WO2015109898A1 - VEGF与PDGFRβ双特异性融合蛋白及其用途 - Google Patents

VEGF与PDGFRβ双特异性融合蛋白及其用途 Download PDF

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WO2015109898A1
WO2015109898A1 PCT/CN2014/093555 CN2014093555W WO2015109898A1 WO 2015109898 A1 WO2015109898 A1 WO 2015109898A1 CN 2014093555 W CN2014093555 W CN 2014093555W WO 2015109898 A1 WO2015109898 A1 WO 2015109898A1
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seq
fusion protein
pdgfrβ
bispecific fusion
vegf
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PCT/CN2014/093555
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French (fr)
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屈向东
曹国庆
叶鑫
陈侃
张蕾
许志宾
袁纪军
张连山
潘琴
管雁宾
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上海恒瑞医药有限公司
江苏恒瑞医药股份有限公司
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Priority to CN201480010804.8A priority Critical patent/CN105026433B/zh
Publication of WO2015109898A1 publication Critical patent/WO2015109898A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/49—Platelet-derived growth factor [PDGF]
    • 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

Definitions

  • the present invention relates to a VEGF and PDGFR ⁇ bispecific fusion protein, comprising a pharmaceutical composition thereof and use thereof.
  • Age-related macular degeneration is a disabling, blinding disease. It mainly affects the central region of the macular area or the retina responsible for high visual acuity and is the cause of irreversible vision loss in the elderly. According to statistics, more than 30 million people worldwide suffer from the disease. As China's population ages, the disease has seriously jeopardized the lives and health of our people.
  • AMD comes in two forms, dry AMD and wet AMD. Although dry AMD is more common, it generally results in a lighter, slower loss of vision. New blood vessels are formed under the retina of patients with wet AMD. When photoreceptor cells and retinal pigment epithelial (RPE) cells slowly degenerate, blood vessels tend to develop from a normal position in the choroid to an abnormal position below the retina. This abnormal new blood vessel growth is called choroidal neovascularization (CNV). These abnormal blood vessels leak and bleed, resulting in bleeding, swelling, scar tissue, and severe vision loss. Only about 10% of AMD patients are of the wet type, but account for about 90% of the total number of blindness caused by AMD. Wet age-related macular degeneration (AMD) has become a leading threat to blindness in the elderly over 65 years of age.
  • AMD age-related macular degeneration
  • laser treatment can sometimes be used to break abnormal blood vessels formed in wet AMD. Only 15% of wet AMD is suitable for laser treatment because the blood vessels cannot be too close to the mid-inner region of the macula.
  • a laser is a beam of light that is absorbed by blood pigments, drugs, and PRE cells and converted into heat to cauterize abnormal blood vessels. Since the stimuli are still not removed, the formation of new blood vessels is often repeated, resulting in loss of vision.
  • Photodynamic therapy is mainly to inject a photosensitive drug by intravenous injection, and then irradiate a choroidal neovascular lesion with a specific wavelength of non-thermal laser to activate the photosensitive drug.
  • photodynamic therapy can only stabilize or reduce the risk of decreased visual acuity in wet AMD. Because it is not the cause of treatment, photodynamic therapy can not prevent the possibility of recurrence, generally requires multiple treatments. Moreover, it should be protected from light for 48 hours after treatment to avoid photoreaction and skin burns. Therefore, it brings a lot of pain to the patient.
  • the platelet-derived growth factor family includes platelet growth factor (PDGF) and vascular endothelial cell factor (VEGF). Each growth factor can be produced by a variety of cells, and its receptors are all tyrosine kinase (RTK) type receptors. Platelet-derived growth factor family members include: PDGFA, PDGFB, PDGFC, PDGFD, Placental growth factor (PGF), and vascular endothelial growth factor (VEGF, VEGF41, VEGFB, VEGFC, FIGF (also known as VEGFD)).
  • PDGF platelet growth factor
  • VEGF vascular endothelial cell factor
  • FIGF also known as VEGFD
  • VEGF is a potent and unique mitogenicogen of vascular endothelial cells that promotes all aspects of the angiogenic process. In several in vivo experiments investigating angiogenesis, VEGF can induce angiogenesis. In experiments with vascular and lymphatic endothelial cells cultured in vitro, VEGF promotes endothelial cell proliferation, migration, and tubular structure formation. VEGF can increase the permeability of capillaries and posterior venules. After skin or muscle injection, continuous endothelial destruction can be induced rapidly (Xie K, Wei D, Huag S. Transcriptional anti-angiogenesis therapy of human pancreatic cancer [J]. Cytokine Growth Factor Rev 2006, 17: 147-156).
  • VEGFR-2 plays a role in regulating endothelial growth, differentiation, and permeability; while VEGFR-1 is involved in regulating endothelial cell migration and aggregation, and inhibits signal transduction through VEGFR-2.
  • VEGFR-3 may also play a role in vascular development, but more uniquely, it is expressed in lymphatic tissue and may play an important role in lymphoid system formation (Joukov V, Pajusola K, Kaipainen A, et al.
  • a novel vascular endothelial growth factor VEGF-C is a ligand for the Flt4 and KDR receptor tyrosine kinases [J]. EMBO J, 2003, 15(2): 290-298).
  • VEGF exerts a biological effect through VEGFR.
  • VEGF binds to VEGFR, dimerizes and autophosphorylates VEGFR, and transmits signals (including MAPK, PI3 kinase, Ras, and phospholipase C pathways) through multiple intracellular pathways, and finally plays a role.
  • VEGF has been found to have three receptors, VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and VEGFR-3 (Flt-4).
  • the first two are mainly expressed in endothelial cells and also in tumor cells, while VEGFR-3 is mainly expressed in lymphatic tissue.
  • the first two receptors have high affinity to VEGF-A and belong to the tyrosine kinase family.
  • Binding to VEGF-A results in its own dimerization, intracellular tyrosine autophosphorylation, and subsequent activation of downstream signaling proteins to exert its physiological functions.
  • the former two are mainly expressed in endothelial cells and also expressed in tumor cells, and VEGFR-3 is mainly expressed in lymphatic tissue (Oh HA novel molecular mechanism involved neuropilin-1 for vascular endothelial growth factor-induced retinal angiogenesis. Nippon Gakkai Zasshi .2003-Nov: 107(11): 651-656).
  • VEGF is known to be a key regulator of neovascularization associated with intraocular conditions.
  • the level of VEGF in eye drops is closely related to the level of active vascular proliferation in diabetic patients and other patients with ischemic retinopathy.
  • Other studies have demonstrated the presence of VEGF in the choroidal neovascular membrane of AMD patients.
  • the currently approved anti-angiogenic drugs for the treatment of wet AMD aim to neutralize the action of VEGF and block angiogenesis from the surface.
  • Pegaptanib the active ingredient of pipeatanitan sodium is a 28-nucleotide "aptamer” whose three-dimensional structure enables it to interact with extracellular Vascular endothelial growth factor receptor (VEGF) binds and inhibits the binding of VEGF to the corresponding receptor. It needs to be administered by intravitreal injection every six weeks;
  • Ranibizumab Rosumab, trade name Lucentis
  • the active ingredient of Lucentis, ranibizumab is an antibody fragment that binds to VEGF and needs to be performed intravitreally every month.
  • Bayer's VEGF-Trap-eye (trade name Eylea), its active ingredient, apocyt, is a polypeptide fragment that binds to VEGF and needs to be injected every month for the first three months, then every two months thereafter. Injection administration.
  • the current standard drug treatment is not effective for a considerable number of patients, such as the current standard Ranibizumab Treatment can only improve the visual acuity of 1/3 of patients, and 10% of patients do not respond to the treatment; in addition, about 70% of patients have no significant increase in visual acuity after anti-VEGF treatment.
  • There is also adaptive resistance in current medical treatment that is, the patient is resistant to the treatment after a period of treatment.
  • each injection not only causes expensive treatment costs, but also causes intraocular infection, bleeding and retinal detachment.
  • the most important complication is intraocular infection. Once infected, the consequences are unimaginable and may be blind. Therefore, the frequency of injection is also important for the development of drugs. Therefore, this field requires more effective treatment, overcomes the resistance to anti-VEGF treatment, and prolongs the duration of anti-VEGF drugs (not requiring one or two months of fundus injection) in order to be more effective and safer.
  • the platelet-derived factor PDGF is one of the early growth factors that occur during wound healing. It plays an important role in the whole process of wound healing, mainly in promoting wound healing.
  • the common PDGF is a homo- or heterodimer formed by two polypeptide chains connected by disulfide bonds, which makes PDGF have various forms of dimer structure, namely PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD.
  • PDGF must bind to the corresponding receptor on the cell membrane to exert its biological effects.
  • the PDGF receptor (PDGFR) is composed of two subunits, ⁇ and ⁇ , and has a molecular weight of 170 to 180 kD.
  • the binding strength of the two to PDGF is very different.
  • the ⁇ unit has a higher affinity with the PDGFa chain and the b chain, while the ⁇ subunit has only a high affinity with the b chain. Therefore, the alpha subunit can bind to PDGF-AA, PDGF-AB and PDGF-BB, and the beta subunit binds only to PDGF-BB and PDGF-AB.
  • PDGFR is a transmembrane glycoprotein with tyrosine protein kinase activity. It consists of a domain specifically recognized by the extracellular N-terminus and PDGF, a transmembrane intermediate hydrophobic domain, and an intracellular C-terminal domain having tyrosine protein kinase activity.
  • PDGF binds to PDGFR on the cell surface, it promotes dimerization of the receptor molecule; activates autophosphorylation of the tyrosine residue in the intracellular domain, or phosphorylates the tyrosine residue that activates a specific target protein; In the afferent cells, the cascaded amplification effect regulates the life activities of the cells (including the division and proliferation of target cells).
  • PDGFR antagonists that block PDGF binding or PDGFR dimerization can be used to treat or prevent diseases associated with the PDGFR pathway.
  • the means of neutralizing VEGF is currently considered to be of limited effectiveness, mainly because the vascular mural cells attached to the blood vessels protect the blood vessels in the absence of VEGF. Therefore, on the basis of neutralizing VEGF, if it can block the protection of vascular cells by pericytes, it can overcome the resistance of anti-VEGF treatment and more effectively treat age-related macular degeneration.
  • the present invention provides a bispecific fusion protein comprising a VEGF-binding peptide and a PDGFR ⁇ -binding peptide.
  • the VEGF-binding peptide can be located upstream of the PDGFR ⁇ -binding peptide; and vice versa.
  • the bispecific fusion protein further comprises an Fc portion of an immunoglobulin.
  • the Fc segment functions to polymerize the fusion protein as a multimerization component.
  • the Fc segment can be located upstream or downstream of the VEGF-binding peptide; can be located upstream or downstream of the PDGFR ⁇ -binding peptide; or between the VEGF-binding peptide and the PDGFR ⁇ -binding peptide.
  • the Fc portion of the immunoglobulin is the Fc portion of an immunoglobulin IgG.
  • the bispecific fusion protein wherein the amino acid sequence of the Fc fragment is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the bispecific fusion protein wherein the VEGF-binding peptide comprises a VEGFR extracellular domain.
  • the VEGFR is Flt-1.
  • the VEGFR is Flk-1.
  • the VEGFR is Flt-4.
  • the VEGFR extracellular domain is an immunoglobulin domain 2 of Flt1, and an immunoglobulin domain 3 of Flk1 or Flt4.
  • amino acid sequence of the VEGFR extracellular domain is set forth in SEQ ID NO: 1.
  • the amino acid sequence of the PDGFR ⁇ binding peptide is selected from one or more of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 are the heavy chain CDR sequences in the PDGFR ⁇ binding peptide
  • SEQ ID NO: 6 is the light chain CDR sequence in the PDGFR ⁇ binding peptide.
  • a combination of SEQ ID NO: 6 and one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 is preferred when selected from a plurality of sequences therein.
  • the bispecific fusion protein further comprises a spacer, and the amino acid sequence of the spacer is selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the bispecific fusion protein further comprises a compartment represented by an amino acid sequence such as (GGGGS)n, wherein n is 1-10, preferably 2-5.
  • the amino acid sequence of the bispecific fusion protein is selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 24.
  • the present invention also relates to a PDGFR ⁇ antibody derivative comprising a PDGFR ⁇ binding peptide and an Fc segment of an immunoglobulin.
  • the PDGFR ⁇ -binding peptide described in the PDGFR ⁇ antibody derivative is as defined above; and the PDGFR ⁇ antibody derivative further comprises a spacer as defined above.
  • amino acid sequence of the PDGFR ⁇ antibody derivative is SEQ ID NO: 18.
  • the present invention also relates to a method for producing a bispecific fusion protein or a PDGFR ⁇ antibody derivative as described above, comprising: constructing an expression vector, transforming the expression vector into a host cell, and expressing the expression vector in the host cell to obtain an expression precursor The expression of the precursor is secreted outside the cell to obtain the bispecific fusion protein.
  • an expression vector which may be any plasmid suitable for expression in a host cell in the prior art, including but not limited to pcDNA3.1. (+).
  • the expression vector is transformed into a host cell by methods well known in the art, such as transformation, transfection, and the like.
  • the host cells are cultured under appropriate culture conditions to obtain expression precursors.
  • the expression precursor is secreted outside the cell to obtain a fusion protein as shown in SEQ ID No. 19-24, or a PDGFR ⁇ antibody derivative as shown in SEQ ID No. 18.
  • the present invention also relates to an expression precursor of a bispecific fusion protein or a PDGFR ⁇ antibody derivative as described above, wherein the expression precursor consists of a signal peptide and the bispecific fusion protein or a PDGFR ⁇ antibody derivative;
  • the amino acid sequence of the signal peptide is selected from the group consisting of SEQ ID NO: 16 or SEQ ID NO: 17; preferably the signal peptide is located at the N-terminus of the bispecific fusion protein or PDGFR ⁇ antibody derivative.
  • the invention further provides a polynucleotide, preferably DNA, encoding an expression product as described above.
  • the invention further provides an expression vector comprising the polynucleotide as described above.
  • the invention further provides a host cell comprising an expression vector as described above.
  • the host cell is a bacterium, preferably E. coli.
  • the host cell is a yeast cell, preferably Pichia pastoris or Saccharomyces cerevisiae.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific fusion protein or PDGFR ⁇ antibody derivative as described above and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is an injectable solution, wherein the bispecific fusion protein, PDGFR beta antibody derivative, or pharmaceutically acceptable carrier is in dissolved form.
  • the invention further relates to the use of a bispecific fusion protein as described above for the manufacture of a medicament for the treatment of a disease or condition associated with the inhibition of VEGF and PDGFR ⁇ dual targets in a mammal.
