An antibody drug conjugate targeting vegfr2 and preparation and application thereof

Abstract

The application provides an antibody conjugate drug targeting VEGFR2 and a preparation and application thereof. The antibody conjugate drug is synthesized by an amide reaction of an antibody fragment anti-VEGFR2 F(ab')2, a connecting bond ketone condensed thioalcohol and a model drug. A kidney disease antibody conjugate drug preparation platform is established by using anti-VEGFR2 F(ab')2 and a ketone condensed thioalcohol bond, and the anti-VEGFR2 F(ab')2 conjugated model drug can improve kidney distribution of the model drug, improve kidney cell uptake, block VEGFR2 and play a kidney disease treatment role, so that multi-target point treatment of the antibody-drug is realized. The antibody conjugate drug provided by the application can be applied to preparation of a kidney disease targeted treatment drug, can significantly improve kidney disease treatment effect of the model drug, and can be widely applied to the fields of life science and pharmacy. A structure of the antibody conjugate drug is shown in formula 1.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to an antibody-drug conjugate targeting VEGFR2 and its preparation and application. It is a type of antibody-drug conjugate targeting VEGFR2 for targeted repair of kidney diseases, its preparation method, and its application in preparing drugs for treating kidney-related diseases using it as a template. Background Technology

[0002] The incidence of kidney disease is increasing year by year, becoming a significant public health issue. The development of highly effective drugs for treating kidney disease remains a huge clinical need. Kidney-targeted drug delivery is a crucial means to improve the efficacy of kidney disease treatments. Antibody-drug conjugates (ADCs) combine the high specificity of monoclonal antibody drugs with the high activity of small molecule drugs, utilizing antibodies to selectively deliver drugs to the lesion site. They are characterized by high specificity and low adverse reactions, and have become a major class of new drugs developed in recent years, widely used in immune and metabolic diseases outside of oncology. However, ADCs are less commonly used in the field of kidney disease, mainly due to their poor renal distribution and low bioavailability, which can lead to side effects. Studies have shown that molecular weight is one of the important factors affecting the renal distribution of drugs or carriers. For IgG type antibodies (composed of F(ab′)2 and Fc), removing the Fc fragment to reduce the molecular weight of the monoclonal antibody can reduce non-specific binding without significantly affecting its antibody-receptor specific binding ability, and this holds promise as a strategy for applying ADCs in the field of kidney disease.

[0003] Overactivation of vascular endothelial growth factor receptor 2 (VEGFR2) is widespread in various chronic kidney diseases associated with inflammation and fibrosis, such as diabetic nephropathy, hyaline occlusion glomerular microangiopathy, glomerulonephritis, and glomerulosclerosis. VEGFR2 is a specific receptor for VEGF and Gremlin. Activation of VEGF / VEGFR2 is the main mechanism stimulating vascular endothelial cells. VEGF, also known as the vascular permeability factor, participates in the progression of chronic kidney disease by interacting with VEGFR2 to alter endothelial cell structure and function, increase endothelial cell migration and leakage, increase glomerular capillary permeability, promote extracellular matrix synthesis, and induce renal hypertrophy. Gremlin activation of VEGFR2 mainly occurs in the tubulointerstitium, contributing to kidney damage by promoting fibrosis through tubulointerstitial injury. Studies have shown that specifically blocking the VEGFR2 pathway with VEGFR2 monoclonal antibodies (anti-VEGFR2) can improve renal function, glomerular damage (mesangial matrix expansion and basement membrane thickening), tubulointerstitial damage, and downregulation of pro-inflammatory mediators. Therefore, anti-VEGFR2 is a candidate antibody for treating inflammation- and fibrosis-related chronic kidney disease. Due to the complex pathological mechanisms of chronic kidney disease, multi-target therapy can help improve treatment efficacy. Therefore, when preparing antibody-drug conjugates, drugs with different targets than anti-VEGFR2 and suitable for treating kidney diseases (small molecules, peptides, nucleic acids, etc.) can be used, such as anti-inflammatory drugs like the glucocorticoids dexamethasone and budesonide, paeoniflorin (a natural product extract that inhibits autophagy and apoptosis), interleukin 8-47 (a peptide that protects endothelial barrier function), mitochondrial-targeting antioxidant peptide SS31, and fibrotic interleukin-11 siRNA. It is worth noting that the aforementioned drugs have shortcomings in application, including strong side effects, poor plasma stability, and poor cell transmembrane ability. Conjugating these drugs to the F(ab′)2 fragment of anti-VEGFR2 (anti-VEGFR2 F(ab′)2) can effectively improve renal delivery efficiency and mitigate these drawbacks. Furthermore, anti-VEGFR-2(Fab′)2 can block VEGFR2, exhibiting certain therapeutic effects, which also provides possibilities for multi-target therapy with antibodies and drugs. Linkers are responsible for connecting antibodies and drugs; through rational design, drug plasma stability can be improved and controlled release can be achieved. In the pathological microenvironment of kidney disease, oxidative stress is a typical feature, manifested as increased intracellular ROS levels. Ketothiols (TK bonds) are ROS-responsive; using them as linkers can release drugs into damaged cells with increased ROS levels, thereby exerting a therapeutic effect. Summary of the Invention

