C5 / VEGF dual specific binding molecule

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to bispecific binding molecules directed against human vascular endothelial growth factor (VEGF / VEGF-A) and complement component 5 (C5), methods for their preparation and therapeutic uses. [Background technology]

[0002] Age-related macular degeneration (AMD) is one of the leading causes of blindness in the elderly (Rein et al., Arch Ophthalmology, 127:533-540, 2009). AMD is typically a disease of the elderly and is one of the leading causes of blindness in individuals over the age of 50 in developed countries. In the absence of adequate preventive or therapeutic treatments, the number of AMD cases associated with vision loss is expected to increase as the population ages.

[0003] AMD is characterized by progressive degeneration of photoreceptors in the macula, the outer retina, and the retinal pigment epithelium. Advanced AMD can occur in two forms: dry (atrophic) AMD and wet AMD. Anti-VEGF agents (e.g., anti-VEGF antibodies, VEGF traps, etc.) are now widely used to treat wet AMD by inhibiting neovascularization and angiogenesis. This reduces the progression of choroidal neovascularization and vision loss due to the downstream effects of neovascularization in most patients and has been observed to reduce some aspects of inflammation in animal models.

[0004] The later stages of AMD are characterized by either choroidal neovascularization or geographic atrophy (GA). GA tends to affect more than 20% of AMD patients. More than 8 million patients worldwide are affected by GA, with AMD patients diagnosed with GA in at least one eye. Currently, there are no effective treatments to treat or slow the progression of GA symptoms, such as the inability to read or missing parts of vision while reading, the need for additional light for reading in the dark or for normal activities, and reduced visual resolution requiring detailed vision (such as difficulty recognizing faces and fading of vision due to the death of retinal pigment epithelium (RPE) cells in the visual center).

[0005] Anti-VEGF therapy is not effective in treating GA (Park, DH et al., Front. Immunol., May 15, 2019). It has even been reported that anti-VEGF therapy may accelerate the progression of GA (Gemenetzi, M. et al., Eye (Lond). 2017 Jan, 31(1):1-9). Complement inhibitors targeting the regulation of complement proteins C3, C5, factor B, factor D, and properdin have been investigated for the potential treatment of GA, but no successful results have been reported yet. 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 significant clinical need for the treatment of AMD and GA, particularly for preventing 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. Methods for producing the multispecific molecules are also disclosed, 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, the multispecific molecule 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 having a length of 1 to 200 amino acids and at least one cysteine ​​residue.

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

[0010] In another aspect, the present disclosure provides isolated polynucleotides encoding the multispecific molecules provided herein.

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

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

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

[0014] In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a C5-related and / or VEGF-related disease, disorder, or condition 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.

[0015] In another aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease, disorder, or condition associated with elevated 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 ocular diseases, cancer, inflammatory diseases, autoimmune diseases, angiogenesis, vascular permeability, edema, and inflammation.

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

[0018] In another aspect, the present 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 present 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 explanation of the drawings]

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

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

[0022] [Figure 3] FIG. 3 is a third structure of a VEGF / C5 bispecific antibody (BSP3).

[0023] [Figure 4] FIG. 4 is a fourth structure of a VEGF / C5 bispecific antibody (BSP4).

[0024] [Figure 5] FIG. 5 is a fifth structure of a VEGF / C5 bispecific antibody (BSP5).

[0025] [Figure 6]FIG. 6 is a sixth structure of a VEGF / C5 bispecific antibody (BSP6).

[0026] [Figure 7] Figures 7 and 8 are dose-response curves for BSP1 and PC1 (aflibercept) in duplicate VEGF-mediated cell proliferation assays. [Figure 8] Figures 7 and 8 are dose-response curves for BSP1 and PC1 (aflibercept) in duplicate VEGF-mediated cell proliferation assays.

[0027] [Figure 9A] Figures 9A and 9B are dose-response curves for BSP1 and PC2 (eculizumab) in duplicate CH50 assays. [Figure 9B] Figures 9A and 9B are dose-response curves for BSP1 and PC2 (eculizumab) in duplicate CH50 assays.

[0028] [Figure 10] FIG. 10 shows the vascular leakage score versus time in a chronic experimental animal / PD model with standard deviation.

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

[0030] [Figure 12] FIG. 12: Progression of vascular leakage over time (0, 12, and 16 weeks) in a chronic experimental animal / PD model.

[0031] [Figure 13] FIG. 13 shows the effect of BSP1 on untreated eyes over time (0, 2, 4, 8, 12, and 16 weeks).

[0032] [Figure 14]FIG. 14 shows the effect of PBS on untreated eyes over time (0, 2, 4, 8, 12, and 16 weeks).

[0033] [Figure 15] FIG. 15 is a SEC-HPLC chromatogram of a sample (BSP1) in a single clone drug stability study at 40° C.

[0034] [Figure 16] FIG. 16 is a CE-SDS-NR electropherogram of sample / BSP1 at 40° C. in a single clone drug stability study.

[0035] [Figure 17] FIG. 17 is an icIEF electropherogram of sample / BSP1 at 40° C. in a single clone drug stability study.

[0036] [Figure 18] FIG. 18 is a SEC-HPLC chromatogram of sample / BSP1 at 5° C. in a single clone drug stability study.

[0037] [Figure 19] FIG. 19 is a CE-SDS-NR electropherogram of sample / BSP1 at 5° C. in a single clone drug stability study.

[0038] [Figure 20] FIG. 20 is an icIEF electropherogram of sample / BSP1 at 5° C. in a single clone drug stability study.

[0039] [Figure 21A] FIG. 21 is a specific sequence disclosed in this disclosure. [Figure 21B] FIG. 21 is a specific sequence disclosed in this disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0040] Before proceeding to a detailed description of the present invention, the following notes and definitions are provided.

[0041] All descriptions provided herein are intended to be illustrative of various embodiments of the present invention provided in this disclosure. Therefore, the specific variations discussed herein should not be construed as limiting the scope of the present disclosure. Those skilled in the art will recognize that various equivalents, changes, and modifications may be made without departing from the scope of the present disclosure, and will understand that such equivalent embodiments are also included herein.

[0042] All references cited in this disclosure (including patent applications, issued patents, published articles, and other publications) are incorporated by reference in their entirety and are intended to provide methodologies that may be used in connection with the description provided herein. With respect to any terms presented in one or more publications that are similar or identical to terms expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure shall control in all respects.

[0043] All technical and scientific terms used in this disclosure, unless expressly defined otherwise, are generally assumed to have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0044] As used herein, i.e., throughout this disclosure, the articles "a," "an," and "one of," unless otherwise specified, are to be construed as meaning "one or more" or "at least one." By way of example, "a molecule" means one molecule or more than one molecule.

[0045] As used herein, terms such as "about," "approximately," or "approximately" refer to an amount, 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 amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, when the terms "about" or "approximately" precede a numerical value, it refers to a range of 15%, 10%, 5%, or 1% plus or minus the stated value.

[0046] As used herein, the terms "comprises," "comprises," "includes," "includes," "contains," "containing," "has," "having," and the like are synonymous, inclusive, and open-ended and do not exclude additional elements, features, steps, acts, operations, etc.

[0047] As used herein, the term "or" is used in its inclusive (and not exclusive) sense, so that, for example, when used to connect 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. I. Definitions and Abbreviations

[0049] This section provides definitions of some general terms. Definitions of other terms may be found in other sections of this disclosure below.

[0050] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to designate a linear series of amino acid residues linked together by peptide bonds, and include proteins, polypeptides, oligopeptides, peptides, and fragments thereof. Proteins may be composed of naturally occurring amino acids and / or synthetic (e.g., modified or non-naturally occurring) amino acids. Thus, "amino acid" or "peptide residue" as used herein refers to both naturally occurring and synthetic amino acids. The terms "polypeptide," "peptide," and "protein" include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusion proteins with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, fusion proteins with a detectable fusion partner (e.g., fusion proteins containing a fluorescent protein, β-galactosidase, luciferase, etc. as a fusion partner), and the like.

[0051] As used herein, the term "amino acid" refers to a building block of a protein, peptide, polypeptide, or amino acid polymer, and 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 amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. As used herein, naturally occurring amino acids include the group of naturally occurring carboxy alpha 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).

[0052] As used herein, the term "domain" refers to a globular structure formed by one or more regions of one or more polypeptide chains, including, for example, beta-pleated sheets and / or peptide loops (e.g., containing 3-4 peptide loops) stabilized by intrachain disulfide bonds. Examples include Fab domains (see below for details). Note that in this disclosure, the terms "domain" and "region" may be used interchangeably.

[0053] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," "polynucleotide," and the like are intended to refer to nucleotide polymers of any length, can include both DNA and RNA, and can be single- or double-stranded.

[0054] 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 reach 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) compared to the reference sequence to be compared by the total number of amino acid residues (or bases) in either 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 the percentage of amino acid (or nucleic acid) sequence identity can be achieved using publicly available tools such as BLASTN, BLASTp (available at the National Center for Biotechnology Information (NCBI) website; see also Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website; see also Higgins D. Get al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize the parameters as appropriate for the alignment, for example by selecting an appropriate algorithm.

[0055] "Conservative substitution" in relation to 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 substitution can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), residues with acidic side chains (e.g., Asp, Glu), amino acid residues with basic side chains (e.g., His, Lys, and Arg), or residues with aromatic side chains (e.g., Trp, Tyr, and Phe).As is known in the art, conservative substitutions usually do not cause significant changes in the conformational structure of proteins, so that the biological activity of proteins can be maintained.

[0056] As used herein, the term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. As used herein, the term "antibody" is broadly interpreted to encompass not only conventional immunoglobulins, which contain two heavy (H) chains and two light (L) chains, but also non-conventional antibodies, such as heavy-chain antibodies, which contain only heavy chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and each heavy chain contains a variable region (V H ) and the first, second, third, and optionally fourth constant regions (C H1 , C H2 , C H3 , C H4 Mammalian light chains are classified as lambda or kappa, and each light chain consists of a variable region (V L) and constant regions. Antibodies are "Y" shaped, with the stem of the Y consisting of the second and third constant regions of two heavy chains linked together by disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable 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 both chains generally contain three hypervariable loops called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3; heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be determined according to the methods described by Kabat, IMGT, Chothia, or Al-Lazikani (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, 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 (second edition), chapter 26, 481-514, (2015)).The three CDRs are flanked by flanking stretches called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exert various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the heavy chain constant region. The five major antibody classes or isotypes are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are further 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), and IgA2 (α2 heavy chain).

[0057] 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 a component of the immune system, e.g., an antibody. As used herein, the term "antigen" includes antigenic epitopes, e.g., fragments of an antigen that are antigenic epitopes.

[0058] 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. The term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0059] As used herein, the term "multispecific molecule" refers to an artificial or engineered molecule that can simultaneously bind to at least two different epitopes. The two epitopes may be present on the same antigen or on two different antigens. A bispecific molecule is essentially a type of multispecific molecule.

