Journal: iScience

Article Title: From byproduct to biotherapeutic: Comparative study of buffy coats and leukoreduction system chambers for NK cell-based immunotherapies

doi: 10.1016/j.isci.2026.114907

Figure Lengend Snippet: Functional characterization of NK cells (A) Degranulation capacity of NK cells following a 2 h co-culture with K562 leukemia cells, measured by surface CD107a expression ( n = 5). (B and C) Europium-based cytotoxicity assay assessing the specific lysis of K562 target cells at E:T ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 on day 7 (B) and day 14 (C) of culture to monitor functional decline over time ( n = 6 for both time points). Here, all comparisons were not significant and are summarized as such (n.s.: p > 0.05). (D) IFN-γ release following 16 h of co-culture of NK cells with K562 cells, quantified via Luminex assay ( n = 5). (E) Kinetic live-cell killing assay using the Incucyte monitoring of Nuclight Red signal loss in K562 cells over 72 h at an E:T ratio of 1:1 ( n = 6). (F) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis thresholds during Incucyte co-culture with K562 cells. (G) CD107a degranulation assay of NK cells with or without pre-incubation with trastuzumab (anti-HER2 antibody) during a 2 h co-culture with MDA-MB-453 cells ( n = 5). (H) Incucyte-based cytotoxicity assay shows the real-time lysis of MDA-MB-453 cells by trastuzumab-loaded NK cells at an E:T ratio of 1:1 over 72 h ( n = 6). (I) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the ADCC assay. (n varies as not all samples reached the benchmark values within the observation period). All ADCC experiments have been performed after 14 days of in vitro NK cell expansion. N/A indicates that statistical analysis was not possible for this group. 9J) CD107a degranulation of unmodified NK cells and ErbB2-CAR-NK cells after 2 h co-culture with MDA-MB-453 cells ( n = 5). (K) Incucyte-based cytotoxicity assay showing killing dynamics of ErbB2-CAR-NK cells against MDA-MB-453 cells at an E:T ratio of 1:1 over 72 h ( n = 6). (L) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the CAR-mediated cytotoxicity assay. (n varies as not all samples reached the benchmark values within the observation period). All CAR-killing experiments have been performed after 14 days of in vitro NK cell expansion. Unless otherwise stated, n refers to biologically independent donor samples and is indicated per group. Data are shown as mean ± SEM. p-values were determined by Student’s t test with Welsh correction (A, D, F, G, I, J, and L) or by one-way ANOVA (B and C) with Tukey post-test (K and L).

Article Snippet: Mouse Monoclonal anti-CD340 (Her2) , Miltenyi Biotec , Cat# 130-124-474.

Techniques: Functional Assay, Co-Culture Assay, Expressing, Cytotoxicity Assay, Lysis, Luminex, Degranulation Assay, Incubation, ADCC Assay, In Vitro

