Anti-angiopoietin-2 antibodies and uses thereof
阅读说明：本技术 抗血管生成素-2抗体及其用途 (Anti-angiopoietin-2 antibodies and uses thereof ) 是由 高圭永 裴点一 金美贞 朴珍成 徐修珍 金载领 朴将烈 金泌汉 吴汪烈 于 2019-02-19 设计创作，主要内容包括：本发明涉及与血管生成素-2(Ang2)结合的小鼠抗体、由其衍生的人源化抗Ang2抗体以及它们的用途。所述抗Ang2抗体具有激活所述Tie2受体以及中和Ang2的双重功能。所述抗Ang2抗体显示出使异常和病理性血管正常化的特性,并且因此针对与异常血管相关的各种疾病和障碍展现出治疗功效。本发明提供了血管生成抑制剂和包含所述抗体作为有效成分的用于预防和治疗与异常Ang2表达和Tie2调节异常相关的疾病的组合物,以及包含所述抗体的用于诊断与Ang2抑制和Tie2激活相关的疾病的组合物。(The present invention relates to mouse antibodies that bind to angiopoietin-2 (Ang2), humanized anti-Ang 2 antibodies derived therefrom, and uses thereof. The anti-Ang 2 antibody has the dual function of activating the Tie2 receptor and neutralizing Ang 2. The anti-Ang 2 antibody exhibits properties that normalize abnormal and pathological blood vessels, and thus exhibits therapeutic efficacy against various diseases and disorders associated with abnormal blood vessels. The present invention provides an angiogenesis inhibitor and a composition for preventing and treating diseases associated with abnormal Ang2 expression and Tie2 dysregulation comprising the antibody as an effective ingredient, and a composition for diagnosing diseases associated with Ang2 inhibition and Tie2 activation comprising the antibody.)
1. An antibody or antigen-binding fragment thereof that specifically binds human angiopoietin-2 and induces Tie2 activation, wherein the antibody or antigen-binding fragment thereof binds to amino acid SEQ ID NO:115, amino acid SEQ ID NO:116, or amino acid SEQ ID NO: 117.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to amino acid SEQ ID NO. 116 or amino acid SEQ ID NO. 117.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to amino acid SEQ ID No. 115.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to human and mouse Ang 2.
5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is polyclonal or monoclonal.
6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is an scFv or Fab.
7. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody is humanized.
8. The antibody or antigen-binding fragment thereof of claim 1, comprising:
(a) complementarity Determining Regions (CDRs) of the heavy chain variable region having the amino acid sequence of HCDR1 of SEQ ID NO. 3, the amino acid sequence of HCDR2 of SEQ ID NO. 4, and the amino acid sequence of HCDR3 of SEQ ID NO. 5; and
(b) CDRs of the light chain variable region comprising the LCDR1 amino acid sequence of SEQ ID NO. 6, the LCDR2 amino acid sequence of SEQ ID NO. 7, and the LCDR3 amino acid sequence of SEQ ID NO. 8.
9. The antibody or antigen-binding fragment thereof of claim 1, comprising:
(a) complementarity Determining Regions (CDRs) of the heavy chain variable region having the amino acid sequence of HCDR1 of SEQ ID NO. 13, the amino acid sequence of HCDR2 of SEQ ID NO. 14, and the amino acid sequence of HCDR3 of SEQ ID NO. 15; and
(b) CDRs of the light chain variable region comprising the LCDR1 amino acid sequence of SEQ ID NO. 16, the LCDR2 amino acid sequence of SEQ ID NO. 17, and the LCDR3 amino acid sequence of SEQ ID NO. 18.
10. The antibody or antigen-binding fragment thereof of claim 1, comprising:
a heavy chain variable region selected from SEQ ID NOs 9, 19, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, or 111; and
11, 21, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108 or 112 light chain variable region selected from SEQ ID NOs.
11. The antibody or antigen-binding fragment thereof of claim 1, which comprises the Complementarity Determining Regions (CDRs) of an antibody produced by the cell line deposited under accession number KCLRF-BP-00417 or KCLRF-BP-00418.
12. A pharmaceutical composition comprising a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof of claim 1 in association with a pharmaceutically acceptable carrier.
13. The pharmaceutical composition of claim 12, further comprising a small molecule inhibitor for use in chemotherapy.
14. The pharmaceutical composition of claim 12, further comprising a Vascular Endothelial Growth Factor (VEGF) antagonist.
15. The pharmaceutical composition of claim 14, wherein the VEGF antagonist is an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
16. A method for inhibiting tumor growth in a patient comprising administering to the patient a pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 12.
17. The method of claim 16, comprising further administering a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy.
18. The method of claim 17, wherein the VEGF antagonist is an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
19. A method for inhibiting choroidal neovascularization, inhibiting ocular vascular leakage, or simultaneously triggering choroidal capillary regeneration in a patient having an eye disease, said method comprising administering to said patient a pharmaceutical composition according to claim 12.
