CN111544600A - Conjugate of anti-human DLL4 humanized antibody and dolastatin derivative MMAE, preparation method and application thereof - Google Patents
Conjugate of anti-human DLL4 humanized antibody and dolastatin derivative MMAE, preparation method and application thereof Download PDFInfo
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- 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
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Abstract
The invention discloses a conjugate of an anti-human DLL4 humanized antibody and a dolastatin derivative MMAE, a preparation method and application thereof, and relates to the technical field of biological pharmacy, wherein a coupling conjugate of the obtained novel anti-human DLL4 humanized antibody THL4 and the dolastatin derivative MMAE is obtained. The method is characterized in that: a preparation method and application of a conjugate synthesized by site-directed coupling of a small molecular toxin dolastatin derivative MMAE on an engineered anti-human DLL4 humanized antibody THL4 are provided. The conjugate HLvM4 is used for promoting the tumor targeting of the toxin molecule MMAE, reducing the toxic effect on normal cells of an organism and achieving a better treatment effect.
Description
Technical Field
The invention relates to the field of antibody conjugate drugs. In particular, the invention relates to site-directed mutagenesis on an anti-human DLL4 humanized antibody H3L2 to obtain an engineered antibody, which is site-directed conjugated to a conjugate of the small toxin molecule dolastatin derivative MMAE.
Background
DLL4(Delta like 4) is an important ligand of a Notch receptor in an evolutionarily conserved Notch signaling pathway, and DLL4 ligand is involved in regulating biological processes such as proliferation, apoptosis, differentiation of adjacent cells and proliferation and renewal of stem cells through binding with adjacent cell surface Notch receptors. Moreover, DLL4 is overexpressed in human tumor tissues and involved in the development of tumor vessels. Until now, researchers observed overexpression of DLL4 in tumor vessels of renal, bladder, colon, brain and breast cancers. In recent years, a plurality of researches find that the DLL4/Notch signal pathway plays an important role in the tumorigenesis development process such as tumorigenesis, tumor angiogenesis, proliferation and renewal of tumor stem cells and the like. Currently, DLL4 fusion proteins and anti-DLL 4 monoclonal antibodies are undergoing laboratory or clinical studies, and some antibodies have entered clinical phase II, including REGN421 anti-DLL 4 antibody, developed by Regeneron corporation. However, OMP-21M18 anti-DLL 4 antibody developed by Oncomed terminated in phase II clinical trials at 2017 at 4, the main reasons for this were that PFS (Progression-Free-Survival, Progression-Free Survival) did not significantly prolong, and that Survival, safety and pharmacokinetic assessments were poor. Although OMP-21M18 was called off in phase II clinical trials, it has made a dramatic progression in inhibiting tumor growth. Aiming at the problems of off-target, poor drug effect and the like of OMP-21M18 in clinic, corresponding measures are taken to solve one by one, so that the research and development of the target DLL4 drug have huge application prospects related to tumor treatment. Thus, the target of the engineered antibody drug conjugates designed and synthesized in the present invention was selected as DLL 4.
The anti-tumor monoclonal antibody is coupled with micromolecular toxin through chemical modification, and the conjugates not only have the capacity of specifically recognizing tumor antigens, but also retain the toxicity of the toxin molecules for killing tumor cells, target at tumor parts and selectively kill the tumor cells. At present, related patents and literature reports of anti-human DLL4 humanized antibody drug conjugates do not exist in China, and only a few reports of application of dolastatin derivative MMAE micromolecule drugs influencing microtubule polymerization to antibody drug coupling exist. Therefore, the inventor hopes that the anti-human DLL4 humanized antibody H3L2 can be coupled with a toxin small molecule drug by the molecular biology technology of site-specific mutagenesis, so that the targeting property of the antibody and the toxicity of the toxin small molecule are fully utilized, the curative effect is improved, and the toxic and side effects are reduced.
The dolastatin derivative MMAE (monomethyyl auristatin E) is an artificially synthesized tubulin inhibitor with high toxicity, can inhibit cell division similar to dolastatin, and has toxicity 10-1000 times that of adriamycin after being connected with maleimidocaproyl-dipeptide (valine-hiruline or vc) joint.
