Efficient elimination of B-lineage lymphomas using sortase A-generated site-specific anti-CD20-MMAE conjugates


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Wen-bin Zhao1,Li-qiang Pan2,Wen-hui Liu1,Shu-qing Chen1
1Zhejiang university,2Harvard Medical School

Abstract

Background

Antibody-drug conjugates (ADCs) delivering cytotoxic drug into the tumor cells via antigen-mediated endocytosis is an effective approach for tumor therapy. CD20 is known as a tumor associated antigen because it is expressed on the surface of most malignant B cells, which is a potential target for ADCs to efficient elimination of B-lineage lymphomas.

Method

Here, we used sortase A-mediated transpeptidation to specifically conjugate triple glycine-modified monomethyl auristatin E (MMAE) to anti-CD20 ofatumumab (OFA) equipped with a short C-terminal LPETG tag at heavy chain. We used multiple bioanalysis methods, including a new in situ quantitative bioanalysis of MMAE-conjugated ADCs we established, to evaluate basic properties of the ADCs. In addition, we examined the internalization of the ADCs by confocal laser scanning, evaluated cell binding affinity, apoptosis-inducing activity and in vitro efficacy of ADCs using multiple CD20+ cell lines. Finally, in vivo efficacy and toxicity were performed on Balb/c athymic nude mice using CD20+ Ramos xenograft.

Results

We generated two C-terminal specifically conjugated antibodies and established a new ligand-based assay (LBA) method based on flow cytometry to assist ADCs bioanalysis. We found that the two ADCs can be effectively internalized into CD20+ cells after binding to cell surface CD20 and were surprisingly effective in eliminating B-lineage lymphoma. The xenograft tumors in mice disappeared after three injections of the ADC at low dose (5 mg kg-1) and remained for the remainder of the experiment (55 days after the first injection). Excitingly, no toxicity was observed even if the mice were treated with 20 mg kg-1 ADCs for 4 times.

Conclusion

We generated uniform and high potent anti-CD20-MMAE conjugates, which can efficiently eliminate B-lineage lymphomas. In addition, we established a new LBA method, which not only extends the scope of application of the conventional LBA methods, but also improved the authenticity of ADC bioanalysis.