  • the mammal is a human.
  • the invention also relates to the use of the bispecific fusion protein, PDGFR ⁇ antibody derivative, for the preparation of a medicament for inhibiting VEGF and PDGF signaling pathways.
  • the invention further relates to the use of a PDGFR beta antibody derivative as described above for the manufacture of a medicament for the treatment of a disease or condition associated with inhibition of a PDGFR beta target in a mammal.
  • the mammal is a human.
  • the invention also relates to the use of the PDGFR beta antibody derivative for the preparation of a medicament for inhibiting the PDGF signaling pathway.
  • the disease or condition described in the pharmaceutical use is a disease of the eye, or any other disease capable of being ameliorated by inhibition of VEGF and/or PDGF signaling pathways, such as tumors and diabetic retinopathy.
  • the disease or condition of the eye described in the pharmaceutical use is age-related macular degeneration, preferably wet age-related macular degeneration.
  • the invention also relates to a method of treating wet AMD comprising administering to a patient in need thereof a therapeutically effective amount A bispecific fusion protein or PDGFR ⁇ antibody derivative as described above, or a pharmaceutical composition as described above.
  • Figure 1 Inhibitory effect of VTEP-0 on PDGFRbb-induced PDGFR ⁇ and AKT phosphorylation.
  • Figure 2 Inhibitory effect of VTEP-13 on PDGFRbb-induced PDGFR ⁇ and AKT phosphorylation.
  • Figure 3 Inhibitory effect of VTEP-15 on PDGFRbb-induced PDGFR ⁇ and AKT phosphorylation.
  • Figure 4 Inhibitory effect of VTEP-17 on PDGFRbb-induced PDGFR ⁇ and AKT phosphorylation.
  • Figure 5 Inhibitory effect of VTEP-22 on PDGFRbb-induced phosphorylation of PDGFR ⁇ and AKT.
  • Figure 6 Inhibition rate of PDGFR-induced PDGFR phosphorylation by VTEP-17 on PDGFR ligand.
  • Figure 7 Inhibition rate of TFEP-17 on PDGFR ligand-induced PDGFRbb-induced AKT phosphorylation.
  • Figure 8 Inhibitory effect of VTEP-13 on VEGF-induced proliferation of human umbilical vein endothelial cells.
  • Figure 9 Inhibitory effect of VTEP-17 on VEGF-induced proliferation of human umbilical vein endothelial cells.
  • Figure 10 Inhibitory effect of VTEP-22 on VEGF-induced proliferation of human umbilical vein endothelial cells.
  • Figure 11 Choroidal neovascularization in the mouse eye of model mice, indicating inhibition of choroidal neovascularization by VTEP-17 in a mouse model of laser-induced choroidal neovascularization.
  • the present invention provides a bispecific fusion protein comprising a first component VEGF-binding peptide and a second component PDGFR ⁇ -binding peptide; more specifically, by providing VEGFR (Flt-1, Flk-1, Flt-4) A novel fusion protein formed by the modification of the modified extracellular domain, the PDGFR ⁇ binding peptide, and the Fc region of IgG.
  • VEGFR Flt-1, Flk-1, Flt-4
  • binding peptide refers to soluble receptors and fragments thereof and analogs thereof that are capable of binding to a target; or antibodies and fragments thereof, and analogs thereof.
  • the VEGF-binding peptide of the present invention is a polypeptide preferably comprising a VEGFR extracellular ligand binding domain (also referred to herein as a VEGFR extracellular domain).
  • the extracellular ligand binding domain is defined as part of the receptor, which is oriented toward the outside of the cell in its native conformation in the cell membrane. It can contact its cognate ligand.
  • the extracellular domain does not include a hydrophobic amino acid associated with the receptor transmembrane domain or any amino acid associated with the receptor intracellular domain.
  • Von Heijone discloses detailed rules that are frequently cited by those skilled in the art for determining whether an amino acid of a particular receptor belongs to the extracellular, transmembrane or intracellular domain (see Von Heijone, 1995, BioEssay 17: 25-30). .
  • websites such as http://ulrec3.unil.cn/software/TMPRED-form.html can Protein chemists provide information on the prerequisites for preparing protein domains.
  • PDGFR ⁇ binding peptide preferably comprises a polypeptide which specifically recognizes and binds to all or a portion of an antigen binding site of an antibody of the target antigen PDGFR ⁇ ; for example, all or part of a heavy chain and/or a light chain variable region, Or at least the HCDR3 region of the heavy chain variable region.
  • the PDGFR ⁇ binding peptide comprises, without limitation, an antibody, or an antibody fragment, and an immunoglobulin-like domain containing all or part of an antigen binding site of the antibody.
  • the relevant definitions in US 61/610905 such as antibody sequences comprising the CDR3 sequence HGGDRSY (SEQ ID NO: 11), may also include the sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 12) or GILPINKTPNYAQRFQG (SEQ ID NO: 13) CDR2 sequence.
  • the "PDGFR ⁇ antibody derivative” includes a PDGFR ⁇ -binding peptide and an Fc segment of an immunoglobulin.
  • the "bispecific fusion protein” as used in the present invention refers to a polypeptide which specifically recognizes and binds to a VEGF epitope and also specifically recognizes and binds to a PDGFR ⁇ epitope.
  • the "bispecific fusion protein” of the present invention may further comprise an Fc fragment of human IgG. Any isotype of IgG can be used, including IgGl, IgG2, IgG3, and IgG4. In order to increase the function of the fusion protein, the Fc segment can also be mutated or increased in the hinge region. Methods for making bispecific antibodies are known in the art.
  • linker peptide refers to one or more molecules, such as nucleic acid, amino acid or polypeptide moieties, which can be inserted between one or more of the component domains.
  • Linker peptide sequences can be used to provide desirable target sites between components to facilitate manipulation.
  • Linker peptides can also be provided to enhance expression of the chimeric plurality of components in the host cell, reducing steric hindrance such that the component can take its best tertiary structure to interact better with its target molecule.
  • the compartment is preferably one or more peptide sequences comprised between one or more components. These polypeptide sequences are between 1 and 50 amino acids in length, preferably between 1 and 25.
  • the "partition” is more preferably (GGGGS)n, wherein n is 1-10, preferably 2-5.
  • the "precursor of the bispecific fusion protein" of the present invention may also comprise a signal peptide, which facilitates secretion of the protein from the endocrine to the extracellular, increasing its yield.
  • an “antibody” as used herein refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, full length antibodies, antibody binding fragments or derivatives. Sources of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, genetically engineered antibodies (eg, bispecific antibodies).
  • the “full length antibody” as used in the present invention refers to an immunoglobulin molecule (for example, IgM) comprising four polypeptide chains (i.e., two heavy chains and two light chains are cross-linked to each other by a disulfide bond to form a multimer).
  • Each heavy chain comprises a heavy chain variable region (VH for short) and a heavy chain constant region, and the heavy chain constant region comprises three domains: CH1, CH2 and CH3.
  • Each light chain comprises a stretch of light chain variable region (VL) and a light chain constant region, and the light chain constant region comprises one domain (CL1).
  • the VH and VL regions may further include hypervariable regions (also complementary in the text) Decision Area (CDR)).
  • CDR Decision Area
  • a more conserved domain interspersed between the complementarity determining regions is called the framework region (FR).
  • antibody-binding fragment or derivative includes any naturally occurring, enzymatically-acquired, synthetic, or genetically engineered polypeptide or sugar which specifically binds to an antigen to form a complex. protein. At least a portion of an antigen binding or variable region (e.g., one or more CDRs) of a parent antibody is typically included that retains at least some of the binding specificity of the parent antibody.
  • An "antibody binding fragment or derivative” may be derived from an antibody, for example by appropriate standard techniques including proteolysis or recombinant genetic engineering techniques, including manipulation and expression of a DNA expressing an antibody variable region and a partial constant region. The full length is modified.
  • Antibody binding fragment or derivative includes but is not limited to: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) (vi) a dAb fragment; and (vii) a minimal recognition unit (eg, an isolated complementarity determining region (CDR)) that mimics the amino acid residue of the hypervariable region of the antibody.
  • CDR complementarity determining region
  • Other engineering molecules such as bivalent antibodies, trivalent antibodies, tetravalent antibodies, and minibodies are also within the scope of "antibody binding fragments or derivatives.”
  • a "Fab fragment” consists of a complete light and heavy chain VH and CH1 functional regions.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a “Fab'fragment” comprises a VH and CH1 functional region of a light chain and a heavy chain, and a region between the CH1 and CH2 domains, such that a chain can be formed between the two heavy chains of the two Fab' fragments. Disulfide bonds to form F(ab') 2 molecules.
  • the "F(ab') 2 fragment” comprises two light chains and two heavy chains containing a partial constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains.
  • the F(ab') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • an "Fv fragment” comprises a variable region VH functional region of a light or/and heavy chain.
  • the PDGFR ⁇ -binding peptide of the present invention is preferably an Fv fragment, a preferred embodiment is a heavy chain variable region of PDGFR ⁇ , and another preferred embodiment is a fusion protein in which a heavy chain variable region is linked to a light chain variable region.
  • an immunoglobulin Fc region refers to a human immunoglobulin chain constant region, particularly a carboxy terminus or a portion thereof of an immunoglobulin heavy chain constant region, which has no antigen-binding activity and is a site where an antibody molecule interacts with an effector molecule and a cell.
  • an immunoglobulin Fc region can include a combination of two or more domains of heavy chains CH1, CH2, CH3, CH4 and an immunoglobulin hinge region.
  • immunoglobulins can be divided into different classes, mainly including five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes) such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2.
  • the "Fc region” preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 regions of IgG. More preferably, it comprises a CH2 domain of IgG1, a CH3 domain and an immunoglobulin hinge region, and the starting amino acid position of the hinge region can be varied.
  • the "Fc segment” has no antigen-binding activity and is a site where an antibody molecule interacts with an effector molecule and a cell.
  • the "hinge region” is used to link the Fab segment and the Fc segment of the antibody.
  • a bispecific fusion protein can be ligated to the Fc segment in the present invention.
  • the first component VEGF-binding peptide of the present invention is abbreviated as VTE; the second component PDGFR-binding peptide is abbreviated as PDX; and the Fc-segment of the third component IGg is abbreviated as Fc.
  • VTE amino acid sequence is upstream of the Fc amino acid sequence, and the Fc amino acid sequence is upstream of the PDX amino acid sequence, such as VTE---Fc---PDX;
  • VTE amino acid The sequence is upstream of the PDX amino acid sequence, the PDX amino acid sequence is upstream of the Fc amino acid sequence, such as VTE---PDX---Fc;
  • the PDX amino acid sequence is upstream of the VTE amino acid sequence, and the VTE amino acid sequence is at the Fc amino acid sequence. Upstream of the sequence, the order is PDX---VTE---Fc.
  • the nucleotide sequence encoding the VEGF-binding peptide is located upstream of the nucleotide sequence encoding the PDGFR-binding peptide. In another embodiment of the invention, the nucleotide sequence encoding the VEGF-binding peptide is located downstream of the nucleotide sequence encoding the PDGFR-binding peptide.
  • the invention provides methods of treatment comprising administering to a subject an effective amount of a bispecific fusion protein of the invention.
  • the fusion protein is substantially purified, i.e., substantially free of substances that limit its effects or produce undesirable side effects.
  • the subject is preferably a mammal, most preferably a human.
  • the pharmaceutical composition of the present invention topically to the area in need of treatment; this can be accomplished, for example, but not by way of limitation, by local perfusion, topical application during surgery, said local perfusion and Topical administration is carried out, for example, by injection, by means of a catheter or by means of an implant, wherein the implant is a porous, non-porous or gel-like substance, including a membrane, such as a sialastic membrane, Fiber or commercial skin substitutes.
  • composition used to practice the methods of the invention may be a solution comprising a solution, suspension or both of the agents of the invention.
  • solution/suspension refers to a liquid composition in which a first portion of the active agent is present in solution and a second portion of the active agent is in the form of particles which are suspended in a liquid matrix.
  • Liquid compositions also include gels.
  • the liquid composition can be aqueous or in the form of an ointment. Further, the composition may be placed in the eye in the form of a solid, such as between the eye and the eyelid or in the conjunctival sac, where the bispecific protein disclosed herein is released.
  • Bispecific proteins can generally be released from the above solids to the cornea or directly to the cornea itself, which is typically in direct contact with the cornea.
  • Solids suitable for implantation into the eye typically consist primarily of bioerodible or non-bioerodible polymers.
  • the aqueous solution and/or suspension may be in the form of an eye drop.
  • the desired dose of active agent can be measured by administering a known number of drops to the eye. For example, for a drop of 50 [mu]l volume, administration of 1-3 drops will deliver 50-150 [mu]l of composition.
  • Aqueous suspensions or solutions/suspensions for carrying out the process of the invention may comprise one or more polymers as suspending agents.
  • Useful polymers include water soluble polymers such as cellulosic polymers, and water insoluble polymers such as crosslinked carboxyl containing polymers.
  • the aqueous suspensions or solutions/suspensions of the invention are preferably viscous or mucoadhesive, or more preferably both are viscous or both are mucoadhesive.
  • the bispecific proteins of the invention are either used diagnostically and/or used in screening methods.
  • the bispecific protein can be used to monitor the levels of VEGF and PDGFR during clinical research to evaluate treatment efficiency.
  • the methods and compositions of the invention can be used to screen individuals having levels of, for example, too high or too low levels of VEGF and PDGFR. It can also be used to screen assays in vivo and in vitro to quantify the amount of unbound VEGF and PDGFR present, for example, in screening methods to identify detection reagents that reduce the expression of VEGF and PDGFR.
  • the bispecific proteins of the invention can be used in any assay or method for quantifying and/or isolating VEGF and PDGFR.
  • compositions comprising the bispecific proteins of the invention.
  • Such compositions comprise a therapeutically effective amount of one or more of the bispecific proteins provided herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to those listed in the Chinese Pharmacopoeia or other pharmacopeia for use in animals and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient that is administered with a drug.
  • Such pharmaceutical carriers can be sterile liquids such as water or oil.
  • compositions for carrying out the methods of the invention have a pH and osmolality compatible with the eye.
  • the invention provides a method of constructing a polynucleotide encoding a fusion protein molecule that is inserted into a vector capable of expressing the fusion protein after introduction into a suitable host cell.
  • Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. Lonza CHO cells are preferred.
  • any method for inserting a DNA fragment into a vector can be used to construct an expression vector that is under the control of a transcription/translation control signal encoding the fusion protein.
  • Such methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination) (see Sambrook et al, Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Molecular Biology Methods, Ausubel Wait, Greene Publishing Association, Wiley Interscience, NY).