20. The method of claim 19, comprising further administering a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy.
21. The method of claim 20, wherein the VEGF antagonist is an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
22. The method of claim 19, wherein the ocular disease is wet age-related macular degeneration (wAMD), Diabetic Macular Edema (DME), or Diabetic Retinopathy (DR).
23. A nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1.
24. An expression vector comprising the nucleic acid of claim 23.
25. A host cell transformed with the expression vector of claim 24.
26. A method for producing an anti-Ang 2 antibody or antigenic fragment thereof, the method comprising culturing the host cell of claim 25.
Technical Field
The present invention includes an anti-Ang 2 antibody or antigen-binding fragment thereof that specifically binds to angiopoietin-2 (Ang2), a ligand known to control angiogenesis and maintenance; a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof; nucleic acid encoding the antibody or antigen-binding fragment thereof; a vector comprising said nucleic acid; a host cell transformed with the vector; and methods for producing the antibodies or antigen-binding fragments thereof.
Background
Angiogenesis occurs dynamically through a variety of regulatory factors during the development, growth, maintenance and homeostasis of an organism. The newly formed blood vessels in this process serve as transport channels for various biological materials, such as nutrients, oxygen, and hormones in the surrounding cells. Dysfunctional and structurally abnormal blood vessels are a direct or indirect cause of the occurrence and progression of various diseases. Tumor vessels exacerbate hypoxia due to their defective function and structure, leading to tumor progression and metastasis to other tissues, and also leading to poor delivery of anticancer drugs to the core of the tumor mass. In addition to cancer, defective blood vessels are also found in a variety of other diseases and conditions. Examples include various eye diseases (e.g., diabetic macular edema, wet age-related macular degeneration), viral infections, and acute inflammatory reactions (e.g., sepsis). Therefore, if a therapeutic agent capable of normalizing pathological blood vessels is available, it can be applied to the treatment of various patients suffering from blood vessel abnormalities.
The angiogenin family plays an important role in the formation and maintenance of blood vessels and consists of four angiogenin classes (Ang1, Ang2, Ang3 and Ang 4). Angiopoietin-1 (Ang1) binds to Tie2 receptors present on the surface of vascular endothelial cells to phosphorylate and activate Tie2 receptors, resulting in vascular stabilization. Angiopoietin-2 (Ang2), on the other hand, binds to the Tie2 receptor but acts as an antagonist to induce inactivation of the Tie2 receptor, leading to vascular instability and vascular leakage. It has been reported that the expression level of Ang2 is greatly increased in the blood of cancer patients, ocular diseases, viral and bacterial infections, and inflammatory diseases (saharanen P et al, 2017, Nature Review Drug Discovery). However, Ang2 is also known to act as an agonist in several processes (including lymphatic formation and maintenance) to induce activation of Tie2 receptors, and thus Ang2 is thought to perform multiple functions depending on the context.
Ang2 binding antibodies have been reported in several documents (e.g., US 7,658,924 and US 8,987,420). Most of Ang2 antibodies reported so far are known to inhibit the binding of Ang2 to Tie2 and thus inhibit the formation of new blood vessels by this Ang2 neutralizing effect. Currently, a variety of Ang2 antibodies are being clinically tested in various cancer patients, but their anti-cancer efficacy is considered to be insufficient. For example, a phase 3 clinical trial by Amgen showed that the anti-cancer efficacy of the Ang2 antibody was not significant in ovarian cancer patients (Marth C et al, 2017, eur.j.
In addition to antibodies, recombinant proteins that bind directly to Tie2 receptor to induce Tie2 phosphorylation and activation have also been reported. Examples include the COMP-Angl (Cho et al, 2004, PNAS) and Vasculotide (David S et al, 2011, Am J Physiol Lung Cell Mol Physiol) peptides, which consist of five angiopoietin-1 protein fragments. However, these proteins are believed to have very short half-lives and unstable physicochemical properties. In addition, there is a phosphatase called VE-PTP which removes a phosphate group from phosphorylated Tie2 to inactivate Tie2, and a low molecular compound (AKB-9778) which indirectly maintains Tie2 activity by inhibiting the activity of the enzyme VE-PTP (Goel S,2013, J NatlCancer Inst) has also been developed. However, a disadvantage of this compound is that it activates other receptors in addition to Tie2 (Frye M,2015, jexp. med, Hayashi M,2013, Nature Communication, Mellberg S et al, 2009, FASEB J.).
Disclosure of Invention
The present invention relates to an antibody or antigen binding fragment thereof that specifically binds human angiopoietin-2 and induces Tie2 activation, wherein said antibody or antigen binding fragment thereof binds to amino acids 289-299 of SEQ ID NO:1, amino acids 316-322 of SEQ ID NO:1 or amino acids 336-353 of SEQ ID NO:1 as determined by the hydrogen/deuterium exchange method.
