Journal: Frontiers in Immunology

Article Title: Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC

doi: 10.3389/fimmu.2024.1365172

Figure Lengend Snippet: Generation of HER2-targeted human UniCAR and conventional CAR (HER2-CAR) T cells and characterization of their in vitro function. (A) Comparison of universal and conventional CARs recognizing HER2. UniCAR: the high-affinity monomeric streptavidin (mSA2) extracellular domain binds the HER2-recognizing biotinylated trastuzumab (BT) linker through a non-covalent interaction. Conventional HER2-CAR: it binds directly to HER2 by an extracellular domain derived from trastuzumab ScFv. Both constructs contain the same CD28 transmembrane and intracellular domains and the CD3ζ effector domain. (B, C) Representative flow cytometry histogram and summary data of CAR expression (N = 4 for each transduction; NT, non-transduced; UNST, unstained control). HER2-specific CAR expression was confirmed by labeling with a HER2-Fc fusion protein followed by Alexa Fluor 647 conjugated anti-human IgG, UniCAR expression was confirmed by labeling with biotinylated goat anti-chicken IgG conjugated with Alexa Fluor 647. (D, E) IFNγ-ELISA assays. T cell products were incubated with surface-adsorbed molecular HER2-Fc target and various BT concentrations (D) or with HER2+/- MDA target cells and a fixed BT concentration (E) . After 24h, IFNγ was determined in the culture supernatant by ELISA (N = 4, assay performed in duplicates). (F) Firefly-Luciferase-based cytotoxicity assay. T cell products were incubated with HER2+ (MDA-HER2) or HER2- (MDA) ffLUC-expressing target cells for 24h in combinations with BT. Target cell survival was determined based on luminescent signal (N = 4; assay was performed in duplicates). (G) Expansion rate of T cell products in rechallenge assay. T cells were counted every 3.5 days and re-plated at the starting density onto freshly coated HER2-Fc target proteins. Fold expansion relative to plating density is plotted. Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 ; n.s., not significant.

Article Snippet: The presence of conventional HER2-CARs or biotinylated trastuzumab-bound UniCAR molecules in the membrane of human T lymphocytes was confirmed by labeling with an Alexa Fluor 647 conjugated monomeric HER2 extracellular domain (Sino Biological Europe GmbH, Eschborn, Germany) in order to achieve comparable signals for the two cell types in a single labeling step.

Techniques: In Vitro, Comparison, Derivative Assay, Construct, Flow Cytometry, Expressing, Transduction, Labeling, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Luciferase, Cytotoxicity Assay

Journal: Frontiers in Immunology

Article Title: Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC

doi: 10.3389/fimmu.2024.1365172

Figure Lengend Snippet: UniCAR T cells infiltrate trastuzumab-resistant tumor spheroids in the presence of biotinylated trastuzumab linker. (A) Representative images (at 24h) for detecting the cytolytic activity of UniCAR T cells ± 10 µg/ml BT against MDA-HER2.ffLUC (eGFP tagged; green) spheroids. Dead cells were visualized by PI uptake (red). (B) Histograms represent the averaged intensity profiles of eGFP and PI signals in cross-sections of spheroids (N =10). (C) Integrated intensities of eGFP (green) and PI (red) signals. Histograms show mean ± SEM; N = 5;***p < 0.001.

Article Snippet: The presence of conventional HER2-CARs or biotinylated trastuzumab-bound UniCAR molecules in the membrane of human T lymphocytes was confirmed by labeling with an Alexa Fluor 647 conjugated monomeric HER2 extracellular domain (Sino Biological Europe GmbH, Eschborn, Germany) in order to achieve comparable signals for the two cell types in a single labeling step.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC

doi: 10.3389/fimmu.2024.1365172

Figure Lengend Snippet: Generation of HER2-directed UniCAR T cells in vivo. Mice received a single i.v. dose of 20 × 10 6 non-transduced (NT), UniCAR or HER2-CAR T cells. The UniCAR+BT mouse received 100 µg biotinylated trastuzumab in 100 µl PBS 15h prior T cell injection. (A) Representative flow cytometry histogram of A647-HER2 recognizing cells. (B, C) Representative images and summary data of frozen sections of the treated lungs 2 minutes after effector cell injection. (B) Excised lungs were fluorescently labeled for CD45 (red), and DNA (blue). (C) Mean cell number in 1 field of view (FOV). Histograms show mean ± SEM; N = 4; *p < 0.05.