[0004] The first objective of this invention is to provide an antibody-drug conjugate targeting VEGFR2, the structure of which is shown in Formula 1:

[0005]

[0006] Where n = 1 to 8.

[0007] This antibody-drug conjugate is synthesized via an amide reaction using an antibody fragment anti-VEGFR2 F(ab′)2 (molecular weight 110 kDa), a linker ketithiolide (NHS-TK-NHS, formula 2), and a model drug. The model drug is a drug containing or modified with a -NH2 group, or a kidney disease treatment drug with synergistic therapeutic effects with VEGFR2 monoclonal antibodies, including SS31 (CAS No. 736992-21-5, molecular formula C). 32 H 49 N9O5, an antioxidant peptide), dexamethasone (CAS No. 4089-36-5, molecular formula C...). 22 H 29 FO5 (a glucocorticoid), budesonide (CAS No. 51333-22-3, molecular formula C50 ... 25 H 34 O6 (glucocorticoid), paeoniflorin (CAS No. 23180-57-6, molecular formula C6) 23 H 28 O 11 It inhibits the natural products of autophagy and apoptosis, and mediastin 8-47 (amino acid sequence shown in SEQ ID NO.1).

[0008] Val-Gly-Cys-Val-Leu-Gly-Thr-Cys-Gln-Val-Gln-Asn-Leu-Ser-His-Arg-Leu-Trp-Gln-Leu-Val-Arg-Pro-Ala-Gly-Arg-Arg-Asp-Ser-Ala-Pro-Val-Asp-Pro-Ser-Ser-Pro-His-Ser-Tyr-NH2 (Disulfide bridge: Cys10-Cys15, a polypeptide protecting endothelial barrier function) and interleukin-11 siRNA (nucleotide sequence SEQ ID NO.2: Sense(5'-3')GCUGUUCUCCUAACCCGAUTT, SEQ ID NO.3: Anti-sense(5'-3')

[0009] AUCGGGUUAGGAGAACAGCTT (inhibits fibrinolytic small interfering RNA).

[0010]

[0011] The second objective of this invention is to provide a method for preparing an antibody-drug conjugate targeting VEGFR2 (anti-VEGFR2 F(ab′)2 antibody-drug conjugate), specifically achieved through the following steps:

[0012] 1. Preparation of anti-VEGFR2 F(ab′)2 antibody fragment

[0013] Take 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 25–15:1 and incubate at 37°C for 5–7 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify by ultrafiltration and centrifugation to obtain F(ab′)2 (… Figure 1 The molecular weight of F(ab′)2 was determined to be approximately 110 kDa using SDS-PAGE. Mass spectrometry analysis of the anti-VEGFR2 F(ab′)2 after TCEP reduction showed the disappearance of the molecular ion peak of the heavy chain containing the Fc segment, confirming the effective removal of the Fc segment of anti-VEGFR2 and thus demonstrating the successful preparation of F(ab′)2.