[0060] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the entire native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), diabodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen as the parent antibody.

[0061] "Fab," in reference to an antibody, refers to the portion of an antibody that consists of a single light chain (both variable and constant regions) joined by a disulfide bond to the variable region and first constant region of a single heavy chain.

[0062] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0063] "F(ab')2" refers to a Fab' dimer.

[0064] "Fv" with respect to an antibody refers to the minimum fragment of an antibody that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0065] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains, i.e., two VH moieties linked by peptide linkers (e.g., long flexible linkers) and connected to two VL moieties via disulfide bridges, respectively. In some embodiments, a dsFv-dsFv' is bispecific, with the disulfide-paired heavy and light chains each having a different antigen specificity.

[0066] A "single-chain Fv antibody" or "scFv" refers to a multispecific molecule consisting of a light chain variable region and a heavy chain variable region linked to each other either directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)).

[0067] "Fc," with respect to an antibody (e.g., of the IgG, IgA, or IgD isotype), refers to the portion of the antibody consisting essentially of the second and third constant domains of a first heavy chain linked via disulfide bonds to the second and third constant domains of a second heavy chain. For antibodies of the IgM and IgE isotypes, the Fc further comprises a fourth constant domain. The Fc portion of an antibody performs various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0068] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to a multispecific molecule consisting of an scFv linked to the Fc region of an antibody.

[0069] "Camelized single domain antibody," "heavy chain antibody," or "HCAb" refers to an antibody that contains two VH domains and no 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 are originally derived from camelids (camels, dromedaries, and llamas). Although camelized antibodies lack light chains, they have authentic antigen-binding repertoires (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) is the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November; 21(13):3490-8. Epub 2007 Jun 15(2007)).

[0070] "Nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant domains (CH2 and CH3).

[0071] "Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, which comprise a VH domain linked to a VL domain (VH-VL or VL-VH) on the same polypeptide chain (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8(1993), EP404097, WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thereby forming two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In one embodiment, a "bispecific dsdiabody" is a diabody that targets two different antigens (or epitopes). In one embodiment, an "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv) in which one VH-VL moiety (linked by a peptide linker) is dimerized with another VH-VL moiety such that one VH moiety cooperates with the other VL moiety to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In another embodiment, an "scFv dimer" is a bispecific diabody in which VH1-VL2 (linked by a peptide linker) associates with VL1-VH2 (also linked by a peptide linker) such that VH1 and VL1 cooperate and VH2 and VL2 cooperate, with each cooperated pair having a different antigen specificity.

[0072] A "domain antibody" refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH domains are covalently linked using a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

[0073] As used herein, the term "vector" refers to a vehicle into which a genetic element may be operably inserted to cause expression of the genetic element (such as for production of a protein, RNA, or DNA encoded by the genetic element) or replication of the genetic element. Vectors may be used to transform, transduce, or transfect host cells to cause expression of the genetic element carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, 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. Vectors may contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Vectors may also contain an origin of replication. Vectors may also contain materials that aid in cell entry. These materials include, but are not limited to, viral particles, liposomes, or protein coatings. Vectors may be expression vectors or cloning vectors. The present disclosure provides vectors (e.g., expression vectors) comprising a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0074] As used herein, the phrase "host cell" refers to a cell into which exogenous polynucleotides and / or vectors can be or have been introduced.

[0075] The phrase "operably linked" refers to a juxtaposition allowing the components to function normally. II.C5 / VEGFR multispecific molecule

[0076] In one aspect, the present disclosure provides a multispecific molecule comprising a fusion protein, the multispecific molecule 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.

[0077] As used herein, a fusion protein can be a single polypeptide chain or a polypeptide complex comprising two or more polypeptide chains associated with one another. A. Antigen-binding fragment

[0078] 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.

[0079] C5 is a component of the complement system, a part of the innate immune system that plays an important role in inflammation, host homeostasis, and host defense against pathogens. The C5 protein contains the C5α and C5β chains linked by disulfide bridges. The C5 protein can be proteolytically processed to generate multiple protein products, including the C5α chain, C5β chain, C5a anaphylatoxin, and C5b.

[0080] An "anti-C5 antibody" is an antibody capable of specifically binding to C5, e.g., human C5. The anti-C5 antibody can be a conventional IgG antibody or can be a single domain antibody, such as a camelized single domain antibody comprising a heavy chain variable region (VH), or a llama anti-C5 single domain antibody.

[0081] In certain embodiments, the anti-C5 antibody comprises or is derived from eculizumab.

[0082] In some 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 light chain variable region of eculizumab. In some embodiments, the antigen-binding fragment of an anti-C5 antibody comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in the heavy chain variable (VH) region comprising the amino acid sequence set forth in SEQ ID NO: 1, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in the light chain variable (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0083] In some embodiments, the antigen-binding fragment of an anti-C5 antibody comprises the six CDRs of eculizumab. In some embodiments, the antigen-binding fragment of an anti-C5 antibody provided herein comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0084] [Table 1]

[0085] In certain embodiments, the antigen-binding fragment of an anti-C5 antibody is derived from or comprises a variant of eculizumab, including, for example, but not limited to, an affinity variant of eculizumab or a glycosylation variant of eculizumab.

[0086] The term "variant" with respect to a parent antibody refers to any antibody that has a structure or sequence derived from the parent antibody and whose structure / sequence is sufficiently similar to that of the parent antibody. Modifications to obtain a variant include, for example, the addition, deletion, and / or substitution of one or more amino acid residues. A variant may have one or more conservative amino acid substitutions.

[0087] In some embodiments, the antigen-binding fragment of the anti-C5 antibody is derived from a variant of eculizumab and contains one or more amino acid residue substitutions or modifications compared to eculizumab, while retaining binding specificity and / or affinity for C5. The substitutions or modifications can occur in one or more CDR sequences and / or VH and / or VL sequences of eculizumab.

[0088] In some embodiments, antigen-binding fragments of anti-C5 antibodies provided herein contain one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences of eculizumab. In some embodiments, antigen-binding fragments of anti-C5 antibodies provided herein contain no more than a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the CDR and / or FR sequences listed in Table 1A.

[0089] In one embodiment, a variant of eculizumab, or an antigen-binding fragment derived from such a variant, has similar or improved affinity for human C5 compared to the parent antibody, eculizumab.

[0090] As used herein, the term "affinity" refers to the strength of non-covalent interaction between an antibody or its fragment and an antigen.The affinity of an antibody to an antigen can be measured by the equilibrium dissociation constant KD using methods well known in the art (see generally Davies et al., Ann.Rev.Biochem.1990,59:439-15 473).For example, Biacore is a typical device for detecting molecular interactions based on surface plasmon resonance (SPR) technology, and is the gold standard for determining antibody affinity.It is also well known in the art that enzyme-linked immunosorbent assay (ELISA) is a common means for determining binding affinity.

[0091] In some embodiments, the eculizumab variant or antigen-binding fragment derived from such a variant is capable of specifically binding to C5 with a K value of 10 nM or less (e.g., 8 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, or 300 pM or less) as measured by Biacore.

[0092] Methods for generating and obtaining affinity variants of a parent antibody are known in the art. For example, variants can be screened for binding affinity to their intended target (e.g., human C5) to identify variants with high affinity for the antigen.

[0093] In some embodiments, the eculizumab variants of the present invention or fusion proteins derived from such variants have improved druggability properties, e.g., when expressed in mammalian cells such as CHO cells, have one or more properties selected from: (i) better expression than wild-type eculizumab or its fusion protein, (ii) greater feasibility of purification to high purity, and (iii) greater stability.

[0094] In some embodiments of the present invention, the eculizumab variant or fusion protein thereof exhibits an increased expression level compared to wild-type eculizumab or its fusion protein. In some embodiments, the increased expression level occurs in a mammalian cell expression system. The expression level can be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of eculizumab variant or its fusion protein in a cell culture supernatant, preferably after one-step affinity chromatography purification. For example, the amount of eculizumab variant or its fusion protein in a 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 is increased by at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold or more compared to wild-type eculizumab or its fusion protein.

[0095] In some embodiments, the eculizumab variants or fusion proteins thereof of the present invention exhibit higher purity than wild-type eculizumab or fusion proteins thereof. In some embodiments, the purity of the protein is determined by SEC-HPLC, CE-SDS-NR, or icIEF techniques. In some preferred embodiments, after purification, the eculizumab variants or fusion proteins thereof can reach a purity of more than 65%, 70%, 75%, 80%, or 85%, preferably more than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 99%.

[0096] In one embodiment, the eculizumab variant or fusion protein thereof has improved thermal stability in a temperature range of -20°C to 60°C. In one embodiment, the eculizumab variant or fusion protein thereof does not have significant aggregation or degradation in a temperature range of -20°C to 60°C. In one embodiment, the eculizumab variant or fusion protein thereof does not have significant aggregation or degradation at 5°C. In one embodiment, the eculizumab variant or fusion protein thereof does not have significant aggregation or degradation in a temperature range of 40°C. In one embodiment, the eculizumab variant or fusion protein thereof exhibits aggregation or degradation of 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% after storage at 5°C for 14 days.

[0097] In certain embodiments, variants of eculizumab provided herein comprise an HCDR1 with no more than three, two, or one amino acid substitutions in SEQ ID NO:3, an HCDR2 with no more than three, two, or one amino acid substitutions in SEQ ID NO:4, an HCDR3 with no more than three, two, or one amino acid substitutions in SEQ ID NO:5, an LCDR1 with no more than three, two, or one amino acid substitutions in SEQ ID NO:6, an LCDR2 with no more than three, two, or one amino acid substitutions in SEQ ID NO:7, and / or an LCDR3 with no more than three, two, or one amino acid substitutions in SEQ ID NO:8.

[0098] In one embodiment, an antigen-binding fragment of an anti-C5 antibody provided herein comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 27, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 41 or SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 30 or SEQ ID NO: 35.

[0099] In one embodiment, an antigen-binding fragment of an anti-C5 antibody provided herein comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0100] In one embodiment, an antigen-binding fragment of an anti-C5 antibody provided herein comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0101] In one embodiment, an antigen-binding fragment of an anti-C5 antibody provided herein comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0102] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0103] In one embodiment, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 1, or a homologous sequence thereof having at least 80% sequence identity with SEQ ID NO: 1 while retaining binding specificity to C5.

[0104] In one embodiment, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 2, or a homologous sequence thereof having at least 80% sequence identity with SEQ ID NO: 2 while retaining binding specificity to C5.

[0105] In one embodiment, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 28, or a homologous sequence thereof having at least 80% sequence identity with SEQ ID NO: 28 while retaining binding specificity to C5.

[0106] In one embodiment, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 31, or a homologous sequence thereof having at least 80% sequence identity with SEQ ID NO: 31 while retaining binding specificity to C5.

[0107] In one embodiment, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 36, or a homologous sequence thereof having at least 80% sequence identity with SEQ ID NO: 36 while retaining binding specificity to C5.

