biotinylated cd33 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: Chimeric Antigen Receptors Transmit Piconewton Forces that are Coupled with T Cell Function

doi: 10.1101/2025.10.23.684052

Figure Lengend Snippet: A. Proposed mechanism of action for dasatinib in CAR T cells which leads to Lck inhibition. B . Representative RICM and tension images of CAR T cells treated with escalating doses of dasatinib. C and D. Plots quantifying CAR T cell forces and cell spread area as a function of dasatinib treatment. N ≥ 162 from 5 independent transductions. ***= 0.0001, ****<0.0001. E . Plot showing cytotoxicity as a function of dose-dependent dasatinib CAR T cell treatment. Data is averaged from two technical replicates using the same batch of CAR T cells. F . Relationship between CAR T cell tension and cytotoxicity showing correlation. G . Schematic showing three additional engineered CARs with 4-1BB costimulatory domain, ITAM mutants lacking key tyrosine residues, and finally CAR lacking a cytoplasmic domain along with representative RICM and 8 pN locked tension (Cy3B) images. H . Plot of single cell tension levels for each CAR T cell construct tested. N ≥ 162 from 5 independent transductions. One-way ANOVA. ***= 0.0001 , ****<0.0001. I . Representative RICM and 8 pN tension images showing the tension and spread area of cells on CD19 and CD33 antigens. J. Quantification of cellular tension levels from n≥90 cells/condition from 3 separate transductions. Each color indicates a biological replicate from a unique transduction using the same human donor. The larger data points indicate the mean of each replicate while the small data points represent the single cell measurements. N ≥ 106 cells/condition from 3 independent transductions. Student’s Two-Tailed Paired T Test, ****<0.0001.

Article Snippet: Biotinylated CD19 protein (Cat#CD9-H82E9-25ug), biotinylated CD33 protein (Cat#CD3-H82E7-25ug), and AlexaFluor 647 Anti-FMC63 Ab (Cat#FM3-AM534-25tests) were purchased from AcroBioSystems (Newark, DE).

Techniques: Inhibition, Construct, Transduction, Two Tailed Test

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

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: Tmod can be adapted for blood cancer. (A) Diagram for Tmod system showing the two receptors that comprise the NOT gate targeting HLA loss of heterozygosity (LOH) in solid tumors. (B) Diagram for Tmod utilizing tandem receptors for blood cancer. (C) CD33 and CD16b mRNA expression in primary AML and healthy blood cells including T cells, neutrophils, monocytes, and hematopoietic stem cells (HSC) (data from sources shown; see Supplementary Table 1 ). (D) mRNA expression of targets in AML cell lines (n=43; DepMap).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: CD33 | CD16b Tmod functions robustly in Jurkat and primary T cells. (A) Diagram of functional screen in Jurkat reporter cell line cocultured with K562 target cells transfected with different amounts of CD16b mRNA. Tmod transgene expression in Jurkat cells was detected by staining with recombinant human (rh) CD16b and CD33. (B) Diagram of functional parameters estimated from the Jurkat cell assay data. (C) Functional readout from 8-point mRNA titration curves. Three CARs combined with 4 blockers, that were selected for further analysis, are shown in color. Data are shown as mean ± standard deviation of technical replicates (n=2), normalized to each sample’s maximum activation. (D) Left, diagram of non-viral construct-screening in primary T cells using PiggyBac transposase and single vectors (BA vectors). Right, metrics used to quantify the potency and selectivity of the Tmod pair. (E) Flow cytometry analysis of stable integrants via staining with labeled recombinant human CD33 (see Methods). (E, F) T cell cytotoxicity curves generated from GFP signal at 48 hour time point with each well normalized to the zero time point. Tumor (CD33(+)CD16(-)) target-cell curves are shown with dashed lines and “normal” (CD33(+)CD16(+)) target-cell curves with solid lines. Black lines are CAR constructs and colored lines are Tmod constructs. Tumor cells are K562 cells engineered with CD33 and normal cells are K562 cells engineered to overexpress CD33 and CD16b. Data are shown as mean ± standard deviation of technical replicates (n=3). (G) Potency calculated as ET50 of Tmod cells cocultured with tumor cells. (H) Selectivity ratios are calculated as ET50 on normal cells divided by ET50 on tumor cells. (I) Kinetic cytotoxicity analysis of the most selective and potent construct compared to the CAR-T. GFP(+) area was used as proxy for target cell viability. Data are shown as mean ± standard deviation of technical replicates (n=3).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Functional Assay, Transfection, Expressing, Staining, Recombinant, Titration, Standard Deviation, Activation Assay, Construct, Flow Cytometry, Labeling, Generated

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: CD33 | CD16b Tmod cells selectively kill tumor but not “normal” cells in vivo . (A) Schema for in vivo experiment. 2 million MV-4-11 AML cells or MV-4-11 cells that overexpress CD16b were injected into NSG-SGM3 mice and 6 days later 7.5 million T cells were injected. (B) Selectivity in vitro using MV-4-11 cells. Surrogate normal cells were generated by overexpression of CD16b in the AML cells. E:T cytotoxicity curves were generated from firefly luciferase bioluminescence at 48 hours. Data are shown as mean ± standard deviation of technical replicates (n=3). Inset: ET50 values of depicted curves. Data shown are interpolated values with 95% CI. (C) Flow cytometry analysis of construct expression by staining with labeled recombinant human CD16b and CD33. (D) Bioluminescence imaging (BLI) at 20 days post target-cell injection. (E) Flow cytometry analysis of MV-4-11 cells in the bone marrow 27 days post target-cell injection. (F) Quantification of data shown in panel. (E) Statistics were calculated using a non-parametric Kruskal-Wallis H test; *: 0.01 < adjusted p < 0.05; **: adjusted p value < 0.01; ns: not significant (adjusted p > 0.05).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: In Vivo, Injection, In Vitro, Generated, Over Expression, Luciferase, Standard Deviation, Flow Cytometry, Construct, Expressing, Staining, Labeling, Recombinant, Imaging

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: Tandem Tmod constructs for blood cancer. (A) Diagram of a Tmod cell with bispecific activator to target AML (CD33) and other blood cancers (SPN) and bispecific blocker to protect HSCs (CLEC9A) and neutrophils (CD16b). (B) Target expression in primary blood cancers and healthy blood cells (data from sources shown; see Supplementary Table 1 ). (C) Target expression in blood cancer cell lines (DepMap). (D) Jurkat cell (SPN KO) functional readout of SPN | CD16b Tmod with blocker titration curves. (E) Jurkat cell (SPN KO) functional readout of SPN-CD33 tandem CAR activation and blocking by CD16b blocker in the presence of SPN and CD33 antigens. (F) Jurkat functional readout of binders cloned as CARs with titration of primary HSCs. (G) Jurkat functional readout of CD33 CAR4 blocked by tandem CLEC9A-CD16b blocker. (H) Jurkat functional readout of CD33 and/or SPN monospecific or bispecific activators paired with CD16b and/or CLEC9A monospecific or bispecific blockers. Data are shown as mean ± standard deviation of technical replicates (n=2).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Construct, Expressing, Functional Assay, Titration, Activation Assay, Blocking Assay, Clone Assay, Standard Deviation