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: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell therapy response. A) Schematic diagram of the tandem anti-CD19/CD22 CAR structure. B) Flow chart of the study. C) Swimmer plot showing clinical responses after tandem anti-CD19/CD22 CAR T-cell product infusion. D) PET-TC imaging of patient P9 before (A.1 and A.2) and 28 days after (B.1 and B.2) tandem anti-CD19/CD22 CAR T-cell infusion. E) Event free survival (EFS) Kaplan–Meier curve in all patients (n = 10). F) Overall survival (OS) Kaplan–Meier curve in all patients (n = 10). For E-F, black dots on the curve represent censored observations. HSCT, haematopoietic stem cell transplantation. MRD, minimal residual disease. EMR, extramedullary relapse. PD, progression of disease. ∗ For P6, 6 reinfusions were performed (every two weeks), the last with 3 doses. ∗∗P4 joined a clinical trial with carfilzomib after relapse, with PD shortly after.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Imaging, Transplantation Assay

Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell product immunophenotype and analysis. A) Tandem anti-CD19/CD22 CAR T-cell expansion in all products manufactured in the CliniMACS Prodigy closed system. B) CD19-CAR and CD22-CAR expression in all products manufactured by flow cytometry. C) CD4 + and CD8 + cell populations in all 10 products manufactured. D) PD-1 receptor expression on product cells. In C and D, graphs show box and whisker plots (vertical bars, min to max points; box, first to third quartile, with median as horizontal bar). E) Memory subpopulations determined by flow cytometry. Central memory cells (CD45RA − CD27 + ), effector memory cells (CD45RA − CD27 − ), naïve cells (CD45RA + CD27 + ) and TEMRA cells (CD45RA + CD27 − ) are represented. F) Specific-lysis by tandem anti-CD19/CD22 CAR T-cells against SEM cell line determined by 4-h Europium-BATDA assay at different E:T ratios. G) Degranulation assay against SEM cell line determined by CD107a expression after 4 h of co-culture at 1:1 or 1:2 E:T ratios (see Methods). A–D and F and G , in green, living patients; in purple, relapsed patient; in black, patients who died.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Expressing, Flow Cytometry, Whisker Assay, Lysis, Degranulation Assay, Co-Culture Assay

Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell persistence in patients after product infusion. A) Tandem anti-CD19/CD22 CAR expression was determined with anti-CD19 CAR gated on CD3 + cells. Left panel, absolute numbers of peripheral blood CAR T-cells per μl in infused patients detected by flow cytometry. Right panel, percentage of CAR + cells within the T cell compartment. B) Copies per ml as detected using real-time qPCR (see Methods) (16). C) IL-6 levels from serum after CAR T-cell infusion. D) Peak IL-6 levels were upregulated in patients with ICANS or severe CRS. Box and whisker plot shows all points (vertical bars, min to max points; box, first to third quartile, with median as horizontal bar). # No CRS group included patients with mild phenotype (I-II grade); CRS group included patients with III-IV grade. In green, living patients; in purple, relapsed patient; in black, patients who died.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Expressing, Flow Cytometry, Whisker Assay

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) In-silico analysis of CAR-T cell-treated patients (n=4,219) 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=2,916). (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: 1,452 CARs were transduced into primary T cells and analyzed for signal-1 (activation), signal-2 (exhaustion), and signal-3 (cell death). (D) Categorization of CARs into low (L), medium (M3), and high (H) levels based on fluorescence intensity cutoffs determined by monospecific CD19 CARs. (E) Distribution of 1,452 screened CARs across L-, M-, and H-CARMSeD categories using the CAR-Mediated Self-Destruction (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 CAR-Mediated Self-Destruction (CAR-MSED) scores using 1,452 CAR constructs (G) 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 CAR-MSED scores for training (R 2 = 0.87) and validation (R 2 = 0.83) sets. (I) Predicted versus measured CAR-MSED scores on the validation set, categorized into low (L-CARMSED, blue), medium (M-CARMSED, orange), and high (H-CARMSED, green) groups. (J) Box plot of predicted signal scores for 9,372 unknown sequences, classified as L-CARMSED (2,749 sequences), M-CARMSED (1,468 sequences), and H-CARMSED (5,155 sequences). (K) Molecular dynamics simulation of CAR constructs with varying linker lengths, assessing CAR-CAR 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 the both constructs, respectively, indicating structural stability and conformational changes. (L) In vitro receptor binding affinity validation for top humanized scFvs of CD19, CD20, and CD22 CARs (n=6).