[0108] In one embodiment, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 39, or a homologous sequence thereof having at least 80% sequence identity with SEQ ID NO: 39 while retaining binding specificity to C5.

[0109] As used herein, the terms "homologous," "substantially homologous," and "substantial homology" refer to a sequence of amino acids that has at least 50%, 60%, 70%, 80%, or 90% identity when comparing one sequence of amino acids to a reference sequence. The percentage of sequence identity or homology is calculated by comparing one sequence to the other when aligned to the corresponding portion of the reference sequence.

[0110] In one embodiment, the antigen-binding fragment of the anti-C5 antibody 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.

[0111] In one embodiment, the antigen-binding fragment of the anti-C5 antibody 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.

[0112] In one embodiment, the antigen-binding fragment of an anti-C5 antibody 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:36.

[0113] In one embodiment, the antigen-binding fragment of the anti-C5 antibody 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:39.

[0114] Without wishing to be bound by any theory, it has been found that certain variants of eculizumab achieve unexpected benefits, for example, in stability and purification, which are useful in manufacturing, particularly in scale-up manufacturing and process development. For example, eculizumab variant #1 as provided herein is more stable than eculizumab, exhibits higher purity in stability tests (see Example 4 for details), and exhibits much higher protein production yields in recombinant expression (see Example 4 for details).

[0115] In some embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a Fab domain, which in some embodiments comprises a heavy chain polypeptide comprising a VH domain and a light chain polypeptide comprising a VL domain, wherein the VH and VL domains associate to form a C5-binding domain.

[0116] In one embodiment, a 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 one embodiment, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, a Fab domain has a heavy chain comprising the amino acid sequence of SEQ ID NO: 21.

[0117] In one embodiment, a 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 one embodiment, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, a Fab domain has a heavy chain comprising the amino acid sequence of SEQ ID NO: 32.

[0118] In one embodiment, a 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 one embodiment, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, a Fab domain has a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0119] In one embodiment, a 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 one embodiment, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, a Fab domain has a light chain comprising the amino acid sequence of SEQ ID NO: 33.

[0120] In one embodiment, a 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 one embodiment, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, a Fab domain has a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0121] In one embodiment, a 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 one embodiment, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, a Fab domain has a light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0122] In one embodiment, 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 and light chains associate to form a C5-binding domain.

[0123] In one embodiment, 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 and light chains associate to form a C5-binding domain.

[0124] In one embodiment, 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 and light chains associate to form a C5-binding domain.

[0125] In one embodiment, 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 and light chains associate to form a C5-binding domain.

[0126] In some embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a single-chain Fab. In some embodiments, the single-chain Fab comprises a heavy chain polypeptide provided herein and a light chain polypeptide provided herein, operably linked via a linker. In some 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.

[0127] In one embodiment, 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 and light chain polypeptides associate to form a C5-binding domain.

[0128] In one embodiment, 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 and light chain polypeptides associate to form a C5-binding domain.

[0129] In one embodiment, 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 and light chain polypeptides associate to form a C5-binding domain.

[0130] In some embodiments, the antigen-binding fragment of an anti-C5 antibody comprises a single-chain Fv (scFv) domain, which 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.

[0131] In one embodiment, the scFv domain 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, operably linked via a linker.

[0132] In one embodiment, the scFv domain 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: 36, operably linked via a linker.

[0133] In one embodiment, 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: 39, operably linked via a linker.

[0134] 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.

[0135] In certain embodiments, the C5 binding domain comprises a VHH domain. B.VEGF-R

[0136] In certain embodiments, the fusion protein in the multispecific molecules provided herein comprises a vascular endothelial growth factor (VEGF) binding domain.

[0137] VEGF is a key 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).

[0138] VEGF receptors have been identified, including VEGFR-1 (fms-like tyrosine kinase, Flt-1), VEGFR-2 (fetal liver kinase 1-mouse 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 neuropilins, but also on neurons.

[0139] VEGFR has seven immunoglobulin-like domains within its extracellular domain. As used herein, the "extracellular" domain is the portion of a cell surface receptor that is on the outside of the surface of the cell and typically contains the ligand binding site.

[0140] The immunoglobulin-like domains of VEGFRs are oriented extracellularly, allowing them to bind VEGF in their unique conformation within the cell membrane. Each VEGFR has seven extracellular Ig domains, numbered from N- 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.

[0141] Upon binding to VEGF, VEGFR undergoes intracellular dimerization and ligand-dependent tyrosine phosphorylation, resulting in mitogenic, chemotactic, and prosurvival signals. Blocking the binding of VEGF to its receptor VEGFR has been shown to be effective in treating pathological angiogenesis. Blocking antibodies against VEGF or soluble VEGF receptor fragments can disrupt VEGF-initiated signal transduction and pathological angiogenesis caused by high VEGF expression by inhibiting VEGF binding to VEGFR on vascular endothelial cells.

[0142] As used herein, a VEGF-binding domain can be any suitable domain capable of binding to VEGF, including, for example, a VEGF-binding domain derived from an anti-VEGF antibody or derived from a VEGF receptor.

[0143] In certain embodiments, the VEGF binding domain comprises one or more extracellular immunoglobulin-like (Ig) domains of one or more VEGF receptors (VEGFRs).

[0144] In certain embodiments, the VEGFR is selected from the group consisting of VEGFR-1, VEGFR-2, and VEGFR-3.

[0145] In certain embodiments, the Ig domain of a VEGFR may be selected from the group consisting of Ig domain 1, Ig domain 2, Ig ​​domain 3, and Ig domain 4.

[0146] In certain embodiments, the VEGF binding domain comprises two or more different Ig domains of two or more different VEGFRs.

[0147] In certain embodiments, the VEGF binding domain comprises a first Ig domain of a first VEGFR operably linked, either directly or via a first linker, to the N-terminus of a second Ig domain of a second VEGFR.

[0148] In certain embodiments, the first Ig domain is Ig domain 2 and the second Ig domain is Ig domain 2 or Ig domain 3.

[0149] In one embodiment, the first VEGFR is VEGFR-1 and the second VEGFR is VEGFR-2.

[0150] In one embodiment, the VEGF-binding domain comprises Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2. In one embodiment, 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.

[0151] In certain embodiments, Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2 are joined directly or via a first linker.

[0152] In certain embodiments, Ig domain 2 of VEGFR-1 is operably linked, either directly or via a first linker, to the N-terminus of Ig domain 3 of VEGFR-2.

[0153] The first linker connects the two Ig domains of VEGFR. In some embodiments, the first linker comprises a peptide linker. The peptide linker can be a synthetic peptide or a peptide derived from a naturally occurring polypeptide.

[0154] In one embodiment, the first linker comprises the amino acid sequence of SEQ ID NO: 18 (TNTII).

[0155] In some embodiments, the VEGF binding domain comprises a VEGF trap, such as aflibercept, which competes with naturally occurring VEGF cell receptors to inhibit VEGF.Aflibercept is an angiogenesis inhibitor that has been developed as a therapeutic agent for treating angiogenesis-related diseases.In some embodiments, the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 11.

[0156] In one embodiment, the VEGF binding domain comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto while retaining binding specificity for VEGF.

[0157] [Table 3] C. Peptide Linker Sequence

[0158] In certain embodiments, in the multispecific molecules provided herein, the C5-binding domain is operably linked, either directly or via a second linker, to the N-terminus of the VEGF-binding domain. An example is shown in Figures 1, 2, or 3.

[0159] In one embodiment, the C5-binding domain comprises a Fab domain comprising a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide is operably linked to the N-terminus of the VEGF-binding domain. An example is shown in Figure 1.

[0160] In certain embodiments, the C5-binding domain comprises a Fab domain comprising a heavy chain polypeptide and a light chain polypeptide, wherein the light chain polypeptide is operably linked to the N-terminus of the VEGF-binding domain.

[0161] In one embodiment, the C5-binding domain comprises an scFv domain operably linked to the N-terminus of the VEGF-binding domain. An example is shown in Figure 2. In one embodiment, the scFv domain comprises a VL region operably linked to the N-terminus of a VH region.

[0162] In one embodiment, the C5-binding domain comprises a VHH domain operably linked to the N-terminus of the VEGF-binding domain. An example is shown in Figure 3.

[0163] In some embodiments, 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 its effect on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and controlled degradation), viscosity, rigidity, flexibility, immunogenicity, modulation of antibody binding, and ability to be incorporated into micelles or liposomes.

[0164] In some embodiments, the peptide linker may be a GS linker. As used herein, a "GS linker" is a peptide linker containing 1, 2, 3, 4, or more repeats of glycine (G) or serine (S). In some embodiments, the GS linker may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats of SEQ ID NO: 13 (GGGS), SEQ ID NO: 14 (GGGGS), or SEQ ID NO: 15 (GGGGSGGGGSGGGGS). D. Multimer-forming components

[0165] In certain embodiments, the fusion protein in the multispecific molecules provided herein comprises a multimerizing component.

[0166] As used herein, a "multimerizing component" refers to a component that has the ability to associate with another multimerizing component to form a homodimer or heterodimer.

[0167] In one embodiment, the multimerizing component comprises a polypeptide fragment, the polypeptide fragment comprising one or more amino acid residues and having at least one cysteine ​​residue that can dimerize to form a disulfide bond, thereby allowing the formation of a dimer.

[0168] In some embodiments, the multimerizing component comprises a polypeptide having a length of 1 to 200 amino acids and at least one cysteine ​​residue, hi some embodiments, the multimerizing component comprises a polypeptide having a length 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.

[0169] Any suitable polypeptide can be used as a multimerizing component, including those that contain cysteine ​​residues capable of forming interchain disulfide bonds, or those that can associate with each other via electrostatic interactions, hydrogen bonds, hydrophobic interactions, etc. Examples of multimerizing components include, but are not limited to, Fc domains and leucine zipper motifs.

[0170] In some embodiments, the multimerizing component comprises an antibody Fc domain or a fragment thereof. For example, the multimerizing component may comprise an immunoglobulin CH3 domain. As another example, the multimerizing component may comprise 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.

[0171] Without wishing to be bound by any theory, it is believed that the Fc domain serves to increase the half-life or stability of the multispecific molecule.

[0172] In some embodiments, the Fc domain comprises a human Fc domain.

[0173] In certain embodiments, the Fc domain is derived from a human immunoglobulin (Ig).

[0174] In certain embodiments, the Fc domain is derived from human IgG, optionally human IgG1, IgG2, IgG3, or IgG4.

[0175] In one embodiment, the Fc domain is derived from human IgG1.

[0176] In one embodiment, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto while retaining the ability to form multimers.

[0177] In certain embodiments, the Fc domain is mutated. In some embodiments, the Fc domain comprises substitutions, deletions, insertions and / or additional mutations to improve aggregation.

[0178] In certain embodiments, the Fc domain comprises mutations at positions 235 and / or 309 of human IgG1 according to the EU numbering system.

