The Mechanisms Underlying The Synergistic Antitumor Activity Of Multi-targeted Tyrosine Kinase Inhibitor Famitinib And The Third-generation EGFR Inhibitor HS-10296 As Well As The Antitumor Activity Of The Novel Antibody-drug Conjugates Targeting CD79b

Targeted tumor therapy aims at developing drugs targeting abnormal targets in tumor cells or tissues,blocking signaling pathways and metabolic processes to kill tumor cells.Targeted tumor therapy has become a research hotspot because of its strong specificity,significant therapeutic effects and less side effects.In this present study,we focused on the combined antitumor effect of epidermal growth factor receptor（EGFR）inhibitors and multi-targeted tyrosine kinase inhibitors（TKI）in the treatment of EGFR-mutant non-small cell lung carcinoma（NSCLC）and explored the mechanism.In addition,we investigated the antitumor effect and mechanism of HR089 and A1912,antibody drug conjugates targeting CD79b.The first part is investigating the mechanism of synergistic antitumor effect by combining with HS-10296,a third-generation EGFR inhibitor,and the multi-targeted TKI Famitinib.Lung cancer is a highly heterogeneous disease,and the use of single targeted drugs may not be sufficient to achieve the best clinical effects.It has been reported that both parallel and reciprocal pathways exist between the EGFR and vascular endothelial growth factor receptor（VEGFR）,and these pathways are clearly linked in tumors.Therefore,using combined drugs to inhibit VEGFR-dependent pathological angiogenesis or EGFR-dependent tumor proliferation may produce synergistic antitumor effects and be a potential treatment strategy for NSCLC.HS-10296 strongly inhibited the proliferation of NSCLC cells expressing EGFR^E746_A750del,EGFR^L858R,EGFR^L858R/T790M.,while it inhibited the proliferation of EGFR^WT tumor cells with much weaker activity,with a 6-70fold difference in IC₅₀,showing certain selectivity.The anti-proliferation activity of Famitinib was weak in vitro,which was not related to EGFR genotype.The combination of HS-10296 and Famitinib showed synergistic anti-proliferation effect on NCI-H1975,PC-9,NCI-H3255 and HCC827 cells,with the combination index CI less than 1,and synergistically induced cells apoptosis.In addition,HS-10296 combined with Famitinib synergistically inhibited HUVEC proliferation and migration,probably resulting in inhibition of tumor angiogenesis.To better understand the mechanism of synergistic anti-proliferation and pro-apoptosis on NSCLC cells,we next measured the combined effect on PI3K-AKT-m TOR and Ras-Raf-MAPK signaling pathways.Combined treatment with HS-10296 and Famitinib synergistically inhibited AKT and ERK phosphorylation.The synergistic antitumor activity of HS-10296 and Famitinib was also verified in animal experiments.In NCI-H1975 and PC-9 xenografts,HS-10296 combined with Famitinib led to significantly enhanced antitumor activity compared with either agent alone,probably through synergistic inhibition of AKT and ERK phosphorylation and tumor angiogenesis.In summary,HS-10296 combined with Famitinib exerted significantly synergistic antitumor effects in EGFR-mutated NSCLC cells both in vitro and in vivo,which provided experimental basis for the clinical study of the combination of the third generation EGFR-TKIs and angiogenic inhibitors.The second part is about the antitumor activity of the novel antibody-drug conjugate HR089 and A1912 in vitro.Antibody-drug conjugates（ADC）,a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules（payloads）through chemical linkers,have potent killing activity to target cancer cells.The development of ADC provides a novel therapeutic strategy for B-cell lymphoma.CD79b is an ideal target for ADC-based therapy given its high expression on most types of B-cell lymphoma and limited expression on normal cells.Polatuzumab vedotin（Polivy）is currently the only FDA-approved ADC targeting CD79b,in combination with bendamustine and rituximab for patients with relapsed/refractory diffuse large B-cell lymphoma.Therefore,the research and development of ADC targeting CD79b with better anti-tumor activity than Polivy remain a research hotspot.HR089 and A1912 are two new ADCs,obtained through rationally designing and screening.HR089 and Polivy have the same cytotoxic payloads,monomethyl auristatin E（MMAE）,a microtubule-disrupting antimitotic agent,while A1912 carries9106-IM-2,a topoisomerase I inhibitor.In cell proliferation assays,HR089 and A1912 strongly inhibited the proliferation of B-cell lymphoma cells with high expression of CD79b.However,HR089 and A1912 inhibited the proliferation of CD79b negative tumor cells with much weaker activity,showing certain selectivity.Moreover,the anti-proliferation activity of HR089 and A1912 were related to the cytotoxicity of the payloads.Compared with A1912,HR089 had stronger anti-proliferation and pro-apoptotic effects in vitro,both of which were stronger than Polivy.Subsequently,we selected DOHH2 cells（high expression of CD79b）to study the anti-tumor mechanism of HR089 and A1912.HR089 and A1912 bound to DOHH2 cells in a concentration-dependent manner,and the binding was localized on cell membrane,which also led to activation of the BCR signaling pathways.Then,the ADC-antigen complex entered into cells by endocytosis and were transported to lysosome.In lysosome,the linker of HR089 was degraded to release MMAE,inhibiting microtubule dynamics and resulting in DOHH2 cells arrest in the G₂/M phase.Similarly,the linker of A1912 was degraded to release 9106-IM-2,interfering with DNA replication and resulting in cells arrest in the S phase.Therefore,we concluded that HR089 and A1912 interfered with the cell cycle process by releasing small molecule toxins,resulting in the inhibition of cell proliferation and inducing cell apoptosis.Meanwhile,HR089 and A1912 also had bystander killing effects,and the small molecule toxins released could be transferred to the tumor microenvironment to inhibit the growth of neighboring CD79b negative tumor cells.In conclusion,HR089and A1912 are novel ADCs targeting CD79b with good selectivity,and exert strong killing activity in vitro mainly through releasing cytotoxic payloads.HR089 and A1912 are worthy of further study.