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

Journal: Cell Reports Methods

Article Title: Sequence enrichment profiles enable target-agnostic antibody generation for a broad range of antigens

doi: 10.1016/j.crmeth.2023.100475

Figure Lengend Snippet:

Article Snippet: HER2 , Sino Biological , Cat# 10004-H08H.

Techniques: Produced, Recombinant, Sequencing, Software

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: A, HCC1954 B, MCF-7-HER2 or C, MCF-7 cells were treated with 2 μM Lapatinib or vehicle (DMSO) for 4 days. Cells were harvested and 100,000 cells/well were plated into 6 well ultra-low attachment plates containing mammosphere forming medium-supplemented with 5 μM MRK-003 GSI or vehicle (DMSO). After 7 days of plating, the mammospheres were collected and counted in order to determine the mammosphere forming efficiency (MFE). Scale bars = 100 μm. Bar graphs show mean %MFE ± S.D. from at least three independent experiments. Statistical significance was calculated using ANOVA with a Tukey post-hoc test for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P <0.0001. Lysates from each treated cell lines were harvested for total protein and subjected to Western blotting to detect phosphorylated forms of HER2 and EGFR. Actin was detected as a loading control.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: HER2+ HCC1954 (A), MCF-7-HER2 (B), MDA-MB-453 (C), and HER2 wild type expressing MCF-7 (D) cells were treated for four days with 2 μM Lapatinib or vehicle (DMSO). Cells were harvested and stained for Jagged1 followed by flow cytometry. E. HCC1954 cells were stained for Jagged1 and then sorted by flow cytometry on the basis of Jagged1 surface expression. The Jagged1 -/low or high population was sorted from both vehicle and lapatinib treated cells. The schematic shows the gating for the cells that were sorted. 35,000 HCC1954 cells were sorted into a well of a 24 well ultra-low attachment plate containing mammosphere forming medium. After 7 days, the mammospheres were harvested and %MFE was determined. Scale bars = 100 μm.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Expressing, Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: HCC1954 (A) or MCF-7-HER2 (B) cells were treated with DMSO (Vehicle) or lapatinib for 4 days followed by flow cytometry to detect cell surface expression of Jagged1. Cells were sorted for low versus high Jagged1 expressing cells and plated onto low attachment plates-containing mammosphere forming medium-supplemented with DMSO (Control) or 5μM MRK-003 GSI. %MFE was assessed at day 7. Scale bar = 100μm. *Denotes statistical significance of P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P <0.0001. One way ANOVA was performed followed by a Tukey post-hoc test.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Flow Cytometry, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: A, Tissue microarray (TMA) was performed on the Nottingham cohort of 145 primary, invasive stage II-III HER2+ breast cancer tissues. Jagged1 staining was performed by immunohistochemistry and Jagged1 was assessed to be localized near the perinucleus, cytoplasm or cell membrane. Jagged1 staining was scored 0.0 for low/negative staining and 1.0 for high/positive staining. A Kaplan-Meier curve was plotted for overall survival and statistics was performed using the log-rank (Mantel-Cox) test. The panels are representative of negative staining, strong membrane + cytoplasmic, cytoplasmic, or perinuclear + cytoplasmic staining. The upper panels are representative images at low magnification with enlarged regions (lower panels) expanded from boxed areas. B. Working model of the effects of anti-HER2 therapy on bulk breast cancer cells to enrich for Jagged1-high expressing CSCs, possibly responsible for resistance and tumor recurrence. A combination approach of anti-HER2 and anti-Jagged1 could potentially prevent this enrichment and drug resistance.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Microarray, Staining, Immunohistochemistry, Negative Staining, Expressing