Multispecific ligand-binding molecules comprising complement inhibitory domains and their use

Abstract

The present invention relates to a ligand-binding molecule comprising a complement inhibitory domain and a growth factor binding domain, a pharmaceutical composition comprising such a ligand-binding molecule, and methods and uses for producing the ligand-binding molecule.

Description

Technical Field

[0001] The present invention relates to the field of proteins. Specifically, the present invention relates to a ligand-binding molecule capable of binding to multiple growth factors, a pharmaceutical composition of such ligand-binding molecules, a nucleic acid sequence encoding such molecules, and methods for their production and uses. Background Art

[0002] Age-related wet macular degeneration (wAMD) and diabetic macular edema (DME) are widespread among patients, with a rapid disease progression and serious consequences (vision loss), being the leading cause of blindness. Geographic atrophy (GA) is one of the final development forms of wAMD. Currently, the main treatment for wAMD and DME is targeting VEGF-A, which has significantly improved efficacy compared to traditional treatment methods (such as laser photocoagulation, etc.), and corresponding targeted drugs (such as ranibizumab, aflibercept, etc.) have also achieved great market success. However, targeting VEGF-A alone still cannot completely overcome these diseases. For example, after AMD treatment, only one-third of the patients can benefit, the vision of the remaining patients does not increase significantly, and one-third of the patients still develop into geographic atrophy with vision approaching blindness (Rofagha S. et al., Seven-Year Outcomes in Ranibizumab-Treated Patients in ANCHOR, MARINA, and HORIZON: A multicenter cohort study (SEVEN UP). J. Ophthalmology. 2013, 120(11):2292-2299).

[0003] In addition, in treatment follow-up studies, it has been found that continuous targeting of VEGF-A treatment also increases the risk of developing geographic atrophy and is a risk factor for geographic atrophy (Grunwald J.E. et al., Risk of Geographic Atrophy in the Comparison of Age-related Macular Degeneration Treatments Trials. J. Ophthalmology. 2014, 121(1):150-161) (Mitchell P. et al., Ranibizumab (Lucentis) in neovascular age-related macular degeneration: evidence from clinical trial. Br. J. Ophthalmol. 2010, 94:2-13).

[0004] On the other hand, the activation of the complement system and the generation of the membrane attack complex (MAC) are considered to be the initial etiologies of AMD and DME. Early studies found that products of complement activation could be detected in the blood of patients, and several complement-related gene mutations have also been listed as risk factors for the disease.

[0005] The activation of the complement system is a cascade reaction controlled by multiple regulatory molecules. The degree of its activation and the activity of individual components are both carried out under a biological feedback mechanism, thus limiting the expansion of activation to maintain the balance of complement levels. The regulatory effects include two aspects, namely self-inactivation and the inactivation effect of some inhibitors. The former refers to the unstable structure of the activated complement molecules. If they do not bind to the target cell membrane in time, they will decay and inactivate. The role of inhibitors means that certain molecules can bind to the active components of the complement, inactivate them or block their binding to other components, thereby inhibiting the assembly of the membrane attack complex. Among them, CD59 (also known as protectin) is a kind of complement inhibitor.

[0006] The present invention provides a recombinant ligand-binding molecule that simultaneously contains a growth factor-binding domain and a complement inhibitory domain, so that it can both bind one or more growth factors including VEGF-A, VEGF-B, and molecules of the PlGF family, and prevent the generation of the membrane attack complex (MAC) on the cell surface, which has important clinical significance for the treatment of neovascularization-related diseases. Summary of the Invention

[0007] Broadly speaking, the present disclosure relates to compounds, methods, compositions, and articles that provide multispecific ligand-binding molecules (such as fusion protein molecules). Such ligand-binding molecules can neutralize and block the binding of various growth factors such as vascular endothelial growth factor to receptors on the membrane surface, thereby playing a synergistic role in the lesion microenvironment. It is beneficial not only to control pathological conditions such as angiogenesis and leakage, but also to intervene in the over-activated complement system at the etiology, thus overcoming the limitations and adverse reactions of single-target growth factor therapy. The benefits provided by the present disclosure are widely applicable to the fields of treatment and diagnosis and can be used in combination with various other drugs.

[0008] In some aspects, the present disclosure provides a ligand-binding molecule that contains at least one growth factor-binding domain and at least one complement inhibitory domain, wherein the growth factor-binding domain can recognize, bind, target, and / or antagonize one or more growth factors including VEGF-A, VEGF-B, and PlGF (preferably human growth factors). In some embodiments, the complement inhibitory domain contains an amino acid sequence derived from the CD59 protein (preferably human CD59 protein).

[0009] In some embodiments, the growth factor binding domain comprises an amino acid sequence derived from any one or more of the proteins selected from VEGFR1, VEGFR2, and VEGFR3.

[0010] In some specific embodiments, the growth factor binding domain comprises an amino acid sequence having at least 90% identity (such as at least 95% identity, such as 100% identity, i.e., the same) with any one of the amino acid sequences selected from:

[0011] (a) The amino acid sequence defined by positions 132 - 230 of SEQ ID NO:1;

[0012] (b) The amino acid sequences defined by positions 117 - 218, 117 - 327, 123 - 327, 117 - 421, 23 - 327, 225 - 327, 23 - 421 of SEQ ID NO:2;

[0013] (c) The amino acid sequence defined by positions 47 - 210 of SEQ ID NO:3; and

[0014] (d) Any combination of at least two of the above amino acid sequences (also referred to as a chimeric amino acid sequence), wherein the amino acid sequences are operably linked to each other. For example, the amino acid sequence defined by positions 132 - 230 of SEQ ID NO:1 is operably linked to the amino acid sequence defined by positions 225 - 327 of SEQ ID NO:2.

[0015] In some embodiments, the growth factor binding domain comprises an amino acid sequence having at least 90% identity with the amino acid sequence defined by positions 132 - 230 of SEQ ID NO:1 (i.e., SEQ ID NO:9). For example, the growth factor binding domain comprises an amino acid sequence having no more than 10 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid mutation compared to the amino acid sequence shown at positions 132 - 230 of SEQ ID NO:1. The mutations can be selected from insertions, substitutions, and deletions. In one embodiment, the growth factor binding domain comprises the sequence shown in SEQ ID NO:9.

[0016] In some embodiments, the growth factor binding domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence defined by positions 225-327 of SEQ ID NO:2 operably linked to an amino acid sequence having at least 90% identity to the amino acid sequence defined by positions 132-230 of SEQ ID NO:1. In some specific embodiments, the growth factor binding domain comprises the sequence shown in SEQ ID NO:10. In some other embodiments, the growth factor binding domain comprises an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:10.

[0017] In some embodiments, the complement inhibitory domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence defined by positions 26-102 of SEQ ID NO:4. For example, the growth factor binding domain comprises an amino acid sequence having no more than 10, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid mutations compared to the amino acid sequence shown in positions 26-102 of SEQ ID NO:4. The mutations can be selected from insertions, substitutions, and deletions.

[0018] In some embodiments, the complement inhibitory domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence defined by SEQ ID NO:11. For example, the growth factor binding domain comprises an amino acid sequence having no more than 10, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:11. The mutations can be selected from insertions, substitutions, and deletions.

[0019] In some embodiments, the ligand-binding molecule comprises one or more complement inhibitory domains operably linked to the N-terminus of one or more growth factor binding domains. Alternatively, the ligand-binding molecule comprises one or more complement inhibitory domains operably linked to the C-terminus of one or more growth factor binding domains. The operable linkage can be a direct linkage or a linkage via a linker, such as a peptide linker comprising a (G4S)n peptide linker, where n = 1-5. In one specific embodiment, the ligand-binding molecule comprises or consists of one complement inhibitory domain operably linked to the N-terminus of one growth factor binding domain.

[0020] In some embodiments, the ligand-binding molecule comprises or consists of the sequence shown in SEQ ID NO:6 or 7 or 8.

[0021] In some embodiments, the ligand-binding molecule further comprises an Fc region of an immunoglobulin selected from IgA, IgM, IgE, IgD, and IgG, including IgG1, IgG2, IgG3, and IgG4. The Fc region can be the Fc region of human IgG1 or a variant thereof.

[0022] In some embodiments, the ligand-binding molecule comprises:

[0023] (a) a complement inhibitory domain operably linked to the N-terminus of a growth factor-binding domain, and the C-terminus of the growth factor-binding domain operably linked to an Fc region;

[0024] (b) a growth factor-binding domain operably linked to the N-terminus of a complement inhibitory domain, and the C-terminus of the complement inhibitory domain operably linked to an Fc region;

[0025] (c) a complement inhibitory domain operably linked to the N-terminus of an Fc region, and the C-terminus of the Fc region operably linked to a growth factor-binding domain; or

[0026] (d) a growth factor-binding domain operably linked to the N-terminus of an Fc region, and the C-terminus of the Fc region operably linked to a complement inhibitory domain.

[0027] The operable linkage can be a direct linkage or a linkage through a linker (e.g., a peptide linker can comprise or consist of a G4S series or a GPG peptide).

[0028] In some embodiments, the ligand-binding molecule comprising an Fc is a dimer and comprises two identical chains or different chains depending on whether the two chains of the Fc are different (e.g., when a knob into hole structure is included, the two chains of the Fc are different). In some embodiments, the ligand-binding molecule comprises an amino acid sequence as set forth in SEQ ID NO: 12, 13, or 14.

[0029] In some embodiments, the ligand-binding molecule is capable of inhibiting or blocking the binding of at least one of VEGF-A, VEGF-B, and PlGF to its corresponding receptor.

[0030] In some specific embodiments, the ligand-binding molecule can block the binding of VEGF-A to VEGFR2 with an EC 50 lower than 3.5 nM, lower than 3 nM, or lower than 2.5 nM, as measured by ELISA.

[0031] In some embodiments, the ligand-binding molecule further comprises a signal peptide.

[0032] In some embodiments, the ligand-binding molecule further comprises a polyethylene glycol moiety, optionally attached to the amino terminus of the molecule.

[0033] In some aspects, the present invention also provides a fusion protein comprising the ligand-binding molecule disclosed above operably linked to a heterologous peptide.

[0034] In some additional embodiments, the heterologous peptide is selected from the following: additional ligand-binding molecules such as the extracellular domain (ECD) of common human receptor proteins or Traps formed by truncation and recombination thereof; antibody variable region fragments and combinations thereof, such as antibody Fab, single-chain antibody (scFv), nanobody, human heavy chain variable region single-domain antibody (V H ), bioactive polypeptides, and protein aptamers.

[0035] In some embodiments, the heterologous peptide is a heterologous peptide targeting human VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD, Ang-2, TGF-β, FGF (fibroblast growth factor), CTGF (connective tissue growth factor), EGF (epidermal growth factor), S1P, Galectin, or Decorin.

[0036] In some embodiments, the heterologous peptide may be an antibody targeting human VEGF-A or an antigen-binding fragment thereof (e.g., Fab), such as bevacizumab (Avastin) or ranibizumab (Lucentis).

[0037] In some embodiments, the ligand-binding molecule is directly linked to the N-terminus of the heterologous peptide by a peptide bond at the C-terminus, or directly linked to the C-terminus of the heterologous peptide by a peptide bond at the N-terminus. In some embodiments, the linker is a polypeptide or a chemical group.