[0014] The reaction formula is as follows:

[0015]

[0016] 2. Drug conjugation technology

[0017] Anti-VEGFR2 F(ab′)2-TK-drug was synthesized by linking anti-VEGFR2 F(ab′)2 and a drug via an amide reaction using NHS-TK-NHS (synthesized according to the reference). 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to PBS (pH 8–9) or DMF solution containing the drug (containing or modified with -NH2 groups) at a molar ratio of 2–10:2.4–24:1. After reacting for 15 min at room temperature, F(ab′)2 in PBS (pH 8–9) solution was added, and the reaction was continued for 30 min at room temperature. The amide reaction was terminated by adding lysine, and F(ab′)2-TK-drug was purified by ultrafiltration and centrifugation. Disulfide bonds in F(ab′)2-TK-drug were reduced using TCEP to obtain light chains or light chains linked with model drugs. Mass spectrometry was used to detect and record mass spectra in the range of 18-30 kDa. The drug-antibody molar ratio (drug-antibody ratio, DAR) of F(ab′)2-TK-drug was calculated to be 1-8 based on the peak area of ​​light chains linked with different numbers of SS31.

[0018] A third objective of this invention is to provide the application of the aforementioned antibody-drug conjugate as a template in the preparation of targeted therapies for kidney diseases. VEGFR2 overexpression and activation are present in various kidney diseases, including diabetic nephropathy, hyaline occlusion glomerular microangiopathy, glomerulonephritis, and glomerulosclerosis. anti-VEGFR2 F(ab′)2 can target VEGFR2 to enhance the uptake of bound therapeutic drugs (dexamethasone, budesonide, paeoniflorin, interleukin 8-47, SS31, and interleukin-11 siRNA) by renal cells, and can also block VEGFR2 to exert therapeutic effects by inhibiting inflammation and fibrosis. Anti-VEGFR2F(ab′)2-TK-drug, through antibody-drug multi-target therapy, significantly improves the therapeutic effect of kidney diseases.

[0019] This invention utilizes an anti-VEGFR2 F(ab′)2 fragment, an NHS-TK-NHS linker, and a model drug to synthesize an anti-VEGFR2 F(ab′)2-TK-drug antibody-drug conjugate. Conjugating the model drug with anti-VEGFR2 F(ab′)2 improves the renal distribution of the model drug, enhances renal cell uptake, and simultaneously blocks VEGFR2 from exerting its therapeutic effect on kidney diseases, thereby achieving multi-target antibody-drug therapy. The antibody-drug conjugate provided by this invention can significantly improve the therapeutic effect of the model drug on kidney diseases and can be widely applied in the life sciences and pharmaceutical fields.

[0020] This invention establishes an antibody-drug conjugate (ADC) preparation platform for kidney diseases using anti-VEGFR2 F(ab′)2 and TK bonds. It can rationally select model drugs, including small molecule chemical drugs, peptides, and nucleic acids, in combination with specific disease types, thereby improving the renal cell targeted uptake of drugs and reducing side effects. Combined with the VEGFR2 blocking ability of anti-VEGFR-2(Fab′)2, it can achieve multi-target treatment of kidney diseases. Attached Figure Description

[0021] Figure 1 This is the mass spectrum of anti-VEGFR2 F(ab′)2 after TCEP reduction. The detection range is 10-60 kDa. LC + LC 2+ and HC + These represent light chains with single protonation and single charge, light chains with two protonation and two charge, and heavy chains with single protonation and single charge, respectively.

[0022] Figure 2 This is the mass spectrum of anti-VEGFR2 F(ab′)2-TK-SS31(DAR 2) after TCEP reduction. The detection range is 18-30 kDa. LC +[LC+2×SS31] + and [LC+3×SS31] + These represent light chains with single protonation and single charge, light chains with single protonation and single charge coupling of two SS31 light chains, and light chains with single protonation and single charge coupling of three SS31 light chains, respectively.

[0023] Figure 3 This shows the distribution of anti-VEGFR2 F(ab′)2 in the major organs of diabetic nephropathy mice.

[0024] Figure 4 This method uses a double-antigen sandwich assay to detect the receptor binding capacity of anti-VEGFR2 F(ab′)2-TK-SS31 with different drug-antibody ratios.

