recombinant human siglec  (R&D Systems)

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Histogram showing in-silico analysis of CAR T cell-treated patients ( n = 4219) revealed a high relapse rate, with 42.11% ( n = 216 of n = 513 overall relapse patients) experiencing CD19-negative recurrence after monospecific CAR Therapy ( n = 2916). b Schematic overview of the CAR design strategy showing mono, bi, and trispecific constructs targeting CD19, CD20, and CD22. c Experimental workflow illustrating CAR screening: 1452 CARs were transduced into primary T cells and analyzed for signal-1 (activation), signal-2 (exhaustion), and signal-3 (cell death). Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . d Categorization of CARs into low (L), medium (M), and high (H) levels based on fluorescence intensity cutoffs determined by CD19 CARs as reference. e bar graph showing the distribution of 1452 screened CARs across L-, M-, and H-CARMSeD categories using the CARMSeD scoring system. f AI model development pipeline for CAR dysfunction risk prediction, based on 1,452 CAR constructs with an 80:20 split for training and testing. g–j Performance metrics of AI model predicting CARMSeD scores using 1452 CAR constructs. g ML learning curve of model accuracy over 50 epochs, achieving a training accuracy of 0.98 and validation accuracy of 0.95. h Scatter plot comparing measured versus predicted CARMSeD scores for training ( R 2 = 0.87) and validation ( R 2 = 0.83) sets. i Predicted versus measured CARMSeD scores on the validation set, categorized into low (blue), medium (orange), and high (green) CARMSeD. j Box plots show the median (center line), the 25th–75th percentiles (box), and whiskers extending to the minimum and maximum non-outlier values; individual points denote outliers. Numbers above each box indicate sequence counts. k Molecular dynamics simulation of CAR constructs with varying linker lengths, assessing scFv-scFv interaction. Structural conformations at 0 ns, 50 ns and 200 ns for different CAR scFv arrangements highlighting CDR regions (surface transparency 30%), Root Mean Square Deviation (RMSD) plots over 200 ns for both constructs, respectively, indicating structural stability and conformational changes. l Bar graph showing in vitro receptor binding affinity validation for top humanized scFvs of CD19, CD20, and CD22 CARs ( n = 6 biologically independent samples). Data represent mean ± SEM. ** p < 0.01; **** p < 0.001; ns: not significant. A non-parametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: In Silico, Construct, Activation Assay, Fluorescence, Biomarker Discovery, Sequencing, In Vitro, Binding Assay

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (red), and CD22 (yellow) antigens. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. c–f Line graph of cytotoxicity assays showing antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR T cells ( n = 3 biologically independent samples). g Heatmap showing comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR T cells, represented as fold expansion up to Day 17 with respect to the baseline at the time of cell seeding. h Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (red), and CD22 (yellow) antigens, edited using CRISPR-Cas9 to generate knockout variants. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . i , j Line graph of cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR T cells in eliminating antigen-negative Raji variants, compared to m19 CARs ( n = 5 biologically independent samples). k Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji WT ). Gray circles represent initial engraftment and monitoring phases, pink circle shows the first incubation with Raji WT , while purple circles indicate the timing of the RajiCD19 −/− rechallenge. l Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis (1:10; T: E), and the number of CAR T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge ( n = 5 biologically independent samples). Data represent mean ± SEM. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Construct, Comparison, CRISPR, Knock-Out, Incubation, Lysis

