The Synthesis And Antitumor Activity Of Anti-DOG1 Antibody-Maytansine Diriverties Conjugates

BackgroundThe Antibody-Drug Conjugates(ADC)are compounds obtained by chemical coupling of an anti-tumor antibody with a potent cytotoxic drug(bullet drug)via a Linker.They are the latest direction of drug development.Now,there are two antibody-drug conjugates have achieved FDA approval.And there are more than 20 antibody-drug conjugates are in clinical phase 2 or 3.The ADCs consists of three components:antibodies to tumor antigens,potent bullet drugs,and linkers connecting antibodies to the bullets.The main function of the antibody is the drug carrier,but also to control ADC targeting.The bullet drug is the executor of tumor killing effect for ADC.The ideal level for IC50(Half Maximal Inhibitory Concentration)value are nM-pM.An antibody molecule coupled with 2-4 molecules of drugs can play efficient killing effect.In addition,linkers connecting antibodies and bullet are also very important,and are closely related to the stability of ADC,metabolism,pharmacodynamics and toxicity.Maytansine is one compound belonging to benzoansamacrolide antibiotics,originally isolated from the bark of the African shrub Maytenus Ovatus.Maytansine can combine with the β-tublin to prevent microtubule polymerization,block cell cycle and induce apoptosis of tumor cells.L-DM1 and L-DM4 are derivatives of maytansines,with higher cell toxicity than maytansines.In vivo,the metabolism of L-DM1/L-DM4 produces active metabolites L-DMlSMe/L-DM4SMe.The IC50 of L-DM1SMe and L-DM4SMe in KB tumor cell lines are 0.029 nM and 0.0011 nM,the IC50 in SK-Br-3 tumor cell lines are 0.014 nM and 0.0032 nM.The Trastuzumab Emtansine(T-DM1,Kadcyla)with DM1 as a cytotoxic drug was approved by FDA in 2013.Currently there are more than 10 ADCs in clinical trials using L-DM1 or L-DM4 as small molecule cytotoxic drugs.DOG1(Discovered On Gastrointestinal Stromal Tumor-1),also known as TMEM16A,Anoctamin-1,is a kind of calcium activated chloride channel protein.DOG1 is involved in physiological processes such as cell membrane potential,secretion of body fluid,olfactory formation,nerve impulse conduction and smooth muscle contraction.DOG1 was highly expressed in gastrointestinal stromal tumors(GIST),and the positive rate was as high as 99%,In addition,DOG1 is also highly expressed in a variety of gastrointestinal tumors,including leiomyoma,esophageal squamous cell carcinoma,adenocarcinoma of the stomach,lung adenocarcinoma and so on.But the expression in the normal gastrointestinal smooth muscle cells was very low.In this study,we found that anti-DOG1 antibody was rapidly internalized by GIST-882 cells and the results further demonstrated the potential of DOG1 as a therapeutic target for ADC.The aim of this study was to prepare a novel ADC complex with DOG1 as the target,DM4 as the warhead,and SPDB as the linker,and to investigate its antitumor effect.This Study has two parts:First,the synthesis of compounds DM1-SMCC(composed by DM1 and linker SMCC)and DM4-SPDB(composed by DM4 and linker SPDB);Second,we connected the bullet drug DM4 and anti DOG1 antibody through the linker SPDB using chemical method to produce anti DOG1 antibody-DM4 complexes.The pharmacological activities of these complexes on Gastrointestinal stromal tumor,colon cancer,two kinds of digestive tract tumors were evaluated in vitro and in vivo.Chapter 1 The synthesis of Maytansinoid DM1/DM4Materials and Methods1.The Ansamitocin P3 was obtained by fermentation of actinomycetes by our group.Under anhydrous and anaerobic condition,the LiAl(OCH3)3H was prepared by using methanol and lithium aluminum hydride.And the Maytansinol was obtained after hydrolysis of Ansamitocin P3 catalysted by LiAl(OCH3)3H.2.With mercaptopropionic acid as raw materials,S-Methyl methanethiolsulfonatemethyl