WO2019134686A1 - 一种长效低毒的重组抗vegf人源化单克隆抗体及其生产方法 - Google Patents

一种长效低毒的重组抗vegf人源化单克隆抗体及其生产方法 Download PDF

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WO2019134686A1
WO2019134686A1 PCT/CN2019/070479 CN2019070479W WO2019134686A1 WO 2019134686 A1 WO2019134686 A1 WO 2019134686A1 CN 2019070479 W CN2019070479 W CN 2019070479W WO 2019134686 A1 WO2019134686 A1 WO 2019134686A1
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antibody
vegf
amino acid
acid sequence
seq
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PCT/CN2019/070479
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French (fr)
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吴晓云
徐臣超
王志刚
李胜峰
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百奥泰生物制药股份有限公司
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Priority to EP19735980.5A priority Critical patent/EP3736288A4/en
Priority to JP2020537130A priority patent/JP2021509813A/ja
Priority to US16/959,922 priority patent/US20210079081A1/en
Publication of WO2019134686A1 publication Critical patent/WO2019134686A1/zh

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    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention belongs to the field of biomedicine; more specifically, the present invention relates to an antibody for reducing human vascular endothelial growth factor (VEGF/VEGF-A), and to a method for preparing the same and application thereof .
  • VEGF/VEGF-A vascular endothelial growth factor
  • Angiogenesis is the proliferation and reconstitution of neovascularization by vascular endothelial cells from preexisting vascular networks.
  • Angiogenesis is essential for normal human proliferation processes, including wound healing and organ development and differentiation.
  • angiogenesis is also involved in the formation of a variety of pathological diseases such as age-related macular degeneration, tumors, rheumatoid arthritis and psoriasis.
  • pathological diseases such as age-related macular degeneration, tumors, rheumatoid arthritis and psoriasis.
  • Angiogenesis is a cascade process that involves the following processes: degradation of extracellular matrices at local sites following release of protease; proliferation of capillary endothelial cells; migration of capillary tubules to angiogenic stimuli.
  • VEGF is considered to be the most critical factor in physiological and pathological angiogenesis.
  • VEGF is essential for angiogenesis and angiogenesis in embryos.
  • VEGF is a multi-directional growth factor that exhibits a variety of organisms in endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis, and calcium influx. Effects, for example, have been reported to have a mitogenic effect on retinoic pigment endothelial cells and neuromembrane cells.
  • Angiogenesis is the proliferation and reorganization of vascular endothelial cells to form new blood vessels.
  • VEGF plays a key role in the development of diseases involving pathological angiogenesis, VEGF mRNA is overexpressed in most human tumors, and the concentration of VEGF in aqueous humor is associated with diabetes or other ischemic retinal diseases.
  • Vascular hyperplasia found in patients is highly correlated, especially in choroid plexus neovascularization in patients with age-related macular degeneration (AMD).
  • AMD age-related macular degeneration
  • VEGF is a glycosylation-secreting polypeptide growth factor, a homodimeric glycoprotein with a molecular weight between 46 and 48 kDa. It acts directly on vascular endothelial cells and induces vascular endothelial cell proliferation and angiogenesis.
  • Members of the VEGF family have type 6 including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factors, of which VEGF-A is most important in the eye.
  • the VEGF-A gene is located at p12 to p21 of chromosome 6, and consists of 8 exons and 7 introns.
  • VEGF-A 121 Seven protein forms such as VEGF-A 121 , VEGF-A 145 , VEGF-A 148 , VEGF-A 165 , VEGF-A 183 , VEGF-A 189 and VEGF-A 206 were produced due to different splicing patterns of mRNA. Among them, five protein forms can increase vascular permeability, promote vascular endothelial cell proliferation, and induce neovascularization.
  • the vascular endothelial growth factor receptor (VEGFR) has three kinds of VEGFR1 (Flt-1), VEGFR2 (KDR) and VEGFR3 (Flt-4), and VEGF binds to its receptor, thereby exerting its biological effects. High levels of VEGF expression were found in both experimental models and pathological angiogenesis of wet AMD. There are also a number of reports in the literature that by neutralizing VEGF, angiogenesis and vascular leakage can be suppressed.
  • VEGF plays an important role in angiogenesis in pathological conditions
  • measures have been taken to block VEGF activity. These include antibodies against inhibitory anti-VEGF receptors, soluble receptor constructs, antisense strategies, and low molecular weight VEGF receptor tyrosine kinase inhibitors that interfere with the VEGF pathway and the like.
  • neutralizing antibodies against VEGF have been shown to inhibit the growth of various human tumor cell lines in nude mice and also inhibit intraocular angiogenesis in a model of ischemic retinopathy. Therefore, anti-VEGF monoclonal antibodies or inhibitors of VEGF action are effective candidates for the treatment of solid tumors and various intraocular neovascular disorders.
  • Macular degeneration is a medical condition found primarily in the elderly where the center of the lining of the eye known as the macular area of the retina is thinned, atrophied, and in some cases bleeding. This can result in a loss of central vision, which prevents the patient from seeing more detailed content.
  • macular degeneration is the leading cause of central vision loss (blindness) in the elderly.
  • AMD age-related macular degeneration
  • Age-related macular degeneration begins with a characteristic yellow deposit (called drusen) in the macula (a central region of the retina that provides detailed central vision, called the fovea) between the retinal pigment epithelium and the choroid below it.
  • drusen a characteristic yellow deposit
  • the macula a central region of the retina that provides detailed central vision, called the fovea
  • AMD retinal pigment epithelium
  • Late AMD leading to deep visual loss has two forms: dry and wet.
  • Central atrophy the dry form of advanced AMD, is caused by atrophy of the retinal pigment epithelial layer under the retina, which causes visual loss by loss of photoreceptors (rods and cones) in the central portion of the eye.
  • Age-related macular degeneration is a disease in which irreversible vision loss or loss is caused by retinal pigment epithelial cells and neuroretinal degeneration. Occurred in patients over the age of 50, both eyes have a simultaneous or simultaneous onset, and progressive damage to vision, is a fundus lesion that seriously threatens the visual function of the elderly. With the aging of the population, it has become the first blinding eye disease in Western countries, and its incidence rate is gradually increasing in Asia.
  • a specific humanized anti-VEGF antibody Bevacizumab, and other humanized anti-VEGF antibodies, Ranibizumab and Aflibercept, are treated with high affinity.
  • Bevacizumab (Avastin) is another humanized IgG1 anti-VEGF monoclonal antibody developed by Genentech. It is a mammalian cell expression system using recombinant DNA technology in Chinese hamster ovary cells (CHO). Production is carried out, followed by purification of human anti-tumor monoclonal antibodies using a process including viral inactivation and removal steps.
  • Ranibizumab (Lucentis) is a humanized monoclonal antibody Fab fragment produced by E. coli by recombinant DNA technology developed by Genentech, targeting VEGF-A. High affinity binds to various subtypes of VEGF-A, such as VEGF-A 121 , VEGF-A 165 , VEGF-A 110 .
  • the binding of ranibizumab to VEGF-A prevents VEGF-A from binding to its receptor on the surface of endothelial cells (VEGFR-1, VEGFR-2), prevents vascular endothelial proliferation, reduces vascular leakage, and inhibits retinal neovascularization (CNV). ) generation.
  • ranibizumab and bevacizumab are derived from the same murine parent antibody A4.6.1.
  • A4.6.1 is a mouse anti-VEGF monoclonal antibody produced by a hybridoma cell line produced by a mouse immunized with human VEGF-A 165 protein.
  • the A4.6.1 antibody gene was humanized by recombinant DNA technology.
  • the humanized Fab fragment retains the murine CDR sequence and is combined with the human Fc fragment to obtain a full length antibody, bevacizumab.
  • Bevacizumab has the same antigenic affinity as its parent antibody, but is less immunogenic and has a longer half-life in vivo.
  • Ranibizumab is a high-affinity antibody screened by amino acid site-directed mutagenesis based on the Fab region of bevacizumab. There are 6 amino acids in the Fab region of ranibizumab and bevacizumab. These 6 amino acid mutations make the affinity of ranibizumab for VEGF-A 5 to 20 times higher than that of bevacizumab. The experiment confirmed that the intensity of inhibition of HUVEC proliferation by ranibizumab was 5.2 times that of bevacizumab.
  • the ranibizumab is a Fab fragment with a small molecular weight that completely penetrates the entire retina within 1 h.
  • ranibizumab is a small antigen-binding fragment, does not contain an Fc region, does not trigger a complement-mediated immune response, has a short systemic half-life, and thus has a small systemic adverse reaction.
  • Fc domain-containing antibodies with longer half-lives are also used in the treatment of ocular diseases.
  • Aflibercept produced by Bayer is an Fc fusion protein.
  • the antibody of the present invention is a humanized full-length anti-VEGF IgG1 antibody specifically designed for the treatment of fundus lesions.
  • the antibody of the present invention has antigen affinity similar to that of ranibizumab, but the antibody of the present invention has no CDC and ADCC effects, is safer for intravitreal injection, and has a longer half-life in the vitreous after intravitreal injection, and the therapeutic effect is expected to be better.
  • the invention provides a full length humanized anti-VEGF antibody having (a) two immunoglobulin light chains; and (b) two immunoglobulin heavy chains, said heavy chain
  • the variable region and the constant region are included from the N-terminus to the C-terminus; the antibody specifically recognizes an antigen-binding site of VEGF.
  • the heavy chain contains an amino acid sequence having at least 80%, at least 85% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1; preferably, the heavy chain contains Amino acid sequence identity to SEQ ID NO: 1 is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% amino acid sequence; more preferably, the heavy chain contains at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.1% identical to the amino acid sequence shown in SEQ ID NO: 1.
  • the light chain comprises an amino acid sequence having at least 80%, at least 85% or at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 2; preferably, the light chain comprises SEQ ID NO :2 shows an amino acid sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% amino acid. More preferably, the light chain contains at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, identical to the amino acid sequence set forth in SEQ ID NO: 2.
  • the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 1
  • the light chain comprises the amino acid sequence set forth in SEQ ID NO: 2.
  • the antibody of the invention is a monoclonal antibody, and further, the antibody is human IgG1.
  • the antibodies provided herein bind to human VEGF with a KD value similar to bevacizumab and are capable of inhibiting binding of VEGF to a VEGF receptor. More specifically, it is possible to bind human VEGF at a KD value of 10 nM or lower at 30 ° C and to inhibit binding of VEGF to a VEGF receptor. According to another embodiment, the KD value is 1 nM or less. According to another embodiment, an antibody of the invention binds VEGF with a KD value of no more than about 500 pM.
  • an antibody of the invention has an on rate (k on ) of 1.0 or higher (10M -1 S -1 ) binding to human and/or mouse VEGF.
  • the rate of association is 4.0 or higher (10M -1 S -1 ).
  • the antibody of the invention binds human VEGF with desirable affinity, further binds to VEGF-A, and further binds to VEGF-A165 but does not bind to human VEGF-B, human VEGF-C and human VEGF-D Any or all of the VEGF-related homologues that make up.
  • the VEGF receptor whose binding to VEGF is inhibited may be VEGF receptor 1 (Flt-1), VEGF receptor 2 (Flt-1) or both.
  • the monoclonal antibody of the invention contacts a 20 s helix VEGF. According to another embodiment, the monoclonal antibody of the invention contacts the 80 s loop of VEGF. According to another embodiment, the monoclonal antibodies of the invention are contacted with a 20 s helix and an 80 s loop of human VEGF.
  • Another aspect of the invention provides nucleic acids encoding the heavy and/or light chains of the antibodies of the invention.
  • the invention also provides an expression vector comprising a nucleic acid of the invention, which expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.
  • the invention also provides a host cell comprising the vector for use in the production of an antibody of the invention.
  • the host cell is eukaryotic or the host cell is prokaryotic.
  • the invention also encompasses a method for producing an antibody, characterized in that the nucleic acid of the invention is expressed in a prokaryotic or eukaryotic host cell and the antibody is recovered from the host cell or host cell culture.
  • the present invention also encompasses antibodies obtained by the method for producing a full length antibody.
  • the invention also provides an affinity purification reagent.