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic timeline of in vivo lymphoma model for evaluation of monospecific and bispecific CAR T cells. Mice were xenografted with RajiWT cells (expressing CD19, CD20, and CD22) (day 0), followed by administration of m19 or b20/19 CAR T cells on day 5 and subsequent RajiCD19 −/− TR on day 12, 19 and 26. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bioluminescent imaging and ( c ) stacked area plot showing tumor burden quantification show effective tumor control by b20/19 CAR T cells versus m19 CARs ( n = 5). d CAR T cell survival over time ( n = 5 mice). e Kaplan-Meier survival curves showing survival outcomes over 70 days ( n = 5 mice). f Analysis of residual tumor CD19 or CD20 tumor cells over time ( n = 5 mice). g , h Bar plot showing Granzyme B and IFN-γ secretion from human CD8 + CAR T cells isolated b20/19 post-treatment to confirm functional cytotoxicity of b20/19 against CD19⁻ targets ( n = 5). The CAR T cells isolated from mice that received conventional monospecific (m)CD19 CAR T cells served as the control for comparison. i , j TR induced upregulation of exhaustion markers PD-1 and LAG-3 ( n = 5 mice). k Immunophenotyping of CAR T cells post-TR shows loss of central memory (T cm ) populations and increased PD-1 expression, consistent with functional exhaustion and limited engraftment ( n = 5 mice). Data represents mean ± SEM. ** p < 0.01; *** p < 0.005; **** p < 0.001. A non-parametric t-test was used for statistical analysis between groups, and for ( k ), a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: In Vivo, Expressing, Imaging, Control, Isolation, Functional Assay, Comparison

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. b Relative mRNA expression levels (normalized to beta actin; ACTB) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR T ( n = 6 biologically independent samples). c Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR T cells after TR with RajiCD19 −/− cells. d Histogram analysis of the flow cytometry plots ( n = 10 biologically independent samples). e Bar graph shows the percentage of CD8 + CAR T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions ( n = 5 biologically independent samples). f Survival of CAR T cells over 15 days under various conditions ( n = 4 biologically independent samples). g Violin plots showing the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown elevated mTOR activity ( n = 6 biologically independent samples). h Bar plots show the percentage of MFI of autophagy from autophagic flux assay ( n = 8 data points from three independent experiments). i Dot plot showing ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from OXPHOS to glycolysis ( n = 12 data points from three independent experiments). j Similarly, OCR with FOXO4 knockdown decreases mitochondrial respiration. Individual data points are shown for each condition ( n = 12 data points from three independent experiments). k Box-and-whisker plot showing percentage of expression of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker ( n = 63 patient samples). l Bar graph showing the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. m Schematic illustration of K562 WT and CD20 expressing K562 stable cells transduced with different MOIs to obtain three populations: CD20 L (low), CD20 M (medium), and CD20 H (high), which were further FACS sorted. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . n Violin plots showing the percentage of CD20 expression (% CD20 expression) in the sorted CD20-expressing K562 cell populations, confirming distinct expression levels ( n = 10 data flow cytometry points from three independent experiments). o Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show CD20 (red) in K562-CD20 L (low), K562-CD20 M (medium), and K562-CD20 H (high) cell. p–r Survival curves of K562 cells expressing varying CD20 expression levels under CAR T cell treatments. The line graph shows the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) ( n = 4 biologically independent samples). s Survival of CAR T cells with varying CD20-targeting CAR constructs over 15 days ( n = 5). Data represents mean ± SEM. **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For e , f and s , a Two-way ANOVA followed by post-hoc testing was applied. Scale bar: 5 μm. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Whisker Assay, Marker, Transduction, Super-Resolution Microscopy, Construct

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic of the strategy for trispecific CAR T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). b Correlation of expression of nbCD3, nbCD22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin, and data were obtained using intracellular flow cytometry ( n = 7 data points from three independent experiments). c Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. d Dose-dependent T cell activation (CD69 expression) in response to culture supernatants (used at various ratios with culture media) with nbCD3/22, using db coated with CD3 for validation ( n = 6 data points from three independent experiments). e Line graph of HEK293T synNotch reporter assay showing dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR T cell supernatants, confirming BiTE functionality under two condition 1 and condition 2. f Experimental timelines for in vitro T cell engineering, transduction, and co-culture with Raji cells (WT or knockout for CD19, CD20, or CD22). Anti-tumor assays were performed on days 9, 11, and 13. g , h Functional assay of CAR T cells against Raji cells (WT or knockout for CD19, CD20, or CD22) demonstrates that b20/19AKT3 PROTAC CAR T cells co-expressing nbCD3/22 exhibit stronger antitumor activity compared to b20/19-AKT3 PROTAC or mCD19 CAR T cells at Day 7 and Day 14. Data represent mean ± SEM. **** p < 0.001; ns: not significant. A nonparametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vitro, Transduction, Co-Culture Assay, Knock-Out, Functional Assay, Activity Assay

