biotinylated cd22 protein  (ACROBiosystems)

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

Journal: bioRxiv

Article Title: MiniCARbids: Minimalistic human binding domains specifically tailored to CAR T applications

doi: 10.1101/2025.09.09.675083

Figure Lengend Snippet: (A) The K D values of CD22-miniCARbids were determined by titrations of soluble CD22-miniCARbids on NALM6 cells. (B) A representative example of titrations of miniCARbids 22_1611 and 22_1317 on NALM6 cells is shown. The binding intensity was assessed via anti-His-tag staining by flow cytometry. Data were fitted with a 1:1 binding model (solid lines) for the calculation of the respective K D values illustrated in (A) (average ± SD, n=3 or 4, biological replicates). (C) Thermostability of CD22-miniCARbids and their parental protein 5UMR was assessed using DSC (average ± SD of 3 independent measurements, technical replicates). (D) Aggregation properties of CD22-miniCARbids were assessed using SEC-HPLC. One representative analysis (n=3, technical replicates) of CD22-miniCARbids and their parental protein 5UMR is shown. (E) Binding specificity was assessed by incubating NALM6, Raji or Jurkat (CD22-negative) cells with 250 nM CD22-miniCARbid, followed by flow cytometric analysis (one of three biological replicates is shown).

Article Snippet: Selection campaigns started with magnetic bead selections using Dynabeads Biotin Binder (Thermo Fisher Scientific) as described previously., Yeast display selections for miniCARbids against CD22 were based on a soluble, biotinylated CD22 protein (AcroBiosystems, SI2-H82E3).

Techniques: Binding Assay, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: MiniCARbids: Minimalistic human binding domains specifically tailored to CAR T applications

doi: 10.1101/2025.09.09.675083

Figure Lengend Snippet: (A) CAR architecture used for the in vitro assessment of CAR activity. (B) Expression of CARs based on ten CD22-specific miniCARbids and scFvs HA22, m971-1xG 4 S and m971-4xG 4 S as benchmarks in Jurkat Nur77 reporter cells was assessed via anti-MAP-tag staining by flow cytometry (average ± SD, n=3, biological replicates). (C) Activation of CD22-specific CARs in Jurkat Nur77 reporter cells in the presence or absence of a 2-fold excess of NALM6 target cells was assessed via the expression of mKO2 by flow cytometry (average ± SD, n=3, biological replicates). (D) Cytotoxicity of CD22-specific CAR T cells and mock T cells (no CAR) against Raji cells (E:T 2:1, average ± SD, n=4, biological replicates). (E and F) Release of IFN-γ (E) and IL-2 (F) analyzed via ELISA. The cytokines were analyzed in the supernatants of co-cultures with Raji cells (E:T 2:1, average ± SD, n=4, biological replicates). (G) Cytotoxicity of CD22-specific CAR T cells and mock T cells (no CAR) against NALM6 cells (E:T 2:1, average ± SD, n=4, biological replicates). (H and I) Release of IFN-γ (H) and IL-2 (I) analyzed via ELISA. The cytokines were analyzed in the supernatants of co-cultures with NALM6 cells (E:T 2:1, average ± SD, n=4, biological replicates). Statistical analysis was performed using a repeated measure One-Way ANOVA with a Tukey post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). The statistical analysis for the cytokine concentration was performed using log-transformed values. Parts of this figure were created with BioRender.com.

Article Snippet: Selection campaigns started with magnetic bead selections using Dynabeads Biotin Binder (Thermo Fisher Scientific) as described previously., Yeast display selections for miniCARbids against CD22 were based on a soluble, biotinylated CD22 protein (AcroBiosystems, SI2-H82E3).

Techniques: In Vitro, Activity Assay, Expressing, Staining, Flow Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transformation Assay

Journal: Experimental Hematology & Oncology

Article Title: Improving CAR-T cell function through a targeted cytokine delivery system utilizing car target-modified extracellular vesicles

doi: 10.1186/s40164-025-00701-z

Figure Lengend Snippet: Targeted delivery EV to CD22 CAR-T cells and targeted delivery IL-2 EV to CD19 CAR-T cells. ( A ) Schematic experimental workflows of EV production and quality control node. ( B - D ) 3 × 10⁵ CAR-T cells (cell density: 1*10 6 /mL) were mixed with Raji cells at an effector-to-target ratio of 1:1 and treated with PBS, control EVs, rhIL-12 and IL-12 EVs respectively ( n = 3 donors). ( B ) Cytokine secretion by CAR-T cells was detected by ELISA after 24 h of coculture. ( C ) CD107a expression in CD8 + CAR-T cells was detected by flow cytometry. ( D ) Raji cell and k562 cell death were determined using PI (BD Pharmingen) and analyzed by using flow cytometry after 24 h. ( E ) Subsets were detected via flow cytometry in CAR-T cells after 7 days of treatment. ( F ) Quantification of IL-2 concentration in EVs by ELISA (independent experiments with n = 3). Mean ± SEM. ( G ) CD19 CART cells labeled with CFSE were cocultured with various types of EVs for 96 h ( n = 3 donors). * p < 0.05. Protein concentration of EVs is 167 µg/mL, and rhIL-12 concentration is 667 pg/mL

Article Snippet: For CD19 CAR expression assays, cells were stained with PE-labeled (Acro Biosystems, Cat.CD9-HP2H3) or FITC-labeled human CD19 protein (Acro Biosystems, Cat. CD9-HP2H3) or APC-conjugated anti‐human EGFR (clone: AY13; BioLegend) For CD22 CAR expression analysis, cells were stained with FITC-labeled (Acro Biosystems, Cat. No. CD2-HF254) or APC-labeled recombinant human CD22 protein (Acro Biosystems, Cat. No. SI2-HA2H4).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Concentration Assay, Labeling, Protein Concentration