[0025] Figure 5 Proteinuria levels in diabetic nephropathy mice treated with anti-VEGFR2 F(ab′)2-TK-SS31 (DAR 2). Detailed Implementation

[0026] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0027] Example 1: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0028] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0029] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which showed that it was approximately 110 kDa, significantly lower than that of the intact antibody anti-VEGFR2 (~150 kDa). Mass spectrometry was used to examine whether the Fc fragment of anti-VEGFR2 was effectively removed to further confirm the successful preparation of F(ab′)2. Since the Fc segment consists of the constant region of the antibody heavy chain, the F(ab′)2 disulfide bonds (two between the light and heavy chains and two between the heavy chains) were first reduced using tris(2-carboxyethyl)phosphine (TCEP). Mass spectra in the mass-to-charge ratio range of 10-60 kDa were then detected and recorded using a Lex TOF / TOF mass spectrometer (Bruker). Figure 1 The results showed that the light chain proton peak of anti-VEGFR2 was retained, but the heavy chain proton peak almost disappeared, indicating that the Fc fragment of anti-VEGFR2 was effectively removed by pepsin hydrolysis, and anti-VEGFR2F(ab′)2 was successfully obtained.

[0030] 2. Drug conjugation technology

[0031] Using the antioxidant peptide SS31 as a model drug, anti-VEGFR2 F(ab′)2-TK-SS31 was synthesized by linking anti-VEGFR2 F(ab′)2 and SS31 via an amide reaction using NHS-TK-NHS (synthesized according to the reference). 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to SS31 in PBS (pH 8–9) solution at a molar ratio of 2:2.4:1 (NHS-TK-NHS:SS31:anti-VEGFR2 F(ab′)2). After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8–9) solution was added, and the reaction was continued at room temperature for 30 min. The amide reaction was terminated by adding lysine, and anti-VEGFR2F(ab′)2-TK-SS31 was purified by ultrafiltration and centrifugation. The disulfide bonds of anti-VEGFR2 F(ab′)2-TK-SS31 were reduced using TCEP to obtain light chains or light chains linked with SS31. Mass spectra in the mass-to-charge ratio range of 18-30 kDa were detected and recorded using mass spectrometry. The DAR of anti-VEGFR2 F(ab′)2-TK-SS31 was calculated to be 1 based on the peak areas of light chains linked with different numbers of SS31. Molecular weight was investigated using SDS-PAGE.

[0032] Example 2: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0033] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0034] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was approximately 110 kDa as determined by SDS-PAGE. Mass spectrometry was used to detect the TCEP-reduced anti-VEGFR2 F(ab′)2 peaks. The disappearance of the molecular ion peak of the heavy chain containing the Fc fragment confirmed that the Fc fragment of anti-VEGFR2 was effectively removed, thus proving the successful preparation of anti-VEGFR2 F(ab′)2.

[0035] 2. Drug conjugation technology

[0036] Using the antioxidant peptide SS31 as a model drug, anti-VEGFR2 F(ab′)2-TK-SS31 was synthesized by linking anti-VEGFR2 F(ab′)2 and SS31 via an amide reaction using NHS-TK-NHS (synthesized according to the reference). 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to SS31 in PBS (pH 8–9) at a molar ratio of 12:14.4:1. After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8–9) was added, and the reaction was continued for 30 min at room temperature. The amide reaction was terminated by adding lysine, and anti-VEGFR2F(ab′)2-TK-SS31 was purified by ultrafiltration and centrifugation. The disulfide bonds of anti-VEGFR2 F(ab′)2-TK-SS31 were reduced using TCEP to obtain light chains or light chains linked with SS31. Mass spectra in the mass-to-charge ratio range of 18-30 kDa were detected and recorded using mass spectrometry. The DAR of anti-VEGFR2 F(ab′)2-TK-SS31 was calculated to be 8 based on the peak areas of light chains linked with different numbers of SS31. Molecular weight was investigated using SDS-PAGE.

[0037] Example 3: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0038] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0039] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was approximately 110 kDa as determined by SDS-PAGE. Mass spectrometry was used to detect the TCEP-reduced anti-VEGFR2 F(ab′)2 peaks. The disappearance of the molecular ion peak of the heavy chain containing the Fc fragment confirmed that the Fc fragment of anti-VEGFR2 was effectively removed, thus proving the successful preparation of anti-VEGFR2 F(ab′)2.

