Novel complement system inhibitory antibodies

Abstract

Novel anti-C3 and anti-C5 antibodies are provided that can modulate complement activity by specifically binding to human complement factors C3, C3a, C3b, and / or C5. Recombinant AAVs (rAAVs) containing variant adeno-associated virus (AAV) capsids and transgenes encoding anti-C3 and / or anti-C5 antibodies are also described herein. Methods for delivering transgenes to the retina by contacting retinal cells with rAAVs, as well as methods for treating atrophic age-related macular degeneration and geographic atrophy secondary to age-related macular degeneration disorders, are also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 503,745 and 63 / 503,768, both filed on May 23, 2023, the entire contents of each of which are incorporated herein by reference. Submission of Sequence Listing via EFS - WEB

[0002] A computer - readable XML file named "090400 - 5023 - WO Sequence Listing", created on May 21, 2024, having a file size of approximately 249,282 bytes, contains the sequence listing of this application, the entire contents of which are incorporated herein by reference.

Background Art

[0003] Background of the Invention Some human diseases are caused by complement dysregulation that leads to complement - mediated self - tissue injury. Complement dysregulation can result from somatic or germline mutations in complement regulatory factors or regulator - related genes, and these regulators may no longer function properly. 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 have focused on the development of reagents such as monoclonal antibodies (mAbs), peptides, or other small molecules that bind to and block specific complement components of the alternative or terminal pathways. A clinically validated example is eculizumab, a humanized mAb against complement C5, which is approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Other approaches described include mAbs against factor B (fB), factor D (fD), or properdin (fP), and cyclic peptides that bind to and inhibit C3. The limitations of these approaches are that they require repeated and inconvenient intravenous (IV) dosing in patients. Additionally, since these treatments block the alternative or terminal pathways, they carry a risk of compromising host defense. Indeed, patients receiving eculizumab therapy need to be vaccinated against strains of bacteria that can cause lethal meningitis, and these patients are also placed on prophylactic antibiotic therapy before being treated with the approved mAb drug.

[0005] In other approaches, recombinant regulatory proteins such as proteins containing the minimal domains of soluble DAF, CR1, CRIg, and the fluid-phase regulator fH (N-terminal short consensus repeats [SCR] 1-5 and C-terminal SCR19-20), or a fusion protein between fH and CR2 (TT30) have been tested. However, large-scale heterologous expression of such proteins as therapeutic agents requires significant effort, and animal studies have shown that their in vivo clearance rate after administration is rapid, making such treatment strategies cumbersome and less practical due to the need for multiple and frequent administrations of such protein drugs. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] There is still a need in the art for compositions useful for treating complement-mediated diseases with higher and longer-lasting efficacy. MEANS FOR SOLVING THE PROBLEMS

[0007] Summary of the Invention Polypeptides comprising a single variable domain (VHH or nanobody) on a heavy chain, which can modulate complement activity by specifically binding to human complement factors C3, C3a, C3b, C5, C5a and / or C5b, are described herein. Preferably, the polypeptide comprises a VH or VHH domain of or derived from a camelid heavy chain antibody.

[0008] In some embodiments, the VH or VHH domain includes one or more complementarity-determining regions (CDRs) as described in Tables 1 and 2. In preferred embodiments, the VH or VHH domain includes CDRs (CDR1, CDR2, and CDR3) of one or more antibodies selected from the group consisting of SEQ ID NOs: 1-14 and SEQ ID NOs: 57-65. In other preferred embodiments, the CDR1, CDR2, and CDR3 are separated by frame regions FR1, FR2, FR3, and FR4 of the VH or VHH domain.

[0009] In some preferred embodiments, the VH or VHH domain comprises or consists of one of the amino acid sequences described in SEQ ID NOs: 1 to 14 or one of the amino acid sequences described in SEQ ID NOs: 57 to 65.

[0010] In other preferred embodiments, the VH or VHH domain is humanized and comprises or consists of one of the amino acid sequences described in SEQ ID NOs: 94-136 or one of the amino acid sequences described in SEQ ID NOs: 137-163.

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

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

[0013] Also provided are recombinant AAV (rAAV) virions comprising a variant AAV capsid sequence that encapsulates a heterogeneous nucleic acid containing a nucleotide sequence encoding an anti-C3 and / or anti-C5 antibody as described herein. Preferably, the nucleotide sequence encoding the antibody is operably ligated to an expression control sequence.

[0014] The rAAV variant AAV capsid protein comprises a capsid protein containing a peptide insert ("heterogeneous peptide" or "peptide insert") of about 7 to 20 amino acids in the GH loop of the capsid protein, preferably in the surface-exposed region of the GH loop, compared to the corresponding parent AAV capsid protein, wherein the peptide insert contains the amino acid sequence ISDQTKH (SEQ ID NO: 168). In some preferred embodiments, the peptide insert contains 1 to 3 spacer amino acids (Y1 to Y3) at the amino and / or carboxyl terminals of the amino acid sequence ISDQTKH (SEQ ID NO: 168), each of Y1 to Y3 independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. In a particularly preferred embodiment, the peptide insert contains, essentially consists of, or consists of the amino acid sequence LAISDQTKHA (SEQ ID NO: 169). In certain preferred embodiments, the peptide is inserted after any of the amino acids at positions 584-591 in VP1 of AAV2 or at the corresponding position in another AAV serotype (i.e., the insertion site is between amino acids 587 and 588 of VP1 of AAV2, or between amino acids 588 and 589, 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 of AAV2, 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 for the VP1 capsid of AAV2, or one or more corresponding substitutions in another AAV serotype, preferably the capsid protein further comprises a P34A amino acid substitution for the VP1 capsid of AAV2, or the 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 described herein to a mammalian subject, comprising administering to the mammal an effective amount of the rAAV described herein or a pharmaceutical composition comprising the same, preferably the rAAV or pharmaceutical composition being administered by intravitreal injection. In some embodiments, the heterologous nucleic acid is delivered to target retinal cells, e.g., target photoreceptor cells (e.g., rods; cones), 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, detectable plasma levels of antibodies 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 after administration of rAAV to the subject. In certain preferred embodiments, rAAV is administered to the subject by intravitreal administration.

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

[0018] In other embodiments, a method is provided for treating complement-related disorders by delivering to a subject rAAV or a pharmaceutical composition comprising rAAV as described herein. Complement-related disorders 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 microangiogenic hemolytic anemia of AMD, thrombocytopenia, acute renal failure, paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, ischemic stroke, and / or bacterial infections caused by the recruitment of bacterial pathogens.

[0019] In a further embodiment, a method is provided for treating atrophic age-related macular degeneration (AMD) (e.g., late atrophic AMD) in a subject by delivering an effective amount of the rAAV described herein or a pharmaceutical composition comprising rAAV to a subject in need. In a related embodiment, 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 by intravitreal injection. [Brief explanation of the drawing]

[0020] [Figure 1A] Figures 1A-C: Induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPEs) were transduced with the C3-N10 rAAV transgene (encoding the anti-C3 antibody of SEQ ID NO: 14) at three infection multiplicity (MOI) values ​​of 5,000, 10,000, and 20,000. Dose-response behavior was observed for nanobody (VHH) expression (Figure 1A) and MAC formation (Figure 1B). Immunocytochemistry describes the inhibition of complement membrane invasion complex (MAC) deposition in iPSC-RPEs after zymosan (a potent activator of the complement II pathway) and after C3-N10 rAAV transduction (Figure 1C). [Figure 1B] Same as above.

[0021] [Figure 2-1] Complement inhibition by wild-type lead nanobodies in the complement II pathway, the complement lectin pathway, and the classical complement pathway. The complement inhibitory activity of all three wild-type nanobodies was compared using Wieslab's complement II pathway, MBL / complement lectin pathway, and classical complement pathway assay kits. As shown in Figures 2A (II pathway), 2B (lectin pathway), and 2C (classical pathway), C5S2 exhibits similarly potent inhibitory activity against all three pathways, while C3S3 shows activity only against the II pathway. C3N10 is thought to have a more potent inhibitory effect against the II pathway and milder efficacy against the lectin and classical pathways. [Figure 2-2] Same as above.

[0022] [Figure 3-1] IC50 potency of wild-type and humanized nanobodies against the complement II pathway. To evaluate the potency difference between the wild-type and its humanized counterpart, each nanobodies were serially diluted and tested using the Wieslab Complement II Pathway Kit, and their IC50 was calculated using GraphPad Prism. As shown for anti-C3 (Figures 3A and 3B) and anti-5C nanobodies (Figure 3C), all humanized versions retain IC50 activity similar to their original wild-type counterparts. [Figure 3-2] Same as above.

[0023] [Figure 4] IC50 potency of C3N10 and C3N10.3 against the classical (Figure 4A) and lectin (Figure 4B) pathways. To compare the inhibitory activity between wild-type and humanized versions of C3N10 within the classical and lectin complement pathways, these nanobodies were serially diluted and samples were tested using their respective Wieslab assay kits. Both versions exhibited similar IC50 potency against these tested pathways.

[0024] [Figure 5] Figure 5 shows the cross-reactivity of wild-type nanobody leads with mouse serum. The mouse cross-inhibitory activity of wild-type nanobody was tested using Hycult's C3 mouse ELISA kit and the mouse anti-mouse C3 antibody BB5.1 as a control. None of the anti-human C3 or C5 nanobody groups, C3N10, C3S3, or C5S2 showed inhibitory activity against the mouse serum-mediated complement II pathway.

[0025] [Figure 6]IC50 of wild-type and humanized nanobodies against complement II pathway in cynomolgus monkey serum. Both wild-type and its humanized counterparts were serially diluted, and their cynomolgus monkey serum-mediated complement II pathway inhibitory activity was calculated using the respective Wieslab assays. C3N10 WT and C3N10.3 (Figure 6A), along with C3S3 WT and C3S3.2 (Figure 6B), showed activity against cynomolgus monkey complement II pathway, exhibiting similar IC50 values ​​across variants. C5S2 and C5S2.2 showed no inhibitory activity in this NHP serum-mediated assay (data not shown).

[0026] [Figure 7] Functional efficacy of anti-C3 and C5 VHH-Fc dimers in the complement II pathway in human serum. Humanized anti-C3 and C5 nanobodies were re-engineered as G2-hinge-G4-Fc fusion proteins, and their IC50s were measured by serial dilution in the Wieslab complement II pathway assay. Data were graphed, and IC50s were calculated using GraphPad Prism (CS52.2-Fc IC50MAC=9.076nM; C3S3-Fc50MAC=23.80nM; C3N10.3-Fc50MAC=49.31nM).

[0027] [Figure 8] Amino acid interactions between C3N10.3 and human C3 protein. Humanized C3N10.3 was bound to human C3 protein, and cross-linked mass spectrometry (CovalX) was performed to identify potential binding peptide epitopes. Three epitopes within amino acids 1540-1560 of human C3 protein were identified (amino acids 1548-1551, 1568-1573, and 1583-1591).

[0028] [Figure 9]Amino acid interactions between nanobody C3S3.2 molecules and human C3 protein. Humanized C3S3.2 molecules were bound to human C3 protein, and cross-linked mass spectrometry (CovalX) was performed to identify potential binding peptide epitopes. An epitope between amino acids 110–130 (amino acids 114–124) was identified in human C3 protein, and another epitope between amino acids 490–510 (amino acids 497–502 and 502–505) was identified.

[0029] [Figure 10] Figure 10 shows the potential binding domains of C3N10.3 and C3S3.2 to the human C3 protein. Based on cross-linking mass spectrometry data, the binding regions of C3N10.3 and C3S3.2 to the human C3 protein are shown, with the former binding within the C345C region of the alpha chain, and the latter interacting across both the MG1 and MG5 domains of the beta chain.

[0030] [Figure 11] Figure 11 shows that eculizumab and C5S2.2 cross-block the binding of human C5:probe anti-human Fc sandwiches. Using a Gator / BLI instrument, we tested whether C5S2.2 cross-blocks eculizumab, a clinically approved anti-human C5-targeted hIgG4 antibody. Anti-human Fc probes were pre-bound with eculizumab and then washed. Human C5, either alone or at increasing concentrations, pre-bound with C5S2.2, was then bound to antibody-loaded probes. Binding dynamics were observed only with respect to free C5 protein, indicating that pre-bound C5S2.2 VHH blocks the C5-binding epitope of eculizumab. Time (seconds) is on the X-axis, and shift (nm) is on the Y-axis.

[0031] [Figure 12]Figure 12 shows that eculizumab and C5S2.2 cross-block the binding of human C5 to the human C5:probe anti-StepII sandwich. Using a Gator / BLI instrument, we tested whether C5S2.2 cross-blocks eculizumab, a clinically approved anti-human C5-targeted hIgG4 antibody. The anti-StrepII probe was pre-conjugated with Strep-tagged anti-human C5 C5S2.2 VHH. After washing, the nanobody-loaded probe was immersed in human C5-containing binding buffer, and the binding rate was observed. Subsequently, when this pre-conjugated complex was immersed in an eculizumab-containing solution, no interaction was observed, suggesting that the latter's binding epitope was blocked. Time (seconds) is on the X-axis, and shift (nm) is on the Y-axis.

[0032] [Figure 13-1] Figures 13A-C show the binding of C5S2.2 to human and cynomolgus monkey C5 mutants. Eculizumab is refractory to two naturally occurring variants: the R885H human polymorphism and the W917S human vs. cynomolgus monkey variant. Human C5 R885H, human C5 W917S, and cynomolgus monkey C5 S917W mutant proteins were recombinantly produced, and the affinity of C5S2.2 to them was compared with eculizumab using Gator (Figures 14A-C). [Figure 13-2] Same as above. [Figure 13-3] Same as above.

[0033] [Figure 14-1] Figures 14A–C show the binding of eculizumab to human and cynomolgus monkey C5 mutants. Eculizumab is refractory to two naturally occurring variants: the R885H human polymorphism and the W917S human vs. cynomolgus monkey variant. Human C5 R885H, human C5 W917S, and cynomolgus monkey C5 S917W mutant proteins were recombinantly produced, and the affinity of eculizumab to them was reflected using Gator. [Figure 14-2] Same as above. [Figure 14-3] Same as above.

[0034] [Figure 15] Figure 15 shows the percentage of anti-drug antibody (ADA) response in normal human donors to wild-type and humanized C3 and C5 nanobodies (Cap = caplacizumab / Cablivi; Beovu = brolucizumab). Serum from normal human donors was tested for ADA against the enumerated nanobodies and controls using a mesoscale exploratory immunoassay. Samples were diluted 1:100 and added to plate wells passively coated with the respective antibodies. Bound human antibodies were detected using Sulfo-TAG goat anti-human IgG and measured in Meso Sector S600.

[0035] [Figure 16] Figures 16A–C show the functional evaluation of VHH expressed after plasmid transfection with complement cascade-related protein degradation products. Figure 16A: C3 VHH inhibitors, rather than C5 VHH inhibitors, prevent C3 cleavage so that it is detectable by the absence of C3a degradation products. Figure 16B: C3 and C5 VHH inhibitors prevent C5 cleavage so that it is detectable by the absence of C5a degradation products. Figure 16C: C3 VHH inhibitors, rather than C5 VHH inhibitors, prevent Bb cleavage so that it is detectable by the absence of B factor degradation products. v, version; h, humanized; kDa, kilodaltons; NT, untransfected.

[0036] [Figure 17] Figure 17 is a Western blot showing the single product for each VHH variant examined after transduction with VHH-supported AAV. NT, untransduced; kDa, kilodaltons; MOI 20,000; 33 μg of protein loaded per lane; 7 days post-transduction.

[0037] [Figure 18]Figures 18A-C show the expression of secreted VHHs (C3N10 and humanized C3N10, Figure 18A; C3S3 and humanized C3S3, Figure 18B; C5S2 and humanized C5S2, Figure 18C) after AAV transduction, as measured by ELISA, and the observed dose response (MOI of 1,000, 5,000, and 20,000) for each VHH. NT, untransduced; MOI, infection multiplicity; error bars ± standard deviation; n=3 wells / MOI.

[0038] [Figure 19] Figure 19 shows the function of secreted VHHs (C3N10, C3N10h, C353, C353h, C5S2, C5S2h) as measured by complement inhibition, expressed as a percentage. Each VHH showed some level of complement inhibition. NT: non-transduced; h: humanized; MOI: infection multiplicity; error bars ± standard deviation; n=3 wells / MOI. [Modes for carrying out the invention]

[0039] Detailed description of the invention definition

[0040] As used herein, “antibody” in its broadest sense refers to a polypeptide or protein having an immunoglobulin-like domain capable of recognizing and binding to an antigen, and includes full-size antibodies, their individual chains and wholes, domains or fragments thereof (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 includes at least one complementarity-determining region (CDR). This disclosure relates primarily to single-variable-domain antibodies of heavy-chain (VHH) antibodies. As used herein, the terms “single-domain antibody,” “single-variable-domain antibody,” “VHH antibody,” and “nanobody” have the same meaning and refer to the variable region of the heavy chain of an antibody, to construct 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 three or fewer CDRs. Generally, antibodies naturally lacking the light chain and heavy-chain constant region 1 (CH1) are obtained first, and therefore the variable region of the heavy chain of an antibody is cloned to construct a single-domain antibody (VHH) consisting of only one heavy-chain variable region. The VHH antibody is preferably derived from a llama.

[0041] An "antigen" is a molecule containing at least one epitope. Antigens can be, for example, polypeptides, nucleic acids, polysaccharides, proteins, lipoproteins, or glycoproteins.

