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. 2017 Dec 5;114(49):E10578-E10585.
doi: 10.1073/pnas.1710877114. Epub 2017 Nov 20.

Anti-SIRPα antibody immunotherapy enhances neutrophil and macrophage antitumor activity

Nan Guo Ring  1   2 , Dietmar Herndler-Brandstetter  2 , Kipp Weiskopf  1 , Liang Shan  2 , Jens-Peter Volkmer  1 , Benson M George  1 , Melanie Lietzenmayer  2 , Kelly M McKenna  1 , Tejaswitha J Naik  1 , Aaron McCarty  1 , Yunjiang Zheng  2 , Aaron M Ring  2 , Richard A Flavell  3   4 , Irving L Weissman  5
Affiliations

Anti-SIRPα antibody immunotherapy enhances neutrophil and macrophage antitumor activity

Nan Guo Ring et al. Proc Natl Acad Sci U S A. .

Abstract

Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-α (SIRPα) is a myeloid-specific immune checkpoint that engages the "don't eat me" signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human SIRPA knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt's lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRPα antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRPα blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.

Keywords: SIRPA; bispecific antibody; cancer immunotherapy; humanized mouse; myeloid cells.

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Conflict of interest statement

Conflict of interest statement: K.W., A.M.R., and I.L.W. are shareholders of Forty Seven, Inc., and have filed a patent application that describes the human anti-SIRPα antibody KWAR23. I.L.W. is co-inventor of the patent, and co-founder and director of the company that has licensed the antibody.

Figures

Fig. 1.
Fig. 1.
Crystal structure and binding characteristics of the anti-human SIRPα antibody KWAR23. (A) Crystal structure of the KWAR23/SIRPα complex. (B) Overlay of the KWAR23/SIRPα and CD47/SIRPα complexes. KWAR23 Fab is depicted as ribbons, and the CD47/SIRPα complex is depicted as transparent surfaces. (C) Competition assay demonstrating KWAR23 blockade of CD47 binding to THP-1 cells. IC50 = 40.82 ng/mL. SD is shown. (D and E) Surface plasmon resonance analysis of the binding kinetics of KWAR23 Fab fragments to the two most prevalent alleles of the SIRPα IgV domain. (F) Binding of KWAR23 to human B cells, T cells, neutrophils, and monocytes. Koff, rate of dissociation; Kon, rate of association; MFI, mean fluorescence intensity.
Fig. 2.
Fig. 2.
Anti-human SIRPα antibody KWAR23 augments phagocytosis of tumor cells in vitro. (A) Schematic of myeloid cell-mediated phagocytosis of tumor cells by anti-SIRPα antibody and tumor-opsonizing antibodies (e.g., the anti-CD20 antibody rituximab). (B) Representative flow cytometry plot of a phagocytosis assay using human CD206+ macrophages and CFSE-labeled tumor cells. (C) KWAR23 augments macrophage phagocytosis of Burkitt’s lymphoma cells following treatment with two different CD20-targeting antibodies, rituximab and obinutuzumab. (D) KWAR23 increases monocyte phagocytosis of Burkitt’s lymphoma cells opsonized by CD20-targeting antibodies across many Ig isotypes (n = 4). (E) KWAR23 enhances macrophage phagocytosis of DLD-1 colorectal adenocarcinoma cells opsonized by two different EGFR-targeting antibodies, cetuximab and panitumumab. (F) KWAR23 increases macrophage phagocytosis of four colon adenocarcinoma lines, regardless of EGFR pathway mutation status (n = 4). wt, wild type. Mean ± SEM is shown. *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig. 3.
Fig. 3.
KWAR23 augments macrophage- and neutrophil-mediated phagocytosis of tumor cells in vitro. (A) Phagocytosis of Burkitt’s lymphoma cells by human macrophages following treatment with rituximab (green) or rituximab + KWAR23 (blue). Rituximab was provided at different concentrations as indicated on the abscissa (n = 4). (B) Phagocytosis of SK-BR-3 breast cancer cells by human macrophages following treatment with trastuzumab (green) or trastuzumab + KWAR23 (blue; n = 4). (C) Phagocytosis of colorectal adenocarcinoma (DLD-1) cells by human macrophages following treatment with cetuximab (green) or cetuximab + KWAR23 (blue; n = 4). (D) Representative flow cytometry plot of a cytotoxicity assay using human neutrophils and CFSE-labeled tumor cells. Dead cells were stained with DAPI. (E) Neutrophil-mediated killing of Burkitt’s lymphoma cells following treatment with rituximab (green) or rituximab + KWAR23 (blue; n = 4). (F) Neutrophil-mediated killing of SK-BR-3 breast cancer cells following treatment with trastuzumab (green) or trastuzumab + KWAR23 (blue; n = 4). Mean ± SEM is shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 4.
Fig. 4.
Therapeutic activity of KWAR23 in SRG mice. (A) Schematic of the tumor engraftment and immunotherapy in SRG mice. (B) Tumor volume and weight in SRG mice treated with PBS (n = 8), rituximab (n = 3), KWAR23 (n = 4), or rituximab + KWAR23 (n = 14). The color of the stars indicates comparison of the corresponding group with the rituximab + KWAR23 group. (C) Individual tumor growth curves in SRG mice treated with PBS (n = 9) or rituximab + KWAR23 (n = 12). (D) SRG mice that established large tumors (170–300 mm3) were treated for 8 d with rituximab (n = 4) or rituximab + KWAR23 (n = 4). (E) Frequency of tumor-infiltrating mouse CD45+ cells in SRG mice treated with PBS (n = 9) or rituximab + KWAR23 (n = 8). (F) Frequency of Ly6Chi Ly6G− macrophages and Ly6G+ Ly6Cdim neutrophils in the blood and tumor of SRG mice treated with PBS (n = 8) or rituximab + KWAR23 (n = 9). (G) Immunofluorescence images showing the infiltration of human Burkitt’s lymphoma xenografts (human CD45+) in rituximab + KWAR23-treated SRG mice by F4/80+ macrophages and Ly6G+ neutrophils. (H) Frequency of dendritic and myeloid cells in the blood and tumor of SRG mice treated with PBS (n = 8) or rituximab + KWAR23 (n = 9). Mean ± SEM is shown. *P < 0.05; **P < 0.01; ****P < 0.0001 (unpaired two-tailed Student’s t test).
Fig. 5.
Fig. 5.
KWAR23 augments macrophage- and neutrophil-mediated destruction of human tumor xenografts in SRG mice. (A) Schematic of the tumor engraftment, depletion of myeloid cell subsets, and immunotherapy in SRG mice. (B) Impact of the depletion of myeloid cell subsets on tumor growth in SRG mice treated with PBS (n = 8), rituximab + KWAR23 + clodronate (n = 12), rituximab + KWAR23 + anti-Ly6G (n = 8), and rituximab + KWAR23 (n = 9). The color of the stars indicates comparison of the PBS group with the corresponding treatment group. (C) Tumor growth in SRG mice treated with PBS (n = 7), vorsetuzumab (n = 3), vorsetuzumab + KWAR23 (n = 5), or bispecific anti-CD70/KWAR23 (n = 6). The color of the stars indicates comparison of the PBS group with the corresponding treatment group. ns, not significant. (D) Phagocytosis of four different renal carcinoma cells by human macrophages following treatment with vorsetuzumab, vorsetuzumab + KWAR23, bispecific anti-human CD70/KWAR23, and anti-human CD47 antibody (n = 4). Mean ± SEM is shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (unpaired two-tailed Student’s t test).
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