WO2023092324A1 - 多特异性配体结合分子及其应用 - Google Patents

多特异性配体结合分子及其应用 Download PDF

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WO2023092324A1
WO2023092324A1 PCT/CN2021/132735 CN2021132735W WO2023092324A1 WO 2023092324 A1 WO2023092324 A1 WO 2023092324A1 CN 2021132735 W CN2021132735 W CN 2021132735W WO 2023092324 A1 WO2023092324 A1 WO 2023092324A1
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vegf
amino acid
binding molecule
lfv
ligand binding
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French (fr)
Chinese (zh)
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张健行
尹晴晴
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Hk Light Ferry Biomed Ltd
Suzhou Light Ferry Biomed Co Ltd
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Hk Light Ferry Biomed Ltd
Suzhou Light Ferry Biomed Co Ltd
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Priority to EP21965062.9A priority Critical patent/EP4438629A1/en
Priority to JP2024531308A priority patent/JP2024543158A/ja
Priority to PCT/CN2021/132735 priority patent/WO2023092324A1/zh
Priority to US18/713,012 priority patent/US20250002557A1/en
Publication of WO2023092324A1 publication Critical patent/WO2023092324A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the field of proteins, in particular, the present invention relates to ligand-binding molecules, and glycosylated forms, fusion proteins and conjugates of such ligand-binding molecules, nucleic acid sequences encoding such molecules, and methods for their production and uses.
  • vascular endothelial growth factor (also called VEGF) related gene family includes a variety of secreted glycoproteins, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E.
  • VEGF-A is a 45,000Da glycoprotein with extensive angiogenic activity. Its gene consists of 8 exons and 7 introns. It is divided into 5 monomers according to different splicing methods, namely VEGF121 and VEGF145 , VEGF165, VEGF189, VEGF206, among which VEGF165 is one of the most important homologous monomers of VEGF-A.
  • VEGF-A plays an important role in the early formation of blood vessels, and all vascularized tissues can express VEGF-A.
  • many other cells can also express VEGF-A under hypoxic stress conditions, such as macrophages, platelets, dendritic cells (DCs), activated T cells, retinal pigment epithelial cells, retinal Muller cells, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, mesangial cells and tumor cells, etc.
  • hypoxic stress conditions such as macrophages, platelets, dendritic cells (DCs), activated T cells, retinal pigment epithelial cells, retinal Muller cells, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, mesangial cells and tumor cells, etc.
  • VEGF-C and VEGF-D play key roles in embryonic and postnatal lymphangiogenesis. Homozygous mice lacking the VEGF-C gene are embryonic lethal, and heterozygous deficiency causes defects in lymphatic vessel development in postnatal mice. VEGF-C and VEGF-D may also play an important role in angiogenesis, especially during pathological stages, such as tumor growth. VEGF-E is isolated from the oropharynx virus infection group, and it stimulates the proliferation and migration of endothelial cells, and also has the effect of enhancing the permeability of blood vessels (Vladimir Joukov et al. The EMBO Journal 15 (2): 290-98, February 1996).
  • VEGF ligands mediate angiogenic effects through a variety of different receptors (ie, through ligand-receptor interactions).
  • the two receptors were initially confirmed in endothelial cells, and they were special receptor tyrosine kinase (RTK) VEGFR-1 (FIt-1) and VEGFR-2 (FiK-1). It was later confirmed that it is also expressed on various hematopoietic cell lineages in adults.
  • RTK receptor tyrosine kinase
  • angiogenesis In diseases related to angiogenesis, such as tumor growth and ophthalmic angiogenesis, inhibition of angiogenesis is an effective treatment.
  • drugs such as Lucentis (Ranibizumab), Eylea (Aflibercept), Avastin (bevacizumab) monoclonal antibody), Cyramza (Ramucirumab), Inlyta (Axitinib), Stivarga (regorafenib), Cometriq (cabozantinib), OFEV (Nintedanib), Lenvim (envatinib lenvatinib).
  • wAMD diabetic macular degeneration
  • DME diabetic macular edema
  • VEGF-A has significantly improved curative effect compared with traditional treatment methods (laser photocoagulation, etc.), and corresponding targeted drugs (such as ranibizumab, aflibercept, etc.) Huge market success.
  • a single target of VEGF-A still cannot completely overcome these diseases.
  • the present invention proposes a new solution to this requirement.
  • the present invention provides novel molecules capable of targeting VEGF, PlGF and PDGF families, which have important clinical significance for treating diseases related to neovascularization.
  • the present invention relates to ligand binding molecules that can bind one or more growth factors including VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A, PDGF-B, PDGF-C (ie, multispecific ligand-binding molecules), their glycosylated modified form molecules, compositions comprising such ligand-binding molecules, and production methods and uses thereof.
  • the present invention further verifies the effect of the ligand binding molecule in vivo and in vitro, especially the effect of anti-angiogenesis.
  • the present disclosure provides an isolated ligand binding molecule comprising a VEGFR2-derived sequence and having, compared to native VEGFR2 (e.g., the native full VEGFR2 extracellular domain, e.g., the extracellular sequence Met 1-Glu 764) Enhanced to at least one of VEGF-A, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC, more preferably two, more preferably three, more preferably four , more preferably five, more preferably six, most preferably seven binding capacities.
  • the VEGFR2-derived sequence is an amino acid sequence that is at least 95% identical to any of the following amino acid sequences:
  • amino acid sequence defined by positions 123-327 of SEQ ID NO:2 e.g. LFV-C, LFV-G, LFV-H, LFV-I, LFV-J, LFV-K, LFV-L;
  • the VEGFR2-derived sequence is an N-terminal and/or C-terminal truncated fragment of any of (a)-(f), for example, 20, 15, 10, 9 , 8, 7, 6, 5, 4, 3, 2 or 1 amino acid or consist thereof.
  • the VEGFR2-derived sequence comprises an insertion, substitution, or deletion of one or more amino acids compared to the sequence at the corresponding position in the native VEGFR2 extracellular domain, such as an insertion, deletion, or deletion at a glycosylation site. replace.
  • glycosylation site various modifications can be made at the glycosylation site to remove glycosylation, particularly to eliminate known glycosylation sites such as the "NXT” and/or "NXS" triaminoacids, e.g. By mutating N to Q or A.
  • Particular glycosylation sites can be selected, for example, from amino acid positions 46, 66, 96, 143, 158, 245 and 318 corresponding to SEQ ID NO:2.
  • Asn at the above positions is substituted with Gln or Ala or other suitable amino acids.
  • substitutions at glycosylation sites may be selected from the following: N143A, N143Q, N158A, N158Q, N245Q and N318Q.
  • the VEGFR2-derived sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID Nos: 4-15 and 18.
  • the ligand binding molecule comprises a VEGFR1 -derived sequence in addition to a VEGFR2 -derived sequence.
  • the VEGFR1 derived sequence may be selected from the group consisting of:
  • the ligand binding molecule comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in positions 117-327 or 123-327 of SEQ ID NO: 2 operably linked to the VEGFR1 derived sequence.
  • the ligand binding molecule comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID Nos: 16-17 and 19-21.
  • the ligand binding molecule comprises, in addition to a VEGFR2-derived sequence, a VEGFR3-derived sequence.
  • the VEGFR3 derived sequence may be selected from the group consisting of:
  • the ligand binding molecule comprises an operable amino acid sequence at least 95% identical to the amino acid sequence set forth in positions 117-327, 123-327, or 221-312 of SEQ ID NO:2.
  • the ligand binding molecule comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID Nos: 22-30.
  • the ligand binding molecule further comprises an Fc region of an immunoglobulin selected from the group consisting of IgA, IgM, IgE, IgD and IgG, including IgG1, IgG2, IgG3, IgG4, preferably human IgG1
  • the Fc region or variant thereof optionally further comprising a hinge region.
  • the hinge region and Fc region of the immunoglobulin may be operably linked to the C-terminus of the VEGFR2-derived sequence, VEGFR1-derived sequence or VEGFR3-derived sequence via a linker (such as GPG) or without a linker.
  • the Fc region or variant thereof is in a polymeric form comprising macromolecular compounds such as phospholipids.
  • the Fc region is as shown in amino acid sequence 104-330 of SEQ ID No:31.
  • the ligand binding molecules disclosed herein comprise or consist of any one of SEQ ID Nos: 32-59.
  • the ligand binding molecule further comprises a signal peptide.
  • the ligand binding molecule is capable of binding VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC proteins At least one, preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, more preferably at least eight, more preferably all nine .
  • the ligand binding molecule is capable of binding multiple targets, ie is a multispecific ligand binding molecule.
  • the VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC proteins can be from or derived from human, mouse, rat or food crab monkey.
  • the above-mentioned proteins may be of human origin, including processed or unprocessed forms thereof, and phosphorylated-activated or unphosphorylated-activated forms.
  • the ligand-binding molecule can bind at least one of VEGF-A, VEGF-C, VEGF-D, and PDGF- AB with a KD of 5 nM or lower, preferably at least two, more preferably At least three, most preferably all four, as determined by SPR.
  • the ligand binding molecule is capable of binding at 50 pM or less, preferably 40 pM or less, more preferably 30 pM or less, more preferably 20 pM or less, more preferably 10 pM, more preferably 5 pM, more preferably 4 pM , more preferably 3 pM, more preferably 1 pM, or less , binds at least one of VEGF-A and VEGF-C, as determined by SPR.
  • the ligand binding molecule is capable of binding VEGF with a K of 20 nM or less, preferably 15 nM or less, more preferably 10 nM or less, more preferably 5 nM or less, more preferably 3 nM or less -D, as determined by SPR.
  • the ligand binding molecule is capable of binding at 50 nM or less, preferably 40 nM or less, more preferably 30 nM or less, more preferably 28 nM or less, more preferably 26 nM or less, more preferably 24 nM or less Lower, more preferably 22nM or lower, more preferably 20nM or lower, more preferably 18nM or lower, more preferably 15nM or lower, more preferably 10nM or lower KD binding to PDGF-AA, PDGF-AB, PDGF - at least one, preferably at least two, preferably at least three, more preferably all four of BB and PDGF-CC, as determined by SPR.
  • the ligand binding molecule is capable of inhibiting or blocking VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC Binding of at least one of the proteins to the corresponding receptor for that protein.
  • the receptor is selected from VEGFR-1 receptor, VEGFR-2 receptor, VEGFR-3 receptor, PDGFR- ⁇ receptor, and PDGFR- ⁇ receptor.
  • the receptors can be in the form of monomers, homodimers or any heterodimers formed with each other according to the binding form when they play a role in physiology and pathology.
  • the present invention also provides a fusion protein comprising a ligand binding molecule disclosed above operably linked to a heterologous peptide.
