Bispecific antibodies and CAR T cells targeting a TP53 mutation–associated neoantigen show discordant affinity requirements
Sarah R. DiNapoli, Katharine M. Wright, Brian J. Mog, Alexander H. Pearlman, Tushar D. Nichakawade, Nikita Marcou, Emily Han-Chung Hsiue, Michael S. Hwang, Jacqueline Douglass, Qiang Liu, Evangeline Watson, Marco Dal Molin, Joshua D. Cohen, Maria Popoli, Suman Paul, Maximilian F. Konig, Nicolas Wyhs, P. Aitana Azurmendi, Stephanie Glavaris, Jiaxin Ge, Tolulope O. Awosika, Jin Liu, Kathleen L. Gabrielson, Sandra B. Gabelli, Drew M. Pardoll, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Shibin Zhou, Bert Vogelstein
Sarah R. DiNapoli, Katharine M. Wright, Brian J. Mog, Alexander H. Pearlman, Tushar D. Nichakawade, Nikita Marcou, Emily Han-Chung Hsiue, Michael S. Hwang, Jacqueline Douglass, Qiang Liu, Evangeline Watson, Marco Dal Molin, Joshua D. Cohen, Maria Popoli, Suman Paul, Maximilian F. Konig, Nicolas Wyhs, P. Aitana Azurmendi, Stephanie Glavaris, Jiaxin Ge, Tolulope O. Awosika, Jin Liu, Kathleen L. Gabrielson, Sandra B. Gabelli, Drew M. Pardoll, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Shibin Zhou, Bert Vogelstein
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Research Article Immunology Oncology

Bispecific antibodies and CAR T cells targeting a TP53 mutation–associated neoantigen show discordant affinity requirements

Abstract

Mutation-associated neoantigens (MANAs) are highly cancer-specific targets for immunotherapy where peptides derived from intracellular mutant proteins are presented on the cell surface via HLA molecules. T cell–engaging bispecific antibodies and CAR T cells can target MANAs to eliminate cancer cells via T cell activation. However, the low antigen density of MANAs on the cell surface can limit therapeutic efficacy. Here, we investigated whether increasing the affinity of the H2 single-chain variable fragment (scFv) targeting the p53 R175H MANA (HMTEVVRHC presented on HLA-A*02:01) improves its therapeutic effect. We identified higher-affinity H2 variants via phage biopanning and a thiocyanate elution method. Increasing bispecific antibody affinity to the low nanomolar range increased cancer cell killing and tumor control in mouse xenograft models without sacrificing antigen specificity. We next asked how increasing scFv affinity impacts CAR T cell function — a matter of debate. We appended each variant scFv to a CD28z CAR, CD3γ, or the T cell receptor. In striking contrast to the bispecific antibody results, increasing CAR affinity decreased function in each CAR format due to lower T cell activation upon interaction with target cancer cells. These results have important implications for the design of future immunotherapeutic approaches targeting low-density antigens.

Authors

Sarah R. DiNapoli, Katharine M. Wright, Brian J. Mog, Alexander H. Pearlman, Tushar D. Nichakawade, Nikita Marcou, Emily Han-Chung Hsiue, Michael S. Hwang, Jacqueline Douglass, Qiang Liu, Evangeline Watson, Marco Dal Molin, Joshua D. Cohen, Maria Popoli, Suman Paul, Maximilian F. Konig, Nicolas Wyhs, P. Aitana Azurmendi, Stephanie Glavaris, Jiaxin Ge, Tolulope O. Awosika, Jin Liu, Kathleen L. Gabrielson, Sandra B. Gabelli, Drew M. Pardoll, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Shibin Zhou, Bert Vogelstein

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Figure 1

Functional testing of variants selected by thiocyanate enrichment screen.

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Functional testing of variants selected by thiocyanate enrichment screen...
(A and B) Variants were expressed as scDbs. 5 × 104 primary human T cells were cocultured overnight with 2.5 × 104 T2A3 target cells pulsed with (A) 10 nM or (B) varying concentrations of the p53 R175H or R175WT 9mer peptide in the presence of 1 nM scDb. (A) IFN-γ production and (B) percent cytotoxicity compared with no scDb control by CellTiterGlo assay (n = 3 biological replicates, single experiment). (C and D) 5 × 104 T cells were cocultured with 2.5 × 104 luciferase+ KMS26 target cells (p53 R175H or p53 KO) and scDb for 18 hours. (C) IFN-γ production. (D) Percent cytotoxicity measured by SteadyGlo assay (n = 3 biological replicates, single experiment). All data are shown as the mean ± SD. (E and F) Top variants were combined into double-mutant scDbs. (E) scDb was applied to plates coated with CD3ε/δ heterodimer, or R175H/A2 or R175WT/A2 monomer. Relative binding was calculated by normalizing mean monomer binding to mean CD3 binding (n = 3 technical replicates, single experiment). (F) 3 × 104 T cells were cocultured with 1.5 × 104 KMS26 target cells in the presence of scDb for 19 hours. Percent cytotoxicity by SteadyGlo assay (n = 3 biological replicates per condition, single experiment).