  • the affinity purification reagent contains the above-described antibody, and the above antibody can be used for the preparation of a reagent useful for diagnostic analysis of VEGF protein.
  • the antibody can be labeled with a detection molecule. Labeling such as radioisotopes, fluorescent markers, or enzymes.
  • the invention also provides a kit for the diagnosis of VEGF protein, the kit comprising the antibody or polypeptide described above.
  • the invention also provides a pharmaceutical preparation comprising the antibody.
  • the pharmaceutical preparation may further comprise a pharmaceutically acceptable carrier.
  • the invention also provides the use of a medicament comprising the antibody or polypeptide for the manufacture of a medicament for treating a disease associated with VEGF overexpression in a mammal.
  • the therapeutic mammal of the invention comprising administering an effective amount of said antibody to said mammal.
  • the amount of antibody administered will be an effective amount to treat the disease.
  • multiple doses of antibody can be administered to the mammal.
  • a therapeutically effective amount of the antibody is administered to a human patient to treat the disease.
  • the mammal can be a human or a non-human mammal, such as a primate or a phagosome (e.g., mouse, rat or rabbit) suitable for producing preclinical data.
  • a primate or a phagosome e.g., mouse, rat or rabbit
  • the mammal is a human.
  • the mammal can be healthy or can have a disease to be treated with the antibody.
  • the VEGF overexpression-related disease or the disease is a disease associated with abnormal angiogenesis caused by VEGF overexpression; and further a fundus lesion caused by overexpression of VEGF.
  • the antibody of the present invention is used for treating wet (neovascular) age-related macular degeneration (wet-AMD), pathological myopia (PM) secondary to choroidal neovascularization (CNV), diabetic macula Fundus lesions such as edema (DME), diabetic retinopathy (DR), retinal branch vein occlusion (BRVO), and central retinal vein occlusion (CRVO).
  • wet-AMD wet age-related macular degeneration
  • PM pathological myopia
  • CNV choroidal neovascularization
  • DME edema
  • DR diabetic retinopathy
  • BRVO retinal branch vein occlusion
  • CRVO central retinal vein occlusion
  • These antibodies can be used in rhesus monkey models that use lasers to induce choroidal neovascularization. These antibodies can be used to identify anti-VEGF antibodies in treatment age-related by observing or monitoring the area change of macular degeneration treated with the anti-VEGF antibody of the present invention. The method of macular degeneration.
  • the antibodies of the invention can also be used to study and evaluate combination therapies of the anti-VEGF antibodies and other therapeutic agents of the invention.
  • the antibodies of the present invention can be used to study the effects of VEGF in other diseases by administering the antibodies or polypeptides to animals suffering from similar diseases and determining whether one or more symptoms of the disease are alleviated.
  • the invention also provides antibody derivatives, which can be further modified to contain other non-proteinaceous moieties known in the art, such as water soluble polymers.
  • the invention also provides an immunoconjugate (antibody-drug conjugate or ADC) comprising an antibody of the invention conjugated to one or more cytotoxic agents, such as an antibody toxic agent, for example Chemotherapeutic agents, drugs, production inhibitors, toxins or radioisotopes.
  • ADC antibody-drug conjugate
  • cytotoxic agents such as an antibody toxic agent, for example Chemotherapeutic agents, drugs, production inhibitors, toxins or radioisotopes.
  • the antibodies of the present invention are more beneficial to patients suffering from macular degeneration diseases due to the presence of their Fc portion. Compared with smaller antibody fragments that do not have a constant heavy chain region, they have high stability in the vitreous environment and slow diffusion characteristics from the vitreous, ie, prolonged half-life, prolonged drug action time, of which the actual disease Located here and treated here. Thus, the treatment cycle can be extended compared to non-IgG-like antibodies, for example, Fab and (Fab) 2 fragments, and the physiological and psychological conditions of the patient can be improved.
  • non-IgG-like antibodies for example, Fab and (Fab) 2 fragments
  • the antibodies of the invention are full length humanized antibodies.
  • Full-length humanized antibodies have several potential advantages in terms of safety and efficacy compared to murine antibodies and chimeric antibodies.
  • Full length humanized antibodies typically exhibit lower clearance rates than other antibody types. A lower clearance rate may allow for lower doses and frequency of dosing.
  • the antibodies of the invention are synthesized by recombinant methods rather than directly from hybridomas or from antibody sequences derived from hybridomas.
  • the antibody binds hVEGF-A 165 with a KD value of no more than about 2 nM, no more than about 1 nM, no more than about 500 pM.
  • the full length antibody of the present invention exhibits a good pharmacological effect of inhibiting fundus diseases, particularly choroidal angiogenesis.
  • Figure 1 is a comparative experiment of different structures of BAT5906 and Ranibizumab neovascularization, showing that the monoclonal antibody BAT5906 has the best inhibitory effect on angiogenesis, and is superior to the marketed drug Ranibizumab.
  • Figure 2 shows the results of SDS-PAGE electrophoresis (non-reduction) of the monoclonal antibody BAT5906, M: 250 kDa pre-stained protein Marker, lane 1: BAT5906 non-reduced sample.
  • Figure 3 shows the results of SDS-PAGE electrophoresis (reduction) of the monoclonal antibody BAT5906.
  • M 250 kDa pre-stained protein Marker
  • lane 1 BAT5906 reduced sample
  • the results of Figures 2 and 3 show that the size of the monoclonal antibody molecule is consistent with the design size under both reducing and non-reducing conditions.
  • Figure 4 shows the improvement rate (%) of laser fluorescein leakage area in the fundus of each group after administration, showing that the monoclonal antibody BAT5906 plays a role in the rhesus monkey efficacy test, effectively inhibiting fluorescein leakage.
  • Figure 5 shows the number of fluorescent spots of the eyeballs of each group before and after administration, showing that the monoclonal antibody BAT5906 plays a role in the rhesus monkey efficacy test, effectively inhibiting the number of fluorescent spots of class 4, and the listed drug Ranibizumab As a positive control.
  • Figure 6 shows the improvement rate of retinal thickness (%) at the highest point of retinal lesions in each group after administration, showing that the monoclonal antibody BAT5906 plays a role in the monkey efficacy test, effectively inhibiting the retinal thickening of the fundus, and the listed drugs.
  • Ranibizumab was used as a positive control, and the difference in mean was statistically significant (P ⁇ 0.05).
  • Figure 7 is a fluorescing image of the monkey eyeball in the rhesus monkey acute toxicity test, which was detected once before administration and 14 days after intravitreal injection.
  • Fundus fluorescein angiography showed that the retinal vascular perfusion was uniform in the early stage (about 1 minute) and late (about 5 minutes later), the filling time of the arteries and veins was normal, no retinal vein dilatation or leakage was observed, and no retinal non-perfusion area was seen.
  • the performance of masking fluorescence and neovascularization indicates that the antibody of the present invention has high intravitreal injection safety.
  • Figure 8 shows the changes in cell biological activity at 37 °C after dilution of BAT5906 and control ophthalmic drugs, showing that the monoclonal antibody BAT5906 maintains stable cell biological activity under simulated human vitreous injection concentration and can be used in fundus lesions. Inhibits the function of VEGF.
  • Figure 9 shows the change of SEC main peak at 37 °C after dilution of BAT5906 and control ophthalmic drugs, showing that the monoclonal antibody BAT5906 maintains a stable structure under the conditions of simulated human vitreous injection, and can exert the function of inhibiting VEGF in the fundus lesion.
  • compositions and methods include the substance, and does not exclude other substances.
  • a composite includes the important components described but excludes other important components.
  • a composite consisting of the defined important components does not exclude those components which do not significantly affect the basic characteristics and novelty of the present invention.
  • Consisting of means excluding more than trace amounts of ingredients and important method steps. The embodiments defined by these terms are intended to be within the scope of the invention.
  • antibody (Ab) or “antigen binding unit (Abu)” refers to a protein or molecule comprising one or more antigen binding sites.
  • the term encompasses full length antibodies and antibody fragments, but is not limited thereto.
  • the antibody comprises a heavy chain variable region (VH) and/or an antibody light chain variable region (VL), or a pair of VH/VL, and can be a full length antibody or antibody fragment, such as a single chain Fv, VH domain and / or VL domain, Fab, or (Fab) 2 .
  • each antigen binding site comprises an antibody heavy chain variable region (VH) and/or an antibody light chain variable domain (VL), or a pair of antibody light chain variable regions (VL) And the heavy chain variable region (VH) polypeptide of the antibody.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable domain
  • VH heavy chain variable region
  • recombinant human antibody is meant to include all human antibodies prepared, expressed, created or isolated by recombinant methods, eg, antibodies isolated from NSO or CHO host cells, or by expression of human immunity.
  • hypervariable region or "antigen-binding portion of an antibody”, as used herein, refers to the amino acid residues of an antibody responsible for antigen binding.
  • Hypervariable regions include amino acid residues from “complementarity determining regions” or "CDRs".
  • a "framework" or "FR” region is a region of those variable domains other than the hypervariable region residues as defined herein.
  • the light and heavy chains of an antibody include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N- to C-terminus.
  • Such framework amino acids separate the CDRs of each strand.
  • the CDRs of each chain are separated by such framework amino acids.
  • the heavy chain CDR3 is the region that is primarily responsible for antigen binding.
  • the determination of the CDR and FR regions is defined according to Kabat et al. (Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, 15 National Institutes of Health, Bethesda, Md. (1991)).
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all of these names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be exactly the same, due to intentional or unintentional mutations. Variant progeny that have the same function or biological activity screened in the originally transformed cells are included. When referring to different names, it will be clear through the context.
  • transfection refers to the process of transferring a vector/nucleic acid into a host cell. If cells with no insurmountable cell wall barrier are used as host cells, transfection is performed, for example, by calcium phosphate precipitation as described by Graham, FL, van der Eb, AJ, Virology 52 (1973) 546-467. Come on. However, other methods of introducing DNA into cells can also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing a parenchymal cell wall structure are used, for example, one transfection method is calcium treatment using calcium chloride, such as Cohen, SN, et al., PNAS (Proc. Natl. Acad. Sci.) 69 (1972) 2110-2114 Said.
  • expression refers to the process of transcription of a nucleic acid into mRNA and/or the subsequent translation of transcribed mRNA (also referred to as a transcript) into a peptide, polypeptide or protein.
  • a transcript also referred to as a transcript
  • the transcript and the encoded polypeptide are collectively referred to as a gene product. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells can include splicing of the mRNA.
  • a "vector” is a nucleic acid molecule, particularly self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the term encompasses vectors that primarily function to insert DNA or RNA into a cell (eg, chromosomally integrated), a replication vector that functions primarily to replicate DNA or RNA, and an expression vector that functions to transcribe and/or translate DNA or RNA. Also included are vectors that provide more than one of the above functions.
  • an “expression vector” is a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • “Expression system” generally refers to a suitable host cell comprising an expression vector that can function to produce the desired expression product.
  • Treatment of a patient's disease refers to (1) preventing the disease from occurring in a patient who has a predisposition or has not yet manifested symptoms of the disease; (2) inhibiting or preventing the progression of the disease; or (3) reducing the disease or causing it to degenerate.
  • Effective amount means the amount of active compound or agent that results in a biological or medicinal response of a tissue, system, animal, individual, and human being sought by a researcher, veterinarian, medical doctor, or other clinician, including treatment of a disease .
  • the invention provides a novel full length antibody having high affinity and a therapeutic effect against macular degeneration.
  • “Full length antibody” or “intact antibody” refers to an antibody or antibody portion comprising at least two light chains and two heavy chains, and each heavy chain comprising at least one variable region (VH) and three constant regions (eg, CH1, CH2 and CH3).
  • VH variable region
  • CH1, CH2 and CH3 constant regions
  • each light chain, variable region, constant region of IgGl, VH and VL regions of the scFv are human sequences and can be arbitrarily modified.
  • each heavy chain of the full length antibody comprises an amino acid sequence having at least 80%, at least 85%, or at least 90% amino acid sequence identity to SEQ ID NO: 1; preferably, said The heavy chain contains at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97, identical to the amino acid sequence set forth in SEQ ID NO:1. % or at least 98% of the amino acid sequence; more preferably, the heavy chain contains at least 98.1%, at least 98.2%, at least 98.3%, and at least 98.3% identical to the amino acid sequence set forth in SEQ ID NO: 1.