The antibody or antigen binding fragment thereof can bind to human and mouse Ang 2. The antibody may be polyclonal or monoclonal. The antigen binding fragment may be an scFv or Fab. The antibody or fragment thereof may be humanized.
In another aspect, the invention relates to an antibody or antigen-binding fragment comprising:
(a) complementarity Determining Regions (CDRs) of the heavy chain variable region having the amino acid sequence of HCDR1 of SEQ ID NO. 3, the amino acid sequence of HCDR2 of SEQ ID NO. 4, and the amino acid sequence of HCDR3 of SEQ ID NO. 5; and
(b) CDRs of the light chain variable region comprising the LCDR1 amino acid sequence of SEQ ID NO. 6, the LCDR2 amino acid sequence of SEQ ID NO. 7, and the LCDR3 amino acid sequence of SEQ ID NO. 8.
In another aspect, the invention relates to an antibody or antigen-binding fragment comprising:
(a) complementarity Determining Regions (CDRs) of the heavy chain variable region having the amino acid sequence of HCDR1 of SEQ ID NO. 13, the amino acid sequence of HCDR2 of SEQ ID NO. 14, and the amino acid sequence of HCDR3 of SEQ ID NO. 15; and
(b) CDRs of the light chain variable region comprising the LCDR1 amino acid sequence of SEQ ID NO. 16, the LCDR2 amino acid sequence of SEQ ID NO. 17, and the LCDR3 amino acid sequence of SEQ ID NO. 18.
In one aspect, the invention relates to an antibody or antigen-binding fragment thereof comprising the Complementarity Determining Regions (CDRs) of an antibody produced by a cell line deposited under accession number KCLRF-BP-00417 or KCLRF-BP-00418.
In yet another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the above-described antibody, or antigen-binding fragment thereof, in association with a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy. In one aspect, the VEGF antagonist can be an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
In yet another aspect, the invention relates to a method for inhibiting tumor growth in a patient comprising administering to the patient a pharmaceutical composition comprising an antibody or antigen-binding fragment described above. The methods may further comprise administering a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy, either simultaneously or stepwise with the administration of the antibody or fragment thereof of the invention. The VEGF antagonist can be an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
In yet another aspect, the present invention relates to a method for inhibiting choroidal neovascularization, inhibiting ocular vascular leakage, or simultaneously triggering choroidal capillary regeneration in a patient having an ocular disease, said method comprising administering to said patient the above pharmaceutical composition. The methods may further comprise administering a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy, either simultaneously or stepwise with the administration of the antibody or fragment thereof of the invention. The VEGF antagonist can be an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor. The ocular disease is wet age-related macular degeneration (wAMD), Diabetic Macular Edema (DME), or Diabetic Retinopathy (DR).
These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying drawings and the appended claims.
Drawings
The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings, which are given by way of illustration only and thus are not limitative of the present invention, and wherein;
figure 1 Akt phosphorylation induced by anti-Ang 2 antibody. HUVECs were serum starved for 6hr and incubated for 30min with COMP-Ang1(CA1, 0.5 μ g/ml) or anti-Ang 2 antibodies (control, 2C8, 4B9, 2F10, and 4E2, respectively) in the absence or presence of human Ang2(1 μ g/ml). Cell lysates were subjected to SDS-PAGE/Western blotting and blotted with anti-phosphoAkt (S473) or anti-Akt antibodies.
Figure 2. schematic shows the epitope of anti Ang2 antibody, which has been analyzed by hydrogen/deuterium exchange mass spectrometry. The recombinant hAng2-RBD or hAng2-RBD/Ang 2-antibody complex alone was labeled with deuterium. The labeled proteins were digested in a pepsin column and analyzed by mass spectrometry. Deuterium uptake was analyzed for both the hAng2-RBD and hAng2-RBD/Ang-2 antibody complexes alone and the differences in deuterium uptake were compared. Peptides with mass differences in deuterium uptake of more than 0.5-1Da were identified as specific epitopes mediating binding to anti-Ang 2 antibodies. The 2C8 epitope (red) and the 4B9 epitope (green) were visualized in an image of the Ang2-RBD crystal structure (PDB: 2GY7) generated using PyMol software.
Figure 3 dose-dependent Akt phosphorylation by humanized anti-Ang 2 antibodies 4B9H11 and 2C8H11 (pAkt). Serum-starved HUVECs were incubated with human Ang2, anti-Ang 2 antibody or human Ang2, and various concentrations of anti-Ang 2 antibody for 30 min. The cell lysates were subjected to SDS-PAGE/Western blotting.