Article Snippet: The presence of conventional HER2-CARs or biotinylated trastuzumab-bound UniCAR molecules in the membrane of human T lymphocytes was confirmed by labeling with an Alexa Fluor 647 conjugated monomeric HER2 extracellular domain (Sino Biological Europe GmbH, Eschborn, Germany) in order to achieve comparable signals for the two cell types in a single labeling step.

Techniques: In Vivo, Injection, Flow Cytometry, Labeling

Journal: Frontiers in Immunology

Article Title: Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC

doi: 10.3389/fimmu.2024.1365172

Figure Lengend Snippet: Toxicity in animals co-treated with UniCAR T cells and biotinylated trastuzumab. Mice were injected s.c. with 3 × 10 6 MDA-HER2.ffLUC cells to establish tumor xenografts, then they received a single i.v. dose of 2 × 10 6 T cells on day 21 (red arrow). Mice (co-)treated with biotinylated trastuzumab received 100 µg BT in 100 µl PBS i.p. twice weekly from day 21. Tumor growth was followed by bioluminescence imaging. (A) Representative images of MDA-HER2.ffLUC injected animals. Images were taken 10 minutes after i.p. injecting 100 µg D-luciferin in 100 µL PBS by an IVIS Spectrum CT instrument. ( B left panel) Quantitative bioluminescence imaging data of MDA-HER2.ffLUC xenografts (radiance = photons/s/cm2/sr; HER2-CAR vs. all other treatments: ***p < 0.001). ( B right panel) Kaplan-Meier survival curve (HER2-CAR vs. all other treatments: ***p < 0.001).

Article Snippet: The presence of conventional HER2-CARs or biotinylated trastuzumab-bound UniCAR molecules in the membrane of human T lymphocytes was confirmed by labeling with an Alexa Fluor 647 conjugated monomeric HER2 extracellular domain (Sino Biological Europe GmbH, Eschborn, Germany) in order to achieve comparable signals for the two cell types in a single labeling step.

Techniques: Injection, Imaging

Journal: Frontiers in Immunology

Article Title: Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC

doi: 10.3389/fimmu.2024.1365172

Figure Lengend Snippet: On target off tumor toxicity in the lungs of animals co-treated with UniCAR T cells and biotinylated trastuzumab. (A) Hematoxylin-eosin-stained sections from lung tissues of mice at 27 (UniCAR+BT) or 64 days (NT; HER2-CAR; BT; UniCAR) after effector cell injection. Arrows indicate massive cellular infiltration around the vessels. (B) Representative frozen section of a UniCAR+BT co-treated lung 27 days after effector cell injection. Excised lungs were fluorescently labeled for HER2 (red), biotin (magenta), CD8+ human T cells (green), CD4+ human T cells (yellow), and DNA (blue).

Article Snippet: The presence of conventional HER2-CARs or biotinylated trastuzumab-bound UniCAR molecules in the membrane of human T lymphocytes was confirmed by labeling with an Alexa Fluor 647 conjugated monomeric HER2 extracellular domain (Sino Biological Europe GmbH, Eschborn, Germany) in order to achieve comparable signals for the two cell types in a single labeling step.

Techniques: Staining, Injection, Labeling

Journal: Frontiers in Immunology

Article Title: Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC

doi: 10.3389/fimmu.2024.1365172

Figure Lengend Snippet: Tumors from mice co-treated with UniCAR T cells and biotinylated trastuzumab show massive CD4 + T cell infiltration. Frozen sections of excised MDA-HER2.ffLUC tumors were fluorescently labeled for human CD4 + (yellow) or CD8 + T cells (green), and DNA (blue) on day 27 (UniCAR+BT) or day 64 (NT; BT; UniCAR) after effector cell injection.

