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

Journal: Clinical Cancer Research

Article Title: Phase II Trial of HER-Vaxx, a B-cell Peptide-Based Vaccine, in HER2-Overexpressing Advanced Gastric Cancer Patients Under Platinum-Based Chemotherapy (HERIZON)

doi: 10.1158/1078-0432.CCR-24-0742

Figure Lengend Snippet: Detected levels of HER2-specific total IgG antibodies. The HER2-specific total IgG antibodies in patients treated with chemotherapy alone A, and 50 µg of HER-Vaxx plus chemotherapy B, measured by ELISA, are shown. The results are representative of at least two experiments.

Article Snippet: The level of intracellular HER2 phosphorylation in the lysates of the treated cells was evaluated by ELISA, using capture mouse antihuman HER2 (R&D Systems, USA; cat. No. MAB1129, RRID:AB_357477), phospho-HER2/ErbB2 (Tyr1221/1222; 6B12) rabbit mAb (Cell Signaling Technology, cat. No. 2243, RRID:AB_490899), and HRP-conjugated goat antirabbit IgG (Sigma-Aldrich; cat. No. 8275, RRID:AB_258382).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical Cancer Research

Article Title: Phase II Trial of HER-Vaxx, a B-cell Peptide-Based Vaccine, in HER2-Overexpressing Advanced Gastric Cancer Patients Under Platinum-Based Chemotherapy (HERIZON)

doi: 10.1158/1078-0432.CCR-24-0742

Figure Lengend Snippet: Correlation between the induced HER2-specific antibody levels and antitumor effect and mediation of ADCC. The total IgG A, and IgG1 B, antibody levels in all subjects’ available sera after four vaccinations (week 12; ) and the observed antitumor effect (as fold change compared with baseline) in each respective patient are shown. The correlation of the induced HER2-specific IgG C, or IgG1 D, antibody levels in representative sera of low, high, and very high responders [after four vaccinations (week 12) with 50- or 100-µg vaccine dose], and ADCC expressed in percent of the positive control (trastuzumab) are presented. The levels of correlation and significance are indicated in the boxes. The target cells NCI-N87 were incubated with effector cells (i.e., PBMC) after treatment with 50 μg of the examined patients' isolated and purified total IgG antibodies. Trastuzumab as a positive control and serum samples from a healthy individual as a negative control were included in the assays. The results represent at least two experiments.

Article Snippet: The level of intracellular HER2 phosphorylation in the lysates of the treated cells was evaluated by ELISA, using capture mouse antihuman HER2 (R&D Systems, USA; cat. No. MAB1129, RRID:AB_357477), phospho-HER2/ErbB2 (Tyr1221/1222; 6B12) rabbit mAb (Cell Signaling Technology, cat. No. 2243, RRID:AB_490899), and HRP-conjugated goat antirabbit IgG (Sigma-Aldrich; cat. No. 8275, RRID:AB_258382).

Techniques: Positive Control, Incubation, Isolation, Purification, Negative Control

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 1 Schematic representation of the EpiView-D4 mobile pathology platform for breast cancer. The device has a smart- phone base which uses detachable modules for brightfield (top) and fluorescence (bottom) imaging using the cell phone camera. Tumor is first sampled by FNA, and then aspirates are processed for cytology and biomarker evaluation. For cytology, aspirates are smeared on a glass slide and processed with rapid (DiffQuikTM) staining kits and then imaged with the brightfield imaging attachment (top). For biomarker evaluation, aspirates are mixed with lysis buffer and then applied to a D4 immunoassay chip, which quantify an analyte of interest (in this case, HER2). D4 chips are read using the fluorescence imaging attachment. The fluorescence intensity of the cAb spots on the D4 chip correlates with the biomarker expression level.

Article Snippet: HER2 capture/ detection Ab pairs (cat# MAB1129/AF1129, respectively, R&D Systems, Inc.) were then deposited onto POEGMA-coated slides as follows.

Techniques: Imaging, Biomarker Discovery, Staining, Lysis, Expressing

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 2 D4 sandwich immunoassay for quantification of HER2 expression level and its in vitro assessment. a–c, Schematic and operation of D4 immunoassay. a Spots of immobile cAb and an excess of “soluble” fluorescently labeled dAb are printed directly onto POEGMA-coated glass. b Dispensing sample fluid onto chip surface leads to dissolution of soluble dAb spots, followed by diffusion-driven mixing and antibody “sandwich” formation if HER2 is present. HER2 binding is detected by fluorescence imaging. c Representative fluorescence image of D4 assay after exposure to HER2-spiked RIPA buffer. Scale bar, 1 mm. d Representative dose-response curve generated from D4 chips for RIPA buffer spiked with recombinant HER2. Error bars: mean ± s.d. of duplicate assays. e, Assessment of cultured breast cancer cell lines (BT474, BT20, MDA-MB-231, MDA-MB-468) by D4 assay and comparison with western blotting. Inset: Western blot against HER2 for each cell line, with representative D4 cAb spots underneath. Main: D4 signal intensity (mean ± 95% CI) of ≥6 replicates. Significant difference by one-way ANOVA (F(4, 31) = 179.4, p < 0.0001). Bars with different letters indicate different groups (Tukey post hoc test, p ≤0.05). f Concordance analysis of 8 different patient-derived tumor cell lines in culture for HER2 expression by D4 assay vs. ELISA performed by clinical lab; specimens indexed “a” through “h”. D4 results are mean ± s.d. of duplicate assays. Pearson’s r = 0.975 (p < 0.0001, 95% CI: 0.845–0.996).