[0042] The "complementarity-determining region" or "CDR" is a hypervariable region of the antigen-binding domain of an antibody. CDRs are scattered among more conserved regions called framework regions (FRs). Therefore, the antigen-binding domain of an antibody may typically contain one or more CDRs and FRs within each variable domain, three CDRs and four FRs arranged from the amino-terminus to the carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0043] An "epitope" is a determinant that can specifically bind to an antibody. Epitopes may be contained within polypeptides or proteins, for example. Epitopes may be continuous or discontinuous, and a discontinuous epitope is a structural epitope on an antigen that is formed from at least two separate regions in the primary sequence of a protein, nucleic acid, or polysaccharide.

[0044] The term "affinity" refers to the strength of the binding between an antibody and its antigen. Antibody affinity can be defined using the dissociation constant KD, which is an equilibrium constant that measures the tendency of molecular complexes to reversibly separate (dissociate) into molecules that form a complex. In one embodiment, K D is, k off and k on k is the rate constant for the binding and dissociation of molecular complexes, relative to k off / k on It is defined as follows. Preferably, affinity is defined as the dissociation constant K based on the IC50 value. D It is determined by calculating [a certain value]. Therefore, affinity is measured as apparent affinity.

[0045] 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, but the same polynucleotide separated from a neighboring nucleic acid that exists naturally is considered "isolated."

[0046] As used herein, “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. “Stop codons” (TAG, TGA, or TAA) are typically not translated into amino acids, but can be considered part of a coding region; however, any flanking sequences, such as promoters, ribosome-binding sites, transcription terminators, or introns, are not part of a coding region. The boundaries of a coding region are typically determined by a 5' start codon encoding the amino terminus of the resulting polypeptide and a 3' translation stop codon encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions may be present in a single polynucleotide construct, for example, on a single vector, or in separate polynucleotide constructs, for example, on separate (different) vectors. Thus, a single vector may contain only one coding region or two or more coding regions.

[0047] As used herein, the term “regulatory region” refers to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding region that affects the transcription, RNA processing, stability, or translation of the relevant coding region. Regulatory regions may include promoters, translational reader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. When expression of a coding region in eukaryotic cells is intended, polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence.

[0048] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule,” and refers to polymers of nucleotides.

[0049] A gene product, such as a polynucleotide encoding a polypeptide, may include a promoter and / or other transcriptional or translational regulatory elements operably bound to one or more coding regions. In operable binding, a gene product, such as a coding region for a polypeptide, is bound to one or more regulatory regions such that the expression of the gene product is under the influence or control of the regulatory regions. For example, a coding region and a promoter are "operably bound" if the induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and the nature of the binding 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 of the DNA template to be transcribed. Other transcriptional regulatory elements, other than promoters, such as enhancers, operators, repressors, and transcription termination signals, may also be operably bound to a coding region to direct the expression of a gene product.

[0050] A “transcriptional regulatory sequence” refers to a DNA regulatory sequence, such as a promoter, enhancer, or terminator, that provides for the expression of a coding sequence in a host cell. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as, but are not limited to, cytomegalovirus (early promoter linked to intron A), Simian virus 40 (early promoter), and promoter and enhancer segments derived from retroviruses (such as Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit betaglobin, as well as other sequences that can regulate gene expression in eukaryotic cells. Further preferred transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters induced by interferon or interleukin).

[0051] Similarly, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from picornaviruses (in particular, internal ribosome entry sites, also known as IRES or CITE sequences).

[0052] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or 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 polypeptides. Expression produces a “gene product.” As used herein, a gene product may be a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from the transcript. Gene products described herein include, further, nucleic acids having post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, binding to other protein subunits, or proteolytic cleavage.

[0053] The terms "promoter" and "promoter sequence" are used interchangeably and refer to DNA sequences that can control the expression of a coding sequence or functional RNA. Generally, coding sequences are located 3' to the promoter sequence. Promoters may be entirely derived from native genes, or composed of different elements derived from different naturally occurring promoters, or may even include synthetic DNA segments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. Promoters that cause gene expression in many cell types over many times are generally called "constitutive promoters." Promoters that cause gene expression in specific cell types are generally called "cell-specific promoters" or "tissue-specific promoters." Promoters that cause gene expression at specific developmental or cell differentiation stages are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoter that induces gene expression after exposure or treatment of cells with promoter-inducing drugs, biomolecules, chemicals, ligands, light, etc., is generally referred to as an "inducible promoter" or "regulatory promoter." It is further recognized that DNA fragments of different lengths can possess identical promoter activity, as the precise boundaries of regulatory sequences are often not fully defined.

[0054] The term "plasmid" refers to an extrachromosomal element that is not part of the cell's central metabolism and often carries genes, usually in the form of a circular double-stranded DNA molecule. Such elements may be single-stranded or double-stranded DNA or RNA, originating from any source, with several nucleotide sequences ligated or rearranged into a unique construct that can introduce a promoter fragment and a DNA sequence for a selected gene product into the cell, along with a suitable 3' untranslated sequence. These sequences may be autonomous replicating sequences, genomic integration sequences, linear, circular, or supercoiled phages or nucleotide sequences.

[0055] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with respect to another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids is the same when comparing the two sequences. As described in aspects of the present invention, sequence identity is related to sequence homology. Homologous comparisons can be performed visually or, more commonly, with the help of readily available sequence comparison programs. Sequence similarity or sequence homology can be determined in several different ways. Commercial computer programs can also calculate percentage (%) homology between two or more sequences and sequence identity shared by two or more amino acid or nucleic acid sequences. Sequence homology can be generated by any of several 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) package from Madison, Wis., USA. Other alignment techniques are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Alternatively, sequences can be aligned using GAP programs that employ the Needleman and Wunsch alignment methods. See J. Mol. Biol. 48: 443-453 (1970).

[0056] Homology percentage can be calculated on consecutive sequences; that is, one sequence is aligned with the other, and each amino acid or nucleotide in one sequence is directly compared to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "non-gap" alignment. Typically, such non-gap alignments are performed only over a relatively short number of residues.

[0057] While this is a very simple and consistent method, it fails to take into account, for example, that in otherwise identical sequence pairs, a single insertion or deletion can make subsequent amino acid residue alignment impossible, and thus potentially lead to a significant decrease in homology % when overall alignment is performed. As a result, many sequence comparison methods are designed to produce an optimal alignment that takes possible insertions and deletions into account without excessively penalizing the overall homology or identity score. This is achieved by inserting “gaps” during sequence alignment in an attempt to maximize partial homology or identity.

[0058] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, allowing, for example, a sequence alignment with as few gaps as possible for the same number of identical amino acids (reflecting a higher relevance between the two sequences being compared) to achieve a higher score than one with many gaps. Typically, an "affinity gap cost" is used, imposing a relatively high cost for the presence of gaps and a smaller penalty for each subsequent residue within the gap. This is the most commonly used gap scoring system. A high gap penalty can, of course, result in an optimized alignment with fewer gaps. Many alignment programs allow modification of the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0059] Therefore, calculating the maximum homology percentage first requires the production of an optimal alignment that takes gap penalties into account. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (Devereux et al., 1984 Nuc. Acids Research 12 p387). Other software capable of performing sequence comparisons include, but are not limited to, the BLAST 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 GENEWORKS integrated comparison tool. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. A newer tool called BLAST2 Sequences is also available for comparing 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 of the National Institutes for Health).

[0060] While the final homology percentage can be measured in relation to identity, the alignment process itself is not typically based on absolute pairwise comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix, which is the default matrix for the BLAST integration program. The GCG Wisconsin program generally uses either the publicly available default values ​​or, if provided, a custom symbolic comparison table (see the user manual for further details). For some applications, it is preferable to use the publicly available default values ​​for the GCG package, or the default matrix for other software such as BLOSUM62.

[0061] Alternatively, the homology percentage may be calculated using multiple alignment characteristics in DNASIS® (Hitachi Software) based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244). Once the software yields the optimal alignment, the homology %, preferably the sequence identity %, can be calculated. The software typically does this as part of a sequence comparison and generates a numerical result.

[0062] Furthermore, sequences may have silent changes resulting from the deletion, insertion, or substitution of amino acid residues, leading to functionally equivalent substances. Intentional amino acid substitutions may be constructed based on similarities in amino acid properties (such as residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity), and therefore, it is useful to classify amino acids together with their functional groups. Amino acids may also be classified together based solely on the properties of their side chains. However, it is more useful to include mutation data as well. Sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of Venn diagrams (Livingstone CD and Barton GJ (1993) “Protein sequence alignments: a strategy 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). Conservative substitutions may be made, for example, according to the following table which describes the generally accepted Venn diagram classification of amino acids. [Table 13]

[0063] Embodiments of the present invention include sequences (both polynucleotides and polypeptides) that may include homologous substitutions (as used herein, both substitution and replacement mean the exchange of an existing amino acid residue or nucleotide with an alternative residue or nucleotide), that is, in the case of amino acids, homogeneous substitutions such as basic to basic, acidic to acidic, and polar to polar. Non-homologous substitutions may also occur, including the inclusion of non-natural amino acids such as ornithine (hereinafter referred to herein as Z), ornithine diaminobutyrate (hereinafter referred to herein as B), norleucine ornithine (hereinafter referred to herein as O), pyriylalanine, thienylalanine, naphthylalanine, and phenylglycine, for one class of residue to another or as a substitute.

[0064] The variant amino acid sequence may include preferred spacer groups that can be inserted between any two amino acid residues in the sequence, including alkyl groups such as methyl, ethyl, or propyl groups, in addition to amino acid spacers such as glycine or β-alanine residues. Those skilled in the art will be able to understand further variant forms, including the presence of one or more amino acid residues in peptoid form. To avoid doubt, “peptoid form” is used to refer to variant amino acid residues in which the α-carbon substituent is located on the nitrogen atom of the residue rather than on the α-carbon. Processes for preparing peptides in peptoid form are known in the art, e.g., Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0065] The above terms “amino acid substitution” and its synonyms are intended to encompass modifications of an amino acid sequence by replacing one amino acid with another amino acid to be substituted. The substitution may be a conservative substitution. It may also be a non-conservative substitution. In reference to two amino acids, the term “conservative” is intended to mean that those amino acids have a common characteristic recognized by those skilled in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. The common characteristic may also be amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with 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 substituting amino acids are well known to those skilled in the art and are not limited to, but include mutations in the nucleotide sequence encoding the amino acid sequence. In this specification, references to “one or more” are intended to encompass, for example, one, two, three, four, five, six, or more individual embodiments.

[0066] As used herein, the terms “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, in particular humans, and includes (a) preventing the occurrence of the disease (and / or symptoms caused by the disease) in a subject that is predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., stopping its onset; and (c) alleviating the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease), i.e., improving one or more symptoms of the disease.

[0067] As used herein, the term “treating complement factor H deficiency” may encompass reducing, mitigating, and / or improving symptoms associated with complement factor H deficiency, which may manifest as several different phenotypes, including asymptomatic recurrent bacterial infections and renal failure, as well as preventing the development of further symptoms. This typically involves decreased serum levels of factor H, complement component C3, and other terminal complement components, indicating activation of the complement II pathway. Serum levels of complement component C5 may also be decreased. This deficiency is associated with several renal diseases with fluctuating clinical findings and clinical progression, including C3 glomerulopathy and atypical hemolytic uremic syndrome. Furthermore, compositions and methods are provided herein for treating one or more of the following conditions: atrophic age-related macular degeneration (AMD), geographic atrophy secondary to AMD, atypical hemolytic uremic conditions (e.g., including syndromic microangiopathy-associated hemolytic anemia, thrombocytopenia, and acute renal failure), paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, and ischemic stroke, and / or for preventing or treating bacterial infections caused by the recruitment of bacterial pathogens (e.g., Aspergillus spp.; Borrelia burgdorferi; B. duttonii; B. recurrentis; Candida albicans; Francisella tularensis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pyogenes, or one of the five factor H binding proteins of B. burgdorferi (CRASP-1, CRASP-2, CRASP-3, CRASP-4, or CRASP-5)).

[0068] As used herein, the term “treating complement-related disorders” includes not only both of the complement factor H disorders identified above, but also alleviating, reducing, and / or improving the symptoms of other disorders associated with uncontrolled complement pathway II regulation.

[0069] "Complement-mediated disorders" may encompass symptoms associated with complement dysregulation that can manifest as several different phenotypes, including asymptomatic recurrent bacterial infections and, but not limited to, various tissue injuries, including renal disease. Unless otherwise specified, both homozygous and heterozygous subjects are included in this definition. 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 proteins (MCP), or by autoantibodies against them, or by gain-of-function mutations in other complement proteins, including, but not limited to, factors C3, C5, and B (fB). Complement dysregulation is typically, but not always, characterized by decreased serum levels of factor H, complement component C3, fB, and other terminal complement components, indicating activation of the complement II pathway and / or terminal complement pathway.Complement-mediated conditions that can be treated by the compositions and methods of the present invention include, but are not limited to, the following diseases with varying clinical findings and clinical progression: C3 glomerulopathy (formerly called membranoproliferative glomerulonephritis type II or MPGNII), of which there are two known forms - dense deposit disease (DDD) and C3 glomerulonephritis (C3GN); but are not limited to, atypical hemolytic uremic syndrome (aHUS), Shiga-like toxin-producing Escherichia coli HUS (STEC-HUS), and thrombotic microangiopathy (TMA), including thrombotic thrombocytopenic purpura (TTP); age-related macular degeneration (AMD), RPE degeneration, chorioretinal degeneration, photoreceptor degeneration, paroxysmal nocturnal hemoglobinuria (PNH), all organ and conditions including ischemia-reperfusion injury, 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), ANCA-mediated hemorrhagic lung injury and disease, ANCA glomerulonephritis, graft-versus-host disease (GvHD), acute or delayed graft rejection in organ transplantation, Crohn's disease, psoriasis, multiple sclerosis, antiphospholipid syndrome, pre-eclampsia, atherosclerosis, neuromyelitis optica (NMO), autoimmune bullous skin diseases, bullous pemphigoid (BP), retinal degenerative eye diseases including Alzheimer's disease (AD), and bacterial pathogens (e.g., Aspergillus). Examples include bacterial infections caused by the mobilization of spp. (Borrelia burgdorferi, B. duttonii, B. recurrentis, Candida albicans, Fr and sella tularensis, Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes).

[0070] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and are not limited to them, but refer to mammals including primates (e.g., humans; non-human primates including monkeys); sport mammals (e.g., horses); agricultural mammals (e.g., sheep, goats, etc.); companion mammals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0071] As used herein, the term “effective dose” is an amount sufficient to produce a beneficial or desirable clinical outcome. An effective dose may be administered in one or more doses. For the purposes of this disclosure, an effective dose of a compound (e.g., infectious rAAV virion) is an amount sufficient to mitigate, improve, stabilize, reverse, prevent, slow, or delay the progression (and / or associated symptoms) of a particular disease condition (e.g., complement dysfunction and associated disorders). Thus, an effective dose of infectious rAAV virion is the amount of infectious rAAV virion that can efficiently deliver heterologous nucleic acids to target cells (or multiple target cells) of an individual. For example, an effective dose may be preclinically determined by detecting gene products (RNA, proteins) encoded by heterologous nucleic acid sequences in cells or tissues using techniques well understood in the art, such as RT-PCR, Western blotting, ELISA, fluorescence, or other reporter readouts. For example, an effective dose can be clinically determined by detecting changes at the onset or progression of the disease using methods known in the art, such as the 6-minute walk test, left ventricular ejection fraction, handheld kinetics, and Vignos scale, as described herein and known in the art. Detailed explanation

[0072] Novel anti-C3 and anti-C5 antibodies and the nucleic acids encoding them are described herein. rAAV virions containing nucleic acids encoding anti-C3 and / or antibodies are also described. These rAAV virions are characterized by sustained and robust expression of anti-C3 and anti-C5 antibodies in the retina and increased efficacy in treating C3 and C5-related conditions and other complement disorders.

[0073] The delivery of these rAAV virions to subjects requiring them can be achieved by several routes, preferably by intravitreal administration. Methods for using these rAAV virions in regimens for treating complement factor C3 and C5-related disorders, particularly atrophic AMD and geographic atrophy secondary to AMD, are also provided.

[0074] antibody

[0075] Naturally occurring human antibodies are heterotetramers. In one embodiment, the antibodies provided herein contain an antigen-binding site in a single polypeptide. Therefore, the antibodies are referred to herein as “single variable domain” or “VHH” antibodies. Single variable domain antibodies are also known as nanobodies. The single variable domain antibodies disclosed herein, however, in certain embodiments may be bispecific or polyspecific single variable domain antibodies to which the single variable domain antibody is coupled.

[0076] A single variable domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it can selectively bind to a specific antigen. Single variable domain antibodies typically have a much lower molecular weight, ranging from 12 to 15 kDa, compared to the typical 150 to 160 kDa range of common antibodies. Single variable domain antibodies are also smaller than the Fab fragment (approximately 50 kDa) of a heterotetramer antibody containing one light chain and half of a heavy chain.

[0077] Single-domain antibodies can naturally originate from antibodies found, for example, in camelids (VHH) and cartilaginous fish (VNAR). The novel or rhinoceros shark antigen receptor (NAR) protein exists as a dimer of two heavy chains without a bound light chain. Each chain consists of one variable (V) domain and five constant domains. Thus, the NAR protein constitutes a single immunoglobulin variable-like domain. Single-heavy-chain antibodies are also found in camelids such as dromedaries, camels, llamas, and alpacas, where the heavy chain loses one of its constant domains and undergoes modification in the variable domain, both of which are structural elements necessary for light chain binding.

