH was fabricated into AAV within the AAV support plasmid backbone. After transduction at three MOIs in iPSC-RPE, the lead VHH AAV was characterized by expression and activity. Figure 17 A protein blot was created from the supernatant collected after transduction, showing a single product at the expected size of 15 kDa for each VHH examined. Figure 17 ).

[0285] VHH expression in the supernatant was also determined by ELISA (Figure 18). Each VHH showed a dose response, as indicated by an increase in OD at 450 nm with increasing dose.

[0286] Next, the function of each VHH was evaluated using the Wieslab® assay to determine complement pathway inhibition. Figure 19 Complement inhibition was detected at both MOIs tested, although to varying degrees for each specific VHH.

[0287] While the materials and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention.

Claims

1. An isolated polypeptide comprising a VH or VHH domain of C3, preferably a complement factor C3 and / or a proteolytic derivative C3a and / or C3b, specifically binding to C3, wherein the VH or VHH domain comprises one or more complementation-determining regions (CDRs) having at least 75% identity with CDRs selected from SEQ ID No: 18-20, 54-56, 15-17 and 21-53.

2. The isolated polypeptide according to claim 1, wherein the CDR of the VH or VHH domain comprises the following: The sequences selected from SEQ ID No: 18, 54, 15, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, and sequences having at least one, at least two, or at least three amino acid differences relative to SEQ ID No: 18, 54, 15, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, or 51; the sequences selected from SEQ ID No: 19, 55, 16, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, and sequences having at least one, at least two, or at least three amino acid differences relative to SEQ ID No: 19, 55, 16, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, or 52; and sequences selected from SEQ ID No: CDR3 of 20, 56, 17, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, and sequences that differ from SEQ ID NO: 20, 56, 17, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, or 53 by at least one, at least two, or at least three amino acids.

3. The isolated polypeptide according to claim 1 or 2, wherein the CDR is separated by frame regions (FR) FR1, FR2, FR3 and FR4.

4. The isolated polypeptide according to any one of claims 1-3, wherein the VH or VHH domain comprises a CDR sequence of any VH or VHH domain shown in SEQ ID No: 2, 14, 1 and 3-13, or a CDR sequence having at least 75% identity with it, preferably wherein the VH or VHH domain comprises a CDR1 having the sequence: G – F / L – T / S – F / L – S / G / D – P / V / Y / N – Y – A / D – M / I – G / S / A, and / or comprises a CDR2 having the sequence: G / S / C – I – R / D / T / S / N – W / S / G / Q – I / G / P / S – S / V / D / R – G / N – N / T / S – P / A / T / Y – Y / H / A – Y / H, wherein the 11th amino acid is optional.

5. The isolated polypeptide according to claim 4, wherein the VHH domain comprises an amino acid sequence as shown in any one of SEQ ID No: 2, 14, 1 and 3-13, or an amino acid sequence having at least 75% identity with it.

6. The isolated polypeptide according to any one of claims 1-4, wherein the VH or VHH domain is a humanized VH or VHH domain.

7. The isolated polypeptide according to claim 6, wherein the VH or VHH domain comprises an amino acid sequence of any one of SEQ ID NO: 98, 135, 94-97, 99-134 and 136, or comprises an amino acid sequence having at least 75% identity with it, preferably wherein the VH or VHH domain comprises an amino acid sequence of any one of SEQ ID NO: 98, 135, 94-97, 99-134 and 136, or comprises an amino acid sequence having at least 75% identity with it.

8. The isolated polypeptide according to any one of claims 1-7, wherein the VH or VHH domain is coupled to a tag, preferably a Strep or his6 tag, more preferably wherein the his6 tag comprises or is composed of the amino acid sequence shown in SEQ ID NO:93 and is located at the C-terminus of the VH or VHH domain.

9. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1-8.

10. A recombinant adeno-associated virus (rAAV) comprising (i) a variant AAV capsid protein comprising a heteropeptide of 7, 8, 9, 10 or 11 amino acids covalently inserted into the GH ring of the capsid protein relative to the corresponding parental AAV capsid protein, wherein the peptide insertion fragment comprises the amino acid sequence ISDQTKH (SEQ ID NO: 168), and (ii) the nucleic acid according to claim 9.

