CN116726191A - Antibody coupling drug targeting VEGFR2 and preparation and application thereof - Google Patents
Antibody coupling drug targeting VEGFR2 and preparation and application thereof Download PDFInfo
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- CN116726191A CN116726191A CN202310689084.3A CN202310689084A CN116726191A CN 116726191 A CN116726191 A CN 116726191A CN 202310689084 A CN202310689084 A CN 202310689084A CN 116726191 A CN116726191 A CN 116726191A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
- A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/07—Tetrapeptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2073—IL-11
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
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- A—HUMAN NECESSITIES
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- 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
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
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- Microbiology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides an antibody coupling drug targeting VEGFR2, and preparation and application thereof. The antibody coupling medicine is prepared from antibody fragment anti-VEGFR2F (ab') 2 The connecting bond ketal and the model drug are synthesized through amide reaction. Use of anti-VEGFR2F (ab') 2 Establishes a kidney disease antibody coupling drug preparation platform with an ketal thiol bond, and uses anti-VEGFR2F (ab') 2 The coupling model drug can improve the kidney distribution of the model drug, improve the uptake of kidney cells, and simultaneously block VEGFR2 to exert the kidney disease treatment effect, thereby realizing antibody-drug multi-target treatment. The antibody coupling drug provided by the invention can be applied to preparation of drugs for targeted treatment of kidney diseases, can obviously improve the kidney disease treatment effect of model drugs, and can be widely applied to the fields of life sciences and pharmacy. The structural schematic of the antibody coupling drug is shown in formula 1:
Description
Technical Field
The invention belongs to the field of biotechnology medicaments, and in particular relates to an antibody coupling medicament targeting VEGFR2, and preparation and application thereof, which are antibody coupling medicaments targeting the kidney disease targeted repair function of VEGFR2, a preparation method thereof and application of the antibody coupling medicaments serving as templates in preparing medicaments for treating kidney related diseases.
Background
The incidence of kidney disease increases year by year, and has become an important public health problem. Development of high-efficiency kidney disease treatment drugs still faces great clinical demands. Kidney targeted drug delivery is an important means of improving the efficacy of kidney disease treatment drugs. The antibody coupling medicine combines the high specificity of monoclonal antibody medicine with the high activity of small molecular medicine, and utilizes the antibody to selectively target and deliver medicine to focus position, so that it has the characteristics of high specificity and low adverse reaction, and has become a main new medicine developed in recent years, and can be extensively used in immunological and metabolic diseases except tumor. However, antibody-conjugated drugs are less useful in the field of kidney diseases, and the important reason is that they have poor kidney distribution ability and low availability, which in turn cause side effects. Studies have shown that molecular weight is one of the important factors affecting the renal distribution of drugs or carriers. For antibodies of the IgG type (from F (ab') 2 And Fc), the Fc segment is removed to reduce the molecular weight of the monoclonal antibody, so that the nonspecific binding can be reduced while the specific binding capacity of the antibody-receptor is not obviously influenced, and the monoclonal antibody is expected to become a strategy for applying the antibody-coupled drug to the field of kidney diseases.