[0040] 2. Drug conjugation technology

[0041] Using the antioxidant peptide SS31 as a model drug, anti-VEGFR2 F(ab′)2-TK-SS31 was synthesized by linking anti-VEGFR2 F(ab′)2 and SS31 via an amide reaction using NHS-TK-NHS (synthesized according to the reference). 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to SS31 in PBS (pH 8–9) solution at a molar ratio of 4:4.8:1 (NHS-TK-NHS:SS31:anti-VEGFR2 F(ab′)2). After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8–9) solution was added, and the reaction was continued at room temperature for 30 min. The amide reaction was terminated by adding lysine, and anti-VEGFR2F(ab′)2-TK-SS31 was purified by ultrafiltration and centrifugation. The disulfide bonds of anti-VEGFR2 F(ab′)2-TK-SS31 were reduced using TCEP to obtain light chains or light chains linked with SS31. Mass spectrometry was used to detect and record mass spectra in the range of 18-30 kDa. Figure 2 The DAR of anti-VEGFR2 F(ab′)2-TK-SS31 was calculated to be 2 based on the peak area of ​​light chains linking different numbers of SS31. Molecular weight was investigated using SDS-PAGE.

[0042] Example 4: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0043] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0044] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was approximately 110 kDa as determined by SDS-PAGE. Mass spectrometry was used to detect the TCEP-reduced anti-VEGFR2 F(ab′)2 peaks. The disappearance of the molecular ion peak of the heavy chain containing the Fc fragment confirmed that the Fc fragment of anti-VEGFR2 was effectively removed, thus proving the successful preparation of anti-VEGFR2 F(ab′)2.

[0045] 2. Drug conjugation technology

[0046] Using aminated dexamethasone (DXMS) as a model drug, anti-VEGFR2 F(ab′)2-TK-DXMS was synthesized by linking anti-VEGFR2 F(ab′)2 with NHS-TK-NHS (synthesized according to the reference) via an amide reaction. 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to the DXMS DMF solution at a molar ratio of 3:3.6:1 (NHS-TK-NHS:DXMS:anti-VEGFR2 F(ab′)2). After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8–9) solution was added, and the reaction was continued at room temperature for 30 min. The amide reaction was terminated by adding lysine, and the anti-VEGFR2F(ab′)2-TK-DXMS was purified by ultrafiltration and centrifugation. Disulfide bonds in anti-VEGFR2 F(ab′)2-TK-DXMS were reduced using TCEP to obtain light chains or light chains linked with DXMS. Mass spectra in the mass-to-charge ratio range of 18-30 kDa were detected and recorded using mass spectrometry. The DAR of anti-VEGFR2 F(ab′)2-TK-DXMS was calculated to be 2 based on the peak areas of light chains linked with different amounts of DXMS. Molecular weight was investigated using SDS-PAGE.

[0047] Example 5: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0048] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0049] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was approximately 110 kDa as determined by SDS-PAGE. Mass spectrometry was used to detect the TCEP-reduced anti-VEGFR2 F(ab′)2 peaks. The disappearance of the molecular ion peak of the heavy chain containing the Fc fragment confirmed that the Fc fragment of anti-VEGFR2 was effectively removed, thus proving the successful preparation of anti-VEGFR2 F(ab′)2.

[0050] 2. Drug conjugation technology

[0051] Using aminated budesonide (BUD) as a model drug, anti-VEGFR2 F(ab′)2-TK-BUD was synthesized by linking anti-VEGFR2 F(ab′)2 with NHS-TK-NHS (synthesized according to the reference) via an amide reaction. 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to BUD in a DMF solution at a molar ratio of 3:3.6:1 (NHS-TK-NHS:BUD:anti-VEGFR2 F(ab′)2). After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8-9) solution was added, and the reaction was continued at room temperature for 30 min. The amide reaction was terminated by adding lysine, and anti-VEGFR2 was purified by ultrafiltration and centrifugation.

[0052] F(ab′)2-TK-BUD. The disulfide bonds of anti-VEGFR2 F(ab′)2-TK-BUD were reduced using TCEP to obtain light chains or light chains linked with BUD. Mass spectra in the mass-to-charge ratio range of 18-30 kDa were detected and recorded using mass spectrometry. The DAR of anti-VEGFR2 F(ab′)2-TK-BUD was calculated to be 2 based on the peak areas of light chains linked with different numbers of BUD. Molecular weight was investigated using SDS-PAGE.