[0038] In some aspects, the present invention also provides a conjugate comprising the ligand-binding molecule as described above conjugated to at least one module selected from the following: a modification module, a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide element, a luminescent label, a fluorescent label, or an enzyme-substrate label), or a purification module.

[0039] In some aspects, the present invention also provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the ligand-binding molecule or the fusion protein. The nucleic acid molecule may further comprise a promoter sequence linked to the polynucleotide sequence.

[0040] In some aspects, the present invention also provides a vector comprising the nucleic acid molecule. The vector may include, but is not limited to, a lentiviral vector, an adeno-associated virus vector, an adenovirus vector, a liposome vector, and any combination thereof.

[0041] In some embodiments, the vector is a replication-defective adenovirus vector, wherein the nucleic acid molecule is operably linked to a promoter and flanked by adenovirus polynucleotide sequences.

[0042] In some aspects, the present invention also provides a host cell transformed or transfected with the polynucleotide or vector described above. The host cell is preferably a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a human cell.

[0043] In some aspects, the present invention also provides a method for preparing a ligand-binding molecule as disclosed herein, which comprises the following steps:

[0044] Culturing the host cell under suitable conditions to express the ligand-binding molecule; and

[0045] Isolating the ligand-binding molecule from the host cell.

[0046] In some aspects, the present invention also provides a pharmaceutical composition comprising the ligand-binding molecule or fusion protein as disclosed above or a nucleic acid molecule encoding the ligand-binding molecule, and a pharmaceutically acceptable adjuvant or carrier, such as a diluent or excipient. The pharmaceutical composition can be formulated for topical administration or intravitreal injection or implantation. The composition can be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion, or suspension.

[0047] In some aspects, the present invention also provides a method for inhibiting complement overactivation in a subject and diseases or conditions caused by complement overactivation, the method comprising administering to the subject an amount of the ligand-binding molecule or pharmaceutical composition as disclosed herein effective to inhibit complement overactivation in the subject. The subject can be a human or a non-human mammal, such as a mouse, a rat, a rabbit, a monkey, etc.

[0048] In some aspects, the present invention also provides a method for inhibiting neovascularization in a subject and diseases or conditions caused by neovascularization, the method comprising administering to the subject an amount of the ligand-binding molecule or pharmaceutical composition as disclosed herein effective to inhibit neovascularization in the subject.

[0049] In some embodiments, the neovascularization is retinal neovascularization and / or choroidal neovascularization.

[0050] In some embodiments, the disease or condition includes choroidal neovascularization and neoplasia, retinal neovascularization, and ocular conditions associated with fundus leakage. The disease may be selected from, including but not limited to, geographic atrophy of the fundus, age-related macular degeneration, diabetic eye diseases (such as diabetic macular edema), polypoidal choroidal vasculopathy, fundus fibrosis lesions, lesions associated with retinal vein occlusion, retinopathy of prematurity, and macular telangiectasia.

[0051] In some embodiments, the pharmaceutical composition is administered locally to the eye of the subject, or the composition is administered by intravitreal injection, by intravitreal implant, or by topical administration.

[0052] In some aspects, the present invention also provides the use of the ligand-binding molecule or fusion protein as described above in the preparation of a medicament for treating neovascularization and diseases or conditions caused by neovascularization in a subject.

[0053] In some aspects, the present invention also provides the use of the ligand-binding molecule or fusion protein as described above in the preparation of a medicament for treating neovascularization in a subject and simultaneously inhibiting complement overactivation in the subject.

[0054] In some aspects, the present invention provides a kit comprising a ligand-binding molecule or fusion protein or pharmaceutical composition as described herein.

[0055] The above is an overview and thus includes simplifications, generalizations, and omissions of details where necessary; thus, those skilled in the art will recognize that this overview is merely illustrative and not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, uses, and / or other subjects described herein will become apparent from the teachings shown herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Showing the protein structural compositions of the respective molecules.

[0057] Figure 2 Showing the SEC detection chromatograms of the respective molecules after one-step Protein A purification.

[0058] Figure 3 Showing the blocking effect of LFV-035, LFV-036, LFV-050, and the control molecule on the binding between VEGF-A 165 and VEGFR2, as determined by ELISA.

[0059] Figure 4 Showing the binding effect of each molecule on VEGF-B, as determined by ELISA.

[0060] Figure 5 Show the binding of each molecule to PlGF, as determined by ELISA.

[0061] Figure 6A Show the average percentage of the fourth-grade fluorescence spots in the fundus of rats in the laser-induced choroidal neovascularization model experiment for each drug group after 14 days and 27 days of drug administration. *P≤0.05: model control group vs. Eylea group (D14), Eylea + LFV-038 mixture group (D14), LFV-036 group (D27); **P≤0.01: model control group vs. LFV-050 group (D14); ***P≤0.001: model control group vs. Eylea + LFV-038 mixture group (D27), LFV-050 group (D27); ■P≤0.05: LFV-036 group vs. LFV-050 group (D27).

[0062] Figure 6B Show the average fluorescence leakage area of the fundus fluorescence spots in the laser-induced choroidal neovascularization model experiment for each drug group after 14 days and 27 days of drug administration. **P≤0.01: model control group vs. Eylea group (D14, D27); ***P≤0.001: model control group vs. Eylea + LFV-038 mixture group (D14, D27), LFV-050 group (D14, D27); ▲P≤0.05: Eylea vs. LFV-050 group (D27); ●P≤0.05: Eylea + LFV-038 mixture group vs. LFV-050 group (D14, D27). Detailed Description of the Invention

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications, and other publications cited herein are incorporated by reference in their entirety. If the definitions set forth herein conflict with the definitions set forth in the patents, patent applications, and other publications incorporated by reference herein, the definitions set forth herein shall control.

[0064] As used herein, the term "growth factor binding domain" refers to a domain contained within a ligand-binding molecule that is capable of recognizing, binding to, targeting, and / or antagonizing one or more growth factors (preferably human growth factors). The growth factors generally include VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, and PDGF family molecules (e.g., PDGF-A, PDGF-B, PDGF-C).

[0065] In some embodiments, the growth factor binding domain comprises an amino acid sequence derived from a VEGF receptor (e.g., the full-length amino acid sequence of VEGFR1 shown in SEQ ID NO: 1, the amino acid sequence of VEGFR2 shown in SEQ ID NO: 2, and the full-length amino acid sequence of VEGFR3 shown in SEQ ID NO: 3). As disclosed herein, the amino acid sequence derived from a VEGF receptor includes a fragment of the VEGF receptor or a variant thereof, and substantially retains the ability to bind to the corresponding VEGF growth factor. In some embodiments, the growth factor binding domain is a chimera comprising amino acid sequences derived from two or more VEGFRs. The term "chimera" or "chimeric amino acid sequence" refers to amino acid sequences derived from two or more VEGFRs operably linked (directly linked or indirectly linked through a linker) together. For example, the chimera can be a fragment of 132-230 of SEQ ID NO: 1 (VEGFR1) operably linked to the N-terminus or C-terminus of a fragment of 225-327 of SEQ ID NO: 2 (VEGFR2).

[0066] As used herein, the term "complement inhibitory domain" is also referred to herein as "CD59 domain" or "protectin domain", and refers to a domain derived from a complement inhibitory protein (specifically CD59), or a domain having the function of inhibiting the overactivation of complement. In some embodiments, the complement inhibitory domain comprises an amino acid sequence derived from the CD59 protein (e.g., the full-length CD59 protein shown in SEQ ID NO: 4). As disclosed herein, the amino acid sequence derived from the CD59 protein is a fragment of the CD59 protein or a variant thereof, and substantially retains the function of the full-length CD59 protein to inhibit complement activation or disrupt the assembly of the membrane attack complex. In some specific embodiments, the amino acid sequence derived from the CD59 protein is as shown in SEQ ID NO: 11. Those skilled in the art will understand that although the complement inhibitory domain specifically disclosed herein relates to CD59, it can comprise amino acid sequences derived from a variety of other complement inhibitory proteins, including but not limited to, complement regulatory molecules such as CR1, CR2, CR3, CD55, CD46, etc. and their functional fragments. Therefore, the ligand-binding molecule of the present invention can comprise a complement inhibitory domain derived from the CD59 protein and a complement inhibitory domain derived from another complement inhibitory protein.

[0067] As used herein, the term "CD59 (also known as protectin)" refers to differentiation factor 59, a 12KD membrane-bound glycosylated protein that can bind to the site where complement components C8 and C9 interact, block the binding of C8 and C9, and inhibit the final assembly of C9 components on the membrane to form leakage pores. Thus, it is an effective inhibitor of the membrane attack complex. In mice, there are two homologous sequences of protectin, protectin a and protectin b, where protectin b is only expressed in local organs, and protectin a is considered the functional sequence that mainly plays a role. In humans, monkeys, and rats, there is only one protectin sequence. The sequence similarity between human protectin and murine protectin a is 34%.

[0068] As used herein, the term "VEGF" refers to the VEGF family, which consists of VEGF glycoproteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E) and platelet-derived growth factor (PDGF). VEGF-A encompasses any form of VEGF-A, including the native protein of VEGF-A (processed or unprocessed form), full-length VEGF-A, its fragments (such as truncated forms, extracellular / transmembrane domains), or variants (such as splice variants, allelic variants, or artificially engineered variants) and their modified forms (such as glycosylation). There are 5 VEGF-A isoforms in the human body according to different splicing patterns, namely VEGF-A 121 、VEGF-A 145 、VEGF-A 165 、VEGF-A 189 、VEGF-A 206 ,where VEGF-A 165 is the most important homologous monomer of VEGF-A. Similarly, VEGF-B, VEGF-C, and VEGF-D also encompass their native proteins (including processed or unprocessed forms), full-length or fragments (such as truncated forms, extracellular / transmembrane domains), or variants (such as splice variants, allelic variants, or artificially engineered variants) and their modified forms (such as glycosylation).

[0069] As used herein, the term "vascular endothelial growth factor receptor" or "VEGF receptor" includes VEGFR1, VEGFR2, and VEGFR3, which are members of the receptor tyrosine kinase family. The proteins of these three receptors contain a large extracellular region composed of 6 or 7 immunoglobulin (Ig)-like domains (designated D1-D7 respectively), a single transmembrane (TM) helix, and a cytoplasmic region with tyrosine kinase activity, as well as additional regulatory sequences. Examples of the amino acid sequences of the full-length native proteins of human VEGFR1 and VEGFR2 are SEQ ID NO:1 and SEQ ID NO:2 respectively.

[0070] As used herein, "Fc" refers to the following portion derived from an antibody, which comprises the second (C H 2) and third (C H 3) constant regions of the first heavy chain joined to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc region may also include all or part of the hinge region. The Fc portion of an antibody is responsible for various effector functions such as ADCC and CDC, but does not function in antigen binding. The ability of an antibody to initiate and regulate effector functions via its Fc domain is a key part of its protective activity in vivo. Increasing evidence indicates that the in vivo activity of an antibody also highly depends on the interaction between the IgG Fc domain and its associated receptors, the Fc gamma receptors (FcγR) and the FcRn receptor.