  • Expression of a polynucleotide molecule encoding a fusion protein of the invention can be regulated by another nucleotide sequence such that the fusion protein is expressed in a host transformed with the recombinant DNA molecule.
  • expression of a fusion protein of the invention can be controlled by any promoter/enhancer element known in the art.
  • an expression vector capable of replication in a bacterial or fungal host is transfected with a polynucleotide comprising a fusion protein disclosed herein, and thus the expression of said polynucleotide is controlled to produce said The fusion protein is then recovered in a biologically active form.
  • the biologically active form comprises a form that is capable of binding to both VEGF and PDGFR.
  • Expression vectors containing the polynucleotides of the present invention can be identified by three general methods: (a) DNA-DNA hybridization, (b) presence and absence of "marker” gene function, and (c) expression of the inserted sequence.
  • first method the presence of a foreign gene inserted into an expression vector can be detected by DNA-DNA hybridization using a probe containing a sequence homologous to the inserted fusion protein sequence.
  • second method certain "marker" gene functions (eg, resistance against biota, transformed phenotype, etc.) may be identified or selected for recombination based on insertion of a foreign gene on the vector.
  • Vector/host system e.g, resistance against biota, transformed phenotype, etc.
  • the recombinant containing the insert can be identified by the lack of function of the marker gene.
  • a recombinant expression vector can be identified by detecting a foreign gene product expressed by the recombinant. For example, the detection can be based on physical or functional properties of the fusion protein.
  • the cells of the invention may be transient, preferably constitutive and permanently, expressing the fusion protein.
  • the fusion protein can be purified by any method to enable subsequent production of a stable, biologically active fusion protein.
  • the fusion protein may be recovered from the cell as a soluble protein or as an inclusion body.
  • conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration can be used.
  • the present invention provides a bispecific fusion protein comprising a VEGF-binding peptide and a PDGFR ⁇ -binding peptide; more specifically, a modified extracellular domain by VEGFR (Flt-1, Flk-1, Flt-4), A novel fusion protein formed by the PDGFR ⁇ binding peptide and the Fc region of IgG.
  • the amino acid sequence of the VEGF-binding peptide of the invention is referred to as VTE; the second component of the PDGFR antibody, designated PDX; and the Fc region of the third component, IGg, is referred to as Fc.
  • VTE amino acid sequence of the VEGF-binding peptide of the invention
  • PDX second component of the PDGFR antibody
  • Fc Fc region of the third component
  • VTE amino acid sequence is upstream of the Fc amino acid sequence, and the Fc amino acid sequence is upstream of the PDX amino acid sequence, such as VTE---Fc---PDX, abbreviated as VFP;
  • VTE amino acid sequence is upstream of the PDX amino acid sequence, and the PDX amino acid sequence is upstream of the Fc amino acid sequence, such as VTE---PDX---Fc, abbreviated as VPF;
  • the PDX amino acid sequence is upstream of the VTE amino acid sequence, and the VTE amino acid sequence is at the Fc. Upstream of the amino acid sequence, the order is PDX---VTE---Fc, abbreviated as PVF.
  • the components of the fusion protein can be directly linked to each other or linked by a linker peptide.
  • a linker peptide For example, a hinge region of 5-20 amino acids, preferably 5-15 amino acids, is added upstream of the Fc.
  • a VEGF-binding peptide in the present invention refers to a polypeptide fragment containing a VEGFR extracellular domain sequence.
  • the sequence is as follows:
  • the VEGF-binding peptide may also be a VTE plus Fc fragment, abbreviated as VEGF-Trap-Fc:
  • the PDGFR ⁇ -binding peptide in the present invention may be a VH fragment having PDGFR ⁇ -binding activity, and the sequence is as follows (for convenience, named PDX, selected from PDX1, PDX2 or PDX3):
  • PDX1 also known as XB2202VH, referred to as VH in the present invention:
  • the Fc region of the invention comprises the CH2 and CH3 functional regions of an IgG (such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1), the sequence of which is as follows (referred to as Fc for convenience):
  • IgG such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1
  • hinge region in front of the Fc region selected from the original sequence or a mutant thereof, and the sequence is as follows:
  • Fc-H1 DKTHT SEQ ID NO: 10.
  • Fc-H2 EPKSSDKTHT SEQ ID NO: 11.
  • linker peptide There may be a linker peptide between the components, the sequence is as follows:
  • the expression precursor of the bispecific fusion protein of the present invention may also have a secretion signal peptide, for example:
  • SIG1 MEFGLSWLFLVAILKGVQC SEQ ID NO: 16.
  • the bispecific fusion protein of the present invention is expressed by cells to obtain the following preferred sequences:
  • Arrangement is such as XB2708VH-linker-1-VEGF-Tarp-Fc, XB2708 heavy chain variable region (PDX3, SEQ ID NO: 5) is linked to VEGF-Trap Fc fusion protein via a linker peptide (SPAC2), in combination order
  • SPAC2 linker peptide
  • Eukaryotic expression vector pcDNA3.1(+) (Life technologies, Cat. No. V790-20);
  • Nucleotide sequence encoding the VTE protein (SEQ ID NO: 1): synthesized by Gene Synthesis Corporation (Jin Weizhi, Suzhou);
  • Nucleotide sequence encoding the Fc protein fragment (SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9): human antibody heavy chain ⁇ 1 constant region Fc fragment, synthesized by gene synthesis company (Jin Weizhi, Suzhou) );
  • Nucleotide sequence of PDX protein fragment (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6): synthesized by Gene Synthesis Corporation (Jin Weizhi, Suzhou).
  • a restriction endonuclease KpnI site, a Kozak sequence, and a signal peptide sequence were introduced at the 5' end of each expression fragment by PCR; a stop codon TGA and a NotI restriction endonuclease site were introduced at the 3' end of each fragment, respectively. .
  • the resulting DNA sequence is from the 5' to the 3' end: KpnI site-Kozak sequence-signal peptide sequence-expression fragment (structural sequence includes Fc-PDX, VTE-Fc-PDX, PDX-VTE-Fc or VTE- PDX-Fc)-TGA stop codon-NotI site.
  • the signal peptide of VTE-0 is preferably the sequence of SIG2, and other embodiments prefer the sequence of SIG1.
  • Each fragment was inserted into plasmid pcDNA3.1(+) using KpnI and NotI restriction enzyme sites, respectively, to construct an expression vector.
  • the expression vector constructed in Example 2 was transiently transfected using FreeStyle 293 cells (GIBCO, Cat# R79007).
  • FreeStyle 293 cell suspension culture was carried out in medium (Freestyle 293 expression medium, GIBCO, Cat #12338018), and a final concentration of 1% ultra low immunoglobulin fetal bovine serum (Ultra Low IgG Fetal Bovine Serum, GIBCO, Cat# 16250078) was added.
  • the expression vector and transfection reagent PEI (Polysciences, Cat#239662) constructed in Example 2 were prepared, and the expression vector was 100 ⁇ g/100 ml of cells, and the mass ratio of expression vector to PEI was 1:2.
  • the cell density on the day of transfection was 1 ⁇ 10 6 /ml.
  • 1L of FreeStyle 293 was transfected into cells, and 50 ml of Opti-MEM medium (GIBCO, Cat#11058021) was mixed with the expression vector, allowed to stand for 5 min, and filtered; 50 ml of Opti-MEM medium was mixed with PEI and allowed to stand. 5 min, filtered.
  • the expression vector and PEI were mixed and allowed to stand for 15 min.
  • the expression vector and the PEI mixture were slowly added to the cells, placed in a 37 ° C, 8% CO 2 , and shaken in a 130 rpm shaker incubator. After 5 days, the supernatant was collected by centrifugation for protein purification.
  • the cell culture medium was centrifuged at a high speed, and the supernatant was collected, and the first step chromatography was carried out by affinity chromatography.
  • Chromatographic conditions The chromatographic medium is Protein A or a derivative filler that interacts with Fc, such as Ma's Mabselect; the equilibration buffer is 1 ⁇ PBS (NaCl 137 mmol/L, KCl 2.7 mmol/L, Na 2 HPO 4 10 mmol/L). , KH 2 PO 4 2mmol / L, pH 7.4), balance 5 column volume; cell supernatant loading, flow rate control on the sample column retention time ⁇ 1min; after the end of the sample, using 1 ⁇ PBS (pH 7.
  • the first eluted sample was subjected to ultrafiltration and concentration, and subjected to size exclusion chromatography.
  • the exclusion chromatography conditions were as follows: buffer was 1 ⁇ PBS (NaCl 137 mmol/L, KCl 2.7 mmol/L, Na 2 HPO 4 10 mmol/L, KH 2 PO 4 2 mmol/L, pH 7.4), column XK26/60 Superdex 200 (GE), flow rate 4 ml/min, loading volume less than 5 ml.
  • the target protein peak is combined according to UV absorption.
  • Purity detection was performed by SEC-HPLC.
  • the column for detection was TSK-Gel 2000-SWXL (TOSOH), flow rate: 0.7 ml/min, sample loading: 50 ⁇ l, mobile phase: 1 ⁇ PBS (NaCl 137 mmol/L, KCl 2.7 mmol/ L, Na 2 HPO 4 10 mmol/L, KH 2 PO 4 2 mmol/L, pH 7.4).
  • the SEC-HPLC purity is greater than 95%.
  • the fusion protein of the present invention was evaluated by Western blotting to inhibit PDGFR ⁇ and AKT (S473) phosphorylation in Caki cells, and the PDGFR antibody function possessed by the fusion protein of the present invention was tested.
  • VTEP-0 Samples of the invention: VTEP-0, VTEP-13, VTEP-15, VTEP-17 and VTEP-22.
  • Caki cells purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, article number TCHu135;
  • Phospho-PDGF receptor ⁇ (Tyr751) (88H8) Mouse mAb (PDGFR ⁇ antibody): purchased from Cell Signalling Technology, Inc. #3166;
  • Recombinant human PDGF-BB CF (10 UG) (PDGFR ligand PDGFRbb): purchased from R&D, Cat. No. 220-BB-010;
  • Protease inhibitor Complete Mini EDTA-free purchased from Roche, article number 04693159001;
  • Phosphorylase inhibitor PhosSTOP was purchased from Roche, article number 04906837001;
  • McCOY's 5A Medium purchased from Life Technologies, Cat. No. 16600-082;
  • Cell RIPA lysate purchased from Biyuntian Biotechnology Research Institute, item number P0013B.
  • the cells were exchanged for the next day, and 2 ml of serum-free medium was added to each well.
  • concentration of each sample is shown in Figure 1 - Figure 5 1 line.
  • the loading mode of VTEP-13 is 200, 66.7, 22.2, 7.4, 0, 200 nM, 1 ml per well.
  • p-PDGFR ⁇ in Fig. 1 to Fig. 5 indicates that the electropherogram of the line indicates the content of phosphorylated PDGFR ⁇ in the cell lysate after the test is added; p-AKT (S473) indicates that the line is electrophoresed.
  • the sample of the present invention has the function of PDGFR antibody, and can inhibit the phosphorylation of PDGFR ⁇ and AKT (S473), has a significant dose effect on the inhibition of PDGFR ⁇ phosphorylation, and has a certain dose effect on the inhibition of downstream AKT (S473) phosphorylation. .
  • the antibody itself does not have a PDGFR ⁇ phosphorylation agonist effect.
  • the inhibitory rate of GFEP-17 on PDGFR-induced PDGFR phosphorylation was 3.6 nm, as shown in Figure 6.
  • the inhibitory rate of VTEP-17 on PDGFR ligand-induced PDGFRbb-induced AKT (S473) phosphorylation was 3.2. Nm, as shown in Figure 7.
  • Samples of the invention VTEP-13, VTEP-15 and VTEP-22;
  • HUVEC cells human umbilical vein endothelial cells: purchased from ATCC, article number CRL-1730 TM ;
  • Low Serum Growth Supplement purchased from GIBCO, item number S-003-10;
  • Medium 200 medium purchased from GIBCO, item number M-200-500;
  • VEGF165 purchased from R&D, article number 293-VE-010;
  • CCK8 purchased from Tongren Chemical, item number CK04.
  • the Medium200+Low Serum Growth Supplement medium was changed to 0.5% FBS medium 200 medium, 90 ⁇ l per well.
  • the sample was diluted three times with the serum-free medium 200 medium from the original concentration, and a total of 9 points were diluted.
  • the diluted samples were incubated with 200 ng/ml VEGF165, 37 ° C, 5% CO 2 incubator for 1 hour.
  • step 4 Add a mixture of samples of different dilutions and VEGF configured in step 3 to the cells, 10 ⁇ l per well, and incubate the plates in a 37 ° C, 5% CO 2 incubator.
  • reaction affinity of the samples of the invention and the PDGFR[beta] protein was determined using a Biacore, GE instrument.
  • biosensor chip CM5 (Cat.#BR-1000-12, GE);
  • HBS-EP + 10 times buffer solution (Cat. #BR-1006-69, GE) was diluted to 1 time (pH 7.4) with D.I.Water.
  • the human anti-capture antibody was covalently coupled to the CM5 biochip according to the method described in the human anti-capture kit, thereby affinity-captured a certain amount of VTE protein, and then flowed through a series of concentration gradients of PDGFR ⁇ protein on the surface of the chip.
  • the Biacore instrument was used to detect the reaction signal in real time to obtain binding and dissociation curves. After each cycle of dissociation was completed in the experiment, the biochip was washed and regenerated using the regeneration solution disposed in the human anti-capture kit.
  • the PDGFR ⁇ protein was covalently coupled to the CM5 biochip according to the method described in the amino coupling kit, and then a series of concentrations of VTE protein were flowed on the surface of the chip, and then the Biacore instrument was used to detect the reaction signal in real time to obtain binding and dissociation curves.
  • Use GE's BIAevaluation software to 1:1 (Langmuir) The data obtained by combining the model analysis, the affinity Kd values determined by this method are shown in Table 3 below.
  • the sample of the present invention has a high reaction affinity with the PDGFR ⁇ protein, and the results are shown in Table 3.
  • reaction affinity of the samples of the invention and the VEGF protein was determined using a Biacore, GE instrument.
  • Samples of the invention VTEP-17 and VTEP-22.
  • biosensor chip CM5 (Cat.#BR-1000-12, GE);
  • VEGF165/VEGFA (Cat. #11066-HNAB, Sino Biological);
  • HBS-EP + 10 times buffer solution (Cat. #BR-1006-69, GE) was diluted to 1 time (pH 7.4) with D.I.Water.
  • Human anti-capture antibody was covalently coupled to a CM5 biochip according to the method described in the human anti-capture kit, thereby affinity-captured a certain amount of VTE protein, and then flowed through a series of gradient concentrations of VEGF165 protein on the surface of the chip.