FIG. 4 dose-dependent Tie2 phosphorylation by humanized anti-Ang 2 antibodies 4B9H11 and 2C8H11 (pTie 2). The ability of the 4B9H11 (fig. 4A) and 2C8H11 (fig. 4B) antibodies to induce Tie2 phosphorylation was investigated by immunoprecipitation and Western blot (Western) analysis. Serum-starved HUVECs were incubated with human Ang2, anti-Ang 2 antibody alone or human Ang2, and various concentrations of anti-Ang 2 antibody for 30 min. Cell lysates were immunoprecipitated with anti-Tie 2 antibody, followed by SDS-PAGE/western blot analysis.
Figure 5 Tie2 receptor aggregation and FOXO1 translocation induced by humanized Ang2 antibody. HUVECs were serum starved for 6hr and incubated with COMP-Ang1(CA1), Ang2(A2) or Ang2 and anti-Ang 2 antibodies (control Ab, 2C8H11 or 4B9H11) for 30 min. After fixation, HUVECs were stained with DAPI (blue), anti-Tie 2 antibody (green), anti-FOXO 1 antibody (red) and anti-human Fc (cyan) to study Tie2 aggregation at the cell surface, FOXO1 translocation from the nucleus and the presence of humanized Ang2 antibody in the intercellular junction region. Arrows indicate Tie2 and co-localized Ang2 antibodies that are aggregated at intercellular contacts.
Figure 6 time course of Tie2 receptor aggregation, FOXO1 translocation and Ang2 antibody localization in intercellular junctions in HUVECs. Serum-starved HUVECs were incubated with anti-Ang 2 antibody (control Ab or 2C8H11) for various time points (from 10min to 240 min). After cell fixation, aggregated Tie2 receptors and endocytosed Tie2 receptors at the cell surface were studied by staining with anti-Tie 2 antibody. The humanized anti-Ang 2 antibody was probed at the cell surface and cytosol with an anti-human Fc antibody. Arrows indicate Tie2 and co-localized Ang2 antibodies that are aggregated at intercellular contacts.
Figure 7 inhibition of vascular permeability by humanized anti-Ang 2 antibody. HUVECs were seeded on penetrating (transwell) chambers and grown for 3 days. At 100% confluence, HUVECs were pretreated with COMP-Ang1(CA1, 0.5. mu.g/ml), Ang2(A2, 1. mu.g/ml), Ang2 and control Ab (A2+ control Ab, 1. mu.g/ml), 2C8H11(A2+2C8H11, 1. mu.g/ml) or 4B9H11(A2+4B9H11, 1. mu.g/ml) for 30min and TNF-a (100ng/ml) for 22hr into the upper compartment. Vascular permeability was assessed by measuring FITC fluorescence in the lower chamber 20min after addition of FITC-dextran in the upper chamber. Values are mean ± SD. P <0.05, p <0.01, p <0.001 by one-way ANOVA.
FIG. 8 EC of anti-Ang 2 antibody on mouse Ang2 by ELISA50The value is obtained. EC by assay with ELISA50To measure the binding affinity of the humanized anti-Ang 2 antibody to mouse Ang2 (hong 2). Recombinant hong 2 was coated and incubated with serial dilutions of anti-Ang 2 antibody 4B9H11 and 2C8H 11. Next, the plate was reacted with anti-human igg (fab) -HRP secondary antibody. The plate was treated with TMB solution and the absorbance measured at 450 nm. EC was analyzed using the WorkOut 2.5 program from PerkinElmer50The value is obtained.
FIG. 9 humanized 2C8H11 antibody and cisplatin (Cpt) inhibited tumor growth in an LLC tumor model. LLC tumor growth was compared in mice treated as indicated, starting 7 days after tumor implantation. Black arrows indicate injection of antibody, while red arrows indicate single injections of Cpt. For each group, n-7-9. Values are mean ± SD. Relative to Fc, < 0.05; relative to Fc + Cpt, # p < 0.05.
