Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Top panel: Schematic representation of the protocol used to inhibit ITK activity. PSCA CAR-T cells were pre-treated for 2 hours with ITK inhibitors 1μM (BMS-509744, Ibrutinib or GNE-9822) or DMSO as control and then cocultured with HPAC WT cells for 0 or 10 minutes. Left panel: Representative Western blot of CD28 pY218 in CAR-T cells after stimulation with HPAC WT cells. Right panel: Normalized CD28 pY218 intensity with respect to CAR (CD3ζ). Significance was determined using one-way ANOVA. * = P<0.05. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). B. Top panel. Schematic representation of the experimental protocol. ITK expression was disrupted using CRISPR/Cas in Jurkat cells. WT and ITK-KO Jurkat cells were transduced to express a PSCA-specific CAR and cocultured with HPAC WT cells for 0 or 10 min. Left panel. Representative membrane of CD28 pY218 in CAR-T Jurkat cells after stimulation. Right panel. Normalized CD28 pY218 intensity quantified by densitometry (representative plot) and CD28 pY218 at 10 min post-stimulation. Significance was determined by paired t-test. ** = P<0.01. Each symbol represents one of 3 independent experiments, performed with different donor T cells.

Article Snippet: Primary antibodies: rabbit anti-CD28 pY218 (Abcam, ab138404), anti-CD28, rabbit ant-ZAP70 pY319 (Cell Signaling, #2717), rabbit anti-ZAP70 (Cell Signaling, #2705), rabbit anti-PLCγ pY783 (Abcam, ab76155), rabbit anti-PLCγ (Abcam, ab76155), rabbit anti-VAV1 pY174 (Abcam, ab76225), rabbit anti-VAV1 (Abcam, ab97574), rabbit anti-LCK (Abcam, ab32149), ITK (Cell Signaling, 77215), mouse anti-CD3ζ (Santa Cruz, sc-166275), mouse anti-B actin (Santa Cruz, sc-47778).

Techniques: Activity Assay, Control, Western Blot, Standard Deviation, Expressing, CRISPR, Membrane

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Schematic representation of WT and PYRP mutant CAR constructs. In the PYRP mutant, alanine (A) 217 and serine (S) 220 were substituted with prolines (P) to generate a proline-rich domain. B . PSCA-targeted WT and PYRP CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30, or 60 min. CAR molecules were immunoprecipitated using protein-L coated beads and co-immunoprecipitated proteins were analyzed by Western blot. Upper panel: Representative Western blot results showing ITK present in CAR immunoprecipitate. CD3ζ staining of the CAR molecule was used as a bait control. Lower panel: Intensity of ITK with respect a total CAR (CD3ζ) was analyzed by densitometry (representative plot). C. PSCA WT and PYRP CAR-T cells were stimulated with HPAC WT cells for 10 min and phosphorylation of Y218 was analyzed by mass spectrometry. Bar charts represent the intensity of phosphorylated Y218 normalized to the total CAR signal. Significance was determined by one-way Anova. * = P<0.05. Data is represented as the mean ± standard deviation (SD). D and F. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Significance was determined by one-way ANOVA. * = P<0.05, ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. RNA was extracted after 24 hours and IL-2 mRNA production was evaluated using quantitative Time PCR. CAR mRNA was used as normalization control. Significance was determined by one-way Anova. ** = P<0.01, *** = P<0.001. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). G. % of change in tumor volume (compared to baseline) is represented (n=5). Representative example of two independent experiments. Significance was determined by permutation two sample t-test. * = P<0.05.

Article Snippet: Primary antibodies: rabbit anti-CD28 pY218 (Abcam, ab138404), anti-CD28, rabbit ant-ZAP70 pY319 (Cell Signaling, #2717), rabbit anti-ZAP70 (Cell Signaling, #2705), rabbit anti-PLCγ pY783 (Abcam, ab76155), rabbit anti-PLCγ (Abcam, ab76155), rabbit anti-VAV1 pY174 (Abcam, ab76225), rabbit anti-VAV1 (Abcam, ab97574), rabbit anti-LCK (Abcam, ab32149), ITK (Cell Signaling, 77215), mouse anti-CD3ζ (Santa Cruz, sc-166275), mouse anti-B actin (Santa Cruz, sc-47778).