  • the heterologous peptide can be selected from the following: additional ligand binding molecules, such as common human receptor protein extracellular domain (ECD) or its truncated and recombinant polypeptides; antibody variable region fragments and combinations thereof, Such as antibody Fab, single chain antibody (scFv), nanobody (nanobody), human heavy chain variable region single domain antibody (V H ); active polypeptide and protein aptamer.
  • the heterologous peptide is a peptide targeting VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD , Ang-2, TGF- ⁇ , CTGF (connective tissue growth factor), EGF (epidermal growth factor), S1P, Galectin, Decorin, or heterologous peptides of receptors of the aforementioned proteins.
  • the operably linked is directly linked or linked via a linker.
  • the ligand binding molecule is linked directly or via a linker to the N- or C-terminus of the heterologous peptide.
  • the C-terminus is directly linked to the N-terminus of the heterologous peptide through a peptide bond, or the N-terminus is directly linked to the C-terminus of the heterologous peptide through a peptide bond.
  • the linker is a polypeptide or a chemical group.
  • the present invention also provides a conjugate comprising a ligand binding molecule or fusion protein as described above conjugated to at least one module selected from the group consisting of: a modification module, a toxin (e.g. a chemotherapeutic agent) , a detectable label (such as a radioisotope, lanthanide, luminescent label, fluorescent label or enzyme-substrate label) or a purification module.
  • the modification moiety is a polyethylene glycol moiety, optionally the polyethylene glycol moiety is attached to the amino terminus of the ligand binding molecule.
  • the present invention also provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the ligand binding molecule or fusion protein.
  • the nucleic acid molecule may further comprise a promoter sequence or other regulatory sequences linked to the polynucleotide sequence.
  • the present invention also provides a vector comprising the nucleic acid molecule.
  • the vectors may include, but are not limited to, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, liposome vectors and any combination thereof.
  • 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.
  • the present invention also provides a host cell transformed or transfected with the above-mentioned polynucleotide or vector.
  • the host cells are preferably eukaryotic cells, such as Chinese Hamster Ovary (CHO) cells or human cells.
  • the present invention also provides a method of preparing a ligand-binding molecule, comprising the steps of:
  • the host cell as described above is cultured under suitable conditions to express the ligand-binding molecule; and the ligand-binding molecule is isolated from the culture supernatant of the host cell.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the ligand-binding molecule or fusion protein as disclosed above or the nucleic acid molecule encoding the ligand-binding molecule, and a pharmaceutically acceptable adjuvant or a carrier, such as a diluent or excipient.
  • the composition may be formulated for topical administration or intravitreal injection or implant administration.
  • the composition may be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion or suspension.
  • the present invention also provides a method of preventing, inhibiting and/or treating neovascularization and diseases or conditions caused by neovascularization in a subject, said method comprising effectively inhibiting said subject
  • the amount of neovascularization in a subject is administered a ligand binding molecule or pharmaceutical composition as disclosed herein to the subject.
  • the neovascularization is retinal neovascularization and/or choroidal neovascularization.
  • the disease or condition is selected from choroidal vasculopathy or neovascularization, retinal angiogenesis, macular neovascularization, and ocular disorders associated with fundus leak.
  • the composition is administered topically to the subject's eye, or the composition is administered by intravitreal injection, by intravitreal implant, or by topical application.
  • the present invention also provides the use of the ligand binding molecule or fusion protein in the manufacture of a medicament for treating neovascularization and diseases or conditions caused by neovascularization in a subject.
  • the present invention also provides the ligand binding molecules or fusion proteins for use in treating neovascularization and diseases or conditions caused by neovascularization in a subject.
  • the invention provides a kit comprising a ligand binding molecule or fusion protein or a pharmaceutical composition as described herein.
  • Figure 1 shows the results of ELISA for the binding of various fusion protein ligand binding molecules derived from VEGFR2 and VEGFR3 to VEGF-A165 ( Figure 1a), VEGF-C ( Figure 1b) or VEGF-D ( Figure 1c).
  • Figure 2 shows the results of ELISA for the binding of various ligand-binding molecules derived from VEGFR2 to VEGF-C.
  • Figure 3-4 shows the relationship between various ligand-binding molecule pairs VEGFR2-VEGF C ( Figure 3a), VEGFR2-VEGF D ( Figure 3b), VEGFR3-VEGF C ( Figure 4a) and VEGFR3-VEGF D ( Figure 4b). Binding blocking ELISA results.
  • Figure 5 shows the binding ELISA results of various ligand binding molecules derived from VEGFR2 to VEGF-A 165 ( Figure 5a) and VEGF-A 121 ( Figure 5b).
  • Figure 6 shows the blocking ELISA results of various ligand binding molecules to the binding between VEGFR2-VEGF A165 ( Figure 6a) and VEGFR2-VEGF-A121 ( Figure 6b), respectively.
  • Figure 7 shows the pharmacokinetic results of ligand binding molecules in mice.
  • Figure 8 shows the therapeutic effect of ligand binding molecules in a rat CNV model.
  • Figure 9 shows the pairs of ligand-binding molecules removed by glycosylation VEGF-A 165-VEGFR2 (Figure 9a), VEGF C-VEGFR2 ( Figure 9b), VEGF D-VEGFR2 ( Figure 9c), VEGF D-VEGFR3 ( Figure 9a).
  • Figure 9d Blocking ELISA results.
  • Figure 10 shows the pair of ligand binding molecules derived from VEGFR1 and VEGFR2 VEGF-A 165-VEGFR2 (Figure 10a), VEGF-A 165-VEGFR1 ( Figure 10b), VEGF C-VEGFR2 ( Figure 10c), VEGF D-VEGFR2 ( Figure 10d), VEGF D-VEGFR3 ( Figure 10e) blocking ELISA results.
  • Figure 11 shows the binding ELISA results of ligand binding molecules derived from VEGFR1 and VEGFR2 to VEGF-B ( Figure 11a), PlGF ( Figure 11b).
  • Figure 12 shows the blocking ELISA results of VEGF-A 165-VEGFR2 (Figure 12a), VEGF C-VEGFR2 ( Figure 12b), and VEGF D-VEGFR2 ( Figure 12c) by fusion and glycosylation engineered ligand-binding molecules .
  • Figure 13 shows the binding ELISA results of LFV-Q to VEGF-B ( Figure 13a), PlGF ( Figure 13b).
  • Figure 14 shows the SEC single-peak purity detection results after one-step purification before and after removal of LFV-M glycosylation sites.
  • Fig. 15 shows that LFV-Q, LFV-S, LFV-M, LFV-R molecules have the effect on VEGF-A 165-VEGFR2 (Fig. 15a), VEGF-A 165-VEGFR1 (Fig. 15b), VEGF C-VEGFR2 (Fig. 15c) , VEGF D-VEGFR2 ( Figure 15d) blocking ELISA results.
  • Figure 16 shows the binding ELISA results of LFV-Q, LFV-S, LFV-M, LFV-R to VEGF-B ( Figure 16a), PlGF ( Figure 16b).
  • Figure 17 shows LFV-Q, LFV-B, LFV-R, LFV-C to VEGF-A ( Figure 17a), VEGF-C ( Figure 17b), VEGF-D ( Figure 17c) relative to VEGFR2-Fc ELISA binding potency.
  • Figures 18a-18b show the results of LFV-R and LFV-Q's proliferation inhibition experiments on HUVEC cells.
  • Figures 19a-19b show the experimental results of LFV-R and LFV-Q on laser-induced choroidal neovascularization in rats.
  • VEGF refers to the VEGF family, which consists of VEGF glycoproteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E).
  • VEGF-A encompasses any form of VEGF-A, including native protein of VEGF-A (processed or unprocessed form), full-length VEGF-A, fragments thereof (e.g., truncated forms, extracellular/transmembrane domains), or altered variants (eg, splice variants, allelic variants, or engineered variants) and their modified forms (eg, glycosylation).
  • VEGF-A there are five isoforms of VEGF-A in the human body according to different cutting methods, namely VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206, among which VEGF165 is the most important homologous monomer of VEGF-A.
  • VEGF-B, VEGF-C, VEGF-D also encompass their native proteins (including processed or unprocessed forms), full length or fragments (e.g. truncated forms, extracellular/transmembrane domains) or variants (such as splice variants, allelic variants or engineered variants) and their modified forms (eg glycosylation).
  • 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 consisting of 6 or 7 immunoglobulin (Ig)-like domains (referred to as D1-D7, respectively), a single transmembrane (TM) helix and a tyrosine Cytoplasmic domain for acid kinase activity and additional regulatory sequences.
  • Ig immunoglobulin
  • TM transmembrane
  • TM transmembrane
  • Exemplary full-length amino acid sequences of native proteins of human VEGFR2 and VEGFR3 are SEQ ID No: 2 and SEQ ID No: 3, respectively. All subtypes of VEGF-A can bind VEGFR1 and VEGFR2 at the same time, VEGF-B specifically binds and activates VEGFR-1, and the specificity of VEGF-E is related to VEGFR-2.
  • VEGF-C and VEGF-D mainly function by binding VEGFR3, but can also bind VEGFR2, but this binding is weaker than that of VEGFR3.
  • VEGF-A can also bind VEGFR2, but again this binding is weaker than VEGFR1.
  • VEGFR2 can bind to a variety of VEGF molecules, its specificity was considered to be inferior to that of VEGFR1, so the development of aflibercept was mainly based on VEGFR1, and VEGFR2, especially its binding properties to VEGF-C and VEGF-D, has not been known so far. enough attention and research.
  • platelet-derived growth factor belongs to the vascular endothelial growth factor family and is a polypeptide growth factor containing sugar chains with a relative molecular weight of about 30kD.
  • PDGF is basically a homologous or heterologous dimer molecule formed by 4 polypeptide chains A, B, C, and D through disulfide bonds, and there are 5 subtypes of PDGF-AA, BB, AB, CC, and DD.
  • PDGF was first discovered in the alpha granules of platelets.
  • diploid cells such as monocytes, vascular smooth muscle cells, endothelial cells, embryonic cells, mesangial cells (MC) and collecting duct cells
  • PDGF promotes the migration, recruitment and distribution of pericytes.
  • PDGF-B is an extremely important regulatory gene for vascular maturation and stability during body development.
  • PDGF-A, B, and C have a certain role in promoting lymphangiogenesis, especially PDGF-B. It is believed that it has a strong effect of promoting lymphangiogenesis, and has a certain relationship with angiogenesis.
  • PDGF is the earliest discovered connective tissue growth factor, which can promote the growth and differentiation of endothelial cells, glial cells, fibroblasts and vascular smooth muscle cells, and has been clinically used in wound repair treatment.