[0071] As used herein, the term "operably linked" refers to the juxtaposition (with or without a spacer or linker or insert sequence) of two or more biological sequences of interest such that they are in a relationship that permits them to function in the intended manner. When used in reference to polypeptides, it refers to linking polypeptide sequences in such a way that the resulting product has the intended biological function. For example, a ligand-binding molecule may be operably linked to an immunoglobulin constant region so as to provide a stable product with ligand-binding activity. As another example, a ligand-binding molecule may be operably linked to an immunoglobulin constant region via an insert sequence therebetween, and such insert sequence may be a spacer or may contain a longer sequence. In an embodiment of the present invention, the growth factor-binding domain and the complement inhibitory domain are operably linked, for example, via a linker, such that they do not interfere with each other's functions.

[0072] As used herein, when used in reference to an amino acid sequence (such as a peptide, polypeptide or protein), the terms "fusion" or "fused" refer to the combination of two or more amino acid sequences, for example, by chemical bonding or recombination, to form a single amino acid sequence that does not occur naturally. A fusion amino acid sequence may be produced by genetic recombination of two coding polynucleotide sequences and may be expressed by introducing a construct containing the recombinant polynucleotide into a host cell. The term "fusion protein" as used herein refers to a polypeptide having two (or more) moieties covalently linked together, where each moiety is a peptide with different properties. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two moieties may be directly linked by a single peptide bond or linked by a peptide linker containing one or more amino acid residues. Generally, the two moieties and the linker will be linked in a manner consistent with the reading frame.

[0073] As used herein, the terms "derived from" and "derived of" are used interchangeably and refer to variants obtained from a parental sequence by deletion, addition, and / or substitution of one or more amino acid residues. In some embodiments, an amino acid sequence "derived from" a parental amino acid sequence is a fragment of the parental amino acid sequence. For example, when the full-length amino acid sequence of VEGFR1 (as shown in SEQ ID NO:1) is used as the parental amino acid sequence, the amino acid sequence derived from SEQ ID NO:1 may refer to the fragment shown at positions 132-230 of SEQ ID NO:1 or an amino acid sequence consisting thereof; when the full-length amino acid sequence of VEGFR2 (as shown in SEQ ID NO:2) is used as the parental amino acid sequence, the amino acid sequence derived from SEQ ID NO:2 may refer to the fragment shown at positions 225-327 of SEQ ID NO:2 or an amino acid sequence consisting thereof. In some other embodiments, an amino acid sequence "derived from" a parental amino acid sequence is a variant of the parental amino acid sequence or a fragment thereof, such as a mutation at one or more amino acid positions. The mutation may be a mutation at a glycosylation site to eliminate the glycosylation site. For example, when the full-length amino acid sequence of CD59 (as shown in SEQ ID NO:4) is used as the parental amino acid sequence, the amino acid sequence derived from SEQ ID NO:4 may refer to the fragment shown in SEQ ID NO:11 or an amino acid sequence consisting thereof.

[0074] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed by specific mathematical models or computer programs (i.e., "algorithms"). Methods that can be used to calculate the identity of nucleic acids or polypeptides for alignment include those described in Computational Molecular Biology, (Lesk, A.M. ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W. ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M. and Griffin, H.G. eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J. eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0075] As used herein, the term "specifically binds" or "binds specifically" refers to a non-random binding reaction between two molecules, such as between a ligand and a ligand-binding molecule. In certain embodiments, the ligand-binding molecules provided herein bind with a Kd ≤ 10 -6 M (e.g., ≤ 5 x 10 -7 M, ≤ 2 x 10 -7 M, ≤ 10 -7 M, ≤ 5 x 10 -8 M, ≤ 2 x 10 -8 M, ≤ 10 -8 M, ≤ 5 x 10 -9 M, ≤ 4 x 10 -9 M, ≤ 3 x 10 -9 M, ≤ 2 x 10 -9 M, or ≤ 10 -9(K of the binding affinity of (M)) D ) specifically binds to human VEGF-A. The K used herein D refers to the ratio of the dissociation rate to the association rate (k off / k on ), which can be determined by using any conventional method known in the art, including but not limited to surface plasmon resonance method, microscale thermophoresis method, LC-MS method, and flow cytometry (e.g., FACS) method. In certain embodiments, K D value can be appropriately determined by using flow cytometry.

[0076] As used herein, the ability of "blocking binding" refers to the ability of the ligand-binding molecule of the present invention to inhibit the binding interaction between two molecules (e.g., VEGF-A and VEGFR2) to any detectable degree. In certain embodiments, the ligand-binding molecule that blocks the binding between two molecules inhibits the binding interaction between the two molecules by at least 85% or at least 90%. In certain embodiments, the inhibition can be greater than 85%, or greater than 90%. In certain embodiments, the ability of the ligand-binding molecule to block binding is measured by IC 50 (half maximal inhibitory concentration) or EC 50 (half maximal effect concentration).

[0077] As used herein, the term "effective amount" refers to the amount of an active compound or the amount of a material, composition, or dosage containing the active compound, which is effective for producing certain desired therapeutic effects commensurate with a reasonable benefit / risk ratio when administered according to the desired treatment regimen. For example, the effective amount can refer to the amount effective for inhibiting angiogenesis in a subject, the amount effective for inhibiting complement overactivation in a subject, the amount effective for inhibiting at least one of VEGF-A, VEGF-B, and PlGF in a subject (e.g., in the eye), or the amount effective for stimulating at least one of the VEGFR-1 and VEGFR-2 receptor families expressed in ocular cells or eye blood vessels. Those skilled in the art can easily determine the effective amount and treatment regimen for a specific subject according to the specific situation of the subject.

[0078] A ligand-binding molecule comprising a growth factor-binding domain and a complement-inhibiting domain

[0079] The ligand-binding molecule in the present invention simultaneously comprises a growth factor-binding domain and a complement-inhibiting domain, so that while effectively binding to block one or more growth factors, it can also act on the downstream pathway of complement to block the formation of complement-mediated membrane attack complex. The inventors of the present application unexpectedly found that the combination of these two domains produces a synergistic effect, overcomes the limitations of single use of growth factor-targeted therapy, is beneficial to effectively treat angiogenesis-related diseases from the root cause of the disease, and achieves the purpose of ultimate cure.

[0080] Preferably, the growth factor binding domain and the complement inhibition domain are connected by a linker, so that there is a certain distance between the two domains in space and they do not interfere with each other when performing their functions. The linker can generally adopt various linkers for protein engineering well known in the art, preferably peptide linkers, and the length can be from 2 amino acids to dozens of amino acids, such as 20, 30, 40 or more amino acids. In some embodiments, the peptide linker used to connect the two domains includes a G4S series linker, such as (G4S)1, (G4S)2, (G4S)3, etc.

[0081] Growth factor-binding domain

[0082] The growth factor binding domain disclosed herein can specifically bind to at least one of VEGF-A, VEGF-B, and PlGF, more preferably at least two. Most preferably, the ligand binding molecule has specific binding affinity for VEGF-A, VEGF-B, and PlGF.

[0083] As used herein, the expression "specifically binds to..." or "has a specific binding affinity for..." when referring to a growth factor includes not only specific binding to the free, active form of the growth factor, but also binding to other forms of the growth factor. For example, VEGF-A has multiple isoforms, some of which are in circulation and others are associated with heparin sulfate proteoglycans on the cell surface. A ligand binding molecule that specifically binds to VEGF-A binds to at least one circulating isoform, preferably all circulating isoforms, and more preferably binds to other isoforms. Therefore, when it is mentioned that a ligand binding molecule specifically binds to VEGF-A, it is encompassed that it is able to specifically bind to VEGF-A. 121 , VEGF-A 145 , VEGF-A 165 , VEGF-A 189 and VEGF-A 206 .

[0084] In some embodiments, the growth factor binding domain comprises an amino acid sequence derived from one or more of VEGFR1, VEGFR2, and VEGFR3 proteins.

[0085] In some embodiments, at least one growth factor binding domain of the ligand binding molecule comprises an amino acid sequence derived from native VEGFR1, particularly the extracellular region of native VEGFR1.

[0086] In some embodiments, at least one growth factor binding domain of the ligand binding molecule comprises an amino acid sequence derived from native VEGFR2, particularly the extracellular region of native VEGFR2.

[0087] In some embodiments, at least one growth factor binding domain of the ligand-binding molecule comprises an amino acid sequence derived from native VEGFR3, particularly the extracellular region of native VEGFR3.

[0088] Preferably, the ligand-binding molecule has a significantly increased binding affinity for one or more of VEGF-A, VEGF-B, and PlGF as compared to the extracellular region of native VEGFR2 or a control ligand-binding molecule.

[0089] Preferably, the VEGF-A, VEGF-B, and PlGF are from or derived from mammals such as humans, mice, and monkeys, and more preferably are native human VEGF-A, VEGF-B, and PlGF, including their various isoforms, partially processed or unprocessed precursor forms.

[0090] In some embodiments, one or more of the VEGF-A, VEGF-B, and PlGF are presented as free soluble proteins or as an anchored form expressed on the cell surface.

[0091] The present invention also encompasses the ability of the ligand-binding molecule to bind to abnormally highly expressed forms of VEGF-A, VEGF-B, and PlGF, thereby promoting the restoration of normal body functions by binding to these abnormally highly expressed growth factors. As is known in the art, in some pathological states, VEGF / PlGF growth factors are overexpressed, causing these growth factors to exhibit abnormal functions in body tissues.

[0092] In some embodiments, at least one growth factor binding domain of the ligand-binding molecule comprises an amino acid sequence derived from VEGFR1 (particularly the extracellular region of VEGFR1), and the amino acid sequence derived from VEGFR1 is a fragment of VEGFR1 or an amino acid sequence having at least 80% identity thereto. Specifically, the fragment of VEGFR1 can be selected from the amino acid sequence defined by positions 132-230 of SEQ ID NO:1 and its N-terminal or C-terminal truncated or extended fragments (e.g., truncated or extended by no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid).

[0093] In some embodiments, at least one growth factor binding domain of the ligand-binding molecule comprises an amino acid sequence derived from VEGFR3 (particularly the extracellular region of VEGFR3), and the amino acid sequence derived from VEGFR3 is a fragment of VEGFR3 or an amino acid sequence having at least 80% identity thereto. Specifically, the fragment of VEGFR3 can be selected from the following:

[0094] (a) The amino acid sequence defined by positions 47-115 or 25-115 of SEQ ID NO:3;

[0095] (b) The amino acid sequence defined by positions 154-210 or 116-153 of SEQ ID NO:3;

[0096] (c) The amino acid sequence defined by positions 248-314 or 211-247 of SEQ ID NO:3; and

[0097] (d) An N-terminal or C-terminal truncated or extended fragment of (a), (b), or (c) (e.g., truncated or extended by no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid).

[0098] In some embodiments, the at least one growth factor binding domain comprises an amino acid sequence derived from VEGFR1 operably linked to an amino acid sequence derived from VEGFR2. Specifically, it comprises the amino acid sequence defined by positions 132-230 of SEQ ID NO:1 operably linked to the amino acid sequence defined by positions 225-327 of SEQ ID NO:2.

[0099] The operable linkage can be a direct linkage to each other or a linkage via a linker. Linkers used in the field of protein engineering are well known to those skilled in the art and can all be used in the ligand-binding molecules of the present invention. In some embodiments, the linker comprises a G4S series linker and can also be AAA or other natural short peptide sequences. As will be understood by those skilled in the art, the linker can have a wide range of amino acid sequences as long as it can achieve an operable linkage between the two amino acid sequences.