Article Snippet: The presence of conventional HER2-CARs or biotinylated trastuzumab-bound UniCAR molecules in the membrane of human T lymphocytes was confirmed by labeling with an Alexa Fluor 647 conjugated monomeric HER2 extracellular domain (Sino Biological Europe GmbH, Eschborn, Germany) in order to achieve comparable signals for the two cell types in a single labeling step.

Techniques: Labeling, Injection

Journal: bioRxiv

Article Title: Chimeric Antigen Cytotoxic Receptors for In-Vivo Engineering of Tumor-targeting Natural Killer Cells

doi: 10.1101/2023.11.07.565144

Figure Lengend Snippet: Cell surface expression levels of NK-CARs in the presence or absence of cognate signaling adaptors. NK-CAR mRNAs were transfected in Huh7 cells with and without co- transfection with the mRNA for signaling adaptors (A) HER2-NKp30 with and without FcRγ, (B) HER2-NKp46 with and without FcRγ and (C) HER2-NKp44 with and without DAP12. Each panel shows the percentage CAR positive and mean fluorescence intensity 1 or 2 days post transfection. (D) The Specificity Index of each CAR-NK constructs are calculated as the ratio of the surface expression of CAR in the presence of signaling adaptors to the surface expression in the absence of signaling adaptor. Representative data of two independent experiments are shown.

Article Snippet: A human HER2/ERBB2 protein-His tag (10004-H08H-100, Sino Biological) conjugated with Alexa Fluor 647 was used to determine the expression of HER2-CAR in transfected cells, and viability was determined by 7-AAD labeling.

Techniques: Expressing, Transfection, Cotransfection, Fluorescence, Construct

Journal: bioRxiv

Article Title: Chimeric Antigen Cytotoxic Receptors for In-Vivo Engineering of Tumor-targeting Natural Killer Cells

doi: 10.1101/2023.11.07.565144

Figure Lengend Snippet: NK-CARs exhibit tumoricidal activity and elicit cytokine/chemokine response. (A) Peripheral blood NK cells transfected with HER2-targeting NK-CARs were cocultured with HER2+ SKOV3 tumor cells at a 5:1 effector-to-target (E:T) ratio for 20 hours at 37°C. The graph represents the percentage specific killing of SKOV3 cells. Data are representative of three independent experiments. The mean ± SD is plotted, and statistical significance is determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test between mock-transfected cells vs. NK-CAR transfected cells. P values are indicated on the graph. (B) TNF-alpha, IFN- gamma, CCL2, and GM-CSF levels (pg/mL) in supernatant collected from NK and SKOV3 coculture were analyzed by Luminex analysis. The mean ± SD from three replicates is shown. Statistical significance is determined by two-way ANOVA with Dunnett’s multiple comparisons test between mock and SKOV3 coculture vs. NK-CAR and SKOV3 coculture. P values are indicated on the graph. Data are representative of two independent experiments.

Article Snippet: A human HER2/ERBB2 protein-His tag (10004-H08H-100, Sino Biological) conjugated with Alexa Fluor 647 was used to determine the expression of HER2-CAR in transfected cells, and viability was determined by 7-AAD labeling.

Techniques: Activity Assay, Transfection, Luminex

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Top: Sequence of ADAPT ERBB2-1 and ten representative candidates found after phage display affinity maturation compared to ABD before randomization. One sequence (ADAPT ERBB2-mat10 ) contains a substitution of a scaffold residue that was not intentionally diversified in the library. The frequency of each clone in the sequence data (among 438 sequences) and tracks (A–H) where the corresponding sequence was found are indicated to the right. Track D was underrepresented in the sequence data set. Bottom: Sequences of 21 clones identified after rounds three and four of FACS sorting. The total frequency in the sequence data (278 sequences) is shown and the two numbers in brackets indicate the number of times each sequence was observed in selections without or with HSA present, respectively. Nine of the variants contained scaffold substitutions and modifications in the region spanning helix two and three were common in sequences observed after selections in the presence of HSA. 13 of these candidates were also observed after two rounds of FACS (not shown) and two of them had been observed already among the clones obtained after four rounds of phage display (identical sequences are indicated by asterisks).

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Sequencing, Clone Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Affinities and melting temperatures (T m ) for the non-randomized albumin-binding domain (ABD), ADAPT ERBB2-1 and variants of ADAPT ERBB2-1 .