[0078] However, single variable domain antibodies can also be manipulated by recombinant methods. One technique involves splitting a dimeric variable domain derived from a common immunoglobulin G (IgG) from humans or mice into monomers. Single domains derived from the light chain also bind specifically to target epitopes. Therefore, single variable domain antibodies may be derived from any suitable organism.

[0079] Single-domain camelid antibodies are comparable to conventional antibodies in terms of specificity. Single-domain antibodies are readily isolated, for example, by using phage panning procedures. Their smaller size and single-domain structure facilitate the expression of these antibodies as proteins in bacterial cells for large-scale production, making them ideal for commercial use. The antibodies of the present invention are therefore single variable-domain antibodies, preferably derived from camelid antibodies, preferably llama antibodies, comprising a functional homolog, its fragments, and a fusion polymer containing VHH covalently bonded to a glycan, nucleic acid, protein, or non-polymeric chemical group.

[0080] In certain embodiments, the antibodies provided herein are antibody fragments. In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, in particular a Fab fragment. Papain digestion of an intact antibody yields two identical antigen-binding fragments called “Fab” fragments, each containing the respective heavy and light chain variable domains (VH and VL, respectively), as well as the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain. The term “Fab fragment” therefore refers to an antibody fragment containing the light chain containing the VL and CL domains, as well as the heavy chain fragment containing the VH and CH1 domains. A “Fab' fragment” is distinguished from a Fab fragment by the addition of a residue at the carboxyl terminus of the CH1 domain, containing one or more cysteines derived from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue of the constant domain supports a free thiol group. Pepsin treatment yields an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting increased in vivo half-lives, see U.S. Patent No. 5,869,046.

[0081] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., Escherichia coli, CHO).

[0082] In a preferred embodiment, the antibody provided herein is a Fab fragment. In one embodiment, the VH domain of the antibody provided herein comprises a human VH3 framework. In one embodiment, the VL domain of the antibody provided herein comprises a human V-kappa 1 framework. In one embodiment, the CL domain of the antibody provided herein is a kappa isotype. In one embodiment, the CH1 domain of the antibody provided herein is a human IgG1 isotype.

[0083] In preferred embodiments, the antibody provided herein is a Fab fragment comprising a kappa isotype CL domain and a human IgG1 isotype CH1 domain.

[0084] In certain embodiments, the antibodies provided herein are polyspecific antibodies. A "polyspecific antibody" is a monoclonal antibody that has binding specificity to at least two distinct sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a polyspecific antibody has three or more binding specificities.

[0085] Polyspecific antibodies having three or more binding specificities, including the antibodies provided herein, can be provided in an asymmetric form having domain crossover in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO2009 / 080252 and WO2015 / 150447), CH1 / CL domains (see, e.g., WO2009 / 080253), or complete Fab arms (see, e.g., WO2009 / 080251, WO2016 / 016299, and Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). Various further molecular forms for polyspecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

[0086] In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the antibody, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, such as antigen binding.

[0087] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are listed below under the heading "Preferred Substitutions." More substantial substitutions are provided below under the heading "Exemplary Substitutions" and further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC. [Table 14-1] [Table 14-2]

[0088] Amino acids may be classified according to their common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0089] Non-conservative substitutions require replacing a member of one class with a member of another class.

[0090] One type of substitution variant involves substituting one or more CDR residues in a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further testing has modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retains certain biological properties of the parent antibody. Exemplary substitution variants are affinity-mature antibodies that can be readily produced using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, the variant antibody is displayed on a phage, and it is screened for specific biological activity (e.g., binding affinity).

[0091] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within a CDR. Such alterations may, for example, be outside the antigen-contact residue in the CDR. In certain variant VH and VL sequences provided above, each CDR is either unaltered or contains one, two, or three or fewer amino acid substitutions.

[0092] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies a residue or target group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) and replaces them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitutions. Alternatively, the crystal structure of the antigen-antibody complex can be used to identify antibody-antigen contact points. Such contact residues and adjacent residues may be targeted or removed as candidates for substitution. Variants can be screened to determine whether they possess the desired properties.

[0093] Amino acid sequence insertions include amino and / or carboxyl terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminus insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of antibodies to the N or C terminus with enzymes (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or polypeptides that increase the serum half-life of the antibody.

[0094] Glycosylated variant

[0095] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which they are glycosylated. The addition or deletion of glycosylation sites to an antibody can be easily achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0096] If an antibody contains an Fc region, the oligosaccharide bound to it may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides, commonly bound by an N-bonding of the CH2 domain of the Fc region to Asn297. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention may produce antibody variants with improved specific properties.

[0097] In one embodiment, an antibody variant is provided having a non-fucosylated oligosaccharide, i.e., an oligosaccharide structure lacking fucose (directly or indirectly) bound to the Fc region. Such a non-fucosylated oligosaccharide (also referred to as a "defucosylated" oligosaccharide) is in particular an N-linked oligosaccharide lacking a fucose residue bound to a first GlcNAc in the stem of a branched oligosaccharide structure. In one embodiment, an antibody variant is provided in which the proportion of non-fucosylated oligosaccharides in the Fc region is increased compared to the natural or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides compared to the total of all oligosaccharides bound to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in WO2006 / 082515. Asn297 refers to the asparagine residue located around position 297 in the Fc region (EU numbering of the Fc region residue), although Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence changes in the antibody. Such antibodies with an increased percentage of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, in particular improved ADCC function. For example, see US2003 / 0157108;US2004 / 0093621.

[0098] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108; and WO2004 / 056312, see particularly Example 11), and knockout cell lines such as those containing the alpha-1,6-fucosyltransferase gene, FUT8, or knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688). Examples include cells in which the activity of GDP-fucose synthesis or transport factor proteins is reduced or inactivated (see 2006; and WO2003 / 085107), or cells in which the activity of GDP-fucose synthesis or transport factor proteins is reduced or inactivated (see, for example, US2004259150, US2005031613, US2004132140, and US2004110282).

[0099] In a further embodiment, antibody variants having a bifid oligosaccharide are provided, for example, in which a bifid oligosaccharide bound to the Fc region of the antibody is bifid 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); WO99 / 54342; WO2004 / 065540, WO2003 / 011878.

[0100] Antibody variants are also provided that have at least one galactose residue in the oligosaccharide bound to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087;WO1998 / 58964; and WO1999 / 22764.

[0101] Fc region variant

[0102] In certain embodiments, an Fc region variant can be generated by introducing one or more amino acid modifications into the Fc region of an antibody provided herein. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0103] In certain embodiments, the present invention envisions antibody variants having some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (e.g., complement-dependent cell-mediated 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 the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. Primary cells for mediating 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 vivo assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., 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 may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and the CytoTox 96(C) 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, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, e.g., 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, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929 A1).

[0104] Antibodies that resist effector function include those having one or more substitutions among Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include the so-called "DANA" Fc mutant (U.S. Patent No. 7,332,581), which has alanine substitutions at residues 265 and 297, and Fc mutants having two or more substitutions among amino acid positions 265, 269, 270, 297, and 327.

[0105] Certain antibody variants with improved or reduced binding to FcR have been described. (See, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0106] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0107] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that attenuate FcγR binding, e.g., substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further includes D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from the human IgG1 Fc region. (See, for example, WO2012 / 130831). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region.

[0108] In some embodiments, modifications resulting in alterations (i.e., either improvement or attenuation) of C1q binding and / or complement-dependent cell injury (CDC) are made in the Fc region, as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0109] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), 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)) are described in US2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include substitutions of one or more of the Fc domain 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, such as a substitution of Fc domain residue 434 (see, for example, U.S. Patent No. 7,371,826, Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0110] The Fc region residues crucial for mouse Fc-mouse FcRn interactions were identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index numbering) 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 important 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 important for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671), various mutants at residues 248-259, 301-317, 376-382, and 424-437 have been reported and investigated.

[0111] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253 and / or 310 and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from the human IgG1 Fc region. See, for example, Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0112] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310 and / or 433 and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, WO 2014 / 177460 A1).

[0113] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252 and / or 254 and / or 256 (residue EU numbering) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254 and 256. In one embodiment, the substitutions are M252Y, S254T and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patents 5,648,260, 5,624,821 and WO 94 / 29351.

[0114] The C-terminus of the heavy chain of the antibodies reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus from which one or two C-terminal amino acid residues have been removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one embodiment of all the embodiments reported herein, the antibody comprising a heavy chain containing the C-terminal CH3 domain identified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid position). In one embodiment of all the embodiments reported herein, the antibody comprising a heavy chain containing the C-terminal CH3 domain identified herein comprises a C-terminal glycine residue (G446, EU index numbering of amino acid position).

[0115] Cysteine-modified antibody variant

[0116] In certain embodiments, it may be desirable to produce a cysteine-modified antibody, such as a THIOMAB® antibody, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the residues to be substituted are located in an accessible site of the antibody. By substituting these residues with cysteine, a reactive thiol group is thereby positioned in an accessible site of the antibody, which can then be used to conjugate the antibody to another part, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate as further described herein. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0117] Immunoconjugate

[0118] In one embodiment, the present invention also provides an immunoconjugate comprising an antibody provided herein that is conjugated (chemically linked) to one or more agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a protein toxin, a bacterial, fungal, plant or animal-derived enzyme-active toxin, or a fragment thereof), or a radioisotope.

[0119] In one embodiment, the present invention provides an immunoconjugate comprising an antibody provided herein conjugated to a polymer. The term “polymer” as used herein includes chemical polymers and protein polymers. In one embodiment, the immunoconjugate comprises an antibody provided herein conjugated to an extended recombinant polypeptide (XTEN). “Extended recombinant polypeptide” is known in the art and is disclosed, for example, in US20190083577. In one embodiment, the immunoconjugate comprises an XTEN having (a) a sequence selected from GGSPAGSCTSP, GASASCAPSTG, TAEAAGCGTAEAA, and GPEPTCPAPSG, (b) a length of 36 to 3000 L-amino acid residues, and / or (c) a total of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) residues accounting for more than 90% of the total amino acid residues of the XTEN.

[0120] Recombination method and composition

[0121] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding antibodies are provided.

[0122] In one embodiment, isolated nucleic acids encoding the antibody of the present invention are provided. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody), or they can be produced by recombinant methods or obtained by chemical synthesis.

[0123] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (For a description of antibody fragment expression in Escherichia coli, see also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254.) After expression, antibodies may be isolated from bacterial cell paste in the soluble fraction and further purified. In one embodiment, the host cell is an Escherichia coli cell.

[0124] Vertebrate cells may be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7), human fetal kidney cell line (e.g., 293 or 293T cells described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described 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 liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather, JP et al., Annals NY Acad. These include MRC 5 cells and FS4 cells (described in Sci. 383 (1982) 44-68). 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), as well as myeloma cell lines such as Y0, NS0, 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.

[0125] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0 cell, NS0 cell, Sp20 cell). In a preferred embodiment, the host cell is a CHO cell. Production of the antibody of the present invention in CHO cells can improve the passability of the antibody through the injection needle.

[0126] Anti-C3 antibody

[0127] In some embodiments, the anti-C3 antibodies described herein can specifically bind to epitopes of human complement factor C3 and / or proteolytic derivatives C3a and / or C3b. Thus, the anti-C3 antibodies can specifically bind to epitopes in any region of the polypeptide sequence identified by GenBank accession number NP_000055.2, the full details of which are incorporated herein by reference.

[0128] The antibodies described herein include one or more CDRs. In particular, since CDRs can identify the specificity of the antibody, the antigen-binding sites preferably include at least two, and more preferably three, CDRs.

[0129] Therefore, the antibodies provided herein are preferably derived from natural antibodies, such as camelid antibodies. In some embodiments, the antibodies are selected from the following sequences or sequences that are at least 75% identical thereto (CDR sequences are underlined):

[0130] C3S1: [ka] [ka]

[0131] C3S3: [ka]

[0132] C3S4:

change

[0133] C3S5:

change

[0134] C3S6:

change

[0135] C3S7:

change

[0136] C3S8:

change

[0137] C3S9:

change

[0138] C3S10:

change

[0139] C3I3:

change

[0140] C3I4:

change

[0141] C3I8: [ka]

[0142] C3I22: [ka]

[0143] C3N10: [ka]

[0144] The CDRs of the above antibodies are listed in Table 1 below: [Table 1]

[0145] In some embodiments, the antibody comprises a CDR having at least one, at least two, or at least three amino acid differences compared to the CDRs listed in Table 1. In some embodiments, the antibody comprises a CDR1 having the following sequence: GF / LT / SF / LS / G / DP / V / Y / NYA / DM / IG / S / A (wherein the first amino acid is G, the second amino acid is F or L, etc.) and / or a CDR2 having the following sequence: G / S / CIR / D / T / S / NW / S / G / QI / G / P / SS / V / D / RG / NN / T / SP / A / T / YY / H / AY / H (wherein the eleventh amino acid is optional).

[0146] In the relevant embodiments, the antibody comprises CDR1, CDR2, and / or CDR3 selected from the group consisting of CDR sequences listed in Table 1.

[0147] In some preferred embodiments, the antibody comprises a CDR1 selected from SEQ ID NOs: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, and 54, or a CDR1 comprising a sequence that is at least 75% identical to any one of SEQ ID NOs: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, and 54, and / or a CDR2 selected from SEQ ID NOs: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55, if or includes a CDR2 containing a sequence that is at least 75% identical to any one of sequence numbers 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55, and / or includes a CDR3 selected from sequence numbers 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56, or includes a CDR1 containing a sequence that is at least 75% identical to any one of sequence numbers 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In related embodiments, the antibodies include CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 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 CDR1, CDR2, and CDR3 that are at least 75% identical to SEQ ID NOs: 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.

[0148] In a particularly preferred embodiment, the antibody comprises CDR1, CDR2, and CDR3 of SEQ ID NOs. 54-56, respectively, or comprises CDRs that are identical to them by 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%).

[0149] The amino acid sequence of Cynomolgus macaque C3 is as follows: [ka] [ka]

[0150] The amino acid sequence of human C3 is as follows: [ka] [ka]

[0151] In some embodiments, the anti-C3 antibody described herein (e.g., C3N10.3) binds to an epitope in the C3 alpha chain at the C345C domain. In some embodiments, the anti-C3 antibody described herein binds to an epitope within amino acids 1540-1600 of human C3, or an amino acid sequence that is at least 80% identical thereto. In related embodiments, the anti-C3 antibody described herein binds to an epitope containing amino acids 1548-1561 of human C3, or an amino acid sequence that is at least 80% identical thereto, and / or to an epitope containing amino acids 1568-1573 of human C3, or an amino acid sequence that is at least 80% identical thereto, and / or to an epitope containing amino acids 1583-1591 of human C3, or an amino acid sequence that is at least 80% identical thereto.

[0152] In some embodiments, the anti-C3 antibody described herein (e.g., C3S3.2) binds to at least one discontinuous epitope in the C3 beta chain in the MG1 and MG5 domains. In some embodiments, the anti-C3 antibody described herein binds to at least one discontinuous epitope within amino acids 110-130 and within amino acids 490-501 of human C3. In some embodiments, the anti-C3 antibody described herein binds to an epitope containing amino acids 114-124 of human C3 or an amino acid sequence that is at least 80% identical thereto, and / or to an epitope containing amino acids 497-505 of human C3 or an amino acid sequence that is at least 80% identical thereto.

[0153] In some preferred embodiments, the anti-C3 antibody comprises CDR1, CDR2 and / or CDR3 of any one of the anti-C3 antibodies of SEQ ID NOs: 2, 14, 98 and 135, or comprises CDR1, CDR2 and / or CDR3 that are at least 70% identical thereto (or comprising four or fewer amino acid substitutions compared to CDR1, CDR2 and CDR3 of any one of SEQ ID NOs: 2, 14, 98 and 135).

[0154] Anti-C5 antibody

[0155] In some embodiments, the anti-C5 antibodies (e.g., nanobodies) described herein can specifically bind to epitopes of human complement factor C5 and / or proteolytic derivatives C5a and / or C5b. Thus, the anti-C5 antibodies can specifically bind to epitopes in any region of a polypeptide sequence identified by GenBank accession number NP_001304092.1, the entire content of which is incorporated herein by reference.

[0156] The antibodies described herein include one or more CDRs. In particular, the antigen-binding site preferably includes at least two, more preferably three CDRs, as CDRs can specify the specificity of the antibody.

[0157] Therefore, the antibodies described herein are preferably derived from natural antibodies such as camelid antibodies. In some embodiments, the antibody is selected from the following sequences or sequences that are at least 75% identical thereto (CDR sequences are underlined):

[0158] C5S1: [ka]

[0159] C5S2: [ka]

[0160] C5S3: [ka]

[0161] C5S4: [ka]

[0162] C5S5: [ka]

[0163] C5S6: [ka]

[0164] C5S7: [ka]

[0165] C5S8: [ka]

[0166] C5S12: [ka]

[0167] The CDRs of the above antibodies are listed in Table 2 below: [Table 2]

[0168] In some embodiments, the antibody comprises a CDR having at least one, at least two, or at least three amino acid differences compared to the CDRs listed in Table 2. In some embodiments, the antibody comprises a CDR1 having the following sequence: GY / N / S / TIS / FY / H / F / A / P / DD / Q / A / Y / S / ED / S / T / E / Y / WD / K / GMG (where the first amino acid is G and the second amino acid is Y, N, S, or T, etc.) and / or a CDR2 having the following sequence: A / T / GID / T / NV / Y / G / DGG / T / AS / N / ITY / N. In related embodiments, the antibody comprises a CDR1 having the following sequence: GY / N / S / TIFY / H / F / A / P / DD / Q / A / Y / S / ED / S / T / E / Y / WDMG and / or a CDR2 having the following sequence: -A / TIDV / Y / G / DGAS / NTY.

