william sellers  (Addgene inc)

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 isolation of CAR T cells for RNA seq analysis. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bar graph of RNA seq showing pathway analysis of dysregulated genes associated with OXPHOS, mitochondrial metabolism, FOXO signaling, and autophagy in TR group CAR T cells. c Representative flow cytometry histogram showing the fluorescence intensity of FITC-labeled AKT3 (FITC-AKT3) in T cells from different groups. d Bar graph showing the mean fluorescence intensity (MFI) of AKT3 in T cells from the CON and TR groups ( n = 6 mice). e Illustration of the plasmids showing pLenti-AKT3 lentiviral vector used for co-transduction of CAR T cells with CAR vectors. f Schematic representation of the 17-day in vitro experimental timeline for evaluating T cell responses in TR model. T cells were isolated, activated, and transduced on Day 0 and further expanded until Day 7, followed by tumor challenge with Raji WT cells on Day 7. g Line graph comparing the survival of CAR T cells transduced with b20/19 CAR and either a control vector (b20/19 pVEC , blue circles) or an AKT3-expressing vector (b20/19 pAKT3 , orange squares) over 15 days ( n = 5 biologically independent samples). h Histogram analysis and pie chart showing distribution of T em and T cm with PD-1 low or PD-1 high within these subsets ( n = 5 biologically independent samples) at day 17. i Line graph showing Extracellular Acidification Rate (ECAR, mpH/min) over 100 min day 17. j Bar graph of basal ECAR (mpH/min, normalized) between b20/19 pVEC (blue) and b20/19 pAKT3 (orange) groups ( n = 6 biologically independent samples). k , l Similarly line graph showing Oxygen Consumption Rate (OCR, pMoles/min) and corresponding bar graph ( n = 6 biologically independent samples). m Schematic of the lentiviral vector delivering shRNA against AKT3 (shAKT3). The lower panel depicts the lentiviral particle transduced target CAR T cells. n Line graph comparing the survival of CAR T cells over 15 days for three groups. Statistical analysis was done for day 15 data points ( n = 5 biologically independent samples). o Bar graph and pie charts showing the percentage of various subsets of CD8 + CAR T cells across three groups ( n = 5 biologically independent samples). p , q Bar graph of ECAR, and OCR across three groups ( n = 8). Data represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For g , h , and o , a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

Techniques: In Vivo, Isolation, RNA Sequencing, Flow Cytometry, Fluorescence, Labeling, Plasmid Preparation, Transduction, In Vitro, Control, Expressing, shRNA

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 Table listing the sequences of peptides designed using RFdiffusion for PROTAC application, targeting AKT3 (AKT3_P1 to AKT3_P5, AKT3_PC) and a non-targeting peptide (NTP), with AKT3_P2 highlighted as the selected peptide. b Schematic illustration of mCherry-AKT3 CAR T cells induced with PROTAC. The PROTAC consists of the AKT3-targeting peptide (AKT3-P) fused to a cell-penetrating peptide (CPP) and via a linker connected to an E3 ligase ligand. c Line graph showing the dose-dependent effect of PROTAC peptide P2 (blue circles) compared to a non-targeting peptide (NTP, orange circles) on the ratio of endogenous AKT3 to GFP fluorescence in CAR T cells ( n = 4 biologically independent samples). The AKT3 was detected using primary anti-AKT3 antibody (raised in rabbit) followed by detection with secondary anti-rabbit antibody (Alexafluor 546; red). d Representative fluorescence microscopy images of CAR T cells treated with NTP or P2, showing GFP (green), anti-AKT3 (Alexafluor 546; red), and merged channels with regions of interest (ROIs). ROIs highlight reduced AKT3 signal in P2-treated cells compared to NTP-treated cells. e Violin plot comparing the integrated density of AKT3 in P2 and NTP-treated CAR T cells ( n = 15 images). f Schematic of the b20/19 CAR construct, including a signal peptide (SP), furin cleavage (FC), and AKT3 PROTAC (P2A linker), designed to target AKT3 for degradation in CAR T cells. Timeline of the 15-day experiment for evaluating b20/19 CAR T cell responses. g Jittered dot plot showing the relative fluorescence units (RFU) of AKT3 in b20/19 CAR T cells treated with NTP or AKT3 PROTAC ( n = 25 data points from three independent experiments). h Western blot analysis of b20/19 CAR T cells treated with NTP PROTAC or AKT3 PROTAC , probing for endogenous AKT3 using anti-AKT3, and GAPDH as a loading control. i Bar graph showing the densitometry analysis of the blots ( n = 6 biologically independent samples). j Representative fluorescence microscopy images of b20/19 CAR T cells treated with non-targeting peptide (NTP PROTAC ) or AKT3 PROTAC , showing GFP (green), anti-AKT3 (Alexafluor 546; red), and merged channels. k Violin plot comparing the integrated density of AKT3 in NTP PROTAC and AKT3 PROTAC treated b20/19 CAR T cells ( n = 10 images). l Survival curve showing percentage of RajiWT cell survival after co-culture with CAR T cells at different target-to-effector (T:E) ratios ( n = 4 biologically independent samples). m Percentage of patient-derived cancer cell survival after 24-h co-culture with CAR T cells at a target-to-effector (T:E) ratio of 1:2.5. b20/19-AKT3 PROTAC exhibits higher cytotoxicity in patient derived cells ( n = 5 biologically independent samples). n In vitro tumor rechallenge model showing survival of b20/19-AKT3 PROTAC CAR T cells over time ( n = 5 biologically independent samples). o Flow cytometry histogram showing various T cell subsets ( n = 5). p , q Dot plot showing showing ECAR and OCR analysis ( n = 12 data points from three independent experiments). Data represent mean ± SEM. ** p < 0.01; *** p < 0.005; **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For o , Two-way ANOVA followed by post-hoc testing was applied. Scale bar; d : 200 μm; j : 100 μm. Source data are provided as a file.