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: ( A ). ERBB2-binding with and without an excess of HSA. Cells expressing variants from tracks selected without (A, C, E and G) or with (B, D, F and H) 1 µM HSA present were incubated with 50 nM ERBB2 +/− 1 µM HSA and analyzed by flow-cytometry. ERBB2-binding was detected through incubation with streptavidin-R-phycoerythrin (y-axis) and surface expression levels by IgG-Alexa Fluor 647 conjugate (x-axis), data are shown using logarithmic scales. Each diagram presents an overlay of contour plots of the expressing populations of cells from two separate samples from the same batch (A-H) incubated with only ERBB2 (blue) or ERBB2 and HSA (red). The contours represent cell density in the respective regions corresponding to 20, 40, 60 or 80% of the maximum cell density observed. Each sample is represented by 25–30000 events. Corresponding data for ADAPT ERBB2-1 and scaffold ABD are shown in the inlay of panel A to facilitate a comparison. The library each track is derived from, the form of ERBB2 and concentrations used during phage display selection are also indicated. (B ). HSA-binding. Cells from tracks selected without (A, C, E and G) or with (B, D, F and H) 1 µM HSA present were incubated with 50 nM HSA-Alexa Fluor 488 conjugate and analyzed by flow-cytometry. HSA-binding is shown on the y-axis and surface expression level, detected by IgG-Alexa Fluor 647 conjugate, on the x-axis. As controls, cells expressing ADAPT ERBB2-1 or scaffold ABD are included.

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry, Derivative Assay, Selection

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: 21 clones identified after rounds three and four of FACS sorting were screened for ERBB2-binding ( A ), HSA-binding ( B ) and ERBB2-binding in the presence of an excess of HSA ( C ). 5 nM ERBB2 was used in screen A and the binding signal divided by the surface expression level for each clone was normalized against the signal from ADAPT ERBB2-1 . 20 nM HSA was used in screen B and the signals were normalized to scaffold ABD. 5 nM ERBB2 together with 1 µM unlabeled HSA was used in screen C and ADAPT ERBB2-1 was used for normalization. All measurements were done in duplicate on different days and the bars indicate the standard deviation.

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Clone Assay, Binding Assay, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Affinities of selected affinity matured ADAPTs after FACS.

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: ( A ). ERBB2-binding at high concentrations of HSA. Four variants of ADAPT ERBB2-FACS-12 (A8) were displayed at the bacterial surface and incubated with labeled ERBB2 (10 nM) in the presence of varying concentrations of HSA. All mutated variants show a retained ability to bind ERBB2 at very high concentrations of albumin, which is in contrast to the parental ADAPT ERBB2-FACS-12 (A8) . Increasing streptavidin-R-phycoerythrin signals were observed at high concentrations of HSA. This effect was only seen for molecules with retained ERBB2-binding and was also observed for an ERBB2-binding Affibody molecule used as control . Hence, this experimental artifact was not ADAPT-specific. All measurements were done in duplicate on different days and the bars indicate the standard deviation. The albumin concentration is shown on a logarithmic scale, the two highest concentrations were 300 and 600 µM, respectively. ( B ). Competition experiment using ERBB2-binding proteins expressed on staphylococcal cells and flow cytometry. ERBB2-binding (5 nM) to cell-displayed binders was always blocked by a 10-fold excess of the same protein in a soluble form. Only ADAPTs and trastuzumab competed with each other for ERBB2-binding, no competition with the ERBB2-binding Affibody molecule Z ERBB2 was observed. All measurements were done in duplicate on different days and the bars indicate the standard deviation. ( C ). Binding to native ERBB2 on SKOV-3 cells. Cells were incubated with 100 nM biotinylated ADAPT ERBB2-1 or ADAPT ERBB2-FACS-12 (A8) and detected with streptavidin-R-phycoerythrin conjugate. Pre-incubating the cells with an excess of either non-labeled ADAPT ERBB2-FACS-12 (A8) or trastuzumab scFv blocked binding of ADAPT ERBB2-FACS-12 (A8) . A two-sample two-tailed t-test demonstrated that the binding for ADAPT ERBB2-1 was significantly stronger than the signal from the scaffold control (p = 0.0067). All measurements were done in duplicate on different days and the bars indicate the standard deviation. Analyses of non-treated cells were included in all experiments (background).

