CN111494645A - Conjugate of anti-human D LL 4 humanized antibody and maytansine alkaloid DM1, and preparation method and application thereof - Google Patents
Conjugate of anti-human D LL 4 humanized antibody and maytansine alkaloid DM1, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a conjugate of an anti-human D LL 4 humanized antibody and maytansine alkaloid DM1, a preparation method and application thereof, and relates to the technical field of biological pharmacy, wherein the obtained conjugate of a new anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid DM1 is a preparation method and application of the conjugate, which are characterized in that small molecular toxin maytansine alkaloid DM1 is coupled on an engineered anti-human D LL 4 humanized antibody TH L4 in a fixed point manner to synthesize the conjugate, and the conjugate H L mD4 is used for promoting the tumor targeting of toxin molecule DM1, reducing the toxic action on normal cells of an organism and achieving a better treatment effect.
Description
Technical Field
The invention relates to the field of antibody coupling drugs, in particular to an engineered antibody obtained by site-directed mutagenesis on an anti-human D LL 4 humanized antibody H3L 2, which is a conjugate of a small toxin molecule maytansine alkaloid DM1 in a site-directed coupling mode.
Background
D LL (Delta like 4) is an important ligand of Notch receptors in evolutionarily conserved Notch signaling pathway, and D LL ligand is involved in regulating biological processes such as proliferation, apoptosis, differentiation of adjacent cells and proliferation and renewal of stem cells by binding with adjacent cell surface Notch receptors. furthermore, D LL 04 is overexpressed in human tumor tissues and involved in the development of tumor vessels. to date, researchers have observed overexpression of D LL in tumor vessels such as renal cancer, bladder cancer, colon cancer, brain tumor and breast cancer successively. many studies have found that D LL/Notch signaling pathway plays an important role in the development of tumor target such as tumorigenesis, tumorigenesis and proliferation and renewal of tumor stem cells. at present, D LL fusion protein and anti-D634 monoclonal antibody are undergoing laboratory or clinical studies, some antibodies have entered into clinical stage, wherein the development of the anti-D634 fusion protein and anti-D LL monoclonal antibody is evaluated by the drug discovery research company, and development of the drug efficacy of the drug discovery of the relevant clinical trial of the development of D2014-T4 antibody, and the development of the related to the tumor angiogenesis and tumor angiogenesis (OMP) are clearly demonstrated that the development of the relevant clinical trial of the anti-T-E4 antibody is not limited to the development of the clinical trial No.2, the invention, the development of the clinical trial No. 7-T (OM7) and the invention is clearly shows that the relevant clinical trial, and the development of the clinical trial No.2 and the invention is not only has been developed by the clinical trial No.2 and no clinical trial No. 2-T-E research of the clinical trial No. 2.
The Antibody Drug Conjugate (ADC) is an anti-tumor monoclonal antibody which is coupled with small molecular toxin through chemical modification, the conjugates have the capacity of specifically recognizing tumor antigens, retain the toxicity of the toxin molecules for killing tumor cells, target to tumor parts and selectively kill the tumor cells, at present, no related patents and literature reports of anti-human D LL humanized antibody drug conjugates exist in China, and only a small amount of maytansine alkaloid DM1 small molecular drugs which influence microtubule polymerization are applied to antibody drug coupling.
DM1 is a maytansine derivative, which is a natural product separated from African shrub-imprinted maytansine (Maytenus ovatus) in the early 70 th 20 th century, as a precursor molecule of an antitumor drug, the maytansine has the same action site as vinblastine, namely the maytansine is combined with tubulin to inhibit aggregation of the maytansine, so that cell cycle arrest is caused and tumor cell apoptosis is induced.
BMPEO (bismaleimide polyoxyethylene glycol) is a linker molecule with a symmetrical structure, and maleimide thioether groups at two ends respectively connect an antibody and a small molecule to prepare an antibody drug conjugate. The linker has high stability to a circulation system, and a drug release mechanism is clear, namely, after ADCs are endocytosed into cytoplasm, the ADCs are degraded into a lysine-MPEO-drug compound under the action of lysosomal enzyme to induce apoptosis, so that BMPEO is a good linker molecule applicable to ADCs.