  • the heavy chain contains SEQ ID NO:
  • the amino acid sequence identity is at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%. Or at least 99.9% amino acid sequence.
  • each light chain of the full length antibody comprises an amino acid sequence having at least 80%, at least 85%, or at least 90% amino acid sequence identity to SEQ ID NO: 2; preferably, said The light chain contains at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97, in amino acid sequence identity to SEQ ID NO:2.
  • the light chain contains at least 98.1%, at least 98.2%, at least 98.3%, and at least 98.3% identical to the amino acid sequence set forth in SEQ ID NO: 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8% or at least 98.9%, of the amino acid sequence; still more preferably, the light chain comprises SEQ ID NO: 2
  • the amino acid sequence identity is at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%. Or at least 99.9% amino acid sequence.
  • each heavy chain of the full length antibody comprises the amino acid sequence of SEQ ID NO: 1.
  • each of the light chains of the full length antibody comprises the amino acid sequence of SEQ ID NO:2.
  • the invention provides a method of treating a macular degeneration disease or a related condition, which method requires the use of an effective amount of a full length antibody as described herein.
  • the disease is wet (neovascular) age-related macular degeneration (wet-AMD), pathological myopia (PM) secondary to choroidal neovascularization (CNV), diabetic macular edema (DME), diabetes Fundus lesions such as retinopathy (DR), retinal branch vein occlusion (BRVO), and central retinal vein occlusion (CRVO).
  • the heavy and light chain amino acid sequences of the recombinant anti-VEGF humanized monoclonal antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • BAT5906-Fc and human IgG1 showed non-target-dependent inhibition
  • Ranibizumab and BAT5906-Fab were target-dependent inhibition of VEGF
  • BAT5906 had the highest degree of complete molecular inhibition and had two inhibitory effects.
  • the test results are shown in Figure 1.
  • BAT5906 can inhibit angiogenesis by specifically binding to the Fab domain of VEGF to inhibit the VEGF pathway, or inhibit angiogenesis by binding to FcgRI via the Fc domain. Since human IgG1 contains an Fc domain, it can inhibit angiogenesis through the FcgRI pathway even if it does not specifically bind to the Fab domain of VEGF, and it is shown that the FcgRI pathway is also a means of inhibiting angiogenesis.
  • Ranibizumab Compared with Ranibizumab, which only has a Fab domain that specifically binds to VEGF, Ranibizumab inhibits angiogenesis only by inhibiting the VEGF pathway; while BAT5906 has both a Fab domain and an Fc domain that specifically bind to VEGF, inhibiting VEGF pathway and FcgRI Pathway, angiogenesis inhibition effect is better.
  • the recombinant expression plasmid pBAT5906 was constructed based on the amino acid sequence, which contains a GS cDNA element for synthesizing the glutamine synthetase gene, and serves as an amplification screening marker for the stable cell line, thereby allowing a certain amount of L-methionine to be added to the medium. Sulfone (methionine sulphoximine, MSX) was used for screening of stable cell lines.
  • the constructed recombinant expression plasmid pBAT5906 was verified by restriction endonuclease Pvu I/Not I digestion, which was consistent with the expected design results, and proved that the recombinant expression vector pBAT5906 was successfully constructed.
  • the host cell strain for antibody expression is a derivative cell line of CHO-K1 cells, which is grown in suspension in CD-CHO medium.
  • the stable cell line expressed by the BAT5906 antibody was constructed as follows: The host cells in the logarithmic growth phase were centrifuged and resuspended in fresh CD-CHO medium (cell density: 1.43 ⁇ 10 7 /ml). 600 ⁇ L of the above cell suspension and 40 ⁇ g of the linearized plasmid were uniformly mixed and added to an electric rotor for electroporation.
  • the parameters of the Bio-rad electroconverter were set to: capacitance: 960 ⁇ FD, voltage: 300 V, and shock time 15-20 ms.
  • the electroporated cells were immediately resuspended in 500 mL of pre-warmed CD-CHO medium at 37 ° C, 100 ⁇ L per well was dispensed into 96-well plates, and 100 ⁇ L of screening medium was added after 2-3 days. After 2-3 weeks, the antibody concentration in the 96-well plate cell culture supernatant was measured, and the clone with higher expression level was transferred from the 96-well plate to the 24-well plate, and the cells were grown to a certain amount, and the antibody expression level was again high. The cells were transferred to a 6-well plate, and finally 20-30 high-expressing cell lines were transferred to shake flasks for further evaluation. The yield of the fully obtained monoclonal antibody-expressing cell line finally obtained can reach about 3 g/L.
  • the expression and purification process of the monoclonal antibody was as follows: After the cells were cultured for 2 weeks on a large scale, the cells and the medium were separated by low-speed centrifugation, and the harvested supernatant was further centrifuged at a high speed to obtain a clear liquid.
  • the recombinant antibody was purified by affinity chromatography (Protein A) and ion exchange two-step method.
  • the media used in the purification were Mab Select SuRe LX produced by GE, Giga Cap Q-650M by TOSOH, and POROS of ABI. XS.
  • the isolated and purified antibodies were verified for correctness by SDS-PAGE (Fig. 2 and Fig. 3). The results showed that the BAT5906 bands were correct in size under both reducing and non-reducing conditions.
  • the laser-induced choroidal neovascularization model is an ideal model for wet-AMD.
  • Ranibizumab (Lucentis)
  • laser-induced choroidal neovascularization of rhesus monkeys is performed, followed by evaluation of vitreous drug injection test. Drug effect. After successful modeling, group 4 rhesus monkeys.
  • the 0.25, 0.5, 1.25 mg/eye dose of BAT5906 group had a better improvement rate of laser fluorescein leakage area in the fundus of the monkey eye than the Ranibizumab group (83.69%) of 0.5 mg/eye. And the low dose of BAT5906 has a better fluorescein leakage area improvement rate.
  • the retinal thickness of all the lesions of the eyeball was reduced, and some of the pigment epithelium of the eyeball was regular and continuous, and part of the eyeball retina The thickness is close to or at or even below the pre-model level.
  • the improvement rate and reduction of retinal thickness at the highest lesion 28 days after administration, the mean improvement rate were 106.61%, 115.17%, 113.45%, respectively) were significantly higher than the model control group.
  • the BAT5906 group at 0.25, 0.5, 1.25 mg/eye dose improved the retinal thickening of the monkey eye slightly better than the 0.5 mg/eye Ranibizumab group.
  • the VEGF concentration of rhesus monkey aqueous humor was lower than that of the model control group and the positive control Ranibizumab group, and the results showed that the antibody of the present invention had more VEGF to the ocular tissue than the positive control drug Ranibizumab. Strong inhibition effect.
  • the 0.5 mg/eye Ranibizumab group was administered 28
  • the number of fluorescent spots in grade 3+4 was 6 and was significantly reduced compared with before administration (39), but the number of grade 3+4 fluorescent spots in the BAT5906 group at 0.25 mg/eye was 5, 0.5.
  • the number of grade 3 and grade 4 fluorescent spots in the 1.25 mg/eye BAT5906 group was 2 and 3, respectively, and the number of fluorescent spots was less than that of the positive control group Ranibizumab.
  • the target cells (HUVEC cells) were resuspended with endothelial cell complete medium ECM 24 hours in advance, and the cell concentration was adjusted to 2 ⁇ 10 5 /mL, and 50 ⁇ L of the cell suspension was added to each well in a flat-bottomed 96-well plate.
  • the antibody was diluted with DMEM/F12 medium containing 2% FBS and 5% complement (initial concentration 40 ⁇ g/mL, diluted in 1/5 fold dilutions, 3 replicate wells per concentration, 8 gradients were established) .
  • the medium in the above step was blotted with a pipette, and then the diluted target cells in the previous step were added, 50 ⁇ L per well, according to the experimental design, added to a 96-well plate, and then placed at 37 ° C, 5%.
  • the CO 2 incubator is continued for at least 4 hours.
  • Each concentration gradient of antibody was added to a 96-well plate at 50 ⁇ L/well at a final concentration of 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064, 0 ⁇ g/mL, then at 37 ° C, 5% CO 2 Incubate for 30 min in the incubator.
  • the maximum LDH release of the target cells, the spontaneous LDH release wells of the target cells, the volume-corrected control wells, and the medium background wells maximum LDH release wells and volume-corrected control wells were added to 10 ⁇ L of lysate 45 minutes prior to detection), each with 3 replicate wells. Incubate at 37 ° C in a 5% CO 2 incubator for at least 4 h.
  • the 96-well plate was centrifuged, centrifuged at 250 g for 4 minutes, and the supernatant was carefully pipetted into another 96-well plate, 50 ⁇ L of LDH detection reagent was added to each well, and incubated at room temperature for 20-30 minutes in the dark, and then added per well. 50 ⁇ L of stop solution.
  • the absorption value was measured by a microplate reader at a detection wavelength of 490 nm. Calculation of test results: The absorbance of all experimental wells, target cells spontaneous LDH release wells was subtracted from the median background absorbance values, and the absorbance of the target LDH release control was subtracted from the mean of the volume corrected control absorbance values. The above corrected values are then taken into the formula below to calculate the percent cytotoxicity produced by each target ratio.
  • % cytotoxicity (experimental - target cell spontaneous) / (target cell max - target cell spontaneous) x 100%
  • the target cells (HUVEC cells) were resuspended in 1640 medium 24 hours in advance, and the cell concentration was adjusted to 2 ⁇ 10 5 /mL, and 50 ⁇ L of the cell suspension was added to each well in a flat-bottomed 96-well plate.
  • the antibody was diluted with DMEM/F12 medium containing 2% FBS (initial concentration 40 ⁇ g/mL, diluted sequentially at 1/5 fold dilution, 3 replicate wells per concentration, and 8 concentration gradients were established).
  • Each concentration gradient of antibody was added to the sample well at 50 ⁇ L/well.
  • the final concentration of the sample was 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064, 0 ⁇ g/mL, and then cultured at 37 ° C, 5% CO 2 . Incubate in the box for 30 min.
  • the effector cells PBMC in the logarithmic growth phase were centrifuged at 800 rpm for 5 minutes to discard the supernatant.
  • DMEM/F12 medium containing 2% FBS was added, and the supernatant was discarded by centrifugation at 800 rpm for 5 minutes, and counted to adjust the effector cell density to 2 ⁇ 10 5 /mL.
  • the diluted effector cells in the previous step were added to the sample wells with a pipette, 50 ⁇ L per well, and then placed in a 37 ° C, 5% CO 2 incubator for at least 4 hours.
  • target cell maximum LDH release target cell spontaneous LDH release well, volume-corrected control well and medium background well (maximum LDH release well and volume-corrected control well were added to the lysate of 10 ⁇ L test kit 45 minutes before detection), each 3 duplicate holes. Incubate at 37 ° C in a 5% CO 2 incubator for at least 4 h.
  • the 96-well plate was centrifuged, centrifuged at 250 g for 4 minutes, and the supernatant was carefully pipetted into another 96-well plate, 50 ⁇ L of LDH detection reagent was added to each well, and incubated at room temperature for 20-30 minutes in the dark, then each well. 50 ⁇ L of stop solution was added. The absorption value was measured by a microplate reader at a detection wavelength of 490 nm.
  • the above two test results of CDC and ADCC indicate that the antibody of the present invention does not cause complement dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) in vivo, and its use in vivo does not affect the normal function of the immune system.
  • the antibody of the invention can exert its anti-VEGF function normally at the lesion without causing adverse toxicity reaction, and has high safety in human body, and is a low-toxic anti-VEGF monoclonal antibody.
  • the experiment consisted of 2 groups, which were negative control group, recombinant anti-VEGF humanized monoclonal antibody (BAT5906) injection 8.0 mg/eye group, and each group of 3 rhesus monkeys, both male and female. Rhesus monkeys of each group were given a single control (100% sodium chloride injection) and 80 mg/mL recombinant anti-VEGF humanized monoclonal antibody injection in a single double-eye vitreous injection at 100 ⁇ L/eye volume. The day of dosing was defined as the first day of the test.