[0053] Example 6: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0054] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0055] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was approximately 110 kDa as determined by SDS-PAGE. Mass spectrometry was used to detect the TCEP-reduced anti-VEGFR2 F(ab′)2 peaks. The disappearance of the molecular ion peak of the heavy chain containing the Fc fragment confirmed that the Fc fragment of anti-VEGFR2 was effectively removed, thus proving the successful preparation of anti-VEGFR2 F(ab′)2.

[0056] 2. Drug conjugation technology

[0057] Using aminated paeoniflorin (PF) as a model drug, anti-VEGFR2 F(ab′)2-TK-PF was synthesized by linking anti-VEGFR2 F(ab′)2 and PF via an amide reaction using NHS-TK-NHS (synthesized according to the reference). 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to PF in a DMF solution at a molar ratio of 4:4.8:1 (NHS-TK-NHS:PF:anti-VEGFR2 F(ab′)2). After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8–9) solution was added, and the reaction was continued at room temperature for 30 min. The amide reaction was terminated by adding lysine, and anti-VEGFR2 F(ab′)2-TK-PF was purified by ultrafiltration and centrifugation. Disulfide bonds in anti-VEGFR2 F(ab′)2-TK-PF were reduced using TCEP to obtain light chains or light chains linked with PF. Mass spectra in the mass-to-charge ratio range of 18-30 kDa were detected and recorded using mass spectrometry. The DAR of anti-VEGFR2 F(ab′)2-TK-PF was calculated to be 2 based on the peak areas of light chains linked with different numbers of PF. The molecular weight was investigated using SDS-PAGE.

[0058] Example 7: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0059] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0060] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was approximately 110 kDa as determined by SDS-PAGE. Mass spectrometry was used to detect the TCEP-reduced anti-VEGFR2 F(ab′)2 peaks. The disappearance of the molecular ion peak of the heavy chain containing the Fc fragment confirmed that the Fc fragment of anti-VEGFR2 was effectively removed, thus proving the successful preparation of anti-VEGFR2 F(ab′)2.

[0061] 2. Drug conjugation technology

[0062] Using intermedulin 8-47 (IMD8-47) as a model drug, anti-VEGFR2 F(ab′)2-TK-IMD was synthesized by linking anti-VEGFR2 F(ab′)2 and IMD via an amide reaction using NHS-TK-NHS (synthesized according to the reference). 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to IMD in PBS (pH 8–9) solution at a molar ratio of 4:4.8:1 (NHS-TK-NHS:IMD:anti-VEGFR2 F(ab′)2). After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8–9) solution was added, and the reaction was continued at room temperature for 30 min. The amide reaction was terminated by adding lysine, and anti-VEGFR2F(ab′)2-TK-IMD was purified by ultrafiltration and centrifugation. Disulfide bonds in anti-VEGFR2 F(ab′)2-TK-IMD were reduced using TCEP to obtain light chains or light chains linked with IMDs. Mass spectra in the mass-to-charge ratio range of 18-30 kDa were detected and recorded using mass spectrometry. The DAR of anti-VEGFR2 F(ab′)2-TK-IMD was calculated to be 2 based on the peak areas of light chains linked with different numbers of IMDs. The molecular weight was investigated using SDS-PAGE.

[0063] Example 8: Preparation of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate

[0064] 1. Preparation of Anti-VEGFR2 F(ab′)2 antibody fragment

[0065] Take 0.5 mL of 7 mg / mL VEGFR2 monoclonal antibody in PBS (pH 7.0, 0.1 M) (Clone#CD101, Bio X Cell, USA) and replace the PBS solvent with acetate-sodium acetate buffer (pH 3.0–3.5, 0.1 M). Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 20:1 and incubate at 37 °C for 6 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify F(ab′)2 by ultrafiltration and centrifugation. The molecular weight of F(ab′)2 was approximately 110 kDa as determined by SDS-PAGE. Mass spectrometry was used to detect the TCEP-reduced anti-VEGFR2 F(ab′)2 peaks. The disappearance of the molecular ion peak of the heavy chain containing the Fc fragment confirmed that the Fc fragment of anti-VEGFR2 was effectively removed, thus proving the successful preparation of anti-VEGFR2 F(ab′)2.