as sulfur m ethylation reagent,we got 3(methyldithio)propionic acid;EDCI catalysed the NHS activation of 3(methyldithio)propionic acid and N-hydroxysuccinimide ester was produced;Triethylamine provided alkaline environment for catalyzing the coupling between N-hydroxysuccinimide ester and N-methyl-L-alanine.And then we got N-methyl-N-(3-(methyldisulfanyl)propanoyl)alanine,namely DM1 chiral arm.3.DM1 SMe was prepared by the esterification reaction between Maytansinol C3-OH and the carboxylic acid of DM1 chiral arm,zinc chloride as catalyst and DCC as condensing agent.L-DM1 SMe and D-DM1 SMe,two configurations was obtained.We used silica gel column chromatography to separate and purify L-DMISMe and D-DMISMe,and then used the cyano column HPLC to detect the effect of separation and purity.4.Using DTT as reducing agent,L-DM1SMe was reduced in pH=7.5 potassium phosphate buffer-2mM EDTA and MeOH-EA mixed solution,L-DM1 was obtained.L-DM1 and the linker SMCC took the coupling reaction in pH=650mM potassium phosphate-2mM EDTA buffer solution,THF as solvent,at room temperature.DM1-MCC was obtained.5.Isobutylene sulfide as raw material,under anhydrous and anaerobic conditions,n-BuLi catalyzed the ring-opening reduction.4-mercapto-4-methylpentanenitrile was obtained.4-mercapto-4-methylpentanenitrile in ethanol-NaOH mixed solution,were refluxed at 100℃.Cyano was hydrolyzed to get 4-mercapto-4-methylvaleric acid.4-mercapto-4-methylpentanoic acid as raw material,S-Methyl methanethiolsulfonate as sulfur methylation reagent,4-methyl-4-(methylsulfinothioyl)pentanoic acid was obtained;EDCI catalyzed the NHS activation of 4-methyl-4-(methylsulfinothioyl)pentanoic acid.And Nhydroxysuccinimide ester was obtained;Triethylamine provided the alkaline environment ti catalyze the coupling of N-hydroxysuccinimide ester and Nmethyl-L-alanine.N-methyl-N-(4-methyl-4-(methylsulfinothioyl)pentanoyl)-L-alanine,namely DM4 chiral arm,was obtained.6.DM4SMe was prepared by the esteration reaction between C3-OH of Maytansinol with the carboxylic acid of DM4 chiral arm,zinc chloride as catalyst and DCC as condensation agent.The two configurations L-DM4SMe and D-DM4SMe were obtained.We have used silica gel column chromatography to separate and purify L-DM4SMe and D-DM4SMe.And then deter min the effect of separation and purity through the cyano column HPLC.7.DTT as reducing agent,L-DM4SMe was reduced in pH=6 potassium phosphate buffer solution 2mM-EDTA and MeOH-EA mixed solution.L-DM4 was obtained after desulfurization.DM4 and linker SPDB reacted in pH=6.5 potassium phosphate-2mM EDTA buffer solution,THF as solvent.Under room temperature DM4-SPDB was obtained.Results1.Maytansinol was obtained by hydrolysis and reduction of Ansamitocin P3.The HPLC purity of Maytansinol was 99.5%after purification with yield of 80%.2.Using 3-Mercaptopropionic acid as the raw material,DM1 chiral arm used for the synthesis of DM1 SMe was obtained after three steps of reactions.Two configurations,L-DM1SMe and D-DM1SMe were obtained.The configuration selectivity ratio was L-DM4SMe:D-DM4SMe=50:50.We could get the L-type:D-type=60:40 through optimization the experimental conditions.3.By optimizing the separation conditions of silica gel column chromatography,L-DM1SMe and D-DM1SMe could be separated at PE:EA=1:1.The purity was detected by HPLC,L-DM1SMe 99.5%,D-DM1SMe 99.5%.4.L-DM1SMe has 10-100 stronger activity than D-DM1SMe.Using DTT to reduce L-DM1SMe,L-DM1 was obtained.Unless special noted,DM1 refers to L-type DM1.DM1 contains the thiol,chemical properties of which were not stable.DM1 could not be kept for a long time.It should be used in the next step emmediately.5.The sulfydryl of DM1 and the double bond on the maleimide of the linker SMCC took the coupling reaction in pH=6 50mM potassium phosphate-2mM EDTA buffer solution,THF as solvent,at