  • each group was observed daily for 14 consecutive days after administration; body weight was measured on the 8th and 14th day of the test; indirect ophthalmoscopy and slit lamp examination were performed on the first (pre-dose), 2, 4, 8, and 14 days of the test; Intraocular pressure was measured before and 10 to 15 minutes, 1 hour, 24 hours, and 14 days after the administration; fundus color photography and fluoroscopy were performed before administration and on the 14th day of the test; Hematology and blood biochemical tests; on the 15th day of the experiment, euthanasia was performed on all rhesus monkeys after anesthesia, and gross anatomy was observed.
  • test results showed that under the conditions of this experiment, rhesus monkeys were injected with 8.0 mg/eye dose of recombinant anti-VEGF humanized monoclonal antibody injection in a single injection and observed for 14 days. No ocular toxicity and systemic toxicity were observed. The tolerated dose (MTD) was 8.0 mg/eye. Fundus fluorescein examination in the acute toxicity test is shown in Figure 7.
  • Example 7 Eye tissue distribution test of intravitreal injection of monoclonal antibody in rhesus monkey eyes
  • vitreous injection of 8.33 nmol/eye ie 1.25 mg/eye
  • 21 rhesus monkeys, 10 females and 11 males were used.
  • the vitreous injection group was administered with 25 mg/mL recombinant anti-VEGF humanized monoclonal antibody (BAT5906) injection in a single injection of bilateral vitreous in a volume of 50 ⁇ L/eye.
  • BAT5906 recombinant anti-VEGF humanized monoclonal antibody
  • 3 monkeys were taken at 4, 10, 24, 72, 168, 336, and 672 hours after administration, and euthanized (anesthesia with pentobarbital sodium 30 mg/kg, femoral artery bleeding) were dissected and removed.
  • Eyes and optic nerve separation of aqueous humor, cornea, iris, vitreous, lens, retina, choroid, optic nerve.
  • Six monkeys dissected at 336 and 672 hours after administration of the vitreous injection group were administered before and 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours after administration.
  • Serum was collected by blood collection at 168 hours, 336 hours, and 672 hours.
  • the concentration of drug in each eye tissue and serum was measured by ELISA, and the drug metabolism parameters in serum and ocular tissues were calculated.
  • the drug can rapidly spread in various ocular tissues from 4 hours after injection. Drugs were detected in all tissues in the eye, and all ocular tissue drug concentrations except the cornea and optic nerve peaked 4 to 24 hours after administration.
  • the exposure levels of the drugs in the tissues of the eye (AUC last ) from high to low are: vitreous, retina, cornea, aqueous humor, choroid, iris, lens, optic nerve. In the vitreous, aqueous humor, retina, choroid, iris, lens and other major ocular tissues, the elimination half-life is between 73.74 and 143.81 hours, and the half-life in the vitreous is 84.2 hours.
  • the elimination half-life (t 1/2 ) of the main pharmacological parameter of the drug in the monkey vitreous was 84.2 hours.
  • the half-life of the market-positive control drug Ranibizumab after vitreous injection in monkey vitreous was 55.7 hours, and the half-life of Aflibercept vitreous in monkey vitreous was 40-60 hours.
  • the monoclonal antibody of the present invention has a longer drug half-life in the vitreous, can exert a longer-term effect in the eye, and has a better effect of inhibiting fundus angiogenesis.
  • the thermal transition temperature of the antibodies of the invention we used a capillary differential calorimeter to detect the thermal unfolding temperature of each domain of the BAT5906 molecule.
  • the instrument model Nano DSC, the capillary sample cell and the reference cell have a volume of 0.300 mL, a heating rate of 1 ° C / min, a 10 min pre-equilibration time, and a filtration cycle of 10 seconds. From the experimental results in Table 4, it can be seen that the BAT5906 molecule has two melting peaks, the first peak is the CH2 melting peak, the Tm value is 76.07 ° C; the second peak is the Fab/CH3 melting peak, and the Tm value is 83.68 °C.
  • the above data indicates that the overall structure of BAT5906 molecule is relatively stable, and the lowest Tm value in each domain is also 76.07 °C.
  • the thermal unfolding temperature of BAT5906 is higher than that of Bevacizumab, which is more stable than Bevacizumab.
  • VEGF has an activity of promoting proliferation of HUVEC cells, and the monoclonal antibody of the present invention has a function of inhibiting VEGF, so this test detects the comparison of cell biological activities using HUVEC.
  • HUVEC cells (HUVEC cells were passaged to the 5th generation) were resuspended in ECM medium containing 2% FBS to a concentration of 9 ⁇ 10 4 cells/mL, and 96-well cell culture plates were inoculated at 100 ⁇ L per well, and incubated (5 ⁇ 1) h makes the cells adhere.
  • BAT5906 monoclonal antibody and Bevacizumab were pre-diluted to 1000 ng/mL using test medium, then further diluted 1:1.5 down to 9 gradients (10 gradients in total), serial dilutions and 80 ng diluted in equal volume of test medium /mL VEGF-A 165 was mixed and incubated at 37 ° C for (60 ⁇ 10) min.
  • Example 10 5-week stability test at 37 ° C after dilution of the antibody
  • the cell biological activity test method is shown in Example 9.
  • the SEC detection method was as follows: the column was TSKgel G3000SWXL (column size 7.8 ⁇ 300 mm, 5 ⁇ m), 100 mM potassium phosphate containing 10% acetonitrile, 125 mM potassium chloride buffer as mobile phase, column temperature 30 ° C, flow rate 0.5 mL / Min; detection wavelength is 280 nm. Take the reference substance, dilute it with water to 2mg/mL of the sample solution, take 50 ⁇ L into the liquid chromatograph, record the chromatogram, and measure it in parallel three times.
  • the number of theoretical peaks of the main peak of the three results should not be less than 2000, the tailing factor Must not be greater than 2.0, the separation between the main peak and the polymer should not be less than 2.0.
  • the test sample was measured by the same method and calculated by the area normalization method. The average value of the three test results was used as the final test result. The test results are shown in Figures 8 and 9.