  • PDGF-AA As used herein, the terms "PDGF-AA”, “PDGF-AB”, “PDGF-BB” and “PDGF-CC” refer to the dimeric forms AA, AB, BB, CC of PDGF and fragments or variants thereof . They generally bind to and function with PDGF receptors as homodimers or heterodimers fixed by disulfide bonds. PDGF A and B chains can form both homodimers and heterodimers. PDGF C chain mainly functions as a homodimer. Experiments have proved that PDGF is an important mitogenic factor, which has the ability to stimulate the division and proliferation of specific cell groups, and plays an important role in pathological fibrosis of tissues.
  • PDGF receptors are composed of two subunits ⁇ and ⁇ , and there are three dimer forms of ⁇ , ⁇ , and ⁇ , all of which can bind to PDGF molecules.
  • the ⁇ unit mainly recognizes PDGF A chain, B chain and C chain, so ⁇ receptors can bind PDGF-AA, AB, BB and CC.
  • the ⁇ unit primarily recognizes the PDGF B chain, whereby the ⁇ receptor primarily binds PDGF-BB.
  • ⁇ receptors can bind PDGF-AB, BB.
  • PDGF molecules After PDGF molecules bind to PDGF receptors, they will promote the dimerization of receptors, and then transmit signals downstream, and play the role of promoting cell proliferation and stimulating cell chemotaxis in an autocrine and paracrine manner, mainly in vascular smooth muscle cells, function in cells such as fibroblasts and glial cells.
  • operably linked refers to the juxtaposition of two or more biological sequences of interest (with or without a spacer or linker or intervening sequence) such that they are in a position allowing relationship in a way that works.
  • polypeptides refers to linking polypeptide sequences in a manner that allows the product of the link to have the intended biological function.
  • a ligand binding molecule can be operably linked to an immunoglobulin constant region so as to provide a stable product with ligand binding activity.
  • the ligand binding molecule can be operably linked to the immunoglobulin constant region through an intervening sequence therebetween, and such intervening sequence can be a spacer or can comprise a longer sequence.
  • fusion when used in reference to amino acid sequences (such as peptides, polypeptides or proteins) refers to the combination of two or more amino acid sequences, such as by chemical bonding or recombinant means , forming a single amino acid sequence that does not occur in nature. Fusion amino acid sequences can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct comprising the recombinant polynucleotides into a host cell.
  • isolated refers to a state obtained artificially from a natural state. If an "isolated” substance or component occurs in nature, it may be because of a change in its natural environment, or the separation of the substance from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a certain living body, and the same high-purity polynucleotide or polypeptide isolated from this natural state is called an isolated polynucleotide or polypeptide.
  • isolated neither excludes mixed man-made or synthetic substances, nor other impure substances which do not affect the activity of the isolated substance.
  • identity refers to the relationship between the sequences of two or more polypeptides or 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 compared molecules and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by specific mathematical models or computer programs (ie, "algorithms”). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides are included in Computational Molecular Biology, (Lesk, A.M.
  • the term “specifically binds” or “specifically binds” refers to a non-random binding reaction between two molecules, eg, between a ligand and a ligand-binding molecule.
  • the ligand binding molecules provided herein are available at ⁇ 10 -6 M (e.g., ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 - 8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M) binding affinity (K D or written as KD ) specifically binds human VEGF-A and/or human PDGF.
  • Kd is the dissociation constant
  • Ka is the association constant
  • KD is the equilibrium dissociation constant ( Kd /Ka ) .
  • KD can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance methods, microthermophoresis methods, HPLC-MS methods, and flow cytometry (eg FACS) methods.
  • the ability to "block binding” means that a ligand binding molecule of the invention inhibits the binding interaction between two molecules (e.g. VEGF-A, C, D and VEGFR2, VEGF-C, D and VEGFR3) to any detectable extent.
  • a ligand binding molecule that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, inhibition may be greater than 85%, or greater than 90%.
  • the term "effective amount” relates to an amount of an active compound or a material, composition or dosage comprising an active compound which is effective to produce a dose commensurate with a reasonable benefit/risk ratio when administered in accordance with a desired treatment regimen.
  • an effective amount herein may refer to an amount effective to inhibit neovascularization in a subject, effective to inhibit (e.g., ocular) VEGF-A, VEGF-B, The amount required for at least one of VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, or to stimulate VEGFR-1 expressed in ocular cells or blood vessels of the eye , the amount of at least one of the VEGFR-2, VEGFR-3 and PDGF receptor families.
  • Ligand binding molecules comprising amino acid sequences derived from VEGFR2
  • the invention provides an isolated ligand binding molecule capable of specifically binding one or more VEGF/PDGF molecules.
  • the VEGF/PDGF molecule may be selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC.
  • the ligand binding molecule is capable of specifically binding at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC Two, more preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, more preferably at least eight. Most preferably, the ligand binding molecule has specific binding affinity for VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC .
  • the expression “specifically binds to” or “has specific binding affinity for” when referring to a growth factor includes not only the free, active form that specifically binds to that growth factor, but also other forms that bind to that growth factor .
  • VEGF-A has multiple isoforms, some of which are circulating and others are associated with heparin sulfate proteoglycans on the cell surface.
  • a ligand binding molecule that specifically binds VEGF-A binds at least one circulating isoform, preferably all circulating isoforms, more preferably other isoforms.
  • a ligand binding molecule specifically binding to VEGF-A when referring to a ligand binding molecule specifically binding to VEGF-A, it is contemplated that it is capable of specifically binding VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A189 and VEGF-A206.
  • VEGF-C is translated upon translation into a precursor molecule with extended amino-terminal and carboxy-terminal propeptides, which are cleaved to produce a "fully processed" form of VEGF-C.
  • a ligand binding molecule that specifically binds VEGF-C encompasses binding to the fully processed form of VEGF-C, preferably also binding to partially processed and unprocessed forms thereof.
  • the ligand binding molecule comprises one or more derived from natural VEGFR2, and compared to the whole extracellular domain of natural VEGFR2 or the ligand binding molecule of the reference, to VEGF-A, VEGF-B, VEGF - Significantly increased binding affinity for one or more of C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC.
  • the ligand binding molecule has significantly increased binding affinity for one or more of VEGF-A, VEGF-C, and VEGF-D compared to native VEGFR2 full extracellular domain or a control ligand binding molecule.
  • the VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC are from or derived from mammals such as human, mouse, Monkey's, more preferably, human's natural VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, respectively, including various Isoforms, partially processed or unprocessed precursor forms.
  • the one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC are represented by Free soluble protein, or in an anchored form expressed on the cell surface.
  • the invention also encompasses abnormally high expression forms of the ligand binding molecules capable of binding VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC , which can promote the body to restore normal functions by combining these abnormally high-expressed growth factors.
  • the VEGF/PDGF/PlGF growth factors are overexpressed in pathological states, so that these growth factors exhibit abnormal functions in body tissues.
  • the ligand binding molecule preferably binds human VEGF- with a KD of about 1 nM or less (e.g., 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM, 5 pM, 1 pM or less).
  • A, and/or human VEGF-C The ligand binding molecule preferably binds human VEGF-D, human PDGF- AB with a K of about 10 nM or less (e.g., 5 nM, 1 nM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM or less). and at least one of human PDGF-BB.
  • the ligand binding molecule preferably binds human PDGF-AA and /or human PDGF-CC.
  • the ligand binding molecule is capable of binding VEGF with a K of 50 pM or less, preferably 40 pM or less, more preferably 30 pM or less, more preferably 20 pM or less, more preferably 10 pM or less - at least one of A and VEGF-C, as determined by SPR.
  • the ligand binding molecule is capable of binding VEGF with a K of 20 nM or less, preferably 15 nM or less, more preferably 10 nM or less, more preferably 5 nM or less, more preferably 3 nM or less -D, as determined by SPR.
  • the ligand-binding molecule can be present at 1 nM or less, preferably 0.5 nM or less, more preferably 0.4 nM or less, more preferably 0.3 nM or less, more preferably 0.2 nM or less EC50 binds VEGF-C; can be at 1nM or lower, preferably 0.5nM or lower, more preferably 0.4nM or lower, more preferably 0.3nM or lower, more preferably 0.2nM or lower, more preferably 0.1nM or A lower EC50 binds VEGF-A (eg VEGF-A165) as determined by ELISA.
  • VEGF-A eg VEGF-A165
  • the ligand binding molecule is capable of binding at 50 nM or less, preferably 40 nM or less, more preferably 30 nM or less, more preferably 28 nM or less, more preferably 26 nM or less, more preferably 24 nM or less Lower, more preferably 22nM or lower, more preferably 20nM or lower, more preferably 18nM or lower, more preferably 15nM or lower, more preferably 10nM or lower KD binding to PDGF-AA, PDGF-AB, PDGF - at least one, preferably at least two, preferably at least three, more preferably all four of BB and PDGF-CC, as determined by SPR.
  • a ligand binding molecule disclosed herein is capable of blocking the binding of one or more growth factors selected from the group consisting of:
  • the ligand binding molecule is capable of blocking the binding of one or more growth factors selected from the group consisting of:
  • the ligand-binding molecules disclosed herein can inhibit the expression with a lower EC 50 (e.g., less than 1 nM, less than 0.5 nM, less than 0.4 nM) compared to native VEGFR2 molecules or control ligand-binding molecules. Block the binding of VEGF-C or VEGF-D to VEGFR-2 or VEGFR-3.
  • a lower EC 50 e.g., less than 1 nM, less than 0.5 nM, less than 0.4 nM
  • any one or more of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha and PDGFR-beta may bind to VEGF/PDGF growth factors in a homodimeric form And to be activated, it can also be activated by combining with VEGF/PDGF growth factors in any heterodimerization form.
  • the corresponding dimerization forms include, but are not limited to, human VEGFR2-human VEGFR2, human VEGFR2-human VEGFR1, human VEGFR2-human VEGFR3, human VEGFR2-human PDGFR ⁇ , human VEGFR2-human PDGFR ⁇ dimer.
  • blocking the binding between a growth factor and a receptor it is contemplated that blocking the binding of the growth factor to the receptor in its various forms (e.g., monomer, homodimer, or heterodimer, etc.) bonding between bodies.
  • the ligand-binding molecule comprises one or more amino acid sequences derived from VEGFR2 (particularly the extracellular region of VEGFR2), which is a fragment of VEGFR2 or has at least 80 % identity amino acid sequence.
  • the fragment of VEGFR2 can be selected from the following:
  • the ligand binding molecule may comprise two or more amino acid sequences derived from the extracellular domain of VEGFR2.
  • the amino acid sequence derived from the extracellular domain of VEGFR2 is a fragment of the extracellular domain of VEGFR2, preferably human VEGFR2.
  • Two or more amino acid sequences derived from the extracellular region of VEGFR2 may be linked to each other directly or via a spacer, or, where the ligand-binding molecule is a double-stranded dimer, may be in two on separate chains.