  • the Biacore instrument was used to detect the reaction signal in real time to obtain binding and dissociation curves.
  • the data obtained were analyzed using the 1:1 (Langmuir) binding model using GE's BIAevaluation software.
  • the affinity Kd values determined by this method are shown in Table 4 below.
  • the sample of the invention has high reaction affinity with VEGF protein, and the results are shown in Table 4:
  • Animals 180 ⁇ 10 g SD rats, male and female (provided by Sipple-Beikai Experimental Animal Co., Ltd., animal production license number SCXK (Shanghai) 2008-0016), 6 in each group.
  • PDGFR-His an extracellular fragment consisting of PDGFR ⁇ (human CD140b/PDGFRb gene functional region from www.uniprot.org, SEQ ID NO: 25) plus a Flag tag and a his tag (marked by a horizontal line in the sequence), It can be prepared by methods well known in the art, such as establishing a clone, transposing a plasmid, and purifying the protein with a nickel column, and verifying the desired protein by electrophoresis. In this test, it can be used to bind to a fragment having the function of a PDGFR ⁇ antibody, and the sequence is as follows:
  • Goat anti-human IgG peroxidase-conjugated antibody Jackson Cat. No.: 109-035-088;
  • Microplate reader Thermo Scientific.
  • VTEP-17 Six SD rats, male and female, were intraperitoneally injected. Under sterile conditions, VTEP-17 was dissolved in physiological saline to a final concentration of 20 ⁇ g/mL. Each rat was administered IP at a dose of 100 ⁇ g/ Kg;
  • IP group blood was taken from the fundus vein of the rats at 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 11 hr, 24 hr, 48 hr, 72 hr, 200 ⁇ L each time (equivalent to taking 100 ⁇ L of serum).
  • the collected blood samples were allowed to stand at room temperature for half an hour to agglutinate, and then centrifuged at 10,000 x g for 5 minutes at 4 °C. The supernatant was collected and immediately subjected to an experiment or sample aliquoting - 80 ° C storage. Avoid repeated freezing and thawing.
  • the ELISA plate reader reads the OD value at a wavelength of 450 nm.
  • the actual concentration of the sample was calculated from the standard curve equation and the OD value of the sample.
  • Standard curve The VTE-17 concentration (5, 10, 20, 50 and 100 ng/ml) is plotted on the abscissa, and the OD value corresponding to different concentrations of VTE-17 is plotted on the ordinate.
  • the typical standard curve equation is obtained.
  • the linear range of the standard curve is 5-100 ng/ml.
  • Example 2 the VTEP-17 obtained in Example 2 was tested, and the pharmacokinetic calculation was performed using the non-compartment model in Phoenix TM WinNonlin 6.1 software to obtain the in vivo half-life (T 1/2 ).
  • T 1/2 the in vivo half-life
  • the inventors introduced a VH fragment against PDGFR ⁇ on the basis of neutralizing VEGF.
  • the fusion protein has the dual specificity of neutralizing VEGF and blocking the signaling pathway of PDGFR ⁇ , thereby effectively treating people who are not responding to VEGF treatment and overcome the resistance of neutralizing VEGF drug therapy. It is possible to prolong the cycle in which patients need intraocular injections, thereby better treating age-related macular degeneration on an existing basis.
  • Test Example 6 Inhibitory effect of VTEP protein on choroidal neovascularization in a mouse model of laser-induced choroidal neovascularization
  • This test was used to investigate the inhibitory effect of VTEP protein on choroidal neovascularization in a mouse model of laser-induced choroidal neovascularization.
  • Test principle This experiment destroys the local Bruch's membrane in mouse retinal tissue by a certain energy laser, so that the choroidal blood supply system and the retinal blood supply system should be communicated, choroidal capillary endothelial cells, pericytes, fibroblasts and inflammation.
  • Cells enter the subretinal and pigment epithelial layers, accompanied by inflammatory factors, pro-angiogenic factors and changes in extracellular matrix components, breaking the balance between local pro-angiogenic and anti-angiogenic factors, and promoting local neovascularization.
  • Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-mediated endothelial cell growth, and extracellular matrix and pericyte envelopment play an important role in choroidal neovascularization.
  • Argon laser emitter Lumenis Selecta Duet, Lumenis, USA
  • Ophthalmic surgery equipment Huaiyin medical equipment
  • VTEP-17 (SEQ ID NO: 21) and VEGF-Trap (SEQ ID NO: 2) proteins diluted with the required dilution concentration buffer (PBS buffer) according to the designed grouping concentration, and the operation in the ultra-clean bench is completed. Packed in 20ul / tube 4 degrees cryopreservation, ready for use.
  • PBS buffer dilution concentration buffer
  • test samples are grouped as follows:
  • mice 6-8 weeks old C57/BL6 mice were fed with free water and fed in 12 hours of light and dark alternately. They were randomly divided into 5 groups, 8 rats in each group, half male and half female, and anesthetized with intraperitoneal injection of 3.5% chloral hydrate at 0.01 ml/g body weight.
  • the effective laser spot distance from the nipple 1-2PD is located in different quadrants, 4-6 per eye, that is, the modeling is successful.
  • Each group was intraperitoneally injected with chloral hydrate on the first day and the seventh day after model establishment, and the corresponding proteins were administered by microinjection in a vitreous cavity (see experimental group design).
  • anesthetized by intraperitoneal injection of chloral hydrate the thoracic cavity was dissected, the heart was exposed, and 0.4 ml of 25 mg/ml sodium fluorescein was perfused into the heart chamber.
  • the eyeball was taken at 4%. Morin was fixed at room temperature for 5 hours.

Abstract

提供的是VEGF与PDGFRβ双特异性融合蛋白及其用途。更具体地，提供了包含VEGFR胞外配体结合结构域和PDGFRβ抗体片段的双功能融合蛋白，可用于治疗湿性老年性黄斑变性（AMD）以及其他任何能够通过抑制VEGF和PDGF信号通路改善的疾病，比如肿瘤和糖尿病视网膜病变。

Description

VEGF与PDGFRβ双特异性融合蛋白及其用途 技术领域
本发明涉及一种VEGF与PDGFRβ双特异性融合蛋白，包含其药物组合物及其用途。
背景技术
老年性黄斑变性(AMD)是一种致残、致盲性疾病。其主要影响黄斑区或视网膜中负责高视敏度视力的中心区域，并且是导致老年人不可逆性视力丧失的原因。据统计，全世界超过3000万人罹患此病。随着中国人口老龄化逐渐加剧，目前该疾病已严重危害我国人民群众的生命健康。
AMD有两种形式，干性AMD和湿性AMD。虽然干性AMD更常见，但一般导致较轻的、较缓慢的视力丧失。湿性AMD患者的视网膜之下生成新血管。当感光细胞和视网膜色素上皮细胞(RPE)细胞慢慢退化时，血管趋于从脉络膜中的正常位置发展到视网膜之下的异常位置。这种异常的新血管生长被称为脉络膜新生血管(CNV)。这些异常血管渗漏流血，导致出血、肿胀、瘢痕组织和严重的视力丧失。只有10％左右的AMD患者是湿性类型，但是却大约占AMD所导致的失明总数的90％。湿性老年视黄斑变性(AMD)已经成为65岁以上老年人失明的首要威胁。
目前批准用于治疗湿性AMD的治疗方法包括激光凝固法、光动力疗法和用于降低过剩VEGF的水平、或阻断VEGF生成的抗血管药物疗法。
根据部位，有时可以使用激光治疗来破环湿性AMD中形成的异常血管。只有15％的湿性AMD适合采用激光治疗，因为血管不能离黄斑部的中内区域太近。激光是光束，由血液色素、药物和PRE细胞所吸收，并转化为热能而烧灼异常血管。由于刺激物依然没有被去除，新生血管形成时常反复，导致视力丧失。光动力疗法主要通过静脉注入光敏药物，继而采用特定波长的非热能激光照射脉络膜新生血管病灶，将光敏药物活化。用光动力疗法治疗湿性AMD，只能稳定或降低湿性AMD视力下降的风险。由于并非对因治疗，光动力疗法不能阻止复发的可能，一般需要多次治疗。而且，治疗后要避光48小时，以免发生光敏反应，造成皮肤灼伤。因此，给患者带来很多痛苦。
血小板衍生生长因子家族包括血小板生长因子(PDGF)和血管内皮细胞因子(VEGF)。每种生长因子均可由多种细胞产生，其受体均为酪氨酸激酶(RTK)型受体。血小板衍生生长因子家族成员包括：PDGFA、PDGFB、PDGFC、PDGFD、胎盘生长因子(Placental growth factor，PGF)以及血管内皮生长因子(VEGF、VEGF41、VEGFB、VEGFC、FIGF(也作VEGFD))等。
