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

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.

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Publication Details

Journal
Journal of Clinical Investigation
Published
2026-01-15
DOI
https://doi.org/10.1172/jci192885
Primary Topic
Monoclonal and Polyclonal Antibodies Research
Type
article
Field-Weighted Citation Impact
0.00

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article

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

Stephanie Glavaris, Evangeline Watson, Marco Dal Molin, Jacqueline Douglass et al.
Journal of Clinical Investigation
Monoclonal and Polyclonal Antibodies Research
article

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

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

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.

Journal of Clinical InvestigationVol. 136(2)
Howard Hughes Medical Institute (US), Johns Hopkins University (US), Johns Hopkins Medicine (US), Lustgarten Foundation (US), Center for Rheumatology (US), Sidney Kimmel Comprehensive Cancer Center (US)
Amgen, Bristol-Myers Squibb, Gilead Sciences, Johns Hopkins University, Georgetown University, Sidney Kimmel Comprehensive Cancer Center, Ludwig Center at Harvard
Good health and well-being
Openalex Percentile: Top 89%
Monoclonal and Polyclonal Antibodies Research
DOIOpen Access PDFOpenAlex
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