Treatment of atrophic age-related macular degeneration

Abstract

This disclosure provides gene therapy targeting the complement pathway for the treatment of atrophic age-related macular degeneration.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 490,736, filed on 16 March 2023, and U.S. Provisional Application No. 63 / 607,419, filed on 7 December 2023. The disclosures of the two priority applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The .XML copy created on 12 March 2024 is named "122548.WO006.xml" and has a size of 189,331 bytes. [Background technology]

[0003] Age-related macular degeneration (AMD) is the leading cause of vision loss in older adults. There are two types of AMD: exudative and atrophic. Exudative AMD, also known as progressive neovascular AMD, is a less common type of AMD and usually causes faster vision loss. Atrophic AMD, on the other hand, accounts for 85-90% of AMD cases worldwide (Schultz et al., Clin Ther. (2021) 43(10): 1792-818).

[0004] Atrophic AMD typically begins with retinal pigment epithelial (RPE) dysfunction in the macula of the eye, progressing to a progressive stage with RPE cell death, followed by photoreceptor death and eventual blindness. The hallmark of this disease is the accumulation of drusen in the RPE and activation of the complement pathway. This leads to a severe inflammatory response, geographic atrophy, and death of RPE cells and photoreceptors, ultimately resulting in blindness.

[0005] Human genetic variants in multiple complement factors are associated with altered risk of AMD, suggesting dysregulation of both classical and alternative complement pathways as causative factors of the disease. Cumulative damage to the retina due to aging, environmental stress, and other factors triggers inflammation in multiple pathways, including the complement cascade. As with several geographic atrophy-associated genetic risk factors in the complement cascade, impaired regulatory components in these pathways can lead to chronic inflammation, ultimately resulting in the retinal cell death characteristic of geographic atrophy / atrophic AMD. Levels of complement activity and inflammation are increased in patients with intermediate and late atrophic AMD with geographic atrophy (GA). GA is the late stage of atrophic AMD, referring to areas of the retina where cells are depleted and die, resulting in significant bilateral central vision loss.

[0006] Innate immunity mediated by the complement cascade enables the elimination of pathogens or damaged cells through phagocytosis. However, a dysregulated complement cascade can also cause harmful inflammation. There are three pathways for the initiation of the complement cascade: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is initiated by the activation of the C1 complex (C1q, C1r, and C1s) upon binding to IgG or IgM immune complexes, resulting in the cleavage of C4 and C2, which then assemble to form C4b2a and C3 convertase. The lectin pathway is initiated, for example, by the activation of the mannan-binding lectin (MBL) / MBL-associated serine protease (MASP) complex upon oligosaccharide binding, resulting in the cleavage of C4 and C2, which then assemble to form C4b2a. The secondary pathway is constitutively active at a low level and is initiated by hydrolysis of C3 to C3(H2O), which binds to factor B (FB), leading to the formation of liquid-phase C3 procombatase C3(H2O)B. This complex is recognized and cleaved by factor D (FD) to form C3(H2O)Bb, a liquid-phase C3 combbatase.

[0007] All C3 convertases cleave C3 into anaphylatoxin C3a and opsonin C3b. Covalently bound C3b mediates phagocytosis in opsonin-tagged cells. Furthermore, opsonized C3b amplifies the complement response via a secondary pathway, regardless of the initiation pathway. This amplification leads to activation of the terminal pathway via the formation of C5 convertases, which cleave C5 into C5b, a component of C5a (a potent anaphylatoxin), and C5b9, or the membrane invasion complex (MAC) (a complex of large pores that can cause cell lysis).

[0008] To date, most management guidelines focus on reducing risk factors and the use of nutritional supplements (Schutz et al., ibid.). The first treatment for GA, namely a C3 inhibitor (SYFOVRE®; pegcetacoplan injection), was recently approved, but this treatment requires chronic, frequent intravitreal injections, which limits patient adherence and increases the risk of developing neovascular AMD. Furthermore, C3 inhibition does not interfere with complement effector function mediated by upstream activating fragments. Another treatment for GA, a C5 inhibitor (IZERVAY®; abasin captado pegol intravitreal solution), was approved by the FDA a few months after SYFOVRE®, but C5 inhibition has similar drawbacks to C3 inhibition. Therefore, there remains an urgent need to develop an effective one-time treatment for atrophic AMD. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] This disclosure provides an expression construct comprising a first nucleotide sequence encoding an inhibitor of activated complement subcomponent C1s and a second nucleotide sequence encoding an inhibitor of complement factor Bb; or a pair of expression constructs, one comprising the first nucleotide sequence and the other comprising the second nucleotide sequence. Unless otherwise specified herein, activated C1s is also referred to herein as "C1s". Factor Bb is also referred to herein as "FBb" or simply "Bb". [Means for solving the problem]

[0010] In some embodiments, the C1s inhibitor and the Bb inhibitor are antibody fragments, and optionally the antibody fragments are single-chain Fv (scFv) or single-chain Fab (scFab). In some embodiments, the C1s inhibitor is an anti-C1s antibody fragment comprising heavy-chain CDR (HCDR) 1-3 of SEQ ID NO: 7, each optionally containing SEQ ID NOs 1-3, and light-chain CDR (LCDR) 1-3 of SEQ ID NO: 8, each optionally containing SEQ ID NOs 4-6. In some embodiments, the Bb inhibitor is an anti-Bb antibody comprising HCDR 1-3 of SEQ ID NO: 19, each optionally containing SEQ ID NOs 13-15, and LCDR 1-3 of SEQ ID NO: 20, each optionally containing SEQ ID NOs 16-18.

[0011] In some embodiments, the C1s inhibitor has a heavy chain variable domain (V) containing an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 7. H ) and a light chain variable domain (V) containing an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 8. L ) and. In some embodiments, the Bb inhibitor is V containing an amino acid sequence identical to SEQ ID NO: 19 or at least 95% (e.g., at least 96, 97, 98, or 99%) thereto. H and V containing an amino acid sequence identical to SEQ ID NO: 20 or at least 95% (e.g., at least 96, 97, 98, or 99%) thereto. L This includes.

[0012] In some embodiments, the C1s inhibitor comprises a heavy chain (HC) having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 10, and a light chain (LC) having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 11. In some embodiments, the Bb inhibitor comprises an HC having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 22, and an LC having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 23.

[0013] In some embodiments, the C1s inhibitor and the Bb inhibitor each include one or more charge mutations to facilitate pairing between the heavy and light chains of each inhibitor. In some embodiments, the charge mutations in the C1s inhibitor include Q42E and Q292K, where numbering follows Sequence ID No. 12. In some embodiments, the charge mutations in the Bb inhibitor include Q38K and Q288E, and optionally further include S114A, N137K and T434E, where numbering follows Sequence ID No. 24.

[0014] In some embodiments, the C1s inhibitor is an scFv or scFab in which HC and LC are linked by a peptide linker, and optionally the peptide linker comprises one or more G4S (SEQ ID NO: 46) repeats, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the Bb inhibitor is an scFv or scFab in which HC and LC are linked by a peptide linker, and optionally the peptide linker comprises one or more G4S repeats, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0015] In some embodiments, the expression constructs herein include a transgene encoding a fusion protein comprising a C1s inhibitor and a Bb inhibitor linked by a peptide linker, the peptide linker optionally comprising one or more, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10 G4S repeats. In some embodiments, the transgene is operably linked to a minimal chicken β-actin (minCBA) promoter.

[0016] In some embodiments, the expression constructs herein include a bidirectional promoter that directs the expression of a C1s inhibitor and a Bb inhibitor as separate molecules, optionally the bidirectional promoter being a pair of CBA promoters positioned in opposite directions and separated by a CMV enhancer, and further optionally the bidirectional promoter containing a nucleotide sequence identical to SEQ ID NO: 53 or at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) thereto.

[0017] In some embodiments, the expression construct expresses a heterodimer comprising (i) a single-chain anti-C1s antibody fragment fused to the HC or LC of an anti-Bb antibody fragment; and (ii) a heterodimer comprising the LC or HC polypeptide of an anti-Bb antibody fragment, wherein the coding sequence of the fusion protein and the coding sequence of the LC or HC polypeptide of the anti-Bb antibody fragment are separated in frame by the coding sequence of a cleavable peptide, the cleavable peptide optionally comprising a 2A sequence and / or a furin cleavage site, and the expression construct optionally comprising a minCBA promoter.

[0018] In some embodiments, the expression construct expresses a heterodimer comprising (i) a fusion protein containing a single-chain anti-Bb antibody fragment fused to the HC or LC of an anti-C1s antibody fragment, and (ii) a heterodimer containing the LC or HC polypeptide of an anti-C1s antibody fragment, wherein the coding sequence of the fusion protein and the coding sequence of the LC or HC polypeptide of the anti-C1s antibody fragment are separated in frame by the coding sequence of a cleavable peptide, the cleavable peptide may contain a 2A sequence and / or a furin cleavage site, and the expression construct may further contain a minCBA promoter.

[0019] In some embodiments, the expression constructs include: (i) an anti-C1s scFv, (G4S)2 linker, and anti-Bb scFv with an amino acid sequence from the N-terminus to the C-terminus that optionally contains at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 55 (with or without signal peptide) or thereto; (ii) an anti-Bb scFv, (G4S)2 linker, and anti-C1s scFv with an amino acid sequence that optionally contains at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 57 (with or without signal peptide) or thereto; (iii) an anti-C1s scFab, (G4S)3 linker, and anti-Bb scFv with an amino acid sequence that optionally contains at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 26 or 28 (with or without signal peptide) or thereto; scFab; (iv) an anti-Bb scFab, (G4S)3 linker and anti-C1s scFab having an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 30 or 32 (with or without signal peptide) or therein; (v) an anti-C1s scFab, (G4S)2 linker and anti-Bb scFv having an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 34 or 36 (with or without signal peptide) or therein; or (vi) an anti-C1s scFab, (G4S)3 linker and anti-Bb scFv having an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 59 or 61 (with or without signal peptide) or therein; the fusion protein comprising these two sequences.

[0020] In some embodiments, the expression construct optionally encodes an anti-C1s scFab containing an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 12, optionally comprising Q42E and Q292K mutations compared to SEQ ID NO: 12; or optionally encodes an anti-Bb scFab containing an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 14, optionally comprising Q38K and Q288E, and optionally S114A, N137K, and T434E mutations compared to SEQ ID NO: 14.

[0021] In some embodiments, the expression construct comprises (A) a fusion protein comprising (i) anti-C1s LC and (ii) anti-C1s HC fused to αBb scFab, and optionally the expression construct comprises the coding sequence of SEQ ID NO: 39 or an amino acid sequence identical thereto by at least 95% (e.g., at least 96, 97, 98, or 99%); (B) a fusion protein comprising (i) anti-C1s LC and (ii) anti-C1s HC fused to anti-Bb scFab, and optionally the expression construct comprises the coding sequence of SEQ ID NO: 41 or an amino acid sequence identical thereto by at least 95% (e.g., at least 96, 97, 98, or 99%); (C) a fusion protein comprising (i) anti-C1s scFab fused to anti-Bb HC and (ii) anti-Bb A heterodimer comprising LC, optionally containing the coding sequence of SEQ ID NO: 43, or an amino acid sequence identical thereto by at least 95% (e.g., at least 96, 97, 98, or 99%); or (D)(i) a heterodimer comprising a fusion protein containing anti-C1s scFab fused to anti-Bb HC and (ii) anti-Bb LC, optionally containing the coding sequence of SEQ ID NO: 45, or an amino acid sequence identical thereto by at least 95% (e.g., at least 96, 97, 98, or 99%).

[0022] In another aspect, the Disclosure provides isolated nucleic acids comprising a nucleotide sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 54, 56, 58, 60, 62, 79, or 80, or encoding the same amino acid sequence as the selected nucleotide sequence.

[0023] In other embodiments, the Disclosure provides one, two, or more recombinant adeno-associated viruses (rAAVs) comprising expression constructs or isolated nucleic acids as defined herein. In some embodiments, the genome of the rAAVs herein comprises an expression construct adjacent to the AAV2 reverse terminal repeat (ITR). In some embodiments, the genome comprises SEQ ID NOs. 50, 51, or 52; or encodes the same amino acid sequence as SEQ ID NOs. 50, 51, or 52. In some embodiments, the rAAVs herein comprises the AAV2 capsid, optionally wild-type AAV2.

[0024] In one embodiment, the present disclosure provides a pharmaceutical composition comprising the rAAV of this specification and a pharmaceutically acceptable carrier.

[0025] In one embodiment, the present disclosure provides one or more expression constructs or one or more proteins encoded by rAAV as described herein.

[0026] In one embodiment, the present disclosure provides a host cell containing an expression construct, isolated nucleic acid, or rAAV as described herein.

[0027] In one embodiment, the present disclosure provides a method for treating atrophic age-related macular degeneration (AMD) in a patient requiring treatment for AMD, comprising administering an effective amount of the rAAV or pharmaceutical composition of this specification. In some embodiments, the administration is by intravitreal injection. In some embodiments, the patient has geographic atrophy (GA) secondary to atrophic AMD. In some embodiments, the effective amount is 10 7 ~10 15 , arbitrarily 10 8 ~10 14 , 109 ~10 13 、 and further optionally 2×10 9 、 2×10 10 or 2×10 11 vector genome.