11. The rAAV according to claim 10, wherein the insert peptide has 1 to 3 spacer amino acids (Y1-Y3) at the amino terminus and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO:168), preferably wherein the insert peptide is LAISDQTKHA (SEQ ID NO:169).

12. The rAAV according to claim 10 or 11, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 183), or at a corresponding position in the capsid protein of another AAV serotype.

13. The rAAV according to any one of claims 10 to 12, wherein the capsid protein comprises relative to AAV2 (SEQ ID NO). One or more amino acid substitutions in VP1 of NO:183 or one or more corresponding substitutions in the capsid protein of another AAV serotype, preferably one or more of the following amino acid substitutions: MIL, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L and L735Q, more preferably P34A amino acid substitution.

14. The rAAV of claim 13, wherein the capsid protein comprises a P34A amino acid substitution relative to VP1 of AAV2, and comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the entire length of the amino acid sequence shown in SEQ ID NO:170, preferably wherein the capsid protein comprises the amino acid sequence shown in SEQ ID NO:

170.

15. The rAAV according to any one of claims 10 to 14, wherein the rAAV exhibits increased infectivity in retinal cells, preferably at least twice the infectivity, compared to the infectivity of retinal cells of AAV containing the corresponding parental AAV capsid protein.

16. The rAAV according to any one of claims 10 to 15, wherein the variant AAV capsid protein comprises an amino acid sequence having 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

170.

17. The rAAV according to any one of claims 10 to 16, wherein the rAAV comprises a heterologous nucleic acid from 5' to 3' comprising: (a) an inverted terminal repeat, (b) a promoter, (c) a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 8, (d) a polyadenylated sequence and / or a WPRE sequence, and (e) an inverted terminal repeat.

18. The rAAV of claim 17, wherein the reverse end repetition in the rAAV is an AAV2 reverse end repetition.

19. The rAAV according to any one of claims 10 to 18, wherein the promoter is a ubiquitous promoter.

20. The rAAV of claim 19, wherein the promoter is a CAG promoter.

21. The rAAV according to any one of claims 10 to 18, wherein the promoter is a tissue-specific promoter.

22. A host cell comprising rAAV according to any one of claims 10 to 21.

23. A pharmaceutical composition comprising rAAV according to any one of claims 10 to 21, and a pharmaceutically acceptable carrier, diluent, excipient, or buffer.

24. A method for treating dry age-related macular degeneration (dry AMD) in a subject with such need, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 10-21 or the pharmaceutical composition according to claim 23.

25. The method of claim 24, wherein the rAAV or pharmaceutical composition is administered periocularly, intravitreally, suprachoroidally, or subretinally, at a dose of about 10 8 One vector genome (vg) / eye to approximately 10 13 A dose of vg / eye, or preferably about 6 × 10⁻⁶. 9 vg / eye to approximately 6×10 10 A dose of vg / eye was administered to the subject.

26. The method of claim 25, wherein the rAAV or pharmaceutical composition is administered intravitreally at about 1 x 10 9 vg / eye to approximately 1 x 10 10 A dose of vg / eye was administered to the subject.

27. A method for treating geographic atrophy (GA) in a subject with this need, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 10 to 21 or a pharmaceutical composition according to claim 23.

28. The method of claim 27, wherein the rAAV or pharmaceutical composition is administered periocularly, intravitreally, suprachoroidally, or subretinally, at a dose of about 10 8 One vector genome (vg) / eye to approximately 10 13 A dose of vg / eye, or preferably about 6 × 10⁻⁶. 9 vg / eye to approximately 6×10 10 A dose of vg / eye was administered to the subject.

29. The method of claim 28, wherein the rAAV or pharmaceutical composition is administered intravitreally at about 1 x 10 9 vg / eye to approximately 1 x 10 10 A dose of vg / eye was administered to the subject.

30. A method for delivering rAAV according to any one of claims 10 to 21 or a pharmaceutical composition according to claim 23 to the eye of a subject, wherein the rAAV or pharmaceutical composition is administered to the subject via periocular, intravitreal, suprachoroidal, or subretinal administration.