[0169] In the relevant embodiments, the antibody comprises CDR1, CDR2, and / or CDR3 selected from the group consisting of CDR sequences listed in Table 2.

[0170] In a preferred embodiment, the antibody comprises a CDR1 selected from SEQ ID NOs: 66, 69, 72, 75, 78, 81, 84, 87, and 90, or a CDR1 comprising a sequence that is at least 75% identical to any one of SEQ ID NOs: 66, 69, 72, 75, 78, 81, 84, 87, and 90, and / or a CDR2 selected from SEQ ID NOs: 67, 70, 73, 76, 79, 82, 85, 88, and 91, or It includes a CDR2 containing a sequence that is at least 75% identical to one of sequence numbers 67, 70, 73, 76, 79, 82, 85, 88, and 91, and / or a CDR3 selected from sequence numbers 68, 71, 74, 77, 80, 83, 86, 89, and 92, or a CDR1 containing a sequence that is at least 75% identical to one of sequence numbers 68, 71, 74, 77, 80, 83, 86, 89, and 92. In related embodiments, the antibodies include CDR1, CDR2, and CDR3 selected from SEQ ID NOs. 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86, 87-89, and 90-92, or include CDR1, CDR2, and CDR3 that are at least 75% identical to SEQ ID NOs. 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86, 87-89, and 90-92.

[0171] The amino acid sequence of Cynomolgus monkey C5 is provided below: [ka] [ka]

[0172] The amino acid sequence of human C5 is provided below:

[0173] [ka] [ka]

[0174] In some embodiments, the anti-C5 antibodies described herein (e.g., C5S2.2) competitively inhibit the binding of eculizumab to human C5.

[0175] In some preferred embodiments, the anti-C5 antibody comprises CDR1, CDR2, and / or CDR3 of either one of the anti-C5 antibodies of SEQ ID NOs. 58 and 141, or CDR1, CDR2, and / or CDR3 that are at least 70% identical thereto (or comprising four or fewer amino acid substitutions compared to CDR1, CDR2, and CDR3 of either one of the SEQ ID NOs. 58 and 141).

[0176] The antibodies provided herein also include their functional variants. The term “functional variant” means that the antibody includes variants that retain some or essentially all of its ability to selectively bind to its antigen or ligand, such as any of the ligands described below herein. Functional variants include any variant that is at least 75% identical to the antibodies provided herein, for example, any variant that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, e.g., 90, 91, 92, 93, 94, 95, 96, e.g., 97, e.g., 98, e.g., 99, e.g., any variant that is at least 99.5% identical to any of the antibodies provided herein, e.g., those identified by SEQ ID NOs. 1-14 and 57-65. In a preferred embodiment, the antibody comprises the amino acid sequence described in SEQ ID NO. 14, or an amino acid sequence that is at least 75% identical thereto.

[0177] A functional variant comprises any variant antibody containing one or more CDRs that are at least 75% identical to the CDR of the antibody provided herein, for example, at least 99.5% identical to such CDR, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, e.g., 90, 91, 92, 93, 94, 95, 96, e.g., 97, e.g., 98, e.g., 99. The CDRs of each antibody identified by SEQ ID NOs. 1-14 and 57-65 are shown above as SEQ ID NOs. 15-56 and 66-92, respectively. Therefore, an antibody is provided that contains one or more regions having at least 75% identity, for example, at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, for example 90, 91, 92, 93, 94, 95, 96, for example 97, for example 98, for example 99, for example at least 99.5%, with one or more regions identified by any one of SEQ ID NOs.

[0178] Amino acid substitutions include conserved amino acid substitutions, which refer to the substitution of one amino acid with another amino acid residue having a side chain with similar properties. For example, the group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains are serine and threonine; the group of amino acids with amide-containing side chains are asparagine and glutamine; the group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains are lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Where applicable in this specification, the term “conservative amino acid substitution” means that one amino acid may be substituted with another from the group of amino acids listed below in this specification: i) Amino acids with polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gln(Gin), Ser, Thr, Tyr, and Cys), ii) Amino acids with nonpolar side chains (Gly, Ala, Val, Leu, Ile(lie), Phe, Trp, Pro, and Met), iii) Amino acids with aliphatic side chains (Gly, Ala, Val, Leu, Ile), 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, Gln), 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, acidic amino acids (Gln, Asn, Glu, Asp), and xiii) Hydrophobic amino acids (Leu, Ile, Val).

[0179] In one preferred embodiment, the functional variant of the antibody may be a fragment of the antibody, preferably an antigen-binding fragment or a variable region. Examples of antibody fragments useful in the present invention include VHH and VNAR fragments.

[0180] Therefore, antibodies can be obtained by immunization of any suitable organism, particularly camelids, sharks, etc. However, antibodies can also be generated from synthetic libraries having randomized or designed CDRs. In one embodiment, antibodies that can specifically bind to the epitopes of human complement factors C3, C3a and / or C3b are provided. In another embodiment, antibodies that can specifically bind to the epitopes of human complement factors C5, C5a and / or C5b are provided.

[0181] In one embodiment, the antibody is coupled to a histidine tag, such as a his6 tag, at the N or C terminus of the polypeptide. In a preferred embodiment, the antibody is coupled to a non-natural Strep or his6 tag (e.g., WSHPQFEKHHHHHH (SEQ ID NO: 93)) at the C terminus. In another embodiment, the antibody is coupled 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. The additional N-terminal region can be selected from any relevant additional portion depending on the intended application of the antibody and the desired function of the final antibody product. Albumin may be added to increase circulation 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 to the antibody product.

[0182] In some embodiments, the anti-C3 antibodies described herein form a first domain in a fusion protein. In some aspects, the fusion protein is a heterodimeric fusion protein comprising a first domain comprising a polypeptide comprising an antibody described herein and a second domain comprising an immunoglobulin Fc domain. In some aspects, the first domain and the second domain 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 NOs: 178 to 180, or an amino acid sequence that is at least 70% identical thereto. In other aspects, the fusion protein is a homodimeric fusion protein comprising a first domain comprising a polypeptide comprising an antibody described herein and a second domain identical to the first domain. In some aspects, the first domain and the second domain are linked by a linker, such as a poly-glycine-serine (G4S) linker, preferably a (G4S)2 linker. In a preferred embodiment, the fusion protein is a homodimeric fusion protein comprising an amino acid sequence of SEQ ID NO: 181 or 182, or an amino acid sequence that is at least 70% identical thereto.

[0183] In some embodiments, for example, a single peptide chain may comprise two antigen-binding regions separated by a linker sequence, and one of the two antigen-binding regions comprises a VHH domain described herein, and bispecific / multispecific antibodies are provided. In some aspects, the VHH domain described herein is coupled to another VHH domain that forms or comprises a bispecific antibody that consists of (i) a VHH domain described herein and (ii) a VHH domain capable of specifically binding to an epitope of a second target.

[0184] The antibodies disclosed herein may, in a preferred embodiment, include modifications that improve the function of the antibody. For example, it is not always desirable to use non-human antibodies for human therapy, and thus the antibodies provided herein may be humanized antibodies.

[0185] In some embodiments, the humanized antibody is a humanized form of the above-mentioned llama nanobody, and in particular, it is selected from the following amino acid sequences or amino acid sequences that are at least 75% identical thereto (the bold amino acids are the amino acids of the humanized form that have changed from the amino acids of the corresponding llama nanobody):

[0186] C3S1.1:

Chemical Structure

[0187] C3S1.2:

Chemical Structure

[0188] C3S1.3:

Chemical Structure

[0189] C3S3.1:

Chemical Structure

[0190] C3S3.2:

Chemical Structure

[0191] C3S3.3:

Chemical Structure

[0192] C3S4.1:

Chemical Structure

Chemical Structure

[0193] C3S4.2:

change

[0194] C3S4.3:

change

[0195] C3S5.1:

change

[0196] C3S5.2:

change

[0197] C3S5.3:

change

[0198] C3S6.1:

change

[0199] C3S6.2:

change

[0200] C3S6.3:

change

[0201] C3S7.1:

change

[0202] C3S7.2:

change

[0203] C3S7.3:

change

[0204] C3S8.1:

change

[0205] C3S8.2:

change

[0206] C3S8.3:

change

[0207] C3S9.1:

change

[0208] C3S9.2:

change

[0209] C3S9.3:

change

[0210] C3S10.1:

change

[0211] C3S10.2:

change

[0212] C3S10.3:

change

[0213] C3I3.1:

change

[0214] C3I3.2:

change

[0215] C3I3.3:

change

[0216] C3I4.1:

change

[0217] C3I4.2:

change

[0218] C3I4.3:

change

[0219] C3I8.1:

change

[0220] C3I8.2:

change

[0221] C3I8.3:

change

[0222] C3I22.1:

change

[0223] C3I22.2:

change

[0224] C3I22.3:

change

[0225] C3N10.1:

change

[0226] C3N10.2:

change

[0227] C3N10.3:

change

[0228] C3N10.4:

change

[0229] C5S1.1:

change

[0230] C5S1.2:

change

[0231] C5S1.3:

change

[0232] C5S2.1:

change

[0233] C5S2.2:

change

[0234] C5S2.3:

change

[0235] C5S3.1:

change

[0236] C5S3.2:

change

[0237] C5S3.3:

change

[0238] C5S4.1:

change

[0239] C5S4.2:

change

[0240] C5S4.3:

change

[0241] C5S5.1:

change

[0242] C5S5.2:

change

[0243] C5S5.3:

change

[0244] C5S6.1:

change

[0245] C5S6.2:

change

[0246] C5S6.3:

change

[0247] C5S7.1:

change

[0248] C5S7.2:

change

[0249] C5S7.3:

change

[0250] C5S8.1:

change

[0251] C5S8.2:

change

[0252] C5S8.3:

change

[0253] C5S12.1:

change

[0254] C5S12.2: [ka]

[0255] C5S12.3: [ka]

[0256] In some preferred embodiments, the antibody contains any one sequence of sequence numbers 94-163, or contains an amino acid sequence that is at least 75%, for example, 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% identical to them, preferably containing the amino acids shown in bold.

[0257] In addition to the antibodies provided herein, nucleic acids comprising nucleotide sequences encoding the antibodies described herein are also provided. In relevant embodiments, the nucleic acids encoding the antibodies described herein form part of an expression vector in which the nucleotide sequences encoding the antibodies are operably linked to a suitable promoter.

[0258] In one embodiment, RNA and / or cDNA coding sequences are designed for optimal expression in human cells. Codon-optimized coding regions can be designed by a variety of different methods. This optimization may be performed using methods available online, published methods, or companies that provide codon optimization services. One codon optimization method is described, for example, in WO2015 / 012924A2, which is incorporated herein by reference. Briefly, it involves modifying the nucleic acid sequence encoding the product with synonymous codon sequences. Preferably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only fragments of the ORF may be modified. By using one of these methods, frequencies can be applied to any given polypeptide sequence to produce nucleic acid fragments of codon-optimized coding regions encoding the polypeptide.

[0259] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a nucleic acid comprising a nucleotide sequence encoding the antibody described herein, wherein the nucleotide sequence is operably linked to a promoter. In other embodiments, a pharmaceutical composition is provided comprising the antibody described herein and a pharmaceutically acceptable carrier.

[0260] Recombinant AAV virion encoding anti-C3 and / or anti-C5 antibodies

[0261] (i) a capsid as described herein and (ii) a heterologous nucleic acid encoding an anti-C3 and / or anti-C5 antibody as described herein, wherein the nucleic acid sequence encoding the anti-C3 and / or anti-C5 antibody is operably linked to an expression regulatory sequence. Typically, the heterologous nucleic acid is an AAV genome in which the rep and cap genes are deleted and / or replaced with an antibody sequence and its associated expression regulatory sequence. The antibody sequence is typically inserted (i.e., sandwiched) adjacent to one or two AAV TRs or TR elements sufficient for viral replication, in place of the nucleic acid encoding the viral rep and cap proteins (Xiao et al., 1997, J. Virol. 71(2): 941-948). Other suitable regulatory sequences may be included for use in facilitating tissue-specific expression of the antibody y gene sequence in target cells (e.g., retinal cells).

[0262] In some embodiments, anti-C3 antibodies encoded by heterologous nucleic acids of rAAV can prevent C3 binding to C3-converting enzymes in the complement II pathway and the classical pathway, and subsequently inhibit C3 cleavage.

[0263] heterogeneous nucleic acid components of rAAV

[0264] In some embodiments, the rAAV virion comprises a heterogeneous nucleic acid including (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.

[0265] In other embodiments, the rAAV virion comprises a heterogeneous nucleic acid including (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.

[0266] The inverted end repeat (ITR) selected for use in rAAV virions is preferably an AAV sequence, with serotypes 1, 2, 3, 4, 5, and 6 preferred. The ITR may be a synthetic sequence that functions as an AAV inverted end repeat, such as the “double D sequence” described in U.S. Patent No. 5,478,745 by Samulski et al., the full disclosure of which is incorporated herein by reference in whole. Typically, although not required, the TRs are derived from the same parvovirus; for example, both ITR sequences are derived from AAV2.

[0267] In some embodiments, the heterologous nucleic acid encapsulated in the rAAV virion includes a 5'ITR having the following sequence: [ka]

[0268] In a related embodiment, the heterologous nucleic acid encapsulated in the rAAV virion includes a 3'ITR having the following sequence: [ka]

[0269] The antibody gene encoded by rAAV is preferably operably ligated to at least one transcriptional regulatory sequence, preferably a transcriptional regulatory sequence heterogeneous to the nucleic acid. In some embodiments, the transcriptional regulatory sequence includes a cell or tissue-specific promoter that results in cell-specific expression of the nucleic acid in photoreceptor cells, such as the human rod photoreceptor-specific human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter or the human photoreceptor-inter-retinoid-binding protein (IRBP) promoter. In other embodiments, the transcriptional regulatory sequence includes a constitutive promoter that results in similar expression levels of the nucleic acid across many cell types. Suitable constitutive promoters include the CAG promoter containing (C) the cytomegalovirus (CMV) initial enhancer element, (A) the first exon and first intron of the chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene (see Miyazaki et al. (1989) Gene 79(2): 269-277), the cytomegalovirus promoter (CMV) (Stinski et al, (1985) Journal of Virology 55(2): 431-441), the human elongation factor 1α promoter (EF1α) (Kim et al. (1990) Gene 91(2): 217-223), the human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417), and the mitochondrial heavy chain promoter (Loderio et al. (2012) PNAS). Examples include 109(17): 6513-6518) and the ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105).

[0270] In a preferred embodiment, the antibody gene encoded by rAAV is operably ligated to a CAG promoter. In a particular preferred embodiment, the CAG promoter contains the sequence of SEQ ID NO: 166, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto: [ka]

[0271] In some embodiments, the heterologous nucleic acid encapsulated by the rAAV virion contains an SV40 polyadenylated sequence of the following sequence: [ka]

[0272] rAAV capsid component

[0273] A variant AAV capsid of rAAV, which encapsulates a heterologous nucleic acid encoding an antibody or a functional variant thereof, comprises a variant AAV capsid protein containing an insertion of approximately 7 to 20 amino acids ("heterologous peptide" or "peptide insert") within the GH loop of the parent AAV capsid protein, wherein the peptide contains the amino acid sequence ISDQTKH (SEQ ID NO: 168). Preferably, when present in an AAV virion, the variant capsid protein results in increased infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parent capsid protein.

[0274] The "GH loop" or loop IV of the AAV capsid protein refers to the solvent-accessible portion of the AAV capsid protein that is referred to in the art as the GH loop or loop IV. For more information on the GH loop / loop 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 may be located approximately within amino acids 570–611 of AAV2 VP1.

[0275] In some embodiments, the peptide insertion has one to three spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminals of the amino acid sequence ISDQTKH (SEQ ID NO: 168). Examples of spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In certain embodiments, the peptide insertion contains two spacer amino acids at the N-terminus and two spacer amino acids at the C-terminus. In other embodiments, the peptide insertion contains two spacer amino acids at the N-terminus and one spacer amino acid at the C-terminus. In preferred embodiments, the peptide insertion contains or is composed of the amino acid sequence LAISDQTKHA (SEQ ID NO: 169).

[0276] In some embodiments, the variant AAV capsid protein comprises a peptide insert containing the amino acid sequence ISDQTKH (SEQ ID NO: 168) and further comprises one or more amino acid substitutions compared to the corresponding parent AAV capsid protein. A representative example of an amino acid substitution can be found, for example, in column 26, rows 40-65 of U.S. Patent No. 11,576,983, the full content of which is incorporated herein by reference.

[0277] In some preferred embodiments, the variant AAV capsid protein comprises a peptide insert containing the amino acid sequence ISDQTKH (SEQ ID NO: 168), and further comprises a P34A amino acid substitution or a corresponding substitution in another AAV serotype compared to the VP1 capsid of AAV2.

[0278] In other embodiments, 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 column 26, rows 66-29, and row 50 of U.S. Patent No. 11,576,983.

[0279] In a particularly preferred embodiment, the variant capsid protein comprises the following amino acid sequence, or comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the following amino acid sequence. [ka]

[0280] The variant AAV capsid protein of SEQ ID NO: 170 contains the following modifications compared to the native AAV2 capsid: (i) a mutation from proline (P) to alanine (A) at amino acid position 34 located within the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids (leucine-alanine-isoleucine-serine-aspartic acid-glutamine-threonine-lysine-histidine-alanine / LAISDQTKHA (SEQ ID NO: 169)) at amino acid position 588, present in VP1, VP2, and VP3. In some embodiments, the capsid contains a variant capsid protein that has a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 170, and includes a P34A substitution and a LAISDQTKHA (SEQ ID NO: 169) peptide insertion at amino acid position 588.

