Figure shows DLD1 xenograft study comparing Biaryl PBD ADCs hR-13 vs hR-DGN549

Table shows TROP2 receptor quantification on DLD1 and HT29 cells

Figure S1. Characterization of XMT-2056

Table shows Cytotoxicity Data for Platforms 18 vs 19

Figure shows Plasma stability comparing indole-linked ADC T-13 to N10-linked ADC T-BH

Figure S3. A. Cancer cell death induced by XMT-2056, HT19 antibody, or non-binding control ADC, shown as percent viable SKBR3-NR (left) or MDA-MB-175-VII-NR (right) cells in PBMC co-cultures (84 hr time point). Each point represents mean and SD (n=3). B. Cytokine induction by XMT-2056, HT19, or non-binding control ADC measured in supernatants of SKBR3-NR (top) or MDA-MB-175-VII-NR (bottom) cells in PBMC co-cultures (24 hr time point). Each point represents mean and SD (n=3). C. Cancer cell death by XMT-2056, non-binding control ADC, or STING agonist payload shown as percent viable cells in monocultures of MDA-MB-231-NR (left) and SKBR3-NR (right) (84 hr time point). Each point represents mean and SD (n=3).

Figure shows Structures of Platforms 6, and 8-16

Table shows Cytotoxicity comparisons between Biaryl PBD ADCs vs DGN549 ADCs

Figure shows Efficacy in OVCAR-3 xenograft for A) targeted ADCs X-17 and X-19 and B) their corresponding non-binding control ADCs NB-17 and NB-19

Figure S6. FVB/NJ immune competent mice bearing syngeneic mBR9013 subcutaneous tumors were intravenously administered anti-mouse PD-1 (clone RPM1-14) 10 mg/kg twice weekly for 3 weeks (red triangles). Each point represents the mean tumor volume and SEM (n=5).

Figure S4. A. HER2 expression by IHC in SKOV3 xenograft tumors; scale bar, 20 µm. B. Percent changes in body weights of SKOV3 tumor-bearing CB.17 SCID mice administered a single dose (black arrowhead) of XMT-2056, non-binding control ADC, HT19, or STING agonist payload, or 3 doses (orange arrowhead) of the diABZI STING agonist. Each point represents the mean change in body weight and SEM (n=10). C. Normalized counts for mouse mRNA and human mRNA of individual tumor cytokine/chemokines in SKOV3 xenografts harvested 12 hours after treatment. Each point represents the mean and SD (n=2). D. Representative images of IHC for leukocyte marker CD45 and macrophage marker CD68 in SKOV3 tumors after treatment with vehicle, 1 mg/kg XMT-2056, or 1 mg/kg control ADC, and collected 72 hr after treatment. Scale bar, 3 mm. Images are provided for 3 animals per group. The red boxes indicate the tumors shown at higher magnifications in main Figure 3H. E. Representative images of IHC for dendritic cell marker CD11c and murine PD-L1 in SKOV3 tumors collected 12 hrs (CD11c) or 72 hrs (mPD-L1) after treatment. Scale bar, 100 µm.

Figure S5. A. PK profile of XMT-2056 in non-tumor bearing CB.17 SCID mice as a function of total antibody (Total Ab) and conjugated drug concentrations in plasma after a single intravenous administration. Each line represents an individual animal (n=4). B. PK profile of XMT-2056 in cynomolgus monkey as a function of total antibody and conjugated drug concentrations in plasma during Q3W repeat-dose administration indicated by the black arrowheads. 1 male and 1 female animal were dosed; each line represents data from one animal.

Table shows Tabulated cell growth inhibition data for NCI-N87 and MDA-MB231 cells in monoculture and coculture when treated with ADCs T-16, T-17, and T-18

Table shows Impact of Tether length and Hydrophilic Modifier on ADC Aggregation and Hydrophobicity

Figure shows comet assay results

Table S2. EC50 values for binding of XMT-2056 or HT19 antibody to human and nonclinical species HER2 extracellular domain. Highest dose tested is 100 nM by antibody. Values represent mean of 2 independent experiments.

Table shows Payload in vitro potency in various cell lines.

Figure S2. A. STING mediated IRF3 activity of THP1 reporter cells expressing the indicated human STING1 haplotypes or STING1 knockout (KO) after treatment with STING agonist at the indicated doses. Each point represents the mean and SD (n=2). B. Cytokine induction as measured by a multiplex Luminex assay from supernatants of fresh human white blood cells treated for 6 (IFN-β) or 24 (CXCL10, IL-6, TNF-α) hours with indicated concentrations of STING agonist. Bars represent mean value of n=2 data points shown as symbols. C. Structure of the human HER2 extracellular domain showing HT19 epitope mapped to domain IV through shotgun mutagenesis using an Alanine Scanning Mutagenesis. D. Lack of competition with pertuzumab by bio-layer interferometry binning assay (Octet). Pertuzumab was loaded onto the sensor chip, and HER2 ECD and HT19 associations indicated by blue arrows. Additional binding by HT19 antibody indicates non-competitive binding. E. HER2+ NCI-N87 gastric cancer xenograft model treated with Dolaflexin (DF)-based ADCs including conjugates of trastuzumab or non-binding isotype control ADC at the indicated doses. Each point represents mean tumor volume and SEM (n=10). F. Bar graphs of % inhibition in phospho-AKT in SKBR3 cells for HT19, trastuzumab, and lapatinib. Each bar represents mean and SD of 2 independent experiments. G. Binding of XMT-2056, HT19, and hIgG1 isotype control antibody to SKBR3, JIMT-1, MDA-MB-453, SNU-5, and MDA-MB-175-VII cells showing fluorescence intensities measured by flow cytometry. Each point represents mean and SD (n=3). H. Graphical traces of binding kinetics of XMT-2056, HT19, or Fc mutant XMT-2056 to human Fcγ-RI recombinant protein, showing association and dissociation phases of binding by Octet. I. IRF3 reporter activity of THP1 cells in monoculture (Ieft) or cultured in plates in the absence of HER2 antigen (right) treated for 24 hours with XMT-2056, Fc mutant XMT-2056, non-binding control ADC, or free payload. Each point represents mean and SD (n=3).

Figure S8. A, B. BALB/c immune competent mice bearing syngeneic EMT-6-rHER2 tumors were treated with XMT-2056 surrogate ADC, anti-mouse PD-1 (Clone RPM1-14), non-binding control ADC in combination with anti-mouse PD-1, or XMT-2056 surrogate ADC in combination with anti-mouse PD-1. The ADCs were administered once while the mouse anti-PD-1 was administered twice weekly for 2 weeks as indicated by the red triangles. A. Tumor volumes of the individual mice, with the number of complete responders (CR) indicated. B. Percent change in body weight (BW). Each point represents the mean change in BW and SEM (n=10).

Figure shows Cytotoxicity of Mesothelin ADCs M-16, M-17, and M-19 in NCI-N87 cell line