[0066] 2. Drug conjugation technology

[0067] Using interleukin-11 siRNA (siIL11) as a model drug, anti-VEGFR2 F(ab′)2-TK-siIL11 was synthesized by linking anti-VEGFR2 F(ab′)2 and siIL11 via an amide reaction using NHS-TK-NHS (synthesized according to the reference). 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to siIL11 in PBS (pH 8–9) at a molar ratio of 4:4.8:1 (NHS-TK-NHS:siIL11:anti-VEGFR2 F(ab′)2). After reacting at room temperature for 15 min, F(ab′)2 in PBS (pH 8–9) was added, and the reaction was continued for 30 min at room temperature. The amide reaction was terminated by adding lysine, and anti-VEGFR2 F(ab′)2-TK-siIL11 was purified by ultrafiltration and centrifugation. The disulfide bonds of anti-VEGFR2 F(ab′)2-TK-siIL11 were reduced using TCEP to obtain light chains or light chains linked with siIL11. Mass spectra in the mass-to-charge ratio range of 18-30 kDa were detected and recorded using mass spectrometry. The DAR of anti-VEGFR2 F(ab′)2-TK-siIL11 was calculated to be 2 based on the peak areas of light chains linked with different amounts of siIL11. The molecular weight was investigated using SDS-PAGE.

[0068] Example 9: Application of Anti-VEGFR2 F(ab′)2-TK-drug Antibody-Drug Conjugate in the Treatment of Kidney Disease

[0069] 1. Study on the renal targeting of Anti-VEGFR2 F(ab′)2

[0070] Using anti-VEGFR2 as a control, the targeting and distribution ability of anti-VEGFR2 F(ab′)2 in the kidneys of diabetic nephropathy mice was investigated. Anti-VEGFR2 F(ab′)2 was prepared according to Example 1, Section 1. ICR mice were used to establish a diabetic nephropathy mouse model by intraperitoneal injection of streptozotocin. Anti-VEGFR2 or anti-VEGFR2 F(ab′)2 labeled with the fluorescent dye Cy5 was then injected via the tail vein at a dose of 33 nmol / kg. Four hours after administration, the mice were sacrificed, and representative organs (heart, lung, liver, spleen, and kidney) were collected. Fluorescence images of each organ were captured using a Maestro in vivo imaging system, and the relative fluorescence intensity of each organ was semi-quantitatively analyzed. Results are as follows: Figure 3 As shown, the renal fluorescence intensity of anti-VEGFR2 F(ab′)2 was significantly higher than that of the anti-VEGFR2 group, indicating a good renal targeting distribution effect. Therefore, F(ab′)2 fragmentation improves the renal targeting distribution ability of anti-VEGFR2 monoclonal antibodies and may become an important means of modifying antibody drugs for the treatment of kidney diseases.

[0071] 2. Study on the binding affinity of Anti-VEGFR2 F(ab′)2-TK-drug antibody-drug conjugate.

[0072] Using the antioxidant peptide SS31 as a model drug, Examples 1, 2, and 3 were prepared, yielding anti-VEGFR2F(ab′)2-TK-SS31 (DAR 1), anti-VEGFR2 F(ab′)2-TK-SS31 (DAR 8), and anti-VEGFR2F(ab′)2-TK-SS31 (DAR 2), respectively. Using anti-VEGFR2 and anti-VEGFR2 F(ab′)2 as controls, the receptor binding affinity of each antibody-drug conjugate was investigated using a double-antigen sandwich method. The results are as follows: Figure 4As shown, the OD values ​​of the Anti-VEGFR2 F(ab′)2-TK-SS31 (DAR 1) and Anti-VEGFR2 F(ab′)2-TK-SS31 (DAR 2) groups were similar to those of the Anti-VEGFR2 and Anti-VEGFR2 F(ab′)2 control groups, indicating that modifying the antibody with a drug-to-antibody ratio of 1–2 has almost no effect on its binding ability. However, the OD value of the Anti-VEGFR2 F(ab′)2-TK-SS31 (DAR 8) group was lower than that of the Anti-VEGFR2 and Anti-VEGFR2 F(ab′)2 control groups, indicating that the binding ability of the antibody-drug conjugate is reduced when DAR is 8. Therefore, high DAR (DAR 8) can impair the antibody's receptor binding ability. In this study, the preferred DAR ratio was 2. Modifying the antibody with a DAR ratio of 2 had almost no effect on its binding ability, thus ensuring that the anti-VEGFR2 F(ab′)2-TK-SS31 antibody-drug conjugate could exert its therapeutic effect of blocking VEGFR2 and its drug delivery effect of targeting VEGFR2.

