C5 / VEGF bispecific binding molecules

Abstract

The present disclosure provides bispecific binding molecules, including protein sequences, against human vascular endothelial growth factor (VEGF / VEGF-A) and against human complement 5 (C5), methods for their production, pharmaceutical compositions containing the bispecific binding molecules, and uses thereof.

Description

Technical Field

[0001] The present invention relates to a bispecific binding molecule targeting human vascular endothelial growth factor (VEGF / VEGF-A) and complement component 5 (C5), and a preparation method and therapeutic use thereof. Background Art

[0002] Age-related macular degeneration (AMD) is the main cause of blindness in the elderly (Rein et al., Arch Ophthalmology, 127:533-540, 2009). AMD is a typical disease of the elderly and the main cause of blindness in people over 50 years old in developed countries. In the absence of adequate preventive or therapeutic measures, it is expected that the number of AMD cases with vision loss will increase as the population ages.

[0003] The feature of AMD is that the photoreceptors at macula, outer retina and retinal pigment epithelium degenerate gradually. Late stage AMD occurs with two forms: dry (atrophic) and wet AMD. Anti-VEGF agents (such as anti-VEGF antibodies, VEGF training etc.) are widely used in treating wet AMD by suppressing new blood vessels and angiogenesis now. For most patients, this can reduce the progress of choroidal neovascularization and the vision loss caused by the downstream effect of new blood vessels, and has observed that it can reduce the inflammation of some aspects in animal models.

[0004] Late-stage AMD is characterized by choroidal neovascularization or geographic atrophy (GA). GA tends to affect more than 20% of AMD patients. Globally, more than 8 million patients are affected by GA or have at least one eye diagnosed with GA in AMD patients. Currently, there is no effective treatment to treat or even slow the progression of GA symptoms, such as missing some letters or losing partial vision when reading, needing additional light to read or perform normal activities in the dark, and losing visual resolution that requires detailed vision (facial recognition and visual color fading due to the death of retinal pigment epithelial (RPE) cells in the central visual field).

[0005] Anti-VEGF therapy is ineffective for treating GA (Park, DH et al., Front. Immunol., May 15, 2019). There are even reports that anti-VEGF treatment may increase the development of GA (Gemenetzi, M., et al., Eye (Lond). 2017 Jan;31(1):1-9). Complement inhibitors targeting the complement proteins C3, C5, factor B, factor D, and properdin are being studied for their potential to treat GA, but have not yet been successful. In a phase II clinical trial (COMPLETE, NCT00935883), the anti-C5 antibody eculizumab failed to significantly reduce the growth rate of GA.

[0006] Therefore, there is a great clinical need for the treatment of AMD and GA, especially to prevent the progression of AMD to geographic atrophy (GA). Summary of the Invention

[0007] The present disclosure provides multispecific molecules capable of inhibiting VEGF and / or C5. In some embodiments, the multispecific molecules are designed to inhibit C5-related and / or VEGF-related diseases. Also disclosed are methods for producing multispecific molecules, including processes involving nucleic acids, vectors, expression vectors, and host-vector systems.

[0008] In one aspect, the present disclosure provides a multispecific molecule comprising a fusion protein comprising: (a) a complement component 5 (C5) binding domain, (b) a vascular endothelial growth factor (VEGF) binding domain, and (c) a multimerization component; wherein: the C5 binding domain comprises an antigen-binding fragment of an anti-C5 antibody, the VEGF binding domain comprises one or more extracellular immunoglobulin-like (Ig) domains of one or more VEGF receptors (VEGFRs), and the multimerization component comprises a polypeptide between 1 and 200 amino acids in length and having at least one cysteine residue.

[0009] In another aspect, the present disclosure provides a pharmaceutical composition comprising a multispecific molecule provided herein, and one or more pharmaceutically acceptable carriers.

[0010] In another aspect, the disclosure provides an isolated polynucleotide encoding a multispecific molecule provided herein.

[0011] In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide provided herein.

[0012] In another aspect, the present disclosure provides a host-expression system comprising a vector provided herein.

[0013] In another aspect, the disclosure provides a method of expressing a multispecific molecule provided herein, the method comprising culturing a host expression system provided herein under conditions for expressing a vector provided herein.

[0014] In another aspect, the present disclosure provides a method of treating, preventing, or ameliorating a C5-related and / or VEGF-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein and / or a pharmaceutical composition provided herein.

[0015] In another aspect, the present disclosure provides a method for treating, preventing, or ameliorating a disease, disorder, or condition associated with increased levels and / or activity of C5 and / or VEGF in a subject, the method comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein and / or a pharmaceutical composition provided herein.

[0016] In another aspect, the disease, disorder or condition is selected from the group consisting of an ocular disease, cancer, an inflammatory disease, an autoimmune disease, angiogenesis, vascular permeability, edema, and inflammation.

[0017] In another aspect, the disclosure provides a method of modulating C5 and / or VEGF activity in a cell, the method comprising exposing the cell to a multispecific molecule provided herein.

[0018] In another aspect, the disclosure provides a multispecific molecule provided herein and / or a pharmaceutical composition provided herein for use in treating, preventing, or ameliorating a C5-related and / or VEGF-related disease, disorder, or condition in a subject.

[0019] In another aspect, the disclosure provides use of a multispecific molecule provided herein and / or a pharmaceutical composition provided herein in the manufacture of a medicament for treating, preventing, or ameliorating a C5-related and / or VEGF-related disease, disorder, or condition in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The first structure of the VEGF / C5 bispecific antibody (BSP1, BSP1a) is depicted.

[0021] Figure 2 The second structure of the VEGF / C5 bispecific antibody (BSP2) is depicted.

[0022] Figure 3 The third structure of the VEGF / C5 bispecific antibody (BSP3) is depicted.

[0023] Figure 4 The fourth structure of the VEGF / C5 bispecific antibody (BSP4) is depicted.

[0024] Figure 5 The fifth structure of the VEGF / C5 bispecific antibody (BSP5) is depicted.

[0025] Figure 6 The sixth structure of the VEGF / C5 bispecific antibody (BSP6) is depicted.

[0026] Figure 7 and 8 Shown are dose-response curves for BSP1 and PC1 (Aflibercept) in replicates of a VEGF-mediated cell proliferation assay.

[0027] Figure 9A and 9B Shown are the dose-response curves for BSP1 and PC2 (eculizumab) in replicates of the CH50 assay.

[0028] Figure 10 Shown are the changes in vascular leakage scores and their standard deviations over time in chronic animal / PD models.

[0029] Figure 11 Shown is the progression of vascular leakage over time (0, 2, 4, and 8 weeks) in a chronic animal / PD model.

[0030] Figure 12 Shown is the progression of vascular leakage over time (0, 12, and 16 weeks) in a chronic animal / PD model.

[0031] Figure 13 The effects of BSP1 over time (0, 2, 4, 8, 12, and 16 weeks) are shown compared to untreated eyes.

[0032] Figure 14 The effects of PBS over time (0, 2, 4, 8, 12, and 16 weeks) are shown compared to untreated eyes.

[0033] Figure 15 The SEC-HPLC chromatogram of a sample (BSP1) in the monoclonal drug stability test at 40°C is shown.

[0034] Figure 16 The CE-SDS-NR electrophoresis pattern of sample / BSP1 at 40°C in the monoclonal drug stability test is shown.

[0035] Figure 17 The icIEF electropherogram of sample / BSP1 at 40°C is shown in the stability test of the monoclonal drug.

[0036] Figure 18 The SEC-HPLC electropherogram of sample / BSP1 at 5°C in the monoclonal drug stability test is shown.

[0037] Figure 19 The CE-SDS-NR electrophoresis pattern of samples / BSP1 at 5°C in the monoclonal drug stability test is shown.

[0038] Figure 20 The icIEF electropherogram of sample / BSP1 at 5°C in the monoclonal drug stability test is shown.

[0039] Figure 21 Certain sequences disclosed in this disclosure are shown. DETAILED DESCRIPTION

[0040] Before providing a detailed description of the present invention, the following are noted and defined.

[0041] All descriptions provided herein are intended only to illustrate the various embodiments of the present invention provided in this disclosure. Thus, the specific modifications discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes and modifications may be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments should be included herein.

[0042] All references cited in this disclosure, including patent applications, issued patents, published articles, or other publications, are hereby incorporated by reference in their entirety for the purpose of providing methods that can be used in conjunction with the description provided herein. For any terms that appear in one or more publications that are similar or identical to terms explicitly defined in this disclosure, the definitions of those terms explicitly provided in this disclosure shall prevail in all respects.

[0043] Unless explicitly defined otherwise, all technical and scientific terms used in the present disclosure are generally understood to have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0044] As used herein, i.e., throughout the disclosure, unless otherwise indicated, the articles "a," "an," and "the" are to be interpreted as meaning "one or more" or "at least one." For example, "molecule" means one molecule or more than one molecule.

[0045] As used herein, the terms "about," "approximately," "around," and the like refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In specific embodiments, the term "about" or "approximately" when preceding a numerical value indicates a range of plus or minus 15%, 10%, 5% or 1% of the value.

[0046] As used herein, the terms "comprise," "comprises," "comprising," "include," "includes," "including," "contain," "contains," "containing," "have," "has," "having," etc. are synonymous and are used in an inclusive and open-ended manner and do not exclude other elements, features, steps, actions, operations, etc.

[0047] As used herein, the term "or" is used in its inclusive sense (and not in its exclusive sense), so that, for example, when used to link a list of elements, the term "or" means one, some, or all of the elements in the list.

[0048] As used herein, the phrase "at least one" means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0049] I. Definitions and Abbreviations

[0050] In this section, definitions of some general terms are provided. Definitions of other terms can be found in other sections of this disclosure that follow.

[0051] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a linear series of amino acid residues interconnected by peptide bonds, including proteins, polypeptides, oligopeptides, peptides, and fragments thereof. Proteins can be composed of naturally occurring amino acids and / or synthetic (e.g., modified or non-naturally occurring) amino acids. Thus, as used herein, "amino acid" or "peptide residue" means both naturally occurring and synthetic amino acids. The terms "polypeptide," "peptide," and "protein" include fusion proteins, including but not limited to, fusion proteins having heterologous amino acid sequences; fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue; immunolabeled proteins; fusion proteins with detectable fusion partners, for example, fusion proteins comprising fluorescent proteins, β-galactosidase, luciferase, etc. as fusion partners; and the like.

[0052] As used herein, the term "amino acid" refers to the building blocks of proteins, peptides, polypeptides, or amino acid polymers, and the term "amino acid" further refers to naturally occurring or synthetic amino acids, as well as any amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. As used in this application, naturally occurring amino acids include the group of naturally occurring carboxy α-amino acids, including alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0053] As used herein, the term "domain" refers to a globular structure formed by one or more regions of one or more polypeptide chains including peptide loops (e.g., including 3 to 4 peptide loops), which are stabilized, for example, by β-pleated sheets and / or intrachain disulfide bonds. Examples may include Fab domains (see below for more details). Note that in this disclosure, the two terms "domain" and "region" can be used interchangeably.

[0054] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," "polynucleotide," and the like are to be interpreted as referring to nucleotide polymers of any length, and may include DNA and RNA, and may be single-stranded or double-stranded.

[0055] As used herein, the term "percentage (%) of sequence identity" is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues are not considered identical residues. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the US National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology). Enzymology), 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software implementation. Those skilled in the art can use the default parameters provided by the tool or can customize the parameters according to the needs of the comparison, for example by selecting a suitable algorithm.

[0056] "Conservative substitution" with respect to an amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.

[0057] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. As used herein, the term antibody is broadly interpreted to cover conventional immunoglobulins comprising two heavy (H) chains and two light (L) chains, as well as unconventional antibodies comprising only heavy chains, such as heavy chain antibodies. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each of which consists of a variable region (V H ) and the first constant region, the second constant region, the third constant region, and optionally the fourth constant region (respectively C H1 、C H2 、C H3 、C H4 ); mammalian light chains are classified as either λ or κ, and each light chain consists of a variable region (V L) and constant regions. Antibodies are "Y" shaped, where the stem of the Y consists of the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains typically contain three highly variable loops, called complementarity determining regions (CDRs) (light chain CDRs, including LCDR1, LCDR2 and LCDR3, heavy chain CDRs, including HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, IMGT, Chothia, or Al-Lazikani rules (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol. 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest. Interest), 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514, (2015). The three CDRs are interposed between flanking extensions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit a variety of effector functions. Antibodies are divided into multiple classes based on the amino acid sequence of their heavy chain constant regions.The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0058] As used herein, "antigen" refers to a compound, composition, peptide, polypeptide, protein or substance (e.g., a polypeptide, carbohydrate, nucleic acid, lipid or other naturally occurring or synthetic compound) that can be specifically recognized and bound by components of the immune system (e.g., antibodies). As used herein, the term "antigen" encompasses antigenic epitopes, such as antigenic fragments that are antigenic epitopes.

[0059] As used herein, the term "bivalent" refers to an antibody or antigen-binding fragment that has two antigen-binding sites; the term "monovalent" refers to an antibody or antigen-binding fragment that has only a single antigen-binding site; and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. In some embodiments, the antibody or antigen-binding fragment thereof is bivalent.

[0060] As used herein, the term "multispecific molecule" refers to an artificial or engineered molecule that can bind to at least two different epitopes simultaneously. The two epitopes can be present on the same antigen, or on two different antigens. Bispecific molecules are essentially a type of multispecific molecule.

