DOI: 10.5061/dryad.vdncjsz83
title: Bispecific antibodies and CAR T cells targeting a TP53 mutation-associated neoantigen show discordant affinity requirements

description: 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.
description: # Bispecific antibodies and CAR T cells targeting a TP53
 mutation-associated neoantigen show discordant affinity requirements Dryad
 DOI:
 [https://doi.org/10.5061/dryad.vdncjsz83](https://doi.org/10.5061/dryad.vdncjsz83) This dataset contains the NGS sequencing data used to score the affinity maturation of the H2 single chain variable fragment (scFv) targeting the p53 R175H MANA (HMTEVVRHC presented on HLA-A*02:01). ## Description of the data and file structure This dataset contains three files as follows: 1. "DiNapoliSequencingBarcodeKey_20251016Corrected.xlsx" is an MS Excel workbook with two worksheets. One each for a sequencing run containing the affinity maturation data from two separate experiments. The name of the worksheets corresponds to the names of the run folders and contains a list of which parts of reads to use to cover the scFv altered regions and a list of the barcode indexes corresponding to the affinity maturation samples. 2. "032020_Sarah_PhageSeq_9822SP-A.zip" is a sequencing run folder containing the compressed (*.gz) files for the index read ("Undetermined_S0_L001_I1_001.fastq.gz") and 6 reads ("Undetermined_S0_L001_R1_001.fastq.gz" to "Undetermined_S0_L001_R6_001.fastq.gz") covering the regions altered in the affinity maturation experiment "032020_Sarah_PhageSeq_9822SP-A". 3. "200821_M01208_0724_000000000-CRBLG.zip" is a sequencing run folder containing the compressed (*.gz) files for the index read ("10075SP-A-082120_S1_L001_I1_001.fastq.gz") and 6 reads ("10075SP-A-082120_S1_L001_R1_001.fastq.gz" to "10075SP-A-082120_S1_L001_R6_001.fastq.gz") covering the regions altered in the affinity maturation experiment "200821_M01208_0724_000000000-CRBLG". ## Sharing/Access information None ## Code/Software None