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: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Binding affinities to human HER2 and CD3 at 37 °C by surface plasmon resonance. Data are K D ( n = 12 technical replicates of a single experiment for HER2-XPAT protein and HER2(1x-N); n = 8 for HER2(1x-C) and uTCE). Surface plasmon resonance sensorgrams for these data are provided in Supplementary Figs. – . b – d , In vitro tumor cytotoxicity of HER2-XPAT protein and its metabolites following a 48-h incubation with co-cultures of huPBMCs and the high HER2-expressing human tumor cell lines (1:1 effector–target ratio) SKOV3 ( b ), BT-474 ( c ) or the medium-low HER2-expressing MCF7 cell line ( d ). e , In vitro cytotoxicity of HER2-XPAT protein and its metabolites against BT-474 cells co-cultured with huPBMCs (1:1 effector–target ratio). f , Impact of the protease-cleavable linker on in vitro cytotoxicity versus BT-474 cells co-cultured with huPBMCs. g , h , CD69-positive T cells ( g ) and IL-2 secretion ( h ) following 72-h incubation of huPBMC/SKOV3 co-cultures with HER2-XPAT protein or its unmasked form (uTCE). i , Target-dependent T-cell activation with HER2-XPAT protein and its metabolites. CD3-expressing Jurkat reporter T cells were incubated with BT-474 cells at a 5:1 effector–target ratio for 6 h, followed by quantification of NFAT -induced luciferase activity and measured in relative luminescence units (RLUs). Mean data for n = 2 technical replicates within one single experiment ( b – i ). Extended Data Table provides a summary of EC 50 values for the different forms of the HER2-XPAT proteins in the cytotoxicity and reporter T-cell activation assays.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Binding Assay, SPR Assay, In Vitro, Incubation, Expressing, Cell Culture, Activation Assay, Luciferase, Activity Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: CD3 + Jurkat reporter T cells were incubated with or without BT-474 cells at a 5ː1 effector-target ratio for 6 hours, followed by quantification of NFAT -induced luciferase activity. The graph shows individual data points from a single representative experiment. There were n = 2 biological repeats at each concentration tested within the same experiment for each cell line. HER2, human epidermal growth factor receptor 2; RLU, relative luminescence unit; uTCE, unmasked T-cell engager; XPAT proteins, TCE fused to XTEN polypeptides.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Incubation, Luciferase, Activity Assay, Concentration Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , TGI ± s.e.m. ( n = 8 mice for each concentration tested within one single experiment) promoted by the i.v. administration of equimolar doses (every week (QW) for 3 weeks) of HER2-XPAT protein (2.1 mg kg −1 ) or uTCE to NOG mice bearing established (maximum tolerated volume (MTV) ~185 mm 3 ) BT-474 human tumors. The dependence of tumor-resident proteases for activity was demonstrated by the lack of significant TGI in mice treated with HER2-XPAT-NoClvSite. The average body weight of the mice bearing tumor xenografts remained generally stable for the duration of the experiment following dosing with the HER2-XPAT proteins or the vehicle control. b ,TGI ± s.e.m. ( n = 8 mice for each concentration tested within one single experiment) in established human HT-55 xenografts (MTV ∼150 mm 3 ) following i.v. administration of HER2-XPAT protein (QW for 4 weeks) and HER2-uTCE (0.9 mg kg −1 three times a week (TIW) for 4 weeks). HER2-XPAT-NoClvSite (QW for 4 weeks) had no impact on tumor growth. The average body weight ± s.e.m. of the mice bearing tumor xenografts remained generally stable for the duration of the experiment following dosing with the HER2-XPAT proteins or the vehicle control. c , T-cell activation in BT-474 human tumor xenografts and peripheral blood evaluated by flow cytometry on day 18 following equimolar TIW i.v. dosing with HER2-XPAT protein and HER2-uTCE (day 40 following tumor inoculation). HER2-XPAT and HER2-uTCE induced robust and comparable activation of intratumoral CD4 + and CD8 + T cells, whereas no trends for T-cell activation were apparent in blood samples in which HER2 was not present. Statistical differences in TGI and T-cell activation for test compounds versus vehicle were assessed using mixed-effects multiple comparison analyses followed by Tukey’s post hoc test.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Concentration Assay, Activity Assay, Control, Activation Assay, Flow Cytometry, Comparison

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Fluorescent dye-labeled HER2-XPAT or EpCAM-XPAT protein was used to track proteolytic cleavage in PDX tumor-bearing mice. b , A representative SDS–PAGE gel showing XPAT protein cleavage forms arising in a single PDX-bearing mouse, after imaging by LI-COR Biosciences. Four bands representing HER2-XPAT protein and its three unmasked forms are visible in the tumor sample (green channel) while the other (red channel) predominantly shows the XPAT protein form. c , Concentration of XPAT protein forms in tumors and healthy tissues. Results are the mean ± s.d. from 20 mice within one single experiment, with tumors consisting of ten different PDX (Supplementary Table ); mice were injected with fluorescent dye-labeled HER2-XPAT or EpCAM-XPAT protein. Note that tissues for which ≥14 samples were below the limit of quantification (BLQ) were reported as ‘BLQ.’ Tissues with averages calculated using some samples with BLQ readings are marked with an asterisk.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Labeling, SDS Page, Imaging, Concentration Assay, Injection