  • the ligand binding molecule comprises an N-terminal truncated fragment of (b), e.g., the amino acid sequence defined by positions 123-218 of SEQ ID NO:2.
  • the ligand molecule comprises both the amino acid sequences of (b) and (c), for example, the amino acid sequence of (b) is directly connected to the amino acid sequence of (c), i.e. the position of SEQ ID NO:2 117-327 defined amino acid sequence.
  • the ligand molecule comprises both the N-terminal truncated fragment of (b) and the amino acid sequence of (c), e.g., the N-terminal truncated fragment of (b) is directly linked to the amino acid of (c) Sequence, i.e. the amino acid sequence defined by positions 123-327 of SEQ ID NO:2.
  • a ligand binding molecule described herein comprises an amino acid sequence set forth in positions 20-116 of SEQ ID NO: 2, an amino acid sequence set forth in positions 117-218 of SEQ ID NO: 2, an amino acid sequence set forth in positions 117-218 of SEQ ID NO: 2, The amino acid sequence shown in the position 219-327 of SEQ ID NO: 2, the amino acid sequence shown in the position 328-421 of SEQ ID NO: 2, or at least 80%, or at least 85%, or at least 90% of the above sequence %, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence.
  • a ligand binding molecule described herein comprises an amino acid sequence set forth in positions 117-327 of SEQ ID NO: 2, an amino acid sequence set forth in positions 23-327 of SEQ ID NO: 2, an amino acid sequence set forth in positions 23-327 of SEQ ID NO: 2, The amino acid sequence shown in position 117-421 of SEQ ID NO:2, the amino acid sequence shown in position 23-421 of SEQ ID NO:2, or at least 80%, or at least 85%, or at least 90% of the above sequence %, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence.
  • the amino acid sequence having identity is the amino acid sequence shown in positions 20-116 of SEQ ID NO: 2, the amino acid sequence shown in positions 117-218 of SEQ ID NO: 2, the amino acid sequence shown in SEQ ID NO: 2
  • the truncation or extension is no more than 40 amino acids, such as no more than 30 amino acids, no more than 20 amino acids, no more than 15 amino acids, no more than 10 amino acids, no more than 8 amino acids Amino acids, no more than 6 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.
  • the ligand binding molecules described herein comprise the amino acid sequence shown in positions 123-218 of SEQ ID NO:2 or the amino acid sequence shown in positions 123-327 of SEQ ID NO:2 .
  • the amino acid sequence having identity is compared to the amino acid sequence shown in positions 20-116 of SEQ ID NO: 2, the amino acid sequence shown in positions 117-218 of SEQ ID NO: 2, the amino acid sequence shown in positions 117-218 of SEQ ID NO: 2, The amino acid sequence shown in the position 219-327 of SEQ ID NO:2, the amino acid sequence shown in the position 328-421 of SEQ ID NO:2, the amino acid sequence shown in the position 117-327 of SEQ ID NO:2, An amino acid sequence as shown in positions 23-327 of SEQ ID NO:2, an amino acid sequence as shown in positions 117-421 of SEQ ID NO:2, or an amino acid sequence as shown in positions 23-421 of SEQ ID NO:2
  • a sequence contains one or more amino acid modifications, such as amino acid substitutions, insertions, deletions, and the like.
  • the amino acid modification may be a mutation at a glycosylation site to eliminate a glycosylation site, for example to eliminate an N-glycosylation site or an O-glycosylation site.
  • the modification is a mutation at one or more of positions Asn46, Asn66, Asn96, Asn143, Asn158, Asn245, and Asn318 of SEQ ID NO:2.
  • Ligand binding molecules comprising amino acid sequences derived from VEGFR2 as well as from other receptors
  • the ligand-binding molecules of the invention comprise amino acid sequences derived from other receptors in addition to amino acid sequences derived from VEGFR2 for improved response to VEGF-A, VEGF-B, VEGF-C, VEGF- D. Binding or blocking of at least one of PlGF, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC.
  • Other receptors may be other receptor tyrosine kinases such as VEGFR-1, VEGFR-3, PDGFR-alpha and/or human PDGFR-beta.
  • a ligand binding molecule of the present disclosure comprises one or more amino acid sequences derived from VEGFR2 and one or more amino acid sequences derived from VEGFR3 (eg, human VEGFR3, particularly the extracellular region of VEGFR3).
  • said amino acid sequence derived from VEGFR2 is operably linked to an amino acid sequence derived from VEGFR3.
  • Said operably linked may be directly linked to each other or linked via a spacer.
  • the amino acid sequence derived from VEGFR3 is a fragment of VEGFR3 or an amino acid sequence at least 80% identical thereto.
  • the fragment of VEGFR3 can be selected from the following: the amino acid sequence defined by the position 25-115 of SEQ ID NO:3; the amino acid sequence defined by the position 154-210 of SEQ ID NO:3; the position of SEQ ID NO:3 Amino acid sequence defined by 248-314; amino acid sequence defined by position 25-210 of SEQ ID NO:3; amino acid sequence defined by position 154-314 of SEQ ID NO:3; amino acid sequence defined by position 25-314 of SEQ ID NO:3 Amino acid sequence; and N-terminal or C-terminal truncated and/or extended fragments of any of the above (eg truncated or extended by no more than 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4 , 3, 2 or 1 amino acid).
  • the ligand binding molecule may comprise two or more amino acid sequences derived from the extracellular domain of VEGFR3, preferably human VEGFR3.
  • the amino acid sequence derived from the extracellular domain of VEGFR3 is a fragment of the extracellular domain of human VEGFR3.
  • the said ligand binding molecules may be linked directly or via a spacer, or in the case where the ligand-binding molecule is a double-stranded dimer In the case of , it can be on two separate chains.
  • a ligand binding molecule described herein comprises the amino acid sequence set forth in positions 25-210 of SEQ ID NO:3, the amino acid sequence set forth in positions 25-314 of SEQ ID NO:3, or 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 to the above sequence amino acid sequence.
  • the amino acid sequence having identity is the amino acid sequence shown in positions 25-210 of SEQ ID NO:3 or the N-terminal of the amino acid sequence shown in positions 25-314 of SEQ ID NO:3 or C-terminal truncated fragment.
  • the truncation is not more than 40 amino acids, such as not more than 30 amino acids, not more than 20 amino acids, not more than 15 amino acids, not more than 10 amino acids, not more than 8 amino acids, not more than More than 6 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.
  • the ligand binding molecules described herein comprise the amino acid sequence shown in the amino acid sequence shown in positions 47-210 of SEQ ID NO:3.
  • the amino acid sequence having identity comprises a or multiple amino acid modifications, such as amino acid substitutions, insertions, deletions, etc.
  • a spacer can take a variety of forms, as is known in the art.
  • the spacer is a peptide sequence between 1-100 amino acids, preferably 1-50 amino acids in length.
  • the spacer is derived from a linker sequence at the corresponding position in native VEGFR-2 or VEGFR-3 (ie, linker sequences at both ends of the sequence of the derived fragment).
  • the spacer comprises at least 80%, or at least 85%, or at least 90%, or at least 92%, the native peptide sequence or portion thereof linked to a fragment in native human VEGFR-2 or human VEGFR-3. Or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical amino acid sequences.
  • the spacer may or may not be used, and may be a linker sequence commonly used in the art, such as (G4S)n, AAA or other natural short peptide sequences.
  • the spacer can have a wide range of amino acid sequences.
  • the amino acid sequence derived from human VEGFR3 is linked to the amino acid sequence derived from human VEGFR2 via a spacer, wherein the amino acid sequence derived from human VEGFR3 is defined by positions 25-210 of SEQ ID NO:3 Amino acid sequence, the amino acid sequence derived from human VEGFR2 is the amino acid sequence defined by the positions 219-327 of SEQ ID NO:2, and the spacer region comprises the positions 211-231 and the positions 211-231 of SEQ ID NO:3 or the positions 211-327 of SEQ ID NO:2
  • the amino acid sequence defined by 220 is 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% identical Sexual amino acid sequence.
  • the amino acid sequence derived from human VEGFR3 may be at the N-terminal or C-terminal of the amino acid sequence derived from human VEGFR2.
  • the ligand binding molecules of the present disclosure comprise one or more amino acid sequences derived from VEGFR2 and one or more amino acid sequences derived from VEGFR1 (e.g., human VEGFR1, particularly the extracellular region of VEGFR1) .
  • VEGFR2 e.g., human VEGFR1, particularly the extracellular region of VEGFR1
  • amino acid sequence derived from VEGFR2 is operably linked to the amino acid sequence derived from VEGFR3.
  • Said operably linked may be directly linked to each other or linked via a spacer.
  • the amino acid sequence derived from VEGFR1 is a fragment of VEGFR1 or an amino acid sequence at least 80% identical thereto.
  • the fragment of VEGFR1 can be selected from the following: the amino acid sequence defined by the position 132-230 of SEQ ID NO: 1 and its N-terminal and/or C-terminal truncated and/or extended fragments (such as truncated or an extension of no more than 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid).
  • the amino acid sequence having identity comprises one or more amino acid modifications, such as amino acid substitutions, insertions, deletions, etc., compared to the amino acid sequence shown at positions 132-230 of SEQ ID NO:1.
  • the ligand binding molecule comprises one or more amino acid sequences derived from VEGFR2 and one or more amino acid sequences derived from VEGFR1.
  • said amino acid sequence derived from VEGFR2 is operably linked to an amino acid sequence derived from VEGFR1. Said operably linked may be directly linked to each other or linked via a spacer.
  • the amino acid sequence derived from VEGFR1 is a fragment of VEGFR1 or an amino acid sequence at least 80% identical thereto.
  • the fragment of VEGFR1 is selected from the amino acid sequence shown in positions 132-230 of SEQ ID NO: 1, or a truncated and/or extended fragment of the N-terminal and/or C-terminal thereof.
  • Ligand binding molecules comprising an Fc region and optionally a signal peptide
  • the ligand-binding molecules of the present invention may be monomers (ie, single chains), dimers (ie, double chains) or multimers. In dimeric or multimeric structures, the individual chains of the ligand-binding molecule are covalently or non-covalently linked to each other. In some embodiments, linkage occurs between VEGFR-2-derived sequences of the ligand binding molecule.
  • the ligand binding molecule provided by the invention further comprises an immunoglobulin constant domain sequence, such as a human IgG constant domain sequence, more specifically may comprise a hinge region and an Fc region, such as IgG1, IgG2, IgG3 , IgG4 Fc region sequence.
  • Said immunoglobulin constant domain sequences may be linked via a linker to a sequence derived from VEGFR1 and/or VEGFR2 and/or VEGFR3.
  • Fc refers to the part of an antibody consisting of the second and third constant regions of the first heavy chain of the antibody bound via disulfide bonding to the second and third constant regions of the second heavy chain,
  • the Fc region also comprises all or part of the hinge region.
