glomax discover plate reader software version 3.2.3  (Promega)

Journal: Clinical Cancer Research

Article Title: XMT-2056, a HER2-Directed STING Agonist Antibody–Drug Conjugate, Induces Innate Antitumor Immune Responses by Acting on Cancer Cells and Tumor-Resident Immune Cells

doi: 10.1158/1078-0432.CCR-24-2449

Figure Lengend Snippet: XMT-2056 elicits potent antigen-dependent and Fc-R–mediated activation of monocytes in cocultures. A, Schematic of the XMT-2056 mechanism, which includes HER2-dependent ADC uptake into tumor cells and Fcγ-R–expressing myeloid cells. B, Chemical structure of XMT-2056. C, Cytokine induction as measured by a multiplex Luminex assay from supernatants of fresh human WBCs treated for 6 (IFN-β) or 24 (CXCL10, IL-6, and TNF-a) hours. Bars represent mean value of n = 2 data points shown as symbols. D, Competition with trastuzumab by biolayer interferometry (Octet): trastuzumab was loaded onto the sensor chip, and HER2 ECD or HT19 antibody associations are indicated by blue arrows. Additional binding by HT19 indicates noncompetitive binding. E, Competition with trastuzumab by cell-based flow cytometry. HT19 hIgG1 or mIgG2a formats detected with either Alexa Fluor anti-hIgG1 (h647) or Alexa Fluor anti-mIgG2a (m647) secondary antibodies in the presence or absence of trastuzumab–mIgG2a. Each point represents the mean and SD ( n = 3). F, Binding of XMT-2056 or HT19 antibody to HCC1954 cells showing fluorescence intensities measured by flow cytometry. Each point represents the mean and SD ( n = 2). G, CXCL10 cytokine induction in HCC1954 monocultures treated for 24 hours with XMT-2056, nonbinding control ADC, HT19, or the free payload. Each point represents the mean and SD ( n = 2). H, Binding of XMT-2056 or Fc-mutant XMT-2056, HT19, and nonbinding control ADC to SKOV3 cells showing fluorescence intensities measured by flow cytometry. Each point represents the mean and SD ( n = 3). I and J, IRF3 reporter activity of THP1 cells in coculture with SKOV3 cells ( I ) or cultured on recombinant HER2 antigen-coated plates ( J ) treated for 24 hours with XMT-2056 or Fc-mutant XMT-2056, nonbinding control ADC, or STING agonist payload. Each point represents the mean and SD ( n = 3). When noted, EC 50 and B max values represent mean of two independent experiments. gMFI, geometric mean fluorescence intensity; RLU, relative light units. ( A, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/s16c825 ; G, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/s16c825 ; I and J, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/s16c825 .)

Article Snippet: Image analysis was conducted with HER2 algorithms built with HALO multiplex IHC software, version 3.2.3 (Indica Labs Inc.).

Techniques: Activation Assay, Expressing, Multiplex Assay, Luminex, Binding Assay, Flow Cytometry, Fluorescence, Control, Mutagenesis, Activity Assay, Cell Culture, Recombinant

Journal: Clinical Cancer Research

Article Title: XMT-2056, a HER2-Directed STING Agonist Antibody–Drug Conjugate, Induces Innate Antitumor Immune Responses by Acting on Cancer Cells and Tumor-Resident Immune Cells

doi: 10.1158/1078-0432.CCR-24-2449

Figure Lengend Snippet: XMT-2056 induces killing of HER2-high and HER2-low cancer cells and induces concomitant immune-mediated killing of HER2-negative cancer cells in vitro . A and B, Cancer cell death induced by XMT-2056, nonbinding control ADC, or STING agonist payload, shown as percent viable SKBR3–NR ( A ) or MDA-MB-175-VII–NR ( B ) cells in PBMC coc ultures (84 hours time point). Each point represents the mean and SD ( n = 3). C and D , Cytokine induction by XMT-2056, nonbinding control ADC, or STING agonist payload in supernatants of SKBR3–NR ( C ) or MDA-MB-175-VII–NR ( D ) cells in PBMC cocultures (24 hours time point). Each point represents the mean and SD ( n = 3). Note that the y-axis scales vary. E, Schematic of concomitant antigen-dependent, immune-mediated killing of HER2-negative cancer cells. F, Flow cytometry of HER2 expression on SKBR3 and nuclear red expressing MDA-MB-231–NR. Each point represents the mean and SD ( n = 4). G, Cancer cell death mediated by XMT-2056, nonbinding control ADC, or STING agonist payload, shown as percent viable MDA-MB-231–NR cells in coculture with PBMCs and HER2-positive SKBR3 at the indicated ratios or in the absence of SKBR3 (84 hours time point). Each point represents the mean and SD ( n = 3). ( E, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/k38t564 .)

Article Snippet: Image analysis was conducted with HER2 algorithms built with HALO multiplex IHC software, version 3.2.3 (Indica Labs Inc.).