[0179] In certain embodiments, the Fc domain comprises substitutions at positions 235 and / or 309 of human IgG1 according to the EU numbering system.

[0180] In one embodiment, the Fc domain comprises an L235K and / or an L309K mutation according to the EU numbering system.

[0181] In one embodiment, 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 retaining the ability to form multimers and the L235K and / or L309K mutations according to the EU numbering system.

[0182] In certain embodiments, the Fc domain comprises substitutions, deletions, insertions and / or additional mutations, e.g., to reduce or eliminate one or more effector functions or to improve pH-dependent binding to the neonatal Fc receptor (FcRn).

[0183] In certain embodiments, the Fc domain provided herein has reduced effector function and comprises one or more amino acid substitutions at positions selected from the group consisting of 234, 235, 237, 238, 268, 297, 309, 330, and 331 of IgG1 according to the EU numbering system. In certain embodiments, the Fc domain provided herein is of the IgG1 isotype and comprises 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.

[0184] 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 (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S), according to the EU numbering system.

[0185] 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, and any combination thereof, according to the EU numbering system.

[0186] In certain embodiments, the Fc domain contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants have an extended pharmacokinetic half-life because they bind to FcRn at acidic pH, allowing them to escape degradation in lysosomes and subsequently be transported outside the cell for release. Methods for engineering antibodies and antigen-binding fragments thereof to improve their 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, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0187] In certain embodiments, the multimerizing component is operably linked to the C-terminus of the VEGF binding domain, either directly or through a third linker.

[0188] In some embodiments, the third linker comprises a peptide linker. The peptide linker may be a synthetic peptide or a peptide derived from a naturally occurring polypeptide. In some embodiments, the third linker comprises a fragment derived from the hinge region of an antibody. In some embodiments, the third linker comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.

[0189] In certain embodiments, the peptide linker may be a GS linker. E. Multispecific molecules

[0190] 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, are at least as good as, or even better than, parent anti-C5 antibodies such as eculizumab and parent VEGF-binding molecules such as aflibercept.

[0191] In certain embodiments, the multispecific molecules described herein comprise a fusion protein comprising a C5-binding domain that is 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.

[0192] In some 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 provided herein). In some 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 provided herein). In some 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 and second polypeptides associate to form the C5-binding domain. In some 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 and second polypeptides associate to form the C5-binding domain.

[0193] [Table 4-1] [Table 4-2] [Table 4-3]

[0194] In some embodiments, the heavy chain polypeptide is operably linked to the C-terminus of the multimerizing component, either directly or through a linker (e.g., a second linker provided herein). In some embodiments, the multimerizing component is operably linked to the C-terminus of the VEGF-binding domain, either directly or through a linker (e.g., a third linker provided herein). An example is shown in Figure 4.

[0195] In certain embodiments, the fusion protein comprises a C5 binding domain comprising an scFv having a VH region and a VL region operably linked via a linker.

[0196] In some embodiments, the fusion protein comprises an scFv operably linked, either directly or via a linker, to the N-terminus of a VEGF-binding domain. In some embodiments, the fusion protein comprises an scFv operably linked, either directly or via a linker, to the C-terminus of a multimerizing component, which is operably linked, either directly or via a linker, to the C-terminus of the VEGF-binding domain. An example is shown in Figure 5.

[0197] In one embodiment, the fusion protein comprises a C5 binding domain comprising a VHH domain.

[0198] In some embodiments, the fusion protein comprises a VHH domain operably linked, either directly or via a linker, to the N-terminus of a VEGF-binding domain. In some embodiments, the fusion protein comprises a VHH domain operably linked, either directly or via a linker, to the C-terminus of a multimerizing component, which is operably linked, either directly or via a linker, to the C-terminus of the VEGF-binding domain. An example is shown in Figure 6.

[0199] Without wishing to be bound by any theory, it appears that the potency of a fusion protein is affected by the positioning of the C5-binding domain. Specifically, it is demonstrated that when the C5-binding domain is operably linked to the C-terminus of the multimerizing component (see, e.g., Figures 4-6), the potency of the fusion protein is reduced. In contrast, when the C5-binding domain is linked to the N-terminus of the VEGF-binding domain (see, e.g., Figures 1-3), its potency appears to be enhanced. In certain embodiments, the multispecific molecule comprises a dimer of a fusion protein in which the multimerizing components in the fusion protein associate to form a dimer.

[0200] In certain embodiments, the multispecific molecules provided herein are capable of specifically binding to C5 with a K value 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, or 220 pM or less as determined by BIACORE, as described in Example 2 of the present disclosure.

[0201] In certain embodiments, the multispecific molecules provided herein bind to VEGF with a KD 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 BIACORE, as described in Example 2 of the present disclosure.

[0202] 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.

[0203] 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 hemolysis assay described in Example 3.2 of this disclosure.

[0204] In certain embodiments, the multispecific molecules provided herein have superior effects (such as the ability to inhibit vascular leakage, and the prolonged duration of regression of leaky vessels) compared to aflibercept. F. Polynucleotides and Recombinant Methods

[0205] The present disclosure provides isolated polynucleotides encoding the multispecific molecules provided herein. Unless otherwise indicated, a particular polynucleotide sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences in addition to the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0206] Polynucleotides encoding the multispecific molecules disclosed herein may be generated using methods known in the art. In certain embodiments, the sequence of the polynucleotide may be obtained based on the amino acid sequence of the multispecific molecule, or the nucleic acid may be generated using synthetic methods. Alternatively, the polynucleotides provided herein may be obtained from another available nucleic acid that encodes a polypeptide having a sequence homologous to a polypeptide in the multispecific molecules disclosed herein. DNA manipulation processes can then be applied to manipulate the sequence of the nucleic acid encoding the parent multispecific molecule (e.g., introduce mutations, insertions, deletions, etc.) to obtain a nucleic acid encoding a multispecific molecule disclosed herein.

[0207] The isolated polynucleotides encoding the multispecific molecules can be inserted into one or more vectors for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, 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.

[0208] The present disclosure provides a vector comprising the isolated polynucleotide provided herein. In some embodiments, the polynucleotide provided herein encodes a multispecific molecule having at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to a nucleic acid sequence and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, M13 phage, and the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, and pGEMEX. , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLex A, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0209] Vectors containing polynucleotide sequences encoding multispecific molecules can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive bacteria, such as Enterobacteriaceae (such as Escherichia, e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella, as well as Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis), Pseudomonas (e.g., Pseudomonas aeruginosa), and Streptomyces.

[0210] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding multispecific molecules. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is most commonly used. However, as used herein, other host organisms, such as Schizosaccharomyces pombe, Kluyveromyces hosts (e.g., Kluyveromyces lactis, Kluyveromyces fragilis, etc. (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces spp., are also contemplated. Many other genera, species, and strains of fungi, such as M. marxianus, Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora, Schwanniomyces (e.g., Schwanniomyces occidentalis), and filamentous fungi (e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus (Aspergillus nidulans and Aspergillus niger)), are also commonly available and useful.

[0211] Suitable host cells for expression of the glycosylated multispecific molecules provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants, as well as corresponding permissive insect host cells, have been identified from hosts such as the armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm, Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori, are publicly available, and such viruses may be used as viruses herein according to the present invention, particularly for transfection of armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

[0212] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (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 CCL10), 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 CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), and human cervical carcinoma cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC5 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 their derivatives.

[0213] Host cells are transformed with the above-described expression or cloning vectors for production of multispecific molecules and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, multispecific molecules may be produced by homologous recombination, as known in the art. In certain embodiments, host cells are capable of producing the multispecific molecules provided herein.

[0214] The present disclosure also provides a method of expressing the multispecific molecules provided herein, the method comprising culturing a host cell provided herein under conditions in which a vector of the present disclosure is expressed. The host cells used to produce the multispecific molecules provided herein may be cultured in a variety of media. Commercially available 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. Also, any of the media described in Ham et al., Meth. Enz. 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. Pat. Reissue No. 30,985 may be used as a culture medium for the host cells. These media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine, thymidine), antibiotics (e.g., gentamicin), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0215] 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, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating multispecific molecules secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the multispecific molecule is secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be added to any of the foregoing steps to inhibit proteolysis, and antibiotics may be added to prevent the growth of adventitious contaminants.

[0216] The multispecific molecules prepared from the 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 the preferred purification technique.

[0217] In one embodiment, solid-phase-immobilized protein A is used for immunoaffinity purification of multispecific molecules containing Fc domains. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domains 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., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for higher flow rates and shorter processing times than agarose. When the multispecific molecule contains a CH3 domain, Bakerbond ABX is often used. TM Resins (JT Baker, Phillipsburg, NJ) are useful for purification. Depending on the polyspecific molecules to be recovered, fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, heparin SEPHAROSE, etc. may be used. TM Other protein purification techniques are also available, such as chromatography on HCl, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0218] After the preliminary purification step, the mixture containing the multispecific molecule of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer of about pH 2.5-4.5, preferably performed at a low salt concentration (e.g., about 0-0.25 M salt). III. Pharmaceutical Formulations and Administration

[0219] The present disclosure also provides pharmaceutical compositions comprising the multispecific molecules provided herein and one or more pharmaceutically acceptable carriers.

[0220] The present disclosure further provides pharmaceutical compositions comprising a polynucleotide encoding a multispecific molecule provided herein and one or more pharmaceutically acceptable carriers.

[0221] The present disclosure further provides pharmaceutical compositions comprising an expression vector containing a polynucleotide encoding a multispecific molecule provided herein and one or more pharmaceutically acceptable carriers. In some 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 viral (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, etc. In some embodiments, the expression vector is suitable for gene therapy in humans. Vectors suitable for gene therapy include, for example, adeno-associated viral (AAV) vectors or adenoviral vectors. In some embodiments, the expression vector comprises a DNA vector or an RNA vector. In some embodiments, the pharmaceutically acceptable carrier is a polymeric excipient, including, but not limited to, microspheres, microcapsules, polymeric micelles, dendrimers, etc. The polynucleotides or polynucleotide vectors of the present disclosure may be encapsulated, attached, or coated in polymer-based components by methods known in the art (see, e.g., W. Heiser, Nonviral gene transfer techniques, Humana Press, 2004; U.S. Pat. No. 6,025,337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0222] As used herein, the term "pharmaceutical composition" refers to a formulation containing active ingredients in a form suitable for administration to a subject.

[0223] As used herein, the term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, salt and / or vehicle is chemically and / or physiologically compatible with the other ingredients, such as the active ingredients (i.e., the multispecific molecules disclosed herein), that generally comprise the formulation, and physiologically compatible with the subject to which the pharmaceutical composition is administered.

[0224] "Pharmaceutically acceptable carrier" refers to an ingredient other than the active ingredient in a pharmaceutical formulation that is physiologically acceptable and non-toxic to a subject. In the context of the present disclosure, pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or various combinations thereof known in the art.