FIG. 10 normalization of tumor vasculature by humanized 2C8H11 antibody PDGFR β on tumors was compared in LLC subcutaneous tumor model+Pericyte coverage and CD31 in intratumoral regions+BV. Scale bar, 100 μm. For each group, n is 5. Values are mean ± SD. Relative to Fc,. about.p<0.05; relative to Fc + Cpt, # p<0.05。
Figure 11-decreased hypoxia perfusion increase in tumor vessels by humanized 2C8H11 antibody. Lectin perfusion and hypoxic probes of tumor vessels were analyzed and compared in LLC tumors+Area of hypoxia. Hypoxic probe+Area is expressed as a percentage of the total cross-sectional area. Scale bar, 100 μm. For each group, n is 5. Values are mean ± SD. Relative to Fc,. about.p<0.05; relative to Fc + Cpt, # p<0.05。
FIG. 12 enhancement of Cpt drug delivery to the tumor core by the humanized 2C8H11 antibody. Using anti-Cpt modified DNA antibodies, Cpt was tested in tumors harvested on
Figure 13 inhibition of CNV regression and vascular leakage by intravitreal injection of 2C8H11 antibody in a laser-induced CNV model. Intravitreal administration of the antibody was performed 7 days after laser photocoagulation. Measuring CD31+CNV volume and the area of leakage around the CNV was calculated as the total superfluorescent area measured in the FA image at 6 and/or 14 days after laser photocoagulation divided by the total CNV area measured in the ICGA image. Scale bar, 100 μm. For each group, n is 11. Values are mean ± SD. By one-way ANOVA, followed by student-neman-keels post-hoc test<0.001; through the t-test of the paired students,###p<0.001。
figure 14 CNV regression and choroidal capillary regeneration by intravitreal injection of 2C8H11 antibody. Intravitreal administration of the antibody was performed 7 days after laser photocoagulation. CNV volume (area outlined by white dashed box) and avascular space surrounding the CNV (area outlined by yellow dashed box) were measured by OCTA imaging of the
Figure 15.2 co-localization of C8H11 antibody and CD31 in CNV endothelial cells. Subcutaneous administration of 2C8H11 antibody was performed 1 day after laser photocoagulation. Co-localization of 2C8H11 antibody and CD31 in CNV endothelial cells was detected directly by anti-human IgG antibody at 2, 4 and 8 days after laser photocoagulation.
Figure 16 CNV inhibition by subcutaneously injected 2C8H11 antibody. Subcutaneous administration of 2C8H11 antibody was performed 1 day after laser photocoagulation.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the terminology used herein is well known in the art and is used in a generic manner.
In this application, "a" and "an" are used to refer to both single and multiple objects.
In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that specifically binds human angiopoietin-2 and induces Tie2 activation, wherein the antibody or antigen-binding fragment thereof binds to amino acid SEQ ID NO:115, amino acid SEQ ID NO:116, or amino acid SEQ ID NO: 117.
The amino acids of SEQ ID NO. 115 correspond to amino acids 336-353 of SEQ ID NO. 1, the amino acids of SEQ ID NO. 116 correspond to amino acids 289-299 of SEQ ID NO. 1 and the amino acids of SEQ ID NO. 117 correspond to amino acids 316-322 of SEQ ID NO. 1.
As used herein, the term "antibody that specifically binds to Ang 2" refers to an antibody that binds to Ang2 resulting in the inhibition of the biological activity of Ang2, and is used interchangeably with "anti-Ang 2 antibody", "Ang 2 binding antibody".
As used herein, an "antibody" is an immunoglobulin molecule that immunoreacts with a specific antigen, and means a protein molecule that serves as a receptor specifically recognizing an antigen, and may include all of polyclonal antibodies, monoclonal antibodies (single monoclonal antibodies), whole antibodies, and antibody fragments. In addition, the antibodies can include chimeric antibodies (e.g., humanized murine antibodies) and bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies.
A whole antibody has a structure comprising two full-length light chains and two full-length heavy chains, and each light chain may be linked to a heavy chain by a disulfide bond. Whole antibodies include IgA, IgD, IgE, IgM, and IgG is a subtype and includes IgG1, IgG2, IgG3, and
In the present disclosure, the antibody or antigen binding fragment thereof can bind to human and mouse Ang 2.
Antibody fragment means a fragment that retains the antigen-binding functionAnd includes Fab, Fab ', F (ab')2scFv, Fv, and the like.
The Fab has the structure of the variable regions of the light and heavy chains and the constant regions of the light and first constant regions of the heavy chains (CH1 domain), and has one antigen binding site. Fab 'differs from Fab in that Fab' has a hinge region that includes one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F (ab')2Antibodies are generated by effecting disulfide bonding of cysteine residues in the hinge region of Fab'.
Fv (variable fragment) refers to the smallest antibody fragment having only the variable regions of the heavy and light chains. In the two-chain fv (dsfv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond. In single chain fv (scfv), the heavy chain variable region and the light chain variable region are typically linked by a covalent bond using a peptide linker. These antibody fragments can be obtained by using a proteolytic enzyme (for example, Fab can be obtained by restriction cleavage of a whole antibody with papain, and F (ab')2 fragment can be obtained by cleavage with pepsin), and can be constructed by recombinant DNA techniques (for example, by amplification by a PCR (polymerase chain reaction) method using a DNA encoding a heavy chain of an antibody or a variable region thereof and a DNA encoding a light chain or a variable region thereof as templates and using a primer pair, and amplification by a combination of DNAs encoding peptide linkers of the primer pair, whereby both ends of the peptide linkers are linked to the heavy chain or the variable region thereof and the light chain or the variable region thereof, respectively).
In the present disclosure, the antibody or antigen binding fragment thereof may be humanized. Preferably, the anti-Ang 2 antibody according to the present invention may be a fully human antibody selected from a human antibody library, but is not limited thereto.
