Preparation And Pharmacodynamics Research Of Antibody Drug Conjugate AC133-L1-MMAF

Cancer stem cells(CSCs) have been identified as root cells of cancer proliferation, metastasis and treatment resistence which possess capacity of self-renew and differentiation. Clinical research exposed that CSCs maybe the source of tumor progression. Therefore, CSCs should be killed selectively while dealing with cancer and specific marker in surface of CSCs could help to kill cancer cells without rephrase. There would be a promise future to select antibody as anti-tumor drugs on the basis of reaction between antigen and antibody. On the other hand, anti-tumor drugs maybe present enough cytotoxic but is always accompanied with serious side effect without targeting. Importantly, the antibody drug conjugate(ADC) consisted of antibody and drug possess targeting and cytotoxic and is greatly improving the anti-tumor effect. In this work, we have successfully synthesized an anti-colon CSCs ADC. MMAF, a derivate of auriststin which can inhibit the construct of mircrotubes to prevent cancer cells mitosis, was choosed as a conjugated agent. CD133, specific marker of colon cance stem cells was selected to linked with MMAF under consideration of morbidity. After synthesization and purification of anti-CD133-L1-MMAF, the linked structure was characterized. We next detected cytotoxic of AC133-L1-MMAF on colon cancer cells CT-26 in vitro. At last the activity of AC133-L1-MMAF was evaluated in CB-17/SCID mouse in vivo. Main contents and results of this work are listed as follows:(1) The specific marker of colon CSCs CD133 was selected to be linked with MMAF, a derivates of auriststin to synthesize ADC. SDS-PAGE was applied to characterize molecular weight and RP-HPLC was used to analysize linking structure of ADC. Experiment results indicated that thw weight of AC133 increased slightly after conjugated with MMAF. RP-HPLC results showed that Light and heavy chain of AC133 were detected at 22.95 min point and 29.01 min, respectively. Then ADC was measured at 220 nm and 280 nm. Light and heavy chain of ADC were also determined at 280 nm, however, absorption of ADC at 220 nm was also observed and showed two peaks with postponed retention time compared with AC133 at 280 nm. Retention time of light chain of ADC was 23.96 min while heavy chain was 29.06 min. Delay of retention time between AC133 and ADC could be explained that conjugated agents delayed retention time of AC133 because of increased molecular weight.(2) The cytotoxic of ADC, DOX and L1-MMAF was detected by MTT analysis on CT-26 cells. Results indicated that IC50 value of ADC after 72 h was 0.182±0.004 nM and L1-MMAF was 2.642±0.179 uM and DOX was 8.986±0.163 uM, respectively. The growth of CT-26/CD133+ and rat pheochromocytoma pc12 cells/CD133- were exposed to ADC, L1-MMAF and DOX. In vitro cytotoxicity comparison indicated that ADCs have a potent pesticide effect on CD133+ than CD133- cells. CT-26 cells were incubated with AC133 showed exiguous fluorescence but the cells which didn't incubate with AC133 presented fluroresecene, revealing that this conjugate synthesized by AC133 and L1-MMAF could provide enough affinity and cytotoxity targeted to CD133+ cells. FCm results indicated the percentage of CD133 positive cells in the CT-26 cell population was decreased about 0.7% in ADC-treated cells compared with remained approximately constant ratio of CD133+ in DOX-treated cells ranged from 1.71% to 1.74%, suggesting that DOX could not kill CD133+ cells specifically.(3) We built a subcutaneous transplantation tumor model with SCID mice with 90% of tumor formation. The cytotoxic of ADC, DOX and L1-MMAF in vivo were characterized with mice weight change, tumor volume, tumor inhibition ratio and survival time. Results indicated the body weight became stable and showed a good tumor inhibition at dose of 4mg/kg ADC.At the end of the experiment, 4 mg/kg of ADC greatly decreased tumor size and almost clear the tumors in >80 d and 2 mg/kg did not significantly differ from DOX and L1-MMAF group(p<0.05) even if prolonged mice survival days under this dosing. Throughout these studies the mice remained in good health and no weight loss or other overt toxicity was observed, indicating that ADCs were capable of inhibiting growth and proliferation of tumor and prolong life of CB-17/SCID mice. HE staining results present cell morphology and density could indicate that this tumor issue presented homogeneous and compact but not necrotic and malign in the control group and the overall pattern of nucleus presented irregular distribution and the nuclear membrane might be dissolved. However, IHC results didn't showed any positive expression in tumor section.