[0225] The carrier may be a solvent, dispersion medium, isotonic agent, etc. The carrier may be a liquid, semi-solid, or solid carrier. In some embodiments, the carrier may be water, saline solution, or other buffers (such as serum albumin and gelatin), carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates, including glucose, sucrose, trehalose, mannose, mannitol, sorbitol, or dextrins), gels, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, antioxidants (including ascorbic acid and methionine), chelating agents (such as EDTA), salt-forming counterions (such as sodium), non-ionic surfactants (such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG), or combinations 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 well known in the art.

[0226] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, dispersing agents, etc. Prevention of the presence of microorganisms may be ensured by the aforementioned sterilization procedures and the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Additionally, the absorption of injectable dosage forms may be delayed by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0227] 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.

[0228] The 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 dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

[0229] The composition may comprise an ophthalmic depot formulation containing an active agent for subconjunctival administration. The ophthalmic depot formulation comprises microparticles of essentially pure active agent, such as a bispecific antibody of the present invention. Microparticles containing the bispecific antibody of the present invention may be embedded in a biocompatible, pharmaceutically acceptable polymer or lipid encapsulating agent. The depot formulation may be adapted to release substantially all of the active agent over an extended period of time. The polymer or lipid matrix, if present, may be adapted to degrade sufficiently to be transported away from the administration site after release of all or substantially all of the active agent. The depot formulation may be a liquid formulation comprising a pharmaceutically acceptable polymer and an active agent dissolved or dispersed therein. Upon injection, the polymer forms a depot at the injection site, for example, by gelation or precipitation.

[0230] In embodiments, the pharmaceutical composition is formulated into an injectable composition.The injectable pharmaceutical composition may be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for forming a liquid solution, suspension, or emulsion.The injectable preparation may include a sterile liquid and / or non-pyrogenic solution for injection, a sterile dry soluble product (including hypodermic tablets), such as a lyophilized powder that is combined with a solvent immediately before use, a sterile suspension for injection, a sterile dry insoluble product that is combined with a vehicle immediately before use, and a sterile and / or non-pyrogenic emulsion.The solution may be aqueous or non-aqueous.

[0231] In some embodiments, the unit dose parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile and pyrogen-free, as known and practiced in the art.

[0232] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving the multispecific molecule as disclosed herein in a suitable solvent. The solvent may contain excipients or other pharmacological ingredients that improve the stability of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may contain a buffer (such as citric acid, sodium phosphate, potassium phosphate, or other such buffers known to those of skill in the art) at a near-neutral pH. The solution is then sterile filtered and lyophilized under standard conditions known to those of skill in the art to obtain the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial can contain a single dose or multiple doses of the polypeptide conjugate. To facilitate accurate sampling and dosing, it is acceptable to slightly overfill the vial with the amount required for a single or multiple dose (e.g., about 10%). The lyophilized powder can be stored under appropriate conditions, such as at a temperature from about 4° C. to room temperature.

[0233] The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration.In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder for reconstitution.The exact amount depends on the selected treatment and can be empirically determined.

[0234] In certain embodiments, there is further provided a composition comprising a pharmaceutically acceptable carrier, diluent or adjuvant, and an active ingredient, which may be a multispecific molecule disclosed herein. IV. Kit

[0235] 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 configurations of the kit will be apparent to those skilled in the art and are within the scope of the present invention.

[0236] Such kits may optionally further include one or more of a variety of conventional pharmaceutical kit components, as would be readily apparent to one of skill in the art, e.g., containers containing one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions (either inserts or labels) indicating the amounts of components to be administered, guidelines for administration, and / or guidelines for mixing of the components may also be included in the kit.

[0237] In some embodiments, treatment kits of the invention may include one or more doses of the multispecific molecules present in the pharmaceutical compositions described herein, a device suitable for intravitreal injection of the pharmaceutical composition, and instructions detailing suitable subjects and protocols for performing the injection. In these embodiments, the compositions are typically administered to a subject in need of treatment by intravitreal injection. V. Medical Use

[0238] In another aspect, the present invention provides a method for treating, preventing or alleviating a condition in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a multispecific molecule disclosed herein, or a polynucleotide encoding a multispecific molecule provided herein, or a pharmaceutical composition provided herein.

[0239] As used herein, the terms "subject," "individual," "animal," or "patient" refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or disorder. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, and bears.

[0240] As used herein, "treating" a condition may include alleviating the condition, slowing the onset or rate of development of the condition, slowing the development of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or a combination thereof.

[0241] As used herein, the terms "disorder," "disease," "condition," and the like refer to a condition affecting a subject that would benefit from treatment with a multispecific molecule.

[0242] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when administered in a suitable manner by a subject, can provide a sufficient therapeutic effect to the subject. As with other therapeutic agents, it is understood that the therapeutically effective amount of a multispecific molecule as provided herein will be affected by various factors known in the art, such as body weight, age, medical history, currently administered medications, the subject's health, cross-reactions, allergies, sensitivities, potential side effects, as well as the route of administration and the extent of disease progression. Dosages may be proportionally reduced or increased by a skilled artisan (e.g., a physician or veterinarian) depending on these and other circumstances or requirements.

[0243] In certain embodiments, the disease or disorder is a C5- and / or VEGF-related disease or disorder. In some embodiments, the disease, disorder, or condition is selected from the group consisting of ocular diseases, cancer, inflammatory diseases, autoimmune diseases, angiogenesis, vascular permeability, edema, and inflammation.

[0244] In certain embodiments, the C5-related and / or VEGF-related disease or disorder is an ocular disease.

[0245] In some embodiments, the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), macular edema, post-retinal vein occlusion macular edema (RVO), diabetic macular edema (DCME), diabetic retinopathy (DR), central retinal vein occlusion, corneal neovascularization (CNV), retinitis pigmentosa (RP), ocular neovascularization (ocular neovascularization affecting the choroid, cornea, or retinal tissue), retinopathy of prematurity (ROP), pathological myopia, vascular glaucoma, retinoblastoma, retinal vein occlusion, uveitis, and neuromyelitis optica.

[0246] In some embodiments, the eye disease is age-related macular degeneration (AMD). AMD is a disease characterized by progressive degenerative abnormalities in the macula, a region in the center of the retina. Age-related macular degeneration is a complex, gradually progressing eye disorder that can lead to distortion and / or blind spots (scotoma), changes in dark adaptation (diagnostic of rod cell health), changes in color interpretation (diagnostic of cone cell health), decreased visual acuity, or irreversible blindness.

[0247] In some embodiments, the AMD is dry AMD. Non-exudative AMD is a non-neovascular ("dry") form of the disease ("dry AMD"). Dry AMD accounts for approximately 90% of all AMD cases. Dry AMD is characterized by macular degeneration, which, if continued to progress over several years, may eventually lead to central retinal atrophy (also known as geographic atrophy (GA)) with central vision loss. Dry AMD is an important cause of moderate and severe central vision loss, and is bilateral in most patients. In dry AMD, thinning of retinal pigment epithelial cells (RPE) develops in the macula, and age-related changes also occur in adjacent retinal tissue layers.

[0248] In some embodiments, AMD is wet AMD.Choroidal neovascularization can be the initial symptom of wet AMD.When neovascularization occurs in non-exudative AMD and begins to leak, this disease is called exudative AMD (neovascular ("wet") form of disease ("wet AMD")), and patients still have non-exudative AMD and may progress.Wet AMD, especially if not treated, can often cause sudden and serious central vision loss.

[0249] In some embodiments, the eye disease is geographic atrophy. Geographic atrophy (GA) is a chronic, progressive degeneration of the macula that is part of the late stage of AMD. The macula is the central part of the retina, the "membrane" that lines the inside of the eyeball. In GA, areas of the retina undergo cell death (atrophy). These areas may enlarge, causing dim or blind spots in vision. GA often first develops near the fovea, the center of the macula, the central, clearest part of vision. GA can lead to progressive and permanent vision loss. If GA develops in one eye, the other eye is more likely to develop GA as well. GA is characterized by localized, well-defined atrophy of the outer retinal tissue, retinal pigment epithelium, and capillary tissue.

[0250] It is estimated that over 8 million people worldwide suffer from GA, and there is currently no cure for it.

[0251] The inventors have unexpectedly discovered that combining VEGF and C5 inhibition provides a synergistic effect in chronic animal models of GA. This cannot be achieved by inhibiting either target alone, for example, with VEGF traps (e.g., aflibercept) or anti-C5 antibodies such as erlizumab. The multispecific molecules provided herein demonstrate significantly improved therapeutic efficacy and longer duration of such therapeutic efficacy compared to aflibercept.

[0252] In certain embodiments, the subject has been treated with a VEGF antagonist or a VEGF receptor antagonist, hi certain embodiments, the subject has developed resistance to the VEGF antagonist or VEGF receptor antagonist.

[0253] The present invention provides methods for treating or preventing ocular disease, or for inducing regression or elimination of, or inhibiting the progression of, at least one sign or symptom of ocular disease in a subject in need thereof, by administering to the subject a therapeutically effective amount of the above-described combination. In one embodiment, the subject is afflicted with ocular disease and is experiencing one or more signs or symptoms of ocular disease.

[0254] Exemplary signs or symptoms of ocular disease include, for example, an increased rate of vision loss, ocular drusen (e.g., in subjects with dry AMD), visual acuity loss, gradual loss of central vision (e.g., in subjects with non-exudative macular degeneration), distorted vision, difficulty adapting to low light levels, distortion of central vision, blurring of central and / or overall vision, pigmentary changes in the eye, astigmatism (e.g., metamorphopsia, in which straight grids appear wavy and sections of the grid appear blank), exudative changes (e.g., hemorrhage in the eye, hard exudates, subretinal / subretinal pigment epithelial / intraretinal fluid), slow recovery of visual function after exposure to bright light (e.g., light striations), and / or ocular ocular hypertrophy. Signs of macular degeneration include: early and / or geographic atrophy (as determined by the Loess test), significant decrease in visual acuity (e.g., two or more levels, e.g., from 20 / 20 to 20 / 80), changes in preferential hyperacuity perimetry (e.g., in subjects with wet AMD), blurred vision, rapid onset of vision loss (e.g., due to leakage and hemorrhage of abnormal blood vessels in subjects with exudative macular degeneration), central scotoma (shadow or visual field defect), difficulty in color discrimination (e.g., especially dark colors against other dark colors and / or light colors against other light colors), decreased contrast sensitivity, and / or the appearance of curved lines in Amsler grids.

[0255] In certain embodiments, the subject is a human.

[0256] In certain embodiments, the multispecific molecules provided herein may be administered in a therapeutically effective amount of about 1 mg to about 20 mg (or 2 mg to 20 mg, 4 mg to 20 mg, or 4 mg to 12 mg) per intravitreal (IVT) injection. The dosing regimen may be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, the multispecific molecules may be administered once or in multiple doses over time.

[0257] The multispecific molecules disclosed herein may be administered by any route known in the art, such as, for example, 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.