[0028] Also provided herein are the recombinant AAV or pharmaceutical composition of the present specification for use in treatment by the treatment method of the present specification in patients requiring treatment for atrophic age-related macular degeneration (AMD), and the use of the recombinant AAV or pharmaceutical composition of the present specification for the manufacture of a medicament for treatment in patients requiring treatment for atrophic age-related macular degeneration (AMD).

[0029] In another aspect, the present disclosure provides a mammalian promoter comprising a sequence that is at least 85%, optionally at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, or 100% identical to SEQ ID NO: 83.

[0030] In another aspect, the present disclosure provides a bidirectional mammalian promoter comprising a pair of chicken β-actin promoters arranged in opposite directions and separated by a CMV enhancer, and optionally, the bidirectional mammalian promoter comprises a sequence that is at least 85%, optionally at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, or 100% identical to SEQ ID NO: 53.

[0031] Other features, objects, and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that this detailed description, while indicating embodiments and aspects of the present invention, is not limiting and is provided by way of example only. Various modifications and variations within the scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] [Figure 1A]This figure shows an exemplary monocistronic construct for expressing linked anti-C1s(αC1s) antibody fragments and anti-Bb(αBb) antibody fragments (for example, via a G4S linker as shown). minCBA: Minimal chicken β-actin promoter. scFab: Single-chain antibody fragment. scFv: Single-chain antibody variable domain. BGH: Bovine growth hormone. [Figure 1B] This figure shows an exemplary bicistronic construct using a bidirectional promoter (modified minCBA) that enables the expression of two distinct antibody fragments in opposite directions. [Figure 1C] This figure shows an exemplary recombinant AAV genome including expression cassettes for expressing anti-Bb antibody fragments and anti-C1s antibody fragments, as shown in Figure 1A or Figure 1B. ITR: Reverse terminal repeat sequence. [Figure 2A] This is a panel of figures showing ligated anti-C1s / anti-Bb antibody fragments generated from four exemplary configurations (#5-#8) of the monocistronic construct. Heavy chain variable domain: VH. Light chain variable domain: VL. Heavy chain constant region: CH. Light chain constant region: CL. [Figure 2B] A pair of figures (#9 and #10) showing two exemplary configurations (#9 and #10) of a construct having a bidirectional ("BiDir") promoter that drives the expression of two independent antibody fragments. [Figure 2C] This figure shows a pair of exemplary ligated anti-C1s / anti-Bb scFab antibody fragments (#11 and #12) having charge mutations ("CM"; Δ) intended to promote the pairing of congeneral heavy and light chains. In the figures herein, "Δ" indicates the presence of a charge mutation and does not mean that the exact location or number of charge mutations in the antibody fragment is indicated. [Figure 2D] This is a pair of figures showing exemplary linked anti-C1s / anti-Bb antibody fragments for αC1s scFab-(G4S)2-αBb scFv with or without charge mutations (#14) and (#13). [Figure 2E] This is a pair of figures showing exemplary linked anti-C1s / anti-Bb antibody fragments for αC1s scFab-(G4S)3-αBb scFv with or without charge mutations (#16). [Figure 2F] This panel of figures shows exemplary linked anti-C1s / anti-Bb antibody fragments containing the self-cleaving peptide F2A or GT2A between the heavy and light chains of an αC1s Fab fragment (#17: αC1s F2A Fab-(G4S)3-αBb scFab; and #18: αC1s GT2A Fab-(G4S)3-αBb scFab) or between the heavy and light chains of an αBb Fab fragment (#19: αC1s scFab-(G4S)3-αBb F2A Fab; and #20: αC1s scFab-(G4S)3-αBb GT2A Fab). F2A: A self-cleaved peptide containing a furin cleavage site linked to the foot-and-mouth disease virus 2A peptide by the SGSG (SEQ ID NO: 81) linker (Fuchs et al., PLOS One (2016) doi:10.1371 / journal.pone.0158009). GT2A: A self-cleaved peptide containing a furin cleavage site linked to the Thosea asigna virus 2A peptide by the GSG linker. [Figure 2G] A pair of diagrams illustrating exemplary configurations of constructs having a bidirectional promoter that drives the expression of two independent antibody fragments distinct from constructs #9 and #22 by having charge mutations (#21 and #10). [Figure 2H] Figure 2D shows construct #14 (αC1s scFab-(G4S)2-αBb scFv-CM) in association with an AAV vector plasmid containing AAV2 ITR. "aC1s": αC1s. "aBb": αBb. [Figure 2I] This figure shows the αC1s scFab-BiDir-αBb scFab constructs with or without charge mutations (#21; Figure 2G) in relation to AAV vector plasmids containing AAV2 ITR. "aC1s": αC1s. "aBb": αBb. [Figure 2J] This figure shows the construct αβb scFab-(G4S)3-αC1s scFab-CM (construct #12 in Figure 2C) in relation to the AAV vector plasmid containing AAV2 ITR. "aC1s": αC1s. "aBb": αBb. [Figure 3] This is a representative biolayer interferometry (BLI) sensorgram showing that the protein expressed from construct #19 in Figure 2F can simultaneously bind to both C1s and Bb. [Figure 4A] This plot shows dose-dependent inhibition of complement activation by recombinant anti-C1s Fab expressed by construct #2 in Figure 2A and purified protein, under conditions where both CP and AP are simultaneously activated in vitro. [Figure 4B] This plot shows dose-dependent inhibition of complement activation by recombinant anti-Bb Fab expressed by construct #4 in Figure 2A and purified protein, under conditions where both CP and AP are simultaneously activated in vitro. [Figure 4C] This plot shows dose-dependent inhibition of complement activation by an equimolar mixture of recombinant anti-Bb Fab and anti-C1s Fab, tested with an equimolar mixture of purified proteins expressed by constructs #2 and #4 in Figure 2A, under conditions where both CP and AP are simultaneously activated in vitro. [Figure 5] This is a panel of photographs showing representative vector-in-situ hybridization of mouse retinas three weeks after administration of AAV2#9. A vector-specific probe targeting the vector genome was used. [Figure 6] This is a panel of graphs showing the combined inhibition of CP and AP against ARPE19 cells in a CRP-mediated complement activation model of atrophic AMD. The data shown are the mean of 12 replicates, along with the standard deviation of each condition across two independent experiments. "NHS": Normal human serum. "CRP": C-reactive protein. ****p<0.0001. [Figures 7A-7B] Figures 7A and 7B are graphs showing cell ELISA data illustrating complement deposition on induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE) in a cell model of AMD. Treatment with anti-Bb scFab and anti-C1s scFab significantly inhibited the deposition of complement products C3d (Figure 7A) and C5b9 (Figure 7B) on iPSC-RPE compared to the CRP control. Error bars represent standard deviation. ****p<0.0001. [Figure 8A-8B] Figures 8A and 8B show immunofluorescence staining of C5b9 in iPSC-RPE. Figure 8A is a panel of confocal microscope images showing C5b9 deposition (red) on iPSC-RPE. Figure 8B is a graph showing the quantitative analysis of the images in Figure 8A. [Figure 9] This heatmap shows the results of eye examinations based on the Preclinical Ocular Toxicology Scoring (SPOTS) system. The heatmap shows clinical indicators of ocular inflammation and irritation in the control before and after LPS treatment; it also shows the median severity scored during eye examinations using the SPOTS system. [Modes for carrying out the invention]

[0033] This disclosure is based on the finding that dual targeting of the classical and alternative complement pathways can be used to treat ocular diseases associated with dysregulation or hyperactivation of the complement system in the eye. This disclosure provides a gene therapy that delivers both an inhibitor of activated complement component 1 subcomponent (aC1s, or simply "C1s" herein) and an inhibitor of activator B (also known as Bb fragment, FBb, or Bb) to the eye of a patient in need. The gene therapy can use a viral vector, such as recombinant adeno-associated virus (AAV, e.g., AAV2), as a vehicle for delivering the transgene that directs the expression of the C1s and Bb inhibitors. In some embodiments, the C1s inhibitor and Bb inhibitor are antibody fragments such as single-stranded Fab (scFab) or single-stranded Fv (scFv). The C1s inhibitor and Bb inhibitor may be expressed as a single protein or as two distinct proteins.

[0034] In some embodiments, the ocular disease to be treated is atrophic AMD, including associated geographic atrophy. In some embodiments, the patient has a dysregulated / hyperactivated complement system at the RPE choroidal interface. In some embodiments, the therapy delivers the recombinant expression construct (e.g., recombinant AAV2) to retinal ganglion cells (RGCs) (e.g., intravitreal or subretinal). Intravitreal delivery of rAAV2 transduces RGCs in the retina and promotes the secretion of inhibitory proteins for broader retinal distribution. For example, intravitreal delivery of rAAV2 in a patient with geographic atrophy (GA) secondary to atrophic AMD can reduce the growth of retinal GA lesion size over 12 months and prevent inevitable vision loss. In addition to its potential as a one-time treatment for GA, the gene therapy of this disclosure may have improved efficacy compared to therapeutic approaches targeting downstream components of the complement pathway. This is because this therapy broadly inhibits both proximal and terminal mediators of inflammation, phagocytosis, and membrane attack complex-mediated cytolysis.

[0035] Approved or currently under-investigation therapies involve repeated administration of complement inhibitors (e.g., monthly or bimonthly). A single treatment via exogenous intravitreous delivery of a recombinant vector offers a best-in-class approach. Furthermore, in other therapies, complement inhibitors block all complement pathways. In contrast, the bifunctional complement inhibitor of this invention targets the upstream activation steps in the complement pathways (AP and CP) that are involved as driving factors in the pathogenesis of atrophic AMD, rather than targeting downstream convertases common to all three initiation pathways. This approach leaves the C1q and lectin pathways intact, thus maintaining immune surveillance. Moreover, this approach has a superior mode of action for inhibiting not only membrane attack complexes (MACs) but also complement amplification loops and terminal events mediated by upstream activating fragments such as inflammation, opsonization, and phagocytosis. Because the inhibitor targets activating enzymes that are often present at much lower levels compared to intact proenzymes, this approach can also reduce targeted drug placement (TMDD).

[0036] I.C1s and Bb inhibitors This gene therapy involves introducing both a C1s inhibitor and a Bb inhibitor into the patient's affected eye, either in a linked or unlinked configuration.

[0037] Prior to processing and activation, the human C1s polypeptide may have SEQ ID NO: 65 (UniProt.P09871), where amino acids 1-15 constitute a signal peptide. Upon activation, the C1s polypeptide is cleaved to form a disulfide heterodimer with the heavy chain corresponding to amino acids 16-437 of SEQ ID NO: 65 and the light chain corresponding to amino acids 438-688 of SEQ ID NO: 65. Unless otherwise specified, C1s inhibitors as used herein refer to inhibitors of this activated form of C1s.

[0038] Prior to processing and activation, the human factor B polypeptide may have SEQ ID NO: 66 (UniProt.P00751), where amino acids 1-25 constitute a signal peptide. Upon activation, the polypeptide is cleaved into two subcomponents: factor Ba, corresponding to amino acids 26-259 of SEQ ID NO: 66, and factor Bb, corresponding to amino acids 260-764 of SEQ ID NO: 66. In this specification, factor Bb is also simply referred to as "Bb".

[0039] The C1s inhibitors and Bb inhibitors described herein may be recombinantly linked with or without a peptide linker (e.g., recombinantly expressed as fusion proteins). When these proteins are introduced into cells via an expression vector, they may also be called "vectorized" proteins (e.g., "vectorized" antibody fragments).

[0040] In some embodiments, C1s inhibitors and Bb inhibitors are antigen-binding fragments of a complete antibody. A complete “antibody” (Ab) or “immunoglobulin” (Ig) refers to a tetrameric protein containing two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (V H ) and heavy chain constant region (C HIt consists of ). Each light chain is composed of a light chain variable domain (V L ) and light chain constant region (C L ) consists of V H and V L The domain can be further subdivided into highly variable regions called "complementarity-determining regions" (CDRs), which are interspersed with more conserved regions called "framework regions" (FRs). H or V L It consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The assignment of amino acids to each region may follow the definition of IMGT® (Lefranc et al., Dev Comp Immunol. (2003) 27(1):55-77; or Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J.Mol.Biol. (1987) 196:901-917; or Chothia et al., Nature (1989) 342:878-83). Further CDR definition systems include the AbM system and the Martin system (e.g., Abhinandan and Martin, Mol Immunol. (2008) 45(14):3832-9).

[0041] The terms "antibody fragment," "antigen-binding fragment," or similar terms refer to a portion of an intact antibody containing amino acid residues that interact with an antigen and confer its specificity and affinity to the antigen to the fragment. An antibody fragment is a portion of an antibody linked by a short peptide linker. H and V LThe fusion protein may be a single-stranded variable fragment (scFv); a diabody which is a non-covalent dimer of scFv (Zapata et al., Protein Eng. (1995) 8(10):1057-62); or a Fab fragment containing a single-stranded Fab (scFab) fragment. The "Fab" fragment consists of a constant domain of the light chain and a first constant domain (C) of the heavy chain. H1 ) include. Other non-limiting examples of antibody antigen-binding fragments include Fd fragments, Fv fragments, dAb fragments, and minimal recognition units consisting of amino acid residues that mimic the antibody's hypervariable domain. In certain embodiments, the antibody fragment is scFv, Fab, or scFab.

