505 A novel anti-PD-L1/anti-VEGF bispecific VHH antibody exhibits a synergistic anti-tumor effect compared to the monospecific VHH antibodies

<h3>Background</h3> Clinical research has demonstrated that combining immunotherapy anti-PD-1/PD-L1 antibodies with anti-angiogenic VEGF/VEGFR blockers improves cancer treatment outcomes. These combined therapies have received approval for several cancer types. A bispecific antibody targeting both PD-L1 and VEGF could enrich the antibody concentration in tumor tissue by specifically binding to PD-L1 expressed in the tumor, thereby increasing VEGF blockade in the local tumor area. Currently, there is no approved monotherapy targeting both PD-L1 and VEGF pathways. IMM2510 and PM8002 are two early-stage drug candidates with dual anti-PD-L1 and anti-VEGF properties. They exhibited promising efficacy against advanced solid tumors and favorable tolerance in clinical trials. <h3>Methods and Results</h3> We developed a novel bispecific anti-PD-L1/anti-VEGF antibody, BB-203. BB-203 was engineered using humanized anti-PD-L1 and anti-VEGF VHH fragments fused with a human IgG1 Fc. BB-203 maintained the high binding affinities and potent biological functions of its parental VHH-Fc antibodies. Results from whole-cell binding assays showed that BB-203 had a higher binding affinity to PD-L1 than Avelumab. BB-203 also exhibited higher potency in blocking human PD-L1 compared to Avelumab, IMM2510, and PM8002 in PD1/PD-L1 blockade assay. The potency of BB-203 in blocking human VEGFA was similar to Avastin, IMM2510, and PM8002 in VEGF bioassay. BB-203 showed remarkable stability in both accelerated stability tests and serum stability tests. Pharmacokinetic studies revealed that BB-203 had a PK profile similar to Avelumab, a conventional IgG1 antibody. The <i>in vivo</i> efficacy of BB-203 was evaluated using a MC38-hPD-L1 model with MC38 cancer cells genetically modified to express the human PD-L1 to replace the mouse PD-L1. The study demonstrated that BB-203 had robust anti-tumor activity, achieving an 81% tumor growth inhibition (TGI), significantly higher than that of its parental monospecific VHH-Fc antibodies, which had TGIs of 24% or 30%, respectively. This highlights the synergistic anti-tumor effect of the two arms of BB-203. Additionally, BB-203 exhibited significantly greater tumor suppression compared to an equimolar dose of IMM2510, which achieved a TGI of 56%. The superior efficacy of BB-203 compared to IMM2510 may be attributed to its more potent anti-PD-L1 activity and higher tumor tissue penetration due to its smaller size (105 KDa) compared to IMM2510 (170 KDa). No weight loss or other obvious side effects were observed. <h3>Conclusions</h3> The novel anti-PD-L1/anti-VEGF bispecific VHH antibody BB-203 has potent target-blocking activities <i>in vitro</i> and strong anti-tumor efficacy <i>in vivo</i>. Together with its excellent developability, BB-203 potentially is the best-in-class anti-PD-L1/anti-VEGF bispecific antibody drug candidate.