Investigation On The Antitumor Activity Of T Cell-engaged Bispecific Antibody In Hepatocellular Carcinoa And Small Cell Lung Cancer

Tumor is one of the major killers that seriously threaten human health.The incidence and mortality of tumor increase every year,yet its treatment is still facing great challenges.In recent years,with the progression of basic research on tumor immunology and translational medicine,tumor immunotherapy,represented by CAR-T,immune checkpoint inhibitor and bispecific antibody drugs,has brought revolutionary progress on tumor treatment,which is considered by scholars as the fourth treatment modality of cancer after the traditional surgery,radiotherapy,and chemotherapy.Liver cancer and small cell lung cancer(SCLC)are two types of solid tumors with very high malignancy and very poor clinical treatment outcomes.Hepatocellular carcinoma(HCC)is the major subtype of liver cancer,accounting for about 85% of all liver cancers.Its incidence and mortality are ranked sixth and fourth,respectively.More than 700,000 people die of liver cancer each year in the world,and more than half of these patients are in China.Up to now,there is no curative treatment for liver cancer.Sorafenib and levatinib,the first-line chemotherapy drugs for liver cancer,have some effects on patients,but strong resistance and relapse are the major problems for the patient at the advanced stage.Similarly,in terms of the strong drug resistance,SCLC is the most lethal and aggressive subtype of lung cancer,accounting for about 15% of all lung cancer cases.Most patients with SCLC are treated with radiotherapy and chemotherapy,but the high recurrence rate in SCLC patients indicates a poor therapeutic effect.Both CAR-T and bispecific antibody immunotherapy require tumor specific target.Glypican 3(GPC3)and Delta like Ligand 3(DLL3)are appealing tumor biomarkers suitable for immunotherapy of liver cancer and SCLC,respectively.GPC3 belongs to the heparan sulfate proteoglycan family,and is highly expressed in HCC,but not,or minimally,expressed in normal tissues.Two representative antibodies of GPC3,HN3 and h YP7,are ideal antibodies for the study of liver cancer immunotherapy.The h YP7 antibody recognizes the C-terminal of GPC3 protein and the HN3 antibody recognizes the N-terminal functional region of GPC3,both of which have a strong affinity for GPC3.DLL3 is an attractive target for immunotherapy of SCLC.DLL3,one of the Notch receptor ligands,is usually expressed on the surface of SCLC cells at high level,but not in normal tissues.SC16 antibody is specific for DLL3 and used to make antibody-drug conjugate(ADC)Rova-T for SCLC.In the current study,a series of T cell-engaged bispecific antibodies were prepared using h YP7 and HN3 antibody targeting GPC3 and SC16.15 antibody targeting DLL3.The anti-tumor activity of T cell-engaged bispecific antibodies was assessed by in vitro tests and in vivo experiments using NSG mice.The results are as follows:(1)GPC3 protein is highly expressed in G1 cells(a stable cell line overexpressing GPC3)and HCC cell lines Hep G2,Hep3 B and Huh7.Co-incubation of cells with recombinant GPC3 protein can effectively inhibit the proliferation of HCC cells,which also indicates that GPC3 is the functional protein in HCC cells.(2)Based on HN3 and h YP7 antibody,six GPC3/CD3 bispecific antibodies(HN3-KH,h YP7-KH,HN3-OKT3-h Fc,h YP7-OKT3-h Fc,HN3-h Fc-OKT3,h YP7-h Fc-OKT3)were constructed using three structures: knob-into-hole(KH),sc Fv-sc Fv-h Fc,and sc Fvh Fc-sc Fv.Enzyme linked immunosorbent assay(ELISA)experiments showed that the six bispecific antibodies have high affinity for GPC3 protein.Flow cytometry also proved that the six bispecific antibodies have high affinity for G1 and HCC cell lines,and have affinity for CD3-positive Jurkat cells and human peripheral blood mononuclear cells(PBMC).(3)In vitro cell killing experiments showed that when a ratio of unstimulated human PBMC cells to HCC cells at 20:1 or activated human PBMC cells to HCC cells at 2:1,had maximal killing of cancer cells in the presence of the six GPC3/CD3 bispecific antibodies.Among them,h YP7-OKT3-h Fc is the most potent antibody,followed by h YP7-KH and HN3-KH,while h YP7-h Fc-OKT3,HN3-OKT3-h Fc and HN3-h Fc-OKT3 are less active.(4)In vivo dose escalating experiment showed that 0.2 mg/kg was the most effective and tolerable dose for GPC3/CD3 bispecific antibody.All the six GPC3/CD3 bi-specific antibodies have similar and effective inhibitory effects on tumor growth in xenograft mouse model.Among them,h YP7-OKT3-h Fc has a slightly better effect.(5)Based on SC16.15 antibody,three DLL3/CD3 bispecific antibodies(SC16.15-KH,SC16.15-OKT3-h Fc,SC16.15-h Fc-OKT3)were prepared using knob-into-hole,sc Fv-sc Fv-h Fc and sc Fv-h Fc-sc Fv structures.Flow cytometry experiment showed that the three DLL3/CD3 bispecific antibodies had moderate affinity for SCLC cells lines H446 and H196,and high affinity for CD3-positive human PBMC.(6)Three DLL3/CD3 bisspecific antibodies showed strong cytotoxic activity in SCLC cells in vitro,and could also potently inhibit tumor growth in H446 xenograft mouse model,among which SC16.15-KH exhibited the strongest anti-tumor activity.(7)In vivo studies showed that h YP7-OKT3-h Fc combined with PD-1 antibody had slightly enhanced anti-tumor activity compared with h YP7-OKT3-h Fc alone in Hep G2 xenograft mouse model,while SC16.15-KH combined with PD-1 antibody significantly increased the anti-tumor activity in H446 xenograft mouse model.(8)In vivo studies showed that the h YP7-OKT3-h Fc combined with Irinotecan significantly increased the anti-tumor activity and showed temporal tumor elimination in80% of G1 xenograft mouse model and 20% of Hep3 B xenograft mouse model.SC16.15-KH combined with Irinotecan also showed obvious enhancement of anti-tumor activity,and 20% of H446 xenograft mouse model showed complete tumor elimination within a period of time.These results provided evidence that combination of bispecific antibody and chemotherapy drugs is effective in tumor therapy,which is worthwhile to be further evaluated in translational studies.