Investigation On The Immunotherapy Of Small Cell Lung Cancer By DLL3 Tareted Bispecifc Antibody And CAR-T

Small cell lung cancer(SCLC)accounts for about 15% of lung cancer cases and is usually closely related to smoking.Small cell lung cancer is classified as very aggressive lung cancer.The treatment of SCLC begins with chemotherapy and radiotherapy.SCLC is divided into two stages,limited disease and extensive disease(ED).About 70% of patients with SCLC are diagnosed as an extensive stage.DLL3 is considered an ideal target for immunotherapy of SCLC because it is highly expressed in patients with SCLC,but almost not in normal tissues.Currently,immune checkpoint inhibitors and DLL3 targeted antibody-drug conjugate(ADC)are the major immunotherapy methods of SCLC.The bispecific antibody can bridge T cells with tumor cells,thereby activate T cells and kill tumor cells.CAR-T cells are generated by expressing antibody fragments on the surface of T cells,which improves the recognition and killing activity of T cells to tumor cells.In the current project,we constructed bispecific antibodies and CAR-T targeting DLL3 and studied their killing activity on small cell lung cancer.The construction of the bispecific antibody was based on the classic knob into hole structure,which adopted the DLL3-targeted antibody fragment SC16.15 that was fused with h Fc(knob),and CD3-targeted OKT3 that was fused with h Fc(hole).CAR-T cells were constructed with the third generation structure,which consisted of SC16.15,CD8 transmembrane region,CD28,4-1BB,and CD3 z.The antitumor activity of the DLL3 bispecific antibody and CAR-T was tested at the cell level and in vivo transplanted tumor mice model.The binding activity of the bispecific antibody was tested on DLL3 negative A431 cells and DLL3 positive small cell lung cancer cell line,T-lymphoma cell line Jurkat,and primary PBMC cells,respectively.The antitumor activity of the bispecific antibody and CAR-T was tested by luciferase method.Firstly,luciferase reporter gene was introduced into the above-mentioned cell lines by lentivirus vector,and the in vitro killing activity of bispecific antibody and CAR-T was determined by testing the decrease of luciferase activity.The in vivo anti-tumor activity of bispecific antibody and CAR-T was tested in NSG mice.After subcutaneous inoculation of SCLC cells into NSG mice,the antitumor effect was observed by intraperitoneal injection of PBMC cells and tail vein injection of bispecific antibody or intraperitoneal injection of CAR-T cells.The results of the in vitro killing showed that DLL3 positive SCLC cell lines H446,H196,H82,and DLL3-overexpressing A431 cell lines could be rapidly killed by DLL3 targeted bispecific antibody and CAR-T.In vivo antitumor activity test showed that bispecific antibody and CAR-T had a significant inhibitory effect on tumor growth.The combination of the PD-1 checkpoint inhibitory antibody can improve the antitumor activity of DLL3 bispecific antibody,but not the CAR-T cells.Taken together,the results of the current study showed that DLL3 and CAR-T have strong antitumor activity on SCLC.Besides,the DLL3 bispecific antibody and PD-1 blockade have a better antitumor effect on SCLC.