Journal: bioRxiv

Article Title: Sequence and structural determinants of efficacious de novo chimeric antigen receptors

doi: 10.64898/2025.12.12.694033

Figure Lengend Snippet: (a) Schematic of CD22 target antigen, with the arrow highlighting the preferred binding pocket as a hydrophobic patch. (b) Summary of YSD screening of de novo designed proteins from two campaigns against CD22. (c) Identification of four hits from the BindCraft campaign via sequencing. (d) CAR co-culture of four de novo CD22 binders (D1-D4) compared to m971 (clinical CAR). Shown is the %CD69 + Jurkats among GFP + cells. (e) Validation of CD22 expression of cell lines in the CAR co-culture. Arrow highlights the absence of CD22 expression in RPMI 8226. (f) Cocultures of three CARs with variable effector to target (E:T) ratios. Statistical test: Wald test of linear regression comparing D1 de novo binder to m971 clinical CAR, adjusting for E:T ratio. (g) Diversifying CD22 binder sequences given a single binder (D1). (h) Triplicate CAR Jurkat co-cultures with variable CAR binders. Statistical test: Two-sided Student’s t test. (i) Summary of diversified CD22 sequences in CAR co-culture. “X” highlights off-target activation from parental binder, D1. (j) Activation scores from scRNA-seq profiles of five CAR binders cultured against two different cell lines. Statistical test: two-sided Mann-Whitney U test. (k) Primary CAR T killing curves against RPMI 8226 (CD22 - ) showing off-target-specific killing in the de novo D1 binder. Statistical test: Wald test of linear regression interaction term between D1.N0 binder and time compared to D1, adjusting for time and binder.

Article Snippet: The following day, cells were washed once with 1× PBS-B (0.25% BSA) and incubated with varying concentrations of biotinylated recombinant antigen BCMA (Sino Biological, Cat. 10620-H40H-B), CD22 (Sino Biological, Cat. 11958-H49H-B), or CD19 (Sino Biological, Cat. 11880-H49H-B) for 1 hour at room temperature.

Techniques: Binding Assay, Sequencing, Co-Culture Assay, Biomarker Discovery, Expressing, Activation Assay, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Sequence and structural determinants of efficacious de novo chimeric antigen receptors

doi: 10.64898/2025.12.12.694033

Figure Lengend Snippet: (a) Summary of mutations introduced to each of the CARPNN diversified CD22 D1 binder. Red residue index denotes interface residues while blue index denotes non-interface residues. (b) Comparison of CAR activation of the evolved CD22 D1 binders in CD22 - RPMI 8226 cell lines and CD22-overexpressing K562 cell lines. (c) Summary of diversified sequences from antigen CAR flow (top) and co-cultures with variable cell lines (bottom). (d) Representative Incucyte killing assays showing the cytolytic activity of CAR T cells expressing either CD22-specific minibinder- or scFv-based receptors. Time-course plots showing normalized red calibrated unit (%RCU) intensity relative to time 0h for each construct. (e) Cytokine productions from CD22-specific CAR T cells in co-cultures with CD22 + and CD22 - target cell lines. Heatmap shows mean cytokine levels across triplicates, revealing elevated cytokine release specifically in response to CD22-expressing targets, consistent with antigen-specific activation and killing. (f) Representative images at 0h and 72h for NB and at 72h for each binder condition to illustrate target-cell killing. Green fluorescence denotes CAR T cells, and red fluorescence denotes the corresponding target cell line. (g) Characterization of CAR antigen binding at variable CD22 concentrations. (h) Identification of plausible candidates of D1 off-target interaction via subsetting HPA surfaceome and GTEx overlap. (i) Comparison of average cofolding ipSAE score between parental D1 to all plausible off-target genes and the evolved D1.N0 binder to plausible off-target genes. (j) Predicted binding site of parental D1 binder towards CXCR4 aligned to a solved structure of CXCR4 (PDB: 8U4R).

Article Snippet: The following day, cells were washed once with 1× PBS-B (0.25% BSA) and incubated with varying concentrations of biotinylated recombinant antigen BCMA (Sino Biological, Cat. 10620-H40H-B), CD22 (Sino Biological, Cat. 11958-H49H-B), or CD19 (Sino Biological, Cat. 11880-H49H-B) for 1 hour at room temperature.

Techniques: Residue, Comparison, Activation Assay, Activity Assay, Expressing, Construct, Fluorescence, Binding Assay