  • the test results showed that the antibody of the present invention was placed at 37 ° C in a simulated human intravitreal injection concentration, the biological activity of the cells was relatively stable, and the percentage change of the main peak of SEC was small, indicating that the antibody of the present invention has better stability under the conditions of the test. It is an anti-VEGF monoclonal antibody that exerts stable biological function at the bottom of the eye after intravitreal injection.

Abstract

本发明涉及一种可特异性结合VEGF的全长人源化单克隆抗体。所述的抗体可抑制VEGF与VEGFR-1和VEGFR-2的结合，从而抑制VEGF的信号传导。所述的抗体对VEGF具有很强的亲和力且半衰期较长，玻璃体注射后安全性较高。本发明的抗体可以有效治疗与VEGF过表达相关的疾病，尤其是VEGF过表达导致的血管生成异常相关的疾病。

Description

一种长效低毒的重组抗VEGF人源化单克隆抗体及其生产方法 技术领域
本发明属于生物医药领域；更具体地，本发明涉及一种用于减少人血管内皮生长因子(vascular endothelial growth factor，VEGF/VEGF-A)的抗体，还涉及所述抗体的制备方法及其应用。
背景技术
血管生成是由血管内皮细胞从先前存在的血管网络增殖并重组成新生血管。血管生成对人体正常增殖过程是必不可少的，包括伤口愈合和器官发育及分化。同时血管生成也涉及多种病理疾病的形成，如年龄相关性黄斑变性、肿瘤、类风湿性关节炎和牛皮癣。鉴于重要的生理病理重要性，有人认为血管生成过程由促血管生成分子和抗血管生成分子之间的平衡来进行调节，并在病症中出现异常调节。血管生成是级联过程，包括下述过程：释放蛋白酶后局部位置的细胞外基质的降解；毛细内皮细胞的增殖；毛细小管向血管生成刺激物的迁移。
新生血管生成的过程是多因素和高度复杂的，但是VEGF被认为是在生理和病理血管生成中最为关键的因子。VEGF对胚胎的脉管生成和血管生成是必不可少的。除了是血管生成和脉管生成中的血管生成因子，VEGF还是多向性的生长因子，在内皮细胞存活、血管渗透性和血管舒张、单核细胞趋化性和钙流入中表现出多种生物效应，例如有报道称VEGF对视网膜色素内皮细胞和神经膜细胞有促进细胞分裂效应。血管生成是由血管内皮细胞增殖、重组形成新的血管，有证据表明血管供给的发育对正常和病理性增殖是必不可少的。大量数据显示VEGF在涉及病理性血管生成的疾病发展中起到关键作用，VEGF mRNA在大部分人类肿瘤中被过表达，VEGF在房水中的浓度与在患有糖尿病或其它缺血性视网膜疾病的患者中发现的血管活性增生高度相关，尤其是在年龄相关性黄斑变性(AMD)患者中脉络丛新血管形成膜中更甚。
VEGF是一种糖基化分泌性多肽生长因子，是一种同源二聚体糖蛋白，分子量在46～48kDa间，它直接作用于血管内皮细胞，能够诱导血管内皮细胞增生和血管新生。VEGF家族成员有6型，包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E和胎盘生长因子，其中VEGF-A在眼睛中最重要。VEGF-A基因定位于6号染色体的p12～p21，由8个外显子 和7个内含子构成。由于mRNA不同的剪切方式，产生出VEGF-A 121、VEGF-A 145、VEGF-A 148、VEGF-A 165、VEGF-A 183、VEGF-A 189和VEGF-A 206等7种蛋白形式。其中有5种蛋白形式可以增加血管通透性、促进血管内皮细胞增殖、诱导新生血管生成。血管内皮生长因子受体(VEGFR)有VEGFR1(Flt-1)、VEGFR2(KDR)和VEGFR3(Flt-4)三种，VEGF通过与其受体结合，从而发挥其生物学效应。无论是在实验模型还是在湿性AMD的病理性血管生成中，都发现存在高水平的VEGF表达。同时也有相当数量的文献报道，通过中和VEGF，血管新生和血管渗漏可以得到抑制。
认识到VEGF在病理状况中的血管生成发挥重要作用后，人们采取很多措施来阻断VEGF活性。包括用抑制性抗VEGF受体的抗体、可溶性受体构建体、反义策略和低分子量VEGF受体酪氨酸激酶抑制物来干扰VEGF通路等。据相关文献报道，抗VEGF的中和性抗体已经显示出抑制多种人类肿瘤细胞系在裸鼠中的生长，也抑制了缺血性视网膜病症模型中的眼内血管生成。所以抗VEGF单克隆抗体或VEGF作用抑制物是治疗实体瘤和多种眼内新生血管病症的有效候选物。
黄斑变性是一种主要在老年人中发现的医学病症，其中已知为视网膜黄斑区域的眼睛的内衬中心变薄、萎缩，并且在一些情形中出血。这可能导致中央视觉的丧失，这使得患者不能看到更详细的内容。根据美国眼科学会的报道，黄斑变性是老年人的中央视觉丧失(失明)的主要原因。尽管一些影响年轻人的黄斑营养不良(macular dystrophies)有时也称为黄斑变性，但是该术语通常是指年龄相关性黄斑变性(age-related macular degeneration,AMD)。
年龄相关性黄斑变性从位于视网膜色素上皮与其下的脉络膜之间的黄斑(提供详细的中央视觉的视网膜中央区域，称为小窝)中的特征性的黄色沉积(称为玻璃疣)开始。具有玻璃疣的人们会继续发展晚期AMD。当玻璃疣变大并且变多，并且与在斑下的色素细胞层中的紊乱相关时，危险相当高。
导致深入的视觉丧失的晚期AMD具有两种形式：干性和湿性。中央萎缩，即干性形式的晚期AMD，由在视网膜下的视网膜色素上皮层的萎缩导致，其通过丧失在眼睛中央部分的光感受器(杆状和锥体)而引起视觉丧失。
年龄相关黄斑病变是视网膜色素上皮细胞和神经视网膜退化造成的一种不可逆性视力下降或丧失的疾病。多发生于50岁以上的患者，双眼先后或同时发病，且进行性损害视力，是一种严重威胁老年人视功能的眼底病变。随着人口老龄化，在西方国家已成为第一位的致盲性眼病，在亚洲其发病率亦呈逐渐增多的趋势。
现代生物技术的发展使我们可通过重组DNA技术生产抗VEGF单克隆抗体，这种技术已经被大量应用于多种单克隆抗体的生产。可选择用来自噬齿类动物抗体的残基替换人抗体中CDR区或框架区的一些位点，以组合成人源化单克隆抗体，从而降低抗原性。目前已经成功生产出多种人源化抗VEGF单克隆抗体，在体内和体外显示了明显的hVEGF亲和力和抑制活性。例如治疗实体瘤的临床试验中使用一种特异性的人源化抗VEGF抗体Bevacizumab，还有其他人源化抗VEGF抗体雷珠单抗(Ranibizumab)和阿柏西普(Aflibercept)以高亲和力治疗脉络丛新血管生成相关的年龄相关性黄斑变性。
在使用治疗性抗体于人体上前，需在非人哺乳动物中进行临床前研究，以评价所用单克隆抗体的有效性和毒性。理想情况是，进行了这些临床前研究的抗体能够识别并以高效性与宿主动物(小鼠、兔子和非人类灵长类动物)的靶标抗原进行反应。
贝伐珠单抗(Bevacizumab，Avastin)是由基因泰克公司(Genentech)研发的另一个人源化IgG1抗VEGF单克隆抗体，是采用重组DNA技术在哺乳动物细胞表达系统中国仓鼠卵巢细胞(CHO)中进行生产，然后采用包括病毒灭活和去除步骤在内的工艺进行纯化的人用抗肿瘤单克隆抗体。
雷珠单抗(Ranibizumab，Lucentis)是由美国基因泰克公司(Genentech)研发的一种通过重组DNA技术在大肠杆菌中进行生产的人源化单克隆抗体Fab片段，靶向VEGF-A，它能高亲和力结合VEGF-A各种亚型，如VEGF-A 121，VEGF-A 165，VEGF-A 110。雷珠单抗与VEGF-A结合从而阻止了VEGF-A与其位于内皮细胞表面的受体(VEGFR-1，VEGFR-2)结合，阻止血管内皮增生，减少血管渗漏，抑制视网膜新血管(CNV)的生成。
雷珠单抗和贝伐珠单抗都是来源于相同的鼠源母本抗体A4.6.1。A4.6.1是由人VEGF-A 165蛋白免疫小鼠产生的杂交瘤细胞系生产的小鼠抗VEGF的单克隆抗体。为了降低鼠源抗体治疗人体疾病时产生的免疫原性，A4.6.1抗体基因通过重组DNA技术进行了人源化改造。人源化的Fab片段保留了鼠源性CDR序列，再与人源Fc片段组合得到全长抗体，即贝伐珠单抗。贝伐珠单抗具有与其亲本抗体相同的抗原亲和力，但是免疫原性更低，体内半衰期更长。
雷珠单抗是在贝伐珠单抗Fab区的基础上通过氨基酸定点突变筛选得到的高亲和力抗体。雷珠单抗和贝伐珠单抗Fab区一共有6个氨基酸不同，这6氨基酸的突变使得雷珠单抗对VEGF-A的亲和力相比贝伐珠单抗提高了5～20倍，体外实验证实雷珠单抗抑制HUVEC增殖的强度，是贝伐珠单抗的5.2倍。雷珠单抗是Fab片段，分子量小，可在1h内完全渗透视网膜全层，玻璃体内药物的半衰期为3.2天，而贝伐珠单抗不能穿透猴视网膜的内界膜。 此外，由于雷珠单抗是较小的抗原结合片段，不含Fc区，不会触发补体介导的免疫反应，全身半衰期短，因而全身不良反应小。
对于眼部疾病，通常使用通过玻璃体内应用的较小的抗体片段，如Fab或(Fab) 2，原因在于其具有低血清半衰期，并且系统性毒性的危险较低。然而，这种较小的抗体片段会导致较短的玻璃体内半衰期(由于较快地扩散到血清中)，并且必须更频繁地给药，给患者造成治疗中精神和身体上的负担。故具有较长半衰期的含Fc结构域的抗体也被应用于眼部疾病的治疗中，例如拜耳公司生产的Aflibercept就是一种Fc融合蛋白。
发明内容
本发明的目的在于提供一种亲和力强的抗-VEGF抗体。
本发明的目的还在于提供一种体内半衰期长的抗-VEGF抗体。
本发明的目的还在于提供一种免疫原性更低的抗-VEGF抗体。
本发明抗体是专门设计的用于眼底病变治疗的人源化全长抗VEGF的IgG1抗体。本发明抗体具有与雷珠单抗相近的抗原亲和力，但是本发明抗体无CDC和ADCC效应，玻璃体内注射安全性更高，玻璃体内注射后在玻璃体中半衰期更长，预期治疗效果更佳。
一方面，本发明提供了一种全长人源化抗VEGF抗体，所述的抗体具有(a)两条免疫球蛋白轻链；以及(b)两条免疫球蛋白重链，所述重链从N末端到C末端都包括可变区和恒定区；所述抗体特异性地识别VEGF的抗原结合位点。
更进一步地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为80％，至少为85％或至少为90％的氨基酸序列；优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为91％，至少为92％，至少为93％，至少为94％，至少为95％，至少为96％，至少为97％或至少为98％的氨基酸序列；更优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为98.1％，至少为98.2％，至少为98.3％，至少为98.4％，至少为98.5％，至少为98.6％，至少为98.7％，至少为98.8％或至少为98.9％，的氨基酸序列；还更优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为99％，至少为99.1％，至少为99.2％，至少为99.3％，至少为99.4％，至少为99.5％，至少为99.6％，至少为99.7％，至少为99.8％或至少为99.9％的氨基酸序列。
所述的轻链含有与SEQ ID NO:2所述的氨基酸序列同一性至少为80％，至少为85％或至少为90％的氨基酸序列；优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为91％，至少为92％，至少为93％，至少为94％，至少为95％，至少为96％，至少为97％或至少为98％的氨基酸序列；更优选地，所述的轻链含有与SEQ ID NO:2所 示的氨基酸序列同一性至少为98.1％，至少为98.2％，至少为98.3％，至少为98.4％，至少为98.5％，至少为98.6％，至少为98.7％，至少为98.8％或至少为98.9％，的氨基酸序列；还更优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为99％，至少为99.1％，至少为99.2％，至少为99.3％，至少为99.4％，至少为99.5％，至少为99.6％，至少为99.7％，至少为99.8％或至少为99.9％的氨基酸序列。
在一些实施方案中，所述的重链含有SEQ ID NO:1所示的氨基酸序列，所述的轻链含有SEQ ID NO:2所示的氨基酸序列。
在一些实施方案中，本发明的抗体为单克隆抗体，进一步地，所述的抗体是人IgG1。
在一些实施方案中，本发明所提供的抗体，其能以与贝伐珠单抗相近的KD值结合人VEGF并能够抑制VEGF与VEGF受体结合。更具体地，能够在30℃以10nM或更低的KD值结合人VEGF并能够抑制VEGF与VEGF受体结合。根据另一个实施方案，KD值是1nM或更低。根据另一个实施方案，本发明抗体以不超过大约500pM的KD值结合VEGF。
根据一个实施方案，本发明抗体具有1.0或更高(10M -1S -1)的结合速率(on rate)(k on)结合人和/或小鼠VEGF。根据另一个实施方案，该结合速率是4.0或更高(10M -1S -1)。
根据另一个实施方案，本发明抗体以理想的亲和力结合人VEGF，进一步结合VEGF-A，更进一步地，与VEGF-A165结合，但不结合人VEGF-B、人VEGF-C和人VEGF-D组成的任一或所有的VEGF相关的同系物。
与VEGF的结合受抑制的VEGF受体可以是VEGF受体1(Flt-1)，VEGF受体2(Flt-1)或者两者。
根据一个实施方案，本发明单克隆抗体接触20s螺旋(helix)的VEGF。根据另一个实施方案，本发明单克隆抗体接触80s环(loop)的VEGF。根据另一个实施方案，本发明单克隆抗体接触20s螺旋(helix)的和80s环(loop)的人VEGF。
本发明的另一方面提供了编码本发明所述的抗体的重链和/或轻链的核酸。
本发明还提供包含本发明所述的核酸的表达载体，所述表达载体能够在原核或真核宿主细胞中表达所述核酸。
本发明还提供包含所述载体的宿主细胞，所述宿主细胞用于生产本发明所述抗体。