Vascular endothelial growth factor receptor 2 (VEGFR 2) overactivation occurs widely in a variety of and inflammatory conditionsChronic kidney diseases associated with symptoms and fibrosis (e.g., diabetic nephropathy, clear occlusive glomerular microangiopathy, glomerulonephritis, glomerulosclerosis). VEGFR2 is a specific receptor for VEGF and Gremlin. The activation of VEGF/VEGFR2 is a main mechanism for stimulating vascular endothelial cells, and VEGF is also called vascular permeability factor, and the VEGF is involved in the development of chronic kidney diseases through the mechanisms of changing the structure and the function of endothelial cells, improving the migration and the leakage of endothelial cells, increasing the permeability of glomerular capillaries, promoting the synthesis of extracellular matrixes, promoting the hypertrophy of kidneys and the like by acting with VEGFR 2; gremlin activation of VEGFR2 occurs primarily in the tubular stroma, which promotes fibrosis through injury to the tubular stroma to participate in renal injury. Studies have shown that the use of VEGFR2 mab (anti-VEGFR 2) to specifically block the VEGFR2 pathway can improve renal function, glomerular injury (mesangial matrix expansion and basement membrane thickening), tubular interstitial injury, and down-regulation of pro-inflammatory mediators. Therefore, anti-VEGFR2 is a candidate antibody for treating chronic kidney disease associated with inflammation and fibrosis. Because the pathological mechanism of chronic kidney disease is complex, multi-target treatment is beneficial to improving the treatment effect, when the antibody coupling medicament is prepared, medicaments (small molecules, polypeptides, nucleic acids and the like) which have different targets with anti-VEGFR2 and are suitable for treating kidney diseases, such as anti-inflammatory medicaments, namely glucocorticoid dexamethasone and budesonide, are used for inhibiting natural product extracts of autophagy apoptosis, paeoniflorin is used for protecting endothelial barrier function polypeptide mesogen 8-47, mitochondria targeted antioxidant peptide SS31 is used for inhibiting interleukin 11siRNA. It is worth mentioning that the above drugs have disadvantages in application, including strong side effects, poor plasma stability, poor cell transmembrane ability, and F (ab') of anti-VEGFR2 2 Fragment (anti-VEGFR 2F (ab') 2 ) The coupling of the drugs can effectively improve the kidney delivery efficiency of the drugs and improve the defects thereof. In addition, anti-VEGFR-2 (Fab') 2 Can block VEGFR2 and has certain therapeutic effect, which also provides possibility for multi-target treatment of antibodies and medicines. The linker is responsible for linking the antibody and the drug, and can improve the plasma stability of the drug and realize the controllable release through reasonable design. In the pathological microenvironment of kidney disease, oxidative stress is one of the typical features, manifested by an increase in intracellular ROS levels. Ketal thiol bond (TK bond)) Has ROS responsiveness, and can be used as a linker to release drugs in injured cells with increased ROS level to play a therapeutic role.
Disclosure of Invention
The first object of the present invention is to provide an antibody-conjugated drug targeting VEGFR2, the structural schematic is shown in formula 1:
wherein n=1 to 8.
The antibody coupling medicine is prepared from antibody fragment anti-VEGFR2F (ab') 2 (molecular weight 110 kDa), the linkage ketal (NHS-TK-NHS, formula 2) and the model drug were synthesized by amide reaction. The model drug contains or is modified with-NH 2 The group drug or kidney disease therapeutic drug with synergistic therapeutic effect with VEGFR2 monoclonal antibody comprises SS31 (CAS number 736992-21-5, formula C) 32 H 49 N 9 O 5 Antioxidant peptide), dexamethasone (CAS number 4089-36-5, molecular formula C 22 H 29 FO 5 Glucocorticoids), budesonide (CAS number 51333-22-3, molecular formula C) 25 H 34 O 6 Glucocorticoids), paeoniflorin (CAS number 23180-57-6, molecular formula C 23 H 28 O 11 Natural products for inhibiting autophagy apoptosis), and mesogen 8-47 (amino acid sequence shown as SEQ ID NO.1,
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-NH 2 (Disulfiber bridge: cys10-Cys 15), protective endothelial barrier function polypeptide) and interleukin 11siRNA (nucleotide sequence SEQ ID NO.2: sense (5 '-3') GCUGUUCUCCUAACCCGAUTT, SEQ ID No.3: anti-sense (5 '-3')
AUCGGGUUAGGAGAACAGCTT, inhibiting small interfering RNAs that are fibrotic).
A second object of the present invention is to provide antibody conjugated drugs targeting VEGFR2 (anti-VEGFR 2F (ab') 2 Antibody-conjugated drugs), specifically by the following steps:
1.anti-VEGFR2 F(ab′) 2 preparation of antibody fragments
7mg/mL of VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 25-15:1, and incubating for 5-7 h at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 (FIG. 1). Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak, showing the disappearance of molecular ion peak of heavy chain where Fc segment is located, confirms that Fc segment of anti-VEGFR2 is effectively removed, thereby proving F (ab') 2 Successful preparation.
The reaction formula is as follows:
2. drug coupling technology
Ligation of anti-VEGFR2F (ab') by amide reaction using NHS-TK-NHS (synthesized according to the reference) 2 And drugs, synthesis of anti-VEGFR2F (ab') 2 -TK-drug. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS: drug: anti-VEGFR2F (ab') 2 The molar mass ratio is 2-10:2.4-24:1, and the medicine (containing or modified with-NH) is added 2 Radical) PBS (pH 8-9) or DMF at room temperature for 15min, and then adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Stopping the amide reaction by adding lysine, and purifying F (ab') by ultrafiltration and centrifugation 2 -TK-drug. Reduction of F (ab') using TCEP 2 Disulfide bond of TK-drug, light chain or light chain linked with model drug, mass spectrum with mass-to-charge ratio 18-30kDa range is detected and recorded with mass spectrometerFIG. A shows the calculation of F (ab') from the peak areas of light chains linked to different numbers of SS31 2 The drug-to-antibody molar ratio (drug-to-antibody ratio, DAR) is 1 to 8.
