Background Targeting mutation-associated neoantigens (MANAs) is a highly-cancer specific strategy to selectively eliminate cells harboring common driver mutations in genes encoding intracellular proteins such as Ras and p53.^{1, 2} T cell-engaging bispecific antibodies targeting MANAs redirect T cells to kill cancer cells presenting mutant peptides on human leukocyte antigens (pHLA). However, T cell-redirecting therapies’ efficacy can be limited by the low antigen density of these MANAs on the cell surface. Here, we investigate whether increasing the affinity of a T cell-engaging bispecific antibody (clone H2) targeting the p53 R175H MANA (HMTEVVRHC) presented on HLA-A*02:01 (R175H/A2) improves its efficacy in vitro and in vivo.

Methods To identify higher affinity variants, we screened a phage display library consisting of 1159 single-amino acid variants at 61 sites in the six complementarity determining regions of the H2 single chain variable fragment targeting R175H/A2. Variants retaining R175H/A2 specificity were selected over multiple rounds of panning followed by affinity enrichment via thiocyanate elution. Selected variants were compared to the original H2 bispecific antibody in co-cultures with primary human T cells and cancer cell lines expressing endogenous levels of HLA-A*02:01 and the mutant p53-R175H protein or isogenic control cell lines. For in vivo comparison, NSG mice inoculated with KMS26 or Nalm6 cells and human T cells were treated with a continuous infusion of bispecific antibody for 14 days.

Results Three variant bispecific antibodies were identified with higher affinity and retained specificity for R175H/A2 (KD of 12.9 nM, 6.8 nM, 3.3 nM vs. the original KD of 29.5 nM). The highest affinity variant was a double mutant incorporating two top single variants. Each higher affinity variant elicited greater T cell activation as measured by interferon gamma release and cytotoxicity in co-cultures with cell lines expressing endogenous levels of the R175H/A2 pHLA. In vivo testing demonstrated that the higher affinity bispecific antibodies had improved tumor control in xenograft models compared to the lower affinity bispecific, particularly at a lower treatment dose (0.075 mg/kg/d).

Conclusions Increasing affinity for the p53 R175H/A2 pHLA to the low nanomolar range yields increased T-cell activation and cancer cell killing without sacrificing specificity for the R175H mutation.

References

1. Hsiue EH, Wright KM, Douglass J, Hwang MS, Mog BJ, Pearlman AH, Paul S, DiNapoli SR, Konig MF, Wang Q, Schaefer A, Miller MS, Skora AD, Azurmendi PA, Murphy MB, Liu Q, Watson E, Li Y, Pardoll DM, Bettegowda C, Papadopoulos N, Kinzler KW, Vogelstein B, Gabelli SB, Zhou S. Targeting a neoantigen derived from a common TP53 mutation. Science 2021;371:eabc8697.

2. Douglass J, Hsiue EH, Mog BJ, Hwang MS, DiNapoli SR, Pearlman AH, Miller MS, Wright KM, Azurmendi PA, Wang Q, Paul S, Schaefer A, Skora AD, Molin MD, Konig MF, Liu Q, Watson E, Li Y, Murphy MB, Pardoll DM, Bettegowda C, Papadopoulos N, Gabelli SB, Kinzler KW, Vogelstein B & Zhou S. Bispecific antibodies targeting mutant RAS neoantigens. Sci Immunol 2021;6:eabd5515

Ethics Approval Animal studies were approved by the Johns Hopkins University Animal Care and Use Committee, #MO18M79.