Journal: Molecular Therapy Oncology

Article Title: A measles virus encoding a CD19/CD3 bispecific T cell engager shows enhanced preclinical anti-BCP-ALL efficacy without significant toxicity

doi: 10.1016/j.omton.2026.201127

Figure Lengend Snippet: MV-Blina therapy significantly prolongs the survival of ALL bearing mice (A) Treatment scheme. Two PDX models were established in NSG mice by i.v. injection to induce ALL. At a leukemic load of approximately 35%, mice received treatment i.v. with MV (2.5 × 10 5 TCID 50 /g) or control. Two days after infection, mice were injected with healthy human PBMCs. (B) Superior survival of PDX #2 with MV-Blina compared to MV-Edm. PDX #1 and #2 were treated either with MV-Blina and PBMC ( n = 10) or MV-Edm and PBMC ( n = 10) or control with PBMC only ( n = 10) and observed for a maximum of 70 days. (C) MV-Blina treatment decreases leukemic load. Leukemic blasts (msCD45 − huCD19 + huCD45 dim ) and human PBMC (msCD45 − huCD19 + huCD45 bright ) in peripheral blood were monitored by flow cytometry at the indicated time points. (D) Marked reduction of spleen weight in PDX upon MV-Blina treatment. At the time of death, a complete necropsy was performed, and spleen weight was determined. (E) Significant reduction of leukemic blasts by MV-Blina in ALL compartments. At the time of death, leukemic blasts (msCD45 − huCD19 + huCD45 dim ) were detected by flow cytometry in bone marrow, spleen, and meninges. Replication of secBlina was detected by qRT-PCR and calculated by the 2 −ΔΔCt method, displaying the fold change relative to huActin and huGAPDH. (F) MV-Blina reduces leukemic blasts in the spleen by decreasing proliferation and increasing apoptosis. Spleens of untreated mice at therapy start (d0, n = 4) and treated mice (MV-Blina and PBMC, n = 10; MV-Edm and PBMC, n = 9; PBMC-only control, n = 9) were formalin-fixed, paraffin-embedded, and subsequently stained for H&E, CD22, Ki67, or cleaved Caspase-3 (CC3). Positive area was determined using ImageJ. Scale bars 50 μm. Statistical analysis was performed using Mantel-Cox log rank test (B), one-way ANOVA with Tukey’s multiple comparisons test (C-F). ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001.

Article Snippet: Immunohistochemistry for CD22 was performed using a mouse anti-human CD22 antibody (clone OTI1F2, 1:75, TA506404, Origene, Rockville, MD), for Ki67 using a mouse anti-human Ki67 antibody (clone MIB-1, 1:150, M7240, Dako, Waldbronn, Germany) and for cleaved caspase-3 (CC3) using a rabbit-anti human active caspase-3 antibody (1:200, ab13847, Abcam, Cambridge, UK).

Techniques: Injection, Control, Infection, Flow Cytometry, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded, Staining

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Histogram showing in-silico analysis of CAR T cell-treated patients ( n = 4219) revealed a high relapse rate, with 42.11% ( n = 216 of n = 513 overall relapse patients) experiencing CD19-negative recurrence after monospecific CAR Therapy ( n = 2916). b Schematic overview of the CAR design strategy showing mono, bi, and trispecific constructs targeting CD19, CD20, and CD22. c Experimental workflow illustrating CAR screening: 1452 CARs were transduced into primary T cells and analyzed for signal-1 (activation), signal-2 (exhaustion), and signal-3 (cell death). Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . d Categorization of CARs into low (L), medium (M), and high (H) levels based on fluorescence intensity cutoffs determined by CD19 CARs as reference. e bar graph showing the distribution of 1452 screened CARs across L-, M-, and H-CARMSeD categories using the CARMSeD scoring system. f AI model development pipeline for CAR dysfunction risk prediction, based on 1,452 CAR constructs with an 80:20 split for training and testing. g–j Performance metrics of AI model predicting CARMSeD scores using 1452 CAR constructs. g ML learning curve of model accuracy over 50 epochs, achieving a training accuracy of 0.98 and validation accuracy of 0.95. h Scatter plot comparing measured versus predicted CARMSeD scores for training ( R 2 = 0.87) and validation ( R 2 = 0.83) sets. i Predicted versus measured CARMSeD scores on the validation set, categorized into low (blue), medium (orange), and high (green) CARMSeD. j Box plots show the median (center line), the 25th–75th percentiles (box), and whiskers extending to the minimum and maximum non-outlier values; individual points denote outliers. Numbers above each box indicate sequence counts. k Molecular dynamics simulation of CAR constructs with varying linker lengths, assessing scFv-scFv interaction. Structural conformations at 0 ns, 50 ns and 200 ns for different CAR scFv arrangements highlighting CDR regions (surface transparency 30%), Root Mean Square Deviation (RMSD) plots over 200 ns for both constructs, respectively, indicating structural stability and conformational changes. l Bar graph showing in vitro receptor binding affinity validation for top humanized scFvs of CD19, CD20, and CD22 CARs ( n = 6 biologically independent samples). Data represent mean ± SEM. ** p < 0.01; **** p < 0.001; ns: not significant. A non-parametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD22 CAR detection reagents (Miltenyi Biotec, #130-129-550, #130-126-727) and CD20 CAR expression was evaluated using Protein L-APC (CST #29480) or biotinylated anti CD20 antibody (Acro biosystems) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec, #130-110-951).