所述的宿主细胞为真核的或者所述的宿主细胞为原核的。
本发明还包括用于生产抗体的方法，其特征在于在原核或真核宿主细胞中表达本发明所述的核酸，并且从所述宿主细胞或宿主细胞培养物中回收所述抗体。
其中一个实施方案包括下述步骤：
a)用包含编码所述抗体的核酸分子的载体转化宿主细胞；
b)在允许合成所述抗体分子的条件下培养所述宿主细胞；和
c)从所述培养物回收所述抗体分子。
本发明还包括通过所述用于生产全长抗体的方法获得的抗体。
本发明还提供一种亲和纯化试剂。所述的亲和纯化试剂含有上述的抗体，上述的抗体可以用于制备可用于VEGF蛋白的诊断性分析的试剂中的用途。所述的抗体可用检测分子进行标记。如放射性同位素、荧光标志、或酶等进行标记。
本发明还提供了一种用于VEGF蛋白诊断的试剂盒，所述的试剂盒含有上述的抗体或多肽。
本发明还提供了一种含有所述抗体的药物制剂。所述药物制剂还可包含药学上可接受的载体。
本发明也提供了包含所述抗体或多肽用于制备治疗哺乳动物中VEGF过表达相关的疾病的药物的用途。
本发明所述的治疗哺乳动物，其包含给以有效量的所述抗体到所述哺乳动物。抗体的给药量将是治疗所述疾病的有效量。在增加剂量的研究中，可将多种剂量的抗体给药所述哺乳动物。另一个实施方案中，治疗有效量的所述抗体给药人类患者，以治疗疾病。
所述的哺乳动物可以是人，也可以是非人的哺乳动物，例如适合产生临床前数据的灵长类动物或噬齿类动物(例如小鼠、大鼠或兔子)。优选地，所述的哺乳动物为人。
所述哺乳动物可以是健康的，也可以是患有要用所述抗体治疗的疾病。
在发明中，所述的VEGF过表达相关的疾病或者是所述的疾病为VEGF过表达导致的血管生成异常相关的疾病；更进一步为VEGF过表达导致的眼底病变。
作为优选的实施方式，本发明所述的抗体用于治疗湿性(新生血管性)年龄相关性黄斑变性(wet-AMD)、病理性近视(PM)继发脉络膜新生血管(CNV)、糖尿病性黄斑水肿(DME)、糖尿病性视网膜病变(DR)、视网膜分枝静脉阻塞(BRVO)、视网膜中央静脉阻塞(CRVO)等眼底病变。
这些抗体可用于采用激光导致脉络膜新生血管的恒河猴模型中，这些抗体可用于通过观察或监测在用本发明抗VEGF抗体治疗黄斑变性的面积变化情况，来鉴定抗VEGF抗体在治疗年龄相关性黄斑变性的方法。本发明所述抗体也可用于研究并评价本发明抗VEGF抗体及其他治疗剂的联合疗法。本发明所述抗体可通过将所述抗体或多肽给药患有类似疾病的动物并确定是否所述疾病的一种或多种症状会减轻，用于研究VEGF在其他疾病中的作 用。
在一些方面，本发明还提供了抗体衍生物，可以将本发明的抗体进一步修饰以含有本领域公知的其他非蛋白质部分，如水溶性的聚合物。
在一些方面，本发明还提供了免疫偶联物(抗体-药物偶联物或ADC)，其包含本发明所述抗体与一种或多种细胞毒性试剂偶联的抗体，该抗体毒性试剂例如化疗剂、药物、生产抑制剂、毒素或者放射性同位素。
本发明所述的抗体由于其Fc部分的存在，对患有视网膜黄斑变性疾病的患者更有益处。与不具有恒定重链区的较小的抗体片段相比，其在玻璃体内环境中具有高度的稳定性和从玻璃体中的缓慢扩散特性，即半衰期延长，药物作用时间得到延长，其中实际的疾病位于此处并在此处治疗。因此，与非IgG样抗体，例如，Fab和(Fab) 2片段相比，治疗周期可以延长，病患生理和心理状况可得到改善。
本发明所述抗体为全长人源化抗体。全长人源化抗体与鼠源抗体、嵌合抗体相比，在安全性和疗效上都有多个潜在优势。与其他的抗体种类相比，全长人源化抗体通常表现出较低的清除率。较低的清除率可以允许较低的剂量和给药频率。
根据一个优选的实施方案，本发明抗体是通过重组方法来合成而不是直接从杂交瘤产生的或从来自杂交瘤的抗体序列衍生。一个优选的实施方案中，所述抗体以不超过大约2nM，不超过大约1nM，不超过大约500pM的KD值来结合hVEGF-A 165。本发明所述的全长抗体表现出抑制眼底疾病尤其是脉络膜血管新生的良好药效。
附图说明
图1为BAT5906不同结构与Ranibizumab新生血管抑制效果比较试验，显示了所述单克隆抗体BAT5906完整分子抑制血管新生效果最佳，优于上市药物Ranibizumab。
图2显示了所述单克隆抗体BAT5906的SDS-PAGE电泳(非还原)结果，M：250kDa预染蛋白Marker，泳道1：BAT5906非还原样品。
图3显示了所述单克隆抗体BAT5906的SDS-PAGE电泳(还原)结果。M：250kDa预染蛋白Marker，泳道1：BAT5906还原样品，由图2和图3的结果显示所述单克隆抗体分子大小在还原与非还原条件下均与设计大小一致。
图4为给药后各组猴眼底激光斑荧光素渗漏面积改善率(％)，显示了所述单克隆抗体BAT5906在恒河猴药效试验中发挥作用，有效地抑制了荧光素渗漏的面积，上市药物Ranibizumab作为对照，与模型对照组比较，均数的差异有统计学意义(P≤0.05)。
图5为给药前后各组猴眼球4级荧光斑数(个)，显示了所述单克隆抗体BAT5906在恒 河猴药效试验中发挥作用，有效抑制了4级荧光斑数量，上市药物Ranibizumab作为阳性对照。
图6为给药后各组猴视网膜病损最高处视网膜厚度改善率(％)，显示了所述单克隆抗体BAT5906在猴药效试验中发挥作用，有效抑制了眼底视网膜的增厚，上市药物Ranibizumab作为阳性对照，与模型对照组比较，均数的差异有统计学意义(P≤0.05)。
图7为恒河猴急性毒性试验中猴眼球荧光造影图，给药前及眼玻璃体内注射后14天各检测一次。眼底荧光造影检查可见造影后早期(约1分钟以内)及晚期(约5分钟以后)视网膜血管灌注均匀，动、静脉充盈时间正常，未见视网膜静脉扩张、渗漏，亦未见视网膜无灌注区、遮蔽荧光以及新生血管形成等表现，说明本发明抗体玻璃体内注射安全性较高。
图8为BAT5906与对照眼科药物稀释后37℃条件下细胞生物学活性变化，显示了所述单克隆抗体BAT5906在模拟人玻璃体注射浓度条件下仍保持稳定细胞生物学活性，可在眼底病变部位发挥抑制VEGF的功能。
图9为BAT5906与对照眼科药物稀释后37℃条件下SEC主峰变化，显示了所述单克隆抗体BAT5906在模拟人玻璃体注射浓度条件下仍保持稳定结构，可在眼底病变部位发挥抑制VEGF的功能。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
I.定义
如果没有特别指明，本文所涉及的描述和要求依照以下所述来定义。
需要说明的是，除非特别指明，本文所述单数形式包含复数含义。如，当述及一种“复合物”时，同时包含复合物的复数形式。
本文所涉的“大约”应为本领域普通技术人员所理解并且能够简单扩展其应用。如果有术语的应用对于本领域的普通技术人员是不清楚的，则需给出应用的上下文。“大约”指特定值的+10％至-10％、+5％至-5％或+1％至-1％。
本文所述术语，“包括”是指组成和方法包括所述物质，而不排除其他物质。当“主要包括”用于定义组成和方法时，应当意为不包括其他任何重要的组分。例如，一个复合物 包括所述的重要组分但排除其他的重要组分。例如，一个复合物由所述定义的重要组分组成，则不排除那些对本发明的基本特性及新颖性无重大影响的组分。“由……组成”意为排除超过微量的成分和重要方法步骤。这些术语所定义的实施方式限于本发明的范围中。
如本文所用，“抗体(Ab)”或“抗原结合单元(Abu)”是指，其包含一个或多个抗原结合位点的蛋白或分子。该术语包含了全长的抗体和抗体片段，但不限于此。在某一方面，抗体包含重链可变区(VH)和/或抗体轻链可变区(VL)，或一对VH/VL，以及可以是全长抗体或抗体片段，如单链Fv、VH结构域和/或VL结构域、Fab、或(Fab) 2。在某一方面，每一个抗原结合位点包括抗体重链可变区(VH)和/或抗体轻链可变结构域(VL)，也可以是一对由抗体轻链可变区(VL)和抗体的重链可变区(VH)多肽组成。
如本文所用，术语“重组人源抗体”是指包括所有利用重组方法制备、表达、创造或分离的人源抗体，例如，从NSO或CHO宿主细胞中分离出的抗体，或是利用表达人免疫球蛋白基因的转基因动物(例如小鼠)中获得的抗体，或使用重组表达载体，转染到宿主细胞中表达的抗体。
术语“高变区”或“抗体的抗原结合部分”，在本文中使用时，请参阅负责抗原结合的抗体的氨基酸残基。高变区包括从“互补性决定区”或“CDR”氨基酸残基。“框架”或“FR”区域是本文所定义的高变区残基以外的那些可变结构域的区域。因此，抗体的轻链和重链包括从N-到C-末端的结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。这样的框架氨基酸分隔的每个链的CDR。每个链的CDR由这样的框架氨基酸分隔开。特别是，重链CDR3是主要负责抗原结合的区域。CDR和FR区域的确定是根据Kabat等的标准定义(Kabat等，Sequences of Proteins of Immunological Interest，5th ed.，Public Health Service，15 National Institutes of Health，Bethesda，Md.(1991))。
用于本文中时，表述“细胞”、“细胞系”和“细胞培养物”可互换使用，且全部这些名称都包括子代。因此，词语“转化体”和“转化的细胞”包括原代受试细胞和由其来源的培养物，而不考虑转移的次数。还理解所有的子代的DNA含量可能不精确一致，这归因于有意或无意的突变。包括在最初转化的细胞中筛选的具有相同功能或生物学活性的变异子代。意指不同名称时，通过上下文其将是清楚的。
术语“转化”用于本文中时，指将载体/核酸转移到宿主细胞中的过程。如果将无难以克服的细胞壁屏障的细胞用作宿主细胞，则转染例如通过如Graham，F.L.，van der Eb，A.J.，Virology(病毒学)52(1973)546-467所述的磷酸钙沉淀法来进行。然而，还可以使用其他将DNA引入细胞的方法，诸如通过核注射或通过原生质体融合。如果使用原核细胞或包含 实质细胞壁结构的细胞，例如，一种转染方法是利用氯化钙的钙处理，如Cohen，S.N.，等，PNAS(美国科学院院报).69(1972)2110-2114所述。
用于本文中时，“表达”指将核酸转录为mRNA的过程和/或将转录的mRNA(也称为转录物)随后翻译为肽、多肽或蛋白质的过程。转录物和被编码的多肽共同称为基因产物。如果多核苷酸源自基因组DNA，则在真核细胞中的表达可以包括mRNA的剪接。
“载体”是核酸分子，特别是自我复制的，其将插入的核酸分子转移到宿主细胞之中和/之间。该术语包括主要功能为将DNA或RNA插入细胞(例如，染色体整合)的载体，主要功能是复制DNA或RNA的复制载体，和功能是转录和/或翻译DNA或RNA的表达载体。还包括提供多于一种上述功能的载体。
“表达载体”是多核苷酸，其在引入到合适的宿主细胞中时能够被转录和翻译为多肽。“表达系统”通常指包括表达载体的适当宿主细胞，所述表达载体可以起作用产生所需的表达产物。
提供下述实施例、序列表和附图来辅助对本发明的理解，其真正目的是在附上的权利要求书中列出。应该理解，在不背离本发明的精神的前提下，可以在所述的方法中进行修改。
患者疾病“治疗”指的是(1)阻止疾病在有倾向性或还没表现疾病症状的患者中出现；(2)抑制疾病或阻止其发展；或(3)减轻疾病或致其退化。
“有效量”意指活性化合物或药剂的量，其导致研究人员、兽医、医生或其他临床医生正寻求的组织、系统、动物、个体以及人的生物或药用响应，这包含治疗一种疾病。
II.全长抗体
在一个实施例中，本发明提供了一种新的全长抗体，该抗体具有高亲和力和具有黄斑变性疾病治疗效果。
“全长抗体”或“完整抗体”是指包括至少两条轻链和两条重链的抗体或抗体部分，并且每条重链包括至少一个可变区(VH)和三恒定区(例如，CH1、CH2和CH3)。
在一些方面，每条轻链、可变区、IgG1的恒定区、scFv的VH和VL区都是人源序列，可以任意的修改。
在一些方面中，全长抗体中的每一条重链都包含与SEQ ID NO：1的氨基酸序列同一性至少为80％，至少为85％或至少为90％的氨基酸序列；优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为91％，至少为92％，至少为93％，至少为94％，至少为95％，至少为96％，至少为97％或至少为98％的氨基酸序列；更优选地，所述的 重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为98.1％，至少为98.2％，至少为98.3％，至少为98.4％，至少为98.5％，至少为98.6％，至少为98.7％，至少为98.8％或至少为98.9％，的氨基酸序列；还更优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为99％，至少为99.1％，至少为99.2％，至少为99.3％，至少为99.4％，至少为99.5％，至少为99.6％，至少为99.7％，至少为99.8％或至少为99.9％的氨基酸序列。
在一些方面中，全长抗体中的每一条轻链都包含与SEQ ID NO：2的氨基酸序列同一性至少为80％，至少为85％或至少为90％的氨基酸序列；优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为91％，至少为92％，至少为93％，至少为94％，至少为95％，至少为96％，至少为97％或至少为98％的氨基酸序列；更优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为98.1％，至少为98.2％，至少为98.3％，至少为98.4％，至少为98.5％，至少为98.6％，至少为98.7％，至少为98.8％或至少为98.9％，的氨基酸序列；还更优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为99％，至少为99.1％，至少为99.2％，至少为99.3％，至少为99.4％，至少为99.5％，至少为99.6％，至少为99.7％，至少为99.8％或至少为99.9％的氨基酸序列。
在一些方面中，全长抗体中的每一条重链都包含SEQ ID NO：1的氨基酸序列。
在一些方面中，全长抗体中的每一条轻链都包含与SEQ ID NO：2的氨基酸序列。
重链氨基酸序列(SEQ ID NO：1):
Figure PCTCN2019070479-appb-000001
轻链氨基酸序列(SEQ ID NO：2):
Figure PCTCN2019070479-appb-000002
III.治疗方案
