mouse anti her2 mab  (R&D Systems)

Journal: Journal of Translational Medicine

Article Title: Contextual reprogramming of CAR-T cells for treatment of HER2 + cancers

doi: 10.1186/s12967-021-03132-6

Figure Lengend Snippet: A Inducible expression of LdCK—Non-transduced (NT), conventional HER2 CAR, cRB-340-1 and RB-340-1 cells were stimulated at a 1:1 effector to target ratio with beads coated with bovine serum albumin (BSA), low or high densities of HER2 ectodomain (Low HER2 and High HER2) or the HER2 + FaDu cells and tested for expression of the HER2-TEV-linked tag tNGFR or the LdCK-linked tag Q8 at day 3 and 5 following stimulation; percentages of each cell population are displayed in the respective quadrant. B Modulation of cellular function by RB-340-1—PD-1 surface expression, IL-2 secretion and T cell proliferation after exposure of CAR-T cells to the FaDu cell line for 6 days at 1:5 and 1:20 E:T ratios. C Kinetics of RB-340-1 cellular function—representative kinetics of PD-1 expression, IL-2, IFN-γ and TNF-α secretion, cytotoxic activity and CAR-T cell expansion after stimulation with FaDu cells at 1:5 effector-to-target ratio. Similar results were obtained at 1:20 effector to target ratio (not shown). Only RB-340-1-related p values are ranked by the asterisks in all panels, in the lower panel the blue hyphens around the asterisks refer to statistical differences between RB-340-1 and conventional HER2 CAR and the gray between RB-340-1 and cRB-340-1. The complete statistical matrixes are shown by Additional file : Table S1 here and thereafter

Article Snippet: The surface density of HER2 on microbeads was evaluated by flow cytometry using mouse anti-human HER2 antibody (clone 191924, R&D Systems) and QiFiKit (Agilent).

Techniques: Expressing, Cell Function Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Contextual reprogramming of CAR-T cells for treatment of HER2 + cancers

doi: 10.1186/s12967-021-03132-6

Figure Lengend Snippet: LdCK as the active element of RB-340-1 function. A Composition of the released product—the cells used for administration to mice were tested for tNGFR and Q8 expression. To detect LdCK presence, cells were stimulated with HER2 high beads ex vivo for three days. B Transduction efficiency was estimated through VCN analysis by ddPCR adopting LV-specific primers common to all constructs (gray bars) representative of the cumulative transduction rate, primers specific for the CAR-containing constructs (blue bars) and LdCK-specific primers (orange bars). C RB-340-1 effect on tumor growth and survival of individual mice and D cumulative survival (asterisks indicate significance level in survival between RB-340-1 and the other experimental groups)

Article Snippet: The surface density of HER2 on microbeads was evaluated by flow cytometry using mouse anti-human HER2 antibody (clone 191924, R&D Systems) and QiFiKit (Agilent).

Techniques: Expressing, Ex Vivo, Transduction, Construct

Journal: Journal of Translational Medicine

Article Title: Contextual reprogramming of CAR-T cells for treatment of HER2 + cancers

doi: 10.1186/s12967-021-03132-6

Figure Lengend Snippet: Persistence of CAR-T cells in vivo. A Frequency of CAR-T cells in tumors at time of necropsy for each experimental group. B Percent of PD-1 expressing intratumoral hCD45 + CAR-T cells. C PD-L1 occupancy by atezolizumab—ligand occupancy by atezolizumab was determined by lack of detection of PD-L1 by a second anti-PD-L1 antibody in the harvested tumors at the time of necropsy. D Correlation between PD-L1 occupancy and cRB-340-1 plus atezolizumab colonization of tumors. E percent of hCD45 + CAR-T cells among the mixed tumor population three days after IVS with HER2 beads in the treatment groups with sufficient number of CAR-T cells to perform functional assays as per A . F PD-L1 occupancy ex vivo and after 3 days of IVS with HER2 beads without addition of atezolizumab to the culture. G frequency of tNGFR + /Q8 + CAR-T cell after IVS with HER2 beads. H Percent of tNGFR + /Q8 + CAR-T cells ex vivo in the mixed tumor population and three days after IVS with HER2 beads. I percent of HER2-expressing cancer cells ex vivo at the time of necropsy. J tumor composition for every experimental group. Asterisks refer to comparisons between RB-340-1 and other groups displaying significant differences. The complete set of statistical values is presented in Additional file : Table S6

Article Snippet: The surface density of HER2 on microbeads was evaluated by flow cytometry using mouse anti-human HER2 antibody (clone 191924, R&D Systems) and QiFiKit (Agilent).

Techniques: In Vivo, Expressing, Functional Assay, Ex Vivo

Journal: Journal of Translational Medicine

Article Title: Contextual reprogramming of CAR-T cells for treatment of HER2 + cancers

doi: 10.1186/s12967-021-03132-6

Figure Lengend Snippet: A Lentiviral components of RB-340-1–RB-340-1 includes two lentiviral (LV) constructs. LV#1 (HER2-TEV) encodes an anti-HER2 (4D5 clone) scFv combined to the CD28 and CD3ζ co-stimulatory domains, the TEV protease and PD-1sg targeting the TSS of the endogenous PD-1 gene. LV#2 (LdCK) encodes LAT, fused to dCas9-KRAB via a TEV-cleavable sequence (TCS). RB-340-1 contains also two extracellular tags: Q8 part of LdCK and tNGFR part of HER2-TEV. In early experiments, GFP and mCherry were used for detection of LdCK or HER2-TEV respectively. B Figurative representation of RB-340-1–RB-340-1 conditionally suppresses expression of PD-1 upon activation of the CAR-T cells, while respective controls such as conventional HER2 CAR-T or RB-340-1 technical control (cRB-340-1) without targeting guide are not. C Mechanism of activation of HER2-TEV/LdCK CRISPRi platform—activation of HER2 CAR brings TEV in proximity of LdCK releasing dCas9-KRAB for nuclear translocation to the PD-1 TSS and conditionally and reversibly suppress PD-1 expression. The figure represents the CRISPRi logic at steady-state levels of LdCK expression. However, LdCK expression varies according to the physio-metabolic status of individual cells resulting in variable degree of transcriptional activity and consequently different substrate availability for TEV cleavage. This in turn contributes to conditionality as shown in Fig. A

Article Snippet: The surface density of HER2 on microbeads was evaluated by flow cytometry using mouse anti-human HER2 antibody (clone 191924, R&D Systems) and QiFiKit (Agilent).

Techniques: Construct, Sequencing, Expressing, Activation Assay, Control, Translocation Assay, Activity Assay