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: HER2-XPAT protein was administered via short i.v. infusions (∼1–3 h), whereas the unmasked counterpart, due to its short half-life, was administered via 48-h continuous i.v. infusion to provide prolonged systemic exposure. a , A dose-escalation study with a single dose of HER2-XPAT protein administered to each cynomolgus monkey. *At doses below 21 mg kg −1 , a HER2-XPAT prototype was used. b , Dose de-escalation study of uTCE in NHPs administered as a 48-h continuous infusion due to its short half-life. c , Plasma concentrations in NHPs following administration of a single i.v. dose of HER2-XPAT protein (42 mg kg −1 , n = 2 NHPs; 25 mg kg −1 , n = 4 NHPs; 6 mg kg −1 , n = 12 NHPs; 2 mg kg −1 , n = 12 NHPs) or a 48-h continuous infusion of uTCE (0.2 mg kg −1 d −1 , n = 1 NHP; 0.1 mg kg −1 d −1 , n = 1 NHP; 0.06 mg kg −1 d −1 , n = 1 NHP). Data represent mean plasma concentration (±s.d. when n > 3 NHP treated) for the NHPs treated at each dose within one single experiment. d , e , Activation of circulating CD4 + T cells ( d ) and CD8 + T cells ( e ) 24 h after drug administration (HER2-XPAT protein, n = 16 NHPs; HER2-uTCE, n = 5 NHPs). f – h , Highest plasma levels of TNF-α ( f ), IL-6 ( g ) and IFN-γ ( h ) measured 0–24 h following drug administration (HER2-XPAT protein, n = 23 NHPs; HER2-uTCE, n = 8 NHPs). Note that the normal ranges for cytokine levels in NHP are as follows: IL-6 0.3–1.2 pg ml −1 , TNF-α 1–10 pg ml −1 , IFN-γ 1.1–8.0 pg ml −1 (ref. ). Data in d – f represent a single sample from each NHP receiving the test material within each single experiment. TNF, tumor necrosis factor; IFN, interferon.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Concentration Assay, Activation Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Mean plasma concentrations (±s.d.) following a single i.v. administration of HER2-XPAT protein (single plasma samples from n = 6 NHPs) and HER2-XPAT-NoClvSite ( n = 4 NHPs) within one single experiment. b , Plasma concentrations of HER2-XPAT(1x−N) and HER2XPAT(1x−C) relative to the entire HER2-XPAT protein, following a single i.v. dose of HER2-XPAT protein (25 mg kg −1 , n = 4 NHPs; 42 mg kg −1 , n = 4 NHPs) within one single experiment. c , Quantification of cleavage products with a similar molecular weight to the uTCE generated following incubation of fluorescent-labeled HER2-XPAT protein in plasma from cancer patients ( n = 11), patients with inflammatory diseases ( n = 27), healthy donors ( n = 4) and NHPs (with ( n = 6) and without ( n = 4) drug-induced inflammation). The plasma incubation experiment represents a closed system with no clearance mechanisms and will therefore overestimate the accumulation of cleavage products occurring in vivo. Horizontal bars represent median values; dots represent individual observations. P values are based on unpaired t -tests.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Molecular Weight, Generated, Incubation, Labeling, In Vivo

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , An XPAT protein comprises a TCE core with two scFvs, one targeting CD3 and the other, a TAA. Each scFv is masked by a protease-releasable XTEN mask, unstructured, hydrophilic polypeptides that act as modular, tunable masks, in addition to extending the half-life of the TCE. Each XTEN mask connects to the TCE core via a protease-cleavable linker, designed to be cleavable by any of eight proteases from three different classes (matrix metalloproteinases, serine proteases and cysteine proteases) involved in cancer progression . b , Predicted structure of HER2-XPAT protein visualized using AlphaFold2 v.2.0, a machine-learning-based computational method for predicting protein structures with reasonable accuracy . Colors indicate anti-HER2 domain, pale green; anti-CD3 domain, light orange; XTEN masks, blue; protease-cleavable linker, red; linkers, gray. The model represents a static picture showing a plausible conformation of the unstructured XTEN masks and the length of unstructured XTEN relative to the folded antibody domains in an XPAT protein. c , XPAT proteins are expected to remain largely intact in healthy tissues, where protease activity is well controlled by protease inhibitors. XPAT protein unmasking occurs in two steps via one of two potential paths to the fully unmasked state. The two requisite cleavage events can occur in either order and each sequence (either the top or bottom paths shown) is equally likely. In aggregate, both 1x-N and 1x-C partially unmasked forms will exist, depending on the cleavage path. Removal of both XTEN masks liberates the unmasked HER2-TCE (uTCE). d , XPAT proteins are designed to exploit the dysregulated protease activity present in tumors versus healthy tissues and expand the therapeutic index of TCEs through preferential unmasking in the TME. The active uTCE promotes the formation of immunologic synapses between tumor and T cells, resulting in potent cytotoxicity. Notably, the uTCE has a short half-life and should be rapidly cleared, thereby sparing healthy tissues when the uTCE diffuses away from the TME. By design, the molecular weight of the uTCE (∼59 kDa) is sufficiently small to allow rapid kidney filtration .

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Activity Assay, Sequencing, Molecular Weight, Filtration