  • the Fc region herein includes both wild-type Fc regions and variants thereof, with different mutations for various purposes.
  • the variant may comprise one or more amino acid residue modifications, eg substitutions, in the Fc region.
  • the Fc region variant comprises one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • Such a variant may have an extended pharmacokinetic half-life because it binds FcRn at acidic pH enabling it to escape degradation in lysosomes and then be transported and released out of the cell.
  • Methods for engineering antibody molecules to improve 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. People, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al. , J. Immunology, 176:346-356 (2006).
  • the Fc region variant comprises one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC), or complement-dependent cellular cytotoxicity (CDC) by improving or reducing C1q binding and/or CDC .
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cellular cytotoxicity
  • the ligand binding molecule provided herein comprises a human IgG4 constant region, wherein the 228th amino acid residue is altered, such as Ser228Pro (S228P, which can prevent or reduce strand exchange), and/or the 235th amino acid residue Amino acid residues are altered, such as Leu235Glu(L235E, which can alter Fc receptor interaction.
  • the 228th amino acid residue is altered, such as Ser228Pro (S228P, which can prevent or reduce strand exchange)
  • S228P Ser228Pro
  • 235th amino acid residue Amino acid residues are altered, such as Leu235Glu(L235E, which can alter Fc receptor interaction.
  • the ligand binding molecules provided herein comprise one or more amino acid substitutions in the interface of the Fc region to assist and/or promote heterodimerization.
  • modifications include the introduction of 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 facilitate the interaction of the first and second Fc polypeptides to form a heterodimer or complex.
  • Methods for producing protein molecules with these modifications are known in the art, eg, as described in US Pat. No. 5,731,168.
  • the ligand binding molecule provided by the invention comprises the natural Fc sequence of human IgG1, as shown in Asp104-Lys330 of SEQ ID NO:31.
  • the ligand binding molecules provided herein comprise at least 80%, or at least 85%, or at least 90%, or at least 92%, or Amino acid sequences that are at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical or have 100% identity.
  • the ligand binding molecule also includes a signal peptide at the amino terminus to facilitate secretion and purification from cells.
  • ligand binding molecule pairs VEGF-A, VEGF-C and VEGF-D were found comprising one or more amino acid sequences derived from VEGFR2 (and/or amino acid sequences derived from other receptors), in particular It is the ability to bind and block VEGF-C and VEGF-D.
  • the ligand binding molecule has significantly improved binding or blocking ability to VEGF-A, VEGF-C and VEGF-D, wherein the binding or blocking ability to VEGF-A is better than that of bevacizumab
  • the monoclonal antibody (Avastin) is close to or superior to Aflibercept (Eylea), and its ability to bind or block VEGF-C and VEGF-D is significantly better than that of the control OPT.
  • the ligand-binding molecule can effectively inhibit angiogenesis by blocking VEGF-A, VEGF-C, VEGF-D, and PDGF family molecules, and play a role in the treatment of various diseases, especially eye diseases or tumor diseases.
  • the present invention finds that the ligand-binding molecule can block the binding of VEGF-A, VEGF-C, and VEGF-D to receptors, and can also bind PDGF molecules. As verified in the examples:
  • the K D of LFV-B binding to VEGF-D was 7.8 times stronger than OPT, and the K D of LFV-E was slightly better than OPT; in the VEGF-D blocking experiment, the EC of LFV-B 50 is 4.7-7.4 times stronger than OPT (respectively for the blocking experiment of VEGFR2 and VEGFR3 binding to VEGF-D), the EC 50 of LFV-C is 4.6-5.9 times stronger than OPT, and the EC 50 of LFV-E is stronger than OPT 4.8-3.8 times, while the control Avastin and Eylea could not block.
  • the K D of LFV-B binding to VEGF-A is 9.3pM, close to or similar to Eylea; in the VEGF-A 165-VEGFR2 blocking ELISA experiment, the EC 50 of LFV-B is 4.67 nM, between Avastin (6.68nM) and Eylea (3.2nM), the EC 50 of LFV-C is 4.57nM, which is close to LFV-B; in the VEGF-A 121-VEGFR2 blocking ELISA experiment, LFV-B The EC 50 of LFV-C is 7.63nM, which is better than Avastin (22.37nM) and Eylea (9.55nM). The EC 50 of LFV-C is 9.35nM, which is close to Eylea and better than Avastin.
  • novel ligand-binding molecules such as LFV-M, LFV- N, LFV-Q, LFV-R, LFV-S
  • the ligand binding molecule has further improved binding or blocking ability to VEGF-A, while good retention of VEGF-A C has the binding or blocking ability to VEGF-D, and has the binding ability to VEGF-B, PlGF, as verified in the examples:
  • the EC 50 of LFV-M and LFV-N were 1.60 and 1.87nM, respectively, which was better than the original molecule LFV-C (3.18nM) not fused with VEGFR1, It is also superior to the control drugs Eylea (2.23nM) and Avastin (4.58nM); in the VEGF-A 165-VEGFR1 blocking ELISA experiment, the EC 50 of LFV-M and LFV-N are 3.14 and 3.30nM, respectively, which is better than Eylea (4.64nM) and Avastin (7.31nM).
  • the EC 50 of LFV-M and LFV-N were 2.17 and 1.79nM, respectively, close to LFV-C (2.30nM), and significantly better than the control OPT (24.59nM) ;
  • the EC 50 of LFV-M and LFV-N were 2.74 and 2.31nM, respectively, close to LFV-C (2.62nM), and significantly better than the control OPT (25.22nM) .
  • the EC 50 of LFV-R was 1.901nM, which was better than 2.645nM of LFV-M, and better than the control drug Eylea (3.937nM);
  • the EC 50 of LFV-R was 6.972nM, better than 7.797nM of LFV-M, and close to the control drug Eylea (6.959nM).
  • the invention also provides polynucleotides encoding such ligand binding molecules.
  • the polynucleotides can be used to express ligand binding molecules.
  • the polynucleotides may also be used as therapeutic agents in active form for in vivo expression of polypeptide ligand binding molecules.
  • an isolated polynucleotide comprises a nucleotide sequence encoding and/or at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identities of nucleotide sequences, and/or variants thereof with only degenerate substitutions:
  • the polynucleotide further comprises a nucleotide sequence encoding at least one of the following amino acid sequences and/or has at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99%) sequence identities, and/or variants thereof with only degenerate substitutions:
  • Polynucleotides encoding the ligand binding molecules are readily isolated and sequenced using routine procedures known in the art.
  • the polynucleotides can also be obtained synthetically.
  • said polynucleotide is codon optimized for expression in eukaryotic host cells, especially mammalian cells.
  • a polynucleotide encoding a ligand binding molecule can be inserted into a vector for further cloning (amplification of DNA) or expression.
  • Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (eg, SV40, CMV, EF-1 ⁇ ), and a transcription termination sequence.
  • the present disclosure provides vectors (eg, expression vectors) comprising a polynucleotide encoding a ligand binding molecule provided herein, at least one promoter (eg, SV40, CMV, EF-1 ⁇ ), and at least one selectable marker.
  • vectors eg, expression vectors
  • a polynucleotide encoding a ligand binding molecule provided herein
  • at least one promoter eg, SV40, CMV, EF-1 ⁇
  • selectable marker eg, SV40, CMV, EF-1 ⁇
  • vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40 ), phage ⁇ , and phage M13, liposome, 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, P
  • 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, for example Gram-negative or Gram-positive bacteria, such as Escherichia coli.
  • 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 among lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful herein.
  • Suitable host cells for expressing the ligand binding molecules provided herein can also be derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • a number of baculovirus strains and variants from hosts and corresponding permissive insect host cells have been identified. However, the greatest interest has been in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure.
  • useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 or 293 cells grown subclonally in suspension culture, Graham et al.
  • Baby hamster kidney cells BHK, ATCC CCL 10
  • Chinese hamster ovary cells/-DHFR CHO, Urlaub et al., Proc.Natl.Acad.Sci.USA 77 :4216(1980)
  • monkey kidney cells CV1 ATCC CCL 70
  • African green monkey kidney cells VEO-76, ATCC CRL-1587
  • human cervical cancer cells HELA, ATCC CCL 2
  • canine kidney cells MDCK , ATCC CCL 34
  • buffalo rat hepatocytes BL 3A, ATCC CRL 1442
  • human lung cells W138, ATCC CCL 75
  • human hepatocytes Hep G2, HB 8065
  • mouse mammary gland tumors MMT 060562, ATCC CCL51
  • TRI cells Mather et al., Annals N.Y.
  • the host cell is a Chinese Hamster Ovary (CHO) cell.
  • the host cells are other mammalian cell lines, such as human cell lines.
  • Host cells are transformed with the expression or cloning vectors described above for the production of ligand binding molecules and cultured in conventional nutrient media with appropriate modifications for induction of promoters, selection of transformants or amplification of genes encoding desired sequences.
  • Host cells used to produce 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.
  • Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; ; WO 87/00195; or any medium described in US Pat. Re. 30,985 can be used as a medium for the 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 drugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source . Any other necessary supplements may 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 host cells selected for expression and will be apparent to those of ordinary skill.
  • 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
  • the ligand binding molecules can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the ligand-binding molecule is produced intracellularly, the first step, eg, by centrifugation or ultrafiltration, removes particulate debris of the host cells or lysed fragments. Cellular debris can be removed by centrifugation. In cases where ligand-binding molecules are secreted into the culture medium, supernatants from such expression systems are usually first concentrated using commercially available protein concentration filters such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.
  • Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.
  • Ligand binding molecules 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, with affinity chromatography being preferred Purification technology.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of ligand binding molecules.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domains present in the ligand-binding molecule.
  • Protein A can be used to purify antibodies based on human ⁇ 1, ⁇ 2 or ⁇ 4 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 may also be used.
  • Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl)benzene allow faster flow rates and shorter processing times than agarose can achieve.
  • Other protein purification techniques such as fractionation on ion-exchange columns , 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.
  • the invention also provides conjugates comprising a ligand binding molecule and a conjugate moiety conjugated thereto.
  • a conjugate moiety is a moiety that can be attached to a ligand binding molecule. It is contemplated that a variety of conjugate moieties can be attached 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 moieties can be linked to the ligand-binding molecule by methods such as covalent bonding, affinity bonding, intercalation, coordination bonding, complexation, association, incorporation, or addition.
  • the ligand binding molecules disclosed herein can be engineered to contain specific sites outside of the ligand binding moiety that can be used to bind one or more conjugate moieties.
  • sites may include one or more reactive amino acid residues, such as cysteine or histidine residues, to facilitate covalent attachment to the conjugate moiety.
