cd22 protein fc  (R&D Systems)

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