[0061] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain a complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (divalent diabodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen as the parent antibody.

[0062] "Fab" with respect to antibodies refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by disulfide bonds.

[0063] "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0064] "F(ab')2" refers to a dimer of Fab'.

[0065] "Fv" in the context of antibodies refers to the smallest antibody fragment that carries a complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0066] "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable region of a single light chain is connected to the variable region of a single heavy chain by a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains: one connected by a peptide linker (e.g., a long flexible linker) and each linked to two V chains via a disulfide bridge. L Partially combined two V H In some embodiments, the dsFv-dsFv' is bispecific, wherein each pair of disulfide-paired heavy and light chains has a different antigenic specificity.

[0067] A "single-chain Fv antibody" or "scFv" refers to a multispecific molecule composed of a light chain variable region and a heavy chain variable region interconnected directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0068] "Fc" with respect to antibodies (e.g., antibodies of the IgG, IgA or IgD isotype) refers to the portion of the antibody that is essentially composed of the second constant domain and the third constant domain of the first heavy chain bound to the second constant domain and the third constant domain of the second heavy chain via a disulfide bond. The Fc with respect to antibodies of the IgM and IgE isotypes further includes a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0069] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to a multispecific molecule composed of a scFv linked to the Fc region of an antibody.

[0070] A "camelized single domain antibody," "heavy chain antibody," or "HCAb" is a HHeavy chain antibodies are antibodies that contain a heavy chain domain and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies originally came from the Camelidae family (camels, dromedaries, and llamas). Despite the absence of light chains, camelized antibodies have a realistic antigen binding repertoire (Hamers-Casterman C. et al., Nature, June 3; 363(6428): 446-8 (1993); Nguyen VK. et al., Immunogenetics. April; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0071] "Nanobody" refers to an antibody fragment consisting of the VHH domain from a heavy chain antibody and the two constant domains CH2 and CH3.

[0072] "Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, wherein the fragments comprise V and V in the same polypeptide chain. H Domain and V L Domain connection (V H -V L or V L -V H(See, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul 15;90(14):6444-8 (1993); EP 404097; WO 93 / 11161). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen binding sites. The antigen binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes). In certain embodiments, a "scFv dimer" is a bivalent bifunctional antibody or bivalent scFv (BsFv) comprising V H -V L (connected by a peptide linker) to another V H -V L Partial dimerization makes a part of V H With another part of the V L Coordinate and form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody comprising V H1 -V L2 (linked via a peptide linker) to V L1 -V H2 (also linked by a peptide linker) are associated so that V H1 and V L1 Coordinate and make V H2 and V L2 Each ligand pair has a different antigen specificity.

[0073] "Domain antibodies" refer to antibody fragments that contain only the variable region of a heavy chain or a light chain. In some cases, two or more VH domains are covalently joined by a peptide linker to produce a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0074] As used herein, the term "vector" refers to a vehicle into which genetic elements can be operably inserted to achieve expression of the genetic elements, thereby producing the protein, RNA or DNA encoded by the genetic elements, or replicating the genetic elements. A vector can be used to transform, transduce or transfect a host cell to achieve expression of the genetic elements it carries within the host cell. Examples of vectors include plasmids; phagemids; cosmids; and artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage; and animal viruses. A vector can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements and reporter genes. In addition, a vector can contain an origin of replication. A vector can also include materials that facilitate its entry into cells, including (but not limited to) viral particles, liposomes or protein coatings. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof as provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0075] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0076] The phrase "operably linked" refers to a juxtaposition so that the normal functions of the components can be performed.

[0077] II. C5 / VEGFR Multispecific Molecules

[0078] In one aspect, the present disclosure provides a multispecific molecule comprising a fusion protein comprising: (a) a complement component 5 (C5) binding domain, (b) a vascular endothelial growth factor (VEGF) binding domain, and (c) a multimerization component. In certain embodiments, the multispecific molecule is bispecific.

[0079] As used herein, a fusion protein can be a single polypeptide chain or a polypeptide complex comprising two or more polypeptide chains associated together.

[0080] A. Antigen-binding fragment

[0081] In certain embodiments, the fusion protein comprises a complement component 5 (C5) binding domain, wherein the C5 binding domain comprises an antigen-binding fragment of an anti-C5 antibody.

[0082] C5 is a component of the complement system, which is part of the innate immune system and plays an important role in inflammation, host homeostasis, and host defense against pathogens. The C5 protein consists of a C5 α chain and a C5 β chain connected by a disulfide bridge. The C5 protein can be proteolytically processed to produce a variety of protein products, including the C5 α chain, the C5 β chain, and the anaphylatoxins C5a and C5b.

[0083] An "anti-C5 antibody" is an antibody that can specifically bind to C5 (e.g., human C5). The anti-C5 antibody can be a conventional IgG antibody, or alternatively can be a single domain antibody, such as a camelized single domain antibody or a llama anti-C5 single domain antibody comprising a heavy chain variable (VH) region.

[0084] In certain embodiments, the anti-C5 antibody comprises or is derived from eculizumab.

[0085] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises one or more complementarity determining regions (CDRs) contained in the heavy chain variable region and the light chain variable region of eculizumab. In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises: three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable (VH) region, wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 1, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained in a light chain variable (VL) region, wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0086] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises the six CDRs of eculizumab. In certain embodiments, the antigen-binding fragment of an anti-C5 antibody provided herein comprises: a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0087] Table 1A. Eculizumab sequences

[0088]

[0089]

[0090] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody is derived from or comprises a variant of eculizumab, such as, but not limited to, an affinity variant of eculizumab or a glycosylation variant of eculizumab.

[0091] The term "variant" with respect to a parent antibody refers to any antibody having a structure or sequence derived from a parent antibody and having a structure / sequence sufficiently similar to that in the parent antibody. Modifications for obtaining a "variant" include, for example, additions, deletions, and / or substitutions of one or more amino acid residues. The variant may have one or more conservative amino acid substitutions.

[0092] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody is derived from an eculizumab variant and comprises one or more amino acid residue substitutions or modifications relative to eculizumab, while retaining binding specificity and / or affinity for C5. The substitutions or modifications may be in one or more CDR sequences and / or VH and / or VL sequences of eculizumab.

[0093] In certain embodiments, the antigen-binding fragments of the anti-C5 antibodies provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences of eculizumab. In certain embodiments, the antigen-binding fragments of the anti-C5 antibodies provided herein comprise no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in a total of the CDR sequences and / or FR sequences listed in Table 1A.

[0094] In certain embodiments, variants of eculizumab, or antigen-binding fragments derived from such variants, have comparable or improved affinity for human C5 relative to the parent antibody, eculizumab.

[0095] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an antibody or its fragment and an antigen. The affinity of an antibody for an antigen can be measured using methods well known in the art using the equilibrium dissociation constant, K DTo measure (see generally Davies et al., Ann. Rev. Biochem. 1990, 59:439-15473). For example, Biacore is a classic device for detecting molecular interactions based on surface plasmon resonance (SPR) technology and is the gold standard for antibody affinity determination. It is well known in the art that enzyme-linked immunosorbent assay (ELISA) is also a common means of measuring binding affinity.

[0096] In certain embodiments, the eculizumab variants or antigen-binding fragments derived from such variants are capable of binding to an antigen-binding protein of no more than 10 nM (e.g., no more than 8 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 800 pM, no more than 700 pM, no more than 600 pM, no more than 500 pM, no more than 400 pM, or no more than 300 pM) as measured by Biacore. D The value specifically binds to C5.

[0097] Methods for preparing and obtaining affinity variants of parent antibodies are known in the art. For example, variants can be screened for their binding affinity to a desired target (e.g., human C5), and variants with high affinity for the antigen can be identified.

[0098] In some embodiments, the eculizumab variants of the present invention or fusion proteins derived from such variants have improved druggability properties, for example, when expressed in mammalian cells such as CHO cells, have one or more properties selected from the following: (i) better expression than wild-type eculizumab or its fusion protein, (ii) greater feasibility of purification to high purity, and (iii) greater stability.

[0099] In some embodiments of the present invention, eculizumab variant or its fusion protein shows increased expression level compared with wild-type eculizumab or its fusion protein. In some embodiments, increased expression occurs in a mammalian cell expression system. Expression level can be determined by any suitable method, which allows quantitative or semi-quantitative analysis of the amount of eculizumab variant or its fusion protein in the cell culture supernatant, preferably after one-step affinity chromatography purification. For example, the amount of eculizumab variant or its fusion protein in the sample can be assessed by Western blotting or ELISA. In some embodiments, the expression level of eculizumab variant or its fusion protein in mammalian cells increases by at least 1.1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times or at least 30 times compared with wild-type eculizumab or its fusion protein.

[0100] In some embodiments, the eculizumab variants or fusion proteins thereof of the present invention exhibit greater purity relative to wild-type eculizumab or its fusion protein. In some embodiments, protein purity is determined by SEC-HPLC, CE-SDS-NR, or icIEF techniques. In some preferred embodiments, the eculizumab variants or fusion proteins thereof, after purification, can achieve a purity greater than 65%, 70%, 75%, 80%, or 85%, preferably greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 99%.

[0101] In one embodiment, the eculizumab variant or its fusion protein has improved thermal stability in the temperature range of -20°C to 60°C. In one embodiment, the eculizumab variant or its fusion protein has no significant aggregation and degradation in the temperature range of -20°C to 60°C. In one embodiment, the eculizumab variant or its fusion protein has no significant aggregation and degradation at 5°C. In one embodiment, the eculizumab variant or its fusion protein has no significant aggregation and degradation at 40°C. In one embodiment, the degree of aggregation or degradation of the eculizumab variant or its fusion protein after storage at 5°C for 14 days is less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1%.

[0102] In certain embodiments, the eculizumab variants provided herein comprise a HCDR1 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO:3, a HCDR2 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO:4, a HCDR3 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO:5, a LCDR1 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO:6, a LCDR2 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO:7, and / or a LCDR3 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO:8.

[0103] In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises: a HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 29 or SEQ ID NO: 41 or SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 30 or SEQ ID NO: 35.

[0104] In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises: a HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 30.

[0105] In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises: a HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 41, a LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 35.

[0106] In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises: a HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 30.

[0107] Table 1B. Sequences of eculizumab variants

[0108]

[0109]

[0110]

[0111] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 1 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 1 but still retains binding specificity for C5.

[0112] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VL region comprising the amino acid sequence shown in SEQ ID NO: 2 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 2 but still retains binding specificity for C5.

[0113] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 28 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 28 but still retains binding specificity for C5.

[0114] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VL region comprising the amino acid sequence as shown in SEQ ID NO: 31 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 31 but still retains binding specificity for C5.

[0115] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VL region comprising the amino acid sequence shown in SEQ ID NO: 36 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 36 but still retains binding specificity for C5.

[0116] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VL region comprising the amino acid sequence shown in SEQ ID NO: 39 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 39 but still retains binding specificity for C5.

[0117] As used herein, the terms "homologous," "substantially homologous," and "substantial homology" refer to amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical when compared to a reference amino acid sequence. The percentage of sequence identity or homology is calculated by comparing corresponding portions of the reference sequence to each other when aligned.

[0118] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 1, and a VL region comprising the amino acid sequence shown in SEQ ID NO: 2.

[0119] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VH region comprising the amino acid sequence shown in SEQ ID NO:28, and a VL region comprising the amino acid sequence shown in SEQ ID NO:31.

[0120] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VH region comprising the amino acid sequence shown in SEQ ID NO:28, and a VL region comprising the amino acid sequence shown in SEQ ID NO:36.

[0121] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 1, and a VL region comprising the amino acid sequence shown in SEQ ID NO: 39.

[0122] Without wishing to be bound by any theory, it has been found that certain variants of eculizumab achieve unexpected effects, such as in terms of stability and purification, which make them advantageous for manufacturing, particularly scale-up manufacturing and process development. For example, eculizumab mutant #1 as provided herein was found to be more stable than eculizumab, had higher purity in stability testing (see Example 4 for details), and had higher protein production yields in recombinant expression (see Example 4 for details).

[0123] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a Fab domain. In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising a VH domain and a light chain polypeptide comprising a VL domain, wherein the VH domain and the VL domain associate to form a C5 binding domain.

[0124] In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising a VH region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain constant region 1 (CH1). In certain embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the Fab domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21.

[0125] In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising a VH region comprising the amino acid sequence set forth in SEQ ID NO: 28 and a heavy chain constant region 1 (CH1). In certain embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the Fab domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32.

[0126] In certain embodiments, the Fab domain comprises a light chain polypeptide comprising a VL region comprising the amino acid sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL). In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0127] In certain embodiments, the Fab domain comprises a light chain polypeptide comprising a VL region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain constant region (CL). In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 33.

[0128] In certain embodiments, the Fab domain comprises a light chain polypeptide comprising a VL region comprising the amino acid sequence set forth in SEQ ID NO: 36 and a light chain constant region (CL). In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0129] In certain embodiments, the Fab domain comprises a light chain polypeptide comprising a VL region comprising the amino acid sequence set forth in SEQ ID NO: 39 and a light chain constant region (CL). In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0130] In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 21 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17, wherein the heavy chain and the light chain associate to form a C5 binding domain.