The antibody or antigen-binding fragment thereof according to the present invention is characterized by containing a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence of SEQ ID No. 3, heavy chain CDR2 having the amino acid sequence of SEQ ID No. 4, heavy chain CDR3 having the amino acid sequence of SEQ ID No. 5; and a light chain variable region comprising light chain CDR1 having the amino acid sequence of
The antibody or antigen-binding fragment thereof according to the present invention is characterized by containing a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence of SEQ ID No. 13, heavy chain CDR2 having the amino acid sequence of SEQ ID No. 14, heavy chain CDR3 having the amino acid sequence of SEQ ID No. 15; and a light chain variable region comprising light chain CDR1 having the amino acid sequence of SEQ ID NO 16, light chain CDR2 having the amino acid sequence of SEQ ID NO 17, light chain CDR3 having the amino acid sequence of SEQ ID NO 18.
In the present invention, the antibody or antigen-binding fragment thereof is characterized by comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 9, 19, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, or 111; and a light chain variable region comprising, but not limited to, the amino acid sequence of
The amino acid sequence of the antibody may be substituted by conservative substitutions. The "conservative substitution" refers to a modification of a polypeptide, which includes the substitution of at least one amino acid with an amino acid having similar biochemical properties as the corresponding polypeptide, without causing a loss of biological or biochemical function. "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. The art defines classes of amino acid residues with similar side chains. These species include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Antibodies of the invention are expected to have conservative amino acid substitutions while still retaining activity.
In the present invention, the antibody or antigen-binding fragment thereof is characterized by containing the Complementarity Determining Regions (CDRs) of the antibody produced by the cell line deposited under accession No. KCLRF-BP-00417 or KCLRF-BP-00418.
The anti-Ang 2 antibody sequences of the invention can be different from those provided herein. For example, the amino acid sequence may differ from those listed above in that: (a) the variable region may be separated from the constant domain of the light chain, (b) the amino acids may differ from those listed above without thereby significantly affecting the chemical properties of the residues (so-called conservative substitutions), (c) the amino acids may differ from those listed above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. Alternatively, the nucleic acid encoding the antibody may (a) be separate from the constant domain of the light chain, (b) be different from those listed above without thereby altering the encoded residues, or (c) may differ from those listed above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.
In making conservative changes in amino acid sequence, the hydropathic index of amino acids may be considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions on proteins is generally understood in the art. It is recognized that the relative hydrophilic character of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
It is also understood in the art that substitution of like amino acids can be made effectively based on hydrophilicity. For example, the maximum local average hydrophilicity of a protein, as governed by the hydrophilicity of its neighboring amino acids, correlates with the biological properties of the protein. It is understood that an amino acid may be substituted for another amino acid with similar hydrophilicity and result in a biologically or immunologically modified protein. In such variations, substitutions of amino acids having hydrophilicity values within +/-2 are preferred, those within +/-1 are particularly preferred, and those within +/-0.5 are even more particularly preferred.
As noted above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the various aforementioned characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In another aspect, the present invention relates to a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof as an active ingredient.
The pharmaceutical composition is characterized by comprising a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to the present invention and a pharmaceutically acceptable carrier.
The pharmaceutical composition may further comprise a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy. The VEGF antagonist can be an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating an eye disease, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.
In another aspect, the present invention relates to a method for inhibiting choroidal neovascularization, inhibiting ocular vascular leakage, or simultaneously triggering choroidal capillary regeneration in a patient having an ocular disease, said method comprising administering to said patient the above pharmaceutical composition.
The pharmaceutical composition for preventing or treating eye diseases may further comprise a small molecule inhibitor or a Vascular Endothelial Growth Factor (VEGF) antagonist used in chemotherapy. The VEGF antagonist can be an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
The methods may further comprise administering a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy, either simultaneously or stepwise with the administration of the antibody or fragment thereof of the invention. The VEGF antagonist can be an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
The anti-Ang 2 antibody or an antigen-binding fragment thereof has a function of inhibiting abnormal angiogenesis by inhibiting the function of Ang2, and thus has an effect of preventing or treating an eye disease accompanied by vascular abnormality.
As used herein, the term "prevention" refers to any action that inhibits or slows the progression of an ocular disease by administration of a composition according to the present invention, and the term "treatment" refers to inhibiting, reducing or eliminating the development of an ocular disease.
In the present invention, the eye disease is wet age-related macular degeneration (wAMD), Diabetic Macular Edema (DME), or Diabetic Retinopathy (DR), but is not limited thereto.
As used herein, the term "macular degeneration" refers to a condition in which neovascularization abnormally grows, causing damage to the macula and affecting vision. Macular degeneration occurs predominantly over the age of 50 and is classified as either non-exudative (dry) or exudative (wet). Especially in the case of wet AMD, blindness can result. The etiology of AMD has not been elucidated, but the risk factor is known to be age; and environmental factors including smoking, hypertension, obesity, genetic susceptibility, excessive UV exposure, low serum antioxidant concentrations, and the like.
