Background Several PD-1 antibodies have been approved as anti-cancer therapies which work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. However, cross-reactive anti-PD-1 antibodies binding to both human and pre-clinically relevant animal models are still required to predict more precisely the efficacy and toxicity in humans, especially in various combination settings of anti-PD-1 with other treatments.
Methods Therefore, we have developed an anti-PD-1 named GNUV201, a highly selective and interspecies cross-reactive monoclonal antibody, with conventional hybridoma technology.
Results GNUV201 equally binds to mouse and human PD-1 (EC50=32 pM and 28 pM, respectively), therefore successfully suppressed the growth of B16F10 (mouse melanoma) and MC38 (mouse colon cancer) in syngeneic mice models.
According to the results of co-crystal structure and alanine-scanning mutagenesis to examine the interaction mode between GNUV201 and human PD-1, the epitope recognized by GNUV201 is on the “FG loop” region of human PD-1, which is well conserved in both human and mouse PD-1, supporting GNUV201’s interspecies cross-reactivity; this position is distinct from those of Keytruda (“C’D loop”) and Opdivo (N-term). Notably, the structural feature in which the protruding epitope loop fits into GNUV201’s binding pocket supports the enhanced binding affinity due to slower dissociation (8.5 times slower than Keytruda in SPR). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (KD = 0.9 nM at pH 6.0, 5.8 times smaller KD than at pH 7.4), which mimics more closely the hypoxic and acidic tumor micro-environments (TME), especially due to further improved Koff (32.8 times slower dissociation than Keytruda in SPR). This phenomenon is not observed with other anti-PD-1 competitors, implying that GNUV201 achieves selective binding to and better occupancy on PD-1 in tumors compared to its marketed competitors. GNUV201 was also shown to completely block the interaction of hPD-1 with hPD-L1 in vitro at similar single digit nM levels compared with Keytruda and Opdivo, which results in enhanced T cell proliferation and cytokine (IL-2 and INF-g) secretion.
Conclusions In summary, even though similar in vitro efficacy of GNUV201, we can expect GNUV201 to be an excellent antibody candidate with superior target occupancy due to its slow dissociation and preferential binding in low pH tumor microenvironments and whose efficacy and toxicity in the human system can be predicted from preclinical study results due to its human-mouse cross-reactivity.
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