Techniques: In Silico, Construct, Activation Assay, Fluorescence, Biomarker Discovery, Sequencing, In Vitro, Binding Assay

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (red), and CD22 (yellow) antigens. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. c–f Line graph of cytotoxicity assays showing antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR T cells ( n = 3 biologically independent samples). g Heatmap showing comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR T cells, represented as fold expansion up to Day 17 with respect to the baseline at the time of cell seeding. h Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (red), and CD22 (yellow) antigens, edited using CRISPR-Cas9 to generate knockout variants. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . i , j Line graph of cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR T cells in eliminating antigen-negative Raji variants, compared to m19 CARs ( n = 5 biologically independent samples). k Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji WT ). Gray circles represent initial engraftment and monitoring phases, pink circle shows the first incubation with Raji WT , while purple circles indicate the timing of the RajiCD19 −/− rechallenge. l Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis (1:10; T: E), and the number of CAR T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge ( n = 5 biologically independent samples). Data represent mean ± SEM. Source data are provided as a file.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD22 CAR detection reagents (Miltenyi Biotec, #130-129-550, #130-126-727) and CD20 CAR expression was evaluated using Protein L-APC (CST #29480) or biotinylated anti CD20 antibody (Acro biosystems) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec, #130-110-951).

Techniques: Expressing, Construct, Comparison, CRISPR, Knock-Out, Incubation, Lysis

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic timeline of in vivo lymphoma model for evaluation of monospecific and bispecific CAR T cells. Mice were xenografted with RajiWT cells (expressing CD19, CD20, and CD22) (day 0), followed by administration of m19 or b20/19 CAR T cells on day 5 and subsequent RajiCD19 −/− TR on day 12, 19 and 26. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bioluminescent imaging and ( c ) stacked area plot showing tumor burden quantification show effective tumor control by b20/19 CAR T cells versus m19 CARs ( n = 5). d CAR T cell survival over time ( n = 5 mice). e Kaplan-Meier survival curves showing survival outcomes over 70 days ( n = 5 mice). f Analysis of residual tumor CD19 or CD20 tumor cells over time ( n = 5 mice). g , h Bar plot showing Granzyme B and IFN-γ secretion from human CD8 + CAR T cells isolated b20/19 post-treatment to confirm functional cytotoxicity of b20/19 against CD19⁻ targets ( n = 5). The CAR T cells isolated from mice that received conventional monospecific (m)CD19 CAR T cells served as the control for comparison. i , j TR induced upregulation of exhaustion markers PD-1 and LAG-3 ( n = 5 mice). k Immunophenotyping of CAR T cells post-TR shows loss of central memory (T cm ) populations and increased PD-1 expression, consistent with functional exhaustion and limited engraftment ( n = 5 mice). Data represents mean ± SEM. ** p < 0.01; *** p < 0.005; **** p < 0.001. A non-parametric t-test was used for statistical analysis between groups, and for ( k ), a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD22 CAR detection reagents (Miltenyi Biotec, #130-129-550, #130-126-727) and CD20 CAR expression was evaluated using Protein L-APC (CST #29480) or biotinylated anti CD20 antibody (Acro biosystems) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec, #130-110-951).

