Supplementary Figure 7. hB6H12.3, bearing a human IgG1 Fc region elicits robust antibody-dependent cellular cytotoxicity in the presence of human NK cells compared to IgG4 competitor antibodies HuF9-G4 and CC-90002.

Supplementary Figure 6. hB6H12.3, bearing a human IgG1 Fc region elicits stronger phagocytosis of human red blood cells by human monocytes in vitro compared to IgG4 competitor antibodies HuF9-G4 and CC-90002.

Supplementary Figure 5. Anti-CD47 antibody hB6H12.3 is able to kill CD47-expressing cells via caspase-independent apoptosis, as evidence by the upregulation of Annexin V in a dose-dependent manner, as assessed by flow cytometry on MCF-7 cells in vitro.

Supplementary Figure 16. Production of proteases in culture by cancer cells is sufficient to activate SGN-CD47M to restore binding and function.

Supplementary Figure 15. Anti-CD47 antibody mediated FcgRIIa-H131 signaling, assessed using FcγRIIa Jurkat NFAT reporter cells incubated with CD47-expressing WIL2-S target cells and increasing concentrations of anti-CD47 antibodies or isotype control.

Supplementary Figure 12. Hemagglutination activity of hB6H12.3, SGN-CD47M, MMP-cleaved SGN-CD47M, and an IgG1 isotype control on human red blood cells, as assessed by optical measurement of spot size following incubation with increasing concentrations of antibodies.

Stimulator of IFN genes (STING) is an innate immune pathway that activates a type I IFN response upon detection of intracellular DNA from foreign pathogens as well as tumor cells. STING signaling is critical for antiviral immunity and can be co-opted to drive an antitumor immune response. However, STING activation requires careful and controlled agonism to drive immune activation in the tumor microenvironment (TME) while avoiding toxic systemic immune activation. Indeed, nontargeted small-molecule STING agonist therapeutics have shown limited antitumor activity in the clinic, likely due to their short half-life and poor retention within the TME. We hypothesized that targeted delivery of a potent STING agonist payload directly to the TME via an antibody-drug conjugate (ADC) may overcome some of these limitations. In this study, we report the development of a novel STING agonist ADC with a noncleavable linker-payload (ncSTING). Tumor-targeted ADCs employing this linker-payload (ncSTING ADC) elicited robust antitumor activity in a variety of preclinical murine tumor models. We found that Fcγ receptor binding affected antitumor activity as ADCs with a wild-type Fc drove more antitumor activity than ADCs with an Fcγ receptor-binding mutant Fc in a subset of tumor models. Moreover, tumor-targeted ncSTING ADCs elicited tumor regression with reduced systemic immune activation compared with the systemic administration of the released payload. Altogether, these data provide a therapeutic rationale for the targeted delivery of a potent STING agonist payload via an ADC.

Supplementary Table 6. Peak cytokine concentrations elicited by anti-CD47 antibodies in cynomolgus monkeys after IV administration.

Supplementary Figure 13. Assessment of the ability of hB6H12.3, SGN-CD47M, MMP-cleaved SGN-CD47, and a IgG1 isotype control antibody to induce direct apoptosis of THP1 cells, as assessed by flow cytometry of Annexin V cell surface expression.

Supplementary Figure 19. Protease activity of select syngeneic and xenograft tumor models was assessed using an activatable cell-penetrating peptide (ACPP)2 bearing the -IPVSLRSG- cleavage sequenced used in Coil-MMP-mIAP301 and SGN-CD47M antibodies.

Supplementary Figure 18. Statistical analysis of tumor growth inhibition from xenograft tumor model experiments shown in Figure 3 in the main text.

Supplementary Figure 2. Concentration-time profiles of 3H-labeled mIAP301 and Coil-MMP-mIAP301 were profiled in the plasma, liver, and tumors of BALB/c mice bearing A20 lymphoma tumors once tumors reached approximately 250 mm3 in size.

Supplementary Figure 10. Optical assessment was used to characterize the hemagglutination of human RBCs induced by hB6H12.3 and the murine antibody mB6H12.

Supplementary Figure 3. Statistical analysis of tumor growth inhibition from the A20 syngeneic tumor model experiments shown in Figure 1 in the main text.

Supplementary Figure 9. hB6H12.3 induces stronger FcγRIIa-H131 activation than similar anti-CD47 based on the parent mB6H12 murine antibody, as assessed using a Jurkat NFAT reporter assay in the presence of WIL2-S target cells that express CD47.

Supplementary Figure 4. hBH12.3 inhibits binding of SIRPα to CD47 in a flow cytometry-based assay whereas SGN-CD47M has no effect.

Supplementary Figure 11. Saturation binding analysis of hB6H12.3, SGN-CD47M, and MMP-cleaved SGN-CD47M against recombinant human CD47, as assessed by ELISA.

Enfortumab vedotin is a Nectin-4-directed antibody-drug conjugate designed to deliver the microtubule-disrupting agent monomethyl auristatin E (MMAE) to tumor cells. Using preclinical models of urothelial cancer (UC), we expand the understanding of the multifaceted mechanism of action for enfortumab vedotin that includes direct cytotoxicity on Nectin-4-positive tumor cells, indirect bystander effect on neighboring Nectin-4-negative tumor cells, and MMAE-mediated induction of immunogenic cell death (ICD) and associated increase in activated immune cells in the tumor microenvironment. Importantly, vaccination with enfortumab vedotin-treated tumor cells results in protection against tumor rechallenge in mice, consistent with antitumor immunity. MMAE-mediated ICD induction modulates the tumor microenvironment in a complementary manner to immune checkpoint inhibition. Accordingly, enfortumab vedotin plus PD-1 inhibitor shows enhanced antitumor activity in vivo. These preclinical findings provide mechanistic hypotheses that may be relevant to the improved clinical outcomes observed for enfortumab vedotin plus pembrolizumab relative to chemotherapy.