room temperature.DM1-MCC was obtained.Because SMCC has cyclohexane structure,DM1-MCC may have a variety of configuration isomers,but does not affect the activity.6.the DM4 chiral arm was obtained after 5 steps of reaction by using Isobutylene Sulfide as raw material.And it was used to synthesize DM4SMe.Two configurations,L-DM4SMe and D-DM4SMe were obtained.The configuration selectivity ratio was L-DM4SMe:D-DM4SMe=50:50.We could get the L-type:D-type=60:40 through optimization the experimental conditions.7.By optimizing the separation conditions of silica gel column chromatography,L-DM4SMe and D-DM4SMe could be separated at PE:EA=1:1.The purity was detected by HPLC,L-DM1SMe 99.5%,D-DM1SMe 99.5%.L-DM4SMe has 10-100 stronger activity than D-DM4SMe.Using DTT to reduce L-DM4SMe,L-DM4 was obtained.Unless special noted,DM4 refered to L-type DM4.DM4 contains the thiol,chemical properties of which were not stable.DM4 could not be kept for a long time.8.DM4 and linker SPDB in pH=6.5 potassium phosphate-2mM EDTA buffer solution,THF as solvent,took the disulfide exchange reaction with the pyridyldithio group in SPDB at room temperature.DM4-SPDB was obtained.ConclusionIn this study,we have susccessfully synthesed the important maytansinoid,Maytansinol,DM1 and DM4,all of which could be used in the future structural modification and optimization work.These chemical sturctures could be conjugated with the existing linkers or the new linkers.And then the activity evaluation could be carried out in the further to find new cytotoxic maytansinoid.We successfully synthesized the ADC bullet drugs,DM1-SMCC and DM4-SPDB with linker,which could be directly coupled to the a mino acid of the protein antibody.The next step,these structuers could be used in the development of a variety of new ADC drugs,or screening new ADC targets.The synthesis work of DM4 chiral arm involved in sulfide ring opening reaction with great difficulty in synthesis,and multi-step reactions need anhydrous and anaerobic operations which laid a foundation for future development of sulfur-containing compounds.The structural modification work of the chiral arm has been carried out.We would do further research about the influence of the chiral arm structure on the activity of the maytansinol,so as to develop a new type of ADC bullet drug.Chapter 2.The synthesis and the activity study of anti-DOGl antibodyMaytansinoidMaterials and Methods1.The positive expression of DOG1 was detected by immunohistochemistry in the whole digestive tract tumor tissue microarray DGS2081.On this basis,we selected and collected specimens of colon cancer and gastrointestinal stromal tumors in patients to verify the positive expression of DOGlthrough immunohistochemical staining.2.We screened the DOG1 positive tumor cell lines by Fluorescence Quantitative.The results of fluorescence quantitative PCR were validated by Western Blot.3.Flow cytometry was used to detect the binding of anti-DOG1 antibody to tumor cell surface DOG1.4.We designed and constructed the pTT5/Light and pTT5/Heavy expression plasmid of anti-DOG1 antibody.Followed by plasmid expression,purification,verification by double enzyme digestion,and abundantly extraction;5.We have expressioned anti-DOG1 antibody using mammalian transient expression system 293F.The anti-DOG1 antibody was affinity purified using GE affinity chromatography column IgSelect and purified with SDS-PAGE.And the purity of the anti-DOG1 antibody was determined by coomassie brilliant blue.6.The affinity of antibody and antigen was determined using BiaCoreX100.7.Confocal microscopy was used to observe the endocytosis of FITC labeled anti-DOG1 antibody in GIST-882 cell line.8.We prepared of anti-DOG1 antibody-DM4,established the calculation formula of the Drug Antibody Rate(DAR),and detected the DAR of ADC by biological mass