ADC drugs currently suffer from traditional ADC product heterogeneity, heterogeneous drug/antibody coupling ratio (DAR); improper design of the mutation site, if the site is not exposed on the surface of the antibody, small molecules cannot reach or even approach the coupling site, if the site is just connected with a certain sulfydryl group in a chain or between chains in a reaction manner, a wrong intra-chain or inter-chain disulfide bond can be formed, in addition, a free sulfydryl group at the coupling site can be reduced into a disulfide bond, and the like, the coupling rate is reduced under the conditions, and the problem of non-uniformity of DAR can be brought; the drug coupling sites are not exposed on the surface of the antibody, wrong intra-chain or inter-chain disulfide bonds are formed between the coupling sites, coupling drug linker molecules are unstable, the coupling drugs are easy to fall off and dissociate in blood, and the coupling drugs cannot be released (off-target) in target cells, specifically, the spatial structure of the macromolecular antibody and the small molecular compound may have structural mutual influence, for example, the combination of the macromolecular antibody and a receptor is influenced, and the antibody may wrap the small molecular compound and is difficult to release; the small molecules are too strongly bound to the linker and are difficult to dissociate under the action of enzymes or acid and alkali conditions. The antibody binds to the cell surface, and the small molecule is released at an early stage, and cannot enter the cell, so that the small molecule is difficult to act. The above are difficulties faced by ADC drugs.
Conventional ADCs drugs couple cytotoxic molecules to the antibody surface through either the native lysine (L ys) residues on the antibody surface or the thiol group of cysteine (Cys) generated by reducing interchain disulfide bonds, but both methods have difficulty in controlling the location and stoichiometry of drug-antibody coupling, and thus, ADCs drugs prepared by random coupling have severe product heterogeneity and non-uniform drug/antibody coupling ratios (DAR).
In order to overcome the problems, the invention respectively carries out site-specific mutagenesis on the light and heavy chains of the antibody of the anti-human D LL humanized antibody H3L with the proprietary intellectual property right, recombinates to obtain a new antibody coupling toxin molecule DM1, and prepares the antibody coupling drug H L mD 4. H L mD4 with targeting property and certain toxicity, and the design of the antibody coupling drug H L mD 4. H L mD4 opens up a new tumor treatment strategy with the D LL target.
Disclosure of Invention
Object of the Invention
The invention aims to overcome the defects in the prior art, and provides an antibody drug conjugate H L mD4 which is formed by coupling an anti-human D LL humanized antibody H3L 2 and maytansine alkaloid DM1 and has strong special shape and high purity, and a preparation method and application thereof, wherein a linker-drug combination of BMPEO and DM1 and an engineered anti-human D LL humanized antibody are selected for coupling to obtain an antibody drug conjugate H L mD4, the tumor targeting of DM1 is promoted, the curative effect is improved, the toxic and side effects are reduced, the biological safety evaluation index is improved, the antibody used in the invention is applied for patent (No. CN105384819B), the light and heavy chains of the antibody are subjected to site-directed mutagenesis at the nucleic acid level, the amino acid on a characteristic site is mutated into cysteine with a sulfhydryl group, after an engineering plasmid is constructed and expressed in a cell, the obtained antibody is subjected to a tricarboxyethylphosphine TCEP (Tris- (2-carboxythioyl) -phosphine) reduction reaction, the obtained antibody is subjected to a cell expression, the intracellular expression, the obtained antibody is subjected to a reductive reaction by using a Tris- (2-carboxyethylphosphine) phosphine TCEP (TCEP-A) reducing agent, the dehydrogenation reaction, the antibody is subjected to a relatively wide temperature range, the anti-thiol-thiothreitol coupling reaction is further provided, the anti-thioflavin (TCEP-thiohydramine) and the anti-thioflavin reaction is further provided, the anti-human D antibody coupling reaction is provided, the anti-T (EP-T reaction, the anti-T antibody conjugate is provided by a relatively wide anti-T conjugation process, the anti-human D antibody conjugate, the anti-T conjugate, the anti-human D antibody conjugate is provided, the anti-T conjugate, the anti.
