Localized CD47 blockade enhances immunotherapy for murine melanoma

Jessica Ingram

Olga Blomberg

Jonathan Sockolosky

Lestat Ali

K Garcia+2 authors

^{Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA, 02142;}

^{Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, 02115;}

^{Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, 94305;}

^{Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305;}

^{Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, 94305;}

^{Division of Gastroenterology, Department of Medicine, Massachusetts General Hospital, Boston, MA, 02114}

^{To whom correspondence may be addressed. Email:}ude.dravrah.snerdlihc@hgeolp.eddih or gro.srentrap@naguodlm.

Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada, and approved August 10, 2017 (received for review June 14, 2017)

Author contributions: J.R.I., O.S.B., J.T.S., F.I.S., S.K.D., K.C.G., H.L.P., and M.D. designed research; J.R.I., O.S.B., J.T.S., L.A., N.P., C.E., and M.D. performed research; F.I.S. contributed new reagents/analytic tools; J.R.I., O.S.B., J.T.S., L.A., F.I.S., S.K.D., K.C.G., H.L.P., and M.D. analyzed data; and J.R.I., H.L.P., and M.D. wrote the paper.

^{Present address: Program in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, MA 02115.}

Significance

CD47 is a broadly expressed membrane-associated innate immune regulator that acts as a ligand of signal regulatory protein alpha (SIRPα) on antigen-presenting cells to inhibit phagocytosis. In xenograft models, inhibitors of the CD47–SIRPα interaction selectively target tumor-expressed CD47 and improve antibody responses to tumors by enhancing antibody-dependent cellular phagocytosis. In syngeneic settings, however, broad expression of CD47 on cells of the hematopoietic lineage creates a formidable antigen sink and increases toxicity. We find that optimal synergy between anti-CD47 antibodies and several immune therapies, including anti–CTLA-4, requires near-complete blockade of CD47 in the tumor microenvironment. Thus, novel strategies to deliver localized CD47 blockade to tumors may be particularly valuable for immune therapy.

Keywords: T cell, macrophage, cancer, protein engineering, immunotherapy

Significance

Abstract

CD47 is an antiphagocytic ligand broadly expressed on normal and malignant tissues that delivers an inhibitory signal through the receptor signal regulatory protein alpha (SIRPα). Inhibitors of the CD47–SIRPα interaction improve antitumor antibody responses by enhancing antibody-dependent cellular phagocytosis (ADCP) in xenograft models. Endogenous expression of CD47 on a variety of cell types, including erythrocytes, creates a formidable antigen sink that may limit the efficacy of CD47-targeting therapies. We generated a nanobody, A4, that blocks the CD47–SIRPα interaction. A4 synergizes with anti–PD-L1, but not anti-CTLA4, therapy in the syngeneic B16F10 melanoma model. Neither increased dosing nor half-life extension by fusion of A4 to IgG2a Fc (A4Fc) overcame the issue of an antigen sink or, in the case of A4Fc, systemic toxicity. Generation of a B16F10 cell line that secretes the A4 nanobody showed that an enhanced response to several immune therapies requires near-complete blockade of CD47 in the tumor microenvironment. Thus, strategies to localize CD47 blockade to tumors may be particularly valuable for immune therapy.

Abstract

Blockade of the adaptive immune regulators CTLA-4, PD-1, and PD-L1 has shown impressive clinical efficacy across a wide range of human malignancies (1, 2). Despite the success of these adaptive checkpoint inhibitors in a subset of patients, the majority of patients still fail to achieve an adequate clinical response (1, 2). CD47 is an innate checkpoint receptor broadly expressed in normal tissues, including all cells of hematopoietic origin (3 –5). CD47 negatively regulates phagocytosis, primarily through interactions with its receptor SIRP1α on macrophages (6). CD47 is up-regulated in a wide range of human and murine malignancies. Blockade of CD47 dramatically enhances antibody-dependent cellular phagocytosis (ADCP) in vitro and substantially improves antitumor responses in vivo, particularly in xenotransplant models (6 –11). There are only a few examples of CD47 blockade in hosts with an intact immune system; how such interventions can synergize with immune checkpoint inhibition remains to be established (7 –11). We have previously demonstrated that CD47 blockade with an alpaca-derived nanobody, in combination with a PD-L1–blocking antibody (αPD-L1) and the anti-melanoma antibody TA99, acted synergistically in the poorly immunogenic B16F10 melanoma model, while completely avoiding toxicity (7); however, whether CD47 blockade would improve the antitumor activity of alternative immune checkpoint regulators, such as CTLA-4 or PD-1, which may act via distinct mechanisms, remains unknown.

The therapeutic efficacy of αPD-L1 therapy does not rely solely on ADCP, whereas αCTLA-4 antibody therapy requires engagement of the FcγR in murine models (12, 13). In the B16F10 melanoma model, the efficacy of combination therapy with αCTLA-4 antibodies and an autologous GM-CSF–secreting tumor vaccine (GVAX) is strongly correlated with therapy-induced depletion of intratumoral regulatory T cells (Tregs). This effect is completely dependent on FcγR expression by the host (12, 13). The requirement for FcγR has been proposed to be the result of ADCP of CTLA-4–expressing Tregs by macrophages in the tumor microenvironment, although alternative antitumor mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC), may also play a role (13). We hypothesized that expression of CD47 on αCTLA-4 antibody-bound cells may limit the efficacy of Treg-targeted ADCP, and that CD47 blockade may therefore improve the antitumor response.

Our previous work used A4, a high-affinity (∼10 pM) blocking nanobody raised against murine CD47. A4 potently antagonizes the CD47–SIRPa interaction, while avoiding anemia, the principal toxicity of antibody-based CD47-targeting therapeutics (7). Due to their low molecular weight (∼15 kDa), nanobodies have a short circulatory half-life. This expedites renal clearance and might compromise their efficacy in blocking CD47 in vivo (7, 14 –16). To circumvent this pharmacokinetic limitation, we took two different approaches, generating an A4-IgG2aFc fusion protein and a B16 cell line that constitutively secretes A4. The A4-Fc fusion showed dose-limiting toxicity, whereas secretion of A4 by B16 within the tumor microenvironment achieved near-complete CD47 blockade and improved responses to the anti-melanoma antibody TA99. CD47 blockade within the tumor microenvironment also enhanced the efficacy of an anti-melanoma vaccine in combination with αCTLA-4 treatment. Localized CD47 blockade within the tumor microenvironment is therefore sufficient to mediate a therapeutic effect. Furthermore, our results highlight the dichotomy between αPD-L1 and αCTLA-4 responses when combined with CD47 blockade, and establish a valuable preclinical model of αCD47 toxicity in vivo.

Acknowledgments

We thank Monique J. Kauke and K. Dane Wittrup for the TA99 antibody, Mohammad Rashidian for helpful discussions, Elisa Bello for technical assistance, and the staff of the flow cytometry facility at Whitehead Institute. Funding was provided by the Ludwig Cancer Research Postdoctoral Fellowship and the Claudia Adams Barr Program for Innovative Cancer Research (to J.R.I.); Maag, Lever, Darm Stichting, and the Bekker-La Bastide Fonds (to O.S.B.); National Institutes of Health (NIH) Training Grant 5T32 {"type":"entrez-nucleotide","attrs":{"text":"AI072905","term_id":"3399099","term_text":"AI072905"}}AI072905 and a PhRMA Translational Medicine and Therapeutics Postdoctoral Fellowship (to J.T.S.); the Melanoma Research Alliance (to S.K.D.); NIH Grant R01 CA177684, the Howard Hughes Medical Institute, and the Ludwig Foundation (to K.C.G.); NIH Grants R01 AI087879-06, DP1 GM106409-03, and R01 GM100518-04 and the Lustgarten Foundation (to H.L.P.); and NIH Training Grant 1F32CA210568-01 and NIH Center for the Study of Inflammatory Bowel Disease Pilot Grant DK043351 (to M.D.).

Acknowledgments

Footnotes

Conflict of interest statement: K.C.G. is a cofounder of Alexo, a biotechnology company focused on the clinical translation of anti-human CD47 antagonists.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1710776114/-/DCSupplemental.

Footnotes

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