Techniques: In Vivo, Expressing, Imaging, Control, Isolation, Functional Assay, Comparison

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. b Relative mRNA expression levels (normalized to beta actin; ACTB) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR T ( n = 6 biologically independent samples). c Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR T cells after TR with RajiCD19 −/− cells. d Histogram analysis of the flow cytometry plots ( n = 10 biologically independent samples). e Bar graph shows the percentage of CD8 + CAR T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions ( n = 5 biologically independent samples). f Survival of CAR T cells over 15 days under various conditions ( n = 4 biologically independent samples). g Violin plots showing the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown elevated mTOR activity ( n = 6 biologically independent samples). h Bar plots show the percentage of MFI of autophagy from autophagic flux assay ( n = 8 data points from three independent experiments). i Dot plot showing ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from OXPHOS to glycolysis ( n = 12 data points from three independent experiments). j Similarly, OCR with FOXO4 knockdown decreases mitochondrial respiration. Individual data points are shown for each condition ( n = 12 data points from three independent experiments). k Box-and-whisker plot showing percentage of expression of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker ( n = 63 patient samples). l Bar graph showing the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. m Schematic illustration of K562 WT and CD20 expressing K562 stable cells transduced with different MOIs to obtain three populations: CD20 L (low), CD20 M (medium), and CD20 H (high), which were further FACS sorted. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . n Violin plots showing the percentage of CD20 expression (% CD20 expression) in the sorted CD20-expressing K562 cell populations, confirming distinct expression levels ( n = 10 data flow cytometry points from three independent experiments). o Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show CD20 (red) in K562-CD20 L (low), K562-CD20 M (medium), and K562-CD20 H (high) cell. p–r Survival curves of K562 cells expressing varying CD20 expression levels under CAR T cell treatments. The line graph shows the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) ( n = 4 biologically independent samples). s Survival of CAR T cells with varying CD20-targeting CAR constructs over 15 days ( n = 5). Data represents mean ± SEM. **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For e , f and s , a Two-way ANOVA followed by post-hoc testing was applied. Scale bar: 5 μm. Source data are provided as a file.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD22 CAR detection reagents (Miltenyi Biotec, #130-129-550, #130-126-727) and CD20 CAR expression was evaluated using Protein L-APC (CST #29480) or biotinylated anti CD20 antibody (Acro biosystems) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec, #130-110-951).

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Whisker Assay, Marker, Transduction, Super-Resolution Microscopy, Construct

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic of the strategy for trispecific CAR T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). b Correlation of expression of nbCD3, nbCD22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin, and data were obtained using intracellular flow cytometry ( n = 7 data points from three independent experiments). c Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. d Dose-dependent T cell activation (CD69 expression) in response to culture supernatants (used at various ratios with culture media) with nbCD3/22, using db coated with CD3 for validation ( n = 6 data points from three independent experiments). e Line graph of HEK293T synNotch reporter assay showing dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR T cell supernatants, confirming BiTE functionality under two condition 1 and condition 2. f Experimental timelines for in vitro T cell engineering, transduction, and co-culture with Raji cells (WT or knockout for CD19, CD20, or CD22). Anti-tumor assays were performed on days 9, 11, and 13. g , h Functional assay of CAR T cells against Raji cells (WT or knockout for CD19, CD20, or CD22) demonstrates that b20/19AKT3 PROTAC CAR T cells co-expressing nbCD3/22 exhibit stronger antitumor activity compared to b20/19-AKT3 PROTAC or mCD19 CAR T cells at Day 7 and Day 14. Data represent mean ± SEM. **** p < 0.001; ns: not significant. A nonparametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD22 CAR detection reagents (Miltenyi Biotec, #130-129-550, #130-126-727) and CD20 CAR expression was evaluated using Protein L-APC (CST #29480) or biotinylated anti CD20 antibody (Acro biosystems) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec, #130-110-951).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vitro, Transduction, Co-Culture Assay, Knock-Out, Functional Assay, Activity 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) 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