Technical scheme
A conjugate H L mD4 of an anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid DM1 is characterized in that the anti-human D LL 4 humanized antibody TH L4 is coupled with the maytansine alkaloid DM1 through a linker molecule BMPEO, wherein the anti-human D LL 4 humanized antibody TH L4 is obtained by site-directed mutation of valine at 207 position of a light chain and lysine at 121 position of a heavy chain of an anti-human D LL 4 humanized antibody H3L 2 to cysteine.
The conjugate H L mD4 of the anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid DM1 is characterized in that the heavy chain amino acid sequence of the anti-human D LL 4 humanized antibody TH L4 is SEQ ID NO.1, and the light chain amino acid sequence is SEQ ID NO. 2.
The preparation method of the conjugate H L mD4 of the anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid DM1 is characterized by comprising the following steps:
(1) the antibody part is prepared by reducing TH L4 antibody and reducing agent TCEP at a molar ratio of 1:10, oxidizing with antibody and oxidant dhAA at a molar ratio of 1:2, reacting at room temperature for 3h, and desalting the mixture by agarose gel G25 FF column molecular sieve chromatography with 1M DTPA-containing pH7.0 PBS solution;
(2) and (3) a conjugate part, namely, fully dissolving BMPEO and DM1 powder in a DMSO solution respectively, mixing and reacting at a molar ratio of 2:1, and fully mixing and stirring the mpeo-DM1 solution and the antibody product at a molar ratio of 10:1, reacting on ice for 1 hour, and ultrafiltering and desalting the mixture on a low-speed freezing centrifuge by using an ultrafiltration tube to obtain the antibody drug conjugate H L mD 4.
The conjugate H L mD4 of the anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid DM1 is applied to preparation of a cancer treatment drug.
The use of (a), wherein the cancer is esophageal cancer, breast cancer, ovarian cancer, colon cancer, leukemia, gastric cancer, lung cancer, melanoma, renal cancer or classical hodgkin's lymphoma.
In particular, it relates to
The technical scheme of the invention is to provide an antibody drug conjugate of an anti-human D LL 4 humanized antibody H3L 2 and maytansine alkaloid, which is characterized in that the antibody drug conjugate of the anti-human D LL 4 humanized antibody H3L 2 and the maytansine alkaloid is conjugated.
The preparation steps of the conjugate comprise:
firstly, carrying out site-directed mutagenesis on 207 (valine) of a light chain and 121 (lysine) of a heavy chain of an anti-human D LL 4 humanized antibody H3L 2 to cysteine to construct an engineering plasmid, expressing an engineered anti-human D LL 4 antibody TH L4, carrying out Protein A column affinity chromatography purification and agar G25 FF column molecular sieve chromatography to replace an antibody solution system, and then, measuring the antibody concentration by using a BCA method.
Step two, mixing a reducing agent TCEP and an antibody according to a molar ratio of 10:1, reducing an engineered antibody TH L4, opening a disulfide bond, reacting a sample at 4 ℃ for 1 hour, carrying out chromatographic desalting on the sample by using a PH7.0 PBS solution containing 1M DTPA through an Qiongguo G25 FF column molecular sieve, mixing the collected antibody and an oxidizing agent dhAA according to a molar ratio of 1:2, reducing the interchain disulfide bond of the antibody opened in the previous reaction, reacting the sample at room temperature for 3 hours, and carrying out ultrafiltration, desalting and concentration to determine the concentration of the antibody by using a BCA method.
Step three: respectively dissolving linker molecule BMPEO powder and small molecule DM1 powder in DMSO solution, mixing at a molar ratio of 2:1 and at 4 ℃ for 1 hour, and desalting by chromatography with a glucose G25 FF column molecular sieve to obtain the mpeo-DM1 conjugate.
Step four: and mixing the antibody obtained in the second step and the mpeo-DM1 conjugate obtained in the third step in a molar ratio of 1:10 for coupling reaction, placing on ice, and reacting for 1 hour under gentle stirring.