The third object of the invention is to provide the application of the antibody coupling drug as a template in preparing a drug for targeted treatment of kidney diseases. VEGFR2 overexpression and activation are found in various kidney diseases such as diabetic nephropathy, transparent occlusive glomerular microangiopathy, glomerulonephritis, glomerulosclerosis, etc., anti-VEGFR2F (ab') 2 Can target VEGFR2 to enhance renal cell uptake of therapeutic drugs (dexamethasone, budesonide, paeoniflorin, mesogen 8-47, SS31, interleukin 11 siRNA) bound thereto, and can block VEGFR2 to exert therapeutic effects of inhibiting inflammation and fibrosis. Anti-VEGFR2F (ab') 2 TK-drug significantly improves the therapeutic effect of kidney diseases by antibody-drug multi-target treatment.
The invention uses anti-VEGFR2F (ab') 2 The segment, NHS-TK-NHS connecting bond and model drug are synthesized to obtain anti-VEGFR2F (ab') 2 -TK-drug antibody conjugated drug. With anti-VEGFR2F (ab') 2 The coupling model drug can improve the kidney distribution of the model drug, improve the uptake of kidney cells, and simultaneously block VEGFR2 to exert the kidney disease treatment effect, thereby realizing antibody-drug multi-target treatment. The antibody coupling drug provided by the invention can obviously improve the kidney disease treatment effect of the model drug, and can be widely applied to the life science field and the pharmaceutical field.
The invention uses anti-VEGFR2F (ab') 2 The kidney disease antibody coupling drug preparation platform is established with TK bond, and model drugs comprising small molecular chemical drugs, polypeptides, nucleic acids and the like can be reasonably selected in combination with specific disease types, so that the kidney cell targeting uptake capacity of the drugs is improved, side effects are reduced, and anti-VEGFR-2 (Fab') 2 VEGFR2 blocking ability of (c) to achieve multi-target treatment of kidney disease.
Drawings
FIG. 1 is an anti-VEGFR2F (ab') 2 Mass spectrum after TCEP reduction. The detection range is 10-60kDa. LC (liquid Crystal) device + 、LC 2+ And HC + Respectively are provided withRepresenting single protonated, singly charged light chains, double protonated, doubly charged light chains and single protonated, singly charged heavy chains.
FIG. 2 is an anti-VEGFR2F (ab') 2 -mass spectrum of TK-SS31 (DAR 2) after TCEP reduction. The detection range is 18-30kDa. LC (liquid Crystal) device + 、[LC+2×SS31] + And [ LC+3×SS31 ]] + Representing single protonation, single charged light chain, single monoprotization, single charged coupled light chain of two SS31 and single monoprotization, single charged coupled light chain of 3 SS31, respectively.
FIG. 3 is an anti-VEGFR2F (ab') 2 Distribution in major organs of diabetic nephropathy mice.
FIG. 4 is a double antigen sandwich assay for detection of anti-VEGFR2F (ab') 2 Receptor binding ability of TK-SS31.
FIG. 5 via anti-VEGFR2F (ab') 2 -proteinuria levels in diabetic nephropathy mice treated with TK-SS31 (DAR 2).
Detailed Description
The invention is further described with reference to the drawings and examples.