Techniques: In Vitro, Control, Flow Cytometry, Expressing

Journal: Clinical Cancer Research

Article Title: XMT-2056, a HER2-Directed STING Agonist Antibody–Drug Conjugate, Induces Innate Antitumor Immune Responses by Acting on Cancer Cells and Tumor-Resident Immune Cells

doi: 10.1158/1078-0432.CCR-24-2449

Figure Lengend Snippet: XMT-2056 elicits antitumor activity in a broad range of tumor models. A, HER2 expression by IHC in tumor xenograft models (human HER2) and syngeneic tumors (rat HER2); see Supplementary Table S3 for quantitation. Scale bar, 20 µm. B–D, SCID beige mice bearing subcutaneous HCC1954 xenograft tumors ( B ) or CB.17 SCID mice bearing subcutaneous SNU-5 ( C ) or JIMT-1 ( D ) xenograft tumors were intravenously administered a single dose (black arrowhead) of XMT-2056, nonbinding control ADC, or HT19 antibody, or three doses of the diABZI STING agonist (orange arrowheads). Each point represents the mean tumor volume and SEM ( n = 10). E, FVB/NJ immune-competent mice bearing syngeneic mBR9013 subcutaneous tumors derived from a spontaneous tumor in MMTV-ERBB2 FVB mouse expressing rat HER2 were intravenously administered a single dose of XMT-2056 surrogate ADC targeting rat HER2, nonbinding control ADC, or three doses of the diABZI STING agonist. Each point represents the mean tumor volume and SEM ( n = 10). F, BALB/c immune-competent mice bearing syngeneic EMT-6 subcutaneous tumors that were engineered to express rat HER2 (EMT-6–rHER2) were treated as described in E . Each point represents the mean tumor volume and SEM ( n = 10).

Article Snippet: Image analysis was conducted with HER2 algorithms built with HALO multiplex IHC software, version 3.2.3 (Indica Labs Inc.).

Techniques: Activity Assay, Expressing, Quantitation Assay, Control, Derivative Assay

Journal: Clinical Cancer Research

Article Title: XMT-2056, a HER2-Directed STING Agonist Antibody–Drug Conjugate, Induces Innate Antitumor Immune Responses by Acting on Cancer Cells and Tumor-Resident Immune Cells

doi: 10.1158/1078-0432.CCR-24-2449

Figure Lengend Snippet: Observed benefit in combining XMT-2056 with other HER2-targeted agents. A–C, Combination of XMT-2056 and trastuzumab. CB.17 SCID mice bearing subcutaneous SKOV3 ( A ), JIMT-1 ( B ) , or SNU-5 ( C ) xenograft tumors were administered in 3 weekly doses ( A and B ) or a single dose ( C ) of XMT-2056, nonbinding control ADC, trastuzumab, or the combinations indicated. ADCs were administered intravenously, whereas trastuzumab was administered intraperitoneally. Each point represents the mean tumor volume and SEM ( n = 10). D, Combination of XMT-2056 and T-Dxd. CB.17 SCID mice bearing subcutaneous JIMT-1 xenograft tumors were intravenously administered XMT-2056, nonbinding control ADC, T-Dxd, or a combination of XMT-2056 and T-Dxd. STING agonist ADCs were administered as a single dose, whereas T-Dxd was administered twice, 1 week apart (red triangles). Each point represents the mean tumor volume and SEM ( n = 10).

Article Snippet: Image analysis was conducted with HER2 algorithms built with HALO multiplex IHC software, version 3.2.3 (Indica Labs Inc.).

Techniques: Control

Journal: Clinical Cancer Research

Article Title: XMT-2056, a HER2-Directed STING Agonist Antibody–Drug Conjugate, Induces Innate Antitumor Immune Responses by Acting on Cancer Cells and Tumor-Resident Immune Cells

doi: 10.1158/1078-0432.CCR-24-2449

Figure Lengend Snippet: Combination of XMT-2056 with immune checkpoint inhibitor anti–PD-1 elicits tumor clearance and immunologic memory in the EMT-6–rHER2 syngeneic model. A, Normalized counts for PD-L1 human mRNA and mouse mRNA in SKOV3 xenograft tumors. B, Study design including efficacy and rechallenge. Tumor implantations are indicated by circles on the left and right flanks of the mice. C, BALB/c immune-competent mice bearing syngeneic EMT-6–rHER2 engineered tumors were treated with XMT-2056 surrogate ADC targeting rat HER2, anti-mouse PD-1 (clone RPM1-14), or the indicated combinations. The ADCs were administered as single doses, whereas the mouse anti–PD-1 was administered twice weekly for 2 weeks, as indicated by the red triangles. Each point represents the mean tumor volume and SEM ( n = 10). D, Tumor-free animals from the XMT-2056 surrogate ADC (blue) and the combination (green) groups were implanted with EMT-6 parental cells on the left flank (opposite the original EMT-6–rHER2 implantation) and CT26 cells on the right flank. Age-matched untreated mice were included as controls. Right, tumor growth in individual mice in each group after implantation on day 0 plotted relative to age-matched naïve mice. CR, complete response; LF, left flank; RF, right flank. ( B, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/k61r850 .)

Article Snippet: Image analysis was conducted with HER2 algorithms built with HALO multiplex IHC software, version 3.2.3 (Indica Labs Inc.).

Techniques: