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: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of HiBiT to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Activity Assay, High Throughput Screening Assay, Luciferase, Generated, Concentration Assay, Enzymatic Assay

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a ABL1 kinase domain bound to PD166326 inhibitor (dark blue) and myristic acid (red) (PDB: 1OPK), Nagar B et al. b SDR output of imatinib (blue square) or ATP (blue diamond) binding to 0.5 nM (open symbol) or 10 nM (solid symbol) ABL1- N -HiBiT kinase domain or imatinib inhibition of kinase activity (black circle) using Kinase-Glo Plus reagent (KGP). Error bars represent the SD, n = 3 experiments (Imatinib SDR) or n = 4 experiments (ATP SDR and imatinib kinase activity). c Correlation plot comparing the ABL1 KGP assay pIC 50 vs pSDR 50 (10 nM enzyme without ATP) for all inhibitors identified from the 128-member kinase inhibitor library. Colored symbols reference compounds discussed in Results section. For underlying data see Supplementary Data , , and Supplementary Fig. . d , e SDR output for myristate site ligands asciminib (red solid triangle) and GNF-5 (red open triangle) compared to imatinib (blue square) and nilotinib (open square) for ABL1- C- or - N -HiBiT, respectively. f Effect of allosteric ligands on enzyme catalysis (imatinib, black circle, asciminib, solid red triangle, and GNF-5, open red triangle). Error bars for d , e , and f represent the SEM, n = 2 technical replicates. g Protein kinase A (PKA) bound to H-89 inhibitor (dark blue) (PDB: 1YDT), Engh RA et al. h PKA enzyme activity (black circle) and SDR assay (blue square) CRC for H-89. Error bars represent the SD, n = 3 experiments i Correlation plot comparing the PKA KGP assay pIC 50 to PKA SDR assay pSDR 50 (without ATP). H-89 (blue square) and staurosporine (staur., red circle) indicated. See Supplementary Data and Supplementary Fig. for underlying data and compound information. Supplementary Data – supports panels ( c , i ). Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Inhibition, Activity Assay

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a Crystal structures of the apo (PDB 5KGL) and ipglycermide Ce-2d bound (PDB 5KGN) iPGM from Yu H et al. Metal ions are identified as purple spheres. b Saturation binding of Ce-2 to 1 nM (black circle) 0.5 nM (gray circle) or 0.1 nM iPGM (open black circle). c Relative detection sensitivity of the SDR assay (blue circle) compared to a functional couple-enzyme assay (gray/black square) for ipglycermide Ce-2 binding C. elegans iPGM. Error bars for ( b , c ) represent the SEM of 2 experiments. d Correlation plot comparing the binding potencies for ipglycermide analogs from either an FP-based competition binding (10 nM iPGM) or SDR assay (0.5 nM iPGM- C -HiBiT) format. The FP assay uses a fluorescein-labeled Ce-2d analog (Ce-2d-FL). e Correlation plot comparing the binding potencies for ipglycermide analogs between B. malayi and C. elegans iPGM orthologs using the SDR assay. Ipglycermides are identified by symbols to the right. Error bars for ( d , e ) represent the SD, n = 3 experiments. RLU, relative light units; FP, fluorescence polarization; Ce, C. elegans ; Bm, B. malayi . Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Functional Assay, Enzymatic Assay, FP Assay, Labeling, Fluorescence

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a DNA ligase crystal structures illustrating the relative positions of the N - and C -termini, DNA and nucleotide binding sites for the E. coli ligase (MW 75 kDa) complexed with NAD + (PDB 5TT5) from Unciuleac MC et al., with NAD + and DNA (PDB 2OWO) from Nandakumar J et al., and bacteriophage T7 ligase (MW 41.1 kDa) complexed with ATP (1A0I) from Subramanya HS et al. The structure of NAD + is shown. b Agarose gel electrophoresis of Hin dIII-digested λDNA repair by ligases from E. coli and bacteriophage T7 containing N - or C -terminal HiBiT α-peptide. Ligase concentrations used: 1, NEB E. coli ligase, 0.5 U/μL; 2, E. coli N -HiBiT, 1 µM; 3, E. coli C -HiBiT, 100 nM; 4, NEB T7 ligase, 150 U/ μL; 5, T7 N -HiBiT, 100 nM; 6, T7 C -HiBiT, 1 μM. Control ligases were from NEB and used as directed. Gel is representative of 2 replicates. Concentration-response curves obtained for the SDR assay for 0.5 nM E. coli Lig- N -HiBiT (solid circle), 1 nM E. coli Lig- C -HiBiT (open circle) and 1 nM T7 Lig- N / C -HiBiT (solid/open squares, respectively). c , d a 22-mer dsDNA oligo; e , f NAD + ; g , h ATP. Error bars are the SD, n = 3 experiments. Supplementary Table supports panels ( c − h ). kb, kilobase; Lig, ligase. The uncropped gel and source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Agarose Gel Electrophoresis, Control, Concentration Assay

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a DHFR bound to folate (red molecular surface) and NADP + (blue molecular surface) (PDB 4M6K) from Bhabha G et al. with N - and C -termini indicated. b Co-factor dependent SDR saturation binding curves for methotrexate (MTX) binding to 5 nM (solid symbol) and 0.5 nM (open symbol) DHFR- C -HiBiT in the presence (square) or absence (circle) of saturating NADPH. c SDR assay concentration response curves for MTX binding to 0.5 nM DHFR- C -HiBiT for various [NADPH]. Data normalized to 32.5 μM MTX response. d Correlation analysis of antifolate chemotherapeutic affinities as determined by a functional DHFR- C -HiBiT assay (100 nM, 75 μM NADPH) versus SDR assay using DHFR- C -HiBiT (0.5 nM, 5 μM NADPH), respectively. e SDR assay concentration response curves for MTX binding in 1:100 cellular lysate from DHFR C -terminus HiBiT gene edited HEK293 cells for indicated NADPH concentrations. Data normalized to vehicle control. f Correlation analysis of antifolate affinities determined by the SDR assay for a 1:100 lysate (~ 0.2 nM enzyme) without added NADPH or with 1 μM NADPH, respectively. The antifolates tested were methotrexate (M), γ-fluoromethotrexate (F), pralatrexate (Pr), aminopterin (A), trimetrexate (T), and pemetrexed (Pm). Error bars represent the SEM, n = 2 technical replicates, representative of 2 ( b ) or 3 ( c ) experiments, or SD, n = 3 experiments ( d , e , and f ). g Aligned minimal energy solution NMR conformer structures of apo (cyan, PDB: 2L28) and holo MTX-bound (raspberry, PDB: 1AO8) L. casei DHFR illustrating the conformational shift induced by the association of MTX (green). h Aligned solution NMR conformer ensemble structures of apo (cyan, 25 conformers, PDB: 2L28) and holo MTX-bound (raspberry, 21 conformers, PDB: 1AO8) L. casei DHFR illustrating the structural dynamics shift in DHFR upon MTX (green) binding as shown by the coalescence of conformers in the ensemble. RLU, relative light units. Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Concentration Assay, Functional Assay, Control