[0281] Also provided herein are packaging cells, which are contained within “host cells” that can be cultured to produce the packaged viral vectors of the present invention. The packaging cells of the present invention generally include heterologous cells having (1) viral vector function, (2) packaging function, and (3) helper function. The functions of each of these components will be discussed in subsequent sections.

[0282] First, vectors can be prepared by several methods known to those skilled in the art (see, for example, WO2013 / 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. Briefly, efficient transfection of HEK293 cells is used as a starting point, and adherent HEK293 cell lines from a qualified clinical master cell bank are grown in shaking flasks and WAVE bioreactors under suspension conditions free of animal components, enabling rapid and scalable rAAV production. Using the triple transfection method (e.g., WO96 / 40240), the HEK293 cell line suspension yields 10 per cell when collected 48 hours after transfection. 5 Vector genome-containing particles (vg) exceeding 10 per liter of cell culture 14 This generates vg exceeding . More specifically, triple transfection refers to the fact that packaging cells are transfected with three plasmids: one plasmid encoding the AAV rep and cap genes, another plasmid encoding various helper functions (e.g., adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VA RNA), and another plasmid encoding the transgene and its various regulatory elements (e.g., modified GLA gene and CAG promoter).

[0283] To achieve the desired yield, several variables are optimized, including the selection of a suitable serum-free suspension medium that supports both growth and transfection, the selection of transfection reagents, transfection conditions, and cell density. A universal purification strategy based on ion exchange chromatography was also developed, yielding high-purity vector preparations of AAV serotypes 1-6, 8, 9 and various chimeric capsids. This user-friendly process can be completed within one week, resulting in a high empty particle ratio (over 90% full particles) and a post-purification yield (1 × 10⁻⁶) suitable for clinical application.13 resulting in titers exceeding [[X]] vg / L and purity, and being universal for all serotypes and chimeric particles. Using this scalable manufacturing technology, GMP Phase I clinical AAV vectors for retinal angiogenesis (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9) and retinitis pigmentosa (AAV2) administered to patients were produced. Furthermore, overall vector production increases by at least 5-fold by performing a perfusion method that requires recovering rAAV from the culture medium at multiple time points after transfection.

[0284] 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, in which rep and cap are deleted and replaced with a modified GLA sequence and its associated expression control sequences. Viral vector functions include sufficient expression control sequences to effect replication of the viral vector for packaging. Typically, a viral vector includes a portion of a parvovirus genome, such as an AAV genome, in which rep and cap are deleted and replaced with a transgene and its associated expression control sequences. The transgene is typically flanked by two AAV TRs instead of the deleted viral rep and cap ORFs. Appropriate expression control sequences are included, such as other regulatory sequences suitable for use in facilitating tissue-specific expression of the transgene in a tissue-specific promoter and target cells. The transgene is typically a nucleic acid sequence capable of being expressed to produce a therapeutic polypeptide or a marker polypeptide.

[0285] The terminal repeats (TRs) (soluble and insoluble) selected for use in viral vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 preferred. A soluble AAV TR does not need to have a wild-type TR sequence (for example, the wild-type sequence may be modified by insertion, deletion, truncation, or missense mutation) as long as the TR mediates the desired function, such as viral packaging, integration, and / or proviral rescue. The TR may also be a synthetic sequence functioning as an AAV inverted terminal repeat, such as the “double D sequence” described in U.S. Patent No. 5,478,745 by Samulski et al., the full disclosure of which is incorporated herein by reference in whole. Typically, though not required, the TRs are derived from the same parvovirus, for example, both TR sequences are derived from AAV2.

[0286] The packaging function includes the variant capsid components mentioned above.

[0287] The packaged viral vector, referred herein as the “transgene” or “transgene expression cassette,” contains an anti-C3 antibody transgene and an expression regulatory sequence flanked by TR elements, sufficient to package the vector DNA and result in the subsequent expression of the gene sequence in the transduced cell. The viral vector function can be delivered to the cell, for example, as a component of a plasmid or amplicon. The viral vector function may be extrachromosomal within the cell line and / or incorporated into the cell’s chromosomal DNA.

[0288] Any method for introducing a nucleotide sequence carrying viral vector functionality into a cellular host for replication and packaging may be used, but is not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments where viral vector functionality is provided by transfection using the viral vector, standard methods for inducing viral infection may be used.

[0289] The packaging function includes genes for viral vector replication and packaging. Therefore, for example, the packaging function may, as needed, include functions necessary 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. The packaging function may be supplied together or separately to the packaging cell using a gene construct such as a plasmid or amplicon, baculovirus, or HSV helper construct. The packaging function may be extrachromosomal within the packaging cell, but is preferably integrated into the cell's chromosomal DNA. Examples include genes encoding the AAV Rep and Cap proteins.

[0290] Helper functions include helper viral elements required to establish active infection of packaging cells, which is necessary to initiate viral vector packaging. Examples include functions derived from adenoviruses, baculoviruses, and / or herpesviruses, sufficient to result in viral vector packaging. For example, adenovirus helper functions typically include adenovirus components E1a, E1b, E2a, E4, and VA RNA. Packaging functions can be supplied by infection of packaging cells with the required virus. Packaging functions may also be supplied to packaging cells together or separately using gene constructs such as plasmids or amplicons. 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. The packaging function may be located extrachromosomally within the packaging cell, but is preferably integrated into the cell's chromosomal DNA (e.g., E1 or E3 in HEK293 cells).

[0291] Any suitable helper virus function can be used. For example, if the packaging cells are insect cells, a baculovirus may function as a helper virus. Herpesviruses can also be used as helper viruses in AAV packaging methods. Hybrid herpesviruses encoding AAV Rep proteins can advantageously facilitate more scalable AAV vector production schemes.

[0292] Any method for introducing a nucleotide sequence carrying a helper function into a cell host for replication and packaging may be used, but is not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments where the helper function is provided by transfection using a viral vector or infection using a helper virus, standard methods for inducing viral infection may be used.

[0293] Any suitable permissible or packaging cells known in the art may be used in the production of the packaged viral vector. Mammalian or insect cells are preferred. Examples of cells useful for the production of packaging cells in the implementation of the present invention include, for example, VERO, WI38, MRC5, A549, HEK293 cells (expressing functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cell, and human cell lines such as HepG2, Saos-2, HuH7, and HT1080 cell lines. In one embodiment, the packaging cells can be grown in a suspension culture, and more preferably, the cells can be grown in a serum-free culture. In one embodiment, the packaging cells are HEK293 cells grown in a 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 PTA13274. Several rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO2002 / 46359. In another embodiment, packaging cells are cultured in the form of cell stacks (e.g., 10-layer cell stacks seeded with HEK293 cells).

[0294] Examples of cell lines for use as packaging cells include insect cell lines. Any insect cell line capable of replicating AAV and being maintained in culture can be used according to the present invention. Examples include Spodoptera frugiperda, Drosophila spp. cell lines such as Sf9 or Sf21 cell lines, or mosquito cell lines, such as Aedes albopictus-derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for instruction on the use of insect cells for the expression of heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, ed. 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.

[0295] The viral capsid according to the present invention can be produced, for example, by expression from a baculovirus using any method known in the art (Brown et al., (1994) Virology 198:477-488). As a further alternative, the viral vector of the present invention can be produced in insect cells using a baculovirus vector to deliver the rep / cap gene and rAAV template, for example, as described by Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

[0296] In another aspect, the present invention provides a method for producing rAAV in insect cells, wherein a baculovirus packaging system or vector can be constructed by manipulating these genes in the polyhedrin coding region of a baculovirus vector to carry the AAV Rep and Cap coding regions, and by producing a viral recombinant by transfection into a host cell. In particular, when baculovirus production is used for AAV, the AAV DNA vector product is preferably a self-complementary AAV-like molecule without using mutations to AAV ITR. This is considered a byproduct of inefficient AAV repnicking in insect cells, resulting in a self-complementary DNA molecule due to the lack of functional Rep enzyme activity. The host cell is either a baculovirus-infected cell or has additional nucleic acids encoding baculovirus helper functions introduced therein, or contains these baculovirus helper functions therein. These baculoviruses can express the AAV component and subsequently facilitate capsid production.

[0297] During production, packaging cells generally contain one or more viral vector functions, along with helper and packaging functions sufficient to result in viral vector replication and packaging. These various functions may be supplied to the packaging cells together or separately using gene constructs such as plasmids or amplicons, and they may be extrachromosomal within the cell line or incorporated into the cell's chromosomes.

[0298] Cells in which one or more of the described functions have already been incorporated may be supplied, for example, cell lines in which one or more vector functions are incorporated extrachromosomally or into the cell's chromosomal DNA, cell lines in which one or more packaging functions are incorporated extrachromosomally or into the cell's chromosomal DNA, or cell lines in which helper functions are incorporated extrachromosomally or into the cell's chromosomal DNA.

[0299] The rAAV vector may be purified by 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; and U.S. Patent Nos. 6,566,118 and WO 98 / 09657.

[0300] A method for delivering nucleic acids encoding anti-C3 antibodies to the retina.

[0301] In some 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. Using rAAV, antibody-encoding nucleotide sequences can be delivered to retinal cells in vitro, for example, to produce antibody polypeptides or nucleic acids in vitro for ex vivo gene therapy. rAAV is further useful in methods for delivering nucleotide sequences to subjects in need, for example, to express antibodies in subjects in need, such as humans with atrophic AMD or geographic atrophy. In this manner, antibodies can be produced in vivo in subjects to restore complement regulation.

[0302] Accordingly, in one embodiment, a method is provided for delivering a nucleic acid encoding an anti-C3 and / or anti-C5 antibody or an antigen-binding fragment thereof to retinal cells, comprising contacting the retinal cells with the rAAV virions described herein.

[0303] In another embodiment, a method is provided for delivering a nucleic acid encoding an anti-C3 and / or anti-C5 antibody or an antigen-binding variant thereof to retinal cells in a mammalian subject, comprising administering an effective amount of the rAAV virion described herein or a pharmaceutical composition comprising the same to the mammalian subject.

[0304] rAAV may be administered to the target retina by any preferred route. In preferred embodiments, rAAV is administered intraocularly, preferably subretinal, suprachoroidal, and / or intravitreal. In some particularly preferred embodiments, rAAV is administered to the target by intravitreal injection, more preferably by a single intravitreal injection.

[0305] Treatment method

[0306] In certain embodiments, a method is provided for treatment of atrophic AMD in a subject requiring such treatment, comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising a variant AAV capsid protein having a heterologous peptide insert having a length of 7 to 20 amino acids covalently inserted into the GH loop 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 an anti-C3 and / or anti-C5 antibody or a nucleotide sequence encoding the antigen-binding variant thereof as described herein, wherein the nucleotide sequence is operably linked to a promoter; or administering to the subject a pharmaceutical composition comprising the rAAV and a pharmaceutically acceptable carrier, preferably the rAAV or the pharmaceutical composition being administered to the subject by intravitreal injection. Also provided is the use of the rAAV or a pharmaceutical composition containing the same for the treatment of atrophic AMD. The use of rAAV in the manufacture of pharmaceuticals for the treatment of atrophic AMD is also provided.

[0307] In related embodiments, a method is provided for treatment of geographic atrophy in subjects requiring such treatment, comprising administering to the subject a recombinant adeno-associated virus (rAAV) virion comprising (a) a variant AAV capsid protein comprising a heterologous peptide insert having a length of 7 to 20 amino acids covalently inserted into the GH loop 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 an anti-C3 and / or anti-C5 antibody or a nucleotide sequence encoding the antigen-binding variant thereof as described herein, wherein the nucleotide sequence is operably linked to a promoter; or administering to the subject a pharmaceutical composition comprising the rAAV virion and a pharmaceutically acceptable carrier, preferably the rAAV or the pharmaceutical composition being administered to the subject by intravitreal injection. Also provided is the use of the rAAV or a pharmaceutical composition containing the same for the treatment of geographic atrophy. Furthermore, the use of rAAV in the manufacture of pharmaceuticals for the treatment of geographic atrophy is also provided.

[0308] In some embodiments, the variant AAV capsid protein contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence described as SEQ ID NO: 170, or is 100% identical, and contains a P34A substitution and a LAISDQTKHA (SEQ ID NO: 169) peptide insertion at amino acid position 588.

[0309] In some preferred embodiments, the heterogeneous nucleic acid encapsulated by the variant AAV capsid protein comprises a nucleotide sequence encoding an antibody comprising one or more CDRs containing amino acid sequences described in Table 1 or Table 2, or amino acid sequences that are at least 75% identical thereto. In preferred embodiments, the antibody comprises at least one, more preferably at least two, most preferably three CDRs, each containing an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs. 54-56, or an amino acid sequence that is at least 75% identical to an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs. 54-56. In related embodiments, the nucleotide sequence encodes an antibody comprising an amino acid sequence selected from the amino acid sequences described as SEQ ID NOs. 14 and 133-136, or an amino acid sequence that is at least 75% identical (e.g., at least 80%, 90%, at least 95%, at least 98%, or at least 99%) to an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs. 14 and 133-136. In other related embodiments, the nucleotide sequence encoding the nanobody comprises a nucleotide sequence described in any one of SEQ ID NOs. 133-136. In a relevant embodiment, the nucleotide sequence encodes an antibody comprising an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs: 137-163, or an amino acid sequence that is at least 75% (e.g., at least 80%, 90%, at least 95%, at least 98%, or at least 99%) identical to an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs: 137-163.

[0310] In some embodiments, rAAV is administered by periorbital, intravitreal, choroidal and / or subretinal injection, preferably intravitreal, to subjects with atrophic AMD and / or geographic atrophy, at a dose of approximately 1 × 10⁻¹⁴. 8 Vector genome (vg) / eye ~ approximately 1 × 10⁻⁶ 13 vg / eye, approx. 1×10 9 vg / eye ~ approx. 1×10 12 vg / eye, approx. 1×10 9 vg / eye ~ approx. 1×10 11 vg / eye or approximately 6 x 109 vg / eye ~ approx. 6×10 10 It is administered at a dose of VG / eye.

[0311] In some embodiments, a method is provided for treating atrophic AMD and / or geographic atrophy in subjects requiring it, comprising administering to a subject by periorbital, intravitreal, choroidal and / or subretinal injection an effective amount of rAAV virion comprising (i) a capsid protein comprising an amino acid sequence described as SEQ ID NO: 170, and (ii) a heteronucleotide comprising (a) an AAV2 terminal repeat, (b) a CAG or CMV promoter, (c) a nucleotide sequence encoding an amino acid sequence described in any of SEQ ID NOs: 14 and 133-136, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat, directed from 5' to 3'.

[0312] In related embodiments, a method is provided for treating atrophic AMD and / or geographic atrophy in subjects requiring it, comprising administering to a subject by periorbital, intravitreal, choroidal and / or subretinal injection a pharmaceutical composition comprising: (i) a capsid protein comprising an amino acid sequence described as SEQ ID NO: 170; and (ii) an rAAV virion comprising a heterogeneous nucleic acid comprising (a) an AAV2 terminal repeat, (b) a CAG or CMV promoter, (c) a nucleotide sequence encoding an amino acid sequence described in any of SEQ ID NOs: 14 and 133-136, (d) a polyadenylated sequence; and (e) an AAV2 terminal repeat, directed from 5' to 3'. In some embodiments, the pharmaceutical composition comprises approximately 1 × 10⁻¹⁶ 8 vg~approx. 1×10 13 vg, approx. 1×10 9 vg~approx. 1×10 12 vg, approx. 1×10 9 vg~approx. 1×10 11 vg, or approximately 6 × 10 9 vg~approx. 6×10 10 Includes vg

[0313] In some embodiments, a method is provided for treating atrophic AMD and / or geographic atrophy in subjects requiring it, comprising administering to a subject by periorbital, intravitreal, choroidal and / or subretinal injection an effective amount of rAAV virion comprising (i) a capsid protein comprising an amino acid sequence described as SEQ ID NO: 170, and (ii) a heteronucleotide comprising (a) an AAV2 terminal repeat, (b) a CAG or CMV promoter, (c) a nucleotide sequence encoding an amino acid sequence described in any of SEQ ID NOs: 137-163, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat, directed from 5' to 3'.

[0314] In related embodiments, a method is provided for treating atrophic AMD and / or geographic atrophy in subjects requiring it, comprising administering to a subject by periorbital, intravitreal, choroidal and / or subretinal injection a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (i) a capsid protein comprising an amino acid sequence described as SEQ ID NO: 170, and (ii) a heteronucleotide comprising (a) an AAV2 terminal repeat, (b) a CAG or CMV promoter, (c) a nucleotide sequence encoding an amino acid sequence described in any of SEQ ID NOs: 137-163, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat, directed from 5' to 3'. In some embodiments, the pharmaceutical composition comprises about 1 × 10⁻¹⁶ 8 vg~approx. 1×10 13 vg, approx. 1×10 9 vg~approx. 1×10 12 vg, approx. 1×10 9 vg~approx. 1×10 11 vg, or approximately 6 × 10 9 vg~approx. 6×10 10 Includes vg

[0315] A pharmaceutical composition comprising rAAV as described herein is provided. In some embodiments, the pharmaceutical composition comprises about 1 × 10⁻¹⁶ 8 ~Approx. 1×10 14 A vector particle or vector genome, approximately 1 × 10⁶ 8~Approx. 1×10 13 A vector particle or vector genome, approximately 1 × 10⁶ 9 ~Approx. 1×10 12 A vector particle or vector genome, or approximately 1 × 10⁶ 9 , about 2×10 9 , 3 x 10 9 , about 4×10 9 , about 5×10 9 , about 6×10 9 , about 7×10 9 , about 8×10 9 , about 9×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , or approximately 1 x 10 12 It contains vector particles or vector genomes. In some embodiments, the pharmaceutical composition contains about 1 × 10⁶ 9 ~Approx. 1×10 11 Contains vg, preferably about 6 × 10 9 ~about 6×10 10 Contains vg. In some preferred embodiments, the pharmaceutical composition is administered by intravitreal injection to a person having atrophic AMD and / or geographic atrophy. [Examples]

[0316] The following embodiments illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. Although the present invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to those skilled in the art from reading this application. (Example 1)

[0317] Data from human patients with atrophic and exudative age-related macular degeneration (AMD) support the hypothesis that steady-state "idling" of the complement II pathway leads to chronic deposition of complement membrane invasion complexes (MACs) on the choroidal capillary lamina and retinal pigment epithelium (RPE). Unsoluble levels of MAC lead to the production of cytokines and inflammatory molecules. Therefore, preventing MAC deposition provides an effective treatment for atrophic age-related macular degeneration and geographic atrophy.