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.
Based on the theoretical basis and scientific research practices, the invention respectively carries out site-directed mutagenesis on the light and heavy chains of the antibody on the basis of the anti-human DLL4 humanized antibody H3L2 with independent intellectual property rights, recombines the light and heavy chains to obtain a new antibody coupled dolastatin derivative MMAE, and prepares the antibody coupled drug HLvM4 with targeting property and certain toxicity. The design of HLvM4 opens up a new DLL4 target tumor treatment strategy. The research scheme exerts the targeting advantage of the antibody, effectively reduces the toxic and side effects generated by the toxin drug, achieves better treatment effect and occupies the highest point in preclinical research and clinical safety evaluation. Therefore, the development of the antibody drug conjugate HLvM4 with proprietary intellectual property rights provides a novel clinical medication scheme for a tumor treatment system.
Disclosure of Invention
Object of the Invention
The invention aims to overcome the defects in the prior art and provides an antibody drug conjugate HLvM4 with a coupled anti-human DLL4 humanized antibody H3L2 and a dolastatin derivative MMAE, which has strong special shape and high purity, and a preparation method and application thereof. The antibody used in the invention is patented (grant number: CN105384819B), and the light and heavy chains of the antibody are subjected to site-directed mutagenesis on the nucleic acid level, the amino acid on the characteristic site is mutated into cysteine with sulfhydryl groups, an engineering plasmid is constructed and the cell is expressed, the obtained antibody is subjected to reduction of the antibody by phosphine reducer tricarboxyethylphosphine TCEP (Tris- (2-carboxyeth) -phosphine hydrochloride), and is subjected to reoxidation by using dhAA (dehydroascorbic acid), so that 4 sulfhydryl groups are exposed. Compared with the thiol reducing agent Dithiothreitol (DTT) used in the coupling process provided by Pinatuzumab vedotin and related patent documents (EP 2478912 Al), TCEP can be used in a wider pH range, the reaction conditions are mild, the solubility is good, the toxicity is low, and meanwhile, the TCEP does not react with other functional groups in proteins. Based on the above, the coupling process is further optimized, and the antibody coupling drug of the anti-human DLL4 antibody, which has an average coupling ratio of 4, a naked antibody component of less than 10%, a monomer content of more than 90%, and in-vitro and in-vivo drug effects of specifically killing target cells and having certain safety, is prepared.
Technical scheme
A conjugate HLvM4 of anti-human DLL4 humanized antibody THL4 and dolastatin derivative MMAE, characterized in that: anti-human DLL4 humanized antibody THL4 dolastatin derivative MMAE was coupled via Val-Cit; wherein: the anti-human DLL4 humanized antibody THL4 was: the anti-human DLL4 humanized antibody H3L2 valine at 207 of light chain and lysine at 121 of heavy chain were site-directed mutated to cysteine.
The conjugate HLvM4 of the anti-human DLL4 humanized antibody THL4 and the dolastatin derivative MMAE is characterized in that: the heavy chain amino acid sequence of the anti-human DLL4 humanized antibody THL4 is SEQ ID NO.1
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSGYWMQWIKQAPGQGLEWIGAIYPGDGDTRYTQKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCAGGNFFFDYWGQGTLVTVSSASLECSTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
The amino acid sequence of the light chain is SEQ ID NO.2
EIVITQSPDFQSVTPKEKVTITCSVSSSISSSYLHWYQQKPDQSPKPWIYGTSNLASGVPSRFSGSGSGTDYT LTINSLEAEDAATYYCQQWSSSPLTFGQGTKLEIKVDTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTKSFNRG EC
The preparation method of the conjugate HLvM4 of the anti-human DLL4 humanized antibody THL4 and the dolastatin derivative MMAE is characterized in that: the method is realized by the following steps:
the method comprises the following steps: performing site-directed mutagenesis on valine at 207 position of a light chain and lysine at 121 position of a heavy chain of an anti-human DLL4 humanized antibody H3L2 to cysteine to construct an engineering plasmid, and expressing an engineered anti-human DLL4 antibody THL 4;
step two: reducing agent TCEP was mixed with antibody at a ratio of 10:1, reducing the engineered antibody THL4, opening disulfide bonds, reacting the sample at 4 ℃ for 1 hour, desalting, mixing the collected antibody with an oxidant dhAA in a molar ratio of 1:2, reducing the interchain disulfide bonds of the antibody opened in the previous reaction, reacting the sample at room temperature for 3 hours, and desalting;
step three: and (3) sufficiently dissolving toxin MMAE powder with dipeptide linker Val-Cit, namely vc-MMAE, in a DMSO solution, mixing the vc-MMAE solution and the antibody product obtained in the step two according to a molar ratio of 10:1 for coupling reaction, placing on ice, and slightly stirring for reaction for 1 hour.
