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) Yeast surface display plasmid encoding a de novo minibinder flanked by an N-terminal HA-tag and a C-terminal Myc-tag under a GAL1 promoter. (b) E.coli protein expression plasmid encoding de novo minibinder flanked by a 6xHis-tag for purification under a araBad promoter. (c) Representative gating strategy used during yeast display campaigns to identify binder hits. (d) Decoy binding of designed binder campaigns against CD19 antigen (100 nM), demonstrating variable levels of off-target reactivity. (e) Enrichment of individual binders (L10 and L11) from the RFdiffusion 5 campaign across increasing rounds of MACS. (f) Biolayer interferometry (BLI) curves and dissociation constants (Kd) values for L10 and L11 binding to BCMA antigen (100nM) from the RFdiffusion 5 campaign. (g) Enrichment of individual binders from BindCraft1 (BC1) across successive rounds of magnetic cell sorting (MACS) at decreasing concentrations, demonstrating progressive selection of high-affinity binders. (h) BLI curves and estimated Kds for additional sequencing hits from the BC1 campaign.

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: Plasmid Preparation, Expressing, Purification, Binding Assay, FACS, Selection, Sequencing

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) Representative gating strategy for CAR Jurkat and CAR T cell co-culture assays used to quantify proportion of cells with CD69 activation. (b) CAR construct used in CAR Jurkat and CAR T assays containing an EF1α promoter, CD8 signal peptide, de novo minibinder, CD8 hinge, 4-1BB domain, P2A sequence, and GFP reporter. (c) Predicted structures of two de novo CAR binders designed by RFDiffusion. Binders are colored by per-residue AlphaFold2 predicted Local Distance Difference Test (pLDDT) metrics. (d) CAR Jurkat co-cultures expressing L10 and L11 BCMA-specific de novo minibinders with BCMA⁻ and BCMA⁺ cancer cell lines. (e) %CD69 activation was quantified for CARs expressing no binder (Ø, black), C11D5.3 (red), or B5 minibinder (dark red), in either Jurkat cells or primary T-cells in co-cultures with CD19⁻ and CD19⁺ cancer cell lines, comparing Jurkat and primary CAR T responses within each binder condition. P values calculated using Student’s t -test.

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: Co-Culture Assay, Activation Assay, Construct, Sequencing, Residue, Expressing

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) Structural rendering of the FMC63-CD19 complex (PDB: 7URV) aligned to the CD19-CD81 complex (PDB ID: 7JIC). The binding surfaces on CD19 for both proteins are highlighted. (b) Summary of CD19 protein, highlighting residues and binding surfaces. Barplots show the normalized proportion (out of 100 draws) of contacted residues from design campaigns. (c) Summary of yeast surface display binding of de novo designed proteins against CD19. (d) Quantification of YSD enrichment across six campaigns. (e) Quantification of individual binders enriched from the BindCraft 2 (BC2) campaign, highlighting enriched binders selected for further validation. (f) Characterization of CAR antigen binding at variable recombinant CD19 concentrations. (g) Overview of CAR Jurkat activation screening from BindCraft 2 campaign. (h) Summary of CD69 activation via coculture of C1 and C2 CARPNN evolved binders with Ramos cell line, highlighting selected CARs. (i) Same as (h) but for CAR activation via 24 hours of recombinant CD19 incubation. (j) Concentration-specific activation of variable CARs with recombinant CD19 antigen. (k) Schematic of occluded epitope engagement of CD19-directed minibinders from CD81 binding on target cells using the aligned FMC63-CD19-CD81 complex.

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, Biomarker Discovery, Recombinant, Activation Assay, Incubation, Concentration Assay

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 CD19 C1 binder. Red residue index denotes interface residues while blue index denotes non-interface residues. (b) Predicted structure of the C1 binder in complex with CD19. (c) Heatmap of the mutagenized C1 binder variants tested as CARs in co-cultures. Upper panel (blue) displays CAR binding across the indicated antigen concentrations. The lower panel (red) reports %CD69 activation in CD19 - and CD19 + cell lines. (d) Summary of mutations introduced to each of the CARPNN diversified CD19 C2 binder. Red residue index denotes interface residues while blue index denotes non-interface residues. (e) Predicted structure of the C2 binder in complex with CD19 (f) Same heatmap layout as in (c) but shown here for the mutagenized C2 binder variants tested as CARs.

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, Binding Assay, Activation Assay

Journal: Cell Reports Medicine

Article Title: Engineered T cells stimulate dendritic cell recruitment and antigen spreading for potent anti-tumor immunity

doi: 10.1016/j.xcrm.2025.102307

Figure Lengend Snippet: FX-armed CAR-T cells demonstrate superior tumor control in both murine and humanized mouse models (A) Schematic depicting the constructs of mouse B7-H3 and CD19 CARs. (B–D) Average tumor growth curves of B16F10-CD19 (B) and B16F10-B7-H3 (C); survival curves of B16F10-B7-H3 tumor-bearing mice (D) ( n = 5 mice). (E and F) Absolute number of CAR-T cells and endogenous CD8 + T cells in B16F10-CD19 (E) and B16F10-B7-H3 (F) tumors. (G) Average tumor growth curves of the B16F10-B7-H3 model ( n = 8–10 mice). (H) Schematic depicting the humanized B7-H3 CAR constructs. (I and J) Average tumor growth curves of the PC3 tumor model (I) and survival curves of the A375-B7-H3 tumor model in Flt3ko&hFLT3LG NSG mice (J). (K) Schematic of the humanized Flt3KO&hFLT3LG NSG mice model. (L–N) Average tumor growth curves of A375-B7-H3 (L) and PC3 (M). Survival curves of PC3 tumor-bearing Flt3ko&hFLT3LG NSG mice (N). All data represent the mean ± SEM and were analyzed by Student’s t test for (E), (F), and (K) or two-way ANOVA with Tukey’s multiple-comparisons test for (B), (C), (G), (I), (L), and (M) or log rank (Mantel-Cox) test for (D), (J), and (N). Statistical difference is delineated by ns, not significant, ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: Biotinylated Recombinant Human CD19 Protein , Yeasen , Cat#94100ES20.

Techniques: Control, Construct