[0073] 3. Study on the therapeutic effect of anti-VEGFR2 F(ab′)2-TK-drug antibody-drug conjugate on kidney disease

[0074] Using the antioxidant peptide SS31 as a model drug, anti-VEGFR2 F(ab′)2-TK-SS31 (DAR 2) was prepared according to Example 3 to investigate its therapeutic effect on diabetic nephropathy. Anti-VEGFR2 F(ab′)2 and free SS31 served as control groups. Simultaneously, streptozotocin-induced diabetic nephropathy mice were used as model animals. After 5 weeks of tail vein administration, urine was collected from the mice, and urinary protein and creatinine were measured. Results are as follows... Figure 5 As shown, the anti-VEGFR2 F(ab′)2-TK-SS31-treated group of diabetic nephropathy mice had the lowest proteinuria level (urine protein / urine creatinine ratio) compared to the control group, indicating the best therapeutic effect on diabetic nephropathy. Therefore, anti-VEGFR2 F(ab′)2-TK-SS31 can improve the therapeutic effect of SS31 alone in diabetic nephropathy, which is related to the improvement of SS31 renal distribution by anti-VEGFR2 F(ab′)2 and the synergistic therapeutic effect of blocking VEGFR2.

Claims

1. An antibody-drug conjugate targeting VEGFR2, characterized in that, This antibody-drug conjugate is synthesized from the antibody fragment anti-VEGFR2 F(ab′)2, the linking ketithiolide, and the model drug via an amide reaction, as shown in Formula 1: Where: n = 1~8, The model drug is a drug containing or modified with a -NH2 group or a kidney disease treatment drug that has a synergistic therapeutic effect with VEGFR2 monoclonal antibody.

2. The antibody-drug conjugate according to claim 1, characterized in that, The model drugs include the antioxidant peptide SS31, dexamethasone, budesonide, paeoniflorin, interleukin 8-47, and interleukin-11 siRNA, wherein the amino acid sequence of interleukin 8-47 is shown in SEQ ID NO. 1, and the nucleotide sequence of interleukin-11 siRNA is shown in SEQ ID NO. 2 and SEQ ID NO.

3.

3. The method for preparing an antibody-drug conjugate targeting VEGFR2 as described in claim 1, characterized in that, This can be achieved through the following steps: (1) Preparation of anti-VEGFR2 F(ab′)2 antibody fragment Take 7 mg / mL VEGFR2 monoclonal antibody PBS solution and replace the solvent PBS with acetate-sodium acetate buffer. Mix the above VEGFR2 monoclonal antibody with pepsin at a mass ratio of 25~15:1 and incubate at 37℃ for 5~7 h to degrade the monoclonal antibody into F(ab′)2 and Fc fragments. Purify by ultrafiltration and centrifugation to obtain F(ab′)2. The molecular weight of Anti-VEGFR2 F(ab′)2 is about 110 kDa. (2) Drug conjugation Anti-VEGFR2 F(ab′)2-TK-drug was synthesized by linking anti-VEGFR2 F(ab′)2 and a model drug via an amide reaction using NHS-TK-NHS. 3 μL of 100 mg / mL NHS-TK-NHS DMF solution was added to PBS or DMF solution containing or modified with -NH2 groups at a molar ratio of NHS-TK-NHS:drug:anti-VEGFR2 F(ab′)2 of 2–10:2.4–24:

1. After reacting at room temperature for 15 min, PBS solution containing F(ab′)2 was added, and the reaction was continued at room temperature for 30 min. Lysine was added to terminate the amide reaction. The anti-VEGFR2 F(ab′)2-TK-drug was purified by ultrafiltration and centrifugation. The DAR of the anti-VEGFR2 F(ab′)2-TK-drug ranged from 1 to 8.

4. The use of the antibody-drug conjugate targeting VEGFR2 as described in claim 1 in the preparation of a targeted therapy for kidney disease, characterized in that, The kidney disease mentioned is diabetic nephropathy.

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