本发明提供了一种治疗黄斑变性疾病或相关病症的方法，该方法需要使用有效量的如本文所述的全长抗体。在某些方面，这种疾病是湿性(新生血管性)年龄相关性黄斑变性(wet-AMD)、病理性近视(PM)继发脉络膜新生血管(CNV)、糖尿病性黄斑水肿(DME)、糖尿病性视网膜病变(DR)、视网膜分枝静脉阻塞(BRVO)、视网膜中央静脉阻塞(CRVO)等眼底病变。
下面实施例进一步阐述本发明的某些方面和帮助熟悉该技术领域的科技人员施行本发明。这些实施例绝不是限制本发明的范围。
实施例1重组人源化抗VEGF抗体对新生血管抑制机理研究
重组抗VEGF人源化单克隆抗体(抗体BAT5906)的重链和轻链氨基酸序列分别见SEQ ID NO：1和SEQ ID NO：2。
对小鼠双眼进行激光光凝，破坏脉络膜结构，形成血管新生模型。对该模型注射人VEGFA抗原后，脉络膜血管新生会在得到增强；然后再分别注射重组人源化抗VEGF单克隆抗体BAT5906、Ranibizumab(Ran)、BAT5906抗体的Fab结构域(BAT5906-Fab)、Fc结构域(BAT5906-Fc)和人IgG1(hIgG1)后，与对照组(PBS相比)，脉络膜血管新生均得到抑制，推测存在两种不同的血管新生抑制机制。其中BAT5906-Fc和人IgG1表现出非靶点依赖性的抑制作用，Ranibizumab以及BAT5906-Fab为VEGF靶点依赖性抑制，而BAT5906为完整分子抑制程度最高，同时具有两种抑制作用。试验结果如图1所示。
BAT5906既可以通过特异性结合VEGF的Fab结构域进而抑制VEGF通路来抑制血管新生，也可以是通过Fc结构域与FcgRI结合来抑制血管新生。人IgG1由于含有Fc结构域，即使没有特异性结合VEGF的Fab结构域来抑制VEGF通路，也可通过FcgRI通路抑制血管新生，显示出FcgRI通路也是抑制血管新生的一种方式。与只具有特异性结合VEGF的Fab结构域的Ranibizumab相比，Ranibizumab只通过抑制VEGF通路来抑制血管新生；而BAT5906同时具有特异性结合VEGF的Fab结构域和Fc结构域，可抑制VEGF通路和FcgRI通路，血管新生抑制效果更佳。
实施例2重组抗VEGF单克隆抗体(抗体BAT5906)的重组质粒及稳定细胞株构建
根据氨基酸序列构建重组表达质粒pBAT5906，它含有GS cDNA元件，用于合成谷氨酰胺合成酶基因，作为稳定细胞株的扩增筛选标，从而可以通过在培养基中添加一定量 的L-蛋氨酸亚砜(methionine sulphoximine，MSX)进行稳定细胞株筛选。对构建好的重组表达质粒pBAT5906进行限制性内切酶Pvu I/Not I酶切验证，与预期设计结果一致，证明pBAT5906重组表达载体构建成功。
用于抗体表达的宿主细胞株为CHO-K1细胞的一种衍生细胞系，悬浮生长于CD-CHO培养基中。BAT5906抗体表达的稳定细胞株构建过程如下：将处于对数生长期的宿主细胞离心，并重悬于新鲜的CD-CHO培养基(细胞密度为1.43×10 7个/毫升)。取600μL上述细胞悬液和40μg已线性化的质粒混合均匀，加入电转杯中电击转化，Bio-rad电转化仪的参数设定为：电容：960μFD，电压：300V，电击时间15-20ms。把电击后的细胞立即重悬于500mL 37℃预热的CD-CHO培养基，每孔100μL分装于96孔板，2-3天后补加100μL的筛选培养基。2-3周后测定96孔板细胞培养上清中的抗体浓度，并将表达水平较高的克隆从96孔板转移到24孔板，待细胞生长到一定数量，再次把抗体表达量高的细胞转入到6孔板，最后保留20-30个高表达细胞株转入摇瓶做进一步评价。最终确得到的全长单克隆抗体表达细胞株的产量能够达到3g/L左右。
实施例3单克隆抗体的表达及纯化
单克隆抗体的表达及纯化过程如下：细胞大规模培养2周后，通过低速离心使细胞和培养基分离，将收获的上清进一步高速离心得到澄清的料液。重组抗体通过亲和层析法(Protein A)和离子交换两步法进行纯化，纯化中使用的介质分别是GE公司生产的Mab Select SuRe LX、TOSOH公司的Giga Cap Q-650M和ABI公司的POROS XS。分离纯化后的抗体通过SDS-PAGE法验证其大小正确性(图2和图3)，结果显示BAT5906条带在还原和非还原下大小都正确。
实施例4抑制恒河猴眼部新生血管有效性实验
激光致猴脉络膜新生血管模型是wet-AMD的理想模型，为了有效评价本发明抗体与已上市药品Ranibizumab(Lucentis)的药效，采用激光导致恒河猴脉络膜新生血管，然后进行玻璃体药物注射试验评价药效。造模成功后分组，每组4只恒河猴。双眼单次玻璃体注射给予0.25mg/眼(1.67nmol/眼)、0.5mg/眼(3.33nmol/眼)、1.25mg/眼(8.33nmol/眼)的BAT5906，模型对照组给予玻璃体注射0.9％氯化钠注射液，另设置上市药物对照药组，每只眼球注射0.5mg/眼的Ranibizumab(摩尔剂量为10nmol/眼)，上述注射体积均为50μL。给药28天后，对荧光斑渗漏面积改善率、眼底视网膜厚度改善率、房水VEGF含量 以及眼底3级和4级荧光斑个数进行测定，结果如表1所示：
表1眼玻璃体注射BAT5906对恒河猴药效学评估
Figure PCTCN2019070479-appb-000003
由上表1和图4可看出，0.25、0.5、1.25mg/眼剂量的BAT5906组对猴眼底激光斑荧光素渗漏面积改善率均略好于0.5mg/眼的Ranibizumab组(83.69％)，且低剂量的BAT5906具有更佳的荧光素渗漏面积改善率。
本发明单克隆抗体BAT5906的0.25、0.5、1.25mg/眼剂量组猴双眼给药后28天，所有眼球病损最高处视网膜厚度均有所降低，部分眼球色素上皮较规整、连续，部分眼球视网膜厚度已接近或达到甚至低于造模前水平。其病损最高处视网膜厚度改善率及减少量(给药后28天，改善率均数分别为106.61％，115.17％，113.45％)均明显高于模型对照组。由上表1和图6可看出，0.25、0.5、1.25mg/眼剂量的BAT5906组对猴眼底视网膜增厚改善率略好于0.5mg/眼的Ranibizumab组。
玻璃体注射3种剂量的本发明抗体BAT5906后29天，恒河猴房水VEGF浓度均低于模型对照组和阳性对照Ranibizumab组，该结果显示本发明抗体对眼组织VEGF有比阳性对照药物Ranibizumab更强的抑制效果。
由表1和图5可知，在程度最为严重的3级+4级荧光斑(显著荧光素渗漏，渗漏超过光斑边缘)数量变化比较中，0.5mg/眼的Ranibizumab组在给药后28天3级+4级荧光斑数均为6个，与给药前(39个)相比明显减少，但0.25mg/眼的BAT5906组的3级+4级荧光斑数为5个，0.5、1.25mg/眼的BAT5906组的3级+4级荧光斑数分别为2个和3个，荧光斑数少于阳性对照组Ranibizumab。
上述药效学试验结果表明，与Ranibizumab相比，本发明单克隆抗体BAT5906对荧光素渗漏的抑制程度更强，药效略优于Ranibizumab。
实施例5 CDC效应检测实验与ADCC效应检测实验
1.CDC效应检测实验
提前24小时用内皮细胞完全培养基ECM重悬靶细胞(HUVEC细胞)，调整细胞浓 度为2×10 5个/mL，在平底透明的96孔板中，每孔中加入50μL细胞混悬液。用含有2％FBS和5％补体的DMEM/F12培养基稀释抗体(起始浓度40μg/mL，以1/5倍稀释倍数依次稀释，每个浓度设3个复孔，共设立8个梯度)。用移液枪将上述步骤中的培养基吸干，然后加入上一步骤中的稀释好的靶细胞，每孔50μL，按照试验设计，加入到96孔板中，然后放入37℃、5％CO 2培养箱继续培养至少4个小时。
每个浓度梯度的抗体都以50μL/孔加入到96孔板中，样品的终浓度为10、2、0.4、0.08、0.016、0.0032、0.00064、0μg/mL，然后在37℃、5％CO 2的培养箱中培养30min。设立靶细胞最大LDH释放、靶细胞自发LDH释放孔、体积校正对照孔及培养基背景孔(最大LDH释放孔和体积校正对照孔在检测前45分钟加入10μL裂解液)，各3个复孔。置于37℃、5％CO 2培养箱孵育至少4h。4h后将96孔板离心，250g离心4分钟，用移液器小心吸取上清至另一块96孔板中，每孔加入50μL LDH检测试剂，室温避光孵育20-30分钟，然后每孔加入50μL终止液。
以490nm为检测波长，在酶标仪测量其吸收值。试验结果的计算：将所有实验孔、靶细胞自发LDH释放孔的吸光值减去培养基背景吸光值的均值，将靶细胞最大LDH释放对照的吸光值减去体积校正对照吸光值的均值。然后将上述经过校正的值带入下面公式，计算每个效靶比所产生的细胞毒性百分比。
％细胞毒性＝(实验-靶细胞自发)/(靶细胞最大-靶细胞自发)x100％
由以上结果可知，Bevacizumab样品、BAT5906样品在相同的试验条件下在补体的作用下对靶细胞的毒性作用没有明显区别，且对靶细胞的相对毒性作用约为0％，表明BAT5906抗体无CDC效应。以上结果表明，本发明抗体在细胞内不会引起补体依赖的细胞毒性(见表2)。
2.ADCC效应检测实验
提前24小时用1640培养基重悬靶细胞(HUVEC细胞)，调整细胞浓度为2×10 5个/mL，在平底透明的96孔板中，每孔中加入50μL细胞混悬液。
用含有2％FBS的DMEM/F12培养基稀释抗体(起始浓度40μg/mL，以1/5倍稀释倍数依次稀释，每个浓度设3个复孔，共设立8个浓度梯度)。
每个浓度梯度的抗体都以50μL/孔加入到样品孔中，样品的终浓度为10、2、0.4、0.08、0.016、0.0032、0.00064、0μg/mL，然后在37℃、5％CO2的培养箱中培养30min。
取对数生长期的效应细胞PBMC，800转离心5分钟弃上清。加入含有2％FBS的DMEM/F12培养基，800转离心5分钟弃上清2遍，计数，将效应细胞密度调整2×10 5个 /mL。
用移液枪往样品孔中加入上一步骤中的稀释好的效应细胞，每孔50μL，然后放入37℃、5％CO 2培养箱继续培养至少4个小时。
设立靶细胞最大LDH释放、靶细胞自发LDH释放孔、体积校正对照孔及培养基背景孔(最大LDH释放孔和体积校正对照孔在检测前45分钟加入10μL检测试剂盒中的裂解液)，各3个复孔。置于37℃、5％CO 2培养箱孵育至少4h。
培养4h后将96孔板离心，250g离心4分钟，用移液器小心吸取上清至另一块96孔板中，每孔加入50μL LDH检测试剂，室温避光孵育20-30分钟，然后每孔加入50μL终止液。以490nm为检测波长，在酶标仪测量其吸收值。
由以上试验结果可知，本发明抗体BAT5906和Bevacizumab在相同的试验条件下在抗体的作用下对靶细胞的毒性作用没有明显区别，且对靶细胞的相对毒性作用约为0％，表明BAT5906抗体无ADCC效应(见表2)。
以上CDC、ADCC两个试验结果表明，本发明抗体在体内不会引起补体依赖的细胞毒性(CDC)和抗体依赖的细胞毒性(ADCC)，在体内使用不会影响免疫系统的正常作用。本发明抗体可以在病灶处正常发挥其抗VEGF功能而不引起不良毒性反应，在人体内使用安全性较高，是一种低毒的抗VEGF单克隆抗体。
表2 BAT5906和对照品的CDC效应、ADCC效应试验结果
Figure PCTCN2019070479-appb-000004
*—表示无此效应，+表示有此效应
实施例6恒河猴眼玻璃体注射单克隆抗体急性毒性试验
本试验设2组，分别为阴性对照组、重组抗VEGF人源化单克隆抗体(BAT5906)注射液8.0mg/眼组，每组3只恒河猴，雌雄兼有。各组恒河猴按100μL/眼体积单次双眼玻璃体注射给予阴性对照品(0.9％氯化钠注射液)及80mg/mL的重组抗VEGF人源化单克隆抗体注射液。给药当天定义为试验第1天。
给药后连续14天每天观察各组猴一般状况；试验第8、14天测定体重；试验第1(给药前)、2、4、8、14天进行间接检眼镜及裂隙灯检查；给药前及给药后约10～15分钟、 1小时、24小时以及试验第14天测定眼压；给药前及试验第14天进行眼底彩色照相及荧光造影检查；试验第2、14天进行血液学、血生化检测；试验第15天对所有恒河猴麻醉后放血实施安乐死，进行大体解剖观察。
试验结果显示，在本试验条件下，恒河猴双眼玻璃体单次注射8.0mg/眼剂量的重组抗VEGF人源化单克隆抗体注射液并观察14天，未见眼毒性及全身系统毒性，最大耐受剂量(MTD)为8.0mg/眼。急性毒性试验中眼底荧光造影检查见图7所示。
实施例7恒河猴眼玻璃体注射单克隆抗体眼组织分布试验
本试验设置玻璃体注射8.33nmol/眼(即1.25mg/眼)组，使用21只恒河猴，10雌11雄。玻璃体注射组猴按50μL/眼的体积于双侧眼经玻璃体单次注射给予25mg/mL的重组抗VEGF人源化单克隆抗体(BAT5906)注射液。玻璃体注射组分别于给药后4、10、24、72、168、336、672小时各取3只猴施以安乐死后(戊巴比妥钠30mg/kg麻醉，股动脉放血)解剖，取下双眼眼球及视神经，分离眼球房水、角膜、虹膜、玻璃体、晶状体、视网膜、脉络膜、视神经。玻璃体注射组给药后336、672小时解剖的6只猴于给药前及给药后30分钟、1小时、2小时、4小时、10小时、24小时、48小时、72小时、96小时、168小时、336小时、672小时采血分离血清。采用ELISA法检测各眼组织、血清中药物浓度，计算血清和眼组织中的药物代谢参数。
主要结果如下：
恒河猴双眼单次玻璃体注射1.25mg/眼(8.33nmol/眼)的剂量重组抗VEGF人源化单克隆抗体注射液后，药物能在各眼部组织中迅速扩散分布，从注射后4小时起眼内各组织中均可检测到药物，除角膜和视神经以外的所有眼组织药物浓度均于给药后4～24小时达峰。药物在眼内各组织中暴露水平(AUC last)由高至低依次为：玻璃体、视网膜、角膜、房水、脉络膜、虹膜、晶状体、视神经。在玻璃体、房水、视网膜、脉络膜、虹膜、晶状体等主要眼组织消除半衰期在73.74～143.81小时之间，在玻璃体中的半衰期为84.2小时。
两种抗VEGF治疗药物与BAT5906的对比情况见表3，BAT5906的半衰期明显优于目前市场上的两种抗VEGF治疗药物。
表3两种抗VEGF治疗药物与BAT5906性质比较
Figure PCTCN2019070479-appb-000005
Figure PCTCN2019070479-appb-000006
a Gaudreault J,Fei D,Rusit J,et al.Preclinical pharmacokinetics of Ranibizumab(rhuFabV2)after a single intravitreal administration[J].Investigative ophthalmology&visual science,2005,46(2):726-733.