[0042] A. Anti-C1s scFv and scFab In some embodiments, the active C1s inhibitor is an antibody fragment such as scFab or scFv derived from the anti-C1s antibody VH3 / VK2 in International Publication No. 2018 / 071676. International Publication No. 2018 / 071676 or International Publication No. 2016 / 164358 and U.S. Patent Application Publications No. 10,729,767 and 11,246,926 may also be used herein. In some embodiments, the anti-C1s (also referred to herein as "αC1s") scFv or scFab herein comprises a CDR derived from the aforementioned VH3 / VK2 antibody. The CDR may be defined by any one of the well-known systems, including those described above. In some embodiments, the CDR is defined by the Kabat system, the IMGT® system, or the Chothia system, as shown in Table A below (sequence numbers are shown in parentheses).

[0043] [Table 1]

[0044] In some embodiments, the anti-C1s scFab or scFv comprises heavy chain CDRs (HCDRs) 1-3, each containing sequence numbers 1-3, and light chain CDRs (LCDRs) 1-3, each containing sequence numbers 4-6.

[0045] In certain embodiments, anti-C1s scFv or scFab is a V containing an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 7. H ; and V containing an amino acid sequence identical to SEQ ID NO: 8 or at least 95% (e.g., at least 96, 97, 98, or 99%) thereto. L Includes. In a particular embodiment, anti-C1s scFv is V H and V L A peptide linker that connects them, for example, a flexible linker, for example (G4S) n Includes a linker containing (SEQ ID NO: 46) (n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the linker includes SEQ ID NO: 48 (i.e., n=3). H V L It may be N-terminus or C-terminus. In some embodiments, anti-C1s scFv contains an amino acid sequence identical to SEQ ID NO: 9, or at least 95% (e.g., at least 96, 97, 98, or 99%) thereto.

[0046] In certain embodiments, anti-C1s scFab comprises a heavy chain (HC) having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 10, and a light chain (LC) having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 11. In further embodiments, the HC and LC are linked by a peptide linker, such as a flexible linker, such as (G4S). n The linkers (n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) include (SEQ ID NO: 46). In some embodiments, the linker includes SEQ ID NO: 49 (i.e., n=7). HC may be the N-terminus or C-terminus of LC. In some embodiments, αC1s scFab contains an amino acid sequence identical to SEQ ID NO: 12, or at least 95% (e.g., at least 96, 97, 98, or 99%) thereto.

[0047] In some embodiments, the C1s inhibitor is an antibody fragment such as scFab or scFv derived from an anti-C1s antibody disclosed in U.S. Patent Application Publication No. 2022 / 0380483A1. For example, the C1s inhibitor is the heavy and light chain CDR of the parent anti-C1s antibody, or V H and V L It may include.

[0048] B. Anti-Bb scFv and scFab In some embodiments, the Bb inhibitor is an anti-Bb antibody V from U.S. Patent No. 11,242,382 and International Publication No. 2021 / 216458. H This is an antibody fragment such as scFab or scFv derived from 6 / Vκ7-IgG4v2. Antibody fragments derived from variants of this antibody described in International Publication No. 2021 / 216458 may also be used herein. In some embodiments, the anti-Bb (also referred to herein as "αBb") scFv or scFab herein is the aforementioned V H The CDR is derived from the 6 / Vκ7-IgG4v2 antibody. The CDR may be defined by any one of the well-known systems, including those described above. In some embodiments, the CDR is defined by the Kabat system, the IMGT® system, or the Chothia system, as shown in Table B below (sequence numbers are shown in parentheses).

[0049] [Table 2]

[0050] In some embodiments, anti-Bb scFab or scFv comprises heavy chain CDRs (HCDRs) 1-3, each containing sequence numbers 13-15, and light chain CDRs (LCDRs) 1-3, each containing sequence numbers 16-18.

[0051] In certain embodiments, anti-Bb scFv or scFab is a V containing an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 19. H ; and V containing an amino acid sequence identical to SEQ ID NO: 20 or at least 95% (e.g., at least 96, 97, 98, or 99%) thereto. L Includes. In a particular embodiment, the anti-Bb scFv is V H and V L A peptide linker that connects them, for example, a flexible linker, for example (G4S) n Includes a linker containing (SEQ ID NO: 46) (n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the linker includes SEQ ID NO: 48 (i.e., n=3). H V L It may be N-terminus or C-terminus. In some embodiments, anti-Bb scFv contains an amino acid sequence identical to SEQ ID NO: 21, or at least 95% (e.g., at least 96, 97, 98, or 99%) thereof.

[0052] In certain embodiments, anti-Bb scFab comprises a heavy chain (HC) having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 22; and a light chain (LC) having an amino acid sequence identical to or at least 95% (e.g., at least 96, 97, 98, or 99%) of SEQ ID NO: 23. In further embodiments, the HC and LC are linked to a peptide linker, e.g., a flexible linker, e.g., (G4S). n The linkers are linked by a linker containing (SEQ ID NO: 46) (where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, linking HC and LC). In some embodiments, the linker contains SEQ ID NO: 49 (i.e., n=7). HC may be the N-terminus or C-terminus of LC. In some embodiments, Bb scFab contains an amino acid sequence identical to SEQ ID NO: 24, or at least 95% (e.g., at least 96, 97, 98, or 99%) thereto.

[0053] In some embodiments, the Bb inhibitor is an antibody fragment such as scFab or scFv derived from an anti-Bb antibody disclosed in U.S. Patent No. 10,131,706; No. 10,604,563; or No. 7,964,705. For example, the Bb inhibitor is the heavy and light chain CDR of the parental anti-Bb antibody, or V H and V L It may include.

[0054] C. Anti-C1s / Bb bispecific fusion protein In some embodiments, the C1s inhibitor (e.g., anti-C1s scFab or scFv) and the Bb inhibitor (e.g., anti-Bb scFab or scFv) are linked by a peptide linker, such as a flexible linker, such as (G4S). n The peptide is linked by a linker containing (SEQ ID NO: 46) n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the peptide linker is SEQ ID NO: 47 (n=2) or 48 (n=3). The C1s inhibitor may be the N-terminus or C-terminus of the Bb inhibitor. The αC1s / αBb fusion protein may have the following exemplary, non-limiting configurations (N-terminus to C-terminus): αC1s scFab-linker-αBb scFab αC1s scFv-linker-αBb scFab αC1s scFab-linker-αBb scFv αC1s scFv-linker-αBb scFv αBb scFab-linker-αC1 scFab αBb scFv-linker-αC1 scFab αBb scFab-linker-αC1 scFv αBb scFv-linker-αC1 scFv Here, “linker” may be one of the peptide linkers described herein (e.g., flexible linkers described herein), for example, (G4S)2 (SEQ ID NO: 47) and (G4S)3 (SEQ ID NO: 48), and in each stereochemistry, scFab and / scFv may have a heavy chain and a light chain in the order of N-heavy chain-light chain-C or N-light chain-heavy chain-C.

[0055] To promote homogeneous pairing of heavy and light chains within each antigen-binding domain of a fusion protein, each antigen-binding domain may contain charge mutations. Charge mutations refer to substituting a neutral amino acid (e.g., Q) with a positively charged (e.g., K) or negatively charged (e.g., E) amino acid, and substituting a charged amino acid with an amino acid of the opposite charge. To increase the pairing of two polypeptide chains, interacting residues on the two chains can be mutated with amino acid residues of the opposite charge. Exemplary charge mutations that may contribute to homogeneous antibody chain pairing are described, for example, in Tan et al., Biophys J (1998) 75:1473-82; U.S. Patent Application Publication No. 2014 / 0242076A1; and International Publication No. 2020 / 136566. In some embodiments, • Charge mutations in αC1s scFv or scFab are Q42E(V L ) and Q292K(V H ) Includes mutations (numbered by sequence number 12); • The charge mutation in αBb scFv is Q38K(V L ) and Q288E(V H ) including (numbering by sequence number 24) and • The charge mutation in αBb scFab is Q38K(V L ) and Q288E(V H ) and optionally further S114A(C L ), N137K(C L ) and T434E(C H1 ) includes (numbering by sequence number 24).

[0056] In some embodiments, the fusion protein has the structure shown in construct #5 (Figure 2A), and the components of the fusion protein are in the order αC1s scFv-(G4S)2-αBb scFv from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 54 or SEQ ID NO: 55 (with or without signal peptide).

[0057] In some embodiments, the fusion protein has the structure shown in construct #6 (Figure 2A), and the components of the fusion protein are in the order αBb scFv-(G4S)2-αC1s scFv from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 56 or SEQ ID NO: 57 (with or without signal peptide) or therein.

[0058] In some embodiments, the fusion protein has the structure shown in construct #7 (Figure 2A), and the components of the fusion protein are in the order αC1s scFab-(G4S)3-αBb scFab from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 25 or SEQ ID NO: 26 (with or without signal peptide).

[0059] In some embodiments, the fusion protein has the structure shown in construct #8 (Figure 2A), and the components of the fusion protein are in the order αBb scFab-(G4S)3-αC1s scFab from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 29 or SEQ ID NO: 30 (with or without signal peptide).

[0060] In some embodiments, the fusion protein has the structure shown in construct #11 (Figure 2C), and the components of the fusion protein are in the order αC1s scFab-(G4S)3-αBb scFab (with CM) from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 27 or SEQ ID NO: 28 (with or without signal peptide).

[0061] In some embodiments, the fusion protein has the structure shown in construct #12 (Figure 2C), and the components of the fusion protein are in the order αBb scFab-(G4S)3-αC1s scFab (with CM) from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 31 or SEQ ID NO: 32 (with or without signal peptide).

[0062] In some embodiments, the fusion protein has the structure shown in construct #13 (Figure 2D), and the components of the fusion protein are in the order αC1s scFab-(G4S)2-αBb scFv from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 33 or SEQ ID NO: 34 (with or without signal peptide).

[0063] In some embodiments, the fusion protein has the structure shown in construct #14 (Figure 2D), and the components of the fusion protein are in the order αC1s scFab-(G4S)2-αBb scFv-CM (#13, with CM in both αC1s and αBb) from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 35 or SEQ ID NO: 36 (with or without a signal peptide).

[0064] In some embodiments, the fusion protein has the structure shown in construct #15 (Figure 2E), and the components of the fusion protein are in the order αC1s scFab-(G4S)3-αBb scFv from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 58 or SEQ ID NO: 59 (with or without signal peptide).

[0065] In some embodiments, the fusion protein has the structure shown in construct #16 (Figure 2E), and the components of the fusion protein are in the order αC1s scFab-(G4S)3-αBb scFv-CM (with CM) from the N-terminus to the C-terminus. In certain embodiments, this fusion protein contains an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 60 or SEQ ID NO: 61 (with or without signal peptide).

[0066] D. Bispecific heterodimer In some embodiments, the bitargeted complement inhibitors are anti-C1s / anti-Bb bispecific heterodimer proteins. These proteins are encoded by a single open reading frame, but one HC and LC of the antibody fragment are cleaved by intracellular translation and post-translational processing, yielding two distinct polypeptides folded into two antigen-binding domains. Figure 2F illustrates such a configuration. In these illustrated configurations, one HC and LC of the antibody fragment are linked by a cleavable peptide (e.g., a self-cleaving 2A peptide with or without a protease (e.g., furin) cleavage site). See also the discussion in Section II ("Recombinant Expression Constructs").

[0067] In some embodiments, the heterodimer has the structure shown in construct #17 (Figure 2F), where the heterodimer consists of a fusion protein comprising (i) αC1s LC and (ii) αC1s HC fused to αBb scFab. In certain embodiments, this heterodimer comprises an amino acid sequence (with or without two signal peptide sequences) that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to or from the uncleaved SEQ ID NO: 39.

[0068] In some embodiments, the heterodimer has the structure shown in construct #18 (Figure 2F), where the heterodimer consists of a fusion protein comprising (i) αC1s LC and (ii) αC1s HC fused to αBb scFab. In certain embodiments, this heterodimer comprises an amino acid sequence (with or without two signal peptide sequences) that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to or from SEQ ID NO: 41 before cleavage.

[0069] In some embodiments, the heterodimer has the structure shown in construct #19 (Figure 2F), where the heterodimer consists of (i) a fusion protein comprising αC1s scFab fused to αBb HC and (ii) αBb LC. In certain embodiments, this heterodimer contains an amino acid sequence (with or without two signal peptide sequences) that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to or from SEQ ID NO: 43 before cleavage.

[0070] In some embodiments, the heterodimer has the structure shown in construct #20 (Figure 2F), where the heterodimer consists of (i) a fusion protein comprising αC1s scFab fused to αBb HC and (ii) αBb LC. In certain embodiments, this heterodimer comprises an amino acid sequence (with or without two signal peptide sequences) that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to or before cleavage of SEQ ID NO: 45.