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

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

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (yellow), and CD22 (red) antigens. (B) Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. (B) Cytotoxicity assays showing potent and antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR-T cells (n=5). (C) Comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR-T cells. Trispecific constructs showed reduced proliferation, consistent with increased structural rigidity predicted by CARMSeD scoring. (D) Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (yellow), and CD22 (red) antigens, edited using CRISPR-Cas9 to generate knockout variants. (E) Cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR-T cells in eliminating antigen-negative Raji variants, compared to ineffective m19 CARs (n=5). (F) Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji WT ). Gray circles represent initial engraftment and monitoring phases, while purple circles indicate the timing of the Raji CD19-/- rechallenge. (G) Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis, and the number of CAR-T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge (n=5). (H) Schematic timeline of in vivo lymphoma model for evaluation of monospecific and bispecific CAR-T cells. Mice were xenografted with Raji WT cells (expressing CD19, CD20, and CD22) (day 0), followed by administration of m19 or b20/19 CAR-T cells on day 5 and subsequent Raji CD19-/- TR on day 12, 19 and 26. (I–J) Bioluminescent imaging and tumor burden quantification show effective tumor control by b20/19 CAR-T cells versus m19 CARs. (K) CAR-T cell persistence over time. (L) Kaplan-Meier survival curves showing survival outcomes over 70 days (n=5). (M) Analysis of residual tumor CD19 or CD20 tumor cells over time. (N, O) Bar plot showing Granzyme B and IFN-γ secretion from CD8 + CAR-T cells isolated post-treatment with m19 and b20/19 confirm functional cytotoxicity of b20/19 against CD19⁻ targets (n=5). (P–Q) Repeated TR induced upregulation of exhaustion markers PD-1 and LAG-3 (n=5). (R) 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 persistence (n=5). Data represents mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

Techniques: Expressing, Construct, Comparison, CRISPR, Knock-Out, Lysis, In Vivo, Imaging, Control, Isolation, Functional Assay

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

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) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR-T. (C1) Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR-T cells after TR with Raji CD19-/- cells (C2) Histogram analysis of the flow cytometry plots (n=10). (D) Bar graph shows the percentage of CD8 + CAR-T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions. (E) Persistence of CAR-T cells over 15 days under various conditions (n=4). (F) Violin plots show the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown significantly elevated mTOR activity (n=6). (G) Bar plots show the percentage of MFI of autophagy from autophagic flux assay (n=8). (H) ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from oxidative phosphorylation (OXPHOS) to glycolysis (n=12 data points). (I) Similarly, OCR with FOXO4 knockdown decreasing mitochondrial respiration. Individual data points are shown for each condition (n=12 data points). (J1) Percentage of expression (% expression) of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker. (J2) Bar graph displays the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. (K) Schematic illustration of K562 WT cells based on CD20 expression levels, resulting in three populations: CD20 L (low), CD20 M (medium), and CD20 H (high). (L) Violin plots show the percentage of CD20 expression (% CD20 expression) in the sorted K562 WT cell populations, confirming distinct expression levels (n=10). (M) Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show DAPI (blue, nuclear staining) and CD20 (red) in K562-C20 L (low), K562-C20 M (medium), and K562-C20 H (high) cell. Scale bar indicates 10 μm. (N) Survival of K562 cells expressing varying CD20 expression levels under CAR-T cell treatments. Panels N1 (K562-CD20 L ), N2 (K562-CD20 M ), and N3 (K562-CD20 H ) show 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). (O) Persistence of CAR-T cells with varying CD20-targeting CAR constructs over 15 days (N=5). Data represents mean ± SEM. ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups. Scale bar indicates 10 μm.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Phospho-proteomics, Marker, Super-Resolution Microscopy, Staining, Construct

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Schematic of the engineering 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, nb22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin and data was obtained using intracellular flow cytometry. (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 with nbCD3/22, using db coated with CD3 for validation. (E) HEK-293T synNotch reporter assay shows dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR-T cell supernatants, confirming BiTE functionality under two conditions. (F) Experimental timeline for in vivo CAR-T cell therapy study in Raji WT or NALM6 WT model followed by CAR-T cell administration and TR with Raji CD19/CD20-/- or NALM6 CD19/CD20-/- cells (G) 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. (H) Quantified tumor radiance over time, showing sustained tumor control in Raji and NALM6 models with b20/19-AKT3 PROTAC+nbCD3/22 . (I1) 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 (I2) Bar graph of CAR-T cell populations in blood at various time points. (J) 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. (K) Kaplan-Meier survival curves demonstrating improved survival with nbCD3/22-modified CAR-T cells. (L) 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) in all conditions. Data represents mean ± SEM. ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vivo, Imaging, Control, Modification