Article Snippet: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

Techniques: Fluorescence, Microscopy, Construct, Western Blot, Control, Co-Culture Assay, Derivative Assay, In Vitro, Flow Cytometry

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: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

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 timeline of the experiment showing Raji WT cell injection, CAR T cell administration, and Raji CD19 −/− TR. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b In vivo bioluminescence imaging of mice treated with NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 , showing tumor burden (red indicates high tumor load, blue indicates low) over 84 days, with “X” marking deceased mice. c Tumor radiance over 84 days, demonstrating reduced tumor burden in the AKT3 PROTAC+scram group. d Kaplan-Meier survival curves, with the AKT3 PROTAC+scram group exhibiting the highest survival rate. e Line graph shows the percentage of CAR T cells detected in blood (% CAR T cells) over time. f Bar graph displays the percentage of CAR T cells in blood at day 84, with the AKT3 PROTAC+scram group showing detectable levels (~3%), while other groups were not analyzable due to the absence of surviving mice, indicated as not determined (ND) ( n = 5 mice). g Tumor burden assessment till day 56. The line graph shows the number of Raji cells over time. The AKT3 PROTAC+scram group exhibits no detectable Raji cell burden by day 56, while other groups with some surviving mice show some detectable cells ( n = 5 mice). h Bar graph shows the percentage of CD8 CAR T cells (% CD8 T cells) expressing different phenotypes on day 28 post-infusion with corresponding pie charts illustrating the proportional distribution ( n = 5 mice). i , j Dot plot showing ECAR and OCR under various conditions on day 28 ( n = 12 data points). Data represent mean ± SEM. *** p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups. The comparison was made between NTP PROTAC+Scram with NTPP ROTAC+shFOXO4 and AKT3 PROTAC+Scram with AKT3 PROTAC+shFOXO4 . For h , a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a Source Data file.

Article Snippet: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

Techniques: Injection, In Vivo, Imaging, Expressing, 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 Schematic of CLDN scFv and EGFR scFv CAR constructs with ICOS, 4-1BB, CD3ζ, and AKT3 PROTAC domains. b , c Survival curves showing cytotoxicity of CLDN and EGFR CAR T cells with NTP PROTAC or AKT3 PROTAC against AGS (K) and A549 (L) cells at varying T:E ratios ( n = 5 biologically independent samples). d Line graph showing CAR T cell proliferation over 15 days, showing enhanced expansion with AKT3 PROTAC . e Bar graph and pie charts analysis of EGFR-CAR and CLDN-CAR T cells with NTP PROTAC or AKT3 PROTAC , showing the percentage of various T cell subsets ( n = 5 biologically independent samples). Data represent mean ± SEM. ****p < 0.001. A two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

Techniques: 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: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vitro, Transduction, Co-Culture Assay, Knock-Out, Functional Assay, Activity 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 Experimental timeline for in vivo study in Raji WT or NALM6 WT model followed by CAR T cell administration and TR with double knockout Raji CD19/CD20−/− or double knockout NALM6 CD19/CD20−/− cells. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bioluminescence imaging of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 CAR T cells, monitored from Day 7 to Day 84. c Line graph of quantified tumor radiance over time, showing sustained tumor control in Raji and NALM6 models with b20/19-AKT3 PROTAC+nbCD3/22 . d Line graph of percentage of CAR T cells in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days. e Bar graph of CAR T cell populations in blood at Day 56. f Levels of nbCD3/22 (pg/mL) in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days, showing sustained secretion. g Kaplan-Meier survival curves demonstrating improved survival with nbCD3/22-modified CAR T cells. h Bar graph and pie charts compare b20/19-AKT3 PROTAC and b20/19-AKT3 PROTAC+nbCD3/22 , showing various memory T cell subsets over time ( n = 5 mice) in all conditions. Data represent mean ± SEM. **** p < 0.001. A two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

Techniques: In Vivo, Double Knockout, Imaging, Control, Modification