[0318] Nanobodies targeting human complement factor C3 (SEQ ID NOs: 1-14) were isolated by phage display panning against the human C3 protein, and their ability to specifically bind to C3 and inhibit complement was tested. Recombinant AAVs containing the capsid of SEQ ID NO: 107, which encapsulates heterologous nucleic acids encoding representative anti-C3 nanobodies, were generated, and their complement inhibition was evaluated.

[0319] material and method

[0320] Recombinant protein production

[0321] Recombinant proteins for each identified nanobody were produced in HEK293 cells using NeoClone and GeneScript.

[0322] cell culture

[0323] HEK293T cells were grown on tissue culture plastics in DMEM, 10% fetal bovine serum (FBS), and 1% penicillin / streptomycin under normal oxygen conditions with 5% CO2.

[0324] iPSC-RPEs were grown on Matrigel-coated plates and matured in XVIVO-10 medium under normal oxygen conditions with 5% CO2 for 30 days.

[0325] Trait introduction

[0326] iPSC-RPE was transduced at infection multiplicity (MOI, vg / cell) of 5,000, 10,000, or 20,000 cells more than 30 days after sowing. Samples were collected 7 days after infection for analysis.

[0327] Complement Inhibition Assay

[0328] The Wieslab® Complement System II Pathway Kit (Eagle Biosciences Inc. (catalog no. COMPL AP330)) is an enzyme immunoassay for the qualitative determination of the functional complement II pathway in human serum and was performed according to the manufacturer's instructions for use. The Wieslab complement assay combines the specific activation of the pathway with the use of an antibody labeled specifically for the terminal complement complex, C5b-9 neoepitope, which is produced as a result of complement activation (the amount of C5b-9 produced is proportional to the functional activity of the complement system). Briefly, for the Wieslab complement inhibition assay performed in Table 2, normal human or cynomolgus monkey serum (Complement Tech) was diluted 1:20 with 3.75 μg of purified nanobodies. Control and test serums were added to plates provided in 100 μL / well. After incubation at 37°C for 1 hour, the plates were washed three times with washing solution. The prepared conjugate alkaline phosphatase-labeled antibody against C5b-9 was added and incubated at room temperature for 30 minutes. After washing, the substrate solution was added to each well and the absorbance was read at 405 nm.

[0329] For the Wieslab complement inhibition assay performed in Figure 1, the supernatant after transfection, or recombinant purified nanobodies normalized to 1 mg / ml for dose-response curves, were diluted in 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 diluent was added to the plate in three repetitions and mixed on a plate mixer for 5 minutes. After mixing, the serum with the nanobody diluent 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 in the kit. 100 μl of the substrate solution provided in the kit was added to the microtiter plate, and absorbance was read at 405 nm at 5-minute intervals over 60 minutes. For data analysis, the OD405nm at 60 minutes was plotted as a function of concentration with respect to the dose-response curve.

[0330] Immunocytochemistry (ICC)

[0331] iPSC-RPE cells were exposed to either 1% human serum or 1% human serum + 5 μg / ml zymosan to induce the complement II pathway. 24 hours after 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 counterstaining at room temperature for 1 hour.

[0332] rAAV manufacturing

[0333] For in vitro testing, rAAV having the capsid amino acid sequence listed in Sequence ID No. 107 was produced in HEK293 cells by PEI-mediated triple transfection. The cells were cultured in DMEM supplemented with FBS and maintained at 37°C in a 5% CO2 environment.

[0334] Briefly, cells were seeded in CellSTACK containers and transfected with pHelper, the associated Rep-Cap plasmid, and the CAG-nanobody payload plasmid. Three to four days after transfection, cells and supernatant were collected, lysed, treated with endonuclease, clarified by sterile filtration, and frozen. The thawed recovered material was clarified, loaded onto the associated affinity resin, eluted at a low pH, and immediately neutralized. The purified lot was formulated in either a phosphate or Tris-based buffer system containing NaCl and 0.005% Pluronic® F-68. The purified lot was stored at -80°C until use.

[0335] ELISA binding assay

[0336] Antigen proteins were pre-coated in standard ELISA plates at 500 ng / well and then blocked with 1% BSA. Nanobodies were diluted to 10 μg / mL and 150 μL was added to each well. After incubation and washing, any remaining bound nanobodies were detected with HRP-conjugated monoclonal rabbit anti-camelid VHH. A control well free of nanobodies was used as background, and its readings were subtracted from the test wells.

[0337] Octet / Gator binding affinity measurement

[0338] The binding affinity of the nanobodies to their respective antigens was determined using biolayer interferometry with a Gator or Octet instrument. The anti-His probe was initially equilibrated in a 96-well plate in buffer (PBST + 5% BSA) at 37°C and 1000 rpm. After equilibration, 6XHis-tagged nanobodies (loaded samples) were immobilized on the sensor. Once the probe reached 60% volume, the loaded sensor was washed in buffer to remove all nonspecific bindings. Next, for the binding step to obtain "Kon," the nanobodies-loaded probe was immersed in serial dilutions of untagged target antigen protein (analyte sample). The sensor was then transferred to blank buffer to obtain the dissociation rate "Koff" and dissociation rate constant (1 / s). The ratio of Koff / Kon is "KD," which is the equilibrium dissociation rate constant, a direct measure of the binding affinity of the binding pair.

[0339] result

[0340] Nanobody target binding and complement system inhibition [Table 3]

[0341] NB = No bonding; WkB = Weak bonding

[0342] Table 3 lists nanobody clones isolated by phage display panning against human C3 protein, along with their respective characteristics (corresponding amino acid sequences are as indicated). Production yield is calculated by the amount of purified protein extracted from 60 mL of supernatant of transiently transfected HEK293 suspension cell lines. ELISA binding to human or cynomolgus monkey C3 protein is a multiplier above the background signal for each clone compared to no antibody. Wieslab's complement II pathway assay results are determined by whether each nanobody was able to inhibit at least 50% of complement activation at the specified concentration. Finally, KD values ​​for VHH binding to human or cynomolgus monkey C3 protein are calculated using BLI.

[0343] All nanobodies assayed in Table 3, except for C3S5 and C3S10, bound to either human and / or cynomolgus monkey C3 protein (see corresponding ELISA binding and KD values). Similarly, all nanobodies assayed in Table 3, except for C3S4, inhibited MAC formation at either 4 μM and / or 0.1 μM, as shown in their corresponding Wieslab inhibition columns.

[0344] Transduction of rAAV-containing nanobody-introduced genes inhibits MAC formation in iPSC-RPEs.

[0345] Induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPEs) were transduced with rAAV containing the capsid amino acid sequence listed in SEQ ID NO: 107, which carries the C3-N10 transgene (SEQ ID NO: 14), at three different MOIs (Moritosity of Infection) of 5,000, 10,000, and 20,000. Seven days after transduction, nanobody (VHH) protein levels in the supernatant were analyzed by ELISA to observe the dose-response relationship in expression (Figure 1a). MAC formation was determined by evaluating complement inhibition using the supernatant. When the dose-response relationship in MAC formation was observed after transduction at MOIs of 5,000, 10,000, and 20,000 compared to non-transduction, higher MOIs were associated with less MAC formation (Figure 1b). Furthermore, MAC deposition was observed on cells after zymosan, a complement II pathway stimulant, and this was reversed when cells were transduced with C3-N10 rAAV or a positive control for complement II pathway inhibition (Figure 1c). (Example 2)

[0346] Data from human patients with atrophic and exudative age-related macular degeneration (AMD) support the hypothesis that steady-state "idling" of the complement II pathway leads to chronic deposition of complement membrane invasion complexes (MACs) on the choroidal capillary lamina and retinal pigment epithelium (RPE). Unsoluble levels of MAC lead to the production of cytokines and inflammatory molecules. Therefore, preventing MAC deposition provides an effective treatment for atrophic age-related macular degeneration and geographic atrophy.

[0347] Nanobodies targeting human complement factor C5 (SEQ ID NOs: 57-65) were isolated by phage display panning against human C protein, and their ability to specifically bind to C5 and inhibit complement was tested.

[0348] material and method

[0349] Complement Inhibition Assay

[0350] The Wieslab® Complement System II Pathway Kit (Eagle Biosciences Inc. (catalog no. COMPL AP330)) is an enzyme immunoassay for the qualitative determination of the functional complement II pathway in human serum and was performed according to the manufacturer's instructions for use. The Wieslab complement assay combines the specific activation of the pathway with the use of an antibody labeled specifically for the terminal complement complex, C5b-9 neoepitope, which is produced as a result of complement activation (the amount of C5b-9 produced is proportional to the functional activity of the complement system). Briefly, for the Wieslab complement inhibition assay performed in Table 2, normal human or cynomolgus monkey serum (Complement Tech) was diluted 1:20 with 3.75 μg of purified nanobodies. Control and test serums were added to plates provided in 100 μL / well. After incubation at 37°C for 1 hour, the plates were washed three times with washing solution. The prepared conjugate alkaline phosphatase-labeled antibody against C5b-9 was added and incubated at room temperature for 30 minutes. After washing, the substrate solution was added to each well and the absorbance was read at 405 nm.

[0351] ELISA binding assay

[0352] Antigen proteins were pre-coated in standard ELISA plates at 500 ng / well and then blocked with 1% BSA. Nanobodies were diluted to 10 μg / mL and 150 μL was added to each well. After incubation and washing, any remaining bound nanobodies were detected with HRP-conjugated monoclonal rabbit anti-camelid VHH. A control well free of nanobodies was used as background, and its readings were subtracted from the test wells.

[0353] Octet / Gator binding affinity measurement

[0354] The binding affinity of the nanobodies to their respective antigens was determined using biolayer interferometry with a Gator or Octet instrument. The anti-His probe was initially equilibrated in a 96-well plate in buffer (PBST + 5% BSA) at 37°C and 1000 rpm. After equilibration, 6XHis-tagged nanobodies (loaded samples) were immobilized on the sensor. Once the probe reached 60% volume, the loaded sensor was washed in buffer to remove all nonspecific bindings. Next, for the binding step to obtain "Kon," the nanobodies-loaded probe was immersed in serial dilutions of untagged target antigen protein (analyte sample). The sensor was then transferred to blank buffer to obtain the dissociation rate "Koff" and dissociation rate constant (1 / s). The ratio of Koff / Kon is "KD," which is the equilibrium dissociation rate constant, a direct measure of the binding affinity of the binding pair.

[0355] result

[0356] Nanobody target binding and complement system inhibition [Table 4]

[0357] NB = No bonding; WkB = Weak bonding

[0358] Table 4 lists nanobody clones isolated by phage display panning against human C5 protein, along with their respective characteristics. Production yield is calculated by the amount of purified protein extracted from 60 mL of supernatant of transiently transfected HEK293 suspension cell lines. ELISA binding to human C5 or cynomolgus monkey C5a protein is a multiplier above the background signal for each clone compared to no antibody. Wieslab's complement II pathway assay results are determined by whether each nanobody was able to inhibit at least 25% of complement activation at a specified concentration. Finally, the KD value for VHH binding to human C5 protein is calculated using BLI.

[0359] All nanobodies assayed in Table 4, except for C5S12, bound to the human C5 protein (see corresponding ELISA binding and KD values). Similarly, all nanobodies assayed in Table 4, except for C5S3, C5S4, and C5S7, inhibited MAC formation at either 4 μM and / or 0.1 μM, as shown in their corresponding Wieslab inhibition columns. (Example 3)

[0360] Methods and materials

[0361] Production of recombinant complement proteins

[0362] To produce recombinant proteins, both wild-type and mutant complement sequences, along with a C-terminal TEV cleavage site and a 6XHis tag, were cloned into a pcDNA plasmid containing a mouse IgG secretion sequence and transiently transfected into Expi293 cells. Cells were harvested and spun at 250Xg for 30 minutes, and the supernatant was collected. Recombinant proteins were purified by single-step purification using fixed 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 trap column pre-equilibriumated 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-tagged elution buffer (250 nM imidazole, 50 mM Tris, 150 mM NaCl pH 8). The his-eluted material was subjected to TEV protease for 6X His tag cleavage. After cleavage, the 6X His tag was removed by passing the eluate through IMAC resin. The eluate from the IMAC column was passed through size exclusion chromatography, buffer changed in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaH2PO4, 1.4 mM KH2PO4), passed through a 0.4 nm filter, and stored in aliquots at -20°C.

[0363] Production of recombinant VHH

[0364] For recombinant nanobody production, both wild-type and humanized nanobody sequences, along with C-terminal strep II tags and 6X His tags, were cloned into a pcDNA plasmid containing a mouse IgG secretion sequence and transiently transfected into Expi293 cells. Cells were harvested and spun at 250Xg for 30 minutes, and the supernatant was collected. Recombinant nanobody sequences were purified by single-step purification 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 trap column pre-equilibriumated 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-tagged elution buffer (250 nM imidazole, 50 mM Tris, 150 mM NaCl pH 8). The his-eluted material was subjected to buffer exchange in PBS, passed through a 0.4 nm filter, and stored in aliquots at -20°C.

[0365] Production of recombinant VHH-Fc

[0366] For recombinant production of Fc-fusion nanobodies, VHH cDNA was cloned into pCDNA3 together with a C-terminal human IgG2 hinge-human IgG4 Fc molecule, and the nanobodies were expressed as Fc dimers in Expi293 cells along with a mouse IgG signal peptide for secretion into the supernatant. After transient transfection, cells were harvested and centrifuged at 250xg for 30 minutes to settle the cells and supernatant, which were then passed through a 0.45 μm membrane. Recombinant VHH-Fc dimers were passed through a Mabselect sure column in 1×PBS buffer at pH 7 to capture the VHH-Fc fusions. After loading, the column was washed with wash buffer and eluted using low pH buffer, 0.1 M citrate buffer, and 150 mM NaCl pH 3. The eluate was immediately neutralized with 1 M Tris pH 8, then buffer-changed in 1×PBS pH 7 and aliquoted.

[0367] Wieslab complement activation assay

[0368] The Wieslab complement II pathway assay system was used for semi-quantitative estimation of the inhibitory activity of nanobodies. The Wieslab II pathway kit is an enzyme immunoassay for the qualitative determination of the functional complement II pathway in human serum. This assay combines the principle of a hemolytic assay for complement activation with the use of labeled antibodies specific to the novel antigen produced for complement activation. For complement-activated AP, the assay plate is coated with a specific activator of the II pathway activator, and the provided diluent solution contains a specific blocker to ensure activation of only the II pathway. To evaluate the nanobody complementary AP inhibitory effect, normal human serum was diluted 1:20 in the provided diluent and added to a 96-well plate. Recombinant purified nanobodies were spiked into the wells in two replicates along with control and test serum and mixed on a plate shaker at 400 rpm for 5 minutes. After mixing, the contents of the 96-well plate were 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 solution, and a conjugate alkaline phosphatase-labeled antibody against C5b-9 was added. The plate was incubated at room temperature for 30 minutes. Following washing, the substrate solution was added to the wells and incubated at room temperature for 45 minutes. The amount of complement activation correlates with color intensity and is measured in units of absorbance / optical density at 405 nm.

[0369] Classical and lectin pathway inhibition assays

[0370] Furthermore, the complement inhibitory effects of nanobodies were investigated in the classical and lectin pathways using Wieslab's classical & mannan-binding lectin pathway kits. In the classical pathway kit, microplate wells are coated with CP activators such as the C1q / C1 complex, and the supplied diluent contains a specific activator for the classical pathway. Similarly, for the lectin pathway, the MBL kit consists of microwell plates coated with mannose-binding lectins, opsonin, or phycolin that specifically activate the lectin pathway, combined with a diluent buffer that has a specific blocker for AP & CP.

[0371] Primate serum cross-reactivity

[0372] Cross-reactivity with primate serum was also investigated using the Wieslab AP assay kit. In this case, nanobodies were spiked into a 1:20 dilution of normal cynomolgus monkey serum, followed by standard manufacturer guidelines.

[0373] Calculation of IC50

[0374] The formation of membrane-associated complexes (IC50 MACs) at median inhibitory concentrations was determined using Wieslab's second-pathway assay. A dilution series of recombinant purified nanobodies was spiked into 20-fold diluted human serum at various concentrations and added to Wieslab assay plates. After 60 minutes, the incubation was washed and treated with C5b-9-labeled conjugate alkaline phosphatase, followed by incubation for 30 minutes. After the conjugate incubation, the plates were washed and incubated with the substrate solution, with readings taken at 405 nm at 45 minutes. IC50 MAC formation was estimated using curve fitting software.