[0258] Many possible delivery modes can be used, including but not limited to intraocular application or topical application. In one embodiment, application is intraocular, including but not limited to subconjunctival injection, intraocular injection, injection into the anterior chamber via the temporal limbus, intrastromal injection, intracorneal injection, subretinal injection, aqueous humor injection, sub-Tenon injection or sustained delivery, intravitreal injection (e.g., anterior vitreous, mid-vitreous, or posterior vitreous injection). In one embodiment, application is topical, including but not limited to instillation into the cornea.

[0259] In one embodiment, the multispecific molecules or pharmaceutical compositions of the invention are administered intravitreal, e.g., by intravitreal injection. This can be done according to standard procedures known in the art. See, e.g., 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.

[0260] Actual dosage levels of the multispecific molecules in the pharmaceutical compositions of the present disclosure may be varied to provide an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without causing toxicity to the patient. The selected dosage level will depend on various 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 compound being used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition being used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0261] In some embodiments, the multispecific molecules disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents. For example, the multispecific molecules disclosed herein may be administered in combination with one or more additional therapeutic agents or methods for the treatment of one or more ocular diseases described herein.

[0262] In some 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. In some of these embodiments, the multispecific molecule and the additional therapeutic agent may be administered as part of the same pharmaceutical composition. However, a multispecific molecule administered "in combination with" another therapeutic agent does not necessarily need to be administered simultaneously with that therapeutic agent or in the same composition. As used herein, a multispecific molecule administered before or after another agent is considered to be administered "in combination with" that agent, even if the multispecific molecule and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with a multispecific molecule disclosed herein are administered according to the schedule listed on the product information sheet of the additional therapeutic agent, or according to protocols known in the art, such as those in the Physician's Desk Reference 2003 (Physician's Desk Reference, 57th Edition, Medical Economics Company, ISBN: 1563634457 (November 2002)).

[0263] In another embodiment, a multispecific molecule or pharmaceutical composition according to the invention is administered in combination with one or more additional therapeutic agents or methods for the treatment of one or more ocular diseases described herein.

[0264] In another embodiment, the multispecific molecules or pharmaceutical compositions of 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.

[0265] In one embodiment, the combination treatment methods provided herein comprise the sequential administration of a multispecific molecule or pharmaceutical composition according to the invention with one or more additional therapeutic agents for the treatment of one or more ocular diseases described herein.

[0266] Additional therapeutic agents include, but are not limited to, angiogenesis inhibitors (such as a VEGF antagonist, a VEGF receptor antagonist, an anti-inflammatory agent, a neuroprotective agent, a treatment for AMD, or a C5 inhibitor).

[0267] In some embodiments, the angiogenesis inhibitor is a VEGF antagonist or a VEGF receptor antagonist. Examples include, but are not limited to, VEGF variants, soluble VEGF receptor fragments, aptamers that can interfere with VEGF or VEGFR, neutralizing anti-VEGFR antibodies, small interfering RNAs that reduce the expression of VEGFR or VEGF ligands, low molecular weight inhibitors of VEGFR tyrosine kinase, and any combination thereof, including anti-VEGF aptamers (e.g., pegaptanib), soluble recombinant decoy receptors (e.g., VEGF traps).

[0268] In certain embodiments, angiogenesis inhibitors include anti-inflammatory agents, m-Tor inhibitors, rapamycin, everolimus, temsirolimus, cyclosporine, anti-TNF agents, anti-complement agents, and non-steroidal anti-inflammatory agents.

[0269] In certain embodiments, angiogenesis inhibitors include corticosteroids, antiangiogenic steroids, anecortave acetate, angiostatin, endostatin, MMP inhibitors, IGFBP3, SDF-1 inhibitors, PEDF, gamma-secretase, delta-like ligand 4, integrin antagonists (e.g., integrin beta3 function inhibitors), HIF-1 alpha blockers, protein kinase CK2 blockers, and inhibition of stem cell (i.e., endothelial progenitor cell) homing to sites of neovascularization using vascular endothelial cadherin (CD-144) and stromal-derived factor (SDF)-I antibodies.

[0270] In certain embodiments, the additional therapeutic agent is a complement-related agent such as C1q, C3, C5, factor B, factor D, or factor H.

[0271] In certain embodiments, the additional therapeutic agent is a C3 inhibitor, examples of which include, but are not limited to, compstatin and / or its analogs, H17 (monoclonal antibody, EluSys Therapeutics, Pine Brook, NJ), mirococept (a CR1-based protein), sCR1 (a CR1-based protein, Celldex, Hampton, NJ), TT32 (a CR-1-based protein, Alexion Pharmaceuticals, Boston, MA), HC-1496 (a recombinant peptide); CB2782 (an enzyme, Catalyst Biosciences, South San Francisco, CA), APL-2 (a pegylated synthetic cyclic peptide, Apellis Pharmaceuticals, Crestwood, KY), or combinations thereof.

[0272] In certain embodiments, the additional therapeutic agent is a complement factor B inhibitor, examples of which include, but are not limited to, anti-FB siRNA (Alnylam Pharmaceuticals, Cambridge, MA), TA106 (monoclonal antibody, Alexion Pharmaceuticals, Boston, MA), LNP023 (small molecule, Novartis, Basel, Switzerland), SOMAmers (aptamer, SomaLogic, Boulder, CO), bicasiomab (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 combinations thereof.

[0273] In certain embodiments, the additional therapeutic agent is a complement factor D antagonist, eg, an anti-complement factor D antibody, eg, lampalizumab (Roche).

[0274] In certain embodiments, the additional therapeutic agent is an Ang-2 antagonist.

[0275] In some 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.

[0276] In certain embodiments, the additional therapeutic agent is a therapeutic agent for AMD, examples of which include, but are not limited to, PDT, pegaptanib sodium, zinc, or verteporfin in combination with an antioxidant, alone or in any combination.

[0277] In certain embodiments, the additional therapeutic agent is a C5 inhibitor, examples of which include, but are not limited to, eculizumab, ravulizumab, 305LO5, SKY59, pozelimab, tesidolumab, crovalimab, or ABP959, or a biosimilar thereof.

[0278] In another aspect, the present 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.

[0279] In certain embodiments, the cell is selected from the group consisting of a retinal ganglion cell, a rod cell, a cone cell, a glial cell (eg, a Müller glial cell), a bipolar cell, an amacrine cell, and a horizontal cell.

[0280] In another aspect, the present 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.

[0281] In another aspect, the present 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. Working Example:

[0282] The following examples are provided to more clearly 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 do not limit the invention, but are intended merely to illustrate specific embodiments falling within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while remaining within the scope of the invention. The inventors intend such variations to be included within the scope of the invention. Example 1 Gene synthesis, expression and purification of bispecific molecules

[0283] 1.1 Experimental Method:

[0284] a) VEGF / C5 bispecific antibodies BSP1, BSP2, BSP3, BSP4, BSP5, and BSP6

[0285] Nucleic acid sequences encoding the VEGF / C5 bispecific molecules BSP1, BSP2, BSP3, BSP4, BSP5, and BSP6 were designed, optimized, and synthesized. All sequences were subcloned into the pcDNA3.4 vector. Recombinant plasmids encoding each target antibody were transiently co-transfected into suspension HD293F cell cultures (Thermo Fisher Scientific). Plasmid-transfected cells were cultured in medium at 37°C and 8% CO2. After 6 days of culture, the supernatant was collected, centrifuged, and filtered for protein purification. 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 pooled, concentrated, and loaded onto a gel filtration chromatography column at an appropriate flow rate to improve purity. The protein was concentrated to the required concentration. The purified protein was analyzed by SDS-PAGE, Western blot, and HPLC analysis to determine its molecular weight and purity. The concentration was determined by the A280 method.

[0286] Meanwhile, aflibercept (Probio) and mAb eculizumab (Probio) were used as positive controls to evaluate the viability and function of the VEGF / C5 bispecific antibody prepared in this application.

[0287] [Table 5]

[0288] In BSP1 to BSP6, the amino acid sequence of VH (eculizumab) is SEQ ID NO: 1, VL (eculizumab) is SEQ ID NO: 2, VEGFR1 (domain 2) is SEQ ID NO: 9, VEGFR2 (domain 3) is SEQ ID NO: 10, human IgG1 CH1 is SEQ ID NO: 19, human CL(κ) is SEQ ID NO: 20, and human IgG1 Fc is SEQ ID NO: 12. Each of BSP1 to BSP6 is a dimer of a fusion protein as shown in Table 3. A linker is present in the molecule but is not explicitly shown in Table 3.

[0289] b) Affinity Kd maturation / optimization

[0290] Experimental Method:

[0291] i) Amino acid selection for Kd maturation

[0292] FASEBA (rapid screening for expression, biophysical properties, and affinity) was selected for affinity maturation (Probio and GenScript). Based on the parent antibody sequence / BSP1, FASEBA screening was performed to increase antibody affinity to the target antigen.

[0293] The parent mAb, eculizumab, was expressed in CHO cells and purified. Biacore 8K / T200 was used to confirm binding of the mAb to the human complement C5 protein. To determine the amino acids selected for Kd maturation, the parent Fab was constructed in a FASEBA format and binding was verified using SPR. A PML (precise mutagenesis library) library was constructed in the FASEBA format for six CDR residues, and next-generation sequencing (NGS) was performed to confirm the distribution of the PML library.

[0294] ii) Production and characterization of affinity matured antibodies

[0295] DNA encoding the best affinity-matured antibody obtained from PML was synthesized and subcloned into an expression vector for antibody expression in CHO cells. The affinity-matured antibody was purified using a protein A column. The kinetics of antigen interaction between the affinity-matured and wild-type antibodies was studied using Biacore 8K.

[0296] 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 of wild-type and mutant eculizumab with human complement C5 were studied.

[0297] The affinity-matured mutants were found to have significantly improved binding affinities to human C5, from 3- to 10-fold.

[0298] Eculizumab variant #1 is an eculizumab variant containing one mutation in HC and three mutations in LC. Eculizumab variant #1 contains a VH region containing the amino acid sequence set forth in SEQ ID NO: 28 and a VL region containing the amino acid sequence set forth in SEQ ID NO: 31. Eculizumab-WT is the original eculizumab sequence.

[0299] The results are shown in Table 3A. It was confirmed that eculizumab variants #1 to #3 had significantly improved binding affinity to human C5.

[0300] [Table 6]

[0301] Eculizumab variant #1 was selected as the preferred affinity-matured variant based on its superior protein expression yield in 100 mL shake flasks and used to generate the bispecific molecule BSP1a for further studies.

[0302] [Table 7]

[0303] In BSP1a, the amino acid sequence of VH (eculizumab variant) is SEQ ID NO: 28, VL (eculizumab variant) is SEQ ID NO: 31, VEGFR1 (domain 2) is SEQ ID NO: 9, VEGFR2 (domain 3) is SEQ ID NO: 10, human IgG1 CH1 is SEQ ID NO: 19, human CL(κ) is SEQ ID NO: 20, and human IgG1 Fc is SEQ ID NO: 12. BSP1a is a dimer of a fusion protein as shown in Table 3B. The linker in the molecule is not explicitly shown in Table 3 and Table 3B.

