The Pharmacodynamics And Pharmacokinetics Study Of Humanized 5A12 Antibody-Drug Conjugates Targeting HAb18G/CD147-Positive Colorectal Carcinoma

Colorectal carcinoma the third malignant tumor in the world.Approximately 30 percent of the patients with colorectal carcinoma die for tumor recurrence and metastasis after receiving traditional treatment.HAb18G/CD147 is a transmembrane glycoprotein highly expressed in cancer and inflammation.Its expression is reported to be positive correlated with tumorigenesis,progression and metastasis of multiple kinds of carcinoma.h5A12 is a novel humanized monoclonal antibody(m Ab)which showed high sensitivity and specificity in targeting HAb18G/CD147 of colorectal carcinoma tissues.Monomethyl auristatin-E(MMAE)is one kind of small molecule drug which strongly inhibits the cell microtubulin synthesis.The idea of conjugating h5A12 and MMAE with appropriate chemical synthesis methods,which makes h5A12-MMAE antibody-drug conjugates(ADC)both highly tumorspecific and strongly cytotoxic,is potential in treating colorectal carcinoma.This work is supported by National Science and Technology Major Projects for Major New Drugs Innovation and Development.This paper aimed to report the conjugating methods,quality standards,the pharmacodynamics and pharmacokinetics properties of h5A12-MMAE conjugates.Part I: The mAb screening and chemical synthesis of h5A12-MMAE conjugates Firstly,the immunohistochemistry(IHC)showed the sensitivity of 66.9% and specificity of 88.6% of h5A12 monoclonal antibody(m Ab)targeting colorectal carcinoma tissues,which indicated a potent property of targeting colorectal carcinoma.Secondly,we tried many methods and succeed in conjugating h5A12 and MMAE with val-cit linker.Thirdly,with the the conjugating methods optimized,the industrial synthesis technique was determined.The synthesis efficiency(productivity of 82.5%)and yield(single production of 500g)was improved.Part II: The mAb screening and chemical synthesis of h5A12-MMAE conjugatesWe established the basic quality evaluation standard for h5A12-MMAE conjugates and determined several essential parameters,including drug-antibody ratio(DAR),purity and antigen-antibody affinity by techniques such as ultraviolet spectrum,LC-MS/MS,capillary electrophoresis,size-exclusion chromatography and surface plasmon resonance.Take 20160706 as instance,the DAR was 3.60,the purity was greater than 99% and dissociation constant was about 5×10-10.According to the quality control methods,we were able to understand the feature of batch-batch chemical synthesis of that conjugates,which is also an important reference in foundation for the future drug approval.Part III: Pharmacodynamics property of h5A12-MMAE conjugatesAfter we knew the essential parameters of h5A12-MMAE conjugates,we investigated the pharmacodynamics property in vitro and in vivo.The half maximal inhibitory concentration(IC50)of h5A12-MMAE was 1.167 nmol/L、4.340 nmol/L and 10.626 nmol/L for SW480、SW620 and HT-29 respectively.In vivo,nude mice bearing the colon cancer cell SW480 was injected with h5A12-MMAE conjugates.The half effective dose(ED50)of SW480 bearing nude mice was 1.33mg/kg for relative tumor volumn or 1.22mg/kg for relative tumor weight.After repeating the research,ED50 of SW480 bearing nude mice was 1.60mg/kg for relative tumor volumn or1.34mg/kg for relative tumor weight;ED50 of HT-29 bearing nude mice was 1.24mg/kg for relative tumor volumn or1.26mg/kg for relative tumor weight.The weights of nude mice were stable,which indicated that h5A12-MMAE conjugates were very effective and safe.We also investigated mechanism of h5A12-MMAE conjugates,which was h5A12-MMAE conjugates targeting the cytomembrane of CD147-positive cancer cells.Part IV: Pharmacokinetics property of h5A12-MMAE conjugatesADC has a complex pharmacokinetic process due to its complex chemical construction in contrast with common chemotherapeutic drug.We investigated the pharmacokinetics property of h5A12-MMAE conjugates by drug-time curve of total h5A12 m Ab and h5A12-MMAE ADC.Drug distribution and organ shapes were also detected in livers by immunohistochemistry(IHC)staining.Conclusion: Firstly,h5A12 got a high sensitivity and specificity in targeting colorectal carcinoma,which made it an available antibody of ADC.With the optimized methods of h5A12-MMAE conjugates synthesis,the industrial synthesis technique was determined and the efficiency and yield was improved.Secondly,we established a quality standard and determined several essential parameters.Thirdly,h5A12-MMAE conjugates showed potent tumor inhibition both in vitro and in vivo and the mechanism was also investigated.Finally,we obtained the pharmacokinetic rule of h5A12-MMAE and distribution of MMAE.Additionally,this research was essential reference of patent medicine and preclinical tests.