Step four: desalting yielded the product THL4-vc-MMAE, i.e., HLvM 4.
The application of the conjugate HLvM4 of the anti-human DLL4 humanized antibody THL4 and the dolastatin derivative MMAE in preparing a medicine for treating cancer is provided.
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.
Further, the method comprises the following steps:
the technical scheme of the invention provides an antibody drug conjugate of an anti-human DLL4 humanized antibody H3L2 and MMAE, which is characterized in that: anti-human DLL4 humanized antibody H3L2 antibody drug conjugates conjugated with MMAE. The preparation steps of the conjugate comprise:
the method comprises the following steps: the method comprises the steps of carrying out site-directed mutagenesis on 207 (valine) of a light chain and 121 (lysine) of an anti-human DLL4 humanized antibody H3L2 to cysteine, constructing engineering plasmids, expressing an engineered anti-human DLL4 antibody THL4, carrying out Protein A column affinity chromatography purification and agar gel G25 FF column molecular sieve chromatography to replace an antibody solution system, and then, measuring the antibody concentration by a BCA method.
Step two: reducing agent TCEP was mixed with antibody at a ratio of 10:1, reducing the engineered antibody THL4, opening a disulfide bond, reacting the sample at 4 ℃ for 1 hour, desalting by chromatography through an agarose G25 FF column molecular sieve with a PH7.0 PBS solution containing 1M DTPA, mixing the collected antibody with an oxidant dhAA at 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, performing ultrafiltration desalting concentration, and determining the concentration of the antibody by a BCA method.
Step three: and (3) sufficiently dissolving toxin MMAE powder with dipeptide linker Val-Cit, namely vc-MMAE, in a DMSO solution, mixing the vc-MMAE solution and the antibody product obtained in the step two according to a molar ratio of 10:1 for coupling reaction, placing on ice, and slightly stirring for reaction for 1 hour.
Step four: and (3) performing molecular sieve chromatography purification on the product THL4-vc-MMAE obtained in the step three, namely HLvM4 by using an agarose gel G25 FF desalting column to remove impurities in the reaction system.
Step five: concentrating the product HLvM4 in the step five by using an ultrafiltration tube on a low-speed freezing centrifuge to obtain an antibody drug conjugate HLvM 4; the BCA method is used for determining the concentration of the conjugate and then storing the conjugate at-20 ℃.
Preferably, the antibody drug conjugate HLvM4 prepared according to the above method has the following characteristics: the content of the naked antibody component is less than 10 percent, and the content of the monomer is more than 90 percent.
More preferably, the antibody drug conjugate HLvM4 prepared according to the method is applied to targeted therapy of human DLL4 positive tumor drugs.
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 fourth step and the fifth 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 H3L2 molecular sequence is shown in the patent CN 105384819B.
Wherein the vc-MMAE is synthesized by Shanghai Chemicals.
The agar-glucose gel G-25FF desalting column is prepared by mixing glucan gel and agarose gel and then highly crosslinking, has the advantages of good stability, good rigidity, high flow rate, good repeatability, high resolution, uniform particles, no non-specific adsorption, no volume shrinkage after filling and the like, and can completely replace and exceed the traditional glucan gel. The agarose gel is represented by an English letter FF, the cross-linked dextrans with different specifications are represented by an English letter G, the Arabic number behind the G is 10 times of the water value of the gel, the G-25 absorbs 2.5G of water when each gram of the gel swells, and the separation range of the G-25 is 1000-5000Da, which is specially designed for protein desalination.
Compared with the prior art, the invention has the following advantages:
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 technique of coupling by site-directed mutagenesis has a key point, namely, the selection of a mutation site. In the early stage of the invention, site-directed mutagenesis exploratory research is carried out, the charging property, the stability, the surface accessibility, the position in space conformation and other properties of site-directed mutagenesis antibodies are compared on computer-aided software, three coupling sites with potential significance, namely a CL region 207, a CH1 region 121 and an Fc segment 402, on an IgG1 type antibody are found, and systematic comparison is carried out according to a coupling comprehensive value: CL region 207 position > CH1 region 121 position > > Fc segment 402 position. Therefore, in the humanized antibody H3L2 of anti-DLL 4 IgG1 type, which was independently developed by our subject group, the CL region 207 and the CH1 region 121 were selected to realize site-directed mutagenesis, so that the in vivo half-life of the ADCs is prolonged, and the structural stability of the antibody and the affinity of the antibody are maintained to the maximum extent.
The invention obtains the antibody drug conjugate HLvM4 of the coupling of the engineered anti-human DLL4 humanized antibody THL4 and the MMAE by an antibody drug coupling technology, the conjugate not only retains the targeting property of H3L2, but also fully utilizes the microtubule polymerization inhibiting ability of the MMAE, thereby more effectively playing the treatment effect of targeted killing of tumors;
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;
advantageous effects
The antibody drug conjugate HLvM4 obtained by coupling the anti-human DLL4 humanized antibody THL4 and the dolastatin derivative MMAE through the engineered site-directed mutagenesis of the anti-human DLL4 humanized antibody H3L2 can be used for promoting the tumor targeting of DM1, improving the curative effect and reducing the toxic and side effects. The beneficial effects are that (1) the conjugate HLvM4 retains high affinity activity for DLL 4; (2) specifically binds to cells expressing human DLL4, and avoids the killing effect of MMAE on normal cells; (3) can obviously inhibit the growth and proliferation of tumors in vivo and in vitro.
Specifically, the method comprises the following steps:
the invention adopts molecular biology technology, and on the basis of not changing targeting property and affinity, the 207 th (valine) of the light chain and the 121 th (lysine) of the heavy chain on the anti-human DLL4 humanized antibody H3L2 are respectively subjected to site-specific mutation to form cysteine, an engineering plasmid is constructed, and the engineered anti-human DLL4 humanized antibody THL4 is expressed.
| 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 |
The invention obtains the antibody drug conjugate HLvM4 of the coupling of the engineered anti-human DLL4 humanized antibody THL4 and maytansine alkaloid by an antibody drug coupling technology, the conjugate not only retains the targeting property of H3L2, but also fully utilizes the microtubule polymerization inhibiting ability of DM1, thereby more effectively playing the treatment effect of targeted killing of tumors;
the Val-Cit (vc) dipeptide linker used in the invention overcomes the defects of hydrazone bond linker in the aspects of drug effectiveness and safety, and the anti-human DLL4 humanized antibody is connected with a small molecule through maleimide of the linker vc-MMAE to obtain the antibody drug conjugate HLvM 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;
the coupling process is further optimized, the average coupling ratio of the prepared conjugate HLvM4 is 3.92, the content of the naked antibody component is lower than 10%, the content of the monomer is higher than 90%, and in vitro and in vivo drug effects prove that the conjugate can effectively and specifically kill target cells.
Drawings
FIG. 1 is a schematic diagram of the molecular structures of THL4 and HLvM4 and the chemical structural formula of HLvM 4.
Figure 2 is a high performance liquid chromatography analysis of antibody drug conjugate HLvM 4.