31. The method of claim 30, wherein the rAAV or pharmaceutical composition is administered to the eye of the subject via intravitreal administration.

32. An isolated polypeptide comprising a VH or VHH domain of C5, preferably a complement factor C5 and / or a proteolytic derivative C5a and / or C5b, specifically binding to C5, wherein the VH or VHH domain comprises one or more complement-determining regions (CDRs) having at least 75% identity with CDRs selected from SEQ ID No: 69-71, 66-68 and 72-92.

33. The isolated polypeptide according to claim 32, wherein the CDR of the VH or VHH domain comprises: CDR1 selected from SEQ ID No: 69, 66, 72, 75, 78, 81, 84, 87, 90, and a sequence having at least one, at least two, or at least three amino acid differences relative to SEQ ID No: 69, 66, 72, 75, 78, 81, 84, 87, or 90; CDR2 selected from SEQ ID No: 70, 67, 73, 76, 79, 82, 85, 88, 91, and a sequence having at least one, at least two, or at least three amino acid differences relative to SEQ ID No: 70, 67, 73, 76, 79, 82, 85, 88, or 91; and CDR3 selected from SEQ ID No: 71, 68, 74, 77, 80, 83, 86, 89, 92, and a sequence having at least one, at least two, or at least three amino acid differences relative to SEQ ID No: Sequences 71, 68, 74, 77, 80, 83, 86, 89, and 92 have at least one, at least two, or at least three amino acid differences.

34. The isolated polypeptide according to claim 32 or 33, wherein the CDR is separated by frame regions (FR) FR1, FR2, FR3 and FR4.

35. The isolated polypeptide according to any one of claims 32-34, wherein the VH or VHH domain comprises a CDR sequence of any VH or VHH domain as shown in SEQ ID Nos. 58, 57, and 59-65, or a CDR sequence having at least 75% identity with it, preferably wherein the VH or VHH domain comprises a CDR1 having a sequence selected from: G–Y / N / S / T–I –S / F–Y / H / F / A / P / D–D / Q / A / Y / S / E – D / S / T / E / Y / W–D / K / G–M–G and G–Y / N / S / TI–F–Y / H / F / A / P / D–D / Q / A / Y / S / E–D / S / T / E / Y / W–D–M–G; and / or contain CDR2 with sequences selected from: A / T / G–I–D / T / N–V / Y / G / D–G–G / T / A–S / N / I–T–Y / N and A / T–I–D–V / Y / G / D–G–A–S / N–T–Y.

36. The isolated polypeptide according to claim 35, wherein the VH or VHH domain comprises an amino acid sequence as shown in any one of SEQ ID No: 58, 57 and 59-65, or an amino acid sequence having at least 75% identity with it.

37. The isolated polypeptide according to any one of claims 32-35, wherein the VH or VHH domain is a humanized VH or VHH domain.

38. The isolated polypeptide according to claim 37, wherein the VH or VHH domain comprises an amino acid sequence of any one of SEQ ID No: 141, 137-140 and 142-163, or comprises an amino acid sequence having at least 75% identity with it.

39. The isolated polypeptide according to any one of claims 32-38, wherein the VH or VHH domain is coupled to a tag, preferably a Strep or his6 tag, more preferably wherein the his6 tag comprises or is composed of the amino acid sequence shown in SEQ ID NO:93 and is located at the C-terminus of the VH or VHH domain.

40. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 32-39.

41. A recombinant adeno-associated virus (rAAV) comprising (i) a variant AAV capsid protein comprising a heteropeptide of 7, 8, 9, 10, or 11 amino acids covalently inserted into the GH ring of the capsid protein relative to the corresponding parental AAV capsid protein, wherein the peptide insertion fragment comprises the amino acid sequence ISDQTKH (SEQ ID NO: 168), and (ii) the nucleic acid according to claim 40.

42. The rAAV according to claim 41, wherein the insert peptide has 1 to 3 spacer amino acids (Y1-Y3) at the amino terminus and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO:168), preferably wherein the insert peptide is LAISDQTKHA (SEQ ID NO:169).