VEGF是血管内皮细胞强有力和特有的促有丝分裂原，可促进血管生成过程的所有环节。在几个研究血管生成的体内试验中，VEGF均可诱导血管新生。在对体外培养的血管和淋巴管内皮细胞的试验中，VEGF能促进内皮细胞增殖、迁移及管状结构形成。VEGF可以增加毛细血管及后微静脉的渗透性。皮肤或肌肉注射后，能迅速诱导连续性的内皮结构破坏(Xie K,Wei D,Huag S.Transcriptional anti-angiogenesis therapy of human pancreatic cancer[J].Cytokine Growth Factor Rev2006,17:147-156)。各VEGF受体(VEGFR)的作用有所不同。VEGFR-2起着调节内皮生长、分化、渗透性的作用；而VEGFR-1则与调节内皮细胞移动、聚集有关，并且通过VEGFR-2抑制信号的传导。VEGFR-3对于血管发育可能也有作用，但更独特的是其在淋巴管组织中表达，可能对淋巴系统生成有重要作用(Joukov V，Pajusola K，Kaipainen A，et al.A novel vascular endothelial growth factor VEGF-C is a ligand for the Flt4and KDR receptor tyrosine kinases[J].EMBO J，2003，15(2)：290-298)。
VEGF通过VEGFR发挥生物学效应。VEGF与VEGFR结合，使VEGFR二聚体化、自我磷酸化，通过多个细胞内途径传递信号(包括MAPK、PI3激酶、Ras和磷脂酶C途径等)，最终发挥作用。迄今，已发现VEGF有3个受体VEGFR-1(Flt-1)、VEGFR-2(Flk-1)和VEGFR-3(Flt-4)。前两者主要在内皮细胞中表达，在肿瘤细胞中也有表达，而VEGFR-3主要在淋巴管组织中表达。其中前2个受体与VEGF-A亲和力高，属酪氨酸激酶家族。与VEGF-A结合导致其本身二聚化、胞内段酪氨酸自磷酸化、随后激活下游信号蛋白而发挥其生理功能。前两者主要在内皮细胞中表达，在肿瘤细胞中也有表达，而VEGFR-3主要在淋巴管组织中表达(Oh H.A novel molecular mechanism involving neuropilin-1for vascular endothelial growth factor-induced retinal angiogenesis.Nippon Gakkai Zasshi.2003-Nov：107(11)：651-656)。
已知VEGF是与眼内病症有关的新血管生成过程中关键的调节因子。眼液中VEGF水平与糖尿病患者和其它缺血性视网膜病患者的活性血管增生水平密切相关。其它研究证明了在AMD患者脉络膜新生血管膜中存在VEGF。目前批准的用于治疗湿性AMD的抗血管药物，目的在于中和VEGF的作用，从面阻断血管新生。例如：辉瑞的哌加他尼钠(Pegaptanib，商品名Macugen)，其活性成分哌加他尼钠是一种由28个核苷酸组成的“aptamer”，其三维结构使其能与细胞外的血管内皮生长因子受体(VEGF)结合，抑制VEGF与相应的受体结合。需要每六周经玻璃体内注射施用；诺华的兰尼单抗(Ranibizumab，商品名Lucentis)，Lucentis中的活性成分兰尼单抗是一种与VEGF结合的抗体片段，需要每个月进行玻璃体内注射；拜耳的艾力亚(VEGF-Trap-eye，商品名Eylea)，其活性成分阿普西柏是一种与VEGF结合的多肽片段，需要头三个月每个月注射，之后每两个月注射施用。
但目前的标准药物治疗对相当一部分患者无效，比如目前标准的Ranibizumab 治疗只能提高1/3患者的视力，10％患者对该治疗无反应；另外，大概70％患者经过抗VEGF治疗后无明显的视力增加。目前的药物治疗还存在适应性抗性，也就是患者在经过一段时间治疗后对该治疗药物产生了抗性。同时由于目前的治疗是通过眼内注射，每一次注射都不仅产生昂贵的治疗费用，也会引起眼内感染，出血以及视网膜脱离，其中最值得重视的并发症是眼内感染。一旦感染，后果不堪设想，有可能会失明。因此注射频度的降低对于药物的发展亦有重要意义。因此，这个领域需要更有效的治疗，克服抗VEGF治疗的抗性，以及延长抗VEGF药物作用的时间(不需要一个月或者两个月的眼底注射)，以期更有效，更安全。
血小板衍生因子PDGF是创伤愈合过程中较早出现的生长因子之一。在创面愈合的全过程中起重要作用，主要表现在促进创面愈合方面。常见的PDGF是由两条多肽链通过二硫键连接而成的同型或异型二聚体，这使PDGF具有多种形式的二聚体结构，即PDGF-AA、PDGF-BB、PDGF-AB、PDGF-CC以及PDGF-DD。PDGF必须与细胞膜上的相应受体结合后才能发挥其生物学效应。PDGF受体(PDGFR)由两种亚单位α及β构成，其分子量为170～180KD。二者与PDGF结合力相差很大，α单位与PDGFa链及b链有较高的亲和力，而β亚单位仅与b链有高亲和力。所以α亚单位可与PDGF-AA、PDGF-AB及PDGF-BB结合，β亚单位仅与PDGF-BB及PDGF-AB结合。
PDGFR是一种跨膜糖蛋白，具有酪氨酸蛋白激酶活性。由细胞外N端与PDGF特异识别的结构域、跨膜的中间疏水结构域和细胞内C端具有酪氨酸蛋白激酶活性的结构域组成。当PDGF与细胞表面的PDGFR结合后，促使受体分子二聚化；激活细胞内结构域酪氨酸残基自身磷酸化，或促使激活特殊靶蛋白的酪氨酸残基磷酸化；从而将信号传入细胞内，经级联式放大效应调控细胞的生命活动(包括靶细胞的分裂增殖)。由此，阻止PDGF结合或PDGFR二聚化的PDGFR拮抗剂，可用于治疗或预防与PDGFR通路相关的疾病。
目前认为中和VEGF的手段效果有限，主要是因为附在血管上的周细胞(vascular mural cells)在缺少VEGF的情况下保护了血管。因此在中和VEGF的基础上，如果能够阻断周细胞对血管的保护，就能够克服抗VEGF治疗的抗性，更有效的治疗老年黄斑变性。
发明内容
为了达到以上目的，本发明提供一种双特异性融合蛋白，包括VEGF结合肽和PDGFRβ结合肽。VEGF结合肽可以位于PDGFRβ结合肽的上游；反之亦然。
在本发明一个优选的实施方案中，所述的双特异性融合蛋白，其中还包括免疫球蛋白的Fc段。Fc段的作用在于，作为多聚化成分，使得融合蛋白多聚化。Fc段可以位于VEGF结合肽的上游或下游；可以位于PDGFRβ结合肽的上游或下游；或位于VEGF结合肽和PDGFRβ结合肽之间。
在本发明又一个优选的实施方案中，所述免疫球蛋白的Fc段是免疫球蛋白IgG的Fc段。
在本发明又一个优选的实施方案中，所述的双特异性融合蛋白，其中所述的Fc段的氨基酸序列选自SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:9。
在本发明又一个优选的实施方案中，所述的双特异性融合蛋白，其中所述的VEGF结合肽包含VEGFR胞外结构域。
在本发明又一个优选的实施方案中，所述的VEGFR是Flt-1。
在本发明又一个优选的实施方案中，所述的VEGFR是Flk-1。
在本发明又一个优选的实施方案中，所述的VEGFR是Flt-4。
在本发明又一个优选的实施方案中，所述的VEGFR胞外结构域是Flt1的免疫球蛋白结构域2，和Flk1或Flt4的免疫球蛋白结构域3。
在本发明又一个优选的实施方案中，所述的VEGFR胞外结构域的氨基酸序列如SEQ ID NO：1所示。
在本发明又一个优选的实施方案中，所述的PDGFRβ结合肽的氨基酸序列选自SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6中的一个或多个。其中SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5为PDGFRβ结合肽中的重链CDR序列，SEQ ID NO:6为PDGFRβ结合肽中的轻链CDR序列。当选自其中的多个序列时，优选SEQ ID NO:6与SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5之一的组合。
在本发明又一个优选的实施方案中，所述的双特异性融合蛋白还包括隔区，隔区的氨基酸序列选自：SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15。
在本发明又一个优选的实施方案中，所述的双特异性融合蛋白还包括氨基酸序列如(GGGGS)n所示的隔区，其中n为1-10，优选2-5。
在本发明又一个优选的实施方案中，所述的双特异性融合蛋白的氨基酸序列选自：SEQ ID NO:19至SEQ ID NO:24。
本发明还涉及一种PDGFRβ抗体衍生物，包括PDGFRβ结合肽和免疫球蛋白的Fc段。
在本发明又一个优选的实施方案中，PDGFRβ抗体衍生物所述的PDGFRβ结合肽定义如上；PDGFRβ抗体衍生物还包括如上定义的隔区。
在本发明又一个优选的实施方案中，所述的PDGFRβ抗体衍生物氨基酸序列为SEQ ID NO:18。
本发明还涉及一种如上所述的双特异性融合蛋白或PDGFRβ抗体衍生物的制备方法，包括：构建表达载体，将表达载体转化至宿主细胞，表达载体在宿主细胞中进行表达获得表达前体，表达前体分泌到细胞外得到所述的双特异性融合蛋白。在一些实施方案中，将编码如SEQ ID No.19-24所示融合蛋白的多核苷酸，或 编码如SEQ ID No.18所示PDGFRβ抗体衍生物的多核苷酸引入质粒构建表达载体，所述质粒可以是现有技术中任何适于在宿主细胞中表达的质粒，包括但不限于pcDNA3.1(+)。在一些实施方案中，通过现有技术中的公知方法将表达载体转化至宿主细胞，例如转化、转染等。在适当的培养条件下培养宿主细胞，获得表达前体。表达前体分泌到细胞外得到如SEQ ID No.19-24所示的融合蛋白，或如SEQ ID No.18所示的PDGFRβ抗体衍生物。
本发明还涉及一种如上所述的双特异性融合蛋白或PDGFRβ抗体衍生物的表达前体，其中所述表达前体由信号肽和所述双特异性融合蛋白或PDGFRβ抗体衍生物组成；所述信号肽的氨基酸序列选自SEQ ID NO:16或SEQ ID NO:17；优选所述信号肽位于所述双特异性融合蛋白或PDGFRβ抗体衍生物的N端。
本发明进一步提供一种编码如上所述的表达前体产物的多核苷酸，优选DNA。
本发明进一步提供一种含有如上所述的多核苷酸的表达载体。
本发明进一步提供一种包含如上所述的表达载体的宿主细胞。
在本发明一个优选的实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。
在本发明又一个优选的实施方案中，所述的宿主细胞为酵母细胞，优选为毕赤酵母或酿酒酵母。
本发明进一步提供一种药物组合物，其含有如上所述的双特异性融合蛋白或PDGFRβ抗体衍生物和可药用载体。
在本发明一个优选的实施方案中，其中所述的药物组合物为一种可注射的溶液，其中所述的双特异性融合蛋白、PDGFRβ抗体衍生物，或可药用载体是溶解形式的。
本发明还涉及上述的双特异性融合蛋白在制备用于治疗哺乳动物中与抑制VEGF和PDGFRβ双靶点相关的疾病或病症的药物中的用途。所述哺乳动物是人类。
本发明还涉及所述的双特异性融合蛋白、PDGFRβ抗体衍生物在制备抑制VEGF和PDGF信号通路的药物中的用途。
本发明还涉及上述的PDGFRβ抗体衍生物在制备用于治疗哺乳动物中与抑制PDGFRβ靶点相关的疾病或病症的药物中的用途。所述哺乳动物是人类。
本发明还涉及所述的PDGFRβ抗体衍生物在制备抑制PDGF信号通路的药物中的用途。
在本发明一个优选的实施方案中，药物用途中所述的疾病或病症是眼睛的疾病、或其它任何能够通过抑制VEGF和/或PDGF信号通路改善的疾病，比如肿瘤和糖尿病视网膜病变。
在本发明又一个优选的实施方案中，药物用途中所述的眼睛的疾病或病症是年龄相关的黄斑变性，优选湿性老年性黄斑变性。
本发明还涉及一种治疗湿性AMD的方法，其包括给予所需患者治疗有效量的 如上所述的双特异性融合蛋白或PDGFRβ抗体衍生物，或如上所述的药物组合物。
附图说明
图1：VTEP-0对PDGFRbb诱导的PDGFRβ和AKT磷酸化的抑制效果。
图2：VTEP-13对PDGFRbb诱导的PDGFRβ和AKT磷酸化的抑制效果。
图3：VTEP-15对PDGFRbb诱导的PDGFRβ和AKT磷酸化的抑制效果。
图4：VTEP-17对PDGFRbb诱导的PDGFRβ和AKT磷酸化的抑制效果。
图5：VTEP-22对PDGFRbb诱导的PDGFRβ和AKT磷酸化的抑制效果。
图6：VTEP-17对PDGFR配体PDGFRbb诱导的PDGFR磷酸化的抑制率。
图7：VTEP-17对PDGFR配体PDGFRbb诱导的AKT磷酸化的抑制率。
图8：VTEP-13对VEGF诱导的人脐静脉内皮细胞增殖的抑制效果。
图9：VTEP-17对VEGF诱导的人脐静脉内皮细胞增殖的抑制效果。
图10：VTEP-22对VEGF诱导的人脐静脉内皮细胞增殖的抑制效果。
图11：模型小鼠眼部脉络膜新生血管面积，表示VTEP-17对激光诱导脉络膜新生血管生成小鼠模型中脉络膜新生血管生成的抑制作用。
具体实施方式
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
定义
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本发明提供了包括第一种成分VEGF结合肽和第二种成分PDGFRβ结合肽的双特异性融合蛋白；更具体地，提供了通过将VEGFR(Flt-1、Flk-1、Flt-4)的修饰过的胞外结构域、PDGFRβ结合肽、和IgG的Fc区嵌合所形成的新型融合蛋白。
本文所采用的术语“结合肽”是指能够与靶结合的可溶性受体及其片段及其类似物；或抗体及其片段及其类似物。
本发明中的VEGF结合肽是优选含有VEGFR胞外配体结合结构域(本申请中也作VEGFR胞外结构域)的多肽。
胞外配体结合结构域被定义为受体的一部分，它在细胞膜中的天然构象中，是朝着细胞外定向的。它可以接触它的同源配体。所述胞外结构域不包括与受体跨膜结构域相关的疏水性氨基酸或与受体胞内结构域相关的任何氨基酸。Von Heijone公开了被本领域技术人员经常引用的用于测定一种特定受体的氨基酸是否属于胞外、跨膜或胞内结构域的详细规则(参见Von Heijone,1995,BioEssay17:25-30)。另外，网站如http://ulrec3.unil.cn/software/TMPRED-form.html能够向 蛋白质化学家提供有关制备蛋白结构域的先决条件的信息。
本文所采用的术语“PDGFRβ结合肽”优选包含可特异性识别并结合靶标抗原PDGFRβ的抗体所有的或一部分抗原结合位点的多肽；例如重链和/或轻链可变区的全部或部分，或者至少是重链可变区的HCDR3区。PDGFRβ结合肽包含并不限于抗体，或抗体片段，和含有抗体全部或部分抗原结合位点的类免疫球蛋白结构域。更具体地，采用US61/610905中相关定义，比如包括CDR3序列为HGGDRSY(SEQ ID NO：11)的抗体序列，还可以包括序列为GIIPIFGTANYAQKFQG(SEQ ID NO：12)或GILPILKTPNYAQRFQG(SEQ ID NO：13)的CDR2序列。
本发明所述的“PDGFRβ抗体衍生物”包括PDGFRβ结合肽和免疫球蛋白的Fc段。
本发明所述的“双特异性融合蛋白”是指即能特异性识别并结合VEGF抗原表位，也能特异性识别并结合PDGFRβ抗原表位的多肽。本发明所述的“双特异性融合蛋白”还可包含人IgG的Fc段。可以使用IgG的任何同种型，包括IgG1、IgG2、IgG3和IgG4。为了增加融合蛋白的功能，Fc段也可以经过突变，或增加铰链区。用于制备双特异性抗体的方法是本领域已知的。
融合蛋白的组分可直接相互连接或通过隔区(也作连接肽)连接。通常，术语“连接肽”(或隔区、连接区)指一个或多个分子，例如核酸、氨基酸或多肽部分，它们可插入至一个或多个组分结构域之间。连接肽序列可用于在组分之间提供合意的靶标位点来使得操作变得容易。也可以提供连接肽来增强嵌合多个组分在宿主细胞中的表达、减少空间位阻，从而该组分可采取它的最佳三级结构与它的靶标分子更好地相互作用。“隔区”在本发明中用于VEGF结合肽、PDGFR抗体片段和Fc区之间的融合连接，以保证不同功能蛋白的正确折叠和稳定性。在本发明中，隔区优选为包含在一个或多个组分之间的一个或多个肽序列。这些多肽序列在1-50个氨基酸长度之间，优选在1-25之间。“隔区”更优选为(GGGGS)n，其中n为1-10，优选2-5。