As used herein, the term "macular edema" refers to the swelling of the macula of the retina, and the swelling occurs due to fluid leakage from the retinal blood vessels. Blood leaks out of the weakened vessel wall into the local area of the macula, which is the color sensitive nerve endings and where the retinal cone is abundant. The image is then faded to the right of the center or to the center of the center area. Vision gradually decreases over several months. As used herein, the term "diabetic retinopathy" refers to a complication of the eye in which vision is degraded due to retinal microcirculation disturbance caused by peripheral circulatory disturbance caused by diabetes. Initially, it may cause mild problems of vision, but ultimately may cause blindness. Diabetic retinopathy may occur in any person with type 1 diabetes or type 2 diabetes.
The present invention provides pharmaceutical compositions comprising a therapeutically effective amount of an anti-Ang 2 antibody and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" is a material that can be added to the active ingredient to aid in the formulation or stabilization of the formulation, and which does not cause significant adverse toxicological effects to the patient.
By carrier is meant a carrier or diluent that does not inhibit the biological activity and properties of the administered compound and does not irritate the patient. The pharmaceutically acceptable carrier in the composition to be formulated as a liquid solution is sterilized and is suitable for living bodies. Saline, sterile water, ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol may be used as a carrier, or at least one component thereof may be mixed to be used, and other conventional additives such as an antioxidant, a buffer, a bacteriostatic agent, and the like may be added as needed. In addition, the composition may be prepared into injections (such as aqueous solutions, suspensions, emulsions, etc.), pills, capsules, granules or tablets by further adding thereto diluents, dispersants, surfactants, binders and lubricants. Other carriers are described, for example, in [ Remington's Pharmaceutical Sciences (e.w. martin) ]. The compositions may contain a therapeutically effective amount of at least one anti-Ang 2 antibody.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of extemporaneous sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active materials is known in the art. The compositions are preferably formulated for parenteral injection. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases, the composition may include an isotonic agent (e.g., sugars), polyhydric alcohols (e.g., mannitol, sorbitol), or sodium chloride. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of the above ingredients, followed by sterile microfiltration as required. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other desired ingredients from those listed above. Sterile powders for the preparation of sterile injectable solutions are obtained by vacuum drying and freeze-drying (lyophilizing) the powder of the active ingredient and any additional powders of the desired ingredients from previously sterile-filtered solutions.
The pharmaceutical compositions may be administered orally or parenterally, the dose and frequency of which may vary depending on the severity of the patient. The composition may be administered to the patient by bolus injection or by continuous infusion as desired. For example, a bolus administration of an antibody of the invention presented as a Fab fragment may have an amount of 0.0025 to 100mg/kg body weight, 0.025 to 0.25mg/kg, 0.010 to 0.10mg/kg, or 0.10 to 0.50 mg/kg. For continuous infusion, an antibody of the invention presented as a Fab fragment may be administered at 0.001 to 100 mg/kg/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min, or 0.10 to 0.50 mg/kg/min for 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours. When an antibody of the invention is administered as a full length antibody (with intact constant regions), the amount administered may be about 1 to 10mg/kg body weight, 2 to 8mg/kg, or 5 to 6 mg/kg. Full-length antibodies are typically administered by injection lasting from 30 minutes to 35 minutes. The frequency of administration depends on the severity of the condition. The frequency may be 3 to once a week or two weeks.
In addition, the composition may be administered to a patient by subcutaneous injection. For example, the anti-Ang 2 antibody can be administered to a patient with an administration amount of 10 to 100mg weekly, biweekly, or monthly by subcutaneous injection.
As used herein, "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, as well as the combined amounts of anti-Ang 2 antibodies. The precise amount may vary depending on a number of factors including the components and physical characteristics of the therapeutic composition, the intended patient population, individual patient considerations, and the like, but is not so limited and can be readily determined by one of ordinary skill in the art. When these factors are fully considered, it is important to administer a minimum amount sufficient to obtain the maximum effect without side effects, and this dose can be easily determined by the expert in the art.
The dosage of the pharmaceutical composition of the present invention is not particularly limited, but varies according to various factors including the health and weight of a patient, the severity of a disease, and the type of drug, administration route and administration time. The composition may be administered in a single dose or multiple doses per day by a route typically allowed in mammals (including rats, mice, cows, humans, etc.), for example, orally, rectally, intravenously, subcutaneously, intrauterine, or intracerebroventricularly.
In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, containing the antibody or the antigen-binding fragment thereof as an active ingredient.
In another aspect, the invention relates to a method for inhibiting tumor growth and treating cancer in a patient comprising administering to the patient a pharmaceutical composition comprising an antibody or antigen-binding fragment described above. The methods may further comprise administering a small molecule inhibitor or Vascular Endothelial Growth Factor (VEGF) antagonist for use in chemotherapy, either simultaneously or stepwise with the administration of the antibody or fragment thereof of the invention. The VEGF antagonist can be an anti-VEGF antibody, a VEGF-inhibiting fusion protein, or a small molecule kinase inhibitor.