[0304] 1.2 Experimental results of bispecific antibody purity analysis:

[0305] The purified protein was analyzed by SDS-PAGE, Western blot, and HPLC analysis to determine its molecular weight and purity.

[0306] [Table 8]

[0307] The purity of BSP1a purified by SDS-PAGE method is in the range of 94% to 96%, and by SEC-HPLC method is in the range of 95% to 99%. Example 2 Binding Affinity Assay

[0308] 2.1 Experimental Method:

[0309] The binding affinity of the bispecific molecules for the antigen was determined by SPR assay using a Biacore8K (GE Healthcare, Chicago, IL). The binding assay was performed by first attaching BSP1 to a protein A-coated series S sensor, then injecting various concentrations of human recombinant VEGF-165 protein (VE5-H4210, Acro, 1.5625-100 nM) or human recombinant complement C5 protein (CO5-H52Ha, Acro, 0.15625-5 nM) over the BSP-bound surface at a flow rate of 30 μL / min for 120 s. After sample injection, the bound analyte was dissociated from the sensor chip using 10 mM glycine-HCl pH 1.5 (GenScript, lot number 20211113). The binding affinity assay for the BSP1a bispecific molecule was performed in the same manner.

[0310] Antibody binding kinetics, including k (association rate constant), k (dissociation rate constant), and K (dissociation equilibrium constant), were determined using Biacore 8K evaluation software version 3.0 (Chicago, IL). Recording a plot of binding response (RU) versus time allowed visualization and evaluation of the various stages of the binding event.

[0311] 2.2 Experimental results:

[0312] The binding affinity of BSP1 and BSP1a for C5 was measured, and the average KD values ​​for BSP1 and BSP1a for human C5 and VEGF were given as shown in Table 5. PC2 (anti-C5 mAb) had a KD range of 400-500 pM for C5. PC1 (aflibercept) had an average KD of 21.7 pM for VEGF.

[0313] [Table 9] Example 3 In Vitro Cellular Function Assays (Anti-VEGF and Anti-C5)

[0314] 3.1 VEGF-mediated cell proliferation assay (anti-VEGF)

[0315] 3.1.1 Experimental Method:

[0316] VEGF-mediated proliferation of HUVEC (human umbilical vein endothelial cells, C2519A, Lonza, Basel, Switzerland) was selected to measure the cellular function of VEGF molecules. HUVEC cells were harvested by centrifugation and resuspended in cell culture medium. Working solutions of test articles and VEGF protein in 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 the working solution of the test article and VEGF protein mixture was transferred to the 384-well plate. The assay plate was incubated in an incubator (37°C, 5% CO2) for 72 hours before adding CellTiter-Glo to the corresponding wells of the assay plate.

[0317] Luminance signals were recorded using a PHERA Star. Raw data were exported from the PHERA Star FSX system and analyzed using Microsoft Office Excel 2016 and GraphPad Prism 6.

[0318] Dose-response curves were fitted to obtain relative IC50 values ​​using the following four-parameter function, which is characterized by a sigmoidal curve showing the percentage of growth inhibition versus the concentration of the test sample:

[0319] Y=Lower limit + (Upper limit - Lower limit) / (1+10^((LogIC50-X)*HillSlope))

[0320] where X = logarithm of concentration, Y = brightness signal

[0321] Target cells were treated with serial dilutions of test articles. The brightness ± SEM of triplicate replicates within each group was plotted.

[0322] 3.1.2 Experimental results:

[0323] Experimental results showed that both positive control 1 (aflibercept) and BSP1 were able to inhibit HUVEC cell proliferation, with mean IC50 values ​​of 1.55 nM and 1.10 nM calculated from the dose-response curve. No inhibition of HUVEC cell proliferation was observed in the negative control. Both PC1 (aflibercept) and BSP1 had similar growth inhibitory effects in the VEGF-mediated HUVEC proliferation assay. The results of the growth inhibitory effects of the test samples (PC1 and BSP1) on the target cell lines are shown in Figures 7 and 8, Tables 6 and 7.

[0324] BSP1a was also tested in a similar manner and exhibits an IC50 (µg / mL) in the range of 0.1-0.3 for its growth inhibitory effect in the VEGF-mediated HUVEC proliferation assay.

[0325] [Table 10]

[0326] [Table 11]

[0327] BSP1a was also tested in a VEGF-mediated cell proliferation assay, and BSP1a exhibits an IC50 (µg / mL) in the range of 0.1-0.2 for VEGF-mediated cell proliferation inhibition.

[0328] 3.2 Interfering effects of hemolytic assay / CH50 assay (anti-C5)

[0329] 3.2.1 Experimental method

[0330] A hemolytic assay was selected to measure the cellular function of C5 molecules. Sheep red blood cells (SRBCs) were collected by centrifugation and resuspended in GVB++ assay buffer. A working solution of anti-erythrocyte stromal polyclonal antibody was prepared using GVB++ assay buffer and transferred to the SRBC suspension. The mixture of the working solution and SRBC suspension was then mixed thoroughly, and the plate was incubated at 37°C and 5% CO2 for approximately 30 minutes. Similarly, working solutions of PNHS and test items were prepared using GVB++ assay buffer and transferred to the corresponding wells of a 96-well assay plate. The mixture was then mixed thoroughly, and the plate was incubated at room temperature for approximately 30 minutes. The SRBC suspension primed with the anti-RBC stromal antibody was removed, and the cell suspension was transferred to the 96-well assay plate. The assay plate was then 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 shedding was performed with a hemoglobin assay kit. The detection reagent was added to the above 96-well test plate, and then incubated at room temperature for 5 minutes.

[0331] The absorbance (OD400nM) signal was read on a PHERA star. The dose-response curves were fitted to obtain relative EC50 values ​​using the following four-parameter function, which is characterized by a sigmoidal curve showing the percentage of growth inhibition versus the concentration of the test sample:

[0332] Y=Lower limit + (Upper limit - Lower limit) / (1+10^((LogEC50-X)*HillSlope))

[0333] where X = logarithm of concentration and Y = absorbance signal

[0334] SRBCs were treated with serial dilutions of the test article. The absorbance signal SEM of triplicate repeats within each group was plotted (Figure 9A). The hemoglobin content ± SEM of triplicate repeats within each group was plotted (Figure 9B).

[0335] 3.2.2 Experimental results

[0336] In the CH50 assay (C5 target test), both eculizumab (positive control 2 (PC2)) and BSP1 had significant inhibitory effects on complement C5, and dose-response curves were observed. The EC50 values ​​for PC2 (eculizumab) and the test item BSP1 were 21.01 nM and 18.08 nM, respectively (Table 8). BSP1a was tested using a similar method and showed an EC50 (nM) in the range of 18–22 nM for the CH50 assay.

[0337] [Table 12] Example 4 Single clone drug stability assay for BSP1

[0338] 4.1 Experimental method

[0339] To assess the developability of the multispecific molecules, stability assays were performed.

[0340] 4.2 Experimental results:

[0341] 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 taken on days 0, 7, and 14, respectively, and underwent SEC-HPLC, CE-SDS-NR, and icIEF (imaged capillary isoelectric focusing) tests. A summary is given in Tables 9 and 10 below.

[0342] [Table 13]

[0343] [Table 14]

[0344] result:

[0345] The results of the 40°C stability test are shown in Table 11, where the D0 sample is taken as the starting point. In the SEC-HPLC test, the sample showed an increase in the proportion of high molecular weight (HMW), a decrease in the proportion of the main peak, and an increase in the proportion of low molecular weight (LMW). In the CE-SDS-NR test, the sample showed a slight decrease in the proportion of the main peak. In the icIEF test, the sample showed a shift to a low pI (acidic) region with increasing incubation time.

[0346] The results of the 5°C stability test are shown in Table 12, where the D0 sample is taken as the starting point. After 14 days of incubation at 5°C, obvious changes were observed in SEC-HPLC, CE-SDS-NR, and icIEF tests compared to D0.

[0347] The results of the stability assays are shown in Figures 15-20 and Tables 11-12. These results confirm that BSP1 is the most stable compound compared to other orientations (BSP2 to BSP6, data not provided), remaining unchanged at 5°C and without significant aggregation or degradation at 40°C for 14 days, making it suitable for further drug development.

[0348] [Table 15]

[0349] [Table 16]

[0350] Example 5 Chronic Experimental Animal Model of Persistent Retinal Neovascularization (up to 12 months)

[0351] The objective of this animal study was to evaluate the efficacy and safety of intravitreal injection (IVT) of the biologics BSP1, BSP1a, and aflibercept (positive control (PC), a commercially available product) in a rabbit model of persistent retinal neovascularization (PRNV or dl-AAA). The PRNV model will simulate neovascular retinal disease and identify indications in humans where drugs or novel therapies for neovascular retinal disease may be beneficial. The efficacy of BSP1 IVT injection will be compared with that of aflibercept IVT injection by its inhibitory effect on an animal pharmacodynamic (PD) model known as the DLAAA / PRVN model (see C. Patel et al., Exp. Eye Res., 2020, 195:108031 for details).

[0352] 5.1 Experimental Method:

[0353] For this study, Dutch-belted rabbits were selected due to their feasibility of achieving the best results. For each animal, a retinal injury model (dl-AAA, PRNV rate <70%) was established using only one eye for characterization, while the other eye served as a control. For further drug testing, retinal injury was performed on one eye. However, regardless of the presence or absence of disease, compounds were administered to both eyes to investigate drug toxicity / effects. A unique component of this protocol is that ophthalmic endpoints are assessed in conscious rather than anesthetized animals. This eliminates potential interference from anesthetics during testing and further increases the likelihood of animal survival over several years of follow-up.

[0354] [Table 17]

[0355] 5.1.1 Standards for PRNV Models for Pharmacology Studies:

[0356] Persistent retinal neovascularization (PRNV) with FA leakage lasted for more than 3 months before medication. Neovascular leakage area and intensity were defined by a stable angiographic leakage area of ​​more than 2 disc diameters on NaF angiography (0.05 ml, 10%, OCT / FA in 3 fields of view, 55°) and a leakage severity score of 2 or higher (which may vary depending on the study objective).

[0357] 5.1.2 Efficacy of BSP1, BSP1a, and PC on reducing FA leakage and altering retinal contour in the PRNV model (dl-AAA eyes):

[0358] Fundus Photography (FP): Serial fundus photographs are useful for monitoring treatment response, inflammatory changes, and drug distribution.

[0359] Fluorescein angiography (FA): The imaging position for FA is the same as for color images. FA images are taken according to the following schedule: Late FA images are recorded at baseline and after 1, 2, 4, 8, 12, and 16 weeks of treatment (may be extended up to 24 weeks) in both dl-AAA and control eyes. For the treatment of BSP1, BSP1a, and PC, angiographic leakage is observed and evaluated by clinical observation in FA.