[0071] E. peptide linker The peptide linkers of various domains of the antibody fragments and fusion proteins of the present invention may preferably be flexible linkers to allow proper folding, movement, and interaction of the linked domains. In some embodiments, the flexible peptide linkers herein mainly consist of small amino acids (e.g., Gly, Ser, or Thr). In some embodiments, the peptide linkers herein consist mainly of Gly and Ser residues (e.g., more than 50% of the residues) ("GS" linkers). As described above, such peptide linkers may include (G4S)n (SEQ ID NO: 46). By adjusting the copy number "n", the length of the linker can be adjusted to achieve a desired distance of linked functional domains. In some embodiments, the peptide linker may include further amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility. See, for example, Chen et al., Adv Drug Deliv Rev. (2013) 65(10):1357-69.

[0072] II. Recombinant Expression Constructs This disclosure provides recombinant expression constructs for expressing the C1s / Bb inhibitors of this specification. The expression constructs have an expression cassette comprising a coding sequence and a poly(A) signaling sequence of the C1s / Bb inhibitor operably linked to a promoter. The coding sequence may be human codon-optimized to improve expression in human cells. The coding sequence may encode a signal peptide (e.g., an IgGκ-derived signaling peptide) that assists in protein secretion. The expression cassette may also comprise further transcriptional regulatory sequences, such as Kozak sequences and sequences that enhance gene expression or RNA stability (e.g., WPRE elements).

[0073] A. Structure of the expression construct 1. Expression constructs encoding single fusion proteins In some embodiments, the expression constructs herein are monocistronic and contain the coding sequence for an αC1s / αBb fusion protein. See, for example, Figures 1A and 1C. For example, the expression construct may be one of the numbered constructs #5–#8 and constructs #11–#16, the gene product of which is described in the section above.

[0074] 2. Expression constructs encoding two distinct proteins In some embodiments, the expression construct encodes the C1s inhibitor and the Bb inhibitor as two separate proteins. Independent target engagement can eliminate the possibility of steric hindrance.

[0075] For example, the expression construct has two separate expression cassettes, one for each C1s inhibitor (e.g., scFv or scFab) and Bb inhibitor (e.g., scFv or scFab). Each expression cassette has its own transcriptional regulatory sequences, such as promoters and enhancers.

[0076] In an alternative configuration, the expression construct has a bicistronic expression cassette and a single promoter. The coding sequences for the C1s inhibitor and the Bb inhibitor are transcribed together into a single mRNA under the single promoter, and then the RNA sequences of each isoform are translated separately by the use of the internal ribosome entry site (IRES) of the mRNA. In another approach, the coding sequences for the C1s and Bb inhibitors are separated by the coding sequences of self-cleaving peptides and / or protease (e.g., furin) cleavage sites, such that translation and subsequent processing of the mRNA transcripts yield two distinct gene products (C1s inhibitor and Bb inhibitor). An example of a self-cleaving peptide is the 2A peptide, which is a viral peptide with a typical length of 18-22 amino acids. 2A peptides include T2A, P2A, E2A, and F2A. Translation of the transgene may leave a few amino acid residues from the 2A peptide in one or both of the gene products. A furin cleavage site may be included to allow for the removal of excess amino acid residues.

[0077] In yet another configuration, a bicistronic expression construct includes a bidirectional promoter that enables the individual expression of each inhibitor. For example, as shown in Figures 2B and 2G, the expression construct may be one of the numbered constructs #9, #10, #21, and #22 listed below (BiDir: bidirectional promoter). #9: αC1s scFab-BiDir-αBb scFab, produces separate αC1s scFab and αBb scFab. #10: αBb scFab-BiDir-αC1s scFab, produces separate αC1s scFab and αBb scFab. #21: αC1s scFab-BiDir-αBb scFab-CM, produces separate αC1s scFab-CM and αBb scFab-CM. #22: αBb scFab-BiDir-αC1s scFab-CM, produces separate αC1s scFab-CM and αBb scFab-CM.

[0078] In constructs #21 and #22, both anti-C1s and anti-Bb scFab contain charge mutations (CMs) that promote homogeneous pairing of heavy and light chains within each antibody fragment.

[0079] 3. Expression constructs encoding heterodimers In some embodiments, the expression construct encodes a heterodimer consisting of a first single-chain antibody fragment (e.g., scFab or scFv) fused to one of the two strands of a second antibody fragment (e.g., Fab), and this fused polypeptide forms a complex with the other strand of the second antibody fragment. The heterodimer is bispecific and binds to both C1s and Bb.

[0080] Exemplary constructs encoding bispecific heterodimer configurations are shown in Figure 2F and are listed below: • #17: αC1s F2A Fab-(G4S)3-αBb scFab produces a heterodimer composed of a fusion protein containing (i) αC1s LC and (ii) αC1s HC fused to αBb scFab. • #18: αC1s GT2A Fab-(G4S)3-αBb scFab produces a heterodimer composed of a fusion protein containing (i) αC1s LC and (ii) αC1s HC fused to αBb scFab. • #19: αC1s scFab-(G4S)3-αBb F2A Fab produces a heterodimer consisting of (i) a fusion protein containing αC1s scFab fused to αBb HC and (ii) αBb LC. • #20: αC1s scFab-(G4S)3-αBb GT2A Fab produces a heterodimer consisting of (i) a fusion protein containing αC1s scFab fused to αBb HC and (ii) αBb LC.

[0081] In the above construct, the inclusion of coding sequences for cleavable peptides such as F2A and GT2A leads to the production of two distinct polypeptides, which then complex and fold into a single bispecific heterodimer protein. The coding and amino acid sequences for F2A and GT2A are shown in Sequence IDs 38-45. Coding sequences for other cleavable peptides (e.g., those mentioned above) may also be used.

[0082] 4. Separate expression constructs of C1s inhibitors and Bb inhibitors In some embodiments, the C1s inhibitor and the Bb inhibitor may be expressed from two distinct constructs, for example, two distinct recombinant AAVs, as further described below. The two AAVs may be the same or different serotypes.

[0083] B. Transcriptional regulatory sequences In this expression construct, the coding sequences of C1s inhibitors and Bb inhibitors are operably linked to transcriptional regulatory sequences such as promoters and enhancers, enabling the expression of the encoded protein in the target cells of interest.

[0084] In some embodiments, C1s and Bb inhibitors are produced in recombinant host cells. In such cases, the promoter and enhancer are active in the host cells.

[0085] In some embodiments, C1s and Bb inhibitors are delivered via gene therapy and produced in vivo in the eye of a subject (e.g., human, non-human primate, or mouse). In such cases, the promoter may be a constitutive promoter or an inducible promoter that functions in ocular or retinal cells (e.g., RGC and RPE cells, Müller cells, and photoreceptors in the inner and outer granular layers).

[0086] In some embodiments, the promoter is a minCBA promoter comprising a CMV enhancer, a chicken β-actin promoter, and an intron sequence. The minCBA promoter may have at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) or completely identical sequences to SEQ ID NO: 83.

[0087] In some embodiments, the promoter is a bidirectional promoter. A bidirectional promoter may include, for example, a pair of CBA promoters arranged in opposite directions and separated by a CMV enhancer. In certain embodiments, the bidirectional promoter includes at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) or an array identical to sequence number 53.

[0088] In some embodiments, the expression cassette has a poly(A) signaling sequence derived from the bovine growth hormone gene. In certain embodiments, the poly(A) signaling sequence includes at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) or a sequence that is completely identical to the sequence italicized and underlined in Sequence ID No. 51 shown in the following sequence section.

[0089] In some embodiments, the expression cassette includes an enhancer, such as a CMV enhancer. In certain embodiments, the CMV enhancer includes at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) or a sequence that is completely identical to the sequence shown in bold and italics in Sequence ID No. 53 shown in the following sequence section.

[0090] In some embodiments, the expression cassette includes intronic sequences such as chimeric introns. These intronic sequences can increase transgene expression levels by promoting the transport of mRNA out of the nucleus and enhancing mRNA stability.

[0091] C. Recombinant AAV expression vector In some embodiments, a viral vector is used to deliver a vectorized antibody fragment to the patient's eye. In some embodiments, the expression / delivery vector is a recombinant adeno-associated virus (rAAV) expression vector. The expression construct herein may be an rAAV genome. In the case of an rAAV genome, the expression cassette herein may be adjacent to a pair of AAV reverse terminal repeats (ITRs), such as AAV2 ITRs. A non-limiting example of a unidirectional monocistronic AAV2 recombinant genome is shown in Figure 2H. A non-limiting example of a bidirectional bicistronic AAV2 recombinant genome is shown in Figure 2I.

[0092] An exemplary rAAV genome having construct #9 may have a nucleotide sequence that encodes the exemplary nucleotide sequence of SEQ ID NO: 50, or the same amino acid sequence as SEQ ID NO: 50, and contains at least 50% (e.g., at least 60, 65, 70, 75, 80, 85, 90, or 95%) identical sequences to SEQ ID NO: 50.

[0093] An exemplary rAAV genome having construct #12 may have a nucleotide sequence that encodes the exemplary nucleotide sequence of SEQ ID NO: 51, or the same amino acid sequence as SEQ ID NO: 51, and contains at least 50% (e.g., at least 60, 65, 70, 75, 80, 85, 90, or 95%) identical sequences to SEQ ID NO: 51.

[0094] An exemplary rAAV genome having construct #14 may have a nucleotide sequence that encodes the exemplary nucleotide sequence of SEQ ID NO: 52, or the same amino acid sequence as SEQ ID NO: 52, and contains at least 50% (e.g., at least 60, 65, 70, 75, 80, 85, 90, or 95%) identical sequences to SEQ ID NO: 52.

[0095] The rAAV genome can be constructed by inserting the expression cassette described herein into the rAAV genome from which the major rAAV open reading frame has been excised. Other parts of the rAAV genome can also be deleted, as long as a sufficient portion of the ITR remains to enable replication and packaging functions.

[0096] Any suitable AAV serotype can be used. For example, AAV may be AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, or AAVrh10, or a pseudotype or serotype which is a variant, variant, or derivative of one of the AAV serotypes listed herein (i.e., AAV derived from multiple serotypes). AAV may be engineered so that its capsid protein has reduced immunogenicity or enhanced transduction ability in humans or non-human primates.

[0097] In some embodiments, the rAAV described herein has an AAV2 capsid. In certain embodiments, the AAV2 capsid is a wild-type AAV2 capsid. In other embodiments, the AAV2 capsid includes a mutation that improves the potency and production yield of rAAV2.

[0098] The viral vectors described herein may be produced using methods known in the art. Any suitable host cell or packaging cell may be used to produce viral particles. For example, mammalian (e.g., 293 or HeLa) or insect (e.g., Sf9) cells may be used as packaging cell lines. Recombinant AAV vectors may be replicated and packaged into infectious viral particles when introduced into host cells that are infected with a suitable helper virus (or express a suitable helper function) and express the AAV rep and cap gene products (i.e., AAV Rep and capsid proteins). See, for example, U.S. Patent No. 11,261,463.

[0099] D. Transfection of host cells When C1s and Bb inhibitors are delivered directly to the patient, the inhibitors can be produced in recombinant mammalian host cells such as COS, NS0, 293, HeLa, or CHO cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences, as well as for the collection and purification of the inhibitors.

[0100] III. Pharmaceutical Compositions and Uses This disclosure provides pharmaceutical compositions comprising a dual-targeted C1s / Bb inhibitor or a recombinant viral vector such as an AAV vector encoding the inhibitor. The pharmaceutical compositions may comprise pharmacologically, and in particular ophthalmologically, acceptable carriers, diluents, and / or excipients. For example, the compositions may comprise isotonic agents (e.g., sodium chloride, amino acids, sugars, or combinations thereof), surfactants (e.g., polysorbate 20 or polysorbate 80), and / or stabilizers (e.g., methionine).

[0101] The pharmaceutical composition may be delivered by intraocular injection, for example, injection into the anterior chamber via the limbus, superior choroidal injection, anterior chamber injection, intramatrix injection, subretinal injection, or intravitreous injection (for example, anterior, intermediate, or posterior intravitreous injection).

[0102] The pharmaceutical compositions of the present invention may be delivered in therapeutically effective doses to treat atrophic AMD and geographic atrophy (GA) secondary to atrophic AMD. “Therapeutic dose” means a dose sufficient to produce a desired outcome, e.g., improvement of one or more symptoms of the disease being treated (e.g., growth of GA lesions, retinal lesions, or destruction of the retinal layer), and / or delay of disease progression. The desired outcome may also include improvement of one or more functional symptoms; for example, the desired outcome may be reduced visual distortion, improved central vision, improved visual acuity in low-light settings, and / or reduced blurring. “To treat” means improvement of one or more symptoms of the disease and / or delay of disease progression.

[0103] The pharmaceutical composition of the present invention can be delivered in a prophylactic effective dose to prevent the development of atrophic AMD or geographic atrophy (GA) secondary to atrophic AMD. “Prophylactic effective dose” means a dose sufficient to produce the desired outcome, for example, prevention or delay of the development of atrophic AMD and / or GA, and / or prevention or delay of the development of one or more symptoms of atrophic AMD and / or GA. The pharmaceutical composition of the present invention can be administered prophylactically to patients at high risk of developing atrophic AMD, such as patients with a genetic predisposition.