[0100] Complement-inhibiting domain

[0101] As described above, the present disclosure provides a ligand-binding molecule comprising at least one growth factor binding domain and at least one complement inhibitory domain, and the complement inhibitory domain comprises an amino acid sequence derived from the CD59 protein (preferably human CD59 protein).

[0102] Studies have found that in patients with geographic atrophy, the complement overreacts and damages the cells in the macula, which is the central part of the retina responsible for processing central vision and visual details, and this overreaction can cause the patients to gradually develop blindness.

[0103] Human CD59 protein is an inhibitor of the terminal complement pathway, consisting of 128 amino acid residues (as shown in SEQ ID NO: 4), which is widely distributed. It has been demonstrated that it is expressed in skin, liver, kidney, pancreas, lung, salivary gland, nervous system, placenta, and various blood cells (red blood cells, lymphocytes, neutrophils, and platelets). The main physiological function of C59 protein is to prevent the lysis and destruction of allogeneic or autologous cells by MAC, that is, allogeneic restriction, by binding to C7, C8, or C9 to prevent the assembly of MAC. When it binds to the C5b-7 complex, it can prevent it from binding to C8 again; when it binds to C8, it can hinder the spreading of the first C9 molecule bound to MAC, thereby preventing the subsequent binding of C9; and when it binds to C9 in MAC, it can also prevent the further polymerization of C9. In this way, in the presence of CD59, the complete assembly of MAC cannot be successfully completed on the surface of allogeneic or autologous cells, or the C5b-(9)n complex cannot be assembled, thus limiting the lysis of allogeneic or autologous cells. Moreover, CD59 protein has an advantage over complement regulatory molecules acting upstream by inhibiting the complement system downstream.

[0104] The ligand-binding molecule in this article contains a complement inhibitory domain. The complement inhibitory domain may contain amino acid sequences derived from various complement inhibitory proteins, including but not limited to, complement regulatory molecules such as CR1, CR2, CR3, CD55, CD59, CD46, and their functional fragments. Preferably, the complement inhibitory domain contains an amino acid sequence derived from CD59 protein or its functional fragment.

[0105] As used herein, the term "functional fragment" refers to a part of a polypeptide molecule (such as human CD59 protein) that contains at least 60%, 70%, 80%, 90%, 95%, or more of the full length of the polypeptide (such as SEQ ID NO: 4) and retains the function of the full-length polypeptide. That is, the functional fragment described in the present invention can inhibit the overactivation of complement and disrupt the assembly of the membrane attack complex. In some embodiments, the functional fragment is the amino acids at positions 26-102 of SEQ ID NO: 4 or the amino acids shown in SEQ ID NO: 11.

[0106] In some embodiments, the complement inhibitory domain contains an amino acid sequence having at least 90% identity with the amino acid sequence defined by positions 26-102 of SEQ ID NO: 4. For example, the growth factor-binding domain contains an amino acid sequence having no more than 10 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid mutation compared to the amino acid sequence shown at positions 26-102 of SEQ ID NO: 4. The mutations can be selected from insertions, substitutions, and deletions.

[0107] In some embodiments, the complement inhibitory domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence defined by SEQ ID NO:11. For example, the growth factor binding domain comprises an amino acid sequence having no more than 10, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid mutation compared to the amino acid sequence shown in SEQ ID NO:11. The mutations can be selected from insertions, substitutions, and deletions.

[0108] A ligand-binding molecule comprising an Fc region and optionally a signal peptide

[0109] The ligand-binding molecule of the present invention can be a monomer (i.e., single-chain) or a dimer (i.e., double-chain) or a multimer. In a dimer or multimer structure, the individual chains of the ligand-binding molecule are covalently or non-covalently linked to each other. In some embodiments, the linkage occurs between the VEGFR2-derived sequences of the ligand-binding molecule and / or between the two chains of the Fc. For example, the linkage can be a disulfide bond linkage.

[0110] In some embodiments, the ligand-binding molecule provided by the present invention further comprises an immunoglobulin constant domain sequence, such as the constant domain sequence of human IgG, more specifically the Fc region sequence of IgG1, IgG2, IgG3, or IgG4. The ligand-binding molecule can be a homodimer or a heterodimer. For example, when the ligand-binding molecule is a homodimer, it comprises two identical chains, each chain comprising a complement-binding domain operably linked to a growth factor binding domain, and the growth factor binding domain operably linked to a single-chain Fc. Alternatively, each chain comprises a growth factor binding domain operably linked to a complement-binding domain, and the complement-binding domain operably linked to a single-chain Fc. In certain embodiments, the Fc region is operably linked to the growth factor binding domain and / or the complement inhibitory domain via a hinge region. Optionally, the hinge region can be derived from human IgG1, IgG2, or IgG4. In certain embodiments, the hinge region is derived from human IgG1.

[0111] The immunoglobulin constant domain sequence can be linked to the sequence derived from VEGFR1 and / or VEGFR2 and / or VEGFR3 via a linker (such as GPG). As is known in the art, Fc refers to the portion of an antibody composed of the second and third constant regions of the first heavy chain of the antibody bound to the second and third constant regions of the second heavy chain via disulfide bonds, and optionally the Fc region further comprises all or part of the hinge region. The Fc region herein includes both the wild-type Fc region and its variants, which have different mutations for various purposes. The variants can contain one or more amino acid residue modifications, such as substitutions, in the Fc region.

[0112] In certain embodiments, the Fc region variant comprises one or more amino acid substitutions that improve the pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants can have an extended pharmacokinetic half-life because it binds to FcRn at acidic pH allowing it to escape degradation in lysosomes and then be transported and released from the cell. Methods for engineering antibody molecules to improve the binding affinity to FcRn are well known in the art, see, e.g., 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).

[0113] In certain embodiments, the Fc region variant comprises one or more amino acid substitutions that alter antibody-dependent cell cytotoxicity (ADCC), or alter complement-dependent cell cytotoxicity (CDC) by improving or reducing C1q binding and / or CDC.

[0114] In certain embodiments, the ligand-binding molecule provided by the present invention comprises a human IgG4 constant region in which the 228th amino acid residue is altered, such as Ser 228 Pro (S 228 P, which can prevent or reduce chain exchange), and / or the 235th amino acid residue is altered, such as Leu 235 Glu (L 235 E, which can alter Fc receptor interaction).

[0115] In certain embodiments, the ligand-binding molecule provided by the present invention comprises one or more amino acid substitutions in the interface of the Fc region to assist and / or facilitate heterodimerization. These modifications include introducing a protrusion into the first Fc polypeptide and a cavity into the second Fc polypeptide, wherein the protrusion can be located in the cavity to promote the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating protein molecules with these modifications are known in the art, e.g., as described in U.S. Patent No. 5,731,168.

[0116] In certain embodiments, the ligand-binding molecules provided by the present invention comprise the native Fc sequence of human IgG1, such as Asp of SEQ ID NO:5 104 -Lys 330 as shown. In some embodiments, the ligand-binding molecules provided by the present invention comprise an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity or 100% identity with the amino acid sequence at positions 104-330 of SEQ ID NO:5.

[0117] In some embodiments, the ligand-binding molecule further comprises a signal peptide at the amino terminus to facilitate secretion and purification in cells.

[0118] Properties of the ligand-binding molecule

[0119] In the present invention, it has been found that a ligand-binding molecule that simultaneously comprises a growth factor-binding domain and a complement inhibitory domain can direct the targeting of complement inhibition to a desired site, i.e., the site where the growth factor-binding domain functions. The ligand-binding molecule obtained by combining the two can not only protect the retina from damage by the human complement immune response, but also neutralize and block the binding of various growth factors such as vascular endothelial growth factor to receptors on the membrane surface. Therefore, the ligand-binding molecule exerts a synergistic effect in the lesion microenvironment, which is beneficial for controlling pathological conditions such as angiogenesis and leakage, and can also intervene in the over-activated complement system at the etiology, thereby overcoming the limitations and adverse reactions of single-target growth factor therapy.

[0120] The present invention has found that the ligand-binding molecule can block the binding of VEGF-A to the VEGFR receptor, and can also bind or neutralize VEGF-B and PlGF molecules. As verified by ELISA in the examples: the ligand-binding molecule can block the binding of VEGF-A to VEGFR2 with an IC 50 lower than 3.5 nM, lower than 3 nM, or lower than 2.5 nM; can bind PlGF with an EC 50 lower than 2 nM, lower than 1.5 nM, or lower than 1 nM; can bind VEGF-B with an EC 50 lower than 3.5 nM, lower than 3 nM, lower than 2.5 nM, or lower than 2 nM.

[0121] Polynucleotides, vectors, and host cells

[0122] In some aspects, the invention also provides polynucleotides encoding such ligand-binding molecules. The polynucleotides can be used to express ligand-binding molecules. In some embodiments, the polynucleotides can also be used as therapeutically active agents for achieving in vivo expression of polypeptide ligand-binding molecules.

[0123] In certain embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding at least one of the following amino acid sequences and / or a nucleotide sequence having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity therewith, and / or a variant thereof having only degenerate substitutions:

[0124] (a) the amino acid sequence shown in SEQ ID NO:6, 9 or 12;

[0125] (b) the amino acid sequence shown in SEQ ID NO:7, 10 or 13; and

[0126] (c) the amino acid sequence shown in SEQ ID NO:8, 11 or 14.

[0127] The polynucleotides encoding the ligand-binding molecules are readily isolated and sequenced using conventional procedures known in the art. The polynucleotides can also be obtained by synthetic methods. Preferably, the polynucleotides are codon-optimized for expression in eukaryotic host cells, particularly mammalian cells.

[0128] Using known recombinant techniques, the polynucleotides encoding ligand-binding molecules can be inserted into vectors for further cloning (amplification of DNA) or expression. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters (e.g., SV40, CMV, EF-1α) and transcription termination sequences.

[0129] In some embodiments, the present disclosure provides a vector (e.g., an expression vector) comprising a polynucleotide encoding a ligand-binding molecule provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the polynucleotide, 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 virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (e.g., SV40), λ phage, and M13 phage, liposomes, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0130] A vector comprising a polynucleotide sequence encoding a ligand-binding molecule can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive bacteria, such as Escherichia coli. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the provided vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used in lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and useful herein.

[0131] Suitable host cells for expressing the ligand-binding molecules provided herein can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants from the host, as well as the corresponding permissive insect host cells, have been identified. However, the greatest interest has been in vertebrate cells, and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In some preferred embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some additional preferred embodiments, the host cell is another mammalian cell line, such as a human cell line.

[0132] The host cells are transformed with the above-described expression or cloning vectors for the production of ligand-binding molecules and cultured in a conventional nutrient medium appropriately modified for induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence.

[0133] Host cells for producing the ligand-binding molecules provided herein can 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. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent 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. Re. 30,985 can be used as media for host cells. Any of these media can be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements (defined as inorganic compounds that are typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and will be apparent to the ordinary skilled artisan.

[0134] When recombinant techniques are used, the ligand-binding molecule can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the ligand-binding molecule is produced intracellularly, the first step is to remove particulate debris of the host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Cell debris can be removed by centrifugation. In the case where the ligand-binding molecule is secreted into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0135] The ligand-binding molecule prepared from 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, where affinity chromatography is the preferred purification technique.