Article Snippet: HER2 capture/ detection Ab pairs (cat# MAB1129/AF1129, respectively, R&D Systems, Inc.) were then deposited onto POEGMA-coated slides as follows.

Techniques: Expressing, In Vitro, Labeling, Dissolution, Diffusion-based Assay, Binding Assay, Imaging, Generated, Recombinant, Cell Culture, Comparison, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 3 EpiView mobile microscope for brightfield and fluorescence imaging. a–d Optomechanical design of EpiView. Labeled 3D schematic and photographs of the EpiView scope with brightfield (a, b) and epifluorescence (c, d) assemblies. e, f Raw resolution of brightfield imaging from unprocessed images of USAF 1951 test target. Brightfield images of test target (green channel extracted) are shown in e, with red and blue dashed lines in image corresponding to vertical (red) and horizontal (blue) intensity profiles of test target features in f. Comparison of brightfield imaging of FNA cytology from human breast cancer specimen using standard benchtop microscopy (g) and EpiView device (h). Left panels: standard microscope image obtained by a 10× objective lens (top) and native view on EpiView (bottom). Scale bar, 0.2 mm. Right panels: standard microscope image obtained by a 40× objective (top), showing the same ROI outlined with red dashed line in the 10× image, and similar view obtained by digital zoom on EpiView (bottom). Scale bar, 30 µm. i, j Resolution testing with USAF 1951 in a manner similar to e and f but in epifluorescence mode based on extracting green channel image. k Left panel: Widefield view of 200 nm fluorescence beads using EpiView in epifluorescence mode. Scale bar, 0.1 mm. Right panel: Comparison of EpiView versus and benchtop microscope (40× objective, NA = 0.6) of the 200 nm beads outlined by the red and blue ROIs in the widefield image. Images were extracted from green channel. Scale bars, 40 µm. l Dose-response curve of HER2 with D4-EpiView and a conventional glass slide scanner. Limit of detection for this experiment using conventional glass scanner (LODG) is 21 pM, while that of the D4-EpiView (LODE) is 77 pM. Data represent log signal intensity ± s.d for duplicate assays for both EpiView-D4 and the slide scanner.

Article Snippet: HER2 capture/ detection Ab pairs (cat# MAB1129/AF1129, respectively, R&D Systems, Inc.) were then deposited onto POEGMA-coated slides as follows.

Techniques: Microscopy, Imaging, Labeling, Comparison

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 4 Evaluation of solid tumor xenografts from nude mice orthotopically engrafted with human breast cancer cell lines with EpiView- D4. a Schematic of workflow. Tumor aspirates are divided into two aliquots; one aliquot from an aspirate is processed as a cytology specimen on a glass slide using a Diff-QuikTM rapid staining kit and then imaged using the brightfield imaging attachment (top row), The second aliquot is lysed in RIPA buffer and applied to a D4 chip and its HER2 level is quantified using the fluorescence attachment on the EpiView-D4. b Representative western blots against HER2 for each human breast cancer cell line used for xenografting (BT20, BT474, and MDA-MB-453). Vinculin used as loading control. c Results of HER2 credentialing for 16 different solid tumor specimens from BT20, BT474, and MDA-MB-453 xenografts (N = 5, 8, 3, respectively). Each data point represents the D4 fluorescence intensity (average of duplicates) measured by the EpiView-D4 for individual mouse tumors, categorized by xenograft line. Also shown is the mean ± 95% CI fluorescence intensity for each category, which mirrors the western blots shown in panel b. There was a statistically significant difference between groups as determined by one-way ANOVA (F (2, 13) = 76.10, p < 0.0001). Multiple comparison testing showed significant differences between each group (Tukey post hoc test, p ≤0.05). d D4 fluorescence intensity by EpiView-D4 for each mouse tumor from f plotted against corresponding HER2 ELISA. Pearson r = 0.968, p < 0.0001, 95% CI: 0.910–0.989). e–g Representative LPF (main panel) and HPF (inset) images of FNA preparations for each xenograft line confirm malignant cytology (see Supplementary Fig. 4 for representative images with standard microscope). Scale bars for LPF and HPF images are 0.2 mm and 30 µm, respectively.

Article Snippet: HER2 capture/ detection Ab pairs (cat# MAB1129/AF1129, respectively, R&D Systems, Inc.) were then deposited onto POEGMA-coated slides as follows.

Techniques: Staining, Imaging, Western Blot, Control, Comparison, Enzyme-linked Immunosorbent Assay, Microscopy