Example one Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . F (ab') was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 2 The molecular weight of (2) is about 110kDa, which is significantly lower than the intact antibody anti-VEGFR2 (-150 kDa). Whether the Fc segment of anti-VEGFR2 was effectively removed was examined using mass spectrometry to further demonstrate F (ab') 2 Is a successful preparation of (a). Since the Fc segment consists of the constant region of the heavy chain of the antibody, the F (ab') is first reduced using tris (2-carboxyethyl) phosphine (TCEP) 2 Disulfide bonds (2 in light)Between chain and heavy chain, 2 between heavy chain), mass spectra in the range of mass to charge ratio 10-60kDa were detected and recorded using a lex TOF/TOF mass spectrometer (Bruker) (FIG. 1). The results show that the light chain proton peak of anti-VEGFR2 remains but the heavy chain proton peak almost disappears, indicating that the Fc fragment of anti-VEGFR2 is effectively removed by pepsin hydrolysis, and anti-VEGFR2F (ab') is successfully obtained 2 。
2. Drug coupling technology
The antioxidative peptide SS31 is used as a model drug, and the NHS-TK-NHS (synthesized according to the reference) is adopted to connect anti-VEGFR2F (ab') 2 And SS31, synthesis of anti-VEGFR2F (ab') 2 -TK-SS31. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS:SS31:anti-VEGFR 2F (ab') 2 Adding PBS (pH 8-9) solution of SS31 in the molar mass ratio of 2:2.4:1, reacting for 15min at room temperature, and continuously adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2F (ab') 2 -TK-SS31. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-SS31 to obtain light chain or light chain connected with SS31, detecting and recording mass spectrogram with mass-to-charge ratio 18-30kDa range by mass spectrometer, calculating anti-VEGFR2F (ab') according to peak area of light chain connected with different quantity of SS31 2 DAR for TK-SS31 is 1. Molecular weight was examined by SDS-PAGE.
EXAMPLE two Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak showing the weight of the Fc segmentThe molecular ion peak of the chain disappeared, confirming that the Fc segment of anti-VEGFR2 was effectively removed, thus proving that anti-VEGFR2F (ab') 2 Successful preparation.
2. Drug coupling technology
The antioxidative peptide SS31 is used as a model drug, and the NHS-TK-NHS (synthesized according to the reference) is adopted to connect anti-VEGFR2F (ab') 2 And SS31, synthesis of anti-VEGFR2F (ab') 2 -TK-SS31. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS:SS31:anti-VEGFR 2F (ab') 2 Adding PBS (pH 8-9) solution of SS31 in the molar mass ratio of 12:14.4:1, reacting for 15min at room temperature, and continuously adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2F (ab') 2 -TK-SS31. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-SS31 to obtain light chain or light chain connected with SS31, detecting and recording mass spectrogram with mass-to-charge ratio 18-30kDa range by mass spectrometer, calculating anti-VEGFR2F (ab') according to peak area of light chain connected with different quantity of SS31 2 DAR for TK-SS31 is 8. Molecular weight was examined by SDS-PAGE.
EXAMPLE three Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak, showing the disappearance of molecular ion peak of heavy chain where Fc segment is located, confirms that Fc segment of anti-VEGFR2 is effectively removed, thereby proving anti-VEGFR2F (ab') 2 Successful preparation.
2. Drug coupling technology
The antioxidative peptide SS31 is used as a model drug, and the NHS-TK-NHS (synthesized according to the reference) is adopted to connect anti-VEGFR2F (ab') 2 And SS31, synthesis of anti-VEGFR2F (ab') 2 -TK-SS31. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS:SS31:anti-VEGFR 2F (ab') 2 Adding PBS (pH 8-9) solution of SS31 in the molar mass ratio of 4:4.8:1, reacting for 15min at room temperature, and continuously adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2F (ab') 2 -TK-SS31. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-SS31, light chain or light chain linked with SS31, mass spectrum of mass to charge ratio 18-30kDa range was detected and recorded with mass spectrometer (FIG. 2), anti-VEGFR2F (ab') was calculated from peak area of light chain linked with different number of SS31 2 DAR for TK-SS31 is 2. Molecular weight was examined by SDS-PAGE.
Example four Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak, showing the disappearance of molecular ion peak of heavy chain where Fc segment is located, confirms that Fc segment of anti-VEGFR2 is effectively removed, thereby proving anti-VEGFR2F (ab') 2 Successful preparation.
2. Drug coupling technology
The aminated Dexamethasone (DXSS) is used as a model drug, and NHS-TK-NHS (synthesized according to the reference) is adopted to connect anti-VEGFR2F (ab') 2 And, synthesis of anti-VEGFR2F (ab') 2 -TK-DXMS. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS: DAMS: anti-VEGFR2F (ab') 2 Adding DMF solution of DXMS in the molar mass ratio of 3:3.6:1, reacting at room temperature for 15min, and continuously adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2F (ab') 2 -TK-DXMS. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-DXS to obtain light chain or light chain connected with DXS, detecting and recording mass spectrogram with mass-to-charge ratio 18-30kDa range by mass spectrometer, calculating anti-VEGFR2F (ab') according to peak area of light chain connected with different quantity of DXS 2 DAR for TK-DXMS is 2. Molecular weight was examined by SDS-PAGE.