Figure 3 is an ELISA assay of the ability of antibody drug conjugate HLvM4 to bind to human DLL 4.
FIG. 4 is a MTT assay for the inhibition of HUVEC cell growth by drug conjugate HLvM 4.
FIG. 5 is an experimental analysis of the inhibition of MDA-MB-231 tumors by drug conjugate HLvM4 in a tumor volume experiment in a model of transplanted tumors.
FIG. 6 is a tumor body weight experiment analysis of the inhibition of MDA-MB-231 tumors by drug conjugate HLvM4 in a transplanted tumor model.
FIG. 7 is an MDA-MB-231 transplant tumor model survival assay analyzing drug conjugate HLvM 4.
FIG. 8 is a 3D conformation chart of the mutation of amino acid 207 (valine) of the light chain to cysteine.
FIG. 9 is a 3D conformation chart of the mutation of amino acid 121 (lysine) of the heavy chain to cysteine.
Detailed Description
Example 1
Preparation of engineered anti-human DLL4 humanized antibody THL4 obtained by site-directed mutagenesis of anti-human DLL4 humanized antibody H3L2 using molecular biology techniques (wherein anti-human DLL4 humanized antibody H3L2 (naked antibody) preparation method, as in reference [1 ]): (1) determining corresponding nucleotide 207 (valine) and 121 (lysine) of the light chain and the heavy chain of the antibody according to the nucleic acid sequences of the light chain and the heavy chain of the humanized antibody H3L2, designing primers (8 in total), and mutating the primers into the nucleotide corresponding to cysteine by using Overlap-PCR; (2) carrying out T-A cloning, transforming a target fragment to escherichia coli DH5 alpha host strains, selecting a strain monoclonal for DNA sequencing, connecting a sequence with correct sequencing to two mutexpression plasmids of pMH3 (neomycin resistance) and pCA-puro (puromycin resistance) through enzyme digestion and enzyme, and obtaining 4 engineering plasmids which are named as pCA-tH, pMH3-tH, pCA-tL and pMH3-tL after the DNA sequencing is correct; (3) the plasmid is transiently transfected into 293F cells for fermentation, antibody is purified by fermentation liquor through Protein A affinity chromatography, and an antibody solution system is replaced by the purified THL4 antibody through agar gel G25 FF molecular sieve chromatography by PBS with the pH of 7.0; the BCA protein concentration determination method is adopted to determine the concentration of the antibody, SDS-PAGE protein gel electrophoresis is adopted to determine the purity of the product, ELISA is used to determine the affinity of the three antibodies with DLL4, laser micro-confocal detection is carried out on the internalization capability of the antibody to cells, and the aim of the identification experiment is to determine whether the mutated antibody meets the basic condition for developing the antibody part of the antibody drug conjugate.
Engineered anti-human DLL4 humanized antibody THL4 and dolastatin derivative MMAE conjugated antibody drug conjugate HLvM 4: (1) antibody moiety: reducing THL4 antibody with TCEP at the molar ratio of antibody to reducing agent of 1:10, oxidizing with dhAA at the molar ratio of antibody to 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 PBS solution at pH 7.0; (2) conjugate moiety: fully dissolving vc-MMAE powder in a DMSO solution, fully mixing the vc-MMAE solution and the antibody product in a molar ratio of 15:1, stirring slightly, and reacting on ice for 1 hour; and (3) carrying out ultrafiltration desalination on the mixture on a low-speed freezing centrifuge by using an ultrafiltration tube to obtain the antibody drug conjugate HLvM 4. The results of the experiment are shown in FIG. 1.
Example 2
The coupling condition of MMAE and an engineered anti-human DLL4 humanized antibody THL4 is detected by High Performance Liquid Chromatography (HPLC).
Agilent 1200HPLC analysis of conjugation of antibody drug conjugate HLvM 4. The sample detection conditions were as follows: (1) mobile phase A: 20mmol/LPBS (pH7.0) +1.5mol/L ammonium sulfate; (2) mobile phase B: 20mmol/LPBS (ph 7.0)/isopropanol 7.5/2.5; (3) elution gradient: 10-100% B; (4) elution time: 20 min; (5) flow rate: 0.60 mL/min; (6) sample introduction amount: 10 uL; (7) detection wavelength: 280 nm. And calculating the antibody drug coupling ratio (DAR) according to the peak number and each peak area in proportion.