[0136] In certain embodiments, Protein A immobilized on a solid phase is used for the immunoaffinity purification of ligand-binding molecules. The suitability of Protein A as an affinity ligand depends on the class and isotype of any immunoglobulin Fc domain present in the ligand-binding molecule. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices can also be used. Mechanically stable matrices (e.g., controlled pore glass or poly(styrene divinyl)benzene) permit faster flow rates and shorter processing times than those achieved with agarose. Other protein purification techniques, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica gel, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available, depending on the antibody to be recovered. TM Chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available, depending on the antibody to be recovered.

[0137] Conjugates

[0138] In some aspects, the present invention also provides conjugates comprising a ligand-binding molecule and a conjugate module conjugated thereto. The conjugate module is a moiety that can be attached to the ligand-binding molecule. A variety of conjugate modules can be linked to the ligand-binding molecules provided herein (see, e.g., “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate modules can be linked to the ligand-binding molecule by methods such as covalent binding, affinity binding, intercalation, coordination binding, complexation, association, admixture, or incorporation.

[0139] In certain embodiments, the ligand-binding molecules disclosed herein can be engineered to contain specific sites outside of the ligand-binding portion that can be used to bind one or more conjugate modules. For example, such sites can include one or more reactive amino acid residues, such as cysteine or histidine residues, to facilitate covalent linkage to the conjugate module.

[0140] In certain embodiments, the conjugate comprises a linker that connects the conjugate module to the ligand-binding molecule. In some other embodiments, the ligand-binding molecule described herein is directly attached to the N-terminal amino acid of the conjugate module by a peptide bond at the C-terminus, or directly attached to the C-terminal amino acid of the conjugate module by a peptide bond at the N-terminus. It can also be connected to the conjugate module through chemical bonds, including but not limited to amide bonds to form a polypeptide chain. In certain embodiments, the ligand-binding molecule can be indirectly or through a second conjugate module connected to the first conjugate module. For example, the ligand-binding molecule can be conjugated to biotin and then indirectly conjugated to a second conjugate module conjugated to avidin. The conjugate module can be a clearance modifying module, a toxin (such as a chemotherapeutic agent), a detectable label (such as a radioisotope, a lanthanide element, a luminescent label, a fluorescent label or an enzyme-substrate label), or a purification module.

[0141] A "toxin" can be any reagent that is harmful to cells or can damage or kill cells. Examples of toxins include but are not limited to paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarboxazine), alkylating agents (such as methyl ethylamine, thiabendazole phenylbutyrate nitrogen mustard, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (such as daunorubicin (previously known as daunomycin) and doxorubicin), antibiotics (such as actinomycin (previously known as actinomycin D), bleomycin, mithramycin and anthramycin (AMC)), antimitotic agents (such as vincristine and vinblastine), topoisomerase inhibitors and tubulin-binding agents.

[0142] Examples of detectable labels can include fluorescent labels (such as fluorescein, rhodamine, dansyl, phycoerythrin or Texas red), enzyme-substrate labels (such as horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, glucose oxidase or β-D-galactosidase), radioisotopes (such as 123 I, 124 I, 125 I, 131 I, 35 S, 3 H,111 In、 112 In、 14 C、 64 Cu、 67 Cu、 86 Y、 88 Y、 90 Y、 177 Lu、 211 At、 186 Re、 188 Re、 153 Sm、 212 Bi and 32 P, other lanthanide elements), a luminescent label, a chromophore moiety, digitoxin, biotin / avidin, a DNA molecule or gold for detection.

[0143] In certain embodiments, the conjugate module can be a clearance modifier that helps increase the half-life of the ligand-binding molecule. Exemplary illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, copolymers of ethylene glycol / propylene glycol, etc. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the ligand-binding molecule can vary, and if more than one polymer is attached, they can be the same or different molecules. In some specific embodiments, the ligand-binding molecule provided by the present invention is conjugated to at least one polyethylene glycol module, and the polyethylene glycol module can be attached to the amino terminus of the ligand-binding molecule.

[0144] In certain embodiments, the conjugate module can be a purification module, such as magnetic beads.

[0145] Pharmaceutical compositions

[0146] In some aspects, the present invention provides a composition comprising the ligand-binding molecule of the present invention formulated with a pharmaceutically acceptable carrier, such as a pharmaceutical composition. Such compositions comprise at least one ligand-binding molecule, fusion protein or conjugate of the present invention. In some embodiments, the pharmaceutical composition is a formulation suitable for intravitreal injection.

[0147] As used herein, "pharmaceutically acceptable carrier" includes any and all pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffering agents, antioxidants, anesthetics, suspension / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or various combinations thereof, and the like. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, transdermal, intravitreal injection or implantation administration, etc. Depending on the route of administration, the active compound, i.e., the ingredient of the present invention, may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

[0148] The pharmaceutical compositions of the present invention may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0149] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. For example, fluidity can be maintained by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by using surfactants.

[0150] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization methods and by including 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, sodium chloride, etc. in the compositions. In addition, the absorption of injectable drug forms can be prolonged by including substances that delay absorption such as aluminum monostearate and gelatin.

[0151] The pharmaceutical composition may be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion or suspension.

[0152] In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for generating liquid solutions, suspensions, or emulsions. Preparations for injection can include sterile and / or pyrogen-free solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be mixed with a solvent before use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle before use, and sterile and / or pyrogen-free emulsions. The solutions can be aqueous or non-aqueous.

[0153] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Such media and reagents for pharmaceutically active substances are known in the art. Unless any conventional media or reagent is incompatible with the active compound, the use of any conventional media or reagent in the pharmaceutical compositions of the present invention can be contemplated. Supplementary active compounds can also be added to the compositions.

[0154] The pharmaceutical composition must generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium that contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using coatings (such as lecithin), by maintaining the required particle size in the case of dispersions, and by using surfactants, appropriate fluidity can be maintained. In many cases, it is preferred to include in the composition isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride. Prolonged absorption of the injectable composition can be caused by including substances that delay absorption (such as monostearates and gelatin) in the composition.

[0155] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent with one or a combination of the aforementioned components as required, followed by sterile microfiltration. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other required components from the aforementioned substances. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered solution.

[0156] The amount of active ingredient that can be combined with a carrier substance to produce a single dosage form depends on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Typically, calculated as 100%, the amount ranges from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, and most preferably about 1% to about 30% active ingredient in combination with a pharmaceutically acceptable carrier.

[0157] Adjust the dosage regimen to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus dose may be administered, several divided doses may be administered over time, or the dose may be proportionally decreased or increased according to the exigencies of the therapeutic situation. It is particularly advantageous to formulate the pharmaceutical compositions in unit dosage form due to ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable as a single dose for a subject to be treated; each unit contains a predetermined quantity of the active compound combined with the required pharmaceutical carrier to produce the desired therapeutic effect. The specifications of the unit dosage forms of the present invention are dictated by and directly depend on: (a) the unique properties of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of individual sensitivities.

[0158] For the administration of the ligand-binding molecule, the dosage range is from about 0.0001 to 100 mg / kg of the host body weight, and more typically 0.01 to 5 mg / kg. For example, the dose can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight or in the range of 1 - 10 mg / kg. Exemplary treatment regimens include once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to six months.

[0159] Optionally, the ligand-binding molecule can be administered as a sustained-release formulation, in which case a less frequent administration is required. The dosage and frequency vary with the half-life of the substance being administered in the patient. The dosage and frequency of administration can depend on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients are treated continuously for the remainder of their lives. In therapeutic applications, relatively short time intervals and relatively high doses are sometimes required until the progression of the disease is slowed or terminated, and preferably until the patient shows partial or complete improvement of the disease symptoms. Thereafter, a prophylactic regimen can be given to the patient.

[0160]

[0161] ​The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can vary so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular combination of the present invention used, or its esters, salts, or amides, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular combination used, the age, sex, weight, condition, general health, and prior medical history of the patient to be treated, and similar factors well known in the medical arts.

[0162] Applications of the ligand-binding molecule

[0163] In some aspects, the present invention provides a method for inhibiting, preventing, or treating neovascularization in a subject and diseases or conditions caused by neovascularization, the method comprising administering to the subject a ligand-binding molecule, its conjugate, or pharmaceutical composition as described herein. The neovascularization includes retinal neovascularization and / or choroidal neovascularization; the diseases or conditions include polypoidal choroidal vasculopathy, retinal neovascularization, and ocular conditions associated with fundus leakage.

[0164] In some embodiments, the ocular conditions are selected from age-related macular degeneration, diabetic eye conditions, geographic atrophy, diabetic macular edema (DME), polypoidal choroidal vasculopathy, fundus fibrosis lesions, retinal vein occlusion-related lesions, retinopathy of prematurity, and macular telangiectasia. Preferably, the pharmaceutical composition comprising a ligand-binding molecule as described herein is administered locally to the eye of the subject by eye drops, by intravitreal injection, or by an intravitreal implant.

[0165] In some embodiments, the ligand-binding molecule, its conjugate, or pharmaceutical composition disclosed herein can be administered in a single administration or multiple administrations. Preferably, the desired therapeutic effect can be achieved by a single administration without the need to administer in combination with other targeted therapeutic drugs.

[0166] In some embodiments, the ligand-binding molecule, its conjugate, or pharmaceutical composition disclosed herein can be administered alone or in combination with one or more additional therapeutic means or reagents. For example, the ligand-binding molecule, its conjugate, or pharmaceutical composition disclosed herein can be used in combination with additional therapeutic agents, such as chemotherapeutic agents or anticancer drugs, or other treatment methods. For example, in the treatment of ocular diseases, other treatment methods include laser photocoagulation, vitrectomy, etc.

[0167] In embodiments where the ligand-binding molecules described herein are used for treating cancer, said additional therapeutic agents include antibodies, bispecific antibodies, multispecific antibodies, chemotherapeutic agents, cytokines, and also include various chemotherapeutic agents, as well as radiotherapy.

[0168] In certain of these embodiments, the ligand-binding molecules, conjugates or pharmaceutical compositions disclosed herein administered in combination with one or more additional therapeutic agents may be administered simultaneously with, sequentially or alternately with one or more additional therapeutic agents. A ligand-binding molecule administered "in combination" with another therapeutic agent need not be administered simultaneously with or in the same composition as the agent. Even in cases where the ligand-binding molecule and the second agent are administered via different routes, the phrase used herein is considered to mean that the ligand-binding molecule administered before or after the other agent is administered "in combination" with that agent. Where possible, the additional therapeutic agents administered in combination with the ligand-binding combinations disclosed herein are administered according to the schedules listed in the product information sheets for the additional therapeutic agents or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or protocols well known in the art.

[0169] In some aspects, the present invention also provides a kit comprising said ligand-binding molecule, conjugate or pharmaceutical composition.

[0170] Advantages of the present invention

[0171] Age-related macular degeneration (AMD) and diabetic macular edema (DME) may be related to factors such as long-term chronic light damage, genetics, and metabolism in the macula. Factors such as inflammatory immunity, upregulation of VEGF expression, oxidative stress, and complement system dysregulation all have an impact on the occurrence and development of AMD. Geographic atrophy is one of the final development forms of wet AMD. The activation of the complement system and the generation of the membrane attack complex (MAC) have an important impact on the pathogenesis of AMD and DME. In the present invention, the inventors combined a growth factor-binding domain with a specific composition and a complement inhibitory domain to produce a ligand-binding molecule with synergistic technical effects. In terms of growth factor binding and blocking, the ligand-binding molecule of the present invention can not only block VEGF-A, but also bind or neutralize VEGF-B and PlGF. In terms of complement inhibition, the strategy of directly targeting the complement inhibitor to the complement-related disease site (the site requiring VEGF antagonism, such as the macular lesion site) by the ligand-binding molecule of the present invention can improve the efficiency of complement inhibition and avoid side effects caused by systemic and long-term complement inhibition.

