Antitumor Effect Of Bleomycin-conjugated Single Domain Antibody Against Human Nasopharyngeal Carcinoma

Objectives:HNSA039 is a single domain antibody against human nasopharyngeal carcinoma (NPC). In this study, we aimed to identify its characteristics, to express and purify the single domain antibody fused to Escherichia coli thioredoxinA (TrxA) protein and further investigate its targeted anti-tumor effect in a form of TrixA-HNSA039-Bleomycin immunoconjugate.Methods:The specificity of HNSA039 was evaluated by immunocytochemistry using NPC cell lines and other cell lines. The recombinant plasmid pET-21a(+)/TrxA-HNSA039 was constructed and validated by DNA sequencing. Then it was transformed into E.Coli BL21(DE3) and induced for expression of TrxA-HNSA039 with IPTG The expressed TrxA-HNSA039 was purified and identified by SDS-PAGE and ELISA. Subsequently, the fusion protein TrxA-HNSA039 was conjugated to Bleomycin (BLM) by using DextranT40 as an intermediate. Finally, the anti-tumor activities of TrixA-HNSA039-BLM immunoconjugate in NPC cell line (CNE2) and lung cancer cell line (A549) was investigated by flow cytometry, MTT analysis and colony formation assay.Results:The immunocytochemical detection showed that HNSA039 reacted specifically with CNE2, but not or weakly reacted with other human tumor cell lines and normal cell lines. The TrxA-HNSA039 fusion protein was expressed in Escherichia coli as inclusion body. The molecular weight was approximately 23KD as measured by SDS-PAGE. ELISA results revealed TrxA-HNSA039 had a notable immune activity with CNE2. The results of flow cytometry, MTT analysis and colony formation assay indicated that TrixA-HNSA039 immunoconjugate exhibited weaker antitumor effect than the mixture of TrxA-HNSA039 and BLM (P<0.05). The antitumor effects of the immunoconjugate on CNE2 cells and A549 cells had no significant difference (P>0.05).Conclusions:Immunocytochemistry results confirm that HNSA039 can bind specifically with NPC cells. ELISA assay indicates that the fusion protein TrxA-HNSA039 has comparatively intense immunoreactivity with CNE2 cells. However, the immunoconjugate of TrxA-HNSA039-BLM has no obvious targeted anti-tumor effects. We plan to couple two HNSA039 domains or combine HNSA039 with another domain to increase antigen binding affinity, and investigate its targeted anti-tumor effect by directly conjugated with anti-tumor drugs.