[0375] Mouse complement pathway 2 assay

[0376] The cross-functional activity of C3 and C5 nanobodies against mouse complement proteins was checked using mouse C3 / C5 ELISA. The assay provides a microwell plate coated with LPS for specific activation of the second pathway. For this assay, mouse serum was diluted 1:20 in the provided dilution buffer and transferred to a 96-well plate. A serial dilution of nanobodies was spiked into the serum in three replicates along with a positive control mouse BB5.1 antibody. The 96-well plate was agitated on a plate shaker at 400 rpm for 5 minutes, and the contents of the plate were transferred to an LPS-coated microwell plate and incubated at 37°C for 60 minutes. After incubation, the plate was washed four times with the provided wash buffer, biotinylated tracer antibody was added, incubated for another 60 minutes at 37°C, and washed four more times. Streptavidin peroxide conjugate was added, incubated for 60 minutes at 37°C, and washed four more times. The TMB solution was added, incubated at room temperature for 30 minutes, and the reaction was stopped by adding a stop solution. The OD was then measured at 450 nm.

[0377] Gator assay for binding affinity (KD)

[0378] Probes coated with anti-strep II antibody were used for binding affinity determination using Octet / Gator. The anti-strep II antibody probes were immersed in buffer (PBST + 5% BSA) in a 96-well plate for equilibration at 37°C and 1000 rpm. After equilibration, the probes were immersed in wells containing VHH-strep-tagged VHH for loading. Once the probes were approximately 60% loaded, the anti-strep II antibody probes were immersed in buffer wells for a washing step to remove all VHH that had nonspecifically bound to the probes. After washing the probes, for the binding step, they were immersed in serial dilutions of recombinant purified human or cynomolgus monkey C3 or C5 wild-type or C5 single and double mutant proteins (untagged) to obtain the binding rate constant (1 / s), "Kon". After binding, the dissociation step was performed in buffer to obtain the dissociation rate "Koff" and dissociation rate constant (1 / s). The Koff / Kon ratio is "KD," which is the equilibrium dissociation rate constant, a direct measure of the binding affinity of a bond pair.

[0379] Antibody cross-blockage against C5 protein by Gati BLI sandwich assay

[0380] The binding epitopes of the C5S2.2 nanobody were investigated using the Gator BLI platform. Similar to eculizumab, the C5S2.2 nanobody did not show cross-binding to cynomolgus monkey C5 protein in cynomolgus monkey serum and did not exhibit inhibitory complement II pathway activity. Therefore, the binding epitopes were matched to determine whether eculizumab and C5S2.2 cross-block each other against human C5. An anti-human Fc binding probe was used, employing a sandwich method on BLI with eculizumab immobilized for 1–2 minutes. After washing, the pre-loaded probes were immersed for 5–6 minutes in the recommended binding buffer with either untagged C5 only or C5 pre-incubated with C5S2 to enable binding to either C5 or the C5S2-C5 complex.

[0381] Reverse sandwich assay

[0382] A reverse sandwich was performed by immobilizing a diluted series of C5S2 nanobodies onto an anti-strep-II probe. After washing, the pre-loaded probe was immersed in a C5-containing binding solution for a check to ensure that the C5 protein bound to the immobilized nanobodies for 2-3 minutes. Once the C5 protein bound to the probe holding the VHH-C5 complex, it was immersed in a well containing eculizumab for 5-6 minutes for binding.

[0383] Epitope mapping by cross-linked mass spectrometry (XL-MS)

[0384] Epitope peptide mapping of humanized C3 nanobodies against human C3 protein was performed by cross-linking mass spectrometry. Purified nanobodies and recombinant proteins were generated. Each nanobody was capable of binding to a target and then chemically cross-linked using disuccinimidyl suberate. This complex was then treated with a set of overlapping proteases, and the peptides were analyzed by mass spectrometry and compared to the native protein sequence.

[0385] QC check regarding sample integrity

[0386] As part of the initial QC check, high-mass MALDI-MS analysis was performed on both recombinant protein nanobodies, C3 and C5. Furthermore, the same experiment was performed on the cross-linked recombinant protein and compared to verify the integrity and aggregation levels of the samples, which also serve as controls for subsequent experiments.

[0387] Characterization of nanobody / C3 composites

[0388] After confirming the integrity of the samples, C3N10 / C3&C3S3 / C3 was characterized. For characterization, the complex was prepared by incubation at two different concentrations, and then measured using an Autoflex MALDI / TOF mass spectrometer. For the crosslinking experiment, the C3N10 / C3&C3S3 / C3 complex was treated with DDS for 180 minutes and measured on an Autoflex MALDI / TOF. The readouts of the control and crosslinked mass spectrometers were compared for any further peaks.

[0389] Peptide mass fingerprinting

[0390] For peptide mass fingerprinting, hC3 was crosslinked with DDS for 180 minutes and cleaved with five different proteases (trypsin, chymotrypsin, elastase, Asp-N, and thermolysin), and measured for maximum sequence coverage. After cleavage, both the control and crosslinked samples were subjected to liquid chromatography for peptide separation, followed by mass spectrometry on a Q-Exactive Plus mass spectrometer. The resulting peptides were analyzed using CovalX proprietary software to generate peptide fingerprint maps for both the crosslinked and control samples.

[0391] Antidrug antibody response to nanobodies derived from normal human donor serum using mesoscale exploratory immunoassay

[0392] Purified nanobodies and controls were passively coated onto MSD LI5XA-3 plates at 1 μg / mL and then blocked with casein. In-house repositories of serum and plasma from normal human donors were diluted 1:100 and added to each well, then incubated at room temperature for 2 hours with shaking to allow conjugation. After washing, conjugated antibodies were detected using Sulfo-TAG goat anti-human IgG. Purified human IgG was directly coated at multiple concentrations to track assay performance and as a positive control. After washing, diluted read buffer was added to each well, and the plates were read in a Meso Sector S600.

[0393] Plasmid DNA cloning and isolation

[0394] The AAV-assisted vector, containing the CAG promoter and other necessary AAV cassette components, was digested with restriction enzymes to remove unwanted payloads. Custom gBlocks (GB) from Integrated DNA Technologies, containing the desired VHH, were cloned into the main chain.

[0395] Miniprep cultures were grown from colonies obtained after cloning and transformation in Terrific Broth, supplemented with 100 mg / mL kanamycin sulfate and grown 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 sCFH and CFH-positive clones.

[0396] cell culture

[0397] HEK293T cells were grown in DMEM medium containing 1% penicillin / streptomycin and 10% fetal bovine serum. The cells were passaged twice a week and were not used beyond passage 20.

[0398] iPSC-RPEs were 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 normal oxygen conditions and 5% CO2.

[0399] AAV manufacturing

[0400] Recombinant rAAV (containing the capsid of SEQ ID NO: 170) for in vitro testing was produced by PEI-mediated triple transfection into HEK293 cells. The cells were cultured in DMEM supplemented with FBS and maintained at 37°C in a 5% CO2 environment.

[0401] Briefly, cells were seeded in CellSTACK containers and transfected with pHelper, the associated Rep-Cap plasmid, and the CAG-nanobody payload plasmid. Three to four days after transfection, cells and supernatant were collected, lysed, treated with endonuclease, clarified by sterile filtration, and frozen. The thawed recovered material was clarified, loaded onto the associated affinity resin, eluted at a low pH, and immediately neutralized. The purified lot was formulated in either a phosphate or Tris-based buffer system containing NaCl and 0.005% Pluronic® F-68. The purified lot was stored at -80°C until use.

[0402] VHH plasmid transfection

[0403] HEK293T cells, 2 × 10⁶ 5 Cells were seeded in 12-well plates at a rate of one cell / well and transfected with various plasmid versions of C3N10, C3S3, and C5S2. The supernatant was collected and transgene expression was tested using various assays.

[0404] Trait introduction

[0405] iPSC-RPE was transduced at infection multiplicity (MOI, vg / cell) of 1,000, 5,000, or 20,000 cells more than 30 days after sowing. Supernatant samples were collected 7 days after infection for analysis.

[0406] VHH ELISA

[0407] The anti-VHH capture antibody was diluted 1:500 in PBS and incubated overnight at 4°C in a MaxiSorp microtiter plate. The plate was then washed five times with PBST and incubated for 2 hours at room temperature in a blocking solution of 2% BSA in PBS. Plate washing was performed similarly between each of the following steps unless otherwise noted. The supernatant from the transduced cells was diluted in 1% BSA in PBS; the dilution was uniform across all samples of each VHH tested. The diluted samples were added to the microtiter plate and incubated for 2 hours at room temperature. The plate was then incubated for 1 hour at room temperature with HRP-conjugated anti-VHH detection antibody diluted 1:10,000 in 1% BSA in PBST. After incubation with the detection antibody, 100 μl of TMB substrate was added to the plate and the color development was monitored. Five to fifteen minutes after adding the TMB substrate, 100 μl of 450 nM TMB stop solution was added to the plate without a washing step. Optical density (OD) was measured at 450 nm using a Cytation 3 photometer within 15 minutes of adding the stop solution.

[0408] Western blot

[0409] The protein concentration in the cell supernatant was quantified using the Pierce Micro BCA Protein assay according to the manufacturer's instructions. An appropriate volume of supernatant to achieve 33 μg of total protein was then diluted to 15 μl in PBS, mixed with 5.78 μl of 4×LDS buffer and 2.31 μl of 10-fold reducing agent, and incubated at 90°C for 10 minutes. The samples were loaded onto 15-well Bolt 12% Bis-Tris Plus polyacrylamide gels or Novex 10-20% tricine polyacrylamide gels and electrophoresed in 1×MOPS buffer at 160 volts for 45 minutes. The separated proteins were transferred to PVDF membranes using a BioRad TransBlot Turbo device (catalog no. 1704150, Bio-Rad) at 2.5 A for 10 minutes. In iBind Flex solutions containing iBind Flex cards, membranes were probed using HRP-conjugated anti-VHH primary antibody, mouse anti-C3a, rabbit anti-C5a antibody, or, if not conjugated, goat anti-human factor B antibody along with an appropriate HRP secondary antibody. Proteins were visualized using SuperSignal West Pico Plus Chemiluminescent Substrate and imaged on ChemiDoc MP.

[0410] Complement inhibition assay for evaluation of transduced samples

[0411] The Wieslab® Complement II Pathway Kit is an enzyme immunoassay for the qualitative determination of the functional complement II pathway in human serum, and was performed according to the manufacturer's instructions for use. Normal human serum was diluted to 2% in the provided dilution buffer, and 200 μl of 2% serum was transferred to a microtiter plate. 20 μl of supernatant sample from transduced RPE culture was added to the plate and mixed. After mixing, the diluted serum with the supernatant sample was transferred to the assay plate in two repeats at 100 μl per well 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 in the kit. 100 μl of the substrate solution provided in the kit was added to the microtiter plate, and absorbance was read at 405 nm at 5-minute intervals over 90 minutes. The complement inhibition percentage for each sample was calculated according to the following formula and considered semi-quantitative.

[0412]

number

[0413] result

[0414] Wild-type and humanized nanobody binding affinity to C3 and C5 targets

[0415] 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 from transiently transfected human HEK293 suspension cells, purified, and further tested below. Table 5 contains the binding affinities of the wild-type and humanized nanobodies to human and cynomolgus monkey C3 and C5 complement protein targets. KD was calculated in nM using biolayer interferometry (BLI) with Gator. Neither the C3-targeted wild-type nanobodies nor the C5-targeted wild-type nanobodies cross-bound to other antigens, nor did any of them bind to other members of the alpha-2-macroglobulin family: human C4b, alpha-2-macroglobulin, and alpha-2-macroglobulin-like 1 protein (data not shown). [Table 5]

[0416] Mechanism of action: Complement pathway inhibitory activity

[0417] The innate immune complement system is initiated by three distinct pathways: the classical pathway, the lectin pathway, and the secondary pathway. While C3 proteins act as convergence points for the activation of each pathway, cleavage of C5 proteins initiates the formation of the final component of complement activation: the membrane invasion complex. To distinguish their mechanisms of action within the three pathways of the complement cascade, the functional potential of wild-type nanobodies was evaluated in human serum available from Wieslab using classical, secondary, and classical pathway assay kits. As shown in Figure 2, all three nanobodies exhibit inhibitory activity against the secondary pathway. However, while C5S2 exhibits potent inhibitory activity across all three pathways, C3S3 exhibits activity only against the secondary pathway. C3N10 shows the highest inhibitory activity against the secondary pathway and moderate potency against both the classical and lectin pathways.

[0418] IC50 efficacy of wild-type and humanized nanobodies against the second human serum-mediated pathway.

[0419] IC50 was calculated between wild-type and humanized nanobodies, and all were serially diluted 1:2 and retested in the same second-pathway assay to compare potency. The humanized nanobodies exhibited similar potency to their original wild-type counterparts, indicating only slight loss of inhibitory activity between variants. Figures 3A-C show the potency, and Table 6 summarizes these results. IC50 of C3N10 (wild-type) 50 The current was 133.8 nM; IC C3N10.3 50 The saturation was 69.47 nM (Figure 3A). C3S3 (wild-type) IC 50 The current was 34.51 nM; IC C3S3.2 50 The current was 42.86 nM (Figure 3B). C5S2 (wild type) IC 50 The current was 46.18 nM; IC C5S2.2 50 The value was 47.33 nM (Figure 3C).

[0420] IC50 of C3N10 WT and humanized C3N10 in the classical and lectin pathways.

[0421] Since C3N10 showed mild inhibition in both the classical and lectin pathways, the IC50s of wild-type and humanized C3N10.3 were calculated for both pathways in a greater number of serial dilutions, as shown in Figures 4A-B and summarized in Table 6 below. [Table 6]

[0422] Activity of anti-human C3 and C5 wild-type nanobodies against the mouse secondary pathway.

[0423] To test whether human complement pathway II inhibitory nanobodies are cross-functional in mice, wild-type versions were tested using Hycult's mouse C3 / C5 Elisa kit (catalog number HK2002). Positive control mice were enriched with the anti-mouse C5 inhibitor antibody BB5.1. As shown in Figure 5, C3N10, C3S3, and C5S2 did not exhibit observable inhibitory activity in this mouse serum-mediated complement pathway II assay.

[0424] IC50 of anti-human C3 and C5 wild-type and humanized nanobodies against the second pathway activity in non-human primate serum

[0425] Based on their binding affinity to each cynomolgus monkey variant, the IC50 of wild-type and humanized nanobodies was evaluated using the same Wieslab complement II pathway assay with cynomolgus monkey serum, as shown in Figures 6A and 6B and Table 7 below. Both the C3-targeted wild-type and humanized nanobodies showed inhibitory activity, but neither C5S2 nor its humanized version showed Wieslab cynomolgus monkey serum-mediated complement II pathway efficacy (data not shown), reflecting their inability to bind to their cynomolgus monkey C5 protein target, as shown in Table 5. [Table 7]

[0426] Humanized nanobody-human Fc fusion protein inhibits binding rate and complement pathway 2.

[0427] Three humanized nanobodies, C3N10.3, C3S3.2, and C5S2.2, were manipulated and expressed as homodimer fusion proteins with human G2 hinge-G4 Fc domains (also known as C3N10.3-Fc, C3S3.2-Fc, and C5S2.2-Fc) to mimic the divalent structure and function of innate immunoglobulins. Their respective binding kinetics and pathway II inhibitory efficacy against the target were calculated using in vivo interferometry and Wieslab assays, and compared to the original monomers in Tables 8 and 9, respectively. Avidity binding was observed for C3S3.2-Fc and C5S2.2-Fc, but a synergistic pathway II inhibitory activity (more than 2x) is thought to be observed for C5S2-Fc (Figure 7). [Table 8] [Table 9]

[0428] Epitope peptide maps of C3N10.3 and C3S3.2 for human C3 protein

[0429] To further distinguish the mechanisms of action between the two C3-targeted VHHs, cross-linking mass spectrometry (XL-MS, CovalX) was used to identify the C3N10.3 and C3S3.2 binding epitopes. Both humanized nanobodies and purified C3 human proteins were provided. Tables of cross-linking peptides between nanobodies and antigens, along with examples of amino acid interactions, are shown in Table 10 and Figure 8 for C3N10.3 and in Table 11 and Figure 9 for C3S3.2, respectively. Based on these interaction residues, the binding epitope of CN10.3 is thought to be located within the alpha chain of C3 in the C345C domain (1540-1600: GVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKK (SEQ ID NO: 171)), while C3S3.2 is thought to bind to two nonlinear epitopes in the beta chain across the MG1 (110-130: ATFGTQVVEKVVLVSLQSGYL (SEQ ID NO: 172)) and MG5 (490-510: YTYLIMNKGRLLKAGRQVREP (SEQ ID NO: 173)) domains. Figure 10. [Table 10] [Table 11]

[0430] Epitope matching of C5S2.2: Cross-blocking of eculizumab against human C5 protein

[0431] Clinically approved eculizumab, like C5S2, binds to the human C5 protein and inhibits all three complement pathways in human serum, but does not bind to cynomolgus monkey C5 or inhibit the complement pathway in cynomolgus monkey serum (data not shown). Gator was also used to investigate whether eculizumab and C5S2 cross-block each other against human C5.

[0432] In Figure 11, anti-human Fc-binding chips were initially loaded with eculizumab and then washed. These pre-loaded chips were then immersed in the recommended binding buffer with either untagged C5 only or C5 pre-incubated with C5S2. As expected, only the C5 protein rapidly bound to the eculizumab-loaded chips, while the C5 pre-incubated with C5S2 did not bind, indicating that the pre-bound nanobodies in the latter case blocked the epitope of the full-size antibody in the former case.