43. The rAAV according to claim 41 or 42, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 183), or at a corresponding position in the capsid protein of another AAV serotype.

44. The rAAV according to any one of claims 41 to 43, wherein the capsid protein comprises relative to AAV2 (SEQ ID NO). One or more amino acid substitutions in VP1 of NO:183 or one or more corresponding substitutions in the capsid protein of another AAV serotype, preferably one or more of the following amino acid substitutions: MIL, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L and L735Q, more preferably P34A amino acid substitution.

45. The rAAV of claim 44, wherein the capsid protein comprises a P34A amino acid substitution relative to VP1 of AAV2, and comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the entire length of the amino acid sequence shown in SEQ ID NO:170, preferably wherein the capsid protein comprises the amino acid sequence shown in SEQ ID NO:

170.

46. ​​The rAAV according to any one of claims 41 to 45, wherein the rAAV exhibits increased infectivity in retinal cells, preferably at least 2-fold increased infectivity, compared to the infectivity of retinal cells of AAV containing the corresponding parental AAV capsid protein.

47. The rAAV according to any one of claims 41 to 46, wherein the variant AAV capsid protein comprises an amino acid sequence having 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

170.

48. The rAAV according to any one of claims 41 to 47, wherein the rAAV comprises a heterologous nucleic acid from 5' to 3' comprising: (a) an inverted terminal repeat, (b) a promoter, (c) a nucleotide sequence encoding a polypeptide according to any one of claims 32-39, (d) a polyadenylated sequence and / or a WPRE sequence, and (e) an inverted terminal repeat.

49. The rAAV of claim 48, wherein the reverse end repetition in the rAAV is an AAV2 reverse end repetition.

50. The rAAV according to any one of claims 41 to 49, wherein the promoter is a pervasive promoter.

51. The rAAV of claim 50, wherein the promoter is a CAG promoter.

52. The rAAV according to any one of claims 41 to 49, wherein the promoter is a tissue-specific promoter.

53. A host cell comprising rAAV according to any one of claims 41 to 52.

54. A pharmaceutical composition comprising rAAV according to any one of claims 41 to 52, and a pharmaceutically acceptable carrier, diluent, excipient, or buffer.

55. A method for treating dry age-related macular degeneration (dry AMD) in a subject with this need, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 41 to 52 or a pharmaceutical composition according to claim 54.

56. The method of claim 55, wherein the rAAV or pharmaceutical composition is administered periocularly, intravitreally, suprachoroidally, or subretinally, at a dose of about 10 8 One vector genome (vg) / eye to approximately 10 13 A dose of vg / eye, or preferably about 6 × 10⁻⁶. 9 vg / eye to approximately 6×10 10 A dose of vg / eye was administered to the subject.

57. The method of claim 56, wherein the rAAV or pharmaceutical composition is administered intravitreally at about 1 x 10 9 vg / eye to approximately 1 x 10 10 A dose of vg / eye was administered to the subject.

58. A method for treating geographic atrophy (GA) in a subject with this need, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 41 to 52 or a pharmaceutical composition according to claim 54.

59. The method of claim 58, wherein the rAAV or pharmaceutical composition is administered periocularly, intravitreally, suprachoroidally, or subretinally, at a dose of about 10 8 One vector genome (vg) / eye to approximately 10 13 A dose of vg / eye, or preferably about 6 × 10⁻⁶. 9 vg / eye to approximately 6×10 10 A dose of vg / eye was administered to the subject.

60. The method of claim 59, wherein the rAAV or pharmaceutical composition is administered intravitreally at about 1 x 10 9 vg / eye to approximately 1 x 10 10 A dose of vg / eye was administered to the subject.

61. A method for delivering rAAV according to any one of claims 41 to 52 or a pharmaceutical composition according to claim 54 to the eye of a subject, wherein the rAAV or pharmaceutical composition is administered to the subject via periocular, intravitreal, suprachoroidal, or subretinal administration.

62. The method of claim 61, wherein the rAAV or pharmaceutical composition is administered to the eye of the subject via intravitreal administration.