Techniques: Mutagenesis, Construct, Immunoprecipitation, Western Blot, Staining, Control, Phospho-proteomics, Mass Spectrometry, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Top panel: Schematic representation of the protocol used to inhibit ITK activity. PSCA CAR-T cells were pre-treated for 2 hours with ITK inhibitors 1μM (BMS-509744, Ibrutinib or GNE-9822) or DMSO as control and then cocultured with HPAC WT cells for 0 or 10 minutes. Left panel: Representative Western blot of CD28 pY218 in CAR-T cells after stimulation with HPAC WT cells. Right panel: Normalized CD28 pY218 intensity with respect to CAR (CD3ζ). Significance was determined using one-way ANOVA. * = P<0.05. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). B. Top panel. Schematic representation of the experimental protocol. ITK expression was disrupted using CRISPR/Cas in Jurkat cells. WT and ITK-KO Jurkat cells were transduced to express a PSCA-specific CAR and cocultured with HPAC WT cells for 0 or 10 min. Left panel. Representative membrane of CD28 pY218 in CAR-T Jurkat cells after stimulation. Right panel. Normalized CD28 pY218 intensity quantified by densitometry (representative plot) and CD28 pY218 at 10 min post-stimulation. Significance was determined by paired t-test. ** = P<0.01. Each symbol represents one of 3 independent experiments, performed with different donor T cells.

Article Snippet: The following antibodies were used for immune and cancer cell phenotyping: G4S Linker AF488 (Cell signaling, 50515), CD3 BV711(biolegend, 317328), CD4 PE-Cy7(Biolegend, 300512), CD8 BUV395 (BD, 563795), rabbit ITK (Cell Signaling, 77215), anti-rabbit AF488(Cell Signaling 4412S), PSCA PE (Santa Cruz, sc-80654).

Techniques: Activity Assay, Control, Western Blot, Standard Deviation, Expressing, CRISPR, Membrane

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Schematic representation of WT and PYRP mutant CAR constructs. In the PYRP mutant, alanine (A) 217 and serine (S) 220 were substituted with prolines (P) to generate a proline-rich domain. B . PSCA-targeted WT and PYRP CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30, or 60 min. CAR molecules were immunoprecipitated using protein-L coated beads and co-immunoprecipitated proteins were analyzed by Western blot. Upper panel: Representative Western blot results showing ITK present in CAR immunoprecipitate. CD3ζ staining of the CAR molecule was used as a bait control. Lower panel: Intensity of ITK with respect a total CAR (CD3ζ) was analyzed by densitometry (representative plot). C. PSCA WT and PYRP CAR-T cells were stimulated with HPAC WT cells for 10 min and phosphorylation of Y218 was analyzed by mass spectrometry. Bar charts represent the intensity of phosphorylated Y218 normalized to the total CAR signal. Significance was determined by one-way Anova. * = P<0.05. Data is represented as the mean ± standard deviation (SD). D and F. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Significance was determined by one-way ANOVA. * = P<0.05, ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. RNA was extracted after 24 hours and IL-2 mRNA production was evaluated using quantitative Time PCR. CAR mRNA was used as normalization control. Significance was determined by one-way Anova. ** = P<0.01, *** = P<0.001. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). G. % of change in tumor volume (compared to baseline) is represented (n=5). Representative example of two independent experiments. Significance was determined by permutation two sample t-test. * = P<0.05.

Article Snippet: The following antibodies were used for immune and cancer cell phenotyping: G4S Linker AF488 (Cell signaling, 50515), CD3 BV711(biolegend, 317328), CD4 PE-Cy7(Biolegend, 300512), CD8 BUV395 (BD, 563795), rabbit ITK (Cell Signaling, 77215), anti-rabbit AF488(Cell Signaling 4412S), PSCA PE (Santa Cruz, sc-80654).

Techniques: Mutagenesis, Construct, Immunoprecipitation, Western Blot, Staining, Control, Phospho-proteomics, Mass Spectrometry, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: RSC Advances

Article Title: Design, synthesis, and biological evaluation of novel 3-oxo-2,3-dihydropyridazine derivatives as interleukin-2-inducible T-cell kinase (ITK) inhibitors

doi: 10.1039/d5ra06565h

Figure Lengend Snippet: Inhibition of ITK and BTK activity by compounds 9 and 22 (a–d) .

Article Snippet: Recombinant ITK and BTK enzyme activities were assessed using the ITK Kinase Assay Kit (BPS Biosciences, Cat. #78429) and the BTK Kinase Enzyme System (Promega, Cat. #V2941).

Techniques: Inhibition, Activity Assay