And step five, performing molecular sieve chromatography purification on the product TH L4-mpeo-DM 1, namely H L mD4, obtained in the step four by using an agar gel G25 FF desalting column to remove impurities in the reaction system.
Step six, concentrating the product H L mD4 in the step five by using an ultrafiltration tube on a low-speed freezing centrifuge to obtain an antibody drug conjugate H L mD4, and storing at-20 ℃ after the concentration of the conjugate is measured by a BCA method.
Preferably, the antibody drug conjugate H L mD4 prepared according to the above method has the characteristics that the naked antibody component is less than 10% and the monomer content is more than 90%.
More preferably, the antibody drug conjugate H L mD4 prepared by the method is applied to a drug for targeted therapy of human D LL 4 positive tumors.
More preferably, in one of the steps, the site-directed mutagenesis is a molecular biology technique of site-directed mutagenesis at the nucleic acid level by using an Overlap PCR method.
More preferably, in the first step, the second step, the third step, the fifth step and the sixth step, the purification comprises an affinity chromatography column, a desalting column and an ultrafiltration liquid exchange system.
More preferably, the affinity chromatography column is a Protein A column affinity chromatography column.
More preferably, the desalting column is an agarose gel G25 FF desalting column.
More preferably, the ultrafiltration concentration system employs ultrafiltration tubes of a 50kD membrane.
Wherein the PBS is phosphate buffer salt solution, phpsphate-buffered saline.
Wherein, the H3L 2 molecular sequence is described in detail in patent CN 105384819B.
Wherein the agar-glucose gel G-25FF desalting column.
The traditional method of coupling by random reduction of inter-chain disulfide bonds of antibodies allows the coupling product to exist in different DAR forms. Lower DAR may result in a decrease in the activity of the conjugate, while higher DAR may adversely affect pharmacokinetics. Thus DAR has a significant impact on the efficacy of ADCs, and improvements and discovery of conjugation methods are also of particular importance. Therefore, the invention adopts the method of site-directed mutagenesis of amino acid and drug-directed coupling of antibody, one amino acid in antibody molecule is mutated into cysteine, and then the cysteine is utilized to carry out specific coupling with drug to synthesize ADCs, and the site-directed coupling mode can not interfere folding and assembly of immunoglobulin, and can not change the combination mode of antibody and antigen; compared with the ADCs obtained by the traditional disordered coupling, the ADCs using the Thio-antibody has the advantages of maintaining the in-vivo anti-tumor activity, improving the tolerance and reducing the systemic toxicity. Aiming at the problem that the coupling mode easily causes wrong inter-chain or intra-chain disulfide bonds, the method can be used for synthesizing the ADCs with high uniformity by selecting proper antibody oxidation and reduction operations, controlling the material ratio of an oxidant and a reductant to a reactant, determining the reaction time and the reaction temperature, standardizing operation methods and the like.
The invention adopts the research of site-directed mutagenesis exploratory in the earlier stage, compares the charged property, stability, surface accessibility, position in space conformation and other properties of site-directed mutagenesis antibodies on computer-aided software, finds three coupling sites with potential significance on the 207 position of the C L region, the 121 position of the CH1 region and the 402 position of the Fc segment on the IgG1 type antibody, and carries out systematic comparison according to the comprehensive value of coupling, namely, the 207 position of the C L region > the 121 position of the CH1 region > the 402 position of the Fc segment, therefore, the invention selects the 207 position of the C L region and the 121 position of the CH1 region to realize site-directed mutagenesis on the anti-D LL 4 IgG1 type humanized antibody H3L 2 which is independently developed by our subject group, and maintains the structural stability of the antibody and the affinity of the antibody to the maximum extent while prolonging the half-life of ADCs in vivo.