The results of the experiment are shown in fig. 2, and compared to H3L2, where the main peak appears at 9.min, HLvM4 also peaks at 11min, 13.5min, 17min and 19min, respectively, corresponding to 2, 4, 6 and 8 coupling MMAE small molecules, respectively, and corresponding peak area ratios
Example 3
Affinity detection of antibody drug conjugate HLvM4 with human DLL4 antigen: adding 1 mu g/mLDLL4 antigen to a 96-well enzyme label plate by an ELISA method at the concentration of 100 mu L per well, and coating overnight at the temperature of 4 ℃; after three plate washes with PBS, the conjugates HLvM4 of example 1 and H3L2 were added to 96-well plates in a blank set at 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000nM concentration gradients, respectively, and incubated at 37 ℃ for 2 hours; after washing the plate for three times with PBS, adding goat anti-mouse IgG antibody coupled with horseradish peroxidase (HRP), and incubating for 1 hour at 37 ℃; and after washing the plate for three times by PBS, adding TMB color development solution, reacting for 20 minutes at room temperature in a dark place, finally adding stop solution to stop the reaction, and detecting OD450-OD630 by an enzyme-linked immunosorbent assay (ELISA).
The results of the experiment are shown in figure 3, and compared to H3L2, HLvM4 has a slightly lower affinity for DLL4 antigen, but is still concentration-dependent, i.e. the affinity increases with increasing drug dose, and by calculation, EC of H3L2 and HLvM45049.22nM and 40.70nM, respectively, indicate no significant decrease in the affinity of the conjugate.
Example 4
HLvM4 experiment for inhibiting HUVEC cell growth in vitro by adopting MTT colorimetry and adding 1.0 × 10 of logarithmically grown HUVEC cells5Adding into 96-hole culture plate, and culturing for 12 hr; the experiment was divided into 3 groups, blank, H3L2 group and antibody drug conjugate HLvM4 group obtained in example 1; the same volume of DMEM medium containing 5% fetal bovine serum, H3L2 and HLvM4(1nM, 5nM, 10nM, 20nM, 40nM, 80nM, 160nM, 320nM, 640nM, 1280nM) were added, respectively. Setting 3 multiple wells per well, culturing for 72 hours, adding 12 μ L5mg/mLMTT solution per well, acting on 4 mice, adding DMSO, reacting for 10min on a shaker, and measuring absorbance A values at 570nm and 630nm of a microplate reader. And calculating the inhibition rate of each experimental group on the growth of the HUVEC cells, and drawing a curve graph.
The experimental results are shown in fig. 4, and it can be seen that the antibody drug conjugate HLvM4 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
HLvM4 experiment for inhibiting tumor cell growth in vivo: a human breast cancer MDA-MB-231 cell-borne tumor Balb/c nude mouse purchased from Keky organisms is 4-5 weeks old and has the weight of about 18-20g, and is raised in a nude mouse raising room under the SPF condition. In the feeding process, the squirrel cage, padding, drinking water, feed and other articles need to be sterilized, and related articles need to be replaced regularly in an aseptic environment. Nude mice were fed with free water before tumor cell inoculation, and were dosed one week after acclimation. Injecting PBS, H3L2 and HLvM4 into tail vein once for three days, and observing tumor growth and mouse state; from the first day after administration, the longest diameter L of the tumor and the maximum transverse diameter W in the vertical direction in each group of nude mice were precisely measured and recorded every 3 days by using a vernier caliper toThe volume is calculated by the following formula: LW (low-voltage line)2/2. 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 experiment are shown in fig. 5, and fig. 6, and it can be seen that the ADC drug HLvM4 is effective in inhibiting the growth of nude mouse transplantable tumors, compared to H3L2 targeting DLL 4.
