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895 Discovery of a safer 4–1BB agonist by targeting a membrane-proximal epitope combined with bispecific-mediated cross-bridging
  1. Andy Tsun1,
  2. Tianhang Zhai2,
  3. Xiaoniu Miao3,
  4. Weifeng Huang4,
  5. Chao Wang3,
  6. Yifeng Xu3,
  7. Zhijun Yuan3,
  8. Tao Wang3,
  9. Shuang Dai4,
  10. Shaogang Peng4,
  11. Tuling Pang3,
  12. Wenchao Jiang3,
  13. Yuhua Huang3,
  14. Yuefeng Zou3,
  15. Yingda Xu3,
  16. Joanne Sun3,
  17. Xinjiang Gong3 and
  18. Bin Li2
  1. 1Biotheus Inc, Zhuhai, China
  2. 2Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Suzhou, China
  3. 3Biotheus Inc., Zhuhai, China
  4. 4Biotheus (Suzhou) Co., Ltd., Suzhou, China


Background Checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have paved the way for a new frontier of anti-cancer therapies that modulate our pre-existing immune system to fight against malignancies. 4-1BB is a tumor-necrosis superfamily member expressed on NK and T cells, that acts as a co-stimulatory receptor to improve effector/memory responses towards tumors. Early efforts have focused on the generation of agonist antibodies towards 4-1BB that relied on Fc-mediated cross-linking to cluster and induce receptor downstream signaling, but has led to liver- and immune-related toxicities. We have discovered a PD-L1 x 4-1BB bispecific that exhibits conditional agonist activity in the presence of PD-L1 with better safety features.

Methods vHH binders to PD-L1 and 4-1BB were generated from immune libraries derived from camelids and selected via yeast display. Antibody screening was carried out by protein-protein interaction analysis and cell-based binding. Target-related activity was confirmed using luciferase reporter assays. Primary immune cells were also tested for T cell activation via the detection of IL-2 and IFNg secretion. PD-L1-mediated 4-1BB activation via cross-bridging was carried out using target cells expressing PD-L1 co-cultured with effector cells. X-ray crystallography was conducted to resolve the binding sites of both the anti-PD-L1 and anti-4-1BB vHHs. Both tumor efficacy and safety assessment were tested in human knockin mice.

Results The 4-1BB binder of PM1003 was found to interact with the CRD4 domain of 4-1BB, as determined by X-ray crystallography. Binding to this domain does not affect the binding between 4-1BB and its ligand 4-1BBL. The anti-PD-L1 vHH binds to an epitope on PD-L1 that overlaps with the binding region of PD-1, and is thus effective in disrupting the interaction between PD-1 and PD-L1. Using luciferase reporter assays and primary cell assays we found the PM1003 could activate 4-1BB in the context of PD-L1-mediated cross-bridging. Data from human 4-1BB and PD-L1 knockin mice also showed that PM1003 could effectively control tumor growth without observing any toxicity signals.

Conclusions PM1003 is a safe and efficacious bispecific antibody that blocks PD-L1 and concurrently activates 4-1BB receptor. An antibody with mild activity was selected directed against the CRD4 domain of 4-1BB to elicit effective potency while minimizing potential toxicity issues. This was reflected in our results. Thus, PM1003 is a potential next generation therapeutic antibody in the IO space that combines and synergizes two independent signaling pathways to control tumor growth.

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