63. An isolated polypeptide comprising a VH or VHH domain that specifically binds to human complement factor C3 (SEQ ID NO:177), wherein the VH or VHH domain is bound to an epitope within amino acids 1540-1600 of SEQ ID NO:177, or an amino acid sequence having at least 80% identity with it.

64. The isolated polypeptide of claim 63, wherein the VH or VHH domain is bound to an epitope comprising, substantially comprising, or comprising an epitope of amino acids 1548-1561 of SEQ ID NO:177 or an amino acid sequence having at least 80% identity with therewith, and / or bound to an epitope comprising, substantially comprising, or comprising an epitope of amino acids 1568-1573 of SEQ ID NO:177 or an amino acid sequence having at least 80% identity with therewith, and / or bound to an epitope comprising, substantially comprising, or comprising an epitope of amino acids 1583-1591 of SEQ ID NO:177 or an amino acid sequence having at least 80% identity with therewith.

65. The isolated polypeptide according to claim 63 or 64, wherein the polypeptide comprises CDR1 of SEQ ID NO: 54 or an amino acid sequence having at least 70% identity with it, CDR2 of SEQ ID NO: 55 or an amino acid sequence having at least 70% identity with it, and CDR3 of SEQ ID NO: 56 or an amino acid sequence having at least 70% identity with it, preferably wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 70% identity with it.

66. An isolated polypeptide comprising a VH or VHH domain that specifically binds to human complement factor C3 (SEQ ID NO:177), wherein the VH or VHH domain binds to a non-near-neighbor epitope contained within amino acids 110-130 and 430-510 of SEQ ID NO:

177.

67. The isolated polypeptide of claim 66, wherein the VH or VHH domain is bound to an epitope comprising, substantially comprising, or comprising an epitope of amino acids 114-124 of SEQ ID NO:177 or an amino acid sequence having at least 80% identity with therewith, and / or bound to an epitope comprising, substantially comprising, or comprising an epitope of amino acids 497-505 of SEQ ID NO:177 or an amino acid sequence having at least 80% identity with therewith.

68. The isolated polypeptide according to claim 66 or 67, wherein the polypeptide comprises CDR1 of SEQ ID NO:18 or an amino acid sequence having at least 70% identity with it, CDR2 of SEQ ID NO:19 or an amino acid sequence having at least 70% identity with it, and CDR3 of SEQ ID NO:20 or an amino acid sequence having at least 70% identity with it, preferably wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:98 or an amino acid sequence having at least 70% identity with it.

69. An isolated polypeptide comprising a VH or VHH domain that specifically binds to human complement factor C5 (SEQ ID NO:175), wherein the VH or VHH domain competitively inhibits the binding of eculizumab to human C5 protein.

70. The isolated polypeptide according to claim 69, wherein the polypeptide comprises CDR1 of SEQ ID NO: 69 or an amino acid sequence having at least 70% identity with it, CDR2 of SEQ ID NO: 70 or an amino acid sequence having at least 70% identity with it, and CDR3 of SEQ ID NO: 71 or an amino acid sequence having at least 70% identity with it, preferably wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 141 or an amino acid sequence having at least 70% identity with it.

71. A heterodimeric fusion protein comprising a first domain and a second domain, the first domain comprising a polypeptide according to any one of claims 1-8 and 32-39, the second domain comprising an immunoglobulin Fc domain, preferably wherein the Fc domain is an IgG4 Fc domain, wherein the first domain and the second domain are connected by a linker, optionally wherein the linker is an IgG2 hinge.

72. The fusion protein of claim 71, wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NO:178-180, or an amino acid sequence having at least 70% identity with it.

73. A homodimeric fusion protein comprising a first domain and a second domain identical to the first domain, the first domain comprising a polypeptide according to any one of claims 1-8 and 32-39, preferably wherein the first domain and the second domain are connected by a linker, optionally wherein the linker is a polyglycine-serine (G4S) linker, preferably a (G4S)2 linker.

74. The fusion protein of claim 73, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 181 or 182, or an amino acid sequence having at least 70% identity with it.

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