Example five Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak, showing the disappearance of molecular ion peak of heavy chain where Fc segment is located, confirms that Fc segment of anti-VEGFR2 is effectively removed, thereby proving anti-VEGFR2F (ab') 2 Successful preparation.
2. Drug coupling technology
The aminated Budesonide (BUD) is used as a model drug, and NHS-TK-NHS (synthesized according to the reference) is adopted to connect anti-VEGFR2F (ab') 2 And, synthesis of anti-VEGFR2F (ab') 2 -TK-BUD. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS: BUD: anti-VEGFR2F (ab') 2 Adding the DMF solution of BUD according to the molar mass ratio of 3:3.6:1, reacting for 15min at room temperature, and continuously adding F (ab ')') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2
F(ab′) 2 -TK-BUD. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-BUD to obtain light chain or light chain connected with BUD, detecting and recording mass spectrogram with mass-to-charge ratio 18-30kDa range by mass spectrometer, calculating anti-VEGFR2F (ab') according to peak area of light chain connected with different amount of BUD 2 DAR for TK-BUD is 2. Molecular weight was examined by SDS-PAGE.
EXAMPLE six Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak, showing the disappearance of molecular ion peak of heavy chain where Fc segment is located, confirms that Fc segment of anti-VEGFR2 is effectively removed, thereby proving anti-VEGFR2F (ab') 2 Successful preparation.
2. Drug coupling technology
An aminated Paeoniflorin (PF) is used as a model drug, and NHS-TK-NHS (synthesized according to reference) is adopted to connect anti-VEGFR2F (ab') 2 And PF, synthesis of anti-VEGFR2F (ab') 2 -TK-PF. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS:PF:anti-VEGFR 2F (ab') 2 Adding DMF solution of PF according to the molar mass ratio of 4:4.8:1, reacting at room temperature for 15min, and continuously adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2F (ab') 2 -TK-PF. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-PF to obtain light chain or light chain connected with PF, detecting and recording mass spectrum of 18-30kDa range of mass-to-charge ratio by mass spectrometer, calculating anti-VEGFR2F (ab') according to peak area of light chain connected with different quantity of PF 2 DAR for TK-PF is 2. Molecular weight was examined by SDS-PAGE.
EXAMPLE seven Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak, showing the disappearance of molecular ion peak of heavy chain where Fc segment is located, confirms that Fc segment of anti-VEGFR2 is effectively removed, thereby proving anti-VEGFR2F (ab') 2 Successful preparation.
2. Drug coupling technology
Adopts mediator 8-47 (IMD 8-47) as a model drug and adopts NHS-TK-NHS (synthesized according to reference document) to connect anti-VEGFR2F (ab') 2 And IMD, synthesis of anti-VEGFR2F (ab') 2 -TK-IMD. 100mg/mL NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS: IMD: anti-VEGFR2F (ab') 2 Adding PBS (pH 8-9) solution of IMD (IMD) in the molar mass ratio of 4:4.8:1, reacting at room temperature for 15min, and continuously adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2F (ab') 2 -TK-IMD. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-IMD to obtain light chain or light chain connected with IMD, detecting and recording mass spectrogram with mass-to-charge ratio 18-30kDa range by mass spectrometer, calculating anti-VEGFR2F (ab') according to peak area of light chain connected with different number of IMDs 2 DAR for TK-IMD is 2. Molecular weight was examined by SDS-PAGE.
Example eight Anti-VEGFR2F (ab') 2 Preparation of TK-drug antibody coupled drug
1.Anti-VEGFR2 F(ab′) 2 Preparation of antibody fragments
0.5mL of 7mg/mL VEGFR2 mab PBS (pH 7.0,0.1M) solution (clone#CD101, bio X Cell, USA) was taken and replaced with acetic acid-sodium acetate buffer (pH 3.0-3.5,0.1M) in solvent PBS. Mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 20:1, and incubating for 6 hours at the constant temperature of 37 ℃ to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 . Investigation of F (ab') by SDS-PAGE 2 Has a molecular weight of about 110kDa. Detection of TCEP reduced anti-VEGFR2F (ab') by Mass Spectrometry 2 Mass spectrum peak, showing the disappearance of molecular ion peak of heavy chain where Fc segment is located, confirms that Fc segment of anti-VEGFR2 is effectively removed, thereby proving anti-VEGFR2F (ab') 2 Successful preparation.