本发明所述的“双特异性融合蛋白的前体”也可以包含信号肽，便于蛋白由细胞内分泌到细胞外，增加其产量。
本发明所述的“抗体”是指表现出所需生物学活性的任何形式的抗体。因此，它以最广义使用，具体地说，包括但不限于全长抗体，抗体结合片段或衍生物。抗体的来源包括但不限于单克隆抗体、多克隆抗体、基因工程抗体(例如双特异性抗体)。
本发明所述的“全长抗体”是指包含4条多肽链(即2条重链和2条轻链通过二硫键相互交联形成多聚体)的免疫球蛋白分子(例如IgM)。每条重链包含一段重链可变区(简称VH)和一段重链恒定区，重链恒定区包含3个结构域：CH1、CH2和CH3。每条轻链包含一段轻链可变区(简称VL)和一段轻链恒定区，轻链恒定区包含1个结构域(CL1)。VH区和VL区可进一步包括高变区(文中也作互补 决定区(CDR))。各互补决定区之间穿插着更加保守的结构域，称为框架区(FR)。
本发明所述的“抗体结合片段或衍生物”包括任何一种自然发生的、酶催化获得的、合成的、或是通过基因工程得到的，可与抗原特异性结合形成复合物的多肽或糖蛋白。通常包括亲本抗体的至少部分抗原结合区或可变区(例如一个或多个CDR)，其保留亲本抗体的至少某些结合特异性。“抗体结合片段或衍生物”可能由抗体衍生而来，例如通过适宜的标准技术包括蛋白水解或重组基因工程技术(包括对表达抗体可变区和部分恒定区的DNA进行操作和表达)对抗体全长进行改造而得。“抗体结合片段或衍生物”包括但不限于：(i)Fab片段；(ii)F(ab’)2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)；(vi)dAb片段；和(vii)模拟抗体高变区氨基酸残基的最小识别单元(如一个分离的互补决定区(CDR))。其它工程分子如双价抗体、三价抗体、四价抗体和微抗体也在“抗体结合片段或衍生物”范围内。
“Fab片段”由一条完整的轻链和重链的VH和CH1功能区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fab’片段”包含一条轻链和重链的VH和CH1功能区，还包含在CH1与CH2结构域之间的区域，以至于可在两个Fab’片段的两条重链之间形成链间二硫键，以形成F(ab’)2分子。
“F(ab’)2片段”包含二条轻链和含有CH1与CH2结构域之间的部分恒定区的两条重链，以至于在两条重链之间形成链间二硫键。因此，F(ab’)2片段由通过两条重链之间的二硫键保持在一起的两个Fab’片段组成。
“Fv片段”包含轻链或/和重链的可变区VH功能区。本发明PDGFRβ结合肽优选Fv片段，一个优选实施例为PDGFRβ的重链可变区，另一优选实施例为重链可变区与轻链可变区连接的融合蛋白。
术语“Fc段”指的是人免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。例如，免疫球蛋白Fc区可包括重链CH1、CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM。其中一些还可进一步分成亚类(同种型)，如IgG-1、IgG-2、IgG-3、IgG-4；IgA-1和IgA-2。
“Fc区”优选包括至少一个免疫球蛋白绞链区，以及IgG的CH2和CH3区。更优选包括IgG1的一个CH2结构域，一个CH3结构域和一个免疫球蛋白绞链区，铰链区起始氨基酸位置可以变动。“Fc段”无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。
“铰链区”用于连接抗体的Fab段和Fc段。在本发明中可以将双特异性融合蛋白与Fc段连接。
在本发明的实施方案中，本发明的第一种组分VEGF结合肽简称为VTE；第二种组分PDGFR结合肽简称为PDX；第三种组分IGg的Fc段简称为Fc。三种组分的组合顺序有三种：(1)VTE氨基酸序列在Fc氨基酸序列的上游，Fc氨基酸序列在PDX氨基酸序列的上游，顺序如VTE---Fc---PDX；(2)VTE氨基酸序列在PDX氨基酸序列的上游，PDX氨基酸序列在Fc氨基酸序列的上游，顺序如VTE---PDX---Fc；(3)PDX氨基酸序列在VTE氨基酸序列的的上游，VTE氨基酸序列在Fc氨基酸序列的上游，顺序如PDX---VTE---Fc。
相应的，在本发明的实施方案中，编码VEGF结合肽的核苷酸序列位于编码PDGFR结合肽的核苷酸序列的上游。在本发明的另一种实施方案中，编码VEGF结合肽的核苷酸序列位于编码PDGFR结合肽的核苷酸序列的下游。
施用方法
本发明提供治疗方法，包括施用给受试者有效量的本发明的双特异性融合蛋白。在一优选的方面，所述的融合蛋白是充分纯化的，即基本没有限制其效果或产生非期望的副作用的物质。受试者优选地是哺乳动物，最优选是人。
在特定的实施方案中，期望将本发明药物组合物局部施用至需要治疗的区域；这可通过例如，但不以此作为限制，在手术期间局部灌注、局部施用完成，所述的局部灌注和局部施用是例如通过注射、通过导管的方式或通过植入物的方式进行，其中的植入物是多孔的、非多孔的或凝胶状的物质，包括膜，例如硅橡胶(sialastic)膜、纤维或商业的皮肤代用品。
用于实践本发明方法的组合物可以是包含本发明试剂的溶液、悬液或两者的液体。术语“溶液/悬液”指液体组合物，其中第一部分活性剂存在于溶液中而第二部分活性剂表现为颗粒的形式，所述颗粒在液体基质中悬浮。液体组合物也包括凝胶体。液体组合物可以是含水的或是以软膏剂的形式。进一步，组合物可采用固体物的形式可将该固体物放入眼睛中，例如放入眼和眼睑之间或结膜囊中，在这里释放本发明所公开的双特异性蛋白。双特异性蛋白一般可从上述固体物释放至角膜，或直接传至角膜本身，固体物一般与角膜直接接触。适合植入眼睛的固体物通常主要由生物溶蚀或非生物溶蚀聚合物组成。水溶液和/或悬液可以是滴眼液的形式。理想的活性剂的剂量可以通过施用已知数量的滴数至眼中测量。例如，对于50μl体积一滴，施用1-3滴将递送50-150μl组合物。
用于实现本发明方法的含水悬液或溶液/悬液可包含一种或多种作为助悬剂的聚合物。有用的聚合物包括水溶性聚合物，如纤维素聚合物，和不可水溶的聚合物如交联的含有羧基的聚合物。本发明的含水悬液或溶液/悬液优选地是粘性的或粘膜粘附性的，或更优选地两者都是粘性的或两者都是粘膜粘附性的。
诊断和筛选方法
本发明的双特异性蛋白或作诊断性使用和/或用于筛选方法。例如，该双特异性蛋白可用于在临床研究阶段监控VEGF和PDGFR的水平来评价治疗效率。在另 外的实施方案中，本发明方法和组合物可用于筛选具有例如太高或太低VEGF和PDGFR的水平的个体。也可用于在体内和体外筛选检测法来定量存在的非结合的VEGF和PDGFR的量，例如可用于筛选方法来鉴定能降低VEGF和PDGFR的表达的检测试剂。更一般地，本发明的双特异性蛋白可用于定量和/或分离VEGF和PDGFR的任意检测或方法。
药物组合物
本发明也提供包含本发明双特异性蛋白的药物组合物。这种组合物包含治疗有效量的一种或多种本发明所提供的双特异性蛋白和可药用载体。术语“可药用的”指用于动物以及更特别是用于人的中国药典或其它药典中列出的。术语“载体”指与药物一起施用的稀释剂、佐剂、赋形剂。这种药用载体可以是无菌液体，如水或油。这些组合物可采取溶液、悬液、乳剂、片剂、丸剂、胶囊剂、粉剂、缓释剂等形式。
此外，用于实现本发明方法的药物组合物具有与眼睛相容的PH值和摩尔渗透压浓度。
实施例
以下结合实施例进一步描述本发明，但这些实施例并非限制本发明的范围。
本发明实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
一般方法
分子生物学的标准方法已有描述(Maniatis等(1982)Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY；Sambrood和Russell(2001)Molecular Cloning,第3版，Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY；Wu(1993)Recombinant DNA,第217卷，Academic Press，San Diego，CA)。标准方法还见于Ausbel等，(2001)Current Protoclols in Molecular Biology,第1-4卷，John Wiley and Sons,Inc.New York，其描述了在细菌细胞中克隆和DNA诱变(第1卷)、在哺乳动物细胞和酵母中克隆(第2卷)、复合糖和蛋白质表达(第3卷)，以及生物信息学(第4卷)。
本发明提供了构建编码融合蛋白分子的多核苷酸的方法，该核酸分子被插入一种载体上，该载体在导入合适的宿主细胞之后能够表达所述融合蛋白。合适的宿主细胞包括，但不限于细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞。优选Lonza CHO细胞。本领域技术人员所公知的，任何用于将DNA片段插入载体中的方法，都可用于构建编码所述融合蛋白的受转录/翻译控制信号控制的表达载体。所述方法可以包括体外重组DNA和合成技术和体内重组(遗传重组)(参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel 等著，Greene出版协会，Wiley Interscience，NY)。
编码本发明融合蛋白的多核苷酸分子的表达可通过另一核苷酸序列调控，以便该融合蛋白是在用所述重组DNA分子转化过的宿主中表达。例如，本发明的融合蛋白的表达可通过本领域所公知的任何启动子/增强子元件控制。
因此，根据本发明用含有本文所披露的融合蛋白的多核苷酸的能够在细菌或真菌宿主中复制的表达载体转染所述宿主，并因此控制所述多核苷酸的表达，以便产生所述融合蛋白，然后以生物学活性形式回收该融合蛋白。在发明中，生物学活性形式包括能够同时与VEGF和PDGFR结合的形式。
含有本发明所述多核苷酸的表达载体可以通过三种一般方法鉴定：(a)DNA-DNA杂交，(b)“标记”基因功能的有和无，和(c)插入序列的表达。在第一种方法中，插入一种表达载体的外源基因的存在可以用含有与插入的融合蛋白序列同源的序列的探针进行DNA-DNA杂交进行检测。在第二种方法中，可以根据由于在所述载体上插入外源基因所导致的某些“标记”基因功能(例如对抗生表的抗性、转化表型等)有或无鉴定并选择重组载体/宿主系统。例如，如果所述融合蛋白DNA序列被插入所述载体的标记基因序列之内，可以通过该标记基因功能的缺乏鉴定含有该插入片段的重组体。在第三种方法中，可以通过检测由重组体所表达的外源基因产物鉴定重组表达载体。例如，所述检测可以基于所述融合蛋白的物理特性或功能特性。
本发明所述细胞可以是暂时性的，优选组成型并永久地，表达所述融合蛋白。
所述融合蛋白可以用任何方法纯化，以便能够随后制成稳定的、有生物学活性的融合蛋白。例如，但并非是限定，所述融合蛋白可以可溶蛋白形式从细胞中回收，或者作为包涵体形式回收。为了进一步纯化所述融合蛋白，可以使用常规的离子交换层析、疏水性相互作用层析、反相层析或凝胶过滤。
实施例1.双特异性融合蛋白
本发明提供了包括VEGF结合肽和PDGFRβ结合肽的双特异性融合蛋白；更具体地，提供了通过将VEGFR(Flt-1、Flk-1、Flt-4)的修饰过的胞外结构域、PDGFRβ结合肽和IgG的Fc区嵌合所形成的新型融合蛋白。
在本发明的实施方案中，本发明VEGF结合肽氨基酸序列称为VTE；第二种组分PDGFR抗体，称为PDX；第三种组分IGg的Fc区称为Fc。三种组分的组合顺序有三种：
(1)VTE氨基酸序列在Fc氨基酸序列的上游，Fc氨基酸序列在PDX氨基酸序列的上游，顺序如VTE---Fc---PDX，简称VFP；
(2)VTE氨基酸序列在PDX氨基酸序列的上游，PDX氨基酸序列在Fc氨基酸序列的上游，顺序如VTE---PDX---Fc，简称VPF；
(3)PDX氨基酸序列在VTE氨基酸序列的的上游，VTE氨基酸序列在Fc 氨基酸序列的上游，顺序如PDX---VTE---Fc，简称PVF。
融合蛋白的组分可直接相互连接或通过连接肽连接。比如在Fc上游加上5-20个氨基酸，优选5-15个氨基酸的铰链区。
本发明中的VEGF结合肽是指含有VEGFR胞外结构域序列的多肽片段。例如包括Flt-1的胞外结构域D2和Flk-1的胞外结构域D3的序列(为方便命名为VTE)，序列如下：
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID NO：1。
VEGF结合肽也可以是VTE加上Fc片段，简称VEGF-Trap-Fc：
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO：2。
本发明中的PDGFRβ结合肽可以是具有PDGFRβ结合活性的VH片段，序列如下(为方便命名为PDX，选自PDX1，PDX2或PDX3)：
PDX1(又名XB2202VH，本发明中简称VH)：
QVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS
SEQ ID NO：3。
PDX2(XB1115VH)：
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAIHGGDRSYWGQGTLVTVSS
SEQ ID NO：4。
PDX3(XB2708VH)：
QVQLVQSGGGVVQPGGSLRLSCAASGFTSRSYGMHWVRQAPGKGLEWVAFILFDGNNKYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS
SEQ ID NO：5。
PDX4(XB2202VL)：
DVVMTQSPSSLSASVGDRVTITCQASQDISNWLNWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNNVLRTFGQGTKVEIK    SEQ ID NO：6。
本发明的Fc区包含IgG(如IgG1，IgG2，IgG3或IgG4，优选IgG1)的CH2和CH3功能区，序列如下(为方便命名为Fc)：
Fc-1：
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK        SEQ ID NO：7。
Fc-2：
CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK         SEQ ID NO：8。
Fc-3：
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK        SEQ ID NO：9。
在Fc区前可以有铰链区，选自原始序列或其突变体，序列如下：
Fc-H1：DKTHT             SEQ ID NO：10。
Fc-H2：EPKSSDKTHT        SEQ ID NO：11。
各组分之间可以有连接肽，序列如下：
Figure PCTCN2014093555-appb-000001
本发明所述的双特异性融合蛋白的表达前体还可以有分泌信号肽，例如：
SIG1：MEFGLSWLFLVAILKGVQC           SEQ ID NO：16。
SIG2：MDMRVPAQLLGLLLLWFPGSRC        SEQ ID NO：17。
本发明所述的双特异性融合蛋白，经细胞表达得到如下优选序列：
VTEP-0：
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS      SEQ ID NO：18。
序列说明：排列如Fc(hIgG1)-XB2202 VH，Fc(人IgG1，Fc-3，SEQ ID NO：9)通过一个连接肽(SPAC1,n＝2)连接XB2202重链可变区(PDX1,SEQ ID NO： 3)，组合顺序为：Fc-PDX；
VTEP-13：
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSQVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK      SEQ ID NO：19。
序列说明：排列如VEGF-Trap-XB2202-FC，VEGF Trap(VTE)通过连接肽(SPAC1,n＝2)连接XB2202重链可变区(PDX1,SEQ ID NO：3)和Fc(Fc-1，SEQ ID NO：7)，在PDX1与Fc之间有绞链区(Fc-H2，SEQ ID NO：11)，组合顺序为：VTE-PDX-Fc；