The ideal linker molecule should be stable in vitro or in the blood circulation to prevent systemic toxicity due to the early release of effector molecules, while at the same time being able to rapidly release the potent cytotoxic drug to kill cancer cells after entering them. Mylotarg using hydrazone linker molecules has poor stability, greater toxicity than chemotherapy alone, and insignificant efficacy, which was withdrawn by the U.S. FDA in 2010. Inotuzumab and ozogamicin are also caused by improper use of linker molecules, and are not marketed in clinical stage III. Therefore, it is important to select linker molecules that have high stability and can effectively release effector molecules intracellularly. Different from the conventional common maleimide-thioether joint, the BMPEO (bismaleimide polyoxyethylene glycol) joint with a symmetrical structure has higher stability for a circulating system, and a drug release mechanism is clear, namely, after ADCs are endocytosed into cytoplasm, the ADCs are degraded into a lysine-MPEO-drug compound under the action of lysosomal enzyme to induce apoptosis, so that the BMPEO is selected as the ADCs joint.
The invention relates to an antibody drug conjugate H L mD4 which is prepared by coupling an anti-human D LL humanized antibody TH L obtained by engineering site-specific mutagenesis of an anti-human D LL humanized antibody H3L 2 and maytansine alkaloid DM1, and can be used for promoting the tumor targeting of DM1, improving the curative effect and reducing the toxic and side effects.
Specifically, the method comprises the following steps:
1. the invention adopts molecular biology technology, and respectively carries out site-directed mutagenesis on 207 (valine) of a light chain and 121 (lysine) of a heavy chain on the light chain and the heavy chain of an anti-human D LL 4 humanized antibody H3L 2 to form cysteine on the basis of not changing targeting property and affinity, so as to construct an engineering plasmid and express an engineered anti-human D LL 4 humanized antibody TH L4.
Table 1 examples of mutations
| Mutation site | Post-mutation amino acid | 3D conformational constellation diagrams |
| Light chain amino acid 207 (valine) | Cysteine | See fig. 8 |
| Heavy chain amino acid position 121 (lysine) | Cysteine | See fig. 9 |
2. The invention obtains an antibody drug conjugate H L mD4 of an engineered anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid coupled by an antibody drug coupling technology, the conjugate not only retains the targeting property of H3L 2, but also fully utilizes the microtubule polymerization inhibiting ability of DM1, thereby more effectively playing the treatment effect of targeted killing of tumors;
3. the BMPEO (bismaleimide polyoxyethylene glycol) linker used in the invention overcomes the defects of hydrazone bond linkers in the aspects of drug effectiveness and safety, and the antibody and small molecules are connected through maleimide groups at two ends of the linker to obtain the antibody drug conjugate H L mD 4.
4. Compared with the prior coupling process, the invention uses a phosphine reducing agent, namely tricarboxyethylphosphine TCEP (Tris- (2-carboxyethyl) -phosphine hydrochloride) to reduce the antibody in an antibody complete reduction reaction system, the TCEP can be used in a wider PH range, the reaction condition is mild, the solubility is good, the toxicity is small, meanwhile, the TCEP does not react with other functional groups in the protein, and the controllability of the reaction and the consistency of the result can be ensured;
5. the coupling process is further optimized, the average coupling ratio of the prepared conjugate H L mD4 is 3.96, the naked antibody component is lower than 10%, the monomer content is higher than 90%, and the in vivo and in vitro drug effects prove that the conjugate can effectively and specifically kill target cells.
Drawings
FIG. 1 is a schematic representation of the molecular structures of TH L4 and H L mD4 and the chemical structural formula of H L mD 4;
FIG. 2 is a high performance liquid chromatography analysis of antibody drug conjugate H L mD 4;
FIG. 3 is an E L ISA assay for antibody drug conjugate H L mD4 binding to human D LL 4;
FIG. 4 shows the inhibitory effect of MTT assay drug conjugate H L mD4 on HUVEC cell growth;
FIG. 5 is a tumor volume experiment in a model of transplanted tumors analyzing the inhibitory effect of drug conjugate H L mD4 on MDA-MB-231 tumors;
FIG. 6 is a tumor body weight experiment analysis of the inhibition of MDA-MB-231 tumors by drug conjugate H L mD4 in a transplanted tumor model;
FIG. 7 is an MDA-MB-231 transplant tumor model survival assay analyzing drug conjugate H L mD 4;
FIG. 8 is a 3D conformation diagram of the light chain at amino acid 207 (valine) mutation;
FIG. 9 is a 3D conformation map of the amino acid 121 (lysine) mutation in the heavy chain.
