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

Kinzler, Kenneth1; DiNapoli, Sarah1

Research facility: Ludwig Center at Johns Hopkins University
Published Nov 16, 2025 on Dryad. https://doi.org/10.5061/dryad.vdncjsz83

Data files

Nov 16, 2025 version files 2.83 GB

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Abstract

Data deposited here are the sequencing data of scFv from the affMat reported in the study below:

Mutation-associated neoantigens (MANAs) are highly cancer-specific targets for immunotherapy when 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, CD3 gamma, or the T-cell receptor (TCR). 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.