[0172] Specifically verified by the laser-induced rat model experiment, when using the combined molecule of the present invention to simultaneously block the growth factor and the complement activation pathway, compared with using the growth factor antagonist (Eylea) alone, the complement antagonist alone, and the combination of the two, it has significantly better curative effects on the proportion of grade 4 light spots and the area of light spot fluorescence leakage.

[0173] Using the ligand-binding molecule of the present invention or a pharmaceutical composition containing the same has a more excellent curative effect than using the VEGF-A antagonist alone. Thus, on the one hand, it can meet the current gap in treatment methods with no obvious response to single-target VEGF-A, and on the other hand, it can reduce the number of injections due to the better curative effect, significantly reduce the burden on patients while obtaining the optimal curative effect, reduce treatment inconvenience, improve compliance, and reduce the risks such as intraocular infection caused by injections.

[0174] Sequence summary:

[0175] The proteins and ligand-binding molecules involved in the present application are provided in the following table and the sequence listing provided with the specification.

[0176]

[0177]

[0178]

[0179]

[0180] Examples

[0181] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages and parts are by weight.

[0182] Experimental materials and reagents:

[0183] Eylea: Provided by Taizhou Baiying Biologics according to the sequence disclosed in WO0075319

[0184] Example 1 - Design and expression of a ligand-binding molecule and its Fc fusion protein

[0185] Fragments of the CD59 protein (as shown at positions 26 - 102 of SEQ ID NO:4) were respectively constructed together with fragments of VEGFR1 (as shown at positions 132 - 230 of SEQ ID NO:1), amino acid sequences derived from VEGFR2 and VEGFR1 (as shown at positions 132 - 230 of SEQ ID NO:1 and positions 225 - 327 of SEQ ID NO:2), and connected using a single G4S linker (AGGGGSG), thereby obtaining 2 ligand-binding molecules. Further, the CD59 protein sequence was rationally designed (as shown in SEQ ID NO:11), and it was constructed together with amino acid sequences derived from VEGFR2 and VEGFR1 (as shown at positions 132 - 230 of SEQ ID NO:1 and positions 225 - 327 of SEQ ID NO:2) to obtain a new ligand-binding molecule.

[0186] The obtained ligand-binding molecules were each connected via a short artificial linker sequence Gly-Pro-Gly (GPG) to the human immunoglobulin constant domain sequence (Fc, as shown by Asp 104 -Lys 330 in SEQ ID NO:5) to construct a protein sequence of the ligand-binding molecule fused with Fc, named LFV-036, LFV-035, and LFV-050 respectively. Their amino acid sequences are as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 respectively, and their protein structural compositions are as Figure 1 shown.

[0187] In addition, when connecting the CD59 protein fragment to the amino acid sequences derived from VEGFR2 and VEGFR1, linkers of different lengths were also respectively tried to compare and select the production and expression situations. As described above, LFV-035, LFV-036, and LFV-050 used a single G4S linker (AGGGGSG). In addition, linkers without G4S (AAA), 2×G4S linkers, and 3×G4S linkers were respectively constructed, and the resulting proteins were LFV-048, LFV-049, and LFV-041 respectively (see Table 1 below for details).

[0188] The above-mentioned Fc fusion proteins were transiently transfected into CHO cells (purchased from Gibco Life) after gene synthesis, vector construction, and extraction. First, they were eluted by protein A affinity, and then whether to use a molecular sieve for secondary purification was selected according to the SEC purity situation (the entire process was completed by Taizhou Baiying Biotechnology). The information and expression situations of each molecule are shown in Table 1 below:

[0189] Table 1.

[0190]

[0191] As can be seen from the above, using a single G4S linker (AGGGGSG) is a better choice. For the sequences using other linkers (LFV-048, LFV-049, LFV-041), after one-step purification of Protein A, the purity percentage of the target main peak did not reach more than 30%, and the developability was poor. The SEC results of one-step purification of Protein A for each molecule are as Figure 2 shown.

[0192] In addition, as a control molecule in the rat choroidal neovascularization model experiment of Example 5 below, the CD59 protein fragment (shown at positions 26-102 of SEQ ID NO: 4) was also fused with the Fc sequence and expressed and purified in the same way. The constructed molecule was named LFV-038, and its sequence is as shown in SEQ ID NO: 15.

[0193] Example 2 - Experiment on the binding blockade of VEGFR2 by the Fc fusion protein to VEGF-A

[0194] VEGFR2-Fc (purchased from Sinobio) molecules at 500 ng / well were coated overnight at 4°C. The next day, the plate was washed with PBS, and BSA solution was added to block at room temperature for 2 hours. During the blocking period, each fusion protein molecule (using Eylea as a control) was serially diluted from high concentration to low concentration, and then mixed with 60 ng / well of VEGF-A 165 (purchased from Sinobio, with His tag), and incubated at room temperature for 30 minutes. The VEGFR2-Fc plate was washed, and the incubation solution was added thereto, and incubated at room temperature for 1 hour for binding. The plate was washed with PBST, anti-polyhistidine secondary antibody conjugated with horseradish peroxidase was added, and incubated at room temperature for 1 hour for binding. The plate was washed with PBST, TMB was added for color development, and terminated with 5% hydrochloric acid solution. The OD value at 450 nm was read using a microplate reader.

[0195] The results are as Figure 3 shown. It can be seen that each Fc fusion protein has a significant ability to block the binding of VEGF A-VEGFR2. Among them, LFV-035 and LFV-050 are stronger and are close to the control molecule Eylea.

[0196] Example 3 - Experiment on the binding of the Fc fusion protein to VEGF-B

[0197] VEGF-B at 200 ng / well (purchased from ACROBiosystems) was prepared in PBS and coated overnight at 4°C. The next day, the plate was washed with PBS, 1% BSA solution was added and blocked at room temperature for 2 hours. After washing the plate with PBS, the recombinant receptor molecules were serially diluted from high concentration to low concentration and added to each well, and incubated at room temperature with 300 rpm for 1.5 hours. The plate was washed three times with PBST solution, goat anti-human Fc secondary antibody conjugated with horseradish peroxidase was added, and incubated at room temperature with 300 rpm for 1 h. The plate was washed three times with PBST solution, TMB was added for color development, and terminated with 5% hydrochloric acid solution. The OD value at 450 nm was read using an enzyme-linked immunosorbent assay (ELISA) reader.

[0198] The results are as Figure 4 shown. It can be seen that LFV-035, LFV-036, and LFV-050 all showed certain binding ability to VEGF-B, which was basically the same as that of the control molecule Eylea.

[0199] Example 4 - Experiment on the binding of the Fc fusion protein to PlGF

[0200] PlGF at 200 ng / well (purchased from ACROBiosystems) was prepared in PBS and coated overnight at 4°C. The next day, the plate was washed with PBS, 1% BSA solution was added and blocked at room temperature for 2 hours. After washing the plate with PBS, the recombinant receptor molecules were serially diluted from high concentration to low concentration and added to each well, and incubated at room temperature with 300 rpm for 1.5 hours. The plate was washed three times with PBST solution, goat anti-human Fc secondary antibody conjugated with horseradish peroxidase was added, and incubated at room temperature with 300 rpm for 1 h. The plate was washed three times with PBST solution, TMB was added for color development, and terminated with 5% hydrochloric acid solution. The OD value at 450 nm was read using an enzyme-linked immunosorbent assay (ELISA) reader.

[0201] The results are as Figure 5 shown. It can be seen that LFV-035, LFV-036, and LFV-050 all showed certain binding ability to PlGF, among which LFV-035 and LFV-050 had better binding ability, which was basically the same as that of the control molecule Eylea.

[0202] Example 5 - Experiment on the treatment of laser-induced choroidal neovascularization and fibrosis in rats

[0203] Forty-nine Brown Norway rats were randomly divided into 7 groups according to body weight. Except for the model control group and the Avastin group, each with 13 animals, the remaining groups each had 11 animals, with both males and females. They were respectively the model control group (0.9% sodium chloride), the Eylea group, the LFV-038 (CD59) single-use group, the combination treatment group (Eylea + LFV-038 mixture), the LFV-036 treatment group, the Avastin treatment group, and the LFV-050 treatment group. The drug was administered by single injection into the vitreous body of both eyes, with a dosing volume of 4 μL / eye, and the concentration of the drug groups was 10 mg / mL, that is, the dosing dose was 40 μg / eye. Twenty-four hours after administration, the outer Bruch's membrane of the rat retina was damaged by laser to establish a choroidal neovascularization model. The appearance of bubbles indicated that the Bruch's membrane was broken. The first day of drug administration was defined as day 1 of the experiment.

[0204] During the experiment, the general condition of the surviving rats in each group was observed daily. Before drug administration and on days 14 and 27 after drug administration, fluorescein angiography and fundus photography were performed, the number of grade 4 fluorescence spots in each group was counted, and the Heidelberg Eye explorer software was used to calculate the area of each leaking fluorescence or neovascularization, so as to compare the drug effects of each group.

[0205] The data were statistically analyzed using Stata / IC 15.0 (Windows version). The percentage of grade 4 fluorescence spots and the fluorescein leakage area were described by mean ± standard deviation (X±SD). The lower the percentage of grade 4 fluorescence spots and the fluorescein leakage area, the better the therapeutic effect of the candidate drug. All data were statistically analyzed using ANOVA or the K-W method. When the K-W method showed a statistically significant difference (P≤0.05), the M-W method was used to compare the differences between groups pairwise.

[0206] The results are as Figure 6A - 6B shown. It can be seen that the therapeutic effect of LFV-038 (CD59) used alone was not significantly different from that of the model control group. LFV-036 had a certain therapeutic effect compared with the model control group in the statistical results of the proportion of grade 4 fluorescence, but the difference was not obvious in the statistical results of the fluorescein leakage area. When Eylea and LFV-038 were used in combination, most showed a better therapeutic effect than Eylea used alone, and all showed a better therapeutic effect than LFV-038. The LFV-050 group had the best therapeutic effect among all groups, which was not only better than the Eylea group and other single-drug treatment groups, but even better than the combination treatment group.