VTEP-15：
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS         SEQ ID NO：20。
序列说明：排列如VEGF-Tarp-Fc-(G4S)4-XB2202，VEGF-Trap Fc融合蛋白(VF1序列，通过铰链区Fc-H1，SEQ ID NO：10，与Fc-2，SEQ ID NO：6连接融合)，通过连接肽(SPAC1,n＝4)连接XB2202重链可变区(PDX1,SEQ ID NO：3)，组合顺序为：VTE-Fc-PDX；
VTEP-17：
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVVMTQSPSSLSASVGDRVTITCQASQDISNWLNWYQQKPGKAPKL LIYEASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNNVLRTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS    SEQ ID NO：21。
序列说明：排列如VEGF-Tarp-FC-(G4S)5-XB2202VL-Linker-VH，VEGF-TrapFc融合蛋白通过连接肽(SPAC1,n＝4)连接XB2202的轻链可变区(PDX4，SEQ ID NO：6)和重链可变区(PDX1,SEQ ID NO：3)，组合顺序为：VTE-Fc-PDX；VTEP-22：
QVQLVQSGGGVVQPGGSLRLSCAASGFTSRSYGMHWVRQAPGKGLEWVAFILFDGNNKYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSSASTKGPSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK           SEQ ID NO：22。
序列说明：排列如XB2708VH–linker-1-VEGF-Tarp-Fc，XB2708重链可变区(PDX3,SEQ ID NO：5)通过一个连接肽(SPAC2)连接到VEGF-Trap Fc融合蛋白，组合顺序为：PDX-VTE-Fc
VTEP-25：
QVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK          SEQ ID NO：23。
序列说明：排列如XB2202VH–(G4S)5-VEGF-Tarp-Fc，XB2202重链可变区通过一个连接肽(SPAC1,n＝5)连接到VEGF-Trap Fc融合蛋白，组合顺序为：PDX-VTE–Fc；
VTEP-26：
QVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPSSLSASVGDRVTITCQASQDISNWLNWYQQKPGKAPKLLIYEA SNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNNVLRTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK          SEQ ID NO：24。
序列说明：排列如XB2202VH–(G4S)3-VL-(G4S)5-VEGF-Tarp-Fc，为XB2202重链可变区和轻链可变区通过一个连接肽(SPAC1,n＝3)连接到VEGF Trap Fc融合蛋白，组合顺序为：PDX-VTE–Fc。
表1.序列组成
名称 组分 连接肽 组分 连接肽 组分
VTEP-0 Fc-3 SPAC1,n＝2 PDX1    
VTEP-13 VTE1 SPAC1,n＝2 PDX1 Fc-H2 Fc-1
VTEP-15 VTE1 Fc-H1 Fc-2 SPAC1,n＝4 PDX1
VTEP-17 VTE1 Fc-H1 Fc-1 SPAC1,n＝3 PDX4,PDX1
VTEP-22 PDX3 SPAC2 VTE1 Fc-H1 Fc-1
VTEP-25 PDX1 SPAC1,n＝5 VTE1 Fc-H1 Fc-1
VTEP-26 PDX1 SPAC1,n＝3 PDX4 SPAC1,n＝5 VTE1,Fc-1
实施例2载体构建
所有PCR和克隆相关操作均按照分子克隆标准操作进行，详细参考《分子克隆》(Sambrook等，冷泉港实验室)。
实验材料：
真核表达载体pcDNA3.1(+)(Life technologies，货号V790-20)；
编码VTE蛋白(SEQ ID NO：1)的核苷酸序列：由基因合成公司合成(金唯智，苏州)；
编码Fc蛋白片段(SEQ ID NO：7，SEQ ID NO：8或SEQ ID NO：9)的核苷酸序列：人源抗体重链γ1恒定区Fc片段，由基因合成公司合成(金唯智，苏州)；
PDX蛋白片段(SEQ ID NO：3，SEQ ID NO：4，SEQ ID NO：5或SEQ ID NO：6)的核苷酸序列：由基因合成公司合成(金唯智，苏州)。
实验方法：
1、片段拼接
按照VTEP-0、VTEP-13、VTEP-15、VTEP-17、VTEP-22、VTEP-25、VTEP-26各序列中VTE、Fc、PDX的顺序，并加入连接肽修饰序列，翻译成对应的核苷酸序列，进行overlap PCR，将各片段拼接成对应蛋白的核苷酸序列表达片段。
2、引入酶切位点及信号肽序列：
利用PCR方法在各个表达片段的5’端引入限制性内切酶KpnI位点，Kozak序列，信号肽序列；在各个片段的3’端分别引入终止密码子TGA和NotI限制性内切酶位点。最终得到的DNA序列从5’至3’端依次为：KpnI位点-Kozak序列-信号肽序列-表达片段(结构顺序包括Fc-PDX，VTE-Fc-PDX，PDX-VTE-Fc或VTE-PDX-Fc)-TGA终止密码子-NotI位点。在本发明的实施例中，VTE-0的信号肽优选SIG2的序列，其它实施例优选SIG1的序列。
3.构建表达载体
利用KpnI和NotI限制酶位点将各个片段分别插入质粒pcDNA3.1(+)，构建成表达载体。
实施例3.蛋白表达
实验方法：
使用FreeStyle 293细胞(GIBCO,Cat#R79007)瞬时转染表达实施例2中构建的表达载体。
FreeStyle 293细胞悬浮培养在培养基(Freestyle 293expression medium，GIBCO,Cat#12338018)中，添加终浓度为1％的超低免疫球蛋白胎牛血清(Ultra Low IgGFetal Bovine Serum，GIBCO,Cat#16250078)。
准备好实施例2中构建好的表达载体和转染试剂PEI(Polysciences,Cat#239662)，表达载体量为100μg/100ml细胞，表达载体和PEI的质量比为1:2。转染当天细胞密度为1×106/ml。1L的FreeStyle 293待转染细胞，取50ml Opti-MEM培养基(GIBCO,Cat#11058021)与表达载体混匀，静置5min，过滤；另取50ml Opti-MEM培养基与PEI混匀，静置5min，过滤。将表达载体和PEI进行混匀，静置15min。
将表达载体和PEI混合物缓慢加入细胞中，置入37℃，8％CO2，130rpm摇床培养箱中培养。5天后离心收集上清进行蛋白纯化。
实施例4蛋白纯化
实验方法：
1、亲合层析
细胞培养液高速离心后收集上清，利用亲合层析进行第一步层析。层析条件：层析介质为与Fc相互作用的Protein A或者衍生填料，如GE的Mabselect；平衡缓冲液为1×PBS(NaCl 137mmol/L，KCl 2.7mmol/L，Na2HPO410mmol/L，KH2PO42mmol/L，pH7.4)，平衡5倍柱体积；细胞上清上样结合，流速控制在样品柱上保留时间≧1min；上样结束后，利用1×PBS(pH7.4)进行清洗，直至紫外吸收回落到基线；洗脱缓冲液为0.1M甘氨酸(pH3.0)进行层析洗脱，根据紫外吸收峰收集洗脱样品，最后用1M Tris(pH9.0)中和。
2、体积排阻。
将第一步洗脱样品超滤浓缩后进行体积排阻层析，排阻层析条件：缓冲液为1×PBS(NaCl 137mmol/L，KCl 2.7mmol/L，Na2HPO410mmol/L，KH2PO42mmol/L，pH7.4)，层析柱XK26/60Superdex200(GE)，流速4ml/min，上样体积小于5ml。根据紫外吸收合并目的蛋白峰。
3：纯度检测。
纯度检测采用SEC-HPLC，检测用柱为TSK-Gel 2000-SWXL(TOSOH)，流速：0.7ml/min，上样量：50μl，流动相：1×PBS(NaCl 137mmol/L，KCl 2.7mmol/L，Na2HPO410mmol/L，KH2PO42mmol/L，pH7.4)。SEC-HPLC纯度大于95％。
测试例
测试例1 PDGFR磷酸化细胞实验
一、试验目的：
通过Western印迹方法评估本发明的融合蛋白抑制Caki细胞中PDGFRβ和AKT(S473)磷酸化作用，测试本发明的融合蛋白具有的PDGFR抗体功能。
二、试验材料：
本发明样品：VTEP-0，VTEP-13，VTEP-15，VTEP-17和VTEP-22。
Caki细胞：购自中科院上海生命科学研究院细胞资源中心，货号TCHu135；
Phospho-PDGF受体β(Tyr751)(88H8)Mouse mAb(PDGFRβ抗体)：购自Cell SignallingTechnology公司，货号#3166；
重组人PDGF-BB CF(10UG)(PDGFR配体PDGFRbb)：购自R&D公司，货号220-BB-010；
蛋白酶抑制剂：Complete Mini EDTA-free购自罗氏公司，货号04693159001；
磷酸化酶抑制剂：PhosSTOP购自罗氏公司，货号04906837001；
McCOY's 5A培养基：购自Life Technologies，货号16600-082；
细胞RIPA裂解液：购自碧云天生物技术研究所，货号P0013B。
三、试验方法：
1、取4块6孔板，每个孔加0.8×106个Caki细胞，37℃培养6小时，换为McCOY's 5A无血清培养基培养37℃培养过夜；
2、第二天给细胞换液，每个孔加2ml无血清培养基。
3、配制不同浓度样品溶液：VTEP-0，VTEP-13，VTEP-15，VTEP-17，VTEP-22，用McCOY's 5A无血清培养基稀释配制，各样品浓度见图1-图5上的第1行。如VTEP-13的加样方式依次为200、66.7、22.2、7.4、0、200nM，每孔1ml。
4、37℃孵育50分钟后加PDGFR配体PDGFRbb(配体)，终浓度为40ng/ml，37℃孵育10分钟；各样品加配体的情况见图1-图5上的第2行，“+”表示所示样品按前述加入配体，“-”表示所示样品不加入配体；
5、用PBS(pH7.4)洗涤2次，用细胞RIPA裂解液(含蛋白酶和磷酸化酶抑制剂)裂解细胞，BCA蛋白定量；
6、电泳，8％SDS-PAGE，Western印迹。
四、试验结果：
结果如图1-图5所示：图1-图5中p-PDGFRβ表示该行电泳图表示测试加入样品后，细胞裂解液中磷酸化PDGFRβ的含量；p-AKT(S473)表示该行电泳为测试加入样品后，细胞裂解液中磷酸化AKT(S473)的含量；PDGFRβ表示该行电泳为测试加入样品后，细胞裂解液中磷酸化PDGFRβ与非磷酸化PDGFRβ的总含量；GAPDH表示该行电泳为测试加入样品后，细胞裂解液中看家基因GAPDH蛋白的含量。
结果显示，本发明样品具有PDGFR抗体功能，均能够抑制PDGFRβ和AKT(S473)磷酸化，对PDGFRβ磷酸化抑制作用具有明显的剂量效应，对下游AKT(S473)磷酸化抑制作用也有一定的剂量效应。且抗体本身没有PDGFRβ磷酸化激动剂作用。其中VTEP-17对PDGFR配体PDGFRbb诱导的PDGFR磷酸化的抑制率IC50为3.6nm，如图6所示；VTEP-17对PDGFR配体PDGFRbb诱导的AKT(S473)磷酸化的抑制率IC50为3.2nm，如图7所示。
测试例2 HUVEC细胞增殖实验
一、试验目的：
检测本发明样品对HUVEC细胞增殖的抑制作用。
二、试验材料：
本发明样品：VTEP-13，VTEP-15和VTEP-22；
HUVEC细胞(人脐静脉内皮细胞)：购自ATCC，货号CRL-1730TM；
Low Serum Growth Supplement(LSGS，低浓度血清生长补充剂)：购自GIBCO，货号S-003-10；
Medium 200培养基：购自GIBCO，货号M-200-500；
VEGF165：购自R&D，货号293-VE-010；
CCK8：购自同仁化学，货号CK04。
三、试验方法：
1、96孔板中，每孔加入100μl含2000个HUVEC细胞的Medium200+Low Serum Growth Supplement培养基(加LSGS的Medium 200培养基)，培养板放在37℃、5％CO2培养箱中培养。
2、24小时后，将Medium200+Low Serum Growth Supplement培养基换为0.5％FBS的medium200培养基，每孔90μl。
3、将样品从原始浓度开始用无血清medium200培养基三倍稀释，共稀释9个点，稀释后的样品分别与200ng/ml VEGF165，37℃、5％CO2培养箱孵育1小时。
4、将步骤3中配置好的不同稀释度的样品与VEGF的混合物加至细胞中，每孔10μl，将培养板放在37℃、5％CO2培养箱中培养。
5、72小时后，每孔加入10μl CCK8显色，4小时后检测CD450。
四、试验结果：
结果如图8-10所示，3个样品对HUVEC细胞增殖均有不同抑制作用，见表2。
表2：对HUVEC细胞增殖的抑制作用
名称 IC50(ng/ml)
VTEP-13 159
VTEP-17 292
VTEP-22 220
测试例3 样品与PDGFRβ蛋白的反应亲和力测试
一、试验目的：
用Biacore，GE仪器测定本发明样品和PDGFRβ蛋白的反应亲和力(affinity)。
二、实验仪器、材料与试剂
本发明样品：VTEP-0，VTEP-13，VTEP-15，VTEP-17，VTEP-22，VTEP-25和VTEP-26。
实验仪器：Biacore X100，GE；
实验材料：生物传感芯片CM5(Cat.#BR-1000-12，GE)；
实验试剂：
1)、氨基偶联试剂盒(Cat.#BR-1000-50，GE)；
2)、人抗捕获试剂盒(Cat.#BR-1008-39，GE)；
3)、PDGFRβ(Cat.#10514-H08H，Sino Biological)；
4)、盐酸甘氨酸(pH 1.5)再生溶液(Cat.#BR-1003-54，GE)；
5)、HBS-EP+10倍缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1倍(pH 7.4)。
三、试验方法：
实验样品VTEP-0，VTEP-13，VTEP-15：
按照人抗捕获试剂盒说明书中的方法将人抗捕获抗体共价偶联于CM5生物芯片上，从而亲和捕获一定量的VTE蛋白，然后于芯片表面流经一系列浓度梯度下的PDGFRβ蛋白，利用Biacore仪器实时检测反应信号获得结合和解离曲线。在实验中每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液将生物芯片洗净再生。
实验样品VTEP-17，VTEP-22，VTEP-25，VTEP-26
按照氨基偶联试剂盒说明书中的方法将PDGFRβ蛋白共价偶联于CM5生物芯片上，然后于芯片表面流经一系列浓度的VTE蛋白，进而利用Biacore仪器实时检测反应信号获得结合和解离曲线。使用GE的BIAevaluation软件以1:1 (Langmuir)结合模型分析所得数据，以此法测定的亲和力Kd值显示于下表3。
四、实验结果
本发明样品与PDGFRβ蛋白有较高的反应亲和力，结果见表3。
表3：与PDGFRβ蛋白的反应亲和力
编号 Kd(nM)
VTEP-0 5.9
VTEP-13 81
VTEP-15 58
VTEP-17 2.3
VTEP-22 3.1
VTEP-25 22
VTEP-26 49
测试例4样品与VEGF蛋白的反应亲和力测试
一、试验目的：
用Biacore，GE仪器测定本发明样品和VEGF蛋白的反应亲和力(affinity)。