Detailed Description
Example 1
Preparing an engineered anti-human D L04 humanized antibody TH L4 obtained by site-directed mutagenesis of an anti-human D LL humanized antibody H3L 2 by adopting a molecular biology technology, (1) determining the corresponding light chain 207 (valine) and heavy chain 121 (lysine) of the antibody according to the nucleic acid sequences of the light chain and heavy chain of the humanized antibody H3L, (8) mutating the corresponding nucleotides to cysteine by using OVLAP-PCR, (2) carrying out T-A cloning, transforming a target fragment into an escherichia coli DH5 α host strain, selecting a strain monoclonal for DNA sequencing, carrying out enzyme digestion and enzyme connection on the correctly sequenced sequence to two mutexpression plasmids of pMH3 (neomycin resistance) and pCA-puro (puromycin resistance), obtaining 4 engineered plasmids after DNA sequencing is correct, namely pCA-tH, pMH3-tH, pCA-539t 7 and pMH3-T L, (3) transiently transfecting the plasmids into 293F cells, identifying whether the DNA is purified by using a PCR-based on the PCR affinity chromatography of a Protein immobilized protease, determining the concentration of the antibody by adopting a confocal electrophoresis system, determining whether the antibody is subjected to detect the purity of the antibody by using a confocal electrophoresis of a PCR antibody, and determining the PCR affinity chromatography system, and determining the purity of the antibody by using a confocal electrophoresis of a confocal chromatography system, wherein the PCR antibody is determined by using a PCR system, and determining the PCR.
An antibody drug conjugate H L mD4 of an engineered anti-human D LL humanized antibody TH L and maytansine alkaloid DM1 are prepared by (1) reducing TH L antibody with TCEP in the molar ratio of the antibody to the reducing agent of 1:10, oxidizing with dhAA in the molar ratio of the antibody to the oxidizing agent of 1:2, reacting at room temperature for 3H, desalting the mixture by agarose gel G25 FF column molecular sieve chromatography with 1M DTPA-containing PH7.0 PBS solution, dissolving BMPEO and DM1 powder in DMSO solution respectively, mixing them at a molar ratio of 2:1, and (3) mixing the conjugate fraction, mixing the mpo-DM 1 solution with the antibody product at a molar ratio of 10:1, stirring gently, reacting on ice for 1H, and desalting the mixture by ultrafiltration using an ultrafiltration tube on a low-speed refrigerated centrifuge to obtain mH 464 humanized antibody conjugate H L (see the experimental results of the patent document No. 5: 5).
Example 2
The coupling condition of DM1 and an engineered anti-human D LL 4 humanized antibody TH L4 is detected by high performance liquid chromatography HP L C.
Agilent 1200HP L C analyzes the coupling condition of an antibody drug conjugate H L mD4, and the sample detection conditions are as follows, (1) mobile phase A is 20 mmol/L PBS (pH7.0) +1.5 mol/L ammonium sulfate, (2) mobile phase B is 20 mmol/L PBS (pH 7.0)/isopropanol is 7.5/2.5, (3) elution gradient is 10-100% B, (4) elution time is 20min, (5) flow rate is 0.60m L/min, (6) sample feeding amount is 10u L, (7) detection wavelength is 280nm, and antibody drug coupling ratio (DAR) is calculated according to the peak number and peak area in proportion.
The experimental results are shown in fig. 2, and H L mD4 also peaked at 11min, 14min, 16.5min, and 19min, respectively, corresponding to the number of DM1 small molecules coupled, 2, 4, 6, and 8, respectively, and corresponding peak area ratios of 2.1%, 92.8%, 2.2%, and 1.3%, as compared to H3L 2, which shows a major peak at 9.min, and finally calculated to give a DAR value of 3.96.