[0207] Those skilled in the art will further recognize that the present invention may be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the present invention has only disclosed its exemplary embodiments, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims to indicate the scope and content of the present invention. Sequence Listing <110> Suzhou Photometry Biotechnology Co., Ltd. Hong Kong Photometry Biotechnology Co., Ltd. <120> Multispecific Ligand-Binding Molecules Comprising Complement Inhibitory Domains and Their Uses <130> IDC226007 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 1338 <212> PRT <213> Homo sapiens <400> 1 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655 Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670 Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685 Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695 700 Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe Ile Glu Arg 705 710 715 720 Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gln 725 730 735 Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser 740 745 750 Asp Lys Ser Asn Leu Glu Leu Ile Thr Leu Thr Cys Thr Cys Val Ala 755 760 765 Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu Phe Ile Arg Lys Met Lys 770 775 780 Arg Ser Ser Ser Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile Met Asp 785 790 795 800 Pro Asp Glu Val Pro Leu Asp Glu Gln Cys Glu Arg Leu Pro Tyr Asp 805 810 815 Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Leu Lys Leu Gly Lys Ser 820 825 830 Leu Gly Arg Gly Ala Phe Gly Lys Val Val Gln Ala Ser Ala Phe Gly 835 840 845 Ile Lys Lys Ser Pro Thr Cys Arg Thr Val Ala Val Lys Met Leu Lys 850 855 860 Glu Gly Ala Thr Ala Ser Glu Tyr Lys Ala Leu Met Thr Glu Leu Lys 865 870 875 880 Ile Leu Thr His Ile Gly His His Leu Asn Val Val Asn Leu Leu Gly 885 890 895 Ala Cys Thr Lys Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys 900 905 910 Lys Tyr Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe 915 920 925 Phe Leu Asn Lys Asp Ala Ala Leu His Met Glu Pro Lys Lys Glu Lys 930 935 940 Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg Leu Asp Ser Val 945 950 955 960 Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe Gln Glu Asp Lys Ser 965 970 975 Leu Ser Asp Val Glu Glu Glu Glu Asp Ser Asp Gly Phe Tyr Lys Glu 980 985 990 Pro Ile Thr Met Glu Asp Leu Ile Ser Tyr Ser Phe Gln Val Ala Arg 995 1000 1005 Gly Met Glu Phe Leu Ser Ser Arg Lys Cys Ile His Arg Asp Leu 1010 1015 1020 Ala Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile 1025 1030 1035 Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr 1040 1045 1050 Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp Met Ala Pro 1055 1060 1065 Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys Ser Asp Val Trp 1070 1075 1080 Ser Tyr Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Gly Ser 1085 1090 1095 Pro Tyr Pro Gly Val Gln Met Asp Glu Asp Phe Cys Ser Arg Leu 1100 1105 1110 Arg Glu Gly Met Arg Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu 1115 1120 1125 Ile Tyr Gln Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu 1130 1135 1140 Arg Pro Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu 1145 1150 1155 Gln Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile Asn 1160 1165 1170 Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser Thr Pro Ala 1175 1180 1185 Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser Ala Pro Lys Phe 1190 1195 1200 Asn Ser Gly Ser Ser Asp Asp Val Arg Tyr Val Asn Ala Phe Lys 1205 1210 1215 Phe Met Ser Leu Glu Arg Ile Lys Thr Phe Glu Glu Leu Leu Pro 1220 1225 1230 Asn Ala Thr Ser Met Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr 1235 1240 1245 Leu Leu Ala Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 1250 1255 1260 Lys Pro Lys Ala Ser Leu Lys Ile Asp Leu Arg Val Thr Ser Lys 1265 1270 1275 Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe Cys 1280 1285 1290 His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg Phe Thr 1295 1300 1305 Tyr Asp His Ala Glu Leu Glu Arg Lys Ile Ala Cys Cys Ser Pro 1310 1315 1320 Pro Pro Asp Tyr Asn Ser Val Val Leu Tyr Ser Thr Pro Pro Ile 1325 1330 1335 <210> 2 <211> 1356 <212> PRT <213> Human <400> 2 Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu 1 5 10 15 Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro 20 25 30 Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr 35 40 45 Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro 50 55 60 Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser 65 70 75 80 Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn 85 90 95 Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser 100 105 110 Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser 115 120 125 Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys 130 135 140 Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser 145 150 155 160 Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg 165 170 175 Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile 180 185 190 Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser 195 200 205 Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr 210 215 220 Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu 225 230 235 240 Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile 245 250 255 Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu 260 265 270 Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 275 280 285 Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 290 295 300 Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 305 310 315 320 Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met 325 330 335 Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala 340 345 350 Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly 355 360 365 Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr 370 375 380 Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu 385 390 395 400 Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val 405 410 415 Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val 420 425 430 Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr 435 440 445 Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu 450 455 460 Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr 465 470 475 480 Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys 485 490 495 Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys 500 505 510 Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr 515 520 525 Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser 530 535 540 Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln 545 550 555 560 Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser 565 570 575 Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro 580 585 590 Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr 595 600 605 Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile 610 615 620 Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr 625 630 635 640 Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val 645 650 655 Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn 660 665 670 Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys 675 680 685 Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn 690 695 700 Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg 705 710 715 720 Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr 725 730 735 Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe 740 745 750 Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu 755 760 765 Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile 770 775 780 Ile Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly 785 790 795 800 Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His 805 810 815 Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp 820 825 830 Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val 835 840 845 Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr 850 855 860 Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg 865 870 875 880 Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu 885 890 895 Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu 900 905 910 Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu 915 920 925 Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg 930 935 940 Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys 945 950 955 960 Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly 965 970 975 Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Pro 980 985 990 Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr 995 1000 1005 Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys 1010 1015 1020 Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu 1025 1030 1035 Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile 1040 1045 1050 Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro 1055 1060 1065 Leu Lys Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr 1070 1075 1080 Ile Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile 1085 1090 1095 Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu 1100 1105 1110 Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro 1115 1120 1125 Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp 1130 1135 1140 His Gly Glu Pro Ser Gln Arg Pro Thr Phe Ser Glu Leu Val Glu 1145 1150 1155 His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln Asp Gly Lys 1160 1165 1170 Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr Leu Ser Met Glu Glu 1175 1180 1185 Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu 1190 1195 1200 Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala 1205 1210 1215 Gly Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro 1220 1225 1230 Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu 1235 1240 1245 Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val 1250 1255 1260 Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys Leu 1265 1270 1275 Ser Pro Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser 1280 1285 1290 Val Ala Ser Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly 1295 1300 1305 Tyr His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu 1310 1315 1320 Ala Glu Leu Leu Lys Leu Ile Glu Ile Gly Val Gln Thr Gly Ser 1325 1330 1335 Thr Ala Gln Ile Leu Gln Pro Asp Ser Gly Thr Thr Leu Ser Ser 1340 1345 1350 Pro Pro Val 1355 <210> 3 <211> 1363 <212> PRT <213> Person <400> 3 Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly 1 5 10 15 Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu 20 25 30 Asn Ile Thr Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser 35 40 45 Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala 50 55 60 Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val 65 70 75 80 Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu 85 90 95 Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr 100 105 110 Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Ser 115 120 125 Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe Ile Asn Lys Pro Asp 130 135 140 Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp Val Pro Cys Leu Val 145 150 155 160 Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu 165 170 175 Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu 180 185 190 Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr 195 200 205 Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile 210 215 220 Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser Leu 225 230 235 240 Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala 245 250 255 Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln 260 265 270 Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His 275 280 285 Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp 290 295 300 Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg 305 310 315 320 Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu 325 330 335 Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu Val 340 345 350 Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp 355 360 365 Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His Ser Pro His Ala Leu 370 375 380 Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly Thr Tyr Thr Leu Ala 385 390 395 400 Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu 405 410 415 Val Val Asn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro 420 425 430 Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr 435 440 445 Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr 450 455 460 Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Gln 465 470 475 480 Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala Val Thr Thr Gln Asp 485 490 495 Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp Thr Glu Phe Val Glu 500 505 510 Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val 515 520 525 Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu 530 535 540 Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile 545 550 555 560 Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu Leu 565 570 575 Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp Tyr Arg 580 585 590 Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn Pro Leu Leu Leu 595 600 605 Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu 610 615 620 Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr Leu Ser Leu Ser Ile 625 630 635 640 Pro Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln 645 650 655 Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val 660 665 670 Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu 675 680 685 Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala 690 695 700 His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Glu 705 710 715 720 Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln Lys Leu Ser Ile Gln 725 730 735 Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn 740 745 750 Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser 755 760 765 Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val 770 775 780 Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met 785 790 795 800 Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile 805 810 815 Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser 820 825 830 Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly 835 840 845 Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val Val Glu Ala Ser Ala 850 855 860 Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met 865 870 875 880 Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu 885 890 895 Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu 900 905 910 Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu 915 920 925 Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp 930 935 940 Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly Arg Phe 945 950 955 960 Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg Arg Arg Pro Gly Ser 965 970 975 Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala 980 985 990 Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro 995 1000 1005 Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg 1010 1015 1020 Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu 1025 1030 1035 Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile 1040 1045 1050 Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr 1055 1060 1065 Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro 1070 1075 1080 Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp 1085 1090 1095 Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser 1100 1105 1110 Pro Tyr Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu 1115 1120 1125 Arg Asp Gly Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala 1130 1135 1140 Ile Arg Arg Ile Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala 1145 1150 1155 Arg Pro Ala Phe Ser Glu Leu Val Glu Ile Leu Gly Asp Leu Leu 1160 1165 1170 Gln Gly Arg Gly Leu Gln Glu Glu Glu Glu Val Cys Met Ala Pro 1175 1180 1185 Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser Gln Val Ser 1190 1195 1200 Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp Ser Pro 1205 1210 1215 Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn Trp 1220 1225 1230 Val Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly 1235 1240 1245 Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr 1250 1255 1260 Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly Met Val 1265 1270 1275 Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His Arg Gln 1280 1285 1290 Glu Ser Gly Phe Ser Cys Lys Gly Pro Gly Gln Asn Val Ala Val 1295 1300 1305 Thr Arg Ala His Pro Asp Ser Gln Gly Arg Arg Arg Arg Pro Glu 1310 1315 1320 Arg Gly Ala Arg Gly Gly Gln Val Phe Tyr Asn Ser Glu Tyr Gly 1325 1330 1335 Glu Leu Ser Glu Pro Ser Glu Glu Asp His Cys Ser Pro Ser Ala 1340 1345 1350 Arg Val Thr Phe Phe Thr Asp Asn Ser Tyr 1355 1360 <210> 4 <211> 128 <212> PRT <213> Human <400> 4 Met Gly Ile Gln Gly Gly Ser Val Leu Phe Gly Leu Leu Leu Val Leu 1 5 10 15 Ala Val Phe Cys His Ser Gly His Ser Leu Gln Cys Tyr Asn Cys Pro 20 25 30 Asn Pro Thr Ala Asp Cys Lys Thr Ala Val Asn Cys Ser Ser Asp Phe 35 40 45 Asp Ala Cys Leu Ile Thr Lys Ala Gly Leu Gln Val Tyr Asn Lys Cys 50 55 60 Trp Lys Phe Glu His Cys Asn Phe Asn Asp Val Thr Thr Arg Leu Arg 65 70 75 80 Glu Asn Glu Leu Thr Tyr Tyr Cys Cys Lys Lys Asp Leu Cys Asn Phe 85 90 95 Asn Glu Gln Leu Glu Asn Gly Gly Thr Ser Leu Ser Glu Lys Thr Val 100 105 110 Leu Leu Leu Val Thr Pro Phe Leu Ala Ala Ala Trp Ser Leu His Pro 115 120 125 <210> 5 <211> 330 <212> PRT <213> Human <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 6 <211> 182 <212> PRT <213> Artificial sequence <220> <223> LFV-036 <400> 6 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Ala Gly Gly 65 70 75 80 Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 85 90 95 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 100 105 110 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 115 120 125 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 130 135 140 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 145 150 155 160 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 165 170 175 Gln Thr Asn Thr Ile Ile 180 <210> 7 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> LFV-035 <400> 7 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Ala Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro 85 90 95 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg 100 105 110 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp 115 120 125 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly 130 135 140 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys 145 150 155 160 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His 165 170 175 Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly 180 185 190 Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg 195 200 205 Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser 210 215 220 Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser 225 230 235 240 Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val 245 250 255 Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu 260 265 270 Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys 275 280 285 <210> 8 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> LFV-050 <400> 8 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Ala Asn 35 40 45 Asp Val Leu Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Ala Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro 85 90 95 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg 100 105 110 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp 115 120 125 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly 130 135 140 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys 145 150 155 160 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His 165 170 175 Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly 180 185 190 Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg 195 200 205 Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser 210 215 220 Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser 225 230 235 240 Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val 245 250 255 Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu 260 265 270 Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys 275 280 285 <210> 9 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> LFV-036 <400> 9 Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met 1 5 10 15 Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn 20 25 30 Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp 35 40 45 Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn 50 55 60 Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn 65 70 75 80 Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr 85 90 95 Ile Ile <210> 10 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> LFV-035 <400> 10 Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His 1 5 10 15 Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro 20 25 30 Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro 35 40 45 Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser 50 55 60 Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val 65 70 75 80 Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn 85 90 95 Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser 100 105 110 Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn 115 120 125 Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His 130 135 140 Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met 145 150 155 160 Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp 165 170 175 Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys 180 185 190 Asn Ser Thr Phe Val Arg Val His Glu Lys 195 200 <210> 11 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> LFV-050 <400> 11 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Ala Asn 35 40 45 Asp Val Leu Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn 65 70 75 <210> 12 <211> 412 <212> PRT <213> Artificial sequence <220> <223> LFV-036 <400> 12 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Ala Gly Gly 65 70 75 80 Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 85 90 95 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 100 105 110 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 115 120 125 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 130 135 140 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 145 150 155 160 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 165 170 175 Gln Thr Asn Thr Ile Ile Gly Pro Gly Asp Lys Thr His Thr Cys Pro 180 185 190 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 195 200 205 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 210 215 220 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 225 230 235 240 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 245 250 255 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 260 265 270 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 275 280 285 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 290 295 300 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 305 310 315 320 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 325 330 335 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 340 345 350 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 355 360 365 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 370 375 380 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 385 390 395 400 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 <210> 13 <211> 516 <212> PRT <213> Artificial Sequence <220> <223> LFV-035 <400> 13 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Ala Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro 85 90 95 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg 100 105 110 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp 115 120 125 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly 130 135 140 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys 145 150 155 160 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His 165 170 175 Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly 180 185 190 Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg 195 200 205 Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser 210 215 220 Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser 225 230 235 240 Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val 245 250 255 Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu 260 265 270 Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro 275 280 285 Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 290 295 300 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 305 310 315 320 Methionine, Isoleucine, Serine, Arginine, Threonine, Proline, Glutamic acid, Valine, Threonine, Cysteine, Valine, Valine, Valine, Aspartic acid, Valine, Serine 325 330 335 Histidine, Glutamic acid, Aspartic acid, Proline, Glutamic acid, Valine, Lysine, Phenylalanine, Asparagine, Tryptophan, Tyrosine, Valine, Aspartic acid, Glycine, Valine, Glutamic acid 340 345 350 Valine, Histidine, Asparagine, Alanine, Lysine, Threonine, Lysine, Proline, Arginine, Glutamic acid, Glutamic acid, Glutamine, Tyrosine, Asparagine, Serine, Threonine 355 360 365 Tyrosine, Arginine, Valine, Valine, Serine, Valine, Leucine, Threonine, Valine, Leucine, Histidine, Glutamine, Aspartic acid, Tryptophan, Leucine, Asparagine 370 375 380 Glycine, Lysine, Glutamic acid, Tyrosine, Lysine, Cysteine, Lysine, Valine, Serine, Asparagine, Lysine, Alanine, Leucine, Proline, Alanine, Proline 385 390 395 400 Isoleucine, Glutamic acid, Lysine, Threonine, Isoleucine, Serine, Lysine, Alanine, Lysine, Glycine, Glutamine, Proline, Arginine, Glutamic acid, Proline, Glutamine 405 410 415 Valine, Tyrosine, Threonine, Leucine, Proline, Proline, Serine, Arginine, Aspartic acid, Glutamic acid, Leucine, Threonine, Lysine, Asparagine, Glutamine, Valine 420 425 430 Serine, Leucine, Threonine, Cysteine, Leucine, Valine, Lysine, Glycine, Phenylalanine, Tyrosine, Proline, Serine, Aspartic acid, Isoleucine, Alanine, Valine 435 440 445 Glutamic acid, Tryptophan, Glutamic acid, Serine, Asparagine, Glycine, Glutamine, Proline, Glutamic acid, Asparagine, Asparagine, Tyrosine, Lysine, Threonine, Threonine, Proline 450 455 460 Proline, Valine, Leucine, Aspartic acid, Serine, Aspartic acid, Glycine, Serine, Phenylalanine, Phenylalanine, Leucine, Tyrosine, Serine, Lysine, Leucine, Threonine 465 470 475 480 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 485 490 495 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 500 505 510 Ser Pro Gly Lys 515 <210> 14 <211> 516 <212> PRT <213> Artificial Sequence <220> <223> LFV-050 <400> 14 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Ala Asn 35 40 45 Asp Val Leu Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Ala Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro 85 90 95 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg 100 105 110 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp 115 120 125 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly 130 135 140 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys 145 150 155 160 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His 165 170 175 Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly 180 185 190 Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg 195 200 205 Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser 210 215 220 Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser 225 230 235 240 Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val 245 250 255 Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu 260 265 270 Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro 275 280 285 Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 290 295 300 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 305 310 315 320 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 325 330 335 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 340 345 350 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 355 360 365 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 370 375 380 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 385 390 395 400 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 405 410 415 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 420 425 430 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 435 440 445 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 450 455 460 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 465 470 475 480 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 485 490 495 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 500 505 510 Ser Pro Gly Lys 515 <210> 15 <211> 309 <212> PRT <213> Artificial sequence <220> <223> LFV-038 <400> 15 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Glu Pro Lys 65 70 75 80 Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 85 90 95 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 100 105 110 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 115 120 125 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 130 135 140 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 145 150 155 160 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 165 170 175 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 180 185 190 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 195 200 205 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 210 215 220 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 225 230 235 240 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 245 250 255 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 260 265 270 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 275 280 285 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 290 295 300 Leu Ser Pro Gly Lys 305 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 16 Ala Gly Gly Gly Gly Ser Gly 1 5