二、实验仪器、材料与试剂
本发明样品：VTEP-17和VTEP-22。
实验仪器：Biacore X100，GE；
实验材料：生物传感芯片CM5(Cat.#BR-1000-12，GE)；
实验试剂：
1)氨基偶联试剂盒(Cat.#BR-1000-50，GE)；
2)人抗捕获试剂盒(Cat.#BR-1008-39，GE)；
3)VEGF165/VEGFA(Cat.#11066-HNAB，Sino Biological)；
4)HBS-EP+10倍缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1倍(pH 7.4)。
三、试验方法：
按照人抗捕获试剂盒说明书中的方法将人抗捕获抗体共价偶联于CM5生物芯片上，从而亲和捕获一定量的VTE蛋白，然后于芯片表面流经一系列梯度浓度下的VEGF165蛋白，利用Biacore仪器实时检测反应信号获得结合和解离曲线。使用GE的BIAevaluation软件以1:1(Langmuir)结合模型分析所得数据，以此法测定的亲和力Kd值显示于下表4。
四、实验结果
本发明样品与VEGF蛋白有较高的反应亲和力，结果见表4：
表4.与VEGF蛋白的反应亲和力
编号 Kd(pM)
VTEP-17 52
VTEP-22 250
测试例5体内的半衰期检测
一、实验目的：
为了检测本发明VTEP-17在大鼠体内药代动力学参数。
二、实验材料与试剂：
动物：180±10g SD大鼠，雌雄各半(西普尔-必凯实验动物有限公司提供，动物生产许可证号SCXK(沪)2008-0016)，每组6只。
PDGFR-His：组成为PDGFRβ的胞外片段(人源CD140b/PDGFRb基因功能区，来自www.uniprot.org，SEQ ID NO：25)加上Flag标签和his标签(序列中以横线标注)，可用领域内熟知的办法制备，如建立克隆，瞬转质粒和用镍柱纯化蛋白，经电泳验证为所要的蛋白后使用。在本测试中，可用于结合检测具有PDGFRβ抗体功能的片段，序列如下：
LVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHVDRPLSVRCTLRNAVGQDTQEVIVVPHSLPFKVGSSDYKDDDDKHHHHHH           SEQ ID NO：25。
羊抗人IgG过氧化物酶偶联抗体：Jackson Cat.No.：109-035-088；
酶标仪：Thermo Scientific。
三、实验方法：
1、体内给药
SD大鼠6只，雌雄各半，腹腔注射给药；无菌条件下，VTEP-17溶于生理盐水中，终浓度为20μg/mL；每只大鼠IP给药，给药剂量为100μg/kg；
IP组按时间点15min、30min、1hr、2hr、4hr、8hr、11hr、24hr、48hr、72hr大鼠眼底静脉取血，每次200μL(相当于取血清100μL)；
收集的血样在室温下置放半小时至凝集，然后4℃下10000×g离心5分钟。收集上清，立即进行实验或样品等分放置-80℃贮存。避免反复冻融。
2、用步骤1中得到的血清样品进行ELISA检测
1)直接包被100ng/ml的PDGFR，4℃过夜；
2)用300μl含5％脱脂乳的PBST封闭酶标板，37℃恒温封闭2h，同时封闭无包被的空白孔作对照；
3)PBST洗涤3次；
4)每孔加入100μl含VTEP-17的大鼠血清样品(实验前取一个大鼠血清样品，按照不同比例稀释，得到一个血清中抗体浓度正好在标准曲线中间位置的最佳稀 释比例，将血清样品按照最佳稀释比例进行稀释)，37℃恒温孵育2h；
5)PBST洗涤3次；
6)每孔加入100μl羊抗人IgG过氧化物酶偶联抗体(1:2500)，37℃恒温孵育1h；
7)PBST洗涤3次。每孔加入100μl TMB底物，37℃恒温孵育5-10min，随后每孔加入100μl 1.25M H2SO4中止反应；
8)ELISA酶标仪读取450nm波长处的OD值。
根据标准曲线方程和样品的OD值，计算得到样品的实际浓度。
标准曲线：以VTE-17浓度(5,10,20,50和100ng/ml)为横坐标，不同浓度VTE-17对应的OD值为纵坐标，获得典型标准曲线方程，标准曲线的线性范围为5-100ng/ml。
四、实验结果：
按以上方法，对实施例2中得到的VTEP-17进行检测，用PhoenixTM WinNonlin6.1软件中非房室模型进行药动学计算，得到其体内半衰期(T1/2)，结果如下：
表5.药代动力学测试
Figure PCTCN2014093555-appb-000002
发明人在中和VEGF的基础上引入了抗PDGFRβ的VH片段。该融合蛋白具有中和VEGF以及阻断PDGFRβ的信号通路的双重特异性，因此可以使本来对中和VEGF治疗无反应的人群得到有效的治疗，并能够克服中和VEGF药物疗法的抗性，另外有可能延长病人需要眼内注射的周期，从而会在现有基础上更好治疗老年黄斑变性。
测试例6 VTEP蛋白对激光诱导脉络膜新生血管小鼠模型中脉络膜新生血管生成抑制作用的研究
一、试验目的：
本测试例用于研究VTEP蛋白对激光诱导脉络膜新生血管生成小鼠模型中脉络膜新生血管生成的抑制作用。
试验原理：本实验通过一定能量激光破坏小鼠视网膜组织中局部的Bruch’s膜，使得本该阻隔的脉络膜血供系统和视网膜血供系统相沟通，脉络膜毛细血管内皮细胞、周细胞、纤维细胞和炎症细胞等进入视网膜下和色素上皮层内，同时，伴随炎症因子、促血管生成因子产生和细胞外基质成分改变，打破局部促血管-抑血管生成因子间的平衡，促发局部新生血管生成，其中血管内皮生长因子(VEGF)、血小板衍生生长因子(PDGF)介导的内皮细胞生长和胞外基质、周细胞包绕对脉络膜新生血管生成具有重要作用。
二、实验材料与试剂：
实验动物：成年(6-8周龄)C57BL/6J母鼠，购自上海斯莱克试验动物有限责任公司。
试验仪器：
图像分析仪：德国Zeiss公司KS 400型
氩激光发射器:Lumenis Selecta Duet，美国Lumenis公司
裂隙灯：苏州六六
眼科手术器械：淮阴医疗器械
眼科手术显微镜：美国LEICA公司
载玻片、盖玻片：世泰
微量注射仪及进样针：美国Parker公司，Parker Hannifin PICOSPRITZERⅢ
试剂准备：
美多丽眼药水：参天制药有限公司
盐酸丙美卡因滴眼液：Alcon公司
迪可罗眼膏：兴齐制药有限公司
4％多聚甲醛：上海生工
水合氯醛：上海生工
葡聚糖荧光素钠试剂fluorescein-labeled dextran：美国Sigma-Aldrich公司
封片剂(Fluorescent Mounting Medium)：DAKO
VTEP-17(SEQ ID NO：21)和VEGF-Trap(SEQ ID NO：2)蛋白：用提供的溶解稀释缓冲液(PBS缓冲液)按设计分组浓度要求稀释，超净台内操作完成，分装为20ul/管4度低温保存，以备取用。
GONIC粘合剂:美国DOW
测试样品分组如下：
Group1(g1):PBS缓冲液
Group2(g2):VEGF-Trap 10ug
Group6(g6):VTEP-175.1ug
Group7(g7):VTEP-1715.4ug
Group8(g8):VTEP-1746.2ug
三、实验方法：
6-8周龄C57/BL6小鼠自由饮水进食，12小时明暗交替环境饲养，随机分成5组，每组8只，雌雄各半，按0.01ml/g体重腹腔注射3.5％水合氯醛进行麻醉，美多丽滴眼液涂于眼表扩瞳，待麻醉充分后于裂隙灯下行眼底激光光凝(波长532nm,能量120mw,时间0.1s,光斑直径100nm)，以激光后可见气泡生成者计 为有效，其中有效激光斑距视乳头1-2PD，分别位于不同象限，每眼4-6处，即造模成功。各组分别于造模后第1天、第7天腹腔注射水合氯醛麻醉，以玻璃体腔给药方式，用微量注射仪给予相应蛋白(见实验分组设计)。激光后第14天，腹腔注射水合氯醛麻醉，解剖胸腔，暴露心脏，25mg/ml荧光素钠每只小鼠0.4ml行心腔灌注，过量麻醉处死小鼠后，取眼球，于4％福尔马林室温固定5小时。取出固定后眼球，沿角巩缘剪开，去除晶体、角膜、玻璃体，余下眼杯以视乳头为中心做放射状切口剪开，将之展平于载玻片上，小心去除视网膜，滴加封片剂，盖盖玻片完成铺片。组织片于-20度保存，隔天荧光显微镜下观察，20倍放大物镜拍下激光斑图片，以Image pro Plus计数脉络膜新生血管面积。
数据处理：使用SAS 9.0统计学软件进行分析，单因素方差分析(One-way ANOVA)检验，用t-test对两组间均数进行比较，P<0.05为有统计学差异。
四、实验结果：
单因素方差分析显示各组有统计学差异(P<0.02)，其中g1、g2与其余各组均有显著性差异，g6、g7、g8间无显著性差异。结果见图11：模型小鼠眼部脉络膜新生血管面积，柱状图表示均数±标准误。
讨论：本实验中，VEGF-Trap具有中和VEGF作用，VTEP-17具有中和PDGF与中和VEGF的双重作用。实验结果提示这两种蛋白均能有效抑制脉络膜新生血管生成(G1与其余各组比较有统计学差异)，G2(单纯VEGF-Trap)与G6-8(不同浓度VTEP-17)间存在统计学差异，提示VTEP-17抗血管生成作用效果强于VEGF-Trap，但在此浓度跨度内计量-效应关系不明显，不同浓度VTEP-17组间抗血管生成作用无明显差异。

Claims (43)

  1. 一种双特异性融合蛋白，包括VEGF结合肽和PDGFRβ结合肽。
  2. 如权利要求1所述的双特异性融合蛋白，其中还包括免疫球蛋白的Fc段。
  3. 如权利要求2所述的双特异性融合蛋白，其中所述免疫球蛋白的Fc段是免疫球蛋白IgG的Fc段。
  4. 如权利要求3所述的双特异性融合蛋白，其中所述Fc段的氨基酸序列选自SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:9。
  5. 如权利要求1所述的双特异性融合蛋白，其中所述的VEGF结合肽包含VEGFR胞外结构域。
  6. 如权利要求5所述的双特异性融合蛋白，其中所述的VEGFR是Flt-1。
  7. 如权利要求5所述的双特异性融合蛋白，其中所述的VEGFR是Flk-1。
  8. 如权利要求5所述的双特异性融合蛋白，其中所述的VEGFR是Flt-4。
  9. 如权利要求5所述的双特异性融合蛋白，其中所述的VEGFR胞外结构域包含Flt1的免疫球蛋白结构域2，和Flk1或Flt4的免疫球蛋白结构域3。
  10. 如权利要求5所述的双特异性融合蛋白，其中所述的VEGFR胞外结构域的氨基酸序列如SEQ ID NO:1所示。
  11. 如权利要求1所述的双特异性融合蛋白，其中所述的PDGFRβ结合肽的氨基酸序列选自序列SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO: 6中的一个或多个。
  12. 如权利要求1所述的双特异性融合蛋白，其中还包括隔区，所述隔区的氨基酸序列选自：SEQ ID NO:12、SEQ ID NO:12的多个重复、SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15。
  13. 如权利要求1所述的双特异性融合蛋白，其中还包括氨基酸序列如(GGGGS)n所示的隔区，n为1-10，优选2-5。
  14. 如权利要求1至13任一项所述的双特异性融合蛋白，其氨基酸序列选自：SEQ ID NO:19至SEQ ID NO:24。
  15. 一种如权利要求1至14任一项所述的双特异性融合蛋白的制备方法，包括：
    构建表达载体，
    将表达载体转化至宿主细胞，
    在宿主细胞中表达，得到表达前体，
    将双特异性融合蛋白分泌到细胞外，得到所述的双特异性融合蛋白。
  16. 一种用于制备如权利要求1至14任一项所述的双特异性融合蛋白的表达前体，其中所述表达前体由信号肽和如权利要求1至14任一项所述的双特异性融合蛋白组成；所述信号肽的氨基酸序列选自SEQ ID NO:16或SEQ ID NO:17；优选所述信号肽位于所述双特异性融合蛋白的N端。
  17. 一种编码如权利要求16所述的表达前体的多核苷酸。
  18. 一种含有如权利要求17所述的多核苷酸的表达载体。
  19. 一种含有如权利要求18所述表达载体的宿主细胞。
  20. 如权利要求19所述的宿主细胞，其中所述的宿主细胞为真核细胞，优选酵母细胞，更优选毕赤酵母或酿酒酵母。
  21. 一种药物组合物，其含有：
    如权利要求1至14任一项所述的双特异性融合蛋白，和可药用载体。
  22. 如权利要求21所述的药物组合物，其中所述的药物组合物为可注射的溶液，其中所述的双特异性融合蛋白和/或可药用载体是溶解形式的。
  23. 如权利要求1至14任一项所述的双特异性融合蛋白在制备用于治疗哺乳动物中与抑制VEGF和PDGFRβ双靶点相关的疾病或病症的药物中的用途；优选在制备抑制VEGF和PDGF信号通路的药物中的用途；哺乳动物优选人类。
  24. 如权利要求23所述的用途，其中所述的疾病或病症是肿瘤或眼睛的疾病。
  25. 如权利要求23所述的用途，其中所述的疾病或病症是糖尿病视网膜病变 或年龄相关的黄斑变性，优选湿性老年性黄斑变性。
  26. 一种治疗湿性老年性黄斑变性的方法，其包括：
    向患者给予治疗有效量的如权利要求1至14任一项所述的双特异性融合蛋白，或如权利要求21所述的药物组合物。
  27. 一种PDGFRβ抗体衍生物，包括PDGFRβ结合肽和免疫球蛋白的Fc段。
  28. 如权利要求27所述的PDGFRβ抗体衍生物，其中所述的PDGFRβ结合肽的氨基酸序列选自SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6。
  29. 如权利要求27所述的PDGFRβ抗体衍生物，其中还包括隔区，所述隔区的氨基酸序列选自：SEQ ID NO:12、SEQ ID NO:12的多个重复、SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15。
  30. 如权利要求27所述的PDGFRβ抗体衍生物，其中还包括氨基酸序列如(GGGGS)n所示的隔区，n为1-10，优选2-5。
  31. 如权利要求27所述的PDGFRβ抗体衍生物，其氨基酸序列为SEQ ID NO:18。
  32. 一种如权利要求27至31任一项所述的PDGFRβ抗体衍生物的制备方法，包括：
    构建表达载体，
    将表达载体转化至宿主细胞，
    在宿主细胞中表达，得到表达前体，
    将PDGFRβ抗体衍生物分泌到细胞外，得到所述的PDGFRβ抗体衍生物。
  33. 一种用于制备如权利要求27至31任一项所述的PDGFRβ抗体衍生物的表达前体，其中所述表达前体由信号肽和如权利要求27至31任一项所述的PDGFRβ抗体衍生物组成；所述信号肽的氨基酸序列选自SEQ ID NO:16或SEQ ID NO:17；优选所述信号肽位于所述PDGFRβ抗体衍生物的N端。
  34. 一种编码如权利要求33所述的表达前体的多核苷酸。
  35. 一种含有如权利要求34所述的多核苷酸的表达载体。
  36. 一种含有如权利要求35所述表达载体的宿主细胞。
  37. 如权利要求36所述的宿主细胞，其中所述的宿主细胞为真核细胞，优选酵母细胞，更优选毕赤酵母或酿酒酵母。
  38. 一种药物组合物，其含有：
    如权利要求27至31任一项所述的PDGFRβ抗体衍生物，和
    可药用载体。
  39. 如权利要求38所述的药物组合物，其中所述的药物组合物为可注射的溶液，其中所述的PDGFRβ抗体衍生物和/或可药用载体是溶解形式的。
  40. 如权利要求27至31任一项所述的PDGFRβ抗体衍生物在制备用于治疗哺乳动物中与抑制PDGFRβ靶点相关的疾病或病症的药物中的用途；优选在制备抑制PDGF信号通路的药物中的用途；哺乳动物优选人类。
  41. 如权利要求40所述的用途，其中所述的疾病或病症是肿瘤或眼睛的疾病。
  42. 如权利要求41所述的用途，其中所述的疾病或病症是糖尿病视网膜病变或年龄相关的黄斑变性，优选湿性老年性黄斑变性。
  43. 一种治疗湿性老年性黄斑变性的方法，其包括：
    向患者给予治疗有效量的如权利要求27至31任一项所述的PDGFRβ抗体衍生物，或如权利要求38所述的药物组合物。
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