Example 3
The affinity detection of the antibody drug conjugate H L mD4 and the human D LL 4 antigen is carried out by adding 1 microgram/m L D LL 4 antigen to a 96-well enzyme-linked immunosorbent assay plate by adopting an E L ISA method according to 100 microgram L per well, coating overnight at 4 ℃, washing the plate for three times by PBS, adding the conjugate H L mD4 and H3L 2 of example 1 to the 96-well plate respectively according to blank groups and concentration gradients of 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000nM, incubating at 37 ℃ for 2, washing the plate for three times by PBS, adding a goat anti-mouse IgG antibody coupled with horseradish peroxidase (HRP), incubating at 37 ℃ for 1 hour, washing the plate for three times by TMB liquid, reacting for 20 minutes at room temperature in a dark place, terminating the reaction, and finally adding a stop solution to the reaction, and detecting the OD630 OD450-OD630 by adopting an enzyme-linked immunosorbent assay plate reader.
The results of the experiment are shown in fig. 3, and compared to H3L 2, H L mD4 has a slightly lower affinity for the D LL 4 antigen, but is still concentration-dependent, i.e. the affinity increases with increasing drug dose, and by calculation, the EC of H3L 2 and H L mD4 is calculated5049.22nM and 42.57nM, respectively, indicate no significant decrease in the affinity of the conjugate.
Example 4
H L mD4 experiment for inhibiting HUVEC cell growth in vitro by adopting MTT colorimetry and adding 1.0 × 10 to HUVEC cells which grow logarithmically5Adding the mixture into a 96-well culture plate, culturing for 12 hours, dividing experiments into 3 groups, namely a blank group, an H3L 2 group and an antibody drug conjugate H L mD4 group obtained in example 1, respectively, adding DMEM culture medium containing 5% fetal bovine serum, H3L 2 and H L mD4(1nM, 5nM, 10nM, 20nM, 40nM, 80nM, 160nM, 320nM, 640nM and 1280nM) in the same volume, arranging 3 duplicate wells in each well, adding 12 mu L5 mg/m L MTT solution in each well after culturing for 72 hours, adding DMSO after 4 mice are acted, reacting for 10min on a shaker, measuring absorbance A values at 570nM and 630nM of a microplate reader, calculating the inhibition rate of the HUVEC cell growth of each experiment group, and drawing a graph.
The experimental results are shown in fig. 4, and it can be seen that the antibody drug conjugate H L mD4 of example 1 can effectively inhibit the growth of HUVEC cells, thereby blocking blood vessels to provide blood oxygen to tumor tissues, resulting in tumor apoptosis, compared with H3L 2.
Example 5
H L mD4 experiment for inhibiting tumor cell growth in vivo, wherein human breast cancer MDA-MB-231 cell-borne tumor Balb/c nude mice purchased from Katy organisms are 4-5 weeks old and about 18-20g in body weight, and are raised in a nude mouse raising room under the SPF condition, in the raising process, mouse cages, padding, drinking water, feed and other articles need to be sterilized, related articles need to be changed in a sterile environment at regular time, the nude mice freely drink water and eat water before inoculating the tumor cells, the administration is carried out after one week of adaptive raising, PBS, H3L 2, DM1 and H L mD4 are respectively injected into tail veins for once every three days, and the tumor growth is observedThe tumor longest diameter L and the tumor longest diameter W in the vertical direction of each group of nude mice were precisely measured and recorded every 3 days from the first day after administration, and the volume was calculated by the following formula, wherein V is L W2/2. At the end of the experiment, the body weight of the mice was measured and recorded, and tumor-bearing nude mice were sacrificed by means of neck-breaking. And (5) drawing a tumor volume curve according to the tumor volume obtained by calculation and the sacrifice time of the tumor-bearing nude mice.
The results of the experiments are shown in FIG. 5 and FIG. 6, and it can be seen that the ADC drug H L mD4 is effective in inhibiting the growth of nude mouse transplantable tumor compared with H3L 2 targeting D LL 4, and H L mD4 is effective in reducing toxicity compared with DM 1.