[0433] In the alternative experiment shown in Figure 12, the C5S2 nanobody initially bound to the strep-II chip. After washing, this pre-loaded chip was immersed in a C5-containing binding solution, and as expected, rapid binding was observed. However, when this pre-bound C5S2-C5 composite chip was immersed in an eculizumab-containing binding solution, the latter failed to bind, indicating that its binding site was blocked.

[0434] Epitope matching of C5S2.2: Comparison with eculizumab binding to mutant C5 protein.

[0435] Eculizumab is refractory to two naturally occurring variants: the R885H human polymorphism and the W917S human vs. cynomolgus monkey variant (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 produced, and the affinities of C5S2.2 and eculizumab to them were compared using Gator.

[0436] Figures 13A-C show that C5S2.2 had high affinity for wild-type human C5 protein but lost all binding activity to human C5 R885H and W917S mutants. On the other hand, it could not bind to wild-type cynomolgus monkey C5, but C5S2.2 had high affinity for the mutant cynomolgus monkey C5 S917W. This wild-type vs. mutant interaction pattern is reflected, as expected, by eculizumab in Figures 14A-C, indicating that C5S2.2 and eculizumab have similar binding epitopes on human C5.

[0437] Antidrug antibody responses derived from healthy donor serum against wild-type and humanized nanobodies

[0438] To examine the relative potential immunogenicity of wild-type and humanized nanobodies, the seropositivity of pre-existing antigen-specific antibodies in serum samples collected from individual healthy human donors was measured using a sandwich immunoassay on MSDs with passively coated antigens and anti-human IgG detection antibodies. The percentage of donors with detectable pre-existing antibodies against each antigen is plotted in both Table 12 and in graph form in Figure 15. In comparisons of different antigens, pre-existing antibodies against monomeric C3 / 5 nanobodies had lower seropositivity rates in human serum samples than those against (G4S)2-conjugated homodimers, C3N10Di, and C3N10.3Di. The percentage of human samples with pre-existing antibodies against clinically approved Beovu and Cablivi is shown as a control. Beovu is a humanized monoclonal single-chain variable fragment (scFv) that binds to and inhibits vascular endothelial growth factor A, while Cablivi (caplacizumab) is a trialanine-binding homodimer nanobody that targets the A1 domain of von Willebrand factor. [Table 12]

[0439] AAVs carrying VHH DNA lead to the production of functional proteins in human cells.

[0440] Different versions of lead VHH, C3N10, C3S3, and C5S2 were cloned into the AAV support backbone during preparation for AAV production. Prior to AAV production, the plasmid-containing VHH were evaluated for function by Western blotting assays. As depicted in Figure 16, upon activation of the complement II pathway cascade, C3 is degraded into C3a and C3b. C3 degradation triggers activation of C5 degradation, resulting in C5a and C5b. A feedback loop of C3 activation can be initiated by this activation, which results in the degradation of factor B. C3 and C5 VHH inhibitors can be evaluated for their ability to halt these degradation products and can be visualized by Western blotting.

[0441] The supernatant from transfected cells was subjected to Western blotting to examine degradation products indicating the activated complement cascade pathway (Figure 16A-C). All plasmids containing C3 VHH inhibitors blocked the formation of the degradation product C3a, but inhibitors against C5, which acts downstream of C3, did not, as expected. All VHH inhibitors against C3 and C5 blocked the degradation of C5a. Factor B degradation products were then evaluated, and all C3 VHH inhibitors blocked degradation, but C5 VHH inhibitors, which act further downstream, did not.

[0442] Following plasmid evaluation, VHHs within the AAV-supported plasmid backbone were synthesized in AAV. Read VHH AAVs were characterized by transduction expression and activity at three MOIs in iPSC-RPEs. Figure 17 depicts Western blots of the supernatant collected after transduction, showing single products of expected size 15 kDa for each VHH examined (Figure 17).

[0443] VHH expression in the supernatant was also determined by ELISA (Figure 18). Each VHH showed a dose-response, indicated by an increase in OD at 450 nm as the dose increased.

[0444] Next, the function of each VHH was evaluated by determining complement pathway inhibition using the Wieslab® assay (Figure 19). Complement inhibition was detected in both MOIs tested, although the degree of change varied for each specific VHH.

[0445] While the materials and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made 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 complement factor C3 and / or proteolytic derivative C3a and / or C3b, preferably a VH or VHH domain that specifically binds to C3, wherein the VH or VHH domain comprises one or more complementarity-determining regions (CDRs) that are at least 75% identical to CDRs selected from the group consisting of SEQ ID NOs: 18-20, 54-56, 15-17, and 21-53.

2. CDR1 of the VH or VHH domain is selected from sequences having at least one, at least two, or at least three amino acid differences compared to SEQ ID NOs: 18, 54, 15, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 and SEQ ID NOs: 19, 55, 16, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52 and SEQ ID NOs: 19, 55, 16, 22, 25, 28, 31, 34, 3 The isolated polypeptide according to claim 1, comprising: CDR2 selected from sequences having at least one, at least two, or at least three amino acid differences compared to 7, 40, 43, 46, 49, or 52; and CDR3 selected from sequences having at least one, at least two, or at least three amino acid differences compared to SEQ ID NOs: 20, 56, 17, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, and SEQ ID NOs: 20, 56, 17, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, or 53.

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

4. The isolated polypeptide according to any one of claims 1 to 3, wherein the VH or VHH domain comprises one CDR sequence of any of the VH or VHH domains described as SEQ ID NOs 2, 14, 1 and 3-13 or a CDR sequence that is at least 75% identical thereto, preferably the VH or VHH domain comprises CDR1 having the following 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 CDR2 having the following 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 eleventh amino acid is optional.

5. The isolated polypeptide according to claim 4, wherein the VHH domain comprises an amino acid sequence described as any one of SEQ ID NOs: 2, 14, 1, and 3-13, or an amino acid sequence that is at least 75% identical thereto.

6. The isolated polypeptide according to any one of claims 1 to 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 one of the amino acid sequences of SEQ ID NOs: 98, 135, 94-97, 99-134, and 136, or comprises an amino acid sequence identical thereto by at least 75%, preferably the VH or VHH domain comprises one of the amino acid sequences of SEQ ID NOs: 98, 135, 94-97, 99-134, and 136, or comprises an amino acid sequence identical thereto by at least 75%.

8. The isolated polypeptide according to any one of claims 1 to 7, wherein the VH or VHH domain is coupled to a tag, preferably a Strep or his6 tag, and more preferably the his6 tag comprises or consists of the amino acid sequence described as Sequence 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 the polypeptide according to any one of claims 1 to 8.

10. (i) a variant AAV capsid protein comprising a heterologous peptide having a length of 7, 8, 9, 10, or 11 amino acids covalently inserted into the GH loop of the capsid protein compared to a corresponding parent AAV capsid protein, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO: 168), and (ii) a recombinant adeno-associated virus (rAAV) comprising the nucleic acid described in claim 9.

11. The inserted peptide has 1 to 3 spacer amino acids (Y) at the amino and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO: 168). １ -Y ３ The rAAV according to claim 10, which has ) and preferably the inserted 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 587 and 588 of VP1 of AAV2 (SEQ ID NO: 183) or between corresponding positions in the capsid protein of another AAV serotype.

13. The capsid protein has one or more amino acid substitutions compared to VP1 of AAV2 (SEQ ID NO: 183), or one or more corresponding substitutions in the capsid protein of another AAV serotype, preferably the following amino acid substitutions: MIL, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G22 rAAV according to any one of claims 10 to 12, comprising one or more of 6E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L, and L735Q, more preferably comprising the P34A amino acid substitution.

14. The rAAV according to claim 13, wherein the capsid protein contains a P34A amino acid substitution compared to VP1 of AAV2, and contains an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the full length of the amino acid sequence described as SEQ ID NO: 170, and preferably the capsid protein consists of the amino acid sequence described as SEQ ID NO:

170.

15. The rAAV according to any one of claims 10 to 14, which exhibits an increased infectivity of retinal cells, preferably at least a twofold increase, compared to the infectivity of retinal cells by an AAV containing a corresponding parent 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 respect to the amino acid sequence described in SEQ ID NO:

170.

17. The rAAV according to any one of claims 10 to 16, comprising, from 5' to 3', (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) a heterogeneous nucleic acid comprising an inverted terminal repeat.

18. The rAAV according to claim 17, wherein the inverted terminal repeat is an AAV2 inverted terminal repeat.

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

20. The rAAV according to 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 containing 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 atrophic age-related macular degeneration (atrophic AMD) in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 10 to 21 or the pharmaceutical composition according to claim 23.

25. The rAAV or the pharmaceutical composition is about 10 ８ Vector genome (vg) / eye ~ approximately 10 １３ A dose of vg / eye, or preferably about 6 × 10⁻¹⁴ ９ vg / eye ~ approx. 6 x 10 １０ The method according to claim 24, wherein the drug is administered to the subject by periocular, intravitreous, choroidal, or subretinal administration at a dose of vg / ocular.

26. The rAAV or the pharmaceutical composition contains about 1 × 10 ９ vg / eye ~ approx. 1 x 10 １０ The method according to claim 25, wherein the drug is administered to the subject by intravitreous administration at a dose of vg / eye.

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

28. The rAAV or the pharmaceutical composition is about 10 ８ vector genomes (vg) / eye to about 10 １３ vg / eye of dosage, or preferably about 6 × 10 ９ vg / eye to about 6 × 10 １０ vg / eye of dosage and is administered to the subject by periorbital, intravitreal, suprachoroidal or subretinal administration, the method according to claim 27.

29. The rAAV or the pharmaceutical composition contains about 1 × 10 ９ vg / eye ~ approx. 1 x 10 １０ The method according to claim 28, wherein the drug is administered to the subject by intravitreous administration at a dose of vg / eye.

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

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

32. An isolated polypeptide comprising complement factor C5 and / or proteolytic derivative C5a and / or C5b, preferably a VH or VHH domain that specifically binds to C5, wherein the VH or VHH domain comprises one or more complementarity-determining regions (CDRs) that are at least 75% identical to a CDR selected from the group consisting of SEQ ID NOs: 69-71, 66-68, and 72-92.

33. The CDR1 of the VH or VHH domain is selected from sequences having at least one, at least two, or at least three amino acid differences compared to SEQ ID NOs: 69, 66, 72, 75, 78, 81, 84, 87, 90 and SEQ ID NOs: 70, 67, 73, 76, 79, 82, 85, 88, 91 and SEQ ID NOs: 70, 67, 73, 76, 79, 82, 85, 88, The isolated polypeptide according to claim 32, comprising CDR2 selected from sequences having at least one, at least two, or at least three amino acid differences compared to 91; and CDR3 selected from sequences having at least one, at least two, or at least three amino acid differences compared to SEQ ID NOs: 71, 68, 74, 77, 80, 83, 86, 89, 92 and SEQ ID NOs: 71, 68, 74, 77, 80, 83, 86, 89, 92.

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

35. The VH or VHH domain comprises one CDR sequence of any of the VH or VHH domains described as Sequence IDs 58, 57, and 59-65, or a CDR sequence that is at least 75% identical thereto, preferably the VH or VHH domain comprises 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 / T-I-F- An isolated polypeptide according to any one of claims 32 to 34, comprising CDR1 having a sequence selected from Y / H / F / A / P / D-D / Q / A / Y / S / E-D / S / T / E / Y / W-D-M-G, and / or CDR2 having a sequence 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 described as any one of SEQ ID NOs. 58, 57, and 59-65, or an amino acid sequence that is at least 75% identical thereto.

37. The isolated polypeptide according to any one of claims 32 to 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 one of the amino acid sequences of SEQ ID NOs: 141, 137-140, and 142-163, or comprises an amino acid sequence that is at least 75% identical thereto.

39. The isolated polypeptide according to any one of claims 32 to 38, wherein the VH or VHH domain is coupled to a tag, preferably a Strep or his6 tag, and more preferably the his6 tag comprises or consists of the amino acid sequence described as 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 the polypeptide according to any one of claims 32 to 39.

41. (i) a variant AAV capsid protein comprising a heterologous peptide having a length of 7, 8, 9, 10, or 11 amino acids covalently inserted into the GH loop of the capsid protein compared to a corresponding parent AAV capsid protein, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO: 168), and (ii) a recombinant adeno-associated virus (rAAV) comprising the nucleic acid of claim 40.

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

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

44. The capsid protein has one or more amino acid substitutions compared to VP1 of AAV2 (SEQ ID NO: 183), or one or more corresponding substitutions in the capsid protein of another AAV serotype, preferably the following amino acid substitutions: MIL, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G22 rAAV according to any one of claims 41 to 43, comprising one or more of 6E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L, and L735Q, more preferably comprising the P34A amino acid substitution.

45. The rAAV according to claim 44, wherein the capsid protein contains a P34A amino acid substitution compared to VP1 of AAV2, and contains an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the full length of the amino acid sequence described as SEQ ID NO: 170, preferably the capsid protein consists of the amino acid sequence described as SEQ ID NO:

170.

46. The rAAV according to any one of claims 41 to 45, which exhibits an increased infectivity of retinal cells, preferably at least twice, compared to the infectivity of retinal cells by an AAV containing a corresponding parent 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 respect to the amino acid sequence described in SEQ ID NO:

170.

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

49. The rAAV according to claim 48, wherein the inverted terminal repeat is an AAV2 inverted terminal repeat.

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

51. The rAAV according to 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 containing 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 atrophic age-related macular degeneration (atrophic AMD) in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 41 to 52 or the pharmaceutical composition according to claim 54.

56. The rAAV or the pharmaceutical composition is about 10 ８ Vector genome (vg) / eye ~ approximately 10 １３ A dose of vg / eye, or preferably about 6 × 10⁻¹⁴ ９ vg / eye ~ approx. 6 x 10 １０ The method according to claim 55, wherein the drug is administered to the subject by periocular, intravitreous, choroidal, or subretinal administration at a dose of vg / ocular.

57. The rAAV or the pharmaceutical composition contains about 1 × 10 ９ vg / eye ~ approx. 1 x 10 １０ The method according to claim 56, wherein the drug is administered to the subject by intravitreous administration at a dose of vg / ocular.

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

59. The rAAV or the pharmaceutical composition is about 10 ８ Vector genome (vg) / eye ~ approximately 10 １３ A dose of vg / eye, or preferably about 6 × 10⁻¹⁴ ９ vg / eye ~ approx. 6 x 10 １０ The method according to claim 58, wherein the drug is administered to the subject by periocular, intravitreous, choroidal, or subretinal administration at a dose of vg / ocular.

60. The rAAV or the pharmaceutical composition contains about 1 × 10 ９ vg / eye ~ approx. 1 x 10 １０ The method according to claim 59, wherein the drug is administered to the subject by intravitreous administration at a dose of vg / eye.

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

62. The method according to claim 61, wherein the rAAV or the pharmaceutical composition is administered to the eye of the subject by intravitreous 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 binds to amino acids 1540-1600 of SEQ ID NO: 177 or to an epitope in an amino acid sequence that is at least 80% identical thereto.

64. The isolated polypeptide according to claim 63, wherein the VH or VHH domain binds to an epitope containing, essentially, or consisting of amino acids 1548-1561 of SEQ ID NO: 177 or an amino acid sequence at least 80% identical thereto, and / or binds to an epitope containing, essentially, or consisting of amino acids 1568-1573 of SEQ ID NO: 177 or an amino acid sequence at least 80% identical thereto, and / or binds to an epitope containing, essentially, or consisting of amino acids 1583-1591 of SEQ ID NO: 177 or an amino acid sequence at least 80% identical thereto, and / or binds to an epitope containing, essentially, or consisting of amino acids 1583-1591 of SEQ ID NO:

177.

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 identical thereto by at least 70%, CDR2 of SEQ ID NO: 55 or an amino acid sequence identical thereto by at least 70%, and CDR3 of SEQ ID NO: 56 or an amino acid sequence identical thereto by at least 70%, and preferably the polypeptide comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence identical thereto by at least 70%.

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 discontinuous epitopes contained within amino acids 110-130 and 430-510 of SEQ ID NO:

177.

67. The isolated polypeptide according to claim 66, wherein the VH or VHH domain binds to an epitope containing, essentially, or consisting of amino acids 114-124 of SEQ ID NO: 177 or an amino acid sequence at least 80% identical thereto, and / or binds to an epitope containing, essentially, or consisting of amino acids 497-505 of SEQ ID NO: 177 or an amino acid sequence at least 80% identical thereto, or consisting thereto.

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 identical thereto by at least 70%, CDR2 of SEQ ID NO: 19 or an amino acid sequence identical thereto by at least 70%, and CDR3 of SEQ ID NO: 20 or an amino acid sequence identical thereto by at least 70%, and preferably the polypeptide comprises the amino acid sequence of SEQ ID NO: 98 or an amino acid sequence identical thereto by at least 70%.

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 the 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 identical thereto by at least 70%, CDR2 of SEQ ID NO: 70 or an amino acid sequence identical thereto by at least 70%, and CDR3 of SEQ ID NO: 71 or an amino acid sequence identical thereto by at least 70%, and preferably the polypeptide comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence identical thereto by at least 70%.

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

72. The fusion protein according to claim 71, wherein the fusion protein comprises any one of the amino acid sequences of Sequence ID No. 178 to 180 or an amino acid sequence that is at least 70% identical thereto.

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

74. The fusion protein according to claim 73, comprising the amino acid sequence of SEQ ID NO: 181 or 182 or an amino acid sequence that is at least 70% identical thereto.

Images