[0131] In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain and the light chain associate to form a C5 binding domain.

[0132] In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 37, wherein the heavy chain and the light chain associate to form a C5 binding domain.

[0133] In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:21 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:40, wherein the heavy chain and the light chain associate to form a C5 binding domain.

[0134] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a single-chain Fab. In certain embodiments, the single-chain Fab comprises a heavy chain polypeptide as provided herein and a light chain polypeptide as provided herein, operably linked via a linker. In certain embodiments, the single-chain Fab comprises: a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 21 operably linked to a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17, wherein the heavy chain polypeptide and the light chain polypeptide associate to form a C5 binding domain.

[0135] In certain embodiments, the single-chain Fab comprises: a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 operably linked to a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain polypeptide and the light chain polypeptide associate to form a C5 binding domain.

[0136] In certain embodiments, the single-chain Fab comprises: a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 operably linked to a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 37, wherein the heavy chain polypeptide and the light chain polypeptide associate to form a C5 binding domain.

[0137] In certain embodiments, the single-chain Fab comprises: a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 21 operably linked to a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 40, wherein the heavy chain polypeptide and the light chain polypeptide associate to form a C5 binding domain.

[0138] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a single-chain Fv (scFv) domain. In certain embodiments, the scFv domain comprises: a VH region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 2, operably linked via a linker.

[0139] In certain embodiments, the scFv domain comprises: a VH region comprising the amino acid sequence as shown in SEQ ID NO: 28 and a VL region comprising the amino acid sequence as shown in SEQ ID NO: 31, which are operably linked via a linker.

[0140] In certain embodiments, the scFv domain comprises: a VH region comprising the amino acid sequence as shown in SEQ ID NO: 28 and a VL region comprising the amino acid sequence as shown in SEQ ID NO: 36, which are operably linked via a linker.

[0141] In certain embodiments, the scFv domain comprises: a VH region comprising the amino acid sequence as shown in SEQ ID NO: 1 and a VL region comprising the amino acid sequence as shown in SEQ ID NO: 39, which are operably linked via a linker.

[0142] In certain embodiments, the VH region is operably linked to the N-terminus of the VL region. In certain embodiments, the VL region is operably linked to the N-terminus of the VH region.

[0143] In certain embodiments, the C5 binding domain comprises a VHH domain.

[0144] B.VEGFR

[0145] In certain embodiments, the fusion protein in the multispecific molecules provided herein comprises a vascular endothelial growth factor (VEGF) binding domain.

[0146] VEGF is an important pro-angiogenic factor that regulates endothelial cell proliferation, permeability and survival with high potency and specificity (Folkman et al., Science 1987, 235:442; Giampietro et al., Cancer Metastasis Rev. 1994; Ferrara, Endocrine Rev. 2004, 25(4):581-611).

[0147] Three VEGF receptors have been identified, including VEGFR-1 (fms-like tyrosine kinase, Flt-1), VEGFR-2 (fetal liver kinase 1-murine homolog / kinase insert domain-containing receptor-human homolog, KDR / Flk-1), and VEGFR-3 (Flt-4). VEGFR-1 and VEGFR-2 are primarily expressed on endothelial cells. VEGFR-3 is primarily expressed on lymphatic vessels and neuropilin, and is also expressed on neuronal cells.

[0148] VEGFR has seven immunoglobulin-like domains in the extracellular domain. As used herein, the "extracellular" domain is the portion of a cell surface receptor that is located outside the cell surface and generally includes one or more ligand binding sites.

[0149] The immunoglobulin-like domains of VEGFR are oriented outside the cell in the native conformation of VEGFR in the cell membrane, where they can bind to VEGF. Each VEGFR has seven extracellular Ig domains, which are numbered from N-terminus to C-terminus as Ig domain 1, Ig domain 2, Ig domain 3, Ig domain 4, Ig domain 5, Ig domain 6, and Ig domain 7.

[0150] Upon binding to VEGF, VEGFR undergoes dimerization and ligand-dependent tyrosine phosphorylation in cells, generating mitogenic, chemotactic, and pro-survival signals. Blocking the binding of VEGF to its receptor, VEGFR, has been shown to be effective in treating angiogenesis under pathological conditions. Blocking antibodies targeting VEGF or soluble VEGF receptor fragments can inhibit VEGF binding to VEGFR on vascular endothelial cells, thereby blocking VEGF-induced signaling and preventing pathological angiogenesis caused by overexpression of VEGF.

[0151] As used herein, a VEGF binding domain can be any suitable domain that can bind to VEGF. Examples include VEGF binding domains derived from anti-VEGF antibodies or from VEGF receptors.

[0152] In certain embodiments, the VEGF binding domain comprises one or more extracellular immunoglobulin-like (Ig) domains of one or more VEGF receptors (VEGFRs).

[0153] In certain embodiments, the VEGFR is selected from the group consisting of VEGFR-1, VEGFR-2, and VEGFR-3.

[0154] In certain embodiments, the Ig domain of a VEGFR can be selected from the group consisting of: Ig domain 1, Ig domain 2, Ig domain 3, and Ig domain 4.

[0155] In certain embodiments, the VEGF binding domain comprises two or more different Ig domains of two or more different VEGFRs.

[0156] In certain embodiments, the VEGF binding domain comprises a first Ig domain of a first VEGFR operably linked to the N-terminus of a second Ig domain of a second VEGFR, directly or via a first linker.

[0157] In certain embodiments, the first Ig domain is Ig domain 2 and the second Ig domain is Ig domain 2 or Ig domain 3.

[0158] In certain embodiments, the first VEGFR is VEGFR-1 and the second VEGFR is VEGFR-2.

[0159] In certain embodiments, the VEGF binding domain comprises Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2. In certain embodiments, Ig domain 2 of VEGFR-1 comprises the amino acid sequence set forth in SEQ ID NO:9, and Ig domain 3 of VEGFR-2 comprises the amino acid sequence set forth in SEQ ID NO:10.

[0160] In certain embodiments, Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2 are joined directly or via a first linker.

[0161] In certain embodiments, the Ig domain 2 of VEGFR-1 is operably linked to the N-terminus of the Ig domain 3 of VEGFR-2, directly or via a first linker.

[0162] The first linker joins the two Ig domains of the one or more VEGFRs. In one embodiment, the first linker comprises a peptide linker. The peptide linker can be a synthetic peptide or a peptide derived from a naturally occurring polypeptide.

[0163] In certain embodiments, the first linker comprises the amino acid sequence of SEQ ID NO: 18 (TNTII).

[0164] In certain embodiments, the VEGF-binding domain comprises a VEGF trap that competes with a naturally occurring VEGF cell receptor to inhibit VEGF, such as aflibercept. Aflibercept is an angiogenesis inhibitor that has been developed as a therapeutic for angiogenesis-related diseases. In certain embodiments, the VEGF-binding domain comprises the amino acid sequence of SEQ ID NO: 11.

[0165] In certain embodiments, the VEGF binding domain comprises the amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto while retaining binding specificity for VEGF.

[0166] Table 2. Sequences of exemplary VEGF-binding domains in C5 / VEGF bispecific antibodies

[0167]

[0168]

[0169] C. Peptide Linker Sequence

[0170] In certain embodiments, in the multispecific molecules provided herein, the C5 binding domain is operably linked to the N-terminus of the VEGF binding domain, either directly or via a second linker. Examples are shown in Figure 1 、 2 Or 3 in.

[0171] In certain embodiments, the C5 binding domain comprises a Fab domain comprising a heavy chain polypeptide and a light chain polypeptide, and the heavy chain polypeptide is operably linked to the N-terminus of the VEGF binding domain. Examples are shown in Figure 1 middle.

[0172] In certain embodiments, the C5 binding domain comprises a Fab domain comprising a heavy chain polypeptide and a light chain polypeptide, and the light chain polypeptide is operably linked to the N-terminus of the VEGF binding domain.

[0173] In certain embodiments, the C5 binding domain comprises a scFv domain operably linked to the N-terminus of the VEGF binding domain. Examples are shown in Figure 2 In certain embodiments, the scFv domain comprises a VL region operably linked to the N-terminus of a VH region.

[0174] In certain embodiments, the C5 binding domain comprises a VHH domain operably linked to the N-terminus of the VEGF binding domain. Examples are shown in Figure 3 middle.

[0175] In one embodiment, the second linker comprises a peptide linker. The peptide linker can be a synthetic peptide or a peptide derived from a naturally occurring polypeptide. Considerations for the linker include the effect on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (generally stable and planned degradation), viscosity, rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be incorporated into micelles or liposomes, and the like.

[0176] In certain embodiments, the peptide linker can be a GS linker. As used herein, a "GS linker" is a peptide linker that comprises one, two, three, four, or more repeats of glycine (G) or serine (S). In certain embodiments, the GS linker can comprise one, two, three, four, five, six, seven, eight, nine, ten, or more repeats of SEQ ID NO: 13 (GGGS) or SEQ ID NO: 14 (GGGGS) or SEQ ID NO: 15 (GGGGSGGGGSGGGGS).

[0177] D. Multimerization Components

[0178] In certain embodiments, the fusion proteins in the multispecific molecules provided herein comprise a multimerization component.

[0179] As used herein, "multimerizing component" refers to a component that is capable of associating with another multimerizing component to form a homodimer or a heterodimer.

[0180] In certain embodiments, the multimerization component comprises a polypeptide fragment comprising one or more amino acid residues and having at least one cysteine residue. Cysteine residues can dimerize to form disulfide bonds, thereby allowing dimer formation.

[0181] In some embodiments, the multimerization component comprises a polypeptide between 1 and 200 amino acids in length and having at least one cysteine residue. In some embodiments, the multimerization component comprises a polypeptide of 1 to 180, 1 to 150, 1 to 120, 1 to 100, 1 to 80, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 amino acid residues in length.

[0182] Any suitable polypeptide can be used as a multimerization component, including components containing cysteine residues that can form interchain disulfide bonds, or components that can associate with each other through electrostatic interactions, hydrogen bonds, hydrophobic interactions, etc. Examples of multimerization components include, but are not limited to, Fc domains and leucine zipper motifs.

[0183] In some embodiments, the multimeric component comprises an antibody Fc domain or fragment thereof. For example, the multimeric component may comprise an immunoglobulin CH3 domain. For another example, the multimeric component may comprise an immunoglobulin CH2 and CH3 domains. In some embodiments, the IgG Fc domain is selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0184] Without wishing to be bound by any theory, it is believed that the Fc domain contributes to extending the half-life of the multispecific molecule or increasing the stability of the multispecific molecule.

[0185] In some embodiments, the Fc domain comprises a human Fc domain.

[0186] In certain embodiments, the Fc domain is derived from a human immunoglobulin (Ig).

[0187] In certain embodiments, the Fc domain is derived from human IgG, optionally human IgG1, IgG2, IgG3, or IgG4.

[0188] In certain embodiments, the Fc domain is derived from human IgG1.

[0189] In certain embodiments, the Fc domain comprises the amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 12 but still retains the ability to multimerize.

[0190] In certain embodiments, the Fc domain has a mutation. In some embodiments, the Fc domain comprises a substitution, deletion, insertion, and / or addition mutation to improve aggregation.

[0191] In certain embodiments, the Fc domain comprises a mutation at position 235 and / or 309 of human IgGl according to the EU numbering system.

[0192] In certain embodiments, the Fc domain comprises a substitution at position 235 and / or 309 of human IgGl according to the EU numbering system.

[0193] In certain embodiments, the Fc domain comprises an L235K and / or L309K mutation according to the EU numbering system.

[0194] In certain embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 80% sequence identity thereto but still retaining multimerization and having L235K and / or L309K mutations according to the EU numbering system.

[0195] In certain embodiments, the Fc domain comprises substitutions, deletions, insertions, and / or additions, for example, to reduce or eliminate one or more effector functions, or to improve pH-dependent binding to the neonatal Fc receptor (FcRn).

[0196] In certain embodiments, the Fc domains provided herein have reduced effector function and comprise one or more amino acid substitutions in IgG1 at a position selected from the group consisting of: 234, 235, 237 and 238, 268, 297, 309, 330, and 331 according to the EU numbering system. In certain embodiments, the Fc domains provided herein are of the IgG1 isotype and comprise one or more amino acid substitutions selected from the group consisting of: N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, and any combination thereof, according to the EU numbering system.

[0197] In certain embodiments, the Fc domains provided herein are of the IgG2 isotype and comprise one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof according to the EU numbering system (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S).

[0198] In certain embodiments, the Fc domain provided herein is of the IgG4 isotype and comprises one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A according to the EU numbering system, and any combination thereof.

[0199] In certain embodiments, the Fc domain comprises one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have a prolonged pharmacokinetic half-life because they bind to FcRn at acidic pH, thereby preventing degradation in lysosomes and allowing them to be translocated and released outside the cell. Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, Springer Publishing, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunol., 176:346-356 (2006).

[0200] In certain embodiments, the multimerization component is operably linked to the C-terminus of the VEGF binding domain, either directly or via a third linker.

[0201] In one embodiment, the third linker comprises a peptide linker. The peptide linker can be a synthetic peptide or a peptide derived from a naturally occurring polypeptide. In one embodiment, the third linker comprises a fragment derived from the hinge region of an antibody. In certain embodiments, the third linker comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.

[0202] In certain embodiments, the peptide linker can be a GS linker.

