Novel SIRPα Antibodies That Induce Single-Agent Phagocytosis of Tumor Cells while Preserving T Cells

Abstract

The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.

Conflict of interest statement

All authors are employees of Arch Oncology, Inc., and have stocks and/or stock options in Arch Oncology, Inc. G.A., B.J.C., R.J.P., and R.R.H. have filed a patent application that describes the invention of the anti-SIRPα and Abs SIRP-1 and SIRP-2. B.J.C. and R.J.P. have received and filed further patent applications that describe the invention of the anti-CD47 Ab AO-176. The other authors have no financial conflicts of interest.

Copyright © 2021 by The American Association of Immunologists, Inc.

Figures

Graphical abstract

FIGURE 1.

SIRP-1 and SIRP-2 bind to recombinant and cell-expressed human SIRPα. (A) SIRP-1 and SIRP-2 Abs were screened by solid-phase ELISA for binding to human SIRPαV1 (hSIRPαV1) and compared with competitor benchmarks KWAR23 and 18D5. (B) At 10 μg/ml SIRP-1, SIRP-2 and KWAR23 bound to cell-expressed SIRPαV1 and SIRPαV2 on promonocytic cell lines U937 and THP-1, respectively, whereas benchmark 18D5 only bound SIRPαV1. (C) SIRP-1 and SIRP-2 bound to human moMΦs from most-common allelic groups. All panels show mean ± SEM; a representative minimum n = 2 is shown.

FIGURE 2.

SIRP-1 and SIRP-2 Abs induce single-agent phagocytosis of a range of cancer cell lines. (A–C) SIRP-1 and SIRP-2 induced human moMΦs to phagocytose a panel of hematopoietic and solid tumor cell lines. CFSE-labeled (A, C, and D–F) or pHrodo Red–labeled (B) target cells were added to moMΦs and phagocytosis measured as the percentage of moMΦs that had engulfed target cells (CFSE+ or pHrodo+) of the total macrophage population. Single-agent phagocytosis of Jurkat T-ALL cells with SIRP-1 (D) and SIRP-2 used at 10 μg/ml (E) is seen in human moMΦs of all three most common allelic groups of SIRPα. The crosses in (A)–(D) indicate mIgG1 control at 10 μg/ml. (F) SIRP-1 and SIRP-2 did not induce moMΦ phagocytosis of autologous PBMCs in contrast to Jurkat T-ALL cells. All panels show mean ± SEM; a representative minimum n = 2 is shown.

FIGURE 3.

SIRP-1 and SIRP-2 binding to tumor cell lines correlates to their expression of SIRPα/SIRPγ. (A) Expression of CD47, SIRPα/β, and SIRPγ by cancer cell lines used in phagocytosis assays. Representative histograms from one of two technical replicates from one of two independent experiments are shown. (B) The binding of SIRP-1 and SIRP-2 at 10 μg/ml correlates well with SIRPα/β or SIRPγ expression seen by commercial anti-SIRP Abs. Data are expressed as mean ± SEM, representative of n = 2.

FIGURE 4.

SIRP-1 and SIRP-2 act via distinct mechanism of action: internalization and conformational change/declustering of SIRPα. (A) Blocking soluble CD47 binding to human SIRPαV1 (hSIRPαV1) was assessed by ELISA. Reduction of 20 μg/ml soluble CD47 binding to cell-expressed SIRPαV1 and SIRPαV2 by 10 μg/ml SIRP Abs was assessed using (B) promonocytic cell lines U937 and THP-1 and (C) moMΦs from various donor genotypes. (D) SIRP-1, but not mIgG1 control, 18D5, KWAR23, or SIRP-2 at 10 μg/ml reduced surface SIRPα levels in moMΦs in a time-dependent manner. The cells were incubated with Abs for 2 h at 4°C or 2, 4, 6, or 24 h at 37°C. (E) A total of 10 μg/ml SIRP-1 and, to a lesser extent, SIRP-2 caused Ab internalization in a time-dependent manner when measured by the uptake of pHrodo dye–Ab conjugates. (F) SIRPα binds CD47 most efficiently when clustered. When SIRPα Ab clone SE5A5, labeled with PE or allophycocyanin in equimolar concentrations, is added to cells, FRET is observed if SIRPα molecules and Abs bound to them are in close proximity. When a noncompeting anti-SIRP Ab is added that does not alter SIRPα architecture, no reduction in FRET is seen. No FRET is observed when a noncompeting anti-SIRP Ab that disables SIRPα from clustering is added. No FRET also occurs if binding of a noncompeting SIRPα mAb induces a conformation change in SIRPα that increases the distance between SE5A5 epitopes. (G) Treatment of moMΦs with 10 μg/ml SIRP-2, but not SIRP-1, for 2 h under the same conditions as in phagocytosis assays reduced the FRET efficiency between anti-SIRP Abs SE5A5-PE and SE5A5–allophycocyanin, indicating SIRPα conformational change/receptor declustering upon SIRP-2 treatment. (H) Addition of 10 μg/ml blocking Abs against human CD16, CD32, and CD64 inhibits SIRP-1– and SIRP-2–induced phagocytosis of Jurkat and DLD-1 cells by human moMΦs. (I) Function-blocking Abs against CD32 at 10 μg/ml, but not CD16 or CD64, inhibit SIRP-1– and SIRP-2–mediated Jurkat phagocytosis of moMΦs. All panels show mean ± SEM; a representative minimum n = 2 is shown. Statistical differences compared with the mIgG1 control are indicated. *p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.0001.

FIGURE 5.

SIRP-1 and SIRP-2 do not inhibit T cell proliferation. (A) Binding of 10 μg/ml Abs to cell-expressed SIRPγ was assayed using T-ALL cell line Jurkat and compared with benchmarks 18D5 and KWAR23. (B) Binding to naive or 72-h-activated human T cells was determined by cell-based binding assays and compared with SIRPγ-specific Ab clone LSB2.20. (C) A total of 10 μg/ml SIRP-1 or SIRP-2 did not inhibit T cell proliferation in an allogeneic T cell/dendritic cell assay over the course of 6–7 d. Each data point represents mean from a separate donor (shown in different shapes), constituting an average of six technical replicates within a minimum of two independent replicates. The asterisks (*) indicate statistical differences compared with the mIgG1 control group. Unless otherwise stated, all panels show mean ± SEM; a representative minimum n = 2 is shown. *p < 0.05.

FIGURE 6.

SIRP-1 and SIRP-2 combine with tumor-opsonizing Abs to potentiate phagocytosis of cancer cell lines. (A) SIRP-1 and (B) SIRP-2 combine with anti–PD-1 mAb avelumab, anti–epidermal growth factor receptor mAb cetuximab, and anti-CD20 mAb rituximab to potentiate human moMΦ phagocytosis of ES-2, DLD-1, and Raji cell lines, respectively. (C) SIRP-1 also combines with anti-CD47 mAb AO-176 to potentiate phagocytosis of Jurkat cells. All panels show mean ± SEM; a representative minimum n = 2 is shown.