[0104] In some embodiments, the dose of recombinant AAV (rAAV) injected into the eye is 10 7 ~10 15 Vector genome (vg), for example 10 8 ~10 14 , 10 9 ~10 13 , or 10 9 ~10 12 In some embodiments, the dose of rAAV is 2 × 10⁻¹⁰. 9 , 2×10 10 , or 2 × 10 11 It is vg.

[0105] In some embodiments, patients are treated with anti-inflammatory agents (e.g., steroids) before, during, and / or after rAAV injection to prevent or improve a potential immune response to rAAV. In some embodiments, patients may be pre-treated with IgG-degrading enzymes such as IdeS to reduce existing neutralizing antibodies against the AAV capsid. These immunomodulators may be administered topically or systemically. In some embodiments, the modulators may be administered intraocularly (e.g., intravitreously), orally, intravenously, intramuscularly, or subcutaneously.

[0106] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout this specification and the embodiments, the terms “having” and “including,” or variations such as “having,” “having,” “including,” or “containing,” shall be understood to include the integer or group of integers described, but not to exclude any other integer or group of integers. All publications and other references referenced herein are incorporated by reference in their entirety. This specification references several documents, but such references do not constitute an endorsement that any of those documents form part of the common technical knowledge in the art. As used herein, the terms “approximately” or “about” refer to a value similar to the reference value described, when applied to one or more values ​​of interest. In certain embodiments, unless otherwise stated or evident from the context, the term refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than either of the reference values ​​described (greater than or less than).

[0107] As used herein, the percentage of identity between two amino acid sequences (or two nucleic acid sequences) is obtained, for example, by BLAST® using default parameters (available on the website of the U.S. National Library of Medicine's National Center for Biotechnology Information). In some embodiments, the length of the query sequence to be aligned for comparison is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%) of the length of the reference sequence.

[0108] According to this disclosure, backreferences in dependent claims mean abbreviated notation to any direct and obvious disclosure of any combination of claims indicated by the backreference. Any compound disclosed herein may be used in any of the therapeutic methods disclosed herein, the individual being treated as defined elsewhere herein.

[0109] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention. [Examples]

[0110] Example 1: Vectorized antibody and its expression construct This example describes the design of bifunctional expression constructs expressing inhibitors of C1s and Bb, and the characterization of recombinant proteins produced from these constructs. These constructs have the following features: (i) either a unidirectional or bidirectional promoter (e.g., minCBA promoter) to drive constitutive transgene expression; (ii) a transgene (e.g., a transgene containing a human codon-optimized sequence); (iii) a parental anti-Bb IgG4 antibody (e.g., from U.S. Patent No. 11,242,382 and International Publication No. 2021 / 216458). H(iv) Different combinations of antibody fragments derived from 6 / Vκ7-IgG4v2) and parental anti-C1s IgG4 antibody (e.g., VH3 / VK2 from International Publication No. 2018 / 071676) (e.g., scFab-scFab and scFab-scFv); (iv) peptide linkers (e.g., containing G4S repeats between antibody fragments and between the heavy and light chains of each antibody fragment); (v) presence or absence of reasonably designed charge mutations (CMs) that promote accurate heavy / light chain pairing; (vi) polyadenylation sites (e.g., bovine growth hormone (bGH) gene polyadenylation signals).

[0111] A. Generation of bifunctional bicistronic or monocistronic structures The bifunctional monocistronic or bicistronic constructs generated herein include a DNA fragment expressing scFv or scFab, constituent antibody fragments against active C1s and Bb downstream of a ubiquitous minCBA promoter, and a poly(A) signal sequence derived from the bovine growth hormone gene. The entire expression cassette was cloned between wild-type inverted terminal repeat (ITR) sequences derived from AAV serotype 2 (Figure 1A-C). A glycine / serine-rich linker (e.g., a linker with a G4S repeat) was inserted between the heavy and light chains of each single-stranded αC1s and αBb antibody fragment (scFab or scFv), and V H and V L The pair facilitated the proper folding of each antigen-binding domain. In this study, a linker with seven G4S repeats was used to link the heavy and light chains of scFab, and a linker with three G4S repeats was used to link the V of scFab. H and V L They were linked together.

[0112] For monocistronic constructs, exemplary formats included scFab-scFab, scFv-scFab, scFab-scFv, and scFv-scFv (see, e.g., Figures 2A, 2D, and 2E). Glycine / serine-rich linkers (e.g., linkers with G4S repeats such as 2 or 3 repeats) were inserted between the two single-stranded fragments of the bifunctional fusion protein to enable flexibility in the bifunctional fusion protein.

[0113] Further features of some monocistronic constructs included, for example, the inclusion of a standard furin cleavage site (RX(R / K)R) (SEQ ID NO: 82) in linkers F2A and GT2A (Figure 2F). By ligating the heavy chain (HC) and light chain (LC) genes into a single cassette using the 2A peptide, improved control of the LC and HC ratio becomes possible. Inserting a furin recognition site upstream of 2A allows for the removal of 2A residues that would otherwise bind to HC and / or LC (see, for example, Figure 2F).

[0114] For a bidirectional, bifunctional construct, a novel bidirectional promoter was designed based on the ubiquitous minimal chicken β-actin (minCBA) promoter. This promoter supports the co-expression of individual antibody fragments to the C1s and Bb factors. minCBA contains a CBA promoter and a CMV enhancer, but with a shortened intron sequence. The bidirectional promoter contains a pair of CBA promoters positioned in opposite directions and separated by the CMV enhancer (SEQ ID NO: 53). The bidirectional expression construct produces separate anti-C1s and anti-Bb antibody fragments for independent target binding, eliminating the possibility of steric hindrance.

[0115] Monocistronic or bicistronic expression cassettes were cloned between AAV2 ITR sequences (e.g., Figures 1C, 2H, and 2I) for AAV delivery.

[0116] In several experiments, antibody fragments containing charge mutations that facilitated precise pairing between the heavy and light chains of each constituent antibody fragment were used. To generate charge mutants (CMs), specific amino acids were substituted in the variable and / or constant domains of the αC1s and αBb antibody fragments. The following amino acid changes were introduced into the following mutant antibody fragments: • αC1s scFab-CM: Q42E and Q292K (numbering according to sequence number 12) • αBb scFab-CM: Q38K, S114A, N137K, Q288E and T434E (numbering according to sequence number 24)

[0117] The configuration of an exemplary unidirectional structure is shown in Figures 2A and 2C-F, and is listed below: ·#5:αC1s scFv-(G4S)2-αBb scFv ·#6:αBb scFv-(G4S)2-αC1s scFv ·#7:αC1s scFab-(G4S)3-αBb scFab ·#8:αBb scFab-(G4S)3-αC1s scFab • #11: αC1s scFab-(G4S)3-αBb scFab-CM (#7, both αC1s and αBb have CM) • #12: αBb scFab-(G4S)3-αC1s scFab-CM (#8, both αC1s and αBb have CM) ·#13:αC1s scFab-(G4S)2-αBb scFv • #14: αC1s scFab-(G4S)2-αBb scFv-CM (#13, both αC1s and αBb have CM) ·#15:αC1s scFab-(G4S)3-αBb scFv • #16: αC1s scFab-(G4S)3-αBb scFv-CM (#15, both αC1s and αBb have CM) • #17: αC1s F2A Fab-(G4S)3-αBb scFab produces a heterodimer composed of a fusion protein containing (i) αC1s LC and (ii) αC1s HC fused to αBb scFab. • #18: αC1s GT2A Fab-(G4S)3-αBb scFab produces a heterodimer composed of a fusion protein containing (i) αC1s LC and (ii) αC1s HC fused to αBb scFab. • #19: αC1s scFab-(G4S)3-αBb F2A Fab produces a heterodimer consisting of (i) a fusion protein containing αC1s scFab fused to αBb HC and (ii) αBb LC. • #20: αC1s scFab-(G4S)3-αBb GT2A Fab produces a heterodimer consisting of (i) a fusion protein containing αC1s scFab fused to αBb HC and (ii) αBb LC.

[0118] An example of a bidirectional structural building configuration is shown in Figure 2B and is listed below: #9: αC1s scFab-BiDir-αBb scFab, produces separate αC1s scFab and αBb scFab. #10: αBb scFab-BiDir-αC1s scFab, produces separate αC1s scFab and αBb scFab. #21: αC1s scFab-BiDir-αBb scFab-CM, produces separate αC1s scFab-CM and αBb scFab-CM. #22: αBb scFab-BiDir-αC1s scFab-CM, produces separate αC1s scFab-CM and αBb scFab-CM.

[0119] Evaluation of B.Bb and C1s bonds Each DNA construct was transfected into HEK293 cells. The supernatant containing the secreted recombinant protein was collected and purified on Protein L beads. More specifically, the supernatant was incubated with Protein L beads at room temperature for 1 hour. The beads were then washed three times with PBS containing polysorbate 20. The bead column was then eluted with 0.1 M glycine (pH 2.0) at room temperature for 10 minutes. The eluate was neutralized with 15% v / v 1 M Tris (pH 8.5) and then desalted by buffer exchange to PBST.

[0120] The purity of recombinant proteins was evaluated using SDS-PAGE (non-reducible and reduced) and mass spectrophotometer (mass distribution). Protein concentration was measured using NanoDrop® (Thermo Fisher).

[0121] The target involvement and binding affinity of recombinant proteins to complement C1s enzyme (active C1s or "C1s" as used herein) and factor Bb (Bb) were evaluated using biolayer interferometry (BLI) (see Complement Technology, Tyler, TX, USA). C1s and Bb were biotinylated with EZ-Link® Sulfo-NHS-LC-LC-Biotin (Thermo Fisher, Waltham, MA, USA) according to the manufacturer's instructions. Biotinylated C1s or Bb were loaded into Octet® Streptavidin® Biosensors (Sartorius, Goettingen, Germany), followed by loading with purified proteins in a range of concentrations. To evaluate dual target involvement, biotinylated active C1s or Bb were loaded onto the sensor, followed by loading with purified protein ("first association step"), and then followed by loading with an uncaptured complement target (Bb or active C1s, unbiotinylated; "second association step"). The assay was performed at 30°C using PBS containing 0.1% Tween20 as a diluent (Figure 3).

[0122] Furthermore, inhibition of classical and alternative complement pathways was evaluated using the Wieslab® Complement System Classical Pathway and Wieslab® Complement System Alternative Pathway kits (Svar, Malmoe, Sweden). Assays were performed according to the manufacturer's instructions. Serial dilutions of constructs were performed using the respective assay diluents for each assay.

[0123] C. Results To confirm the expression and secretion of vector-derived antibody fragments, supernatant was collected from HEK293 cells transfected with plasmids encoding the transgenes, and kappa light chain-containing antibody fragments were concentrated from the supernatant by affinity purification using protein L beads. Western blot analysis of the concentrated supernatant demonstrated that all transgenes produced antibody fragments.

[0124] Target engagement of antibody fragments from cell supernatant was evaluated using the Octet® binding assay. The data demonstrated that proteins produced from all expression constructs showed dual target engagement against C1s and Bb. Across all tested constructs, the binding affinity of partially purified bifunctional antibody fragments was within 2–10 times that of purified parental anti-C1s and anti-Bb Fab.

[0125] Exemplary data is shown in Figure 3, which indicates that an increase in signal was observed when a partially purified antibody fragment produced by construct #19 (Figure 2F) was added at the first "association" stage (for binding to Bb). A further increase in signal was observed when the second target αC1s was added at the second "association" stage (Figure 3). All antibody fragments produced by the tested bifunctional constructs, with the exception of construct #5 (Figure 2A), showed similar levels of bitarget binding (see Example 2 below).

[0126] The parental monoclonal antibodies used to design bifunctional complement inhibitors have previously been shown to inhibit either the classical complement (CP; see International Publication No. 2016 / 164358) or surrogate (AP; see U.S. Patent No. 11,242,382) pathway without inhibiting the lectin pathway. The ability of bifunctional antibody fragments or antibody fragment pairs to inhibit the activity of both CP and AP was evaluated in vitro using the Wieslab® assay. All tested bifunctional antibody constructs inhibited both IgM-stimulated CP activation and LPS-stimulated AP activation (see Example 2 below). The data show that for all tested constructs, the inhibitory activity was within 4 times that of the parental Fab.

[0127] The results indicate that vector-expressed antibody fragments against complement factors Bb and C1s bind to the target complement factors and inhibit activated complement with similar efficiency to the individual parental Fab proteins. These results were unexpected, as the parental antibody fragments are highly purified Fab expressed in CHO cells, generated using recombinant mAB technology, whereas the antibody fragments generated from the AAV previral plasmid are scFab and scFv fragments, tested as partially purified antibody fragments. Furthermore, the plasmid-derived antibody fragments are monocistronic for some constructs and therefore act like bifunctional antibodies. Despite this design / structural difference with the parental Fab, inhibition of each target was largely conserved.

[0128] Example 2: Functional Characterization of Anti-C1s and Anti-Bb scFab Constructs #2 and #4 were recombinantly expressed, purified to homogeneity as described above, and tested using target binding assays as well as serum-based and cell-based functional assays. Direct target binding was measured using surface plasmon resonance (SPR).