b Biologic License Application(BLA):125387 Orig1s000 PHARMACOLOGY REVIEW(S),Company:REGENERON PHARMACEUTICALS,11/18/2011.
由表3可知，恒河猴经双眼玻璃体注射8.33nmol/眼的本发明单克隆抗体后，药物在猴玻璃体中主要药代参数消除半衰期(t 1/2)为84.2小时。而上市阳性对照药物Ranibizumab玻璃体注射后在猴玻璃体中的半衰期为55.7小时，Aflibercept玻璃体注射后在猴玻璃体中的半衰期为40-60小时。与上述两种上市阳性对照药物相比，本发明单克隆抗体在玻璃体中的药物半衰期更长，可在眼部发挥更长时间的药效，抑制眼底血管新生的作用更佳。
实施例8差式热量扫描仪(Differential scanning calorimetry、DSC)检测
为了研究本发明抗体的热转化温度，我们运用毛细管差式热量扫描仪来检测BAT5906分子各个结构域的热解折叠温度。仪器型号Nano DSC，毛细管样品池和参比池的体积均为0.300mL，加热速率为1℃/min，10min预平衡时间，过滤周期10秒。从表4的实验结果中可以看出BAT5906分子有2个熔解峰，第1个峰为CH2熔解峰，Tm值为76.07℃；第2个峰为Fab/CH3熔解峰，Tm值为83.68℃。以上数据表明BAT5906分子整体结构比较稳定，各个结构域中的最低Tm值也有76.07℃，BAT5906的热解折叠温度高于Bevacizumab，较Bevacizumab更稳定。
表4 BAT5906与对照品的热解折叠温度对比结果
Figure PCTCN2019070479-appb-000007
实施例9细胞生物学活性检测试验
VEGF具有促进HUVEC细胞增殖的活性，本发明单克隆抗体具有抑制VEGF的功能，故本试验检测使用HUVEC进行细胞生物学活性的比较。
首先用含2％FBS的ECM培养基重悬HUVEC细胞(HUVEC细胞代次为第5代)至浓度为9×10 4cells/mL，以每孔100μL接种96孔细胞培养板，孵育(5±1)h使细胞贴壁。使用测试培养基将BAT5906单克隆抗体和Bevacizumab均预稀释至1000ng/mL，然后再1:1.5 向下系列稀释9个梯度(共10个梯度)，系列稀释样品与等体积测试培养基稀释的80ng/mL VEGF-A 165混合后，37℃孵育(60±10)min。孵育结束后，将每孔100μL抗体VEGF-A 165混合物加入至HUVEC接种细胞培养板的对应孔中，置于5％CO 2培养箱中孵育(66±3)h。孵育结束后，加入CCK8每孔20μL并继续孵育(4±0.5)h显色。显色结束后细胞培养板置于室温平衡30min，在酶标仪于450nm处读取吸收值结果。实验结果见表5，BAT5906的细胞生物学活性约为Bevacizumab的3.8倍。
表5 BAT5906与对照品的细胞生物学活性对比结果
Figure PCTCN2019070479-appb-000008
实施例10抗体稀释后37℃条件下5周稳定性试验
通过体外试验模拟人玻璃体内注射抗VEGF单克隆抗体药物后在眼球部位的稳定性变化情况。将药物按照临床使用中浓度稀释后置于37℃，对其稳定性进行研究。具体操作为：分别将本发明抗体BAT5906、雷珠单抗(Ranibizumab)、贝伐珠单抗(Bevacizumab)和阿柏西普(Aflibercept)的浓度稀释至0.3125mg/mL、0.125mg/mL、0.3125mg/mL和0.5mg/mL，放置在37℃恒温培养箱进行连续5周的稳定性试验，首次稀释后留样，后续每周定时留样备用。在第5周时将所有6个时间点的样品进行单克隆抗体的细胞生物学活性和SEC(分子排阻色谱法)主峰百分比变化的检测。
细胞生物学活性试验方法见实施例9。SEC检测方法为：色谱柱为TSKgel G3000SWXL(柱规格7.8×300mm，5μm)，以含10％乙腈的100mM磷酸钾，125mM氯化钾缓冲液作为流动相，柱温30℃，流速为0.5mL/min；检测波长为280nm。取对照品，用水稀释成2mg/mL的上样溶液，取50μL注入液相色谱仪，记录色谱图，平行测定3次，3次结果的主峰理论塔板数都不得低于2000，拖尾因子不得大于2.0，主峰与多聚体的分离度不得小于2.0。同法测定供试品，按面积归一化法计算，以3次检测结果的平均值作为最终检测结果。试验结果如图8和图9所示。
试验结果显示，本发明抗体在模拟人玻璃体内注射稀释浓度下置于37℃进行研究，细胞生物学活性较为稳定，SEC主峰百分比变化很小，表明本发明抗体在本试验条件下稳定性较好，是一种玻璃体内注射后可在眼底部位发挥稳定生物学功能的抗VEGF单克隆抗体。
尽管已经联系具体实施例及其实施例描述了本发明，但明显的是，本领域技术人员能够看出许多替代方案、修改和变动。相应地，旨在涵盖落在所附权利要求的精神和宽范围内的所有这样的替代方案、修改和变动。
本说明书中提到的所有出版物、专利和专利申请均全文经此引用并入本说明书，就像各个出版物、专利或专利申请专门且逐一被指出经此引用并入本文。

Claims (12)

  1. 一种人源化全长抗VEGF的IgG1抗体，包含：
    (a)两条免疫球蛋白轻链；
    以及(b)两条免疫球蛋白重链；
    所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为80％，至少为85％或至少为90％的氨基酸序列；优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为91％，至少为92％，至少为93％，至少为94％，至少为95％，至少为96％，至少为97％或至少为98％的氨基酸序列；更优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为98.1％，至少为98.2％，至少为98.3％，至少为98.4％，至少为98.5％，至少为98.6％，至少为98.7％，至少为98.8％或至少为98.9％，的氨基酸序列；还更优选地，所述的重链含有与SEQ ID NO:1所示的氨基酸序列同一性至少为99％，至少为99.1％，至少为99.2％，至少为99.3％，至少为99.4％，至少为99.5％，至少为99.6％，至少为99.7％，至少为99.8％或至少为99.9％的氨基酸序列；
    所述的轻链含有与SEQ ID NO:2所述的氨基酸序列同一性至少为80％，至少为85％或至少为90％的氨基酸序列；优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为91％，至少为92％，至少为93％，至少为94％，至少为95％，至少为96％，至少为97％或至少为98％的氨基酸序列；更优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为98.1％，至少为98.2％，至少为98.3％，至少为98.4％，至少为98.5％，至少为98.6％，至少为98.7％，至少为98.8％或至少为98.9％，的氨基酸序列；还更优选地，所述的轻链含有与SEQ ID NO:2所示的氨基酸序列同一性至少为99％，至少为99.1％，至少为99.2％，至少为99.3％，至少为99.4％，至少为99.5％，至少为99.6％，至少为99.7％，至少为99.8％或至少为99.9％的氨基酸序列；
    优选地，所述的抗体是人IgG1；
    优选地，所述的抗体与VEGF结合；更优选地，所述的抗体与VEGF-A结合；还更优选地，所述的抗体与VEGF-A165结合；
    优选地，所述抗体与人VEGF结合的KD值不超过10nM；更优选地，所述的抗体与人VEGF结合的KD值不超过1nM；还更优选地，所述的抗体与人VEGF结合的KD值不超过500pM。
  2. 如权利要求1所述的抗体，其中所述的重链含有SEQ ID NO:1所示的氨基酸序列。
  3. 如权利要求1所述的抗体，其中所述的轻链含有SEQ ID NO:2所示的氨基酸序列。
  4. 一种核酸，所述的核酸编码权利要求1-3任一所述的抗体。
  5. 一种载体，所述的载体含有权利要求4所述的核酸。
  6. 一种宿主细胞，所述的宿主细胞含有权利要求5所述的载体。
  7. 一种产生如权利要求1-3任一所述的抗体的方法，包括培养权利要求6所述的宿主细胞，从而核酸被表达出来；
    优选地，所述的方法还包括从宿主或宿主培养物中回收抗体。
  8. 一种亲和纯化试剂，所述的试剂含有权利要求1-3任一所述的抗体。
  9. 如权利要求1-3任一所述的抗体在制备用于VEGF蛋白的诊断分析的试剂中的用途。
  10. 一种用于VEGF蛋白诊断的试剂盒，所述的试剂盒含有权利要求1-3任一所述的抗体。
  11. 一种药物制剂，所述的药物制剂含有权利要求1-3任一所述的抗体；
    优选地，所述的药物制剂还含有药学上可接受的载体。
  12. 如权利要求1-3任一所述的抗体在制备哺乳动物中VEGF过表达相关的疾病的药物中用途；
    优选地，所述的VEGF过表达相关的疾病为VEGF过表达相关的眼底病变；更优选地，所述的VEGF过表达相关的疾病选自年龄相关性黄斑变性、病理性近视继发脉络膜新生血管、糖尿病性黄斑水肿、糖尿病性视网膜病变、视网膜分枝静脉阻塞或视网膜中央静脉阻塞；
    优选地，所述的哺乳动物为人。
PCT/CN2019/070479 2018-01-05 2019-01-04 一种长效低毒的重组抗vegf人源化单克隆抗体及其生产方法 WO2019134686A1 (zh)

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