spectrometry.9.In vitro cytotoxicity test of anti-DOG1-DM4 by CCK-8.10.We established the HT-29 nude mice xenograft model and the GIST PDX nude mouse model,and evaluated the anti-tumor activity of anti-DOG1-DM4 in vivo.Results1.DGS2018 chips screening of all gastrointestinal tumors showed highexpression of DOG1 in gastrointestinal stromal tumor,colon cancer,liver cancer,gastric cancer and esophageal squamous carcinoma,but low expression or no expression in paracancerous tissues.2.Fluorescence quantitative PCR was used to detect the DOG1 related protein mRNA in several tumor cell lines.The results showed thatGIST T1,GIST882,HT-29,HCT-116,AGS,MKN-45 Suspension cell lines had high expression of DOG1 protein related mRNA.3.We have used WB to screen the expression of DOG1 in many kinds of tumor cells.We found that colon cancer HT-29 and Lovo cell lines,hepatocellular carcinoma cell line HepG2,breast cancer cell line Skbr3 had high expression of DOG1,breast cancer cell line MCF-7 had little DOG1 expression,and ovarian cancer cell line SKOV3 had almost no expression.4.We have used flow cytometry to screen tumor cell lines with cell membrane DOG1 possitive expression.The result showed that GIST-882,GIST patients,Hepa3b,Hepg2,HT-29,HCT-116 and AGS had DOG1 expression on cell membrane surface,but SW48 almost no expression of DOG1,and LOVO negative.5.We constructed the expression vector of anti-DOG1 antibody,established the HEK293F transient expression system,prepared and purified anti-DOG1 antibodies.Subsequently,the anti-DOG1 antibody was purified using GE IgSelect affinity purification column.SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining of the purified antibody showed that there was a protein bar at 20 kDa and 50 kDa Band,as the antibody light chain and heavy chain,proved that the purified antibody structure was correct.6.Using the BiaCoreX100 to determine the affinity of the antibody and the antigen,it was found that the KD of the anti-DOG1 antibody bound to the antigen was 1.342 × 10-8.7.Confocal microscopy result showed that the anti-DOG1 antibody can be rapidly internalized by the GIST-882 cells in 1h.8.The anti-DOG1 antibody of ADC was prepared by the combination of NHS on the small molecule drug DM4-SPDB with the Lysine amino of the antibody.The DAR(Drug/Antibody Ratio,DAR)of the ADC was 3.55 detected by differential ultraviolet spectrophotometry and biological mass spectrometry.9.The cytotoxicity of anti-DOG1 antibody-DM4 in vitro was detected by CCK-8 assay.In GIST882 cells,the IC50 values of anti-DOG1 antibody-DM4 and DM4-SMe are 0.362 nM and 0.318 nM.In HT-29 cells,the IC50 values of anti-DOG1 antibody-DM4 and DM4-SMe are 105 nM and 28 nM.In HCT-116 cells,the IC50 values of anti-DOG1 antibody-DM4 and DM4-SMe are 4.032 nM and 1.086 nM.In HT-29 cells,the IC50 values of anti-DOG1 antibody-DM4 and DM4-SMe are 3.129 nM and 2.803 nM.The results showed that the IC50 of ADC drug reached the DM4-SMe level of nM over 72 hours.10.We established the HT-29 xenograft nude mice model and the GIST PDX nude mice model.In the GIST PDX model high dose group(10 mg/mL),tumorcompletely disappeared.In HT-29 model high dose group(10 mg/mL)tumor completely regressed.And there was a dose-dependent relationship for ADC therapy.The results showed that,ADC anti DOG1 positive tumor activity in vivo.ConclusionIn this study,we have developed the methods and systems of synthesizing the maytansinoids including the Maytansinol,DM1/DM4、DM1-SMCC and DM4-SPDB.And we have constructed the anti DOG1 antibody-DM4 conjugates with DOG1 as the anti-tumor target,using DM4 as the bullet drug,with SPDB as the linker constructs.And its effectiveness has been demonstrated through in vivo and in vitro experiments,which laid the foundation for the future research of ADCs based on DM1/DM4 as bullet drug and DOG 1 as target.