[0129] The inhibitory activity of scFab was tested using a serum-based Wieslab® enzyme immunoassay. In commercially available assay kits, the wells of the microtiter strips are coated with specific activators for each pathway of the complement system. Furthermore, the buffers and reagents included in the kit prevent cross-activation of multiple pathways and maintain the specificity of pathway activation. (Test kits for AP are coated with lipopolysaccharide, and test kits for CP are coated with human IgM). The final readout is the detection of neoepitopes on the C5b9 complex generated as a result of complement pathway activation, measured colorimetrically. Recombinant scFab was also tested in a modified Wieslab® assay that allows simultaneous activation of CP and AP by coating the microtiter plate with both heat agglutination (HAGG) IgG and C3b; in this assay, the C5b9 complex generated from the activation of both pathways was also measured colorimetrically.

[0130] Furthermore, recombinant scFab was tested in an in vitro ARPE19 cell line-based model of atrophic AMD. In all functional assays, recombinant scFab was tested individually and as an equimolar mixture representative of the vector-derived product.

[0131] Table 1 below shows the characterization of recombinant scFab and a comparison of them with parent scFab (#2 and #4) and mAb.

[0132] [Table 3]

[0133] These data demonstrate that recombinant scFabs for both C1s and Bb exhibit similar binding and inhibitory properties to their corresponding parent scFabs.

[0134] Example 3: Characteristics of exemplary complement inhibitors having charge mutations Three expression constructs were selected for further study. The first construct, #14 (Figures 2D and 2H), consisted of a unidirectional minCBA promoter driving the expression of a single transcript encoding anti-C1s scFab linked to anti-Bb scFv by a flexible (G4S)2 linker [αC1s scFab-(G4S)2-αBb scFv], followed by a bGH poly(A) signal. The sequence was human codon optimized and included charge mutations to facilitate precise strand pairing.

[0135] The second expression construct, construct #12 (Figures 2C and 2J), consisted of a unidirectional minCBA promoter driving the expression of a single transcript encoding anti-Bb scFab linked to anti-C1s scFab by a flexible (G4S)3 linker [αBb scFab-(G4S)3-αC1s scFab], followed by a bGH poly(A) signal. The sequence was human codon optimized and included charge mutations to facilitate precise strand pairing.

[0136] A third expression construct, construct #9 (Figures 2B and 2I), consisted of a bidirectional minCBA promoter driving the expression of a separate transcript encoding human codon-optimized αBb scFab or αC1s scFab [αC1s scFab + αBb scFab], each followed by a bGH poly(A) signal. In assays performed as described in Example 1, complement-binding antibody fragments expressed from constructs #9 and #14 had binding affinities to both C1s and Bb within 2–6 times that of purified parental Fab, and complement-binding antibody fragments expressed from construct #12 had binding affinities to C1s and Bb within approximately 6–7 times that of purified parental Fab (Table 2).

[0137] [Table 4]

[0138] In the Wieslab® assay, IC of complement inhibitors derived from construct #14 50The IC values ​​were within approximately 6 times those of purified anti-C1s Fab (CP inhibition) and purified anti-Bb Fab (AP inhibition). IC of antibody fragment #12 50 The IC50 levels were within approximately 7 times those of purified anti-Bb Fab (AP inhibition) and within 14 times those of purified anti-C1s (CP inhibition). IC50 of antibody fragment #9 50 The values ​​were within approximately 3 times that of purified anti-Bb Fab (AP inhibition) and within 25 times that of purified anti-C1s Fab (CP inhibition) (Table 3).

[0139] [Table 5]

[0140] Furthermore, constructs #2, #4, #12, and #14 (Figures 2A, 2C, and 2D) were expressed and purified to over 98% purity using chromatography (referred to as recombinant constructs). The functional properties of these constructs were characterized by one-to-one testing with parental anti-C1s and anti-Bb Fab using the assay described in Example 1. Constructors #2 and #4 were selected to represent two scFabs expressed and secreted by bidirectional vector constructor #9.

[0141] The results of these experiments are summarized in Table 4 below (ND: undecided).

[0142] [Table 6]

[0143] Table 5 below summarizes the in vitro binding and functional inhibition results of proteins expressed by #2, #4, #5, #6, #7, #8, #9, #11, #12, #13, #14, #15, #16, #17, #18, #19, and #20 in Figures 2A-2F, compared with recombinant parental anti-C1s Fab and anti-Bb Fab.

[0144] [Table 7]

[0145] In addition to direct target binding (BLI) and Wieslab® EIA assays, we developed another functional assay to evaluate the simultaneous inhibition of both CP and AP by these recombinant constructs. In this assay, ELISA plates were coated with both HAGG (thermally agglutinated gamma globulin) and C3b and incubated with 12% C1s-deficient serum containing 380 ng / ml of proenzyme C1s to simultaneously activate both CP and AP. The assay conditions were optimized to achieve similar levels of CP and AP activation on the plates. The dose-response of constructs #2 and #4 was tested individually or in equimolar mixtures (to represent the expression conditions from construct #9). Equimolar mixtures of parental anti-C1s Fab and anti-Bb Fab were also tested together.

[0146] Under these conditions, constructs #2 and #4 achieved dose-dependent but partial inhibition (70–85%; Figures 4A and 4B). However, when these two constructs were mixed together in equimolar ratios, it resulted in >99% inhibition of complement activation, similar to what was observed for an equimolar mixture of the parent Fab. Observed IC 50 The inhibition levels were within 2–3 times those observed for an equimolar mixture of parent Fabs (Figure 4C). See also Table 6, which summarizes the maximum half-molar inhibitory concentrations for anti-C1s Fab, anti-Bb Fab, proteins expressed from construct #2, proteins expressed from construct #4, or equimolar mixtures of these two, as well as the maximum inhibition achieved under conditions where both CP and AP were simultaneously activated in vitro.

[0147] [Table 8]

[0148] Example 4: In vivo retinal study in mice Based on the in vitro results described above, constructs #9, #12, and #14 were selected for in vivo studies, and their ITR plasmid expression cassettes were packaged in AAV2 for delivery to target cells (e.g., Figures 2H, 2I, and 2J). This example describes in vivo testing of these vectorized antibody constructs in wild-type mouse retina to confirm transduction of retinal ganglion cells (RGCs) and secretion of antibody fragments into the vitreous humor. Antibody fragments secreted into mouse vitreous fluid were evaluated by in vitro assays to demonstrate targeted binding to human complement factors C1s and Bb. Tolerability was assessed by optical coherence tomography (OCT).

[0149] A.AAV injection More specifically, recombinant AAV2 expression constructs #9, #12, and #14 adjacent to the AAV2 ITR were produced. AAV2#14, AAV2#12, and AAV2#9 were administered in three doses [10 8 , 10 9 pieces or 10 10 A vector genome (vg) per eye was administered to C57BL / 6J mice by intravitreal injection, and retinal transduction, transgene expression, antibody secretion, and tolerability were evaluated after 3-4 weeks of lifetime exposure. Recombinant AAV2 encoding the secreted VEGF inhibitor was used as a positive control, with 2 × 10¹⁶ samples per eye. 9 The drug was administered concurrently via VG. Vector-naive mice that had not been injected were used as negative controls.

[0150] B. Vector Transduction Vector transduction was quantified using the TaqMan® assay, and vector-derived bGH poly(A) was detected by quantitative PCR analysis of purified DNA from mouse retina. The data showed that all three vectors successfully transduced the retina, with approximately 10 per 500 ng of DNA. 4 ~10 5 This indicates that vg was achieved. The level of transduction from the bifunctional antibody fragment vector was equivalent to that achieved in the positive control. There was a vector dose-dependent increase in transduction of AAV2#14 (10 10 vs 10 8;p=0.01). AAV2#12(10 9 Several mice administered with vg exhibited relatively low levels of transduction; similar results were observed for this group (which may have been due to technical issues with the dosage). AAV2#9, possessing two copies of bGH poly(A), showed high levels of transduction at all doses.

[0151] Vector transduction and cell targeting in mouse retina were also evaluated using vector-specific probe set in situ hybridization (ISH) analysis of sections from fixed paraffin-embedded eyes. Each probe set contained 40 pairs of probes, each approximately 50 nucleotides long. In eyes administered with each AAV2 vector, vector transduction was detected mainly in retinal ganglion cells (RGCs) and inner granular layer (INL) cells, to a lesser extent in outer granular layer (ONL) cells, and rarely in retinal pigment epithelium (RPE) cells (Figure 5).

[0152] Table 7A summarizes the levels of transduction achieved in mouse retinas 3 weeks after intravitreous administration of AAV2#9, AAV2#12, and AAV2#14 (median ± MAD).

[0153] [Table 9]

[0154] C. Transgene expression Transgene expression in the retina was measured by quantitative RT-PCR analysis of purified RNA from mouse retinas using the TaqMan® assay to detect the vector-derived bGH poly(A) sequence. RNA quality was assessed, and samples with an RNA completeness number (RIN) of less than 6 were not included in the analysis. The data showed that all three AAV vectors resulted in high levels of transgene expression in the retina after 3 weeks of lifetime exposure (approximately 10 per 500 ng of RNA). 5 ~10 6It has been shown to result in (transcripts). Table 7B summarizes the levels of transgene expression (bGH transcripts / 500 ng RNA) achieved in the mouse retina 3 weeks after intravitreal administration of AAV2#9, AAV2#12, and AAV2#14 (median ± MAD).

[0155]

Table 10

[0156] Across all samples, the transcription levels correlated with the levels of the vector genome (p = 0.59), and the expression levels were lower in the AAV2#12 retina with insufficient transduction from the 10 9 vg treatment group. AAV2#9 showed a dose-dependent increase in transgene expression (10 10 vs. 10 8 , p = 0.036; 10 10 vs. 10 9 , p = 0.0495).

[0157] D. Antibody Expression The expression and distribution of vector-derived complement inhibitors in the mouse retina were evaluated by immunohistochemistry (IHC) using an anti-human kappa light chain antibody to detect vector-derived human antibody fragments. The data show that inhibitors produced by all three vectors were detected in cells of the RGC (retinal ganglion cells) and INL (inner nuclear layer).

[0158] E. Antibody Secretion and Target Engagement To demonstrate that the viral vector produced a secreted bifunctional complement inhibitor, inhibitor levels in mouse vitreous fluid were evaluated using ELISA. Vector-derived complement inhibitors present in mouse vitreous fluid were quantified by target engagement ability using C1s and Bb ELISA and purified anti-C1s and anti-Bb scFab as standards. Tables 8A and 8B summarize the ex vivo dual-target engagement results of secreted anti-C1s antibody fragments (Table 8A) and anti-Bb antibody fragments (Table 8B) present in mouse vitreous fluid 3 weeks after intravitreous administration of AAV2#9, AAV2#12, and AAV2#14 (mean ± SD; ng / mL).

[0159] [Table 11]

[0160] [Table 12]

[0161] Overall, C1s and Bb ELISAs demonstrated that all three rAAVs, when delivered into the vitreous humor, resulted in expression and secretion from mouse retinal ganglion cells. Proteins expressed by all three expression vectors were able to bind to C1s and Bb ex vivo. Overall, the data indicate that all three expression vectors yielded comparable levels of anti-C1s and anti-Bb binding activity in mice. It was unexpected that retinal ganglion cells could support the in vivo production of vectorized antibody fragments exhibiting similar binding properties to parental antibodies produced in vitro using established recombinant antibody production methods.

[0162] AAV2#14 treated mice have vitreous levels of the bifunctional antibody ranging from approximately 150 ng / ml to approximately 900 ng / ml. Vitreous levels of the AAV2#12-derived inhibitor showed a slight dose-response across treatment groups, increasing from approximately 80 ng / ml to approximately 140 ng / ml. Intravitreous inhibitor levels in AAV2#9 treated mice increased in a dose-dependent manner, reaching approximately 1100 ng / ml at the peak dose. In addition to quantifying intravitreous inhibitor levels, these data demonstrate ex vivo bitarget binding of the vector-derived antibody fragment.

[0163] The target involvement and efficacy of vector-derived complement inhibitors cannot be evaluated in vivo in mice because these inhibitors bind only to human and non-human primate (NHP) C1s and Bb and do not interact with mouse complement factors.

[0164] In mice administered with AAV2-positive controls (see above), intravitreous secretion of VEGF inhibitors was measured by ELISA. Vitreous levels of VEGF inhibitors averaged approximately 57 ng / ml after 2 weeks of lifetime exposure. Therefore, AAV2#14, AAV2#12, and AAV2#9 all produce higher levels of secreted protein than the positive controls.

[0165] F. Tolerability Photoreceptor damage can be detected as thinning of the photoreceptor. Viral vector tolerance was evaluated by measuring the thickness of the photoreceptor (PR) layer [outer granular layer (ONL) + inner / outer segment (IS / OS)] in optical coherence tomography (OCT) images from the retinas of vector-naive and transduced mice. The photoreceptor thickness of retinas transduced with AAV2#14-, AAV2#12-, and AAV2#9 was compared to that of vector-naive retinas at each dose (10 8 , 10 9 or 10 10 The dose did not decrease even at vg, suggesting that the dosage and time point tested in mice did not affect photoreceptor tolerance.

