Journal: Frontiers in Immunology

Article Title: Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo

doi: 10.3389/fimmu.2023.1072810

Figure Lengend Snippet: Design, pharmacokinetic and pharmacodynamic profiles of novel ACC TA99-HL2-KOA1. (A) Model depiction of engineered cytokine mimic HL2-KOA1 binding to IL-2Rα. IL-2 shown in blue, IL-2Rα shown in grey. Positions in contact with F42 shown as grey spheres. F42 and F42V shown as pink and red spheres respectively. Clash scores provided in total Rosetta energy units (REU). (B) Reducing SDS-PAGE analysis comparing the migration patterns of TA99-WT, TA99-Neo2/15, TA99-HL2-KOA1, pVAX backbone control transfection supernatants. (C) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human Tyrp1 compared to a murine isotype IgG2a control. (D) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rα compared to human IL2-Fc control. (E) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rβ compared to human IL2-Fc control. (F) Geometric mean fluorescence intensity of phosphorylated STAT5 in CD25+ CD4+ CD19- T cells from naïve mice (n=6) treated with indicated ACC. (G) Pharmacokinetic profile of recombinant TA99-WT and TA99-Neo-2/15 antibodies; C57BL/6 mice were injected intraperitoneally with 100µg of ACC constructs (n=5 mice per group). Serum at the indicated timepoints was assessed for TYRP1 binding by ELISA. (H) Total serum cytokine level over 14-day period in terms of area under the curve (AUC) in mice treated with ACC. (I) Timecourse of serum TNFα levels in sera of mice administered 100µg ACC. Two sera pools were used for (H, I) . (J) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-Neo2/15 (50µg) + TriVax (10µg each of Trp2, Gp100 and Tyrp1), or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax. 1x10 5 B16F10 cells subcutaneously into the right flank of C57BL/6 mice on Day 0. Green arrows indicate administration of treatment. (K) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax, or anti-PD1 + TA99 (50µg) + Human IL2 (5µg) + TriVax. Challenge and treatment scheme was performed as in (J) Error bars represent standard deviation; non-parametric Mann Whitney T test compared with TA99-WT used in (F) ; non-parametric Kruskal-Wallis ANOVA was used in (I) ; log-rank test was used to compare between differences in all survival curves; *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: In addition, serially diluted recombinant protein variants of antibody-cytokine chimera or recombinant mouse Fc-tagged IL2 (IL2-Fc) (Molecular Innovations, Cat# MIL2-FC-0.05MG) were used in place of serially diluted mouse sera.

Techniques: Binding Assay, SDS Page, Migration, Control, Transfection, Recombinant, Fluorescence, Injection, Construct, Enzyme-linked Immunosorbent Assay, Standard Deviation, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy

doi: 10.1073/pnas.2312374120

Figure Lengend Snippet: Increasing the affinity of the TROP2-targeting iCAR does not increase inhibition efficiency. ( A ) Comparison of binding affinities between three different antibodies targeting Trop2. Binding affinity kinetics were measured by BLI using biosensors precoated with recombinant TROP2 protein. Antibodies were serially diluted in concentrations ranging from 400 to 12.5 nM. The binding values were obtained and plotted against concentrations of antibody (nM). ( B ) Representative histograms from one experiment that show CAR and iCAR surface expression is similar between all T cell groups being tested. CAR and iCAR expression are measured using flow cytometry with antibodies against the FLAG- and HA-tags on the engineered receptors respectively. ( C ) CAR + /iCAR + T cells with the C3, B11, and H11 scFv show similar levels of cytotoxicity to each other. Representative cytotoxicity curves are displayed from one experiment where the total green object area (µm/well) of GFP + DU145 target cells that express CEACAM5 and/or TROP2 were measured over approximately 160 h. ( D ) Area under the curve analysis of cytotoxicity curves. The delay in inhibition was measured by calculating the area under each cytotoxicity curve. The AUC was normalized against the AUC calculated for untransduced T cells cocultured with target cells. The normalized AUC quantified is the mean ± SD (n = 2) from two independent experiments. The significance values shown are comparisons between a control group. For the CEA – /TROP2 – cell line, values are compared to the untransduced control. For the CEA + /TROP2 – cell line, values are compared to the CAR control. For the CEA LO /TROP2 HI or CEA HI /TROP2 LO cell lines, values are compared to the CAR + /iCAR + (H11-Long) group. Statistics are performed using 1-way ANOVA analysis with Tukey multiple comparison correction. * P value ≤ 0.05, ** P value ≤ 0.01, and *** P value ≤ 0.001.

Article Snippet: The phage library was panned with recombinant TROP2 extracellular domain-Fc chimera (R&D Systems, 650-T2-10).

Techniques: Inhibition, Comparison, Binding Assay, Recombinant, Expressing, Flow Cytometry, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy

doi: 10.1073/pnas.2312374120

Figure Lengend Snippet: DiCARs increases the efficiency of inhibition in the AND-NOT-gate CAR T strategy. ( A ) The models represent the structure of each DiCAR tested. The DiCARs are composed of a TROP2 scFv, the IgG4 Hinge, CH2, and CH3 constant domains, a CD28 TM, the PD-1 inhibitory signaling domain, and the additional inhibitory signaling domains PD-1, BTLA, SIGLEC-9, or LAIR-1. ( B ) The representative histogram indicates that iCAR/DiCAR surface expression level is similar between the groups of DiCARs being compared. The DiCAR surface expression was determined by flow cytometry for an HA-tag located on the N terminus of the iCAR/DiCAR. ( C ) Representative cytotoxicity curves of each CAR T cell population demonstrate that CAR T cell populations with a DiCAR have a reduced delay in inhibition compared to the TROP2-PD1 iCAR. CAR T cells were cocultured with DU145 target cells that express GFP and CEACAM5 and/or TROP2. Presence of target cells was measured by total green object area (µm 2 /well) over time as measured by Incucyte live cell image analysis over 150 h. ( D ) The delay in inhibition of the iCAR was measured by area under the cytotoxicity curve analysis of each cytotoxicity curve and normalized to the coculture with the untransduced T cell group. This AUC is a representative of one experiment in which triplicate wells were analyzed. Three biological replicates were performed and reported in SI Appendix , Figs. S6 and S7 . The significance values shown are comparisons between a control group. For the CEA – /TROP2 – cell line, values are compared to the untransduced control. For the CEA + /TROP2 – cell line, values are compared to the CAR control. For the CEA LO /TROP2 HI or CEA HI /TROP2 LO cell lines, values are compared to the CAR + PD1 iCAR group. Statistics are performed using 1-way ANOVA analysis with Tukey multiple comparison correction. * P value ≤ 0.05, ** P value ≤ 0.01, and *** P value ≤ 0.001.

Article Snippet: The phage library was panned with recombinant TROP2 extracellular domain-Fc chimera (R&D Systems, 650-T2-10).

Techniques: Inhibition, Expressing, Flow Cytometry, Control, Comparison