[0360] Optical coherence tomography (OCT): OCT is an established medical imaging technique that uses light to capture micrometer-resolution, three-dimensional images of retinal and choroidal structures to map and measure their thickness. Analysis and measurement of retinal and choroidal thickness in OCT is correlated with color fundus or FA findings.

[0361] Slit lamp examinations performed to assess safety and tolerability revealed significant inflammatory reactions in the anterior chamber of the eyes of treated rabbits receiving the test article.

[0362] All eyes will be observed by an ophthalmologist immediately after injection and thereafter on days 1 and 2. If there are signs of inflammation, continue with follow-up based on the efficacy and duration of the drug.

[0363] In all studies, the Standardized Uveitis Nomenclature (SUN) was applied. Cells and flare in the anterior chamber were observed using a 1 mm slit-lamp beam cast at an angle of 45–60°. Based on the findings, inflammation could be graded from 0 to 3+ / - (100%).

[0364] 5.2 Experimental results:

[0365] 5.2.1 Effect of test article on angiographic leakage

[0366] The effects of BSP1, BSP1a, and PC1 on angiographic leakage were evaluated by late-phase fluorescein angiography (FA) at 2, 4, 8, 12, and 16 weeks after intravitreal injection (Table 13). A total of 10 rabbits were used in this study. Seven rabbits received BSP1 in both the dl-AAA model eye (OD) and the untreated eye (OS). Three rabbits received PC in the dl-AAA model eye (OD) and PBS as NC in the untreated eye (OS). Representative images of the leakage contours at each time point between the two groups are shown in Figures 11 and 12. Representative images of the untreated eye contours at each time point between the two groups are shown in Figures 13 and 14.

[0367] All PRNV model eyes (n = 10) in both treatment groups showed no angiographic leakage from the PRNV site observed 2 weeks after treatment. Different effects were observed between the two drugs on the duration and intensity of inhibition of PRNV leakage in FA. Recurrence of leakage primarily began at approximately weeks 6–8 in the PC1 (aflibercept) group and weeks 12–16 in the BSP1 or BSP1a groups.

[0368] To evaluate and compare the effects of BSP1, BSP1a, and PC1 on vascular leakage in chronic experimental animals / PD models, the vascular leakage scores of BSP1, BSP1a, and PC1, measured by calculating the percentage of the corresponding vascular leakage area based on FA images, were plotted against time (0, 2, 4, 8, 12, and 16 weeks) in Figure 10. In addition, FA images (Figure 11) of each group were taken at 0, 2, 4, 8, 12, and 16 weeks for the compound-treated vascular leakage evaluation (OD) and the compound-treated non-DLAAA-induced eye (OS) as a control.

[0369] According to Figure 11 and Table 14, BSP1 showed superior vascular leakage inhibition ability, regression of induced leaky vessels, vascular proliferation, and longer duration compared to the group of animals treated with PC1 (aflibercept), a known VEGF inhibitor.

[0370] [Table 18]

[0371] The efficacy of BSP1a was also investigated using the same model. BSP1a also demonstrated superior vascular leakage inhibition, regression of induced leaky vessels, vessel proliferation, and a longer duration of response compared to animals treated with PC1 (aflibercept), a known VEGF inhibitor. As shown in Figures 10, 11, 12, 13, and Table 14, the results of BSP1a are at least comparable to those of BSP1.

[0372] In animal models, BSP1 and BSP1a demonstrated far superior therapeutic effects than aflibercept in inhibiting vascular leakage and reducing inflammation. In particular, BSP1 and BSP1a successfully delayed the onset of leakage until 12 weeks and significantly slowed leakage progression to the point where only an average of less than 30% leakage was observed at 16 weeks. In contrast, animals treated with aflibercept showed obvious leakage at 8 weeks and progressed more rapidly, with an average leakage rate exceeding 66% at 12 weeks. This clearly demonstrates that BSP1 and BSP1a are superior to aflibercept in terms of therapeutic efficacy and long-term duration.

[0373] Example 6 BSP1a was expected to have advantages over BSP1 in terms of purification and recovery.

[0374] Mutations in BSP1a can improve the long-term stability of the protein and allow for scale-up of production yield / recovery at high protein concentrations for further drug processing and development.

[0375] 6.1 Long-term stability

[0376] Using the experimental methods described in Example 4, stability assays of BSP1 and BSP1a were performed to assess their developability.

[0377] The results of the stability assay are shown in Table 15. BSP1a exhibited a superior stability profile and protein expression yield compared to BSP1.

[0378] [Table 19]

[0379] 6.2 Protein production yield

[0380] Both BSP1 and BSP1a constructs were further developed into stable cell lines using CHOK1 cells (Thermos Fisher). The protein production yield of BSP1a was 6.6 g / L, which is superior to that of BSP1 (3.6 g / L).

Claims

1. 1. A multispecific molecule comprising a fusion protein, said multispecific molecule comprising: (a) a complement component 5 (C5) binding domain; (b) a vascular endothelial growth factor (VEGF) binding domain; and (c) a multimer-forming component; 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); A multispecific molecule wherein the multimerizing component comprises a polypeptide having a length of 1 to 200 amino acids 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 multimer-forming component.

4. 10. The multispecific molecule of claim 1, wherein the multimerizing component comprises an antibody Fc domain.

5. The antigen-binding fragment may be Fab, Fab', F(ab) ２ , F(ab') ２ , single chain Fab, VHH, Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ２ , a bispecific dsFv (dsFv-dsFv'), a diabody, a disulfide-stabilized diabody (ds diabody), 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. 10. The multispecific molecule of any one of the preceding claims, wherein said antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable (VH) region comprising the amino acid sequence set forth in SEQ ID NO: 1, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence set forth in SEQ ID NO:

2.

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

8.

8. 10. The multispecific molecule according to any one of the preceding claims, wherein said antigen-binding fragment comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 1, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 1 while retaining binding specificity for C5.

9. 10. The multispecific molecule of any one of the preceding claims, wherein said antigen-binding fragment comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 2, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 2 while retaining binding specificity for C5.

10. 10. The multispecific molecule according to any one of the preceding claims, wherein said antigen-binding fragment 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.

11. the antigen-binding fragment: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable (VH) region comprising the amino acid sequence set forth in SEQ ID NO: 28, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence set forth 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 comprising the amino acid sequence set forth in SEQ ID NO: 28, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 36; or (c) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable (VH) region comprising the amino acid sequence set forth in SEQ ID NO: 1; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within the light chain variable (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 39; The multispecific molecule of any one of claims 1 to 5, comprising:

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

13. 13. The multispecific molecule of any one of claims 1 to 5, 11 and 12, wherein said antigen-binding fragment comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 28, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 28 while retaining binding specificity for C5.

14. 14. The multispecific molecule according to any one of the preceding claims 1 to 5 and 11 to 13, wherein said antigen-binding fragment comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 31, 36 or 39, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 31, 36 or 39 while retaining binding specificity for C5.

15. the antigen-binding fragment: (a) 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; or (b) 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: 36; or (c) 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: 39; 15. The multispecific molecule of any one of claims 1 to 5 and 11 to 14, comprising:

16. 16. The multispecific molecule of any one of claims 6 to 15, wherein the antigen-binding fragment further comprises a substitution or modification of one or more amino acid residues while retaining binding specificity for C5.

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

18. 10. The multispecific molecule of any one of the preceding claims, wherein said Ig domain is selected from the group consisting of Ig domain 1, Ig domain 2, Ig ​​domain 3 and Ig domain 4.

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

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

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

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

24. 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, either directly or via the first linker.

25. 24. The multispecific molecule of claim 23, wherein the Ig domain 2 of VEGFR-1 comprises the amino acid sequence set forth in SEQ ID NO: 9 and the Ig domain 3 of VEGFR-2 comprises the amino acid sequence set forth 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. 27. The multispecific molecule of claim 26, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 18 (TNTII).

28. 10. The multispecific molecule of any one of the preceding claims, wherein the VEGF-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto while retaining binding specificity for VEGF.

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

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

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

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

35. 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. 37. The multispecific molecule of claim 36, wherein the Fc domain comprises a mutation at position 235 and / or 309 of the human IgG1 according to the EU numbering system.

38. 37. The multispecific molecule of claim 36, wherein the Fc domain comprises a substitution at position 235 and / or 309 of the human IgG1 according to the EU numbering system.

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

40. 10. The multispecific molecule according to any one of the preceding claims, wherein the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 26, or an amino acid sequence having at least 80% sequence identity thereto while retaining the ability to form multimers.

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

25.

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

43. 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. (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.

43. The multispecific molecule of claim 42.

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

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

17.

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

33.

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

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

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

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

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

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

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

55. 10. The multispecific molecule of claim 1 , wherein the multispecific molecule comprises a dimer of the fusion protein.

56. 10. A pharmaceutical composition comprising a multispecific molecule according to any one of the preceding claims and one or more pharmaceutically acceptable carriers.

57. 10. An isolated polynucleotide encoding a multispecific molecule according to any one of the preceding claims.

58. 58. A vector comprising the isolated polynucleotide of claim 57.

59. 59. A host expression system comprising the vector of claim 58.

60. 60. The host expression system of claim 59, which is a microbial, yeast or mammalian cell.

61. 56. A method of expressing a multispecific molecule according to any one of claims 1 to 55, said method comprising culturing a host expression system according to any one of the preceding claims under conditions in which a vector according to any one of the preceding claims is expressed.

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

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

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

65. 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, post-retinal vein occlusion macular edema (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), central retinal vein occlusion, corneal neovascularization (CNV), retinitis pigmentosa (RP), ocular neovascularization (ocular neovascularization affecting the choroid, cornea, or retinal tissue), retinopathy of prematurity (ROP), pathologic myopia, vascular glaucoma, retinoblastoma, retinal vein occlusion, uveitis, and neuromyelitis optica.

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

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

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

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

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

71. 71. The method of any one of claims 62-70, further comprising administering a therapeutically effective amount of a second therapeutic agent.

72. 72. The method of claim 71, wherein the second therapeutic agent is selected from the group consisting of an angiogenesis inhibitor, an inflammatory drug, an m-Tor inhibitor, rapamycin, everolimus, temsirolimus, cyclosporine, an anti-TNF agent, an anti-complement agent, and a non-steroidal anti-inflammatory agent.

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

74. 74. The method of claim 73, wherein the cell is selected from the group consisting of a retinal ganglion cell, a rod cell, a cone cell, a glial cell (e.g., a Muller glial cell), a bipolar cell, an amacrine cell, and a horizontal cell.

75. 57. A multispecific molecule according to any one of claims 1 to 55 and / or a pharmaceutical composition according to claim 56 for use in the treatment, prevention or alleviation of a C5-related and / or VEGF-related disease, disorder or condition in a subject.

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

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