[0203] E. Multispecific molecules

[0204] In some embodiments, the multispecific molecules provided herein are capable of specifically binding to both human C5 and human VEGF. The multispecific molecules provided herein retain specific binding affinity for both human C5 and human VEGF, and in certain embodiments have at least comparable or even better affinity than the parent anti-C5 antibody (e.g., eculizumab) and parent VEGF-binding molecule (e.g., aflibercept).

[0205] In certain embodiments, the multispecific molecules described herein comprise a fusion protein comprising a C5 binding domain as a Fab domain. In certain embodiments, the Fab domain comprises a heavy chain polypeptide and a light chain polypeptide. In certain embodiments, the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and / or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32, and / or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33.

[0206] In certain embodiments, the heavy chain polypeptide is operably linked to the N-terminus of the VEGF binding domain, either directly or via a linker (e.g., a second linker as provided herein). In certain embodiments, the VEGF binding domain is operably linked to the N-terminus of the multimerization component, either directly or via a linker (e.g., a third linker as provided herein). In certain embodiments, the fusion protein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17, wherein the first polypeptide and the second polypeptide associate to form a C5 binding domain. In certain embodiments, the fusion protein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 34 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 33, wherein the first polypeptide and the second polypeptide associate to form a C5 binding domain. Table A. Sequences of full-length C5 / VEGF bispecific antibodies

[0207]

[0208]

[0209] In certain embodiments, the heavy chain polypeptide is operably linked to the C-terminus of the multimerization component directly or via a linker (e.g., a second linker as provided herein). In certain embodiments, the multimerization component is operably linked to the C-terminus of the VEGF binding domain directly or via a linker (e.g., a third linker as provided herein). Examples are shown in Figure 4 middle.

[0210] In certain embodiments, the fusion protein comprises a C5 binding domain comprising an scFv comprising a VH region and a VL region operably linked via a linker.

[0211] In certain embodiments, the fusion protein comprises an scFv operably linked directly or via a linker to the N-terminus of a VEGF binding domain. In certain embodiments, the fusion protein comprises an scFv operably linked directly or via a linker to the C-terminus of a multimerizing component, and the multimerizing component is operably linked directly or via a linker to the C-terminus of a VEGF binding domain. Examples are shown in Figure 5 middle.

[0212] In certain embodiments, the fusion protein comprises a C5 binding domain comprising a VHH domain.

[0213] In certain embodiments, the fusion protein comprises a VHH domain operably linked directly or via a linker to the N-terminus of a VEGF binding domain. In certain embodiments, the fusion protein comprises a VHH domain operably linked directly or via a linker to the C-terminus of a multimerization component, and the multimerization component is operably linked directly or via a linker to the C-terminus of a VEGF binding domain. Examples are shown in Figure 6 middle.

[0214] Without wishing to be bound by any theory, it was found that the efficacy of the fusion protein was affected by the positioning of the C5 binding domain. Specifically, when the C5 binding domain was operably linked to the C-terminus of the multimerization component (see, e.g., Figures 4 to 6 ), the fusion protein exhibited reduced efficacy. In contrast, linking the C5 binding domain to the N-terminus of the VEGF binding domain (see, e.g. Figures 1 to 3 In certain embodiments, the multispecific molecule comprises a dimer of a fusion protein, wherein the multimeric components of the fusion protein associate to form a dimer.

[0215] In certain embodiments, the multispecific molecules provided herein are capable of expressing a molecule with a K of 2000 pM or less, 1800 pM or less, 1500 pM or less, 1200 pM or less, 1000 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 220 pM or less, as determined by the BIACORE described in Example 2 of the present disclosure. D The value specifically binds to C5.

[0216] In certain embodiments, the multispecific molecules provided herein have a K of 1000 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 220 pM or less, 200 pM or less, 190 pM or less, 160 pM or less, 130 pM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM or less as determined by the BIACORE described in Example 2 of the present disclosure. D Values are bound to VEGF.

[0217] In certain embodiments, the multispecific molecules provided herein inhibit HUVEC cell proliferation at 50 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, or 6 nM or less as determined by the anti-VEGF cell proliferation functional assay described in Example 3.1 of the present disclosure.

[0218] In certain embodiments, the multispecific molecules provided herein block the effects of C5 activity with an IC50 value of 500 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, or 40 nM or less as determined by the hemolytic assay described in Example 3.2 of the present disclosure.

[0219] In certain embodiments, the multispecific molecules provided herein have superior effects (such as vascular leakage inhibition, regression of leaky vessels, and longer duration) compared to aflibercept.

[0220] F. Polynucleotides and Recombinant Methods

[0221] The present disclosure provides isolated polynucleotides encoding the multispecific molecules provided herein. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acids Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0222] Polynucleotides encoding the multispecific molecules disclosed herein can be generated using methods known in the art. In certain embodiments, the sequence of the polynucleotide can be obtained based on the amino acid sequence of the multispecific molecule, and nucleic acids can be generated using synthetic methods. Alternatively, the polynucleotides provided herein can also be obtained from another available nucleic acid encoding a polypeptide having a sequence homologous to the polypeptide in the multispecific molecules disclosed herein. DNA manipulation processes can then be applied to manipulate the sequence of the parent multispecific molecule encoding nucleic acid, such as by introducing mutations, insertions, deletions, etc., to obtain nucleic acids encoding the multispecific molecules disclosed herein.

[0223] The isolated polynucleotide encoding the multispecific molecule can be inserted into one or more vectors for further cloning (DNA amplification) or for expression using recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), a transcription termination sequence, and one or more other regulatory elements.

[0224] The present disclosure provides vectors comprising isolated polynucleotides provided herein. In certain embodiments, the polynucleotides provided herein encode multispecific molecules having at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to a nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEME X, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p 15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0225] The vector comprising the polynucleotide sequence encoding the multispecific molecule can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in this paper vector are prokaryotic cells, yeast cells or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (Escherichia) (for example, Escherichia coli (E.coli)), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (for example, Salmonella typhimurium (Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0226] In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding multispecific molecules. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species, and strains are in common use and suitable for use herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0227] Suitable host cells for expressing the glycosylated multispecific molecules provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. A variety of baculovirus strains and variants and corresponding receptive insect host cells have been identified, and the receptive insect host cells are derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of transfection virus strains are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0228] However, vertebrate cells have also attracted considerable attention, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture; Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, and derivatives thereof.

[0229] Host cells are transformed with the above-mentioned expression or cloning vectors for the production of multispecific molecules and cultured in conventional nutrient media modified as needed to induce promoters, select transformants, or amplify genes encoding the desired sequences. In another embodiment, multispecific molecules can be produced by homologous recombination as known in the art. In certain embodiments, host cells are capable of producing the multispecific molecules provided herein.

[0230] The present disclosure also provides a method for expressing the multispecific molecules provided herein, the method comprising culturing the host cells provided herein under conditions expressing the vectors disclosed herein. The host cells used to produce the multispecific molecules provided herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Methods in Enzymology 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Rep. 30,985 can be used as culture medium for the host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium chloride, magnesium chloride, and sodium, calcium, or magnesium phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM The expression vector is prepared by mixing a mixture of at least one of the following: a) a 5-10 μg / mL lysine phosphate (drug), b ...

[0231] When using recombinant techniques, multispecific molecules can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the multispecific molecule is produced intracellularly, then the first step is to remove the microparticles of the host cells or dissolved fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a process for isolating multispecific molecules secreted into the periplasmic space of Escherichia coli. In brief, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 min. Cell debris can be removed by centrifugation. In the case where the multispecific molecule is secreted into the culture medium, a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit, is generally first used to concentrate the supernatant from such an expression system. Protease inhibitors, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0232] Multispecific molecules produced by cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0233] In certain embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of multispecific molecules comprising an Fc domain. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the multispecific molecule. Protein A can be used to purify multispecific molecules based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., Journal of Immunological Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., Journal of the European Molecular Biology Association (EMBO J.) 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly(styrene divinyl) benzene, can achieve faster flow rates and shorter processing times than can be achieved with agarose. In the case where the multispecific molecule comprises a CH3 domain, Bakerbond ABX TM Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification include ion exchange column fractionation, ethanol precipitation, reversed-phase HPLC, silica chromatography, heparin SEPHAROSE TMChromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographing, SDS-PAGE, and ammonium sulfate precipitation are also useful, depending on the multispecific molecule to be recovered.

[0234] Following any one or more preliminary purification steps, the mixture comprising the multispecific molecule of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5 and 4.5, preferably at low salt concentrations (e.g., about 0 to 0.25 M salt).

[0235] III. Pharmaceutical Formulations and Administration

[0236] The present disclosure also provides pharmaceutical compositions comprising the multispecific molecules provided herein and one or more pharmaceutically acceptable carriers.

[0237] The present disclosure further provides pharmaceutical compositions comprising a polynucleotide encoding a multispecific molecule provided herein and one or more pharmaceutically acceptable carriers.

[0238] The present disclosure further provides pharmaceutical compositions comprising an expression vector comprising a polynucleotide encoding a multispecific molecule as provided herein and one or more pharmaceutically acceptable carriers. In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, and adenoviral vectors. Examples of non-viral vectors include, but are not limited to, naked DNA, plasmids, exosomes, mRNA, and the like. In certain embodiments, the expression vector is suitable for gene therapy in humans. Suitable vectors for gene therapy include, for example, adeno-associated virus (AAV) or adenoviral vectors. In certain embodiments, the expression vector comprises a DNA vector or an RNA vector. In certain embodiments, the pharmaceutically acceptable carrier is a polymeric excipient, such as, but not limited to, microspheres, microcapsules, polymeric micelles, and dendrimers. The polynucleotides and / or polynucleotide vectors disclosed herein can be encapsulated, adhered or coated on polymeric components by methods known in the art (see, for example, W. Heiser, Nonviral gene transfer technologies, published by Humana Press, 2004; U.S. Patent 6,025,337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0239] As used herein, the term "pharmaceutical composition" refers to a formulation containing the active ingredients in a form suitable for administration to a subject.

[0240] As used herein, the term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient(s), salt(s), and / or culture medium are generally chemically and / or physiologically compatible with the other ingredients, such as the active ingredients (i.e., the multispecific molecules disclosed herein) comprising the formulation, and are physiologically compatible with the subject to whom the pharmaceutical composition will be administered.

[0241] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is biologically acceptable and non-toxic to the subject. In the context of the present disclosure, pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / partitioning agents, sequestrants or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0242] The carrier can be a solvent, a dispersion medium, an isotonic agent, etc. The carrier can be a liquid, a semisolid or a solid carrier. In some embodiments, the carrier can be water, a saline solution or other buffer (such as serum albumin and gelatin), a carbohydrate (such as monosaccharides, disaccharides and other carbohydrates, including glucose, sucrose, trehalose, mannitol, sorbitol or dextrin), a gel, a lipid, a liposome, a resin, a porous matrix, an adhesive, a filler, a coating, a stabilizer, a preservative, an antioxidant (including ascorbic acid and methionine), a chelating agent (such as EDTA), a salt-forming counterion (such as sodium), a nonionic surfactant [such as TWEEN tm 、PLURONICS tm or polyethylene glycol (PEG)], or a combination thereof. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

[0243] The composition can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By the above-mentioned sterilization procedure, and by including various antibacterials and antifungals (for example, parabens, chlorobutanol, phenol, sorbic acid etc.), it is possible to ensure the presence of prevention microorganisms. It may also be desirable to include isotonic agents such as sugar, sodium chloride etc. in the composition. In addition, the absorption of the extension of the injectable drug form can be achieved by including the medicament (such as aluminum monostearate and gelatin) that delays absorption.

[0244] The composition must be sterile and fluid to the extent that the composition is deliverable by syringe.In addition to water, the carrier is preferably an isotonic buffered saline solution.

[0245] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

[0246] The composition can comprise an ophthalmic reservoir formulation comprising an active agent for subconjunctival administration. An ophthalmic reservoir formulation comprises microparticles of substantially pure active agents, such as bispecific antibodies according to the present invention. Microparticles comprising bispecific antibodies according to the present invention can be embedded in biocompatible pharmaceutically acceptable polymers or lipid encapsulating agents. The reservoir formulation can be suitable for releasing all or substantially all active materials over an extended period of time. If present, the polymer or lipid matrix can be suitable for sufficient degradation so as to be transported from the site of administration after releasing all or substantially all active agents. The reservoir formulation can be a liquid formulation comprising a pharmaceutically acceptable polymer and a dissolved or dispersed active agent. After injection, the polymer forms a reservoir at the injection site, such as by gelation or precipitation.

[0247] In an embodiment, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions or solid forms suitable for generating liquid solutions, suspensions or emulsions. Injectable formulations can include sterile and / or pyrogen-free solutions for injection, sterile dry soluble products prepared before use with solvent combinations, such as lyophilized powders, including subcutaneous injection tablets, sterile suspensions prepared for injection, sterile dry insoluble products prepared before use with vehicle combinations, and sterile and / or pyrogen-free emulsions. The solution can be aqueous or non-aqueous.

[0248] In certain embodiments, the unit dose parenteral formulation is packaged in an ampoule, a vial, or a syringe with a needle.As is known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.