2. Drug coupling technology
Interleukin 11siRNA (siIL 11) is used as a model drug, and NHS-TK-NHS (synthesized according to reference) is adopted to connect anti-VEGFR2F (ab') through an amide reaction 2 And siIL11, synthesis of anti-VEGFR2F (ab') 2 -TK-sil 11. 100mg/mL of NHS-TK-NHS DMF solution 3uL was taken according to NHS-TK-NHS: siIL11: anti-VEGFR2F (ab') 2 Adding PBS (pH 8-9) solution of siIL11 in the molar mass ratio of 4:4.8:1, reacting for 15min at room temperature, and continuously adding F (ab') 2 The reaction is carried out for 30min at room temperature in PBS (pH 8-9). Adding lysine to stop amide reaction, ultrafiltering and centrifuging to purify anti-VEGFR2F (ab') 2 -TK-sil 11. Reduction of anti-VEGFR2F (ab') Using TCEP 2 Disulfide bond of TK-siIL11 to obtain light chain or light chain connected with siIL11, detecting and recording mass spectrogram of mass-to-charge ratio 18-30kDa range by mass spectrometer, calculating anti-VEGFR2F (ab') according to peak area of light chain connected with different quantity of siIL11 2 DAR for TK-siIL11 is 2. Molecular weight was examined by SDS-PAGE.
EXAMPLE nine AntiVEGFR 2F (ab') 2 TK-drug antibody conjugated drug for kidney diseasesApplication in disease treatment
1.Anti-VEGFR2 F(ab′) 2 Renal targeting studies of (2)
Consider anti-VEGFR2F (ab') with anti-VEGFR2 as a control 2 Targeted distribution capacity in diabetic nephropathy mouse kidneys. Preparation of anti-VEGFR2F (ab') according to example 1 2 . ICR mice were intraperitoneally injected with streptozotocin to construct diabetic nephropathy mice models, and at a dose of 33nmol/kg, tail vein was injected with fluorescent dye Cy 5-labeled anti-VEGFR2 or anti-VEGFR2F (ab') 2 . Mice were sacrificed 4h after dosing, representative organs (heart, lung, liver, spleen, kidney) were collected, fluorescence photographs of each organ were taken using a Maestro biopsy system, and the relative fluorescence intensities of each organ were semi-quantitatively analyzed. As a result, FIG. 3 shows that anti-VEGFR2F (ab') 2 Compared with the anti-VEGFR2 group, the fluorescence intensity of the kidney is obviously improved, namely, the good kidney targeting distribution effect is shown. Thus, F (ab') 2 The fragmentation improves the kidney targeting distribution capacity of the anti-VEGFR2 monoclonal antibody, and is possibly an important means for modifying antibody drugs for treating kidney diseases.
2.Anti-VEGFR2 F(ab′) 2 Investigation of the binding Capacity of TK-drug antibody conjugated drug
The antioxidant peptide SS31 is used as a model drug to prepare the first, the second and the third examples to obtain anti-VEGFR2F (ab') 2 -TK-SS31(DAR 1)、anti-VEGFR2 F(ab′) 2 -TK-SS31(DAR 8)、anti-VEGFR2F(ab′) 2 TK-SS31 (DAR 2). With anti-VEGFR2 and anti-VEGFR2F (ab') 2 For control, the receptor binding capacity of each antibody-conjugated drug was examined using a double antigen sandwich method, and the results are shown in fig. 4. Anti-VEGFR2F (ab') 2 -TK-SS31 (DAR 1) and anti-VEGFR2F (ab') 2 OD values of the TK-SS31 (DAR 2) group with anti-VEGFR2 and anti-VEGFR2F (ab') 2 The control group was similar, indicating that the drug-resistant ratio modified antibodies of 1-2 had little effect on their binding capacity. And anti-VEGFR2F (ab') 2 OD values of the TK-SS31 (DAR 8) group compared to anti-VEGFR2 and anti-VEGFR2F (ab') 2 The control group decreased, indicating that the binding capacity of the antibody-conjugated drug was decreased at DAR 8. Thus (2)High DAR (DAR 8) disrupts the receptor binding capacity of antibodies. The preferred DAR in this study was 2, and modification of the antibody in the proportion of DAR 2 had little effect on its binding capacity, thus ensuring anti-VEGFR2F (ab') 2 -TK-SS31 antibody conjugated drugs exert therapeutic effects that block VEGFR2 and drug delivery effects that target VEGFR 2.