[0166] Example 5: Inhibition of complement activation in a cell-based model of atrophic AMD. C-reactive protein (CRP) is an acute-phase reactive protein and an activator of the classical complement pathway (CP). CRP binds to dead cells, activates CP, and labels those cells for clearance by phagocytic cells. CRP levels are elevated under inflammatory conditions. Elevated CRP levels are an independent risk factor for the pathogenesis of AMD, and high serum CRP concentrations have been shown to be associated with faster progression of AMD to advanced disease and higher severity of vision loss in other retinal diseases such as retinitis pigmentosa (Chen et al., Trans Vis Sci & Techno. (2021) 10(7):7; Molins et al., Front Immunol. (2018) 9:808; and Murakami et al., Acta Ophthalmol. (2018) 96(2):e174-e179). Furthermore, Bruch's membrane, drusen, and choroidal vascular walls have been shown to stain elevated levels of CRP in the eyes of AMD patients, suggesting that complement activation during the disease is initiated, at least partially, by CRP (Bhutto et al., Br J Ophthalmol. (2011) 95(9): 1323-30).

[0167] To reproduce some of these patient features in vitro in a cell-based model, ARPE19 cells (retinal pigment epithelial (RPE) cell line) were treated with normal human serum (NHS) supplemented with CRP. The degree of complement activation was assessed by monitoring the levels of C3 fragments and C5b9 deposited on the cell surface using an on-cell ELISA protocol. The data showed that treatment of ARPE19 cells with CRP-supplemented NHS resulted in elevated levels of both C3 fragments and C5b9 on the cells compared to treatment with NHS alone, indicating stronger complement system activation in the presence of CRP (Figure 6). When complement inhibitors were included in the treatment, combined inhibition of CP and AP (anti-C1s Fab + anti-Bb Fab) resulted in a stronger decrease in both C3 fragments and C5b9 levels compared to the inhibition levels achieved by anti-C1s Fab (CP) or anti-Bb Fab (AP) individually (Figure 6).

[0168] Example 6: A novel iPSC-derived cell model for AMD This embodiment describes a novel cell model developed to demonstrate CRP-initiated complement activation in AMD. This model measures complement deposition on induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPEs). RPEs play many important roles in the eye and, in addition to many other essential functions, are responsible for phagocytosis of photoreceptor outer segments and the transport of nutrients from the choroid to the retina. Complement activation on RPEs may contribute to inflammation and cell death in AMD. iPSC-RPEs were selected for this model because they maintain the morphology of native RPEs and share similar cellular markers. Therefore, by measuring complement deposition on the surface of these cells, it is possible to model how specific drug treatments limit complement activation in the retina during the course of AMD disease.

[0169] Complement deposition on the surface of iPSC-RPE was measured using cell ELISA. iPSC-RPE (FujiFilm Cellular Dynamics, Madison, WI) was grown in fibronectin-coated black / clear-bottom 96-well plates. CRP (100 μg / mL) (ImmunoPrecise Antibodies, Utrecht, The Netherlands), 10% normal human serum (Complement Technology, Tyler, TX), and a complement inhibitor were added to the cell culture medium and incubated overnight with the iPSC-RPE. The following day, the cells were washed and fixed with 4% paraformaldehyde. After blocking, the cells were incubated with anti-C3d or anti-C5b9 HRP conjugate antibody (Novus Biologicals, Centennial CO). Fluorescence signals were generated using QuantaRed® Enhanced Chemifluorescent HRP Substrate (Thermo Fisher, Waltham, MA) and measured using a plate reader. The data show that individual treatment with either anti-C1s or anti-Bb scFab resulted in a significant reduction in C3d and C5b9 deposition on iPSC-RPEs; however, a combination of both scFabs maximized the reduction in complement product deposition (Figures 7A and 7B).

[0170] A similar method was used for fluorescence imaging of C5b9 deposition on iPSC-RPE cells. In this method, cells were grown on fibronectin-coated 24-well suspension cell culture inserts. Cells were treated overnight with CRP, 10% normal human serum, and a complement inhibitor. Z-stack images were captured at 40x magnification using confocal microscopy. For image quantification, three regions of interest (ROIs) were randomly imaged from each sample. The total area of ​​C5b9 was calculated within each ROI and averaged for each sample. The mean of three replicate experiments was measured for each group, and error bars were calculated from the mean of the standard deviations. Fluorescence imaging experiments were repeated three times using three different iPSC-RPE cell lines. The data similarly indicate that treatment with a combination of anti-C1s and anti-Bb scFab resulted in a significant reduction in C5b9 (red) staining (Figures 8A and 8B).

[0171] In conclusion, results from the iPSC-RPE model suggest that both classical and alternative pathways likely play a role in the pathogenesis of AMD. Blocking each pathway separately reduced complement deposition on RPE cells. However, simultaneous inhibition of both pathways resulted in the greatest reduction in deposition, suggesting that simultaneous inhibition of classical and alternative pathways may be beneficial in AMD.

[0172] Example 7: In vivo retinal study in non-human primates This example describes an exemplary in vivo study of a vectorized antibody construct in non-human primates (NHPs) to confirm transduction and transgene expression in the retina. The activity of the viral vector after intravitreous administration in NHPs was evaluated in two studies: (1) a 6-week dose-range study of AAV2#14 and AAV2#12, and (2) an 8-week single-dose study of AAV2#14 and AAV2#9. In each study, NHPs administered with ophthalmic buffer were used as controls.

[0173] A. Exam 1 In the first test, NHPs were administered intravitreally with an intravitreal injection formulation buffer (N = 2 NHPs), or with AAV2#14 or AAV2#12 at three doses (based on vector titers determined using an assay that detects BGH poly(A), 2×10 9 、2×10 10 or 2×10 11 vg; N = 3 NHPs per treatment group). Animals were evaluated after 6 weeks of lifetime exposure.

[0174] For evaluation of vector transduction, the vector genome level was quantified by using a vector-specific TaqMan™ assay in quantitative PCR analysis of DNA purified from the NHP retina. TUBB1 was used as a reference gene to confirm equivalent DNA input across samples. The data show that both AAV2#12 and AAV2#14 successfully transduced the NHP retina, resulting in a dose-dependent increase in the vector genome level (dose-response AAV2#14 p = 0.0286, AAV2#12 p = 0.0095). Table 9 below summarizes the levels of transduction achieved in the NHP retina 6 weeks after intravitreal administration (median vector genome / 500 ng of genomic DNA).

[0175]

Table 13

[0176] To evaluate transgene expression, vector-derived transgene levels were quantified using a transcript-specific TaqMan® assay in quantitative RT-PCR analysis of RNA purified from NHP retina. RNA quality was assessed, and all samples were shown to have an RNA completeness number (RIN) greater than 7.5. One sample was not included in the RNA analysis due to low RNA input. Transcript levels were quantified by comparison with a double-stranded plasmid DNA standard curve. The data show that transduction of both AAV2#12 and AAV2#14 results in dose-dependent levels of transgene expression in NHP retina (dose-response AAV2#14 p=0.0286, AAV2#12 p=0.0286). Table 10 below summarizes the transcript abundance (median transcript / RNA 500ng) in NHP retina 6 weeks after intravitreous administration.

[0177] [Table 14]

[0178] B. Exam 2 In the second trial, NHP was administered via intravitreous injection ophthalmic buffer (N=2 NHP) or AAV2#14 or AAV2#9, with 2 × 10⁶ doses per eye. 11 NHPs were administered via vg (N=3 per vector treatment group). The vector titer was determined based on an assay detecting BGH poly(A). Animals were evaluated over 8 weeks of lifetime exposure. Due to the presence of serum AAV2 neutralizing antibody (Nab), all two NHPs in each study were administered IgG-degrading enzyme (IdeS) intravitreously 2 days prior to vector administration.

[0179] To evaluate vector transduction, vector genome levels were quantified using a vector-specific TaqMan® assay in quantitative PCR analysis of DNA purified from NHP retina. For AAV2#9, vector genome levels were assessed using two different assays detecting anti-Bb and anti-C1s arms. TUBB was used as the reference gene to confirm equivalent DNA input across samples. Despite potential impairment by existing AAV2 Nab, the data showed that both AAV2#14 and AAV2#9 successfully transduced NHP retina, with AAV2#14 showing approximately 9.3 × 10⁶ levels 8 weeks after intravitreous administration. 3 VG achieved, AAV2#9 is approximately 7.6×10 4 ~Approx. 2.8×10 5 This indicates that the vg level has been achieved (median of vector genome / 500ng genomic DNA).

[0180] To evaluate transgene expression, vector-derived transgene levels were quantified using a transcript-specific TaqMan® assay in quantitative RT-PCR analysis of RNA purified from NHP retina. In the case of AAV2#9, anti-Bb transcripts and anti-C1s transcripts were expressed independently and therefore evaluated separately. RNA quality was assessed, and all samples were shown to have an RNA completeness number (RIN) greater than 7.5. Transcript levels were quantified by comparison with a double-stranded plasmid DNA standard curve. After 8 weeks of lifetime exposure, AAV2#14 was approximately 9.7 × 10⁶. 4 This results in a transfer material abundance level of approximately 1.6 × 10⁻⁶ for AAV2#9. 6 Anti-Bb transfer and approximately 3.3 × 10 5 This resulted in anti-C1s transcripts (median transcripts per 500 ng of retinal RNA).

[0181] Durability testing in C.NHP The pharmacology and persistence of AAV2#9 across multiple dose levels were evaluated in a 16-week life assessment study including a 6-week interim autopsy.

[0182] NHPs (cynomolgus monkeys) were administered either by bilateral intravitreal injection of AAV2#9 at multiple dose levels using a formulation buffer (180 mM NaCl, 5 mM sodium phosphate, 0.01% PS20, pH 7.4) or by droplet digital PCR (ddPCR) analysis using a vector-specific assay targeting the anti-C1s region of AAV2#9 (based on vector titer determined by ddPCR). All NHPs received prophylactic steroids (1 mg / kg oral prednisolone daily) starting two weeks prior to vector administration and continued throughout the study period. Vector genome levels in the NHP retina were quantified using vector-specific C1s and Bb Taqman® assays in quantitative PCR analysis of purified DNA from the right eye.

[0183] C1s and Bb assays detected comparable vector genome levels within each sample at 6 and 16 weeks. At 6 weeks, AAV2#9 transduction resulted in a dose-dependent increase in vector genome levels in the retina. A dose-dependent increase in retinal transduction was also observed at 16 weeks.

[0184] The in vivo distribution of the vector in NHP eyes was evaluated using RNAscope® ISH analysis with an AAV2#9 vector-specific probe set (containing 40 pairs of probes, each approximately 50 base pairs long, designed to detect the sense strand of the vector genome). At weeks 6 and 16, the vector was detected in the retina and iris ciliary body of eyes administered with AAV2#9. The vector was not detected in the optic nerve. In the retina, the vector was found in rare cells of the RGC and INL, often in the foveal and parafoveal regions of the macula.

[0185] Levels of AAV2#9-derived anti-C1s and anti-Bb transcripts in the NHP retina were quantified using C1s and Bb-specific Taqman® assays in quantitative RT-PCR analysis of purified RNA from the right eye. Transcript levels were quantified by comparison with a double-stranded plasmid DNA standard curve. Transcript levels in both the 6-week and 16-week cohorts were highly correlated with vector genome levels (Spearman r ≥ 0.97). At 6 weeks, AAV2#9 transduction resulted in a dose-dependent increase in transcription levels in the retina. A dose-dependent trend in the increase in transcript levels was also observed at 16 weeks.

[0186] To evaluate the dynamics of peak scFab expression and persistence over time, aqueous humor was collected at baseline and at weeks 3, 6, 12, and 16. Vitreous fluid was collected at autopsy. In aqueous humor collected from several NHPs in the 16-week cohort, scFab levels peaked between weeks 3 and 6 and persisted until the end of the study at month 4 (day 113). At month 4, scFab levels in vitreous fluid were similar to or higher than those in aqueous humor.

[0187] Efficacy study to evaluate LPS-induced complement activation and AAV2#9 inhibition of ocular inflammation in D.NHP The ability of AAV2#9-derived scFab to inhibit complement pathway activation in vivo was evaluated using an acute model of endotoxin-induced inflammation.

[0188] NHPs (cynomolgus monkeys) were administered by bilateral intravitreal injection of either a formulation buffer (180 mM NaCl, 5 mM sodium phosphate, 0.01% PS20, pH 7.4) or AAV2#9, followed by bilateral intravitreal lipopolysaccharide (LPS) administration via [0.5 endotoxin units (EU) LPS per eye from Escherichia coli O111:B4; Sigma-Aldrich L4391] on day 41. NHPs were administered prophylactic steroids (1 mg / kg oral prednisolone daily) starting two weeks prior to vector administration and continuing daily for four weeks. NHPs were gradually reduced from prednisolone before LPS administration on day 41. The study endpoint was evaluated two days after LPS treatment (day 43), which induced high levels of ocular inflammation (Figure 9).