[0249] In certain embodiments, a sterile lyophilized powder is prepared by dissolving a multispecific molecule as disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder or other pharmacological components. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art. In one embodiment, the buffer is at approximately neutral pH. The solution is then sterile filtered and lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single or multiple doses of a polypeptide complex or polypeptide complex. Overfilling the vial beyond a small amount (e.g., approximately 10%) required for a single dose or a set of doses is acceptable to facilitate accurate sampling and dosing. The lyophilized powder can be stored under appropriate conditions, such as at approximately 4°C to room temperature.

[0250] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, for reconstitution, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder. The exact amount depends on the selected therapy to be administered and can be determined empirically.

[0251] In certain embodiments, a composition is further provided, comprising a pharmaceutically acceptable carrier, diluent or adjuvant and an active ingredient. The active ingredient may be a multispecific molecule disclosed herein.

[0252] IV. Reagent test kit

[0253] In another aspect, the present invention provides a kit comprising a multispecific molecule as provided herein and instructions for use of the multispecific molecule. The kit may also include a container and, optionally, one or more vials, test tubes, flasks, bottles, or syringes. Other formats of the kit will be apparent to those skilled in the art and are within the scope of the present invention.

[0254] As will be apparent to those skilled in the art, such kits may further include, if necessary, one or more of various conventional pharmaceutical kit components, such as a container with one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions (either as an insert or as a label) indicating the quantities of the components to be administered, directions for administration, and / or directions for mixing the components may also be included in the kit.

[0255] In some embodiments, the therapeutic kits of the invention can contain one or more doses of a multispecific molecule present in a pharmaceutical composition described herein, a suitable device for intravitreal injection of the pharmaceutical composition, and instructions detailing a suitable subject and a protocol for performing the injection. In these embodiments, the composition is typically administered to a subject in need of treatment via intravitreal injection.

[0256] V. Medical uses

[0257] In another aspect, the present invention provides a method for treating, preventing, or ameliorating a disease condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a multispecific molecule of the invention disclosed herein, or a polynucleotide encoding a multispecific molecule provided herein, or a pharmaceutical composition provided herein.

[0258] As used herein, the term "subject" or "individual" or "animal" or "patient" refers to a human or non-human animal, including a mammal or primate, for whom diagnosis, prognosis, amelioration, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo animals, competitive animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0259] As used herein, "treating" of a condition can include alleviating the condition, slowing the rate of onset or development of the condition, delaying the development of symptoms associated with the condition, relieving or ending symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0260] As used herein, the terms "disorder," "disease," "condition," and the like refer to conditions affecting a subject that would benefit from treatment with a multispecific molecule.

[0261] As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount of the therapeutic agent that, when administered to a subject in an appropriate manner, produces a sufficient therapeutic effect on the subject. It should be understood that, as with other therapeutic agents, the therapeutically effective amount of a multispecific molecule as provided herein will be affected by various factors known in the art, such as the subject's weight, age, past medical history, current medications, health status and potential for cross-reactions, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of the disease. A person of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage as indicated by these and other circumstances or requirements.

[0262] In certain embodiments, the disease or disorder is a C5-related and / or VEGF-related disease or disorder.In some embodiments, the disease, disorder or condition is selected from the group consisting of an ocular disease, cancer, an inflammatory disease, an autoimmune disease, angiogenesis, vascular permeability, edema, and inflammation.

[0263] In certain embodiments, the C5-related and / or VEGF-related disease or disorder is an ocular disease.

[0264] In some embodiments, the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), macular edema, macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), central retinal vein occlusion, corneal neovascularization (CNV), retinitis pigmentosa (RP), ocular angiogenesis (ocular neovascularization affecting choroidal, corneal or retinal tissue), retinopathy of prematurity (ROP), pathological myopia, vascular glaucoma, retinoblastoma, retinal vein occlusion, uveitis and neuromyelitis optica.

[0265] In some embodiments, the eye disease is age-related macular degeneration (AMD). AMD is a disease characterized by progressive degenerative abnormalities of the macula (the central part of the retina). Age-related macular degeneration is a complex, gradually progressive eye condition that can lead to distortion and / or blind spots (scotomas), changes in dark adaptation (diagnostic for rod health), changes in color interpretation (diagnostic for cone health), decreased vision, or irreversible blindness.

[0266] In some embodiments, AMD is dry AMD.Non-exudative AMD is the non-neovascular (" dry ") form (" dry AMD ") of the disease.Dry AMD accounts for 90% of all AMD cases.The feature of dry AMD is macular degeneration, and along with the continuous progress of several years, may eventually cause the central retinal atrophy relevant to central vision loss, also referred to as geographic atrophy (GA).Dry AMD is the important reason causing moderate and severe central vision loss, and is all bilateral dry AMD in most patients.In dry AMD, the retinal pigment epithelium (RPE) in the macula can attenuate, and other age-related changes with adjacent retinal tissue layers.

[0267] In some embodiments, AMD is wet AMD. Choroidal neovascularization can be the early stage sign of wet AMD. Once neovascularization occurs in non-exudative AMD and begins to leak, this disease is just referred to as exudative AMD, i.e. the neovascularization (" wet ") form (" wet AMD ") of the disease, wherein the patient still has non-exudative AMD and may be in progress. Wet AMD may cause central vision to be lost suddenly, normally seriously loses, especially in the case of being untreated.

[0268] In some embodiments, the eye disease is geographic atrophy. Geographic atrophy (GA) is a chronic progressive degeneration of the macula and is a part of late AMD. The macula is the central part of the retina, and the retina is the "membrane" lining the inside of the eye. In GA, areas of the retina experience cell death (atrophy). These areas may increase and may cause visual scotoma or blind spots. GA usually first develops near the fovea centralis (i.e., the center of the macula), which is the clearest part of the central part of vision. GA may cause progressive and permanent vision loss. If GA occurs in one eye, GA is more likely to occur in the other eye. GA is characterized by a clear-cut, localized atrophy of the outer retinal tissue, retinal pigment epithelium, and choriocapillaris.

[0269] It is estimated that more than 8 million people worldwide suffer from GA. Currently, there is no cure for GA.

[0270] The inventors of this application unexpectedly discovered that by combining the inhibition of VEGF and C5, a synergistic effect can be achieved in a chronic disease animal model for GA, which cannot be achieved by inhibiting either target alone, for example, by VEGF-trapping agents (e.g., aflibercept) or by anti-C5 antibodies (e.g., eculizumab). The multispecific molecules provided herein exhibit significantly improved therapeutic efficacy and duration of such therapeutic efficacy relative to aflibercept.

[0271] In certain embodiments, the subject has been treated with a VEGF antagonist or a VEGF receptor antagonist. In certain embodiments, the subject has developed resistance to the VEGF antagonist or a VEGF receptor antagonist.

[0272] The present invention provides a method for treating or preventing an ocular disease in a subject in need thereof, or for inducing regression, elimination, or inhibition of progression of at least one sign or symptom of an ocular disease in a subject in need thereof, by administering to the subject a therapeutically effective amount of a combination. In certain embodiments, the subject suffers from an ocular disease and exhibits one or more signs or symptoms of the ocular disease.

[0273] Typical signs or symptoms of eye diseases include, for example: accelerated rate of vision loss; drusen in the eye (e.g., in subjects with dry AMD); loss of vision; gradual loss of central vision (e.g., in subjects with non-exudative macular degeneration); distorted vision; difficulty adapting to low light levels; curvature of central vision; central and / or global blurring of vision; changes in eye pigment; strabismus (e.g., metamorphopsia, where straight lines appear wavy and sections of the grid may appear blank); exudative changes (e.g., intraocular hemorrhages, hard exudates, subretinal / sub-RPE / intraretinal fluid); exposure to bright light Slow recovery of visual function (e.g., as determined on light stress testing); early and / or geographic atrophy; a sharp decrease in visual acuity (e.g., a decrease of two or more grades, e.g., from 20 / 20 to 20 / 80); preemptive superacute perimetry changes (e.g., for subjects with wet AMD); blurred vision; rapid onset of vision loss (e.g., caused by leakage and bleeding from abnormal blood vessels in subjects with exudative macular degeneration); central scotoma (shadows or loss of visual field); difficulty distinguishing colors (e.g., specifically distinguishing dark colors from other dark colors, and / or light colors from other light colors); loss of contrast sensitivity; and / or straight lines appearing curved on an Amsler grid.

[0274] In certain embodiments, the subject is a human.

[0275] In certain embodiments, a multispecific molecule as provided herein can be administered in a therapeutically effective amount of about 1 mg to about 20 mg (or 2 mg to 20 mg, or 4 mg to 20 mg, or 4 mg to 12 mg) per intravitreal (IVT) injection. The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single dose can be administered, or several divided doses can be administered over time.

[0276] The multispecific molecules disclosed herein can be administered by any route known in the art, such as parenteral (e.g., intraocular, intravitreal injection, subcutaneous, intraperitoneal, intravenous (including intravenous infusion), intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, intravitreal injection, sublingual, rectal, or topical) routes.

[0277] Many possible modes of delivery can be used, including but not limited to intraocular application or topical application. In one embodiment, the application is intraocular and includes but is not limited to subconjunctival injection, intracranial injection, injection into the anterior chamber via the temporal limbus, intrastromal injection, intracorneal injection, subretinal injection, aqueous humor injection, sub-Tenon's injection or continuous delivery device injection, intravitreal injection (e.g., anterior vitreous, middle vitreous, or posterior vitreous injection). In one embodiment, the application is topical and includes but is not limited to eye drops to the cornea.

[0278] In one embodiment, the multispecific molecule or pharmaceutical composition according to the invention is administered via intravitreal application, for example, via intravitreal injection. This can be performed according to standard procedures known in the art. See, for example, Ritter et al., J. Clin. Invest. 116 (2006) 3266-76; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.

[0279] Actual dosage levels of the multispecific molecules in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed; the route of administration; the time of administration; the rate of excretion of the particular active compound employed; the duration of treatment; other drugs, compounds, and / or materials used in combination with the particular composition employed; the age, sex, weight, condition, general health, and previous medical history of the patient being treated; and similar factors well known in the medical arts.

[0280] In some embodiments, the multispecific molecules disclosed herein can be administered alone or in combination with one or more additional therapeutic means or agents. For example, the multispecific molecules disclosed herein can be administered in combination with one or more additional therapeutic agents or methods for treating one or more ocular diseases described herein.

[0281] In certain of these embodiments, a multispecific molecule as disclosed herein administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the multispecific molecule and the one or more additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, a multispecific molecule administered "in combination" with another therapeutic agent need not be administered simultaneously with the agent or in the same composition as the agent. As the phrase is used herein, a multispecific molecule administered before or after another agent is considered to be administered "in combination" with the agent, even if the multispecific molecule and the second agent are administered via different routes. Where possible, the additional therapeutic agent administered in combination with the multispecific molecule disclosed herein is administered according to the schedule listed in the product information sheet for the additional therapeutic agent or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)) or a regimen well known in the art.

[0282] In other embodiments, the multispecific molecules or pharmaceutical compositions according to the invention are administered in combination with one or more additional therapeutic agents or methods for treating one or more ocular diseases described herein.

[0283] In other embodiments, the multispecific molecules or pharmaceutical compositions according to the invention are formulated in combination with one or more additional therapeutic agents and administered for the treatment of one or more ocular diseases described herein.

[0284] In certain embodiments, combination therapies provided herein comprise administration of a multispecific molecule or pharmaceutical composition according to the invention, sequentially with one or more additional therapeutic agents for treating one or more ocular diseases described herein.

[0285] Additional therapeutic agents include, but are not limited to, anti-angiogenic agents (such as VEGF antagonists, VEGF receptor antagonists, anti-inflammatory drugs, neuroprotective agents, therapeutics for AMD, or C5 inhibitors).

[0286] In certain embodiments, the anti-angiogenic agent is a VEGF antagonist or a VEGF receptor antagonist. Examples include, but are not limited to, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, small interfering RNA that reduces expression of VEGFR or VEGF ligands, low molecular weight inhibitors of VEGFR tyrosine kinases, and any combination thereof, and examples include anti-VEGF aptamers (e.g., pegaptanib), soluble recombinant decoy receptors (e.g., VEGF traps).

[0287] In certain embodiments, anti-angiogenic agents include anti-inflammatory drugs, m-Tor inhibitors, rapamycin, everolimus, temsirolimus, cyclosporine, anti-TNF agents, anti-complement agents, and nonsteroidal anti-inflammatory agents.

[0288] In certain embodiments, anti-angiogenic agents include corticosteroids, angiostatic steroids, anecortave acetate, angiostatin, endostatin, MMP inhibitors, IGFBP3, SDF-1 blockers, PEDF, γ-secretase, delta-like ligand 4, integrin antagonists (e.g., integrin β3 function inhibitors), HIF-1α blockers, protein kinase CK2 blockers, and inhibitors that inhibit stem cell (i.e., endothelial progenitor cell) homing to neovascularization sites using vascular endothelial cadherin (CD-144) and stromal-derived factor (SDF)-I antibodies.

[0289] In certain embodiments, the additional therapeutic agent is a complement-related agent, such as CIq, C3, C5, Factor B, Factor D, or Factor H.