As used herein, the term "cancer" or "tumor" generally refers to or describes the physiological condition of a mammal characterized by uncontrolled cell growth/proliferation.
The cancer that can be treated with the composition of the present invention is not particularly limited, and includes both solid cancer and hematological cancer. Examples of such cancers include, but are not limited to, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, cancer of the skin, cutaneous or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, cancer of the small intestine, endocrine cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, cancer of the urinary tract, chronic or acute leukemia, lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, and the like.
The composition for preventing or treating cancer includes an anti-Ang 2 antibody, and its constitution is the same as that included in the composition for preventing or treating ocular diseases, and thus the description of each constitution is equally applicable to the composition for preventing or treating cancer.
The application also contemplates the use of the anti-Ang 2 antibodies described herein in conjunction with chemical or radiation therapeutic intervention or other therapy. In particular, combining an anti-Ang 2 antibody with other therapies targeting different aspects of Ang2 function may also prove effective.
In another embodiment, the antibodies of the invention may be linked to at least one agent to form an antibody conjugate to increase the efficacy of the antibody molecule as a diagnostic or therapeutic agent.
In another aspect of the invention, the invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.
The nucleic acids used herein may be present in the cell, cell lysate, or may also be present in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. The nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not comprise an intron sequence.
In yet another aspect of the invention, the invention relates to a recombinant expression vector comprising said nucleic acid.
To express the antibody or fragment thereof, DNA encoding light and heavy chains having partial or full lengths may be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the target antibody), and may be "operably associated with" transcriptional and translational control sequences to be inserted into an expression vector.
The term "operably linked" as used herein may indicate that an antibody gene is ligated into a vector such that transcriptional and translational control sequences in the vector have the intended function of controlling the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to have compatibility with the host cell to be used for expression. The light chain gene of the antibody and the heavy chain gene of the antibody are inserted into separate vectors, or the two genes are inserted into the same expression vector. The antibody is inserted into the expression vector by standard methods (e.g., ligation of antibody gene fragments to complementary restriction enzyme sites on the vector, or blunt-ended ligation when no restriction enzyme sites are present at all). In some cases, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain by the host cell. The antibody chain genes can be cloned into a vector such that the signal peptide binds to the amino terminus of the antibody chain gene according to the framework. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a protein other than an immunoglobulin). In addition, the recombinant expression vector has regulatory sequences that control the expression of the antibody chain gene in the host cell. "regulatory sequences" may include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. One skilled in the art will recognize that the design of the expression vector can be varied by varying the regulatory sequences depending on factors such as the choice of the host cell to be transformed, the level of expression of the protein, etc.
In yet another aspect, the invention relates to a cell transformed with a recombinant expression vector.
The cells used to produce the antibodies of the present disclosure can be prokaryotes, yeast, or higher eukaryotes, but are not limited thereto.
Specifically, strains of the genus Bacillus (e.g., Escherichia coli), Bacillus subtilis, and eubacterium tuligensis), Streptomyces (Streptomyces), Pseudomonas (e.g., Pseudomonas putida), and prokaryotic host cells such as Proteus mirabilis (Proteus mirabilis) and Staphylococcus (e.g., Staphylococcus carnosus) can be used.
Examples of host cell lines of greatest interest and usefulness for animal cells include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI,
The nucleic acid or vector is transfected into a host cell. For "transfection", various commonly used techniques (e.g., electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc.) can be used to introduce exogenous nucleic acid (DNA or RNA) into a prokaryotic or eukaryotic host cell. The antibodies according to the invention may be expressed in eukaryotic cells, preferably in mammalian host cells, in view of their suitability for use in mammalian cells. Mammalian host cells suitable for expression of the antibody may include Chinese Hamster Ovary (CHO) cells (e.g., including DHFR-CHO cells used with DHFR selectable markers), NSO myeloma cells, COS cells, or SP2 cells, among others.
In another aspect, the invention relates to a method for producing an anti-Ang 2 antibody or antigen-binding fragment thereof, comprising culturing a host cell and expressing the antibody or antigen-binding fragment thereof.
When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody can be produced by: the host cell is cultured for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably, for a sufficient period of time to allow the antibody to be secreted into the medium in which the host cell is cultured.
In some cases, the expressed antibody may be isolated from the host cell and purified for homogeneity. The antibody can be isolated or purified by a separation method, a purification method (e.g., chromatography) commonly used for proteins. The chromatography may include, for example, affinity chromatography, ion exchange chromatography, or hydrophobic chromatography including a protein a column and a protein G column. In addition to chromatography, the antibody may also be isolated and purified by further combination with filtration, ultrafiltration, salting out, dialysis, and the like.
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