[0189] Free drug levels of AAV2#9-derived scFab in aqueous humor and vitreous fluid were measured using Bb and C1s targeted capture ELISA. At the end of the study on day 43, the mean levels of free anti-C1s scFab in aqueous humor and vitreous fluid were approximately 50–100 ng / mL (1–2 nM), and the median level of free anti-Bb scFab reached approximately 100–200 ng / mL (2–4 nM). The levels of anti-C1s scFab in both aqueous humor and vitreous fluid were above the equilibrium dissociation constant of anti-C1s scFab for both human and cynomolgus monkey C1s (human K D =0.34nM; Cryptomolgus macaque K D (=0.016nM). Anti-Bb scFab is human Bb(K D Compared to (K = 3.7 nM), it has a lower affinity for cynomolgus monkey Bb. D (=14.8nM), the level of anti-Bb scFab that reached the aqueous humor and vitreous fluid in this test was the K of anti-Bb scFab against cynomolgus monkey Bb. D The level was below the threshold and was not sufficient for Bb inhibition in NHP eyes.

[0190] To evaluate complement pathway activation, multiplex ELISA from Quidel was used to measure the levels of activation fragments of C4a (classical pathway), Ba (alternative pathway), and sC5b9 (terminal pathway) in aqueous humor. Compared to non-LPS-treated control eyes, LPS-treated eyes had elevated levels of Ba, C4a, and sC5b9 in aqueous humor, demonstrating activation of the alternative, classical, and terminal pathways. LPS-treated eyes administered with AAV2#9 had decreased levels of C4a and sC5b9 compared to LPS-treated control eyes, demonstrating inhibition of the classical and terminal pathways. Inhibition of the alternative pathway (Ba) was not detected in AAV2#9-treated eyes, probably due to the low affinity of the anti-Bb scFab for the cynomolgus target.

[0191] Eye examinations performed 2 days after LPS administration detected ocular inflammation in all treatment groups. However, eyes treated with AAV2#9 had reduced severity and frequency of clinical indicators of inflammation scored using the SPOTS system.

[0192] Sequence SEQ ID NO: 1 - HCDR1 of anti-C1s antibody DDYIH SEQ ID NO: 2 - HCDR2 of anti-C1s antibody RIDPADGHTK YAPKFQV SEQ ID NO: 3 - HCDR3 of anti-C1s antibody YGYGREVFDY SEQ ID NO: 4 - LCDR1 of anti-C1s antibody KASQSVDYDG DSYMN SEQ ID NO: 5 - LCDR2 of anti-C1s antibody DASNLES SEQ ID NO: 6 - LCDR3 of anti-C1s antibody QQSNEDPWT SEQ ID NO: 7 - V of anti-C1s antibody H (Underlined with Kabat CDR)

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Claims

1. A single expression construct comprising a first nucleotide sequence encoding an inhibitor of activated complement subcomponent C1s (C1s inhibitor) and a second nucleotide sequence encoding an inhibitor of complement factor Bb (Bb inhibitor); or A pair of expression constructs, one containing a first nucleotide sequence and the other containing a second nucleotide sequence.

2. The expression construct according to claim 1, wherein the C1s inhibitor and the Bb inhibitor are each antibody fragments, and optionally the antibody fragments are single-stranded Fv (scFv) or single-stranded Fab (scFab).

3. (a) The C1s inhibitor is Each of these may include SEQ ID NOs: 1 to 3, and heavy chain CDRs (HCDRs) 1 to 3 of SEQ ID NO: 7 Each of these is an anti-C1s antibody fragment containing the light chain CDR (LCDR) 1-3 of SEQ ID NO: 8, which may include SEQ ID NOs: 4-6, and / or (b) The Bb inhibitor is Each of the following may include HCDR1-3 of SEQ ID NO: 13-15, and The expression construct according to claim 2, which is an anti-Bb antibody containing LCDR1 to 3 of SEQ ID NO: 20, which may each contain SEQ ID NOs: 16 to 18.

4. (a) The C1s inhibitor is SEQ ID NO: 7 or a heavy chain variable domain (V) containing an amino acid sequence that is at least 95% identical thereto. Ｈ ), and A light chain variable domain (V) containing the amino acid sequence of Sequence ID No. 8 or at least 95% identical thereto. Ｌ ) including; and / or (b) The Bb inhibitor is Sequence ID 19 or V containing at least 95% identical amino acid sequence Ｈ , and Sequence ID 20 or V containing at least 95% identical amino acid sequence Ｌ The expression construct according to claim 3, including the expression construct according to claim 3.

5. (a) The C1s inhibitor A heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 10 or at least 95% identical thereto, and A light chain (LC) containing the amino acid sequence of SEQ ID NO: 11 or at least 95% identical thereto, and / or (b) The Bb inhibitor is HC containing the amino acid sequence of SEQ ID NO: 22 or at least 95% identical thereto, The expression construct according to claim 3 or 4, comprising an LC having at least 95% identical amino acid sequence to SEQ ID NO:

23.

6. The expression construct according to any one of claims 2 to 5, wherein the C1s inhibitor and the Bb inhibitor each include one or more charge mutations to promote pairing between the heavy chain and light chain of each inhibitor.

7. (a) The charge mutations in the C1s inhibitor include Q42E and Q292K, where the numbering follows sequence number 12, and (b) The expression construct according to claim 6, wherein the charge mutations in the Bb inhibitor include Q38K and Q288E, and optionally further include S114A, N137K and T434E, where the numbering follows Sequence ID No.

24.

8. The C1s inhibitor is an scFv or scFab in which the HC and LC are linked by a peptide linker, and optionally the peptide linker is one or more of 2, 3, 4, 5, 6, 7, 8, 9, or 10 G ４ An expression construct according to any one of claims 2 to 7, comprising an S (Sequence ID 46) repeat.

9. The Bb inhibitor is an scFv or scFab in which the HC and LC are linked by a peptide linker, and the peptide linker is one or more of 2, 3, 4, 5, 6, 7, 8, 9, or 10 G ４ An expression construct according to any one of claims 2 to 8, comprising an S (Sequence ID 46) repeat.

10. The transgene comprises a transgene encoding a fusion protein containing the C1s inhibitor and the Bb inhibitor linked by a peptide linker, Optionally, the peptide linker may contain one or more G molecules, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10. ４ Includes S (Sequence No. 46) repeat, The single expression construct according to any one of claims 1 to 9, wherein the introduced gene is optionally operably linked to a minimal chicken β-actin (minCBA) promoter.

11. The expression construct includes a bidirectional promoter that directs the expression of the C1s inhibitor and the Bb inhibitor as separate molecules. Optionally, the bidirectional promoter is a pair of chicken β-actin (CBA) promoters arranged in opposite directions and separated by a CMV enhancer. Furthermore, optionally, the bidirectional promoter comprises the nucleotide sequence of Sequence ID No. 53, or at least 85% identical thereto, in the single expression construct according to any one of claims 1 to 9.

12. The expression construct expresses (i) a fusion protein comprising a single-chain anti-C1s antibody fragment fused to the HC or LC of the anti-Bb antibody fragment; and (ii) a dimer comprising the LC or HC polypeptide of the anti-Bb antibody fragment, wherein the coding sequence of the fusion protein and the coding sequence of the LC or HC polypeptide of the anti-Bb antibody fragment are separated in frame by a cleavable peptide coding sequence. Optionally, the cleavable peptide includes the 2A sequence and / or the fulin cleavage site. Furthermore, optionally, the expression construct comprises a minCBA promoter, as described in any one of claims 2 to 9.

13. The expression construct expresses (i) a fusion protein comprising a single-chain anti-Bb antibody fragment fused to the HC or LC of the anti-C1s antibody fragment; and (ii) a dimer comprising the LC or HC polypeptide of the anti-C1s antibody fragment, wherein the coding sequence of the fusion protein and the coding sequence of the LC or HC polypeptide of the anti-C1s antibody fragment are separated in frame by a cleavable peptide coding sequence. Optionally, the cleavable peptide includes the 2A sequence and / or the fulin cleavage site. Furthermore, optionally, the expression construct comprises a minCBA promoter, as described in any one of claims 2 to 9.

14. It codes for a fusion protein, from the N-terminus to the C-terminus, (i) An anti-C1s scFv, (G ４ S) ２ linker, and an anti-Bb scFv, comprising an amino acid sequence that is optionally SEQ ID NO: 55 (with or without a signal peptide) or at least 95% identical thereto; (ii) Optionally containing SEQ ID NO: 57 (with or without signal peptide) or an amino acid sequence at least 95% identical thereto, anti-Bb scFv, (G ４ S) ２ Linker and anti-C1s scFv; (iii) Anti-C1s scFab, (G) optionally containing SEQ ID NO: 26 or 28 (with or without signal peptide) or an amino acid sequence at least 95% identical thereto. ４ S) ３ Linker, and anti-Bb scFab; (iv) Anti-Bb scFab, (G) optionally containing SEQ ID NO: 30 or 32 (with or without signal peptide) or an amino acid sequence at least 95% identical thereto. ４ S) ３ Linker, and anti-C1s scFab; (v) Optionally containing SEQ ID NO: 34 or 36 (with or without signal peptide) or an amino acid sequence at least 95% identical thereto, anti-C1s scFab, (G ４ S) ２ Linker, and anti-Bb scFv; or (vi) Anti-C1s scFab, (G) optionally containing SEQ ID NO: 59 or 61 (with or without signal peptide) or an amino acid sequence at least 95% identical thereto. ４ S) ３ A linker and an anti-Bb scFv are included. A single-expression construct according to any one of claims 2 to 10.

15. Anti-C1s scFab, which optionally includes an amino acid sequence identical to or at least 95% of SEQ ID NO: 12, and optionally includes Q42E and Q292K mutations in the amino acid sequence compared to SEQ ID NO: 12; and Anti-Bb scFab optionally contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 14, and optionally contains mutations in the amino acid sequence of Q38K and Q288E, and optionally S114A, N137K and T434E, compared to SEQ ID NO:

14. An expression construct according to any one of claims 2 to 9 and 11, which codes for

16. (A) comprising a fusion protein comprising (i) anti-C1s LC and (ii) anti-C1s HC fused to αBb scFab, wherein the expression construct optionally contains the coding sequence of SEQ ID NO: 39, or an amino acid sequence that is at least 95% identical thereto; (B) A fusion protein comprising (i) anti-C1s LC and (ii) anti-C1s HC fused to anti-Bb scFab, wherein the expression construct optionally contains the coding sequence of SEQ ID NO: 41 or an amino acid sequence that is at least 95% identical thereto; (C) (i) a fusion protein comprising anti-C1s scFab fused to anti-Bb HC and (ii) anti-Bb LC, wherein the expression construct optionally contains the coding sequence of SEQ ID NO: 43, or an amino acid sequence that is at least 95% identical thereto; or (D) A single expression construct according to any one of claims 2 to 9, 12, and 13, comprising (i) a fusion protein containing anti-C1s scFab fused to anti-Bb HC and (ii) anti-Bb LC, wherein optionally the expression construct encodes a heterodimer containing the coding sequence of SEQ ID NO: 45 or an amino acid sequence identical thereto by at least 95%.

17. An isolated nucleic acid containing a nucleotide sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 54, 56, 58, 60, 62, 79, or 80, or encoding the same amino acid sequence as the selected nucleotide sequence.

18. One, two, or more recombinant adeno-associated viruses (rAAVs) comprising the expression construct according to any one of claims 1 to 16 or the isolated nucleic acid according to claim 17.

19. The rAAV according to claim 18, wherein the genome of the rAAV comprises the expression construct adjacent to the AAV2 reverse terminal repeat (ITR).

20. The rAAV according to claim 19, wherein the genome includes sequence number 50, 51, or 52, or codes for the same amino acid sequence as sequence number 50, 51, or 52.

21. The rAAV according to any one of claims 18 to 20, comprising AAV2, and optionally a capsid of wild-type AAV2.

22. A pharmaceutical composition comprising the rAAV according to any one of claims 18 to 21 and a pharmaceutically acceptable carrier.

23. One or more proteins encoded by the expression construct or rAAV according to any one of claims 1 to 21.

24. A host cell comprising the expression construct, the isolated nucleic acid, or rAAV according to any one of claims 1 to 21.

25. A method for treating atrophic age-related macular degeneration (AMD) in a patient requiring treatment for AMD, comprising administering an effective amount of rAAV according to any one of claims 18 to 21 or the pharmaceutical composition according to claim 22.

26. The method according to claim 25, wherein the administration is performed by intravitreous injection.

27. The method according to claim 25 or 26, wherein the patient has geographic atrophy (GA) secondary to atrophic AMD.

28. The effective amount is 10 ７ ~10 １５ , arbitrarily 10 ８ ~10 １４ , 10 ９ ~10 １３ Furthermore, arbitrarily 2 × 10 ９ , 2 x 10 １０ or 2 x 10 １１ The method according to any one of claims 25 to 27, wherein the vector genome is...

29. A recombinant AAV according to any one of claims 25 to 28, or a pharmaceutical composition according to claim 22, for use in the treatment of atrophic age-related macular degeneration (AMD) in patients requiring treatment for AMD.

30. The method according to any one of claims 25 to 28, wherein the recombinant AAV according to any one of claims 18 to 21 or the pharmaceutical composition according to claim 22 is used to produce a pharmaceutical for treating atrophic age-related macular degeneration (AMD) in a patient requiring treatment for AMD.

31. A mammalian promoter containing sequence number 83 or a sequence that is at least 85% identical thereto.

32. A bidirectional mammalian promoter comprising a pair of chicken β-actin promoters separated by a CMV enhancer and arranged in opposite directions, which may include sequence number 53 or a sequence that is at least 85% identical thereto.

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