[0290] In certain embodiments, the additional therapeutic agent is a C3 inhibitor, including but not limited to compstatin and / or its analog H17 (monoclonal antibody, EluSys Therapeutics, Pine Brook, NJ); mirococept (CR1-based protein); sCR1 (CR1-based protein, Celldex, Hampton, NJ); TT32 (CR-1-based protein, Alexion Pharmaceuticals, Inc., Boston, MA); HC-1496 (recombinant peptide); CB 2782 (enzyme, Catalyst Biosciences, South San Francisco, CA); APL-2 (PEGylated synthetic cyclic peptide, Apellis Pharmaceuticals, Crestwood, KY), or a combination thereof.

[0291] In certain embodiments, the additional therapeutic agent is a complement factor B inhibitor, including but not limited to: anti-FB siRNA (Alnylam Pharmaceuticals, Cambridge, MA); TA106 (monoclonal antibody, Alexion Pharmaceuticals, Inc., Boston, MA); LNP023 (small molecule, Novartis, Basel, Switzerland); SOMAmers (aptamers, Soma Logic, Boulder, CO); bikaciomab (Novelmed Therapeutics, Cleveland, OH); complin (see Kadam et al., J. Immunol. 2010, DOI: 10.409 / jimmunol.10000200); Ionis-FB-LRx (ligand-binding antisense drug, Ionis Pharmaceuticals, Carlsbad, CA); or a combination thereof.

[0292] In certain embodiments, the additional therapeutic agent is a complement factor D antagonist, such as an anti-complement factor D antibody, such as lampalizumab (Roche).

[0293] In certain embodiments, the additional therapeutic agent is an Ang-2 antagonist.

[0294] In certain embodiments, the additional therapeutic agent is a neuroprotective agent that can potentially slow the progression of dry macular degeneration. This class of drugs is also known as neurosteroids and includes, for example, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate, and pregnenolone sulfate.

[0295] In certain embodiments, the additional therapeutic agent is a therapeutic agent for AMD, including but not limited to verteporfin in combination with PDT, pegaptanib, zinc, or one or more antioxidants, alone or in any combination.

[0296] In certain embodiments, the additional therapeutic agent is a C5 inhibitor, including but not limited to eculizumab, ravulizumab, 305LO5, SKY59, pozelimab, tesidolumab, crovalimab, or ABP 959, or a biosimilar thereof.

[0297] In another aspect, the disclosure provides a method of modulating C5 and / or VEGF activity in a cell, the method comprising exposing the cell to a multispecific molecule provided herein and / or a pharmaceutical composition provided herein.

[0298] In certain embodiments, the cell is selected from the group consisting of retinal ganglion cells, rods, cones, glial cells (eg, Müller glial cells), bipolar cells, amacrine cells, and horizontal cells.

[0299] In another aspect, the disclosure provides a multispecific molecule provided herein and / or a pharmaceutical composition provided herein for use in treating, preventing, or ameliorating a C5-related and / or VEGF-related disease, disorder, or condition in a subject.

[0300] In another aspect, the disclosure provides use of a multispecific molecule provided herein and / or a pharmaceutical composition provided herein in the manufacture of a medicament for treating, preventing, or ameliorating a C5-related and / or VEGF-related disease, disorder, or condition in a subject.

[0301] Example:

[0302] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials and methods described below fall within the scope of the invention in whole or in part. These specific compositions, materials and methods are not intended to limit the present invention, but are merely intended to illustrate specific embodiments falling within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without using inventive capabilities and without departing from the scope of the present invention. It should be understood that many changes can be made in the procedures described herein while still remaining within the limits of the present invention. The intention of the inventors of this case is that such changes are all included within the scope of the present invention.

[0303] Example 1 Gene synthesis, expression and purification of bispecific molecules

[0304] 1.1 Experimental methods:

[0305] a) VEGF / C5 bispecific antibodies BSP1, BSP2, BSP3, BSP4, BSP5, and BSP6

[0306] Nucleic acid sequences encoding the VEGF / C5 bispecific molecules BSP1, BSP2, BSP3, BSP4, BSP5, and BSP6 were designed, optimized, and synthesized. The complete sequences were subcloned into the pcDNA3.4 vector. Recombinant plasmids encoding the target antibodies were transiently co-transfected into suspension HD 293F cell cultures (Thermofisher Scientific). The plasmid-transfected cells were cultured in culture medium at 37°C, 8% CO2. After 6 days of culture, the supernatant was collected for protein purification, centrifuged, and filtered. The filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and elution with an appropriate buffer, the eluted fractions were combined, concentrated, and loaded onto a gel filtration column at an appropriate flow rate to increase purity. The protein was concentrated to the desired concentration. The purified protein was analyzed by SDS-PAGE, Western blotting, and HPLC to determine molecular weight and purity. Concentration was determined by the A280 method.

[0307] On the other hand, aflibercept (ProBio) and mAb eculizumab (ProBio) were used as positive controls to evaluate the activity and function of the VEGF / C5 bispecific antibody prepared in this application.

[0308] Table 3. Structures of different VEGF / C5 bispecific antibodies from N-terminus to C-terminus

[0309]

[0310]

[0311] In BSP1 to BSP6, the amino acid sequence of VH (eculizumab) is SEQ ID NO: 1, the amino acid sequence of VL (eculizumab) is SEQ ID NO: 2, the amino acid sequence of VEGFR1 (domain 2) is SEQ ID NO: 9, the amino acid sequence of VEGFR2 (domain 3) is SEQ ID NO: 10, the amino acid sequence of human IgG1 CH1 is SEQ ID NO: 19, the amino acid sequence of human CL (κ) is SEQ ID NO: 20, and the amino acid sequence of human IgG1 Fc is SEQ ID NO: 12. Each of BSP1 to BSP6 is a dimer of the fusion protein shown in Table 3. The linker in the molecule is present but not explicitly indicated in Table 3.

[0312] b) Affinity Kd maturation / optimization

[0313] Experimental methods:

[0314] i) Amino acid selection for Kd maturation

[0315] FASEBA (rapid screening of expression, biophysical properties and affinity) was selected for affinity Kd maturation (Probio and Genscript). Based on the parental antibody sequence / BSP1, FASEBA screening was performed to increase the affinity of the antibody for the target antigen.

[0316] The parent mAb, eculizumab, was expressed in CHO cells. After antibody purification, binding of the mAb to human complement C5 protein was analyzed using Biacore 8K / T200. To determine the amino acids selected for Kd maturation, the parent Fab was constructed using FASEBA format, and binding was verified using SPR. A Precise Mutagenesis Library (PML) was constructed using FASEBA format at six CDR residues, and next-generation sequencing (NGS) was performed to examine the distribution of the PML library.

[0317] ii) Generation and characterization of affinity matured antibodies

[0318] DNA encoding the best affinity-matured antibodies obtained from PML was synthesized and subcloned into an expression vector for antibody expression in CHO cells. The affinity-matured antibodies were purified using a Protein A column. The kinetics of antigen interactions with one or more affinity-matured antibodies and wild-type antibodies were studied using Biacore 8K.

[0319] Mutations were introduced into HCDR1, HCDR3, HFR2, HFR3, and HFR4 in the heavy chain variable region. Additional mutations were introduced into LCDR2, LFR1, and LFR2 in the light chain variable region. The kinetics of the interaction between human complement C5 and wild-type and mutant eculizumab were investigated.

[0320] Affinity-matured mutants were identified that showed significant improvements in binding affinity for human C5, ranging from 3-fold to 10-fold.

[0321] Eculizumab-Mutant #1 is an eculizumab mutant comprising one mutation in the HC and three mutations in the LC. Eculizumab-Mutant #1 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:28 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:31. Eculizumab-WT is the original eculizumab sequence.

[0322] The results are shown in Table 3 A. It was determined that eculizumab-mutants #1 to #3 had significantly improved binding affinity to human C5.

[0323] Table 3A. Affinity of wild-type eculizumab and mutant eculizumab

[0324]

[0325]

[0326] Based on the excellent protein expression yields in 100 mL shake flasks, eculizumab-mutant #1 was selected as the preferred affinity maturation mutant and used to generate the bispecific molecule BSP1a for further study.

[0327] Table 3B. Structures of different VEGF / C5 bispecific antibodies from N-terminus to C-terminus

[0328]

[0329] In BSP1a, the amino acid sequence of VH (eculizumab mutant) is SEQ ID NO: 28, the amino acid sequence of VL (eculizumab mutant) is SEQ ID NO: 31, the amino acid sequence of VEGFR1 (domain 2) is SEQ ID NO: 9, the amino acid sequence of VEGFR2 (domain 3) is SEQ ID NO: 10, the amino acid sequence of human IgG1 CH1 is SEQ ID NO: 19, the amino acid sequence of human CL (κ) is SEQ ID NO: 20, and the amino acid sequence of human IgG1 Fc is SEQ ID NO: 12. BSP1a is a dimer of the fusion proteins shown in Table 3B. The linkers in the molecule are not explicitly indicated in Tables 3 and 3B.

[0330] 1.2 Experimental results of bispecific antibody purity analysis:

[0331] The purified protein was analyzed by SDS-PAGE, Western blotting, and HPLC analysis to determine the molecular weight and purity.

[0332] Table 4. Purity analysis of VEGF / C5 bispecific molecules by SDS-Page and SEC-HPLC

[0333]

[0334] The purity of BSP1a purified by SDS-Page ranged from 94% to 96%, and the purity of BSP1a purified by SEC-HPLC ranged from 95% to 99%.

[0335] Example 2 Binding affinity determination

[0336] 2.1 Experimental methods:

[0337] The binding affinity of the bispecific molecules to the antigen was determined by SPR assay using a Biacore 8K (GE Healthcare, Chicago, IL). Binding assays were performed by first attaching BSP1 to an S-series sensor coated with Protein A. Different concentrations of human recombinant VEGF-165 protein (Acro: VE5-H4210, 1.5625 to 100 nM) or human recombinant complement C5 protein (Acro: CO5-H52Ha, 0.15625 to 5 nM) were then injected onto the BSP binding surface at a flow rate of 30 μL / min for 120 seconds. After sample injection, the bound analyte was separated from the sensor chip using 10 mM glycine-HCl pH 1.5 (GenSript, lot 20211113). Binding affinity assays for the BSP1a bispecific molecule were performed in the same manner.

[0338] Antibody binding kinetics, including ka (association rate constant), kd (dissociation rate constant), and K, were determined using Biacore 8K Evaluation Software version 3.0 (Chicago, IL). D (Dissociation equilibrium constant). The binding response (RU) was recorded as a graph over time, allowing the different stages of the binding event to be visually visualized and evaluated.

[0339] 2.2 Experimental results:

[0340] As shown in Table 5, the binding affinities of BSP1 and BSP1a for C5 were measured, and the K values of BSP1 and BSP1a for human C5 and VEGF are provided. D Average value. PC2 (anti-C5 mAb) K for C5 D The K of PC1 (aflibercept) against VEGF is in the range of 400 to 500 pM. D The average value is 21.7pM.

[0341] Table 5. Binding affinities of VEGF / C5 bispecific molecules and controls for VEGF and C5

[0342]

[0343] Example 3 In vitro cell function assay (anti-VEGF and anti-C5)

[0344] 3.1 VEGF-mediated cell proliferation assay (anti-VEGF)

[0345] 3.1.1 Experimental methods:

[0346] The VEGF-mediated proliferation of human umbilical vein endothelial cells (HUVEC, C2519A, Lonza, Basel, Switzerland) was selected to measure the cellular function of the VEGF molecule. HUVEC cells were collected by centrifugation and resuspended in cell culture medium. Working solutions of the test article, VEGF protein, and assay buffer were prepared and transferred to the corresponding wells of a 96-well plate. The cell suspension was transferred to a 384-well assay plate, and then a working solution of the mixture of the test article and VEGF protein was transferred to the 384-well plate. The assay plate was cultivated in an incubator (37°C and 5% CO2) for 72 hours, and CellTiter-Glo was subsequently added to the corresponding wells of the assay plate.

[0347] Luminescence signals were recorded using a PHERAStar. Raw data were exported from the PHERAStar FSX system and analyzed using Microsoft Office Excel 2016 and GraphPad Prism 6.

[0348] The dose-response curves were fitted using the following four-parameter function and relative IC50 values were obtained, which were characterized by a sigmoidal curve in which the percentage of growth inhibition was related to the concentration of the test sample:

[0349] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0350] Where X = logarithmic concentration and Y = luminescent signal

[0351] Target cells were treated with serial dilutions of the test substances. Luminescence ± SEM of three replicates in each group are plotted.

[0352] 3.1.2 Experimental results:

[0353] The experimental results showed that both positive control 1 (aflibercept) and BSP1 could inhibit HUVEC cell proliferation, and the average IC50 values of the dose-response curve were calculated to be 1.55nM and 1.10nM. No inhibition of HUVEC cell proliferation was observed in the negative control. PC1 (aflibercept) and BSP1 had similar growth inhibitory effects in the VEGF-mediated HUVEC proliferation assay. The growth inhibitory effects of the test samples (PC1 and BSP1) on the target cell lines are shown in Figure 7 、 Figure 8 And Tables 6 and 7.

[0354] BSP1a was also tested using a similar approach and showed a growth inhibitory effect in a VEGF-mediated HUVEC proliferation assay with an IC of 50 (μg / mL) ranged from 0.1 to 0.3.

[0355] Table 6. Summary of best fit values for the first experiment of the VEGF-mediated cell proliferation assay

[0356]

[0357]

[0358] Table 7. Summary of best fit values for the second experiment of the VEGF-mediated cell proliferation assay

[0359]

[0360] BSP1a was also tested using a VEGF-mediated cell proliferation assay, and BSP1a showed an IC for inhibiting VEGF-mediated cell proliferation. 50 (μg / mL) ranged from 0.1 to 0.2.