3.Anti-VEGFR2 F(ab′) 2 Research on renal disease treatment effect of TK-drug antibody coupled drug
anti-VEGFR2F (ab') 2 TK-SS31 (DAR 2), examined for its therapeutic effect on diabetic nephropathy. With anti-VEGFR2F (ab') 2 And free SS31 as control group, and streptozotocin-induced diabetic nephropathy mice as model animals, and urine of mice is collected after 5 weeks of tail vein administration, and urine protein and urine creatinine are detected. As a result, FIG. 5 shows that anti-VEGFR2F (ab') 2 The mice with TK-SS31 administration group had the lowest proteinuria level (urine protein/urine creatinine ratio) than the control group, i.e. showed the best therapeutic effect on diabetic nephropathy. Thus, anti-VEGFR2F (ab') 2 TK-SS31 can enhance the therapeutic effect of SS31 alone on diabetic nephropathy, which is comparable to anti-VEGFR2F (ab') 2 Improving SS31 kidney distribution and blocking VEGFR2 from exerting a synergistic therapeutic effect.
Claims (6)
1. An antibody coupling drug targeting VEGFR2 is characterized in that the antibody coupling drug consists of an antibody fragment anti-VEGFR2F (ab') 2 The connection bond ketal and the model drug are synthesized through amide reaction, and the structural schematic is shown as formula 1:
wherein: n=1 to 8,
the model drug contains or is modified with-NH 2 A group drug or a kidney disease treatment drug with synergistic therapeutic effect with VEGFR2 monoclonal antibody.
2. The antibody conjugated drug of claim 1, wherein the model drug comprises SS31, dexamethasone, budesonide, paeoniflorin, mesogenic 8-47, and interleukin 11siRNA.
3. The antibody conjugated drug of claim 2, wherein the amino acid sequence of interleukin 8-47 is shown in SEQ ID No.1, and the nucleotide sequence of interleukin 11siRNA is shown in SEQ ID No.2 and SEQ ID No. 3.
4. The method for preparing the antibody-conjugated drug targeting VEGFR2 as claimed in claim 1, which is characterized by comprising the following steps:
(1)anti-VEGFR2 F(ab′) 2 preparation of antibody fragments
Taking 7mg/mL of VEGFR2 monoclonal antibody PBS solution, replacing acetic acid-sodium acetate buffer solution with PBS solvent, mixing the VEGFR2 monoclonal antibody and pepsin according to the mass ratio of 25-15:1, and incubating at the constant temperature of 37 ℃ for 5-7 h to degrade the monoclonal antibody into F (ab') 2 And Fc fragment, ultrafiltering, centrifuging and purifying to obtain F (ab') 2 ，Anti-VEGFR2 F(ab′) 2 Molecular weight about 110kDa;
(2) Drug coupling
The NHS-TK-NHS is adopted to connect anti-VEGFR2F (ab ') 2 and model drugs through amide reaction, so as to synthesize anti-VEGFR2F (ab') 2 100mg/mL NHS-TK-NHSDMF solution 3uL was taken as per NHS-TK-NHS: drug: anti-VEGFR2F (ab') 2 The molar mass ratio is 2-10:2.4-24:1, and the mixture is added with or modified with-NH 2 PBS or DMF solution of the radical medicine is reacted for 15min at room temperature, and then F (ab') is continuously added 2 After 30min at room temperature, lysine was added to terminate the amide reaction, and anti-VEGFR2F (ab') was purified by ultrafiltration centrifugation 2 -TK-drug，Anti-VEGFR2 F(ab′) 2 DAR for TK-drug is 1-8.
5. An anti-VEGFR2F (ab') 2 The application of the antibody coupling medicine in preparing medicine for targeted treatment of kidney diseases.
6. The use according to claim 5, wherein said kidney disease is a plurality of inflammation-fibrosis-associated kidney diseases including diabetic nephropathy, clear occlusive glomerular microangiopathy, glomerulonephritis, glomerulosclerosis.
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