Example 6
H L mD4 in vivo survival counting experiment, tumor-bearing mouse model construction and grouping administration mode are the same as above, the number of days from tumor implantation to death of naturally dead hosts, and the number of days from tumor implantation to death of non-dead hosts in tumor volume of more than 2000mm3The patient is sacrificed in an off-neck manner, and the life cycle of the patient is counted.
The experimental result is shown in fig. 7, and it can be seen that compared with H3L 2 targeting D LL 4 and small molecule DM1, ADC drug H L mD4 can effectively prolong the survival time of transplanted tumor mice, which indicates that H L mD4 not only can inhibit tumor proliferation, but also exhibits certain in vivo safety.
Reference to the literature
[1]Jia X.L.et al.Ahumanized anti-DLL4 antibody promotes dysfunctionalangiogenesis and inhibits breast tumor growth.Scientific Reports.6,27985(2016).
Sequence listing
<110> university of Chinese pharmacy
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Claims (5)
1. A conjugate H L mD4 of an anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid DM1 is characterized in that the anti-human D LL 4 humanized antibody TH L4 is coupled with the maytansine alkaloid DM1 through a linker molecule BMPEO, wherein the anti-human D LL 4 humanized antibody TH L4 is obtained by site-directed mutation of valine at 207 position of a light chain and lysine at 121 position of a heavy chain of an anti-human D LL 4 humanized antibody H3L 2 to cysteine.
2. The conjugate H L mD4 of the anti-human D LL 4 humanized antibody TH L4 and the maytansine alkaloid DM1 as claimed in claim 1, wherein the heavy chain amino acid sequence of the anti-human D LL 4 humanized antibody TH L4 is SEQ ID NO.1, and the light chain amino acid sequence is SEQ ID NO. 2.
3. The preparation method of the conjugate H L mD4 of the anti-human D LL 4 humanized antibody TH L4 and the maytansine alkaloid DM1 according to claim 1 or 2, which is realized by the following steps:
(1) the antibody part is prepared by reducing TH L4 antibody and reducing agent TCEP at a molar ratio of 1:10, oxidizing with antibody and oxidant dhAA at a molar ratio of 1:2, reacting at room temperature for 3h, and desalting the mixture by agarose gel G25 FF column molecular sieve chromatography with 1M DTPA-containing pH7.0 PBS solution;
(2) and (3) a conjugate part, namely, fully dissolving BMPEO and DM1 powder in a DMSO solution respectively, mixing and reacting at a molar ratio of 2:1, and fully mixing and stirring the mpeo-DM1 solution and the antibody product at a molar ratio of 10:1, reacting on ice for 1 hour, and ultrafiltering and desalting the mixture on a low-speed freezing centrifuge by using an ultrafiltration tube to obtain the antibody drug conjugate H L mD 4.
4. The use of a conjugate of anti-human D LL 4 humanized antibody TH L4 and maytansine alkaloid DM1, H L mD4, according to claim 1 or 2, in the manufacture of a medicament for the treatment of cancer.
5. Use according to claim 4, characterized in that the cancer is oesophageal cancer, breast cancer, ovarian cancer, colon cancer, leukaemia, stomach cancer, lung cancer, melanoma, kidney cancer or classical Hodgkin's lymphoma.
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| CN107375941A (en) * | 2017-07-17 | 2017-11-24 | 中国药科大学 | A kind of anti-human DLL4 monoclonal antibodies and aplysiatoxin derivative MMAE conjugate |
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| JP2010523469A (en) * | 2007-03-30 | 2010-07-15 | ジェネンテック， インコーポレイテッド | Antibodies and immunoconjugates and methods for their use |
| US20120121615A1 (en) * | 2010-11-17 | 2012-05-17 | Flygare John A | Alaninyl maytansinol antibody conjugates |
| WO2016165580A1 (en) * | 2015-04-17 | 2016-10-20 | 江苏恒瑞医药股份有限公司 | Anti-c-met antibody and anti-c-met antibody-cytotoxic drug conjugate and pharmaceutical use thereof |
| CN105384819A (en) * | 2015-12-17 | 2016-03-09 | 中国药科大学 | Anti-human Delta-like4 humanized antibody and preparation and application thereof |
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