Example 6
HLvM4 in vivo survival counting experiments: the tumor-bearing mouse model construction and the grouping administration mode are the same as those described above. A naturally dead host, the number of days from the implantation of the tumor to the time of death; non-dead host, greater than 2000mm in tumor volume3The patient is sacrificed in an off-neck manner, and the life cycle of the patient is counted.
The experimental results are shown in fig. 7, and it can be seen that compared with H3L2 targeting DLL4, the ADC drug HLvM4 can effectively prolong the survival time of transplanted tumor mice, indicating that HLvM4 not only can inhibit tumor proliferation, but also exhibits certain in vivo safety.
Reference to the literature
[1]Jia X.L.et al.A humanized anti-DLL4 antibody promotesdysfunctional angiogenesis and inhibits breast tumor growth.ScientificReports.6,27985(2016)。
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Claims (6)
1. A conjugate HLvM4 of anti-human DLL4 humanized antibody THL4 and dolastatin derivative MMAE, characterized in that: anti-human DLL4 humanized antibody THL4 dolastatin derivative MMAE was coupled via a linker; wherein: the anti-human DLL4 humanized antibody THL4 was: the anti-human DLL4 humanized antibody H3L2 valine at 207 of light chain and lysine at 121 of heavy chain were site-directed mutated to cysteine.
2. The conjugate HLvM4 of anti-human DLL4 humanized antibody THL4 and dolastatin derivative MMAE of claim 1, characterized in that: the heavy chain amino acid sequence of the anti-human DLL4 humanized antibody THL4 is SEQ ID NO.1, and the light chain amino acid sequence is SEQ ID NO. 2.
3. The conjugate HLvM4 of anti-human DLL4 humanized antibody THL4 and dolastatin derivative MMAE according to claim 1 or 2, characterized in that: the linker is Val-Cit.
4. The preparation method of the conjugate HLvM4 of anti-human DLL4 humanized antibody THL4 and dolastatin derivative MMAE according to any one of claim 3, characterized in that: the method is realized by the following steps:
the method comprises the following steps: performing site-directed mutagenesis on valine at 207 position of a light chain and lysine at 121 position of a heavy chain of an anti-human DLL4 humanized antibody H3L2 to cysteine to construct an engineering plasmid, and expressing an engineered anti-human DLL4 antibody THL 4;
step two: reducing agent TCEP was mixed with antibody at a ratio of 10:1, reducing the engineered antibody THL4, opening disulfide bonds, reacting the sample at 4 ℃ for 1 hour, desalting, mixing the collected antibody with an oxidant dhAA in a molar ratio of 1:2, reducing the interchain disulfide bonds of the antibody opened in the previous reaction, reacting the sample at room temperature for 3 hours, and desalting;
step three: fully dissolving toxin MMAE powder with dipeptide linker Val-Cit, namely vc-MMAE, in a DMSO solution, mixing the vc-MMAE solution and the antibody product obtained in the step two according to a molar ratio of 10:1 for coupling reaction, placing on ice, slightly stirring, and reacting for 1 hour;
step four: desalting yielded the product THL4-vc-MMAE, i.e., HLvM 4.
5. Use of a conjugate HLvM4 of anti-human DLL4 humanized antibody THL4 and dolastatin derivative MMAE in the preparation of a medicament for the treatment of cancer according to any one of claims 1 to 3.
6. 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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| CN113398242A (en) * | 2021-06-04 | 2021-09-17 | 浙江大学 | Antibody coupling medicine and application thereof |
| CN111544600B (en) * | 2020-05-20 | 2022-10-04 | 中国药科大学 | Conjugate of anti-human DLL4 humanized antibody and dolastatin derivative MMAE, preparation method and application thereof |
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| CN106467575A (en) * | 2015-08-18 | 2017-03-01 | 四川百利药业有限责任公司 | Cysteine engineered Antibody-toxin conjugate |
| 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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| CN113181373A (en) * | 2021-05-10 | 2021-07-30 | 深圳安特生物医药科技有限公司 | Antibody drug coupling preparation and preparation method and application thereof |
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