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay, Incubation, Labeling, Standard Deviation, Concentration Assay, Flow Cytometry, Two Tailed Test

Journal: PLoS ONE

Article Title: Interactions between αv-Integrin and HER2 and Their Role in the Invasive Phenotype of Breast Cancer Cells In Vitro and in Rat Brain

doi: 10.1371/journal.pone.0131842

Figure Lengend Snippet: MM2BH breast cancer cells, previously transfected to overexpress HER2, were transduced with lentiviral shRNAs (sR), scrambled or specific to αv-integrin, to create stable knockdown cell lines in combination with high or low HER2 protein. (A) Immunoblotting of protein expression in MM2BH lines with αv integrin knockdown in combination with high or low HER2 expression. (B) Relative expression of αv integrin and HER2 in MM2BH clones quantified by densitometry of immunoblots from three total cell lysates prepared at different culture passages. The band intensity was normalized to β-actin as a loading control. Error bars show SEM and * indicates values P<0.05 in comparison to control cells.

Article Snippet: Cell surface protein expression was evaluated with the following antibodies: anti-HER2/neu-APC (BD340554, BD Biosciences; 2.5uL per reaction), anti-αv-integrin (Q20-R, Santa Cruz Biotechnology; 1:200), and Alexa Fluor 647 conjugated secondary antibody (Life Technologies; 1:500) was used for αv-integrins.

Techniques: Transfection, Transduction, Western Blot, Expressing, Clone Assay

Journal: PLoS ONE

Article Title: Interactions between αv-Integrin and HER2 and Their Role in the Invasive Phenotype of Breast Cancer Cells In Vitro and in Rat Brain

doi: 10.1371/journal.pone.0131842

Figure Lengend Snippet: Lysates from the SKBR3, HCC1954, and αv+H2+ MM2BH cells was incubated separately with antibodies against HER2 (A) and αv-integrin (B). Components of the immunocomplex precipitate were analyzed by Western blotting for αv-integrin and HER2 proteins. Protein levels are shown in whole lysate (pre-IP), and the immunoprecipated protein complex (IP). (C) Flow cytometry confirmation of HER2 and αv-integrin expression (arrows) in HCC1954 and SKBR3 cells. Light gray peak is unstained control and medium grey is secondary antibody only control.

Article Snippet: Cell surface protein expression was evaluated with the following antibodies: anti-HER2/neu-APC (BD340554, BD Biosciences; 2.5uL per reaction), anti-αv-integrin (Q20-R, Santa Cruz Biotechnology; 1:200), and Alexa Fluor 647 conjugated secondary antibody (Life Technologies; 1:500) was used for αv-integrins.

Techniques: Incubation, Western Blot, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Interactions between αv-Integrin and HER2 and Their Role in the Invasive Phenotype of Breast Cancer Cells In Vitro and in Rat Brain

doi: 10.1371/journal.pone.0131842

Figure Lengend Snippet: MM2BH cell clones grown on glass coverslips were fixed, permeabilized and immunostained. (A) Deficiency of αv-integrin results in increased cytoplasmic localization of HER2 receptors. Yellow: HER2, using an antibody specific for the HER2 intracellular domain, red: f-actin, blue: nucleus. (B) HER2 co-localizes with lysosomes in αv-integrin knockdown cells. Yellow: HER2, red: lysosome-associated membrane protein2 (LAMP2), blue: nucleus. The images are representative of three independent experiments. Scale bar = 20μm. (C) Flow cytometry analysis of surface HER2 protein expression in live MM2BH clones.

Article Snippet: Cell surface protein expression was evaluated with the following antibodies: anti-HER2/neu-APC (BD340554, BD Biosciences; 2.5uL per reaction), anti-αv-integrin (Q20-R, Santa Cruz Biotechnology; 1:200), and Alexa Fluor 647 conjugated secondary antibody (Life Technologies; 1:500) was used for αv-integrins.

Techniques: Clone Assay, Flow Cytometry, Expressing