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Abstract
Background Combination treatments using anti-PD-1/PD-L1 antibodies with other VEGF antagonists have shown enhanced clinical antitumor activities.1 The expression of PD-1 and VEGF are found to be frequently upregulated and co-expressed in solid tumors. Importantly, VEGF promotes tumor angiogenesis and suppresses anti-tumor immune response.2 3 Consequently, we characterized the mechanism-of-action of a novel first-in-class anti-PD-1/VEGF bispecific antibody, ivonescimab, designed to simultaneously inhibit PD-1-mediated immunosuppression and block tumor angiogenesis in the tumor microenvironment.
Methods Binding activity of ivonescimab to PD-1/VEGF were assessed by ELISA. Blockade of PD-1/VEGF signaling pathways was determined in luciferase reporter cell assays. Ivonescimab-VEGF complex formation was detected by SEC-HPLC. Cooperative binding of ivonescimab-VEGF complex to PD-1 or ivonescimab-PD-1 complex to VEGF was measured by Octet BLI. The enhanced PD-1 blockade bioactivity of ivonescimab with VEGF was evaluated in hPBMC and engineered cell-line co-culture/luciferase-reporter cell assays. Anti-tumor activity of ivonescimab was investigated in hPBMC-humanized SCID/Beige mice implanted with HCC827 (mEGFR lung adenocarcinoma) or U87MG (glioblastoma). Immuno-safety was assessed by FcγR binding, ADCC, ADCP assays, and reported clinical irAEs.
Results Ivonescimab displayed strong binding activity to human PD-1 and VEGF alone or simultaneously, effectively blocking interactions with ligands and the downstream signaling effects. In presence of VEGF, ivonescimab forms soluble complexes with VEGF dimers, leading to over 10-fold enhanced binding affinity (KD) of ivonescimab to PD-1. The avidity increase was consistent with reduced cell surface PD-1 expression on human T-cell lines, increased potency on blockade of PD-1/PD-L1 signaling and subsequent enhanced T cell activation in-vitro. Likewise, PD-1 binding enhanced ivonescimab binding to VEGF which was associated with enhanced VEGF-signaling blockade. Furthermore, ivonescimab treatment demonstrated statistically significant dose-dependent anti-tumor response in hPBMC-humanized murine HCC827 and U87MG tumor models. Additionally, ivonescimab enhanced anti-tumor response in combination with anti-CD47 (AK117) or anti-CD73 (AK119) in mouse models (figure 1). Finally, ivonescimab contains Fc-silencing mutations abrogating FcgR/IIIa binding and showed significantly reduced ADCC, ADCP activities and cytokine release in-vitro (figure 2). Clinically, this consistent with the safety profile in Phase 1/2 studies of ivonescimab in advanced solid tumors.4 5
Conclusions Ivonescimab is a novel tetravalent anti-PD-1/VEGF bispecific antibody displaying unique cooperative binding to each of its intended targets consistent with increased in vitro functional bioactivities compared with bevacizumab or PD-1 inhibitors alone. Importantly, the Fc-null IgG1 design resulted in reduced FcgR interactions and minimal ADCC, ADCP activities consistent with its clinical immunosafety profile. Ivonescimab is currently in Ph3 NSCLC trials in the US and EU.
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Ivonescimab demonstrates anti-tumor response in both single/combination treatment in mice. (A-B) Each mouse was inoculated subcutaneously at the right hind flank with HCC827 cells. PBMCs and ivonescimab: bevacizumab or isotype control anti-HEL mixture were administered on day 0. Different doses of antibodies were then continuously intravenously injected on day 7, 14, 21, 28, 35. (A) Percentage of tumor growth inhibition (TGI%) on day 60 was calculated with the formula: TGI(%) = [1-(RTV of the treated group)/(RTV of the control group)]×100 (%), The relative tumor volume (RTV) was calculated using the following formula: RTV=(tumor volume on measured day)/(tumor volume on day 0). (B) Body weight was measured over time (n=3–7 for each group). (C) MC38-hPDL 1/hCD73 cells were subcutaneously inoculated into C57BL/6-hPD1/hPDL1/hCD73 transgenic mice. Mice were grouped when tumor volume reached 80–120 mm3 and anti-CD73 (AK119) and/or ivonesimab were then intraperitoneally injected biweekly. Tumor was monitored over time. (D) MDA-MB-231 cells were subcutaneously inoculated in SCID/Beige mice. Mice were grouped when tumor volume reached 100 mm3 and intraperitoneally injected with activated hPBMCs. Anti-CD47 (AK117) was then continuously intravenously injected twice per week for eight times and ivonescimab was administrated weekly for four times. Tumor volume was monitored over time. *p<0.05, **p<0.01, ***p<0.001. Comparisons of tumor growth among groups Mere analyzed using two-way ANOVA followed by Bonferroni’s multiple comparison test and survival rate comparisons among groups were analyzed using Log-rank test
Immunosafety profile of ivonescimab vs nivolumab in-vitro. (A) The binding profiles of ivonesclmab and nivolumab to FCγRI were analyzed by Octet BLI. (B) ADCC of ivonescimab was determined by measuring lactase dehydrogenase (LDH) release in mixed culture of CHO-K1-PD1 cells and human PBMCs (C) ADCP activities of ivonescimab were measured by reporter assays. Jurkat-NFAT-CD64-CD32H cells and CHO-K1-PD1 cells were co- cultured for 5 hrs in the presence of indicated antibodies. (D) The release of inflammatory cytokines IL-1β, TNF-α and IL-6 from human PBMCs when treated with ivonescimab were accessed by ELISA. LPS were used as a positive control.
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