[0361] 3.2 Hemolytic assay / CH50 assay blocking effect (anti-C5)

[0362] 3.2.1 Experimental methods

[0363] Hemolytic assay was selected to measure the cellular function of C5 molecules. Sheep red blood cells (SRBC) were collected by centrifugation and resuspending the cells with GVB++ assay buffer. A working solution of anti-erythrocyte matrix polyclonal antibodies and GVB++ assay buffer was prepared and transferred to a SRBC suspension. Then, the mixture of the working solution and the SRBC suspension was fully mixed, and the plate was incubated at 37°C and 5% CO2 for approximately 30 minutes. Similarly, a working solution of PNHS and test material and GVB++ assay buffer was prepared and transferred to the corresponding wells of a 96-well assay plate. The mixture was then fully mixed, and the plate was incubated at room temperature for approximately 30 minutes. A sheep red blood cell (SRBC) suspension activated by cultivating anti-RBC matrix antibodies was taken out, and the cell suspension was transferred to the above-mentioned 96-well assay plate. The assay plate was incubated at 37°C and 5% CO2 for approximately 1 hour. The assay plate was then removed from the incubator, and the supernatant collected by centrifugation was transferred to a new 96-well test plate. Hemoglobin was removed using a hemoglobin assay kit. The detection reagent was added to the above 96-well assay plate and incubated at room temperature for 5 minutes.

[0364] The absorbance (OD 400 nM) signal was read using PHERA star. The dose-response curve was fitted using the following four-parameter function and the relative EC50 value was obtained, which is characterized by a sigmoidal curve in which the growth inhibition percentage is related to the concentration of the test sample:

[0365] Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)*Hillslope))

[0366] Where X = log concentration and Y = absorbance signal

[0367] SRBC were treated with serial dilutions of the test substance. The absorbance signal SEM ( Figure 9A The hemoglobin levels ± SEM of three replicates in each group were plotted. Figure 9B ).

[0368] 3.2.2 Experimental results

[0369] For the CH50 assay (C5 target test), both eculizumab (positive control 2 (PC2)) and BSP1 had a significant blocking effect on complement C5, and a dose-response curve could be observed. EC values of PC2 (eculizumab) and the test substance BSP1 were50 The values were 21.01 nM and 18.08 nM, respectively (Table 8). BSP1a was tested using a similar method and showed an EC of 21.01 nM and 18.08 nM, respectively (Table 8). 50 (nM) ranged from 18 to 22 nM.

[0370] Table 8. Summary of best fit values for CH50 determinations regarding absorbance signals

[0371] sample EC50 (nM) BSP1 18.08 PC2 21.01

[0372] Example 4: Stability determination of monoclonal drugs targeting BSP1

[0373] 4.1 Experimental methods

[0374] Stability assays are performed to assess the developability of one or more multispecific molecules.

[0375] 4.2 Experimental Results

[0376] BSP1 and BSP1a samples were stored in PBS at a concentration of 40 mg / mL at 40°C or 5°C for 14 days. Samples were collected on days 0, 7, and 14 and subjected to SEC-HPLC, CE-SDS-NR, and imaged capillary isoelectric focusing (icIEF). The results are summarized in Tables 9 and 10 below.

[0377] Table 9. Experimental conditions for sample (BSP1) formulation used in monoclonal drug stability testing

[0378]

[0379] Table 10. Experimental setup used in the stability testing of monoclonal drugs

[0380] condition Time point Test items (three items) D0 D0 A, B, C 40℃ D7, D14 A, B, C 5℃ D7, D14 A, B, C

[0381] result:

[0382] The results of the 40°C stability test are shown in Table 11, with the D0 sample used as the starting point. In the SEC-HPLC test, the samples showed an increase in the percentage of high molecular weight (HMW), a decrease in the percentage of the main peak, and an increase in the percentage of low molecular weight (LMW). In the CE-SDS-NR test, the samples showed a slight decrease in the percentage of the main peak. In the icIEF test, the samples showed a shift to a lower pI (acidic) region with increasing incubation time.

[0383] The results of the 5°C stability test are shown in Table 12, with the D0 sample used as the starting point. Compared to D0, no significant changes were observed in SEC-HPLC, CE-SDS-NR, and icIEF tests after 14 days of incubation at 5°C.

[0384] The stability test results are shown in Figures 15 to 20 As shown in Tables 11 and 12, the results support that at least BSP1 is the most stable compound relative to other orientations (BSP2 to BSP6, data not provided), with no change at 5°C and no significant aggregation and degradation at 40°C within 14 days, and is suitable for further drug development.

[0385] Table 11. Stability test results of BSP1 stable cell line monoclonal drugs at 40°C

[0386]

[0387] Table 12. Results of the monoclonal drug stability test of BSP1 at 5°C

[0388]

[0389]

[0390] Example 5 Chronic Animal Model of Persistent Retinal Neovascularization (up to 12 months)

[0391] The purpose of the animal study was to evaluate the efficacy and safety of the biologics BSP1, BSP1a, and aflibercept (a commercially available positive control (PC)) administered via intravitreal injection (IVT) in a rabbit model of persistent retinal neovascularization (PRNV or dl-AAA). The PRNV model mimics angiogenic retinal diseases to identify indications for drugs or novel therapies that may benefit from human angiogenic retinal diseases. The efficacy of BSP1 IVT injection was compared with that of aflibercept IVT injection by inhibition of the dl-AAA / PRVN model in an animal pharmacodynamic (PD) model (for details, see C. Patel et al., Exp. Eye Res., 2020, 195:108031).

[0392] 5.1 Experimental methods:

[0393] In this study, Dutch black belt rabbits were selected to obtain the best results feasibility. For each animal, only one eye was used to establish a retinal injury model (dl-AAA, PRNV rate is less than 70%) for characterization, and the same side eye was used as a control. In order to carry out further drug testing, one eye was examined for retinal damage. However, the compound will be administered to both eyes to study the drug toxicity / effect when sick and without disease. A unique component of this protocol is that ophthalmological endpoints are evaluated in awake animals rather than anesthetized animals. This eliminates the potential interference of anesthetics during the inspection process and additionally increases the likelihood that the animals will survive and the tracking period can be as long as several years.

[0394] Table 13. Animal study time points and procedures

[0395]

[0396]

[0397] 5.1.1 Standards for PRNV models used in pharmacological studies:

[0398] Persistent retinal neovascularization (PRNV) with FA leakage within 3 months before dosing. The area and intensity of neovascularization leakage were defined by stable angiographic leakage area >2 optic disc diameters and leakage severity ≥2 points on NaF angiography (0.05 mls, 10%, OCT / FA in 3 fields, 55°) (may differ from the study objectives).

[0399] 5.1.2 Effects of BSP1, BSP1a, and PC on FA leakage reduction and retinal contour changes in the PRNV model (dl-AAA eyes) effect:

[0400] Fundus photography (FP): Serial fundus photographs help monitor treatment response, inflammatory changes, and drug distribution.

[0401] Fluorescein Angiography (FA): FA imaging was performed at the same location as for color imaging. FA images were acquired according to the following schedule: Late FA images were recorded for dl-AAA eyes and control eyes at baseline, 1, 2, 4, 8, 12, and 16 weeks after treatment (possibly extended to 24 weeks). Angiographic leakage was observed and evaluated by clinical observation of FA treatments for BSP1, BSP1a, and PC.

[0402] Optical coherence tomography (OCT): OCT is a well-established medical imaging technique that uses light to capture three-dimensional images of retinal and choroidal structures at micrometer resolution, mapping and measuring their thickness. Analysis and measurement of retinal and choroidal thickness in OCT is performed in correlation with fundus color photography or FA studies.

[0403] Slit lamp examinations performed to evaluate safety and tolerability showed severe inflammatory reactions in the anterior chamber of the eye in rabbits treated with the test article.

[0404] All eyes will be observed by an ophthalmologist immediately after injection and on days 1 and 2. If there are signs of inflammation, follow-up will continue based on the effect and duration of the medication.

[0405] All our studies used the Standardized Uveitis Nomenclature (SUN). Using a slit lamp beam with a height and width of 1 x 1 mm at an angle of 45-60°, cells and flares in the anterior chamber were observed. Based on the findings, inflammation was graded from 0 to 3+ / (100%).

[0406] 5.2 Experimental Results

[0407] 5.2.1 Effect of the Test Article on Angiographic Leakage

[0408] The effects of BSP1, BSP1a, and PC1 on angiographic leakage were evaluated by late fluorescein angiography (FA) at 2, 4, 8, 12, and 16 weeks after intravitreal administration (Table 13). A total of 10 rabbits were used in the study. Seven rabbits received BSP1 in both eyes (dl-AAA model eyes (OD) and untreated eyes (OS); three rabbits received PC in the dl-AAA model eyes (OD) and PBS as the NC in the untreated eyes (OS). Figure 11 and 12 Representative images of leakage profiles at each time point between the two groups are shown. Figure 13 and 14 Representative images of the profiles of untreated eyes at each time point between the two groups are shown.

[0409] All PRNV model eyes (n=10) in both treatment groups showed no angiographic leakage at the RNV site 2 weeks after treatment. Different effects were observed between the two agents on the duration and intensity of inhibition of PRNV leakage in the FA. Leakage recurrence primarily began around 6 to 8 weeks in the PC1 (aflibercept) group, and around 12 to 16 weeks in the BSP1 or BSP1a group.

[0410] To evaluate and compare the effects of BSP1, BSP1a, and PC1 on vascular leakage in chronic animal / PD models. Figure 10 The vascular leakage scores of BSP1, BSP1a, and PC1 over time (0, 2, 4, 8, 12, and 16 weeks) were plotted in Figure 2. These scores were measured by calculating their corresponding vascular leakage percentage areas (%) based on FA images. In addition, FA images were taken for each group at 0, 2, 4, 8, 12, and 16 weeks ( Figure 11 ) to evaluate vascular leakage in compound-treated eyes (OD) and compound-treated non-DLAAA-induced eyes (OS) as controls.

[0411] according to Figure 11 and Table 14, compared with the animal group treated with the known VEGF inhibitor PC1 (aflibercept), BSP1 exhibited superior vascular leakage inhibition ability, regression of induced leaky vessels, angiogenesis, and longer duration.

[0412] Table 14. Changes in vascular leakage scores over time (weeks) and their SD (standard deviation) in chronic animal / PD models.

[0413]

[0414] The efficacy of BSP1a was also studied using the same model. Compared to the animal group treated with the known VEGF inhibitor PC1 (aflibercept), BSP1a also showed superior vascular leakage inhibition, regression of induced leaky vessels, angiogenesis, and longer duration of effect. The results of BSP1a were at least comparable to those of BSP1, such as Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 and as shown in Table 14.

[0415] BSP1 and BSP1a demonstrated superior vascular leakage control and anti-inflammatory therapeutic effects compared to aflibercept in animal models. Specifically, BSP1 and BSP1a successfully delayed the onset of leakage until week 12 and significantly slowed the progression of leakage, with an average leakage of less than 30% observed at week 16. In contrast, animals treated with aflibercept showed significant leakage at week 8, and leakage progressed rapidly, reaching an average of over 66% by week 12. This clearly demonstrates the superiority of BSP1 and BSP1a over aflibercept in terms of therapeutic effect and long-term duration of action.

[0416] Example 6. BSP1a is expected to have advantages over BSP1 in terms of purification and recovery

[0417] Mutations in BSP1a can improve long-term protein stability and scale up production yield / recovery at high protein concentrations for further drug processing and development.

[0418] 6.1 Long-term stability

[0419] Using the experimental method described in Example 4, stability assays of BSP1 and BSP1a were performed to assess developability.

[0420] The stability assay results are shown in Table 15. BSP1a has a better stability profile and protein expression yield than BSP1.

[0421] Table 15. Results of monoclonal drug stability testing of BSP1 and BSP1a at 5°C

[0422]

[0423] 6.2 Protein production yield

[0424] Both BSP1 and BSP1a constructs were further developed into stable cell lines using CHOK1 cells (Thermos Fisher). BSP1a produced a protein yield of 6.6 g / L, which was superior to BSP1's protein production yield of 3.6 g / L.

Claims

1. A multispecific molecule comprising a fusion protein, wherein the fusion protein comprises (a) complement component 5 (C5) binding domain, (b) a vascular endothelial growth factor (VEGF) binding domain, and (c) a multimerized component; in: The C5 binding domain comprises an antigen-binding fragment of an anti-C5 antibody, The VEGF binding domain comprises one or more extracellular immunoglobulin-like (Ig) domains of one or more VEGF receptors (VEGFRs), and The multimerization components include polypeptides between 1 and 200 amino acids in length and having at least one cysteine residue.

2. The multispecific molecule of claim 1, wherein the C5 binding domain is operably linked to the N-terminus of the VEGF binding domain.

3. The multispecific molecule of claim 1 or 2, wherein the VEGF binding domain is operably linked to the N-terminus of the multimerization component.

4. The multispecific molecule of any preceding claim, wherein the multimerization component comprises an antibody Fc domain.