Claims

1. A ligand-binding molecule, wherein a complement inhibitory domain is operably linked to the N-terminus of a growth factor-binding domain, and the C-terminus of the growth factor-binding domain is operably linked to the Fc region of an immunoglobulin, wherein: the complement inhibitory domain consists of the amino acid sequence shown in SEQ ID NO: 11, and the growth factor-binding domain consists of a chimeric amino acid sequence derived from VEGFR1 and VEGFR2, the chimeric amino acid sequence being composed of the amino acid sequence defined by positions 132-230 of SEQ ID NO: 1 operably linked to the amino acid sequence defined by positions 225-327 of SEQ ID NO:

2.

2. The ligand-binding molecule according to claim 1, wherein the growth factor-binding domain consists of the amino acid sequence shown in SEQ ID NO:

10.

3. The ligand-binding molecule according to claim 1, wherein the complement inhibitory domain is operably linked to the growth factor-binding domain through a linker.

4. The ligand-binding molecule according to claim 3, wherein the linker is AGGGGS or AGGGGSG.

5. The ligand-binding molecule of any one of the preceding claims, which comprises the amino acid sequence shown in SEQ ID NO:

8.

6. The ligand-binding molecule of claim 1, which comprises an Fc region of an immunoglobulin selected from IgA, IgM, IgE, IgD, and IgG, including IgG1, IgG2, IgG3, and IgG4.

7. The ligand-binding molecule according to claim 6, wherein the Fc region is the Fc region of human IgG1 or a variant thereof.

8. The ligand-binding molecule according to claim 1, wherein the growth factor-binding domain is operably linked to the Fc region of an immunoglobulin through a linker.

9. The ligand-binding molecule according to claim 8, wherein the linker is a peptide linker comprising a G4S series or a GPG peptide.

10. The ligand-binding molecule according to claim 9, wherein the linker has a length of 5-8 amino acid residues and comprises 1 G4S linker.

11. The ligand-binding molecule of claim 1, which consists of the amino acid sequence shown in SEQ ID NO:

14.

12. The ligand-binding molecule of claim 1, wherein the ligand-binding molecule can inhibit or block the binding of at least one of VEGF-A, VEGF-B, and PlGF to its corresponding receptor.

13. The ligand-binding molecule according to claim 12, wherein the VEGF-A, VEGF-B, and PlGF are from or derived from human, mouse, rat, or cynomolgus monkey.

14. The ligand-binding molecule of claim 1, which further comprises a signal peptide.

15. A conjugate comprising the ligand-binding molecule of any one of claims 1-14 conjugated to at least one module selected from: a modification module, a detectable label, or a purification module.

16. The conjugate according to claim 15, wherein the detectable label is a radioisotope, a lanthanide element, a luminescent label, a fluorescent label, or an enzyme-substrate label.

17. The conjugate according to claim 15, wherein the modifying module is a polyethylene glycol module.

18. The conjugate according to claim 17, wherein the polyethylene glycol module is attached to the amino terminus of the ligand-binding molecule.

19. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding the ligand-binding molecule according to any one of claims 1-14.

20. A vector comprising the nucleic acid molecule according to claim 19.

21. The vector according to claim 20, wherein the vector is selected from the group consisting of lentiviral vectors, adeno-associated virus vectors, adenoviral vectors, liposomal vectors, and any combination thereof.

22. The vector according to claim 21, wherein the vector is a replication-deficient adenoviral vector, wherein the nucleic acid molecule is operably linked to a promoter and flanked by adenoviral polynucleotide sequences.

23. A host cell transformed or transfected with the nucleic acid molecule according to claim 19 or the vector according to any one of claims 20-22.

24. The host cell according to claim 23, which is a eukaryotic cell.

25. The host cell according to claim 24, wherein the eukaryotic cell is a Chinese hamster ovary (CHO) cell or a human cell.

26. A method for preparing a ligand-binding molecule, comprising the steps of: - culturing the host cell according to any one of claims 23-25 under suitable conditions to express the ligand-binding molecule; and - isolating the ligand-binding molecule from the host cell.

27. A pharmaceutical composition comprising the ligand-binding molecule according to any one of claims 1-14 or the nucleic acid molecule according to claim 19, and a pharmaceutically acceptable carrier.

28. The pharmaceutical composition according to claim 27, which is in the form of a paste, ointment, gel, aerosol, spray, polymer, film, emulsion, or suspension.

29. The pharmaceutical composition according to claim 27 or 28, which is formulated for topical administration, intravitreal injection, or intravitreal implant administration.

30. Use of the ligand-binding molecule according to any one of claims 1-14 in the preparation of a drug for treating neovascularization and diseases or conditions caused by neovascularization in a subject, wherein the diseases or conditions are selected from choroidal neovascularization or neovascularization and retinal neovascularization.

31. The use according to claim 30, wherein the diseases are selected from geographic atrophy, age-related macular degeneration, diabetic macular edema, polypoidal choroidal vasculopathy, fundus fibrosis lesions, retinopathy of prematurity, and macular telangiectasia.

32. The use according to any one of claims 30-31, wherein the drug is topically administered to the eye of the subject.

33. The use according to claim 32, wherein the drug is administered by intravitreal injection or by intravitreal implant.

34. A kit comprising the ligand-binding molecule according to any one of claims 1-14 or the pharmaceutical composition according to any one of claims 27-29.

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