Abstract 1838: LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody with highly differentiated binding affinity, shows anti-tumor efficacy in a mouse tumor model
Authors: Huang Xiao , Sun Jianming , Qin Yurong , Lin Huan , Guan Jing , Lai Shoupeng , Kang Xiaoqiang , Ling Hong 
Published: July 2021

Objective: 4-1BB is an inducible costimulatory receptor expressed on activated T and NK cells. 4-1BB ligation on T cells triggers a signaling cascade that results in upregulation of antiapoptotic molecules, cytokine secretion, and enhanced effector function. PD-L1, a ligand of PD-1, is mainly expressed on tumor cells, and a key checkpoint inhibitor implicated in tumor immune evasion. A tetravalent bispecific antibody LBL-024, targeting both PD-L1 and 4-1BB, was generated to maximize anti-tumor immunity.

Methods: The bispecific antibody was designed as 2:2 IgG-scFv format with an affinity ratio of approximately 500:1 between two molecules. The binding affinity of LBL-024 to human PD-L1 and 4-1BB was determined by ForteBio. The activity of LBL-024 was determined using several in vitro assays, including PD-1/NFAT, 4-1BB/NF-κB reporter gene and PBMC activation assays. The in vivo anti-tumor activity of LBL-024 was investigated in the PD-L1/4-1BB double knock-in mouse model (Biocytogen) implanted with MC38 cells expressing PD-L1.

Results: The binding affinity of LBL-024 to PD-L1 and 4-1BB was designed with affinity differentiation (~500-fold) between two molecules, with 0.29 nM to PD-L1 and 146 nM to 4-1BB, respectively, which may restrict activating 4-1BB signal on T cells only in PD-L1 expressing tumor microenvironment, rather activating T cells systemically. In vitro LBL-024 could block PD-1-PD-L1 interaction, recovering NFAT reporter signal comparable to anti-PD-L1 antibody. In 4-1BB/NF-κB reporter gene assay, LBL-024 activated the reporter gene system, only in the presence of CHO-K1-PD-L1 cells, suggesting possibly more restricted activation of 4-1BB within tumor microenvironment and potentially less toxicity in patients. The potency of LBL-024 on 4-1BB/NF-κB reporter gene assay was much stronger than Urelumab, a 4-1BB antibody. In pre-activated human PBMC, co-cultured with CHO-K1-PD-L1 cells, IL-2 level was significantly elevated more in LBL-024 treated group than using an anti-PD-L1, anti-4-1BB, or even the combination of anti-PD-L1 and anti-4-1BB was applied. In a model of huPD-L1/hu4-1BB double knock-in mice implanted with MC38-PD-L1 cells. LBL-024 was shown a significant anti-tumor activity (p&lt;0.001), with 81.8% of tumor growth inhibition (TGI), more efficacious than mice dosed with anti-PD-L1 (70.5% of TGI) and anti-4-1BB (46.1% of TGI).

Conclusions: LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody was shown a great synergetic anti-tumor efficacy in a mouse tumor model, more effective than the antibodies targeting either PD-L1 or 4-1BB alone. With highly differentiated binding affinity between two molecules, the efficacy and toxicity could be well balanced, to avoid the potential toxicity of systemic activation of 4-1BB, hence a promising bispecific antibody for further clinical development.

Citation Format: Xiao Huang, Jianming Sun, Yurong Qin, Huan Lin, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody with highly differentiated binding affinity, shows anti-tumor efficacy in a mouse tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1838.