Background Immune-checkpoint inhibitors (ICI) like PD1/PD-L1 blockers have been great successes in cancer immunotherapy. It normalizes anti-tumor immunity by reversing effector T cells (T_eff) exhaustion within TME. However, majority of treated patients still responded poorly, even for melanoma. Insufficient SIRPα-expression (CD47 ligand) on melanoma cells was blamed for the poor response, leading to a new theory and subsequently confirmed at pre-clinical setting: simultaneous targeting tumor-infiltrate (TIL)-CD8⁺ T_eff by PD1 mAb and the tumor-expressing SIRPα are both required for long-term reversal of exhaustion.¹ We thus hypothesized that PD1xCD47-dual targeting BsAb (HX009)could potentially enhance T-cell activation over PD1-mAb, seemingly consistent with our earlier observation that HX009 consistently showed higher T-cell activation (~3x per EC₅₀ value) in a reporter assay over PD1-mAb (HX008),² even with low affinity SIRPa/CD47 binding.

Methods A two-cell reporter system containing PD-1⁺CD47⁺ Jurkat reporter cell (luciferase) and PD-L1-expressing APC was used, where Jurkat cell carries an IL-2 promoter-guided luciferase gene expression that mimics T-cell activation quantitively. Seriel dilutions of HX009/HX008, in the presence of various competing or combining molecules, were added in the co-culture system to investigate T cell activation.

Results We repeated the same reporter assay with the presence of SIRPa-neutralizing mAb, which reduced T-cell activation of HX009 to that of HX008, confirming that the T-cell activation difference between HX009 and HX008 indeed resulted from the T-cell surface CD47 engagement by SIRPa on HX009. In contrast, the addition of CD47 mAb (as equal molar as the neutralizing SIRPa mAb) had no effect on T-cell activation by HX009, suggesting that free CD47 mAb at the tested concentration could not meaningfully engage T-cell surface CD47. Our interpretation is that although with magnitude lower affinity than PD1 mAb, SIRPa’s binding avidity to T-cell CD47 remains high due to ‘cis-binding’ driven by the PD1 mAb high affinity binding. This was further confirmed by that soluble SIRPa (SIRPa-Fc) had no enhanced effect when combined with HX008 (PD1 mAb). In another word, HX009 BsAb is superior to the combo of HX008 and SIRPa-Fc. This was also further confirmed with the observatiions that HX009 can compete with high-affinity CD47 mAb binding to PD1⁺CD47⁺ double positive T-cells, but not to the CD47⁺ single positive cells (no enhanced avidity).

Conclusions HX009 CD47xPD1-BsAb T-cell could have potential superior T-cell activation than PD1-mAb, which could be potentially translated into stronger immunotherapy efficacy.

References

1. Zhou Z, Chen MM, Luo Y, Mojumdar K, Peng X, Chen H, Kumar SV, Akbani R, Lu Y, and Liang H. Tumor-intrinsic SIRPA promotes sensitivity to checkpoint inhibition immunotherapy in melanoma. Cancer Cell 2022; 40 , 1324–1340 e1328. 10.1016/j.ccell.2022.10.012.

2. Ke H, Zhang F, Wang J, Xiong L, An X, Tu X, Chen C, Wang Y, Mao B, Guo S, et al. HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent anti-lymphoma activity in preclinical models. Sci Rep 2023; 13 : 5419. 10.1038/s41598–023-32547-y.