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: THP1 reporter cell lines, THP1-Dual (thpd-nfis), THP1-Dual KI-hSTING-H232 (thpd-h232), THP1-Dual KI-hSTING-R232 (thpd-r232), and THP1-Dual KO-STING (thpd-kostg) were purchased from InvivoGen.
Techniques: Activation Assay, Expressing, Multiplex Assay, Luminex, Binding Assay, Flow Cytometry, Fluorescence, Control, Mutagenesis, Activity Assay, Cell Culture, Recombinant
InvivoGen is a verified supplier 
InvivoGen manufactures this product 
![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 <t>THP1</t> 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 .)](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0966/pmc12010966/pmc12010966__ccr-24-2449_f1.jpg)