  • the conjugate comprises a linker linking the conjugate moiety to the ligand binding molecule.
  • the ligand binding molecules described herein are directly attached at the C-terminus by a peptide bond to the N-terminal amino acid of the conjugate moiety, or at the N-terminus by a peptide bond directly to the N-terminal amino acid of the conjugate moiety. C-terminal amino acid.
  • Polypeptide chains can also be formed by linking conjugate modules with chemical bonds, including but not limited to amide bonds.
  • a ligand binding molecule can be linked to a first conjugate moiety either indirectly or through a second conjugate moiety.
  • the ligand binding molecule can be conjugated to biotin and then indirectly conjugated to a second conjugate moiety that is 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, lanthanide, luminescent label, fluorescent label, or enzyme-substrate label), or a purification module.
  • a "toxin” can be any agent that is harmful to a cell or that can damage or kill a cell.
  • toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, mithromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, Procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, arabinosin glycosides, 5-fluorouracil decarboxyrazine), alkylating agents (
  • detectable labels may include fluorescent labels (such as fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (such as horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase or ⁇ -D-galactosidase), radioactive isotopes (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 lanthanides), Luminescent labels, chromophore moieties, digoxigenin, biotin/avidin, DNA molecules or gold for detection.
  • fluorescent labels such as fluorescein, rh
  • the conjugate moiety can be a clearance modifier that helps increase the half-life of the ligand-binding molecule.
  • Illustrative examples include water soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like.
  • the polymers can be of 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.
  • the ligand binding molecules provided herein are conjugated to at least one polyethylene glycol moiety, which can be attached to the amino terminus of the ligand binding molecules.
  • the conjugate modules can be purification modules, such as magnetic beads.
  • compositions eg, pharmaceutical compositions, comprising a ligand binding molecule of the invention formulated together with a pharmaceutically acceptable carrier.
  • Such compositions comprise at least one ligand binding molecule, fusion protein or conjugate of the invention.
  • the pharmaceutical composition is a formulation suitable for intravitreal injection.
  • pharmaceutically acceptable carrier includes any and all pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, etc. that are physiologically compatible Osmotic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or their respective combination etc.
  • the carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, transdermal, intravitreal injection or implant administration, and the like.
  • the active compound ie, the ingredient of the invention, may be coated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.
  • compositions of the present invention may also include pharmaceutically acceptable antioxidants.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, Such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylene glycol Eminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants Such as ascorbyl palmitate, butylated hydroxyanisole (BHA
  • aqueous and non-aqueous carriers examples 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, etc.
  • vegetable oils such as olive oil And injectable organic esters such as ethyl oleate.
  • proper fluidity can be maintained by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • 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 the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include into the compositions isotonic agents, such as sugars, sodium chloride, and the like. In addition, prolonged absorption of the injectable pharmaceutical forms can be brought about by the inclusion of substances which delay absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical composition may be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion or suspension.
  • the pharmaceutical compositions are formulated as injectable compositions.
  • injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions or solid forms suitable for giving liquid solutions, suspensions or emulsions.
  • Preparations for injection may include sterile and/or non-pyrogenic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be mixed with a solvent just before use, including hypodermic tablets, ready for injection Sterile suspensions, sterile dry insoluble products to be combined with a vehicle before use, and sterile and/or non-pyrolyzable emulsions. Solutions can be aqueous or non-aqueous.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • Such media and agents for pharmaceutically active substances are known in the art. Unless any conventional media or agents are incompatible with the active compounds, any conventional media or agents are contemplated for use in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be incorporated into the compositions.
  • compositions generally must 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 to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols (eg, mannitol, sorbitol) or sodium chloride in the compositions.
  • Prolonged absorption of the injectable compositions can be brought about by including in the compositions substances which delay absorption, for example monostearates and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. .
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form depends upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient and pharmaceutically acceptable combination of carriers.
  • Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound suitable to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • dosages range from about 0.0001 to 100 mg/kg, and more typically 0.01 to 5 mg/kg, of the body weight of the host.
  • dosages may 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 within the range of 1-10 mg/kg.
  • Exemplary treatment regimens include administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.
  • the ligand binding molecule can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary with the half-life of the substance 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 dosages are administered at relatively infrequent intervals over a prolonged period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively short intervals and relatively high dosages are sometimes required until the progression of the disease is slowed or terminated, and preferably until the patient shows partial or complete amelioration of disease symptoms. Thereafter, the patient may be administered a prophylactic regimen.
  • Actual dosage levels of active ingredients and small molecules in the pharmaceutical compositions of the invention may be varied in order to obtain an amount of active ingredient effective to achieve the desired therapeutic response in a particular patient, composition and mode of administration without being toxic to that patient.
  • the selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular combination of the invention employed, or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, Duration of treatment, other drugs, compounds and/or materials used in conjunction with the particular combination used, age, sex, weight, condition, general health and prior medical history of the patient to be treated, and similar factors well known in the medical field .
  • the invention provides a method of inhibiting, preventing or treating neovascularization and diseases or conditions caused by neovascularization in a subject, the method comprising administering to the subject a ligand as described herein Binding molecules, conjugates thereof or pharmaceutical compositions.
  • the neovascularization includes retinal neovascularization and/or choroidal neovascularization; the disease or condition includes polypoidal choroidal vasculopathy, retinal neovascularization, ocular disorders associated with fundus leak.
  • the ocular disorder is selected from age-related macular degeneration, diabetic eye disorder, polypoidal choroidal vasculopathy, fundus fibrotic lesion, retinal vein occlusion-related pathology, retinopathy of prematurity, and macular telangiectasia.
  • a pharmaceutical composition comprising a ligand binding molecule described herein is topically administered to the eye of a subject by eye drops, by intravitreal injection, by intravitreal implant.
  • a ligand binding molecule, conjugate thereof, or pharmaceutical composition disclosed herein can be administered in a single administration or in multiple administrations.
  • the desired therapeutic effect can be achieved through a single administration without the need to administer it together with other targeted therapeutic drugs.
  • a ligand binding molecule, conjugate thereof, or pharmaceutical composition disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents.
  • a ligand binding molecule disclosed herein, a conjugate thereof, or a pharmaceutical composition can be practiced in combination with another therapeutic agent, such as a chemotherapeutic agent, or other treatment methods.
  • another therapeutic agent such as a chemotherapeutic agent, or other treatment methods.
  • other treatment methods include laser photocoagulation, vitrectomy, etc.
  • a ligand binding molecule disclosed herein, a conjugate thereof, or a pharmaceutical composition administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents. Either sequentially or alternately.
  • a ligand binding molecule administered "in combination" with another therapeutic agent need not be administered at the same time or in the same composition as that agent.
  • the phrase as used herein contemplates that a ligand-binding molecule administered either before or after the other agent is administered "in combination" with that agent.
  • kits comprising the ligand binding molecules, conjugates or pharmaceutical compositions thereof.
  • angiogenesis In diseases related to angiogenesis, such as tumor growth and ophthalmic angiogenesis, inhibition of angiogenesis is an effective treatment.
  • VEGF-related signaling Over the past few decades, a great deal of research has been done on VEGF-related signaling, and many drugs are available for treatment.
  • targeting PDGF-BB and VEGF-A at the same time has been placed high hopes. Inhibiting PDGF-BB can cause pericyte apoptosis, thereby breaking the resistance to anti-VEGF-A treatment.
  • PDGF-BB Closely related to tissue fibrosis, fundus fibrosis is the advanced form of AMD and the cause of blindness, and is a major reason for the final failure of VEGF-A therapy (Rofagha S. et al. (2013), J.Ophthalmology.120 (11):2292-2299) (Rosenfeld P.J. et al. (2011), Ophthalmology. 118(3):523-530).
  • the PDGF-BB antagonist E10030 introduced by Novartis and the bispecific antibody ABBV642 developed by AbbVie are more striking.
  • Ranibizumab has a more effective curative effect, and has a more obvious improvement effect on fundus fibrosis.
  • E10030 did not show a comparative advantage compared with single-targeted VEGF-A therapy in a larger phase III clinical trial, but Due to the effective intervention effect on fibrosis, its research on the direction of fibrosis treatment is still ongoing.
  • VEGF-A is obviously better and has a wider response effect, thus introducing the treatment of nAMD and other diseases into the second generation.
  • the therapy also has a good effect on polypoidal choroidal vasculopathy (PCV) patients with high incidence in Asians.
  • PCV polypoidal choroidal vasculopathy
  • Polypoidal choroidal vasculopathy is a subtype of wet macular degeneration, which is different from wet macular degeneration. It has no effective and broad response to targeted VEGF-A therapy, and it has a high incidence in Asia (Imamura Y et al. (2010), Surv.
  • PCV Ophthalmol. 55(6):501-515
  • PCV accounts for a large proportion of newly diagnosed AMD patients in China. The ratio is as high as 33% (LandeA. et al. (2010), Prog Retin Eye Res. 29 (1): 19-29), and Japan reaches 54.7% (Maruko I. et al. (2007), Am J Ophthalmol. 144: 15 -22), due to the large population base in Asia, there is currently a huge treatment gap for PCV.
  • this therapy is a combination therapy, which requires simultaneous injection of two drugs into the vitreous, that is, targeting VEGF-A drug and OPT-302, and the treatment cost is relatively high. Inconvenience, poor patient compliance, increased risk of accidents such as intraocular infection caused by injection.
  • this therapy needs to target VEGF-A, VEGF-C and VEGF-D at the same time, and all of them need to have high affinity (to 10pM and below level), which also hinders the use of currently popular monoclonal antibody technology (An antibody generally only binds to one target, and the affinity is generally at 100pM) to develop its substitutes.
  • ligand binding molecules comprising specific amino acid sequences derived from VEGFR2 (e.g., positions 123-327 or 117-327 of SEQ ID NO: 2) have better binding to VEGF-C or VEGF than OPT -D binding capacity.
  • VEGFR2 e.g., positions 123-327 or 117-327 of SEQ ID NO: 2
  • VEGF Trap molecules containing different fragments from VEGFR2 were constructed and expressed, and their binding and blocking abilities to VEGF-A, VEGF-C, VEGF-D, etc. were studied and characterized in detail.
  • ligand binding molecules comprising amino acid sequences derived from VEGFR2, particularly the amino acid sequences shown in positions 123-327 or 117-327 of SEQ ID NO: 2 exhibit significantly better It has the binding and blocking ability to VEGF-C or VEGF-D, and it also has significantly better binding and blocking ability to VEGF-A.
  • the inventors also unexpectedly characterized the ligand-binding molecule pair PDGF-AA, AB, BB comprising an amino acid sequence derived from VEGFR2, particularly the amino acid sequence shown in positions 117-327 or 123-327 of SEQ ID NO:2 PDGF family growth factors such as , CC, etc. also have a certain binding ability, which is the main structural domain involved in the binding, and is better than the natural VEGFR2 whole extracellular domain recombinant molecule binding to PDGF.
  • PDGF family molecules Since the high expression of PDGF family molecules is considered to be one of the reasons for insufficient response to targeted VEGF-A therapy, resistance and fundus fibrosis after treatment, targeting PDGF family at the same time may benefit more patients , especially for fundus lesions caused by diabetes, it has been reported that PDGF-AA, PDGF-AB, and PDGF-BB are highly expressed in such fundus lesions, and thus have the potential to treat fundus lesions caused by diabetes.
  • the ligand binding molecule comprising the amino acid sequence derived from VEGFR2, especially the amino acid sequence shown in the positions 117-327 or 123-327 of SEQ ID NO: 2, is more effective in the expression and purification process of HEK293 and CHO cells Among them, the initial monomer purity and final yield are significantly better, which means that it can have lower production cost and drug cost than it.
  • novel ligand binding molecules comprising one or more amino acid sequences derived from VEGFR2 operably linked to one or more amino acid sequences derived from VEGFR1 and glycoengineered versions thereof (e.g., LFV-M, LFV-N, LFV-Q, LFV-R (LFV-R HPLC-SEC purity is higher than LFV-M), LFV-S), which have good retention of binding and blocking ability to VEGF-C and VEGF-D
  • VEGF-A especially found to have blocking ability to VEGF A-VEGFR1, and its blocking ability to VEGF A-VEGFR1, VEGF A-VEGFR2 Both are better than the control drugs Eylea and Avastin, and they also have the ability to bind to VEGF-B and PlGF.
  • the present invention can block VEGF-A, VEGF-C, and VEGF-D by using a ligand-binding molecule, and can also bind VEGF-B, PlGF, and PDGF family molecules at the same time, which not only helps to meet the clinical needs.
  • LFV-B and LFV-C have stronger affinity to VEGF-C and VEGF-D than OPT, and their affinity and blocking ability to VEGF-A are also significantly better than Avastin, close to or better than Aflibercept, This helps to extend the interval between injections and reduce the number of injections, thereby further reducing the cost of treatment, while addressing the multiple disadvantages of frequent injections.
  • one drug can be used instead of multiple drugs to meet the vacancy of treatment methods that have no obvious response to VEGF-A targeting, and the number of injections can be reduced by high affinity, Therefore, while obtaining the optimal curative effect, the burden on patients can be significantly reduced, the inconvenience of treatment can be reduced, compliance can be improved, and risks such as intraocular infection caused by injection can be reduced.
  • Embodiment 1- Design and expression of recombinant receptor molecule
  • Trap polypeptides comprising different derivative sequences of VEGFR2 were constructed, as shown in Table 1A below.
  • Some of the Trap polypeptides include fragments derived from VEGFR1 or VEGFR3. These fragments can be operably linked by linkers or linkers.
  • the mutation site numbers used in the present invention such as N143A, N158Q, N318Q, etc., are all relative to the corresponding position of SEQ ID NO:2.
  • the Trap polypeptides designed above can be constructed by linking the human immunoglobulin IgG1 constant domain sequence (fused with the Fc Hinge hinge region) via a short artificial linker sequence Gly-Pro-Gly (GPG). After gene synthesis, vector construction and extraction, the above sequences were transiently transfected into HEK293 or CHO cells (purchased from Gibco Life), and purified by protein A affinity chromatography and molecular sieve secondary purification (of which LFV-D, LFV-F And the LFV-001/V0 molecule has met the purity requirements after one-step purification of protein A, without secondary purification by molecular sieve). The obtained truncated VEGFR2-Fc fusion polypeptide is also called recombinant receptor molecule or ligand binding molecule.
  • Eylea, Avastin, R3A and OPT were used as controls. Eylea according to the sequence published in WO0075319.
  • R3A uses VEGFR3 to use amino acids at positions 25-329 to connect with the human immunoglobulin IgG1 constant domain sequence (fused with the Fc Hinge hinge region)).
  • OPT is constructed as described in WO2015/123715A1, which uses amino acids at positions 25-329, including amino acids at positions 47-314 of VEGFR3, and is glycosylated at positions 104-106 as described in WO2015/123715 A1,
  • the molecule constructed by transforming the N-D-T sequence into a non-Q-D-T sequence and connecting it with the human immunoglobulin IgG1 constant domain sequence (fused with the Fc Hinge hinge region) is called OPT herein.
  • LFV-001 or VO used the sequence in natural VEGFR3 (position 217-227 of SEQ ID NO: 3); sequence; in order to introduce flexibility in length, LFV-002 removed Thr in the connection sequence based on LFV-001; LFV-003 replaced this Thr with Gly to introduce greater flexibility in the main chain structure torsion.
  • LFV-004 to 008 the linker sequence derived from VEGFR3 used by LFV-001 was subjected to a series of mutations to the linker sequence of the corresponding region of VEGFR2 (as shown in Table 1 below), wherein In LFV-008, an Ile derived from VEGFR2 was mutated into Ala of the same polarity with a smaller side chain to reduce the steric clash between the side chains.
  • LFV-B comprising only the VEGFR2 fragment shown in 117-327 of SEQ ID NO:2 and R3A comprising only the VEGFR3 fragment shown in 25-329 of SEQ ID NO:3 were designed.
  • Each of the above trap polypeptides can be constructed by linking the short artificial linker sequence Gly-Pro-Gly (GPG) and the human IgG1 immunoglobulin constant domain sequence (fused with the Fc Hinge hinge region), through gene synthesis, vector construction and After extraction, HEK293 cells (purchased from Gibco Life) were transiently transformed, and then obtained after one-step affinity elution and purification of protein A, which is hereinafter referred to as recombinant receptor molecule, and its expression characteristics are shown in Table 2B below:
  • VEGF-A165, VEGF-C, and VEGF-D polypeptide molecules were purchased from ACROBiosystems. VEGF molecules of 100-300ng/well were configured in PBS, coated overnight at 4 °, washed the plate with PBS the next day, added 1% BSA solution to block at room temperature for 2 hours, washed the plate with PBS, and expressed the recombinant protein obtained in Example 1.
  • Receptor molecules were serially diluted with PBT solution (0.5% BSA dissolved in PBS solution containing 0.05% Tween-20) from high concentration to low concentration, and then added to each well, incubated at room temperature and 300rpm for 1.5 hours, and then used PBST solution (containing 0.05 %Tween-20 in PBS) to wash the plate three times, add goat anti-human Fc secondary antibody coupled with horseradish peroxidase diluted in PBT, incubate at room temperature and 300rpm for 1h, wash the plate three times with PBST, add TMB for color development , terminated with 5% hydrochloric acid solution, and read the OD value at 450 nm using a microplate reader.
  • PBT solution 0.5% BSA dissolved in PBS solution containing 0.05% Tween-20
  • PBST solution containing 0.05 %Tween-20 in PBS
  • the VEGF-C molecule of 100ng/well was prepared in PBS, coated overnight at 4 °, washed the plate with PBS the next day, added 1% BSA solution at room temperature to block for 2 hours, washed the plate with PBS, and expressed the recombinant protein obtained in Example 1.
  • Receptor molecules were serially diluted from high concentration to low concentration and added to each well, incubated at room temperature and 300rpm for 1.5 hours, washed with PBST solution three times, and goat anti-human Fc secondary antibody coupled with horseradish peroxidase was added at room temperature and 300rpm Incubate for 1 h, wash the plate three times with PBST solution, add TMB for color development, stop with 5% hydrochloric acid solution, and read the OD value at 450 nm with a microplate reader.
  • VEGFR2-Fc Metal 1-Glu 764 molecules were coated overnight at 4°, the plate was washed with PBS the next day, and BSA solution was added to block at room temperature for 2 hours.
  • each recombinant receptor molecule was mixed from high concentration to Low-concentration gradient dilution, mixed with 100ng/well VEGF-C or VEGF-D (both with His tags), incubated at room temperature for 30 minutes, washed the VEGFR2-Fc plate, mixed the recombinant receptor molecule with VEGF-C, VEGF Add the incubation solution of -D, incubate at room temperature for 1 hour, wash the plate with PBST, add anti-polyhistidine secondary antibody coupled with horseradish peroxidase, incubate at room temperature for 1 hour, wash the plate with PBST, add TMB for color development , terminated with 5% hydrochloric acid solution, and read the OD value at 450 nm using a
  • VEGFR3-Fc 500ng/well VEGFR3-Fc (Tyr25-IIe776) molecules were coated at 4° overnight, washed the plate with PBS the next day, added BSA solution to block at room temperature for 2 hours, during the blocking period, each recombinant receptor molecule was changed from high concentration to low concentration Serially dilute, mix with 10ng/well VEGF-C or 500ng/well VEGF-D (both with His tags), incubate at room temperature for 30 minutes, wash the VEGFR3-Fc plate, add the incubation solution to it, and incubate at room temperature for binding 1 hour, wash the plate with PBST, add anti-polyhistidine secondary antibody conjugated with horseradish peroxidase, incubate at room temperature for 1 hour, wash the plate with PBST, add TMB for color development, stop with 5% hydrochloric acid solution, use a microplate reader Read the OD value at 450nm.
  • VEGF-A 165 or VEGF-A 121 molecules purchased from ACROBiosystems at 50ng/well were coated overnight at 4°, the plate was washed with PBS the next day, blocked at room temperature for 2 hours, washed with PBS, and the recombinant receptor molecules were removed from high Add to each well after gradient dilution to a low concentration, incubate at room temperature for 1.5 hours, wash the plate three times with PBST solution, add goat anti-human Fc secondary antibody coupled with horseradish peroxidase, incubate at room temperature for 1 hour, wash the plate three times with PBST solution, TMB was added for color development, terminated with 5% hydrochloric acid solution, and the OD value at 450 nm was read using a microplate reader.
  • each VEGFR2-related recombinant receptor molecule has better binding ability to VEGF-A except LFV-A, LFV-D, and LFV-F
  • LFV-B and its truncated Form LFV-C has the best VEGF-A binding ability, which is better than Eylea
  • LFV-E has the VEGF-A binding ability close to Eylea.
  • the binding EC 50 values for VEGF-A 165 and VEGF-A 121 are shown in Table 8 and Table 9 below, respectively.
  • VEGFR2-Fc molecule 500ng/well of VEGFR2-Fc molecule was coated overnight at 4°, the next day the plate was washed with PBS, and BSA solution was added to block at room temperature for 2 hours.
  • each recombinant receptor molecule was serially diluted from high concentration to low concentration, and then mixed with Mix 60ng/well VEGF-A 165 or 120ng/well VEGF-A 121 (both with His tags), incubate at room temperature for 30 minutes, wash the VEGFR2-Fc plate, add the incubation solution, and incubate at room temperature for 1 hour , wash the plate with PBST, add anti-polyhistidine secondary antibody conjugated with horseradish peroxidase, incubate at room temperature for 1 hour, wash the plate with PBST, add TMB for color development, stop with 5% hydrochloric acid solution, and read with a microplate reader OD value at 450nm.
  • LFV-B and its truncated form LFV-C have better blocking ability, reaching a level superior to that of bevacizumab and close to that of Eylea.
  • the EC50 values for blocking VEGFR2 binding of VEGF-A 165 and VEGF-A 121 are shown in Table 10 and Table 11 below, respectively.
  • the surface plasmon resonance method was used to detect the affinity, the instrument was Biacore T200, protein A chip was used to capture recombinant receptor molecules (including LFV-B, LFV-E, with OPT as the control), and the running buffer was 1 ⁇ HBS-EP , inflowing antigens of various concentrations at a flow rate of 30ul/min
  • C57BL/6 mice were divided into 3 groups, 6 in each group, and each group was injected with LFV-B, LFV-E, and reference substance OPT 4mg/Kg respectively in the tail vein, and at 1h, 2h, 12h, 24h, 72h ( 3 days), 168h (7 days), 336h (14 days), 504h (21 days) blood collection, centrifugation to get serum, use anti-human Fc antibody capture ELISA method to quantify target molecules, using WinNonlin (PhoenixTM, version 8.0) and Other relevant software for pharmacokinetic calculations.
  • the resulting PK data are shown in Figure 7 and Table 14 below:
  • N glycosylation sites (Asn46, Asn66, Asn96) in LFV-E and the N glycosylation sites (Asn143, Asn158, Asn245, Asn318) in LFV-B and LFV-C were point mutated to remove N Glycosylation, preferentially mutating Asn to Gln or Ala.
  • the sequence designed by the point mutation is subjected to gene synthesis, vector construction, extraction, cell transfection, expression and purification, and ELISA method is used to detect the binding blocking ability of VEGFR2/R1-VEGFA, VEGFR2/R3-VEGFC and VEGFR2/R3-VEGFD , compared with the original molecule without mutation, combined with the comprehensive evaluation of expression level and single peak purity, several variants were selected.
  • HEK 293 cells were used for transfection expression and purification, and the PK test in mice was performed to compare with the original molecule without mutation.
  • the above-mentioned glycosylated recombinant protein and heterologous peptide are also used for recombination, and related ELISA experiments, cell experiments and animal experiments are carried out for further optimization.
  • VEGF-A 165-VEGFR1 blocking ELISA 200ng/well VEGFR1-Fc (Met1-Asn756) molecules are coated at 4° overnight, the plate is washed with PBS the next day, and BSA solution is added to block at room temperature for 2 hours During the blocking period, each recombinant receptor molecule was serially diluted from high concentration to low concentration, then mixed with 40ng/well VEGF-A 165 (with His tag), incubated at room temperature for 30 minutes, and the VEGFR1-Fc plate was washed.
  • VEGFR1-Fc Metal-Asn756
  • the 132-230 region of VEGFR1 is introduced Into LFV-B or LFV-C molecules or their glycosylated molecules for transient transformation and expression purification of CHO cells.
  • VEGFR2/R1-VEGFA, VEGFR2/R3-VEGFC, VEGFR2/R3-VEGFD use the system described in Examples 8 and 9 to detect the blocking ability of VEGF and PDGF family molecules , to explore whether it has enhanced VEGF-A and receptor binding blocking ability, as well as VEGF-B, PlGF binding ability, while retaining better VEGF-C, VEGF-D and PDGF family molecules and The ability to block receptor binding, and use Biacore to detect its binding ability to PDGF family molecules, and conduct cell or animal experiments on preferred fusion proteins.
  • the 132-230 region of VEGFR1 was linked to the nitrogen and carbon ends of the LFV-C molecule, and the combination of the 132-230 region of VEGFR1 and the 225-327 region of VEGFR2 was constructed into LFV In the -C molecule, the GS linker is used for connection.
  • the full sequences of the constructed molecules LFV-M, LFV-N, and LFV-O are shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:46.
  • LFV-M is the nitrogen-terminal form of VEGFR1 at position 132-230 of LFV-C
  • LFV-N is the carbon-terminal form of VEGFR1 at position 132-230 of LFV-C
  • LFV-O is VEGFR1( 132-230)-VEGFR2 (225-327) and the recombinant form of LFV-C, their expression and purification in CHO cells are shown in Table 16:
  • ELISA method was used to detect their ability to block VEGF-A, VEGF-C, and VEGF-D.
  • the results are shown in Figure 10. It can be seen that LFV-M and LFV-N have the same or slightly superior blocking ability to VEGF-C, VEGF-D and receptor binding as LFV-C, and have significantly higher than LFV-C. Improved blocking ability of VEGF-A binding to receptors, especially in the blocking experiment of VEGF A-VEGFR1, LFV-M and LFV-N showed better blocking ability than Eylea.
  • VEGF-B and PlGF both purchased from ACROBiosystems
  • 200ng/hole VEGF-B or PlGF molecules were configured in PBS, coated overnight at 4 °, and washed with PBS the next day.
  • LFV-B molecule For LFV-B molecule or its truncated or extended form, introduce the glycosylation modified form used by LFV-K and LFV-L molecules, and recombine with the 132-230 region of VEGFR1 to generate a new fusion protein. Expression and purification, and further molecular and cellular level characterization analysis and animal experiments, in order to obtain more effective therapeutic molecules.
  • the LFV-B molecule with the N245Q mutation was named LFV-P as a control, and LFV-P was combined with the 132-230 region of VEGFR1 to generate a new fusion protein, named LFV-Q.
  • the sequence of LFV-P is shown in SEQ ID NO:18
  • the sequence of LFV-Q is shown in SEQ ID NO:19.
  • VEGF-A, VEGF-C, VEGF-D and VEGFR2 The binding blocking results of VEGF-A, VEGF-C, VEGF-D and VEGFR2 are shown in Figure 12. It can be seen that after glycosylation modification, LFV-P has a better binding effect on VEGF-A, VEGF-A, VEGF-B than LFV-B. Blocking ability of VEGF-C, VEGF-D binding to receptors. After LFV-Q is fused with the 132-230 region of VEGFR1, it has the optimal ability to block the binding of VEGF-A to the receptor. The results of binding to VEGF-B and PlGF are shown in Figure 13. It can be seen that after being fused with the 132-230 region of VEGFR1, LFV-Q also has better binding ability to VEGF-B and PlGF.
  • LFV-Q has certain binding ability to PDGF family molecules.
  • LFV-Q uses the deglycosylated form of the N245 position derived from the sequence 117-327 of VEGFR2, that is, N245 is mutated to Q.
  • N245 in LFV-M is also The Q245 position of LFV-Q was mutated back to N for glycosylation. Functional comparison before and after kylation, and the resulting molecule was named LFV-S.
  • LFV-R and LFV-S were transiently expressed and purified. During this process, it was found that LFV-R after removing N245 glycosylation, compared with LFV-M without removal, had a higher expression and protein expression in CHO cells. After A one-step purification, it was unexpectedly found that the SEC single-peak purity increased from 52.1% to 78.7% (as shown in Figure 14). In addition, LFV-R was concentrated in PBS after the second purification by molecular sieves to facilitate later intra-lens injection , it was found that it still showed good solubility when the concentration reached 19.2mg/ml, which reflected the better downstream development potential of LFV-R.
  • ELISA was used to detect the binding blocking ability of these molecules to VEGFR2/VEGFR1 and VEGF-A, VEGFR2 and VEGF-C, and VEGFR2 and VEGF-D.
  • the results are shown in Figure 15. It can be seen that LFV-R has the optimal The blocking ability of VEGFR2 and VEGF-A is significantly better than that of the control drug Eylea.
  • the blocking ability of the four molecules to VEGFR2, VEGF-C and VEGF-D is relatively close, especially the blocking ability of VEGF-D is obvious Better than the reference OPT.
  • LFV-R The binding of LFV-R, LFV-Q, LFV-B, LFV-C molecules to VEGF-A was detected by ELISA method, and VEGFR2-Fc and Eylea of the whole extracellular domain were used as controls.
  • the experimental results are shown in the figure As shown in 17a, it can be seen that LFV-R has the best binding ability to VEGF-A, which is higher than that of the control drug Eylea, LFV-R, LFV-Q, LFV-B, and LFV-C.
  • the binding force was significantly higher than that of VEGFR2-Fc with the whole extracellular domain.
  • LFV-R has clear binding ability to PDGF-AA, AB, BB and CC.
  • VEGF-A plus VEGF-C molecules were used to stimulate the proliferation of HUVEC cells, and at the same time, different concentrations of LFV-R, Eylea, and OPT molecules were added. After the cells were cultured for 72 hours, MTS was used for color development to detect the effect of different drugs on the cells. Inhibition of cell proliferation. The results are shown in Figure 18a. It can be seen that only LFV-R has obvious inhibitory effect, and the effect of complete inhibition is achieved at a concentration of about 3ug/ml. OPT has no inhibitory effect, and Eylea is only at an ultra-high concentration (above 30ug/ml). Only then did it show a complete inhibitory effect.
  • Eylea and OPT were mixed together (1:1 mixture) to detect the proliferation inhibition of HUVEC, and LFV-R and LFV-Q were added for comparison.
  • the results are shown in Figure 18b, which can be seen Compared with single use, the mixture of Eylea and OPT can completely inhibit the proliferation of HUVEC cells.
  • LFV-R not only has a better IC 50 than Eylea+OPT and LFV-Q, but also compares Eylea+OPT and LFV-Q, in Low concentration shows significantly higher inhibitory efficacy, which will help to maintain sustained inhibitory efficacy during the concentration decay period after vitreous administration, thereby improving therapeutic benefits and extending injection intervals.
  • Brown-Norway rats were randomly divided into 4 groups according to body weight, with 6 animals in each group, half male and half male, respectively model control group, LFV-R group, LFV-Q group, and Eylea group.
  • Laser modeling on the third day of modeling, give a single injection of 0.9% sodium chloride, LFV-R, LFV-Q and Eylea to the vitreous body of both eyes, the administration volume is 4 ⁇ L/eye, the administration concentration is 10 mg/mL, and the dose is 40 ⁇ g /eye, and the day of administration was defined as the first day of the test. During the test period, the state of the surviving rats in each group was observed every day. Fundus photography and fluorescein angiography were performed before modeling and on the 18th day after administration to observe the growth and leakage of new blood vessels in each group, and compare the efficacy of each group.

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CN116162148B (zh) * 2021-11-24 2025-09-09 中山光度生物医药有限公司 多特异性配体结合分子及其应用

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