5. The multispecific molecule of any of the preceding claims, wherein the antigen binding fragment is a Fab, a Fab', a F(ab)2, a F(ab')2, a single-chain Fab, a VHH, a Fd, a Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a diabody, a disulfide-stabilized diabody (dsdiabody), a single-chain Fv (scFv), a scFv dimer (bivalent diabody), a camelized single domain antibody, a nanobody, a tetrabody, a domain antibody, or a bivalent domain antibody.

6. The multispecific molecule of any of the preceding claims, wherein the antigen-binding fragment comprises: three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable (VH) region, wherein the VH region comprises the amino acid sequence as shown in SEQ ID NO: 1, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable (VL) region, wherein the VL region comprises the amino acid sequence as shown in SEQ ID NO:

2.

7. The multispecific molecule of any of the preceding claims, wherein the antigen-binding fragment comprises: a HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence shown in SEQ ID NO:

8.

8. The multispecific molecule of any of the preceding claims, wherein the antigen-binding fragment comprises a VH region comprising the amino acid sequence as shown in SEQ ID NO: 1 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 1 but still retains binding specificity for C5.

9. The multispecific molecule of any of the preceding claims, wherein the antigen-binding fragment comprises a VL region comprising the amino acid sequence as shown in SEQ ID NO: 2 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 2 but still retains binding specificity for C5.

10. The multispecific molecule of any of the preceding claims, wherein the antigen-binding fragment comprises: a VH region comprising the amino acid sequence shown in SEQ ID NO: 1 and a VL region comprising the amino acid sequence shown in SEQ ID NO:

2.

11. The multispecific molecule of any one of claims 1 to 5, wherein the antigen binding fragment comprises (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable (VH) region, wherein the VH region comprises the amino acid sequence as shown in SEQ ID NO: 28, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable (VL) region, wherein the VL region comprises the amino acid sequence as shown in SEQ ID NO: 31; or (b) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable (VH) region, wherein the VH region comprises the amino acid sequence as shown in SEQ ID NO: 28, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable (VL) region, wherein the VL region comprises the amino acid sequence as shown in SEQ ID NO: 36; or (c) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable (VH) region, wherein the VH region comprises the amino acid sequence as shown in SEQ ID NO: 1, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained in a light chain variable (VL) region, wherein the VL region comprises the amino acid sequence as shown in SEQ ID NO:

39.

12. The multispecific molecule of any one of claims 1 to 5 and 11, wherein the antigen binding fragment comprises (a) a HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 29 or SEQ ID NO: 41 or SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 30 or SEQ ID NO: 35; or (b) a HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30; or (c) a HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 41, a LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35; or (d) a HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence shown in SEQ ID NO:

30.

13. The multispecific molecule of any one of claims 1 to 5, 11 and 12, wherein the antigen-binding fragment comprises a VH region comprising the amino acid sequence as shown in SEQ ID NO: 28 or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 28 but still retains binding specificity for C5.

14. The multispecific molecule of any one of claims 1 to 5, 11 to 13, wherein the antigen binding fragment comprises a VL region comprising an amino acid sequence as shown in SEQ ID NO: 31, 36 or 39, or a homologous sequence thereof that has at least 80% sequence identity to SEQ ID NO: 31, 36 or 39 but still retains binding specificity for C5.

15. The multispecific molecule of any one of claims 1 to 5, 11 to 14, wherein the antigen binding fragment comprises (a) a VH region comprising the amino acid sequence shown in SEQ ID NO: 28, and a VL region comprising the amino acid sequence shown in SEQ ID NO: 31; or (b) a VH region comprising the amino acid sequence shown in SEQ ID NO: 28, and a VL region comprising the amino acid sequence shown in SEQ ID NO: 36; or (c) a VH region comprising the amino acid sequence shown in SEQ ID NO: 1, and a VL region comprising the amino acid sequence shown in SEQ ID NO:

39.

16. The multispecific molecule of any one of claims 6 to 15, wherein the antigen-binding fragment further comprises one or more amino acid residue substitutions or modifications but still retains binding specificity for C5.

17. The multispecific molecule of any one of the preceding claims, wherein the VEGFR is selected from the group consisting of VEGFR-1, VEGFR-2, and VEGFR-3.

18. The multispecific molecule of any of the preceding claims, wherein the Ig domains are selected from the group consisting of: Ig domain 1, Ig domain 2, Ig domain 3, and Ig domain 4.

19. The multispecific molecule of any one of the preceding claims, wherein the VEGF binding domain comprises two or more different Ig domains of two or more different VEGFRs.

20. The multispecific molecule of any of the preceding claims, wherein the VEGF binding domain comprises a first Ig domain of a first VEGFR, wherein the first Ig domain is operably linked directly or via a first linker to the N-terminus of a second Ig domain of a second VEGFR.

21. The multispecific molecule of claim 20, wherein the first Ig domain is Ig domain 2, and the second Ig domain is Ig domain 2 or Ig domain 3.

22. The multispecific molecule of claim 20, wherein the first VEGFR is VEGFR-1 and the second VEGFR is VEGFR-2.

23. The multispecific molecule of any of the preceding claims, wherein the VEGF binding domain comprises Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2.

24. The multispecific molecule of claim 23, wherein the Ig domain 2 of VEGFR-1 is operably linked to the N-terminus of the Ig domain 3 of VEGFR-2 directly or via the first linker.

25. The multispecific molecule of claim 23, wherein the Ig domain 2 of VEGFR-1 comprises the amino acid sequence as shown in SEQ ID NO:9, and the Ig domain 3 of VEGFR-2 comprises the amino acid sequence as shown in SEQ ID NO:

10.

26. The multispecific molecule of any one of claims 20 to 25, wherein the first linker comprises a peptide linker.

27. The multispecific molecule of claim 26, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 18 (TNTII).

28. The multispecific molecule of any of the preceding claims, wherein the VEGF binding domain comprises the amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto but still retaining binding specificity for VEGF.

29. The multispecific molecule of any of the preceding claims, wherein the C5 binding domain is operably linked to the VEGF binding domain directly or via a second linker.

30. The multispecific molecule of any preceding claim, wherein the second linker comprises a peptide linker, optionally a GS linker.

31. The multispecific molecule of claim 30, wherein the second linker comprises a GS linker.

32. The multispecific molecule of claim 31, wherein the GS linker comprises one, two, three, four, or more repeats of SEQ ID NO: 13 (GGGS) or SEQ ID NO: 14 (GGGGS).

33. The multispecific molecule of any one of claims 4 to 32, wherein the Fc domain is derived from a human immunoglobulin (Ig).

34. The multispecific molecule of claim 33, wherein the Fc domain is derived from human IgG, optionally human IgG1, IgG2, IgG3 or IgG4.

35. The multispecific molecule of claim 34, wherein the Fc domain is derived from human IgG1.

36. The multispecific molecule of any one of claims 4 to 32, wherein the Fc domain is mutated.

37. The multispecific molecule of claim 36, wherein the Fc domain comprises a mutation at position 235 and / or 309 of the human IgGl according to the EU numbering system.

38. The multispecific molecule of claim 36, wherein the Fc domain comprises a substitution at position 235 and / or 309 of the human IgGl according to the EU numbering system.

39. The multispecific molecule of claim 36, wherein the Fc domain comprises L235K and / or L309K mutations according to the EU numbering system.

40. The multispecific molecule of any of the preceding claims, wherein the Fc domain comprises the amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 26, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 26 but still retains the ability to multimerize.

41. A multispecific molecule according to any of the preceding claims, wherein the multimerization component is operably linked to the C-terminus of the VEGF binding domain directly or via a third linker, optionally, the third linker is a peptide linker, further optionally, the third linker comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:

25.

42. The multispecific molecule of any of the preceding claims, wherein the fusion protein comprises the C5 binding domain, wherein the C5 binding domain comprises a Fab domain, wherein the Fab domain comprises a heavy chain polypeptide and a light chain polypeptide.

43. The multispecific molecule of claim 42, wherein the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and / or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

17.

44. The multispecific molecule of claim 42, wherein (a) the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32, and / or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33; or (b) the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32, and / or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 37; or (c) the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and / or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

40.

45. The multispecific molecule of claim 42, wherein the heavy chain polypeptide is operably linked to the N-terminus of the VEGF binding domain directly or via the second linker.

46. The multispecific molecule of claim 42, wherein the fusion protein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

17.

47. The multispecific molecule of claim 42, wherein the fusion protein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 34; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

33.

48. The multispecific molecule of claim 42, wherein the heavy chain polypeptide is operably linked directly or via a linker to the C-terminus of the multimerization component, and the multimerization component is operably linked directly or via a linker to the C-terminus of the VEGF binding domain.

49. The multispecific molecule of any of the preceding claims, wherein the fusion protein comprises the C5 binding domain, the C5 binding domain comprises an scFv comprising the VH region and the VL region operably linked via a linker.

50. The multispecific molecule of claim 49, wherein the fusion protein comprises the scFv, wherein the scFv is operably linked to the N-terminus of the VEGF binding domain directly or via a linker.

51. The multispecific molecule of claim 50, wherein the fusion protein comprises the scFv, wherein the scFv is operably linked directly or via a linker to the C-terminus of the multimerization component, and the multimerization component is operably linked directly or via a linker to the C-terminus of the VEGF binding domain.

52. The multispecific molecule of any of the preceding claims, wherein the fusion protein comprises the C5 binding domain, wherein the C5 binding domain comprises a VHH domain.

53. The multispecific molecule of claim 52, wherein the fusion protein comprises the VHH domain operably linked to the N-terminus of the VEGF binding domain directly or via a linker.

54. The multispecific molecule of claim 53, wherein the fusion protein comprises the VHH domain, wherein the VHH domain is operably linked directly or via a linker to the C-terminus of the multimerization component, and the multimerization component is operably linked directly or via a linker to the C-terminus of the VEGF binding domain.

55. The multispecific molecule of any of the preceding claims, wherein the multispecific molecule comprises a dimer of the fusion protein.

56. A pharmaceutical composition, wherein the pharmaceutical composition comprises the multispecific molecule according to any one of the preceding claims, and one or more pharmaceutically acceptable carriers.

57. An isolated polynucleotide, wherein the isolated polynucleotide encodes the multispecific molecule of any one of the preceding claims.

58. A vector, wherein the vector comprises the isolated polynucleotide according to claim 57.

59. A host-expression system, wherein the host-expression system comprises the vector according to claim 58.

60. The host expression system of claim 59, wherein the host expression system is a microorganism, yeast or mammalian cell.

61. A method of expressing a multispecific molecule according to any one of claims 1 to 55, wherein the method comprises culturing a host expression system according to any one of the preceding claims under conditions that express a vector according to any one of the preceding claims.

62. A method of treating, preventing or ameliorating a C5-related and / or VEGF-related disease, disorder or condition in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the multispecific molecule of any one of claims 1 to 55 and / or the pharmaceutical composition of claim 56.

63. A method for treating, preventing or ameliorating a disease, disorder or condition associated with increased levels and / or activity of C5 and / or VEGF in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the multispecific molecule of any one of claims 1 to 55 and / or the pharmaceutical composition of claim 56.

64. The method according to claim 62 or 63, characterized in that The disease, disorder or condition is selected from the group consisting of an ocular disease, cancer, an inflammatory disease, an autoimmune disease, angiogenesis, vascular permeability, edema and inflammation.

65. The method of claim 64, wherein the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), macular edema, macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), central retinal vein occlusion, corneal neovascularization (CNV), retinitis pigmentosa (RP), ocular angiogenesis (ocular neovascularization affecting choroidal, corneal or retinal tissue), retinopathy of prematurity (ROP), pathological myopia, vascular glaucoma, retinoblastoma, retinal vein occlusion, uveitis and neuromyelitis optica.

66. The method of claim 64, wherein the ocular disease is AMD or GA.

67. The method of claim 66, wherein the AMD is wet AMD.

68. The method of claim 66, wherein the AMD is dry AMD.

69. The method of any one of claims 62 to 68, wherein the subject is a human.

70. The method of any one of claims 62 to 69, wherein the administration is via intraocular, intravitreal injection, topical, subcutaneous, intraperitoneal, intravenous (including intravenous infusion), intramuscular, intradermal injection, oral, intranasal, intraocular, intravitreal injection, sublingual or rectal administration.

71. The method of any one of claims 62 to 70, wherein the method further comprises administering a therapeutically effective amount of a second therapeutic agent.

72. The method of claim 71, wherein the second therapeutic agent is selected from the group consisting of anti-angiogenic agents, inflammatory drugs, m-Tor inhibitors, rapamycin, everolimus, temsirolimus, cyclosporine, anti-TNF agents, anti-complement agents, and nonsteroidal anti-inflammatory agents.

73. A method of modulating C5 and / or VEGF activity in a cell, wherein the method comprises exposing the cell to a multispecific molecule according to any one of claims 1 to 55.

74. The method of claim 73, wherein the cells are selected from the group consisting of retinal ganglion cells, rods, cones, glial cells (eg, Müller glial cells), bipolar cells, amacrine cells, and horizontal cells.

75. The multispecific molecule of any one of claims 1 to 55 and / or the pharmaceutical composition of claim 56 for use in treating, preventing or ameliorating a C5-related and / or VEGF-related disease, disorder or condition in a subject.

76. Use of the multispecific molecule of any one of claims 1 to 55 and / or the pharmaceutical composition of claim 56 in the preparation of a medicament for treating, preventing or ameliorating a C5-related and / or VEGF-related disease, disorder or condition in a subject.

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