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: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Inhibition of growth and HER2 signaling by HER2Ab-NSCs cells. (A) Inhibition in growth of BT474 cells using supernatant of HER2Ab-NSCs. (B) Co-Culture assay using BT474 cells with either vector control or HER2Ab-NSCs cells. (C-D) Inhibition of PI3K-AKT signaling using purified anti-HER2Ab released by NSC. Right panel in C and D demonstrates significant decrease in relative densitometric units. Trastuzumab was used as positive control in A, C and D. The experiments were repeated three times. * indicates p<0.05 and ** indicates p<0.01.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Co-culture Assay, Plasmid Preparation, Control, Purification, Positive Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Binding of anti-HER2Ab to HER2 overexpressing breast cancer cells. (A) BT474Br were stained with trastuzumab as a positive control (B-F) MCF 7, MDA-MB-361, BT474Br, ZR-75-30 and SKBR3 cells stained with anti-HER2Ab secreted by NSC. The experiments were repeated two times.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Staining, Positive Control

Journal: International journal of molecular sciences

Article Title: Tumor Site-Specific Cleavage Improves the Antitumor Efficacy of Antibody-Drug Conjugates.

doi: 10.3390/ijms241311011

Figure Lengend Snippet: Figure 2. Characterization of the conjugates. (A) SDS—PAGE of the purified ZHER2-MMAE, Herceptin-MMAE, and Fc-U-ZHER2-MMAE. Lane 1, uncoupled raw proteins; Lane 2, conjugates. (B) Western blot analysis of ZHER2-MMAE, Herceptin-MMAE, and Fc-U-ZHER2-MMAE using HRP- labeled mouse anti-IgG Fc antibody, HRP-labeled mouse anti-His antibody, or HRP-labeled mouse anti-MMAE antibody. (C) ELISA assays of the binding ability of the conjugates with HER2 receptor. The human HER2 protein was coated on the plates and incubated with different concentrations of Herceptin-MMAE, ZHER2-MMAE or Fc-U-ZHER2-MMAE at 37 ◦C for 2 h, and then analyzed with mouse anti-MMAE monoclonal antibody by ELISA. Data are expressed as the mean ± SEM (n = 3). (D) EC50 data calculated with GraphPad Prism 5.0 software.

Article Snippet: Briefly, microtiter plates were coated with human HER2 protein (ACRO biosystems, Cat # HE2-H521y, Beijing, China) in PBS overnight at 4 ◦C.

Techniques: SDS Page, Western Blot, Labeling, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Software