CD47 is a cell-surface glycoprotein that is expressed on normal human tissues and plays a key role as a marker of self. Tumor cells have co-opted CD47 overexpression to evade immune surveillance, and thus blockade of CD47 is a highly active area of clinical exploration in oncology. However, clinical development of CD47-targeted agents has been complicated by its robust expression in normal tissues and the toxicities that arise from blocking this inhibitory signal. Furthermore, pro-phagocytic signals are not uniformly expressed in tumors, and antibody blockade alone is often not sufficient to drive antitumor activity. The inclusion of an IgG1 antibody backbone into therapeutic design has been shown to not only serve as an additional pro-phagocytic signal but also exacerbate toxicities in normal tissues. Therefore, a need persists for more selective therapeutic modalities targeting CD47. To address these challenges, we developed SGN-CD47M, a humanized anti–CD47 IgG1 mAb linked to novel masking peptides through linkers designed to be cleaved by active proteases enriched in the tumor microenvironment (TME). Masking technology has the potential to increase the amount of drug that reaches the TME while concomitantly reducing systemic toxicities. We demonstrate that SGN-CD47M is well tolerated in cynomolgus monkeys and displays a 20-fold improvement in tolerability to hematologic toxicities when compared with the unmasked antibody. SGN-CD47M also displays preferential activation in the TME that leads to robust single-agent antitumor activity. For these reasons, SGN-CD47M may have enhanced antitumor activity and improved tolerability relative to existing therapies that target the CD47–signal regulatory protein α interaction.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
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Supplementary data

Supplementary Table 1

Supplementary Table 1. Sequences of anti-CD47 antibodies used in these studies.

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Supplementary Table 2

Supplementary Table 2. Assessment of the protease specificity for cleavage of the -IPVSLRSG- cleavable linker contained in Coil-MMP-mIAP301 and SGN-CD47M.

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Supplementary Table 3

Supplementary Table 3. MMP2 and MMP9 quantification from select tumor models.

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Supplementary Table 4

Supplementary Table 4. SGN-CD47M and hB6H12.3 bind to cynomolgus macaque CD47.

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Supplementary Table 5

Supplementary Table 5. Pharmacokinetic parameters of gTAb in monkey plasma following a single IV dose of hB6H12.3 (1 mg/kg) or SGN-CD47M (10 or 20 mg/kg) as determined by non-compartmental analysis.

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Supplementary Table 6

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

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Supplementary Figure 1

Supplementary Figure 1. Example mass spectrometry data for MMP2 re-activated masked antibody.

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Supplementary Figure 2

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.

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Supplementary Figure 3

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

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Supplementary Figure 4

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

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Supplementary Figure 5

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.

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Supplementary Figure 6

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.

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Supplementary Figure 7

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.

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Supplementary Figure 8

Supplementary Figure 8. hB6H12.3, bearing a human IgG1 Fc region demonstrates enhanced T cell responsiveness compared to IgG4 competitor antibodies HuF9-G4 and CC-90002.

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Supplementary Figure 9

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.

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Supplementary Figure 10

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

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Supplementary Figure 11

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

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Supplementary Figure 12

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.

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Supplementary Figure 13

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.

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Supplementary Figure 14

Supplementary Figure 14. Induction of ADCC, assessed using FcγRIIIa-V158 Jurkat NFAT reporter cells incubated with CD47-expressing WIL2-S target cells and increasing concentrations of anti-CD47 antibodies or isotype control.

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Supplementary Figure 15

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.

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Supplementary Figure 16

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

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Supplementary Figure 17

Supplementary Figure 17. F4/80 staining of select xenograft tumor models used in the study.

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Supplementary Figure 18

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

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Supplementary Figure 19

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.

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