Fig 1.
ALX148 binds human CD47 and prevents its interaction with SIRPα.
(A) Structure of ALX148 molecule. Lines indicate disulfide bonds between ALX148 monomers. (B) Flow cytometry analysis of ALX148 binding (solid white histograms), negative control protein ALX180 binding (dashed histograms), or background fluorescence of unstained cells (filled histograms) for indicated human and mouse tumor cell lines. Cell number is normalized to mode. Results are representative of at least three independent experiments. (C) Flow cytometry analysis of wild-type SIRPα binding to Jurkat cells. Geometric mean fluorescence intensity is indicated on the y-axis. Cells were pre-incubated with proteins indicated on the x-axis prior to SIRPα incubation. Bkgd = background fluorescence in the absence of labeled wild-type SIRPα. Error bars indicate standard deviation of triplicates. Results are representative of three independent experiments.
Table 1.
Apparent affinity of ALX148 for CD47 of various species.
Table 2.
Apparent affinity ALX148 for human Fcγ receptors.
Fig 2.
ALX148 does not affect hematologic parameters in vitro or in vivo.
(A) Hemagglutination of human erythrocytes incubated with titrations of ALX148, the indicated antibodies against CD47, or PBS. Results shown are from the same experiment and are representative of three independent experiments. (B) CD-1 mice were administered 30mg/kg ALX377 or ALX148 intravenously. Each time point consists of 4–5 mice. RBC, platelet (PLT) and WBC counts for each mouse were normalized to their predose values. The mean normalized values for all mice at each time point and standard error mean (SEM) are indicated. ****p<0.0001, ***p<0.001. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
Fig 3.
ALX148 enhances antibody-dependent cellular phagocytosis.
In vitro phagocytosis experiments with human monocyte-derived macrophages and tumor cell lines. Percent of macrophages that engulfed tumor cells is indicated on the y-axis. On y-axis, open circle indicates background phagocytosis observed with isotype control antibody and open square indicates cells treated with antitumor antibody alone. Antitumor antibody concentrations were constant and ALX proteins were titrated. Cells were treated with antitumor antibody plus ALX148 (solid black line) or antitumor antibody plus ALX180 (solid grey line). Cells were then treated with control antibody plus ALX148 (dotted black line) or control antibody plus ALX180 (dotted grey line). Tumor cell line and antibody combinations were (A) OE19 cells and trastuzumab, (B) DLD-1 GFP Luciferase cells and cetuximab, (C) MM1.R cells and daratumumab, and (D) Daudi cells and obinutuzumab. Error bars represent standard deviation of triplicates. For all antitumor antibodies tested, 100 nM ALX148 significantly enhanced antibody-dependent cellular phagocytosis (****p<0.0001 one-way ANOVA, Tukey-Kramer) compared to antibody alone. Results are representative of at least three independent experiments.
Fig 4.
ALX148 enhances antitumor therapy in vivo.
(A) Z138 B-cell mantle cell lymphoma and (B) OE19 gastric tumor cells were implanted subcutaneously on the right flank of NOD-SCID mice. Mice with established tumors (average of 240 mm3 for Z138 and 140 mm3 for OE19) were randomized and treated i.p. with vehicle, ALX148, obinutuzumab for Z138 or trastuzumab for OE19 or combination of ALX148 and anti-cancer antibody. Graphs show mean tumor growth ± SEM of n = 10 mice. ALX148 in combination with obinutuzumab or trastuzumab showed significant inhibition of tumor growth as compared to obinutuzumab or trastuzumab monotherapy, **p<0.01, day 32 and **p<0.01, day 36 (two-tailed student’s t-test), respectively. (C) MC38 and (D) CT26 colon carcinoma cells were implanted subcutaneously on the right flanks of C57BL/6 and BABL/C mice, respectively. When tumors reached an average of 70–75 mm3, mice were randomized into groups and treated. (C) Mice bearing MC38 tumors were treated with PBS, ALX148, anti-PD-L1 or ALX148 + anti-PD-L1. Graphs show tumor growth ± SEM of n = 10 mice (left panel) and survival curves (right panel). ALX148 + anti-PD-L1 shows significant inhibition of tumor growth as compared to anti-PD-L1 monotherapy, (*p< 0.05, day 24 two-tail student’s t-test). Survival of ALX148 + anti-PD-L1 and anti-PD-L1 alone are significantly increased compared to PBS alone (***p<0.001 and **p<0.01, respectively, log-rank (Mantel-Cox) test). (D) Mice bearing CT26 tumors were treated with PBS, ALX148, anti-PD-1 or ALX148 + anti-PD-1. Graphs show tumor growth ± SEM of n = 9 mice (left panel) and survival curves (right panel). ALX148 + anti-PD-1 shows significant inhibition of tumor growth as compared to anti-PD-1 monotherapy, (*p< 0.05, day 25 two-tail student’s t-test) and significant increased survival as compared to PBS alone (*p<0.05, log-rank (Mantel-Cox) test). ns, not significant.
Fig 5.
ALX148 reduces myeloid-driven immune suppression in tumor.
(A) Schematic of dosing schedule and tissue harvest for immune phenotyping. CT26 colon carcinoma cells were implanted subcutaneously in BALB/c mice and treated on day 10 post-implantation with a single dose of PBS, ALX148, anti-PD-1 or ALX148 + anti-PD-1. 10 days post-treatment, spleen and tumor were harvested for immune phenotyping. (B) Ratio of M1/M2 TAMs in tumor. M1 is defined as CD45+CD11b+CD38+EGR2- and M2 is defined as CD45+CD11b+CD38-EGR2+. Results are representative of two independent experiments of n = 5–7 mice/group. (C) Percent mMDSC in tumor. mMDSC is defined as CD45+CD11b+Ly6ChiMHCII-. Results are representative of two independent experiments of n = 5–6 mice/group. *p<0.05, **p<0.01. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
Fig 6.
ALX148 induces splenic dendritic cell activation.
Spleen of CT26 tumor-bearing mice, 10 days post single-dose of PBS, ALX148, anti-PD1 or ALX148 in combination with one dose of anti-PD1. (A) Percent splenic CD8+ DC of CD45+ cells. (B) Percent splenic CD8- DCs of CD45+ cells. (C) Geometric MFI of CD86+ splenic CD8+DCs (left panel) and CD8-DCs (right panel). Results are representative of at least two independent experiments of n = 5–7 mice/group. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
Fig 7.
ALX148 activates adaptive immune response in the spleen.
Spleen of CT26 tumor-bearing mice, 10 days post single-dose of PBS, ALX148, anti-PD-1 or ALX148 in combination with one dose of anti-PD-1. (A-B) Percent effector memory (CD44+CD62L-) and central memory (CD44+CD62L+) CD4+ T cells. (C-D) Percent effector memory and central memory CD8+ T cells. (E) KLRG1 expression on CD8+ T cells. (F) Percent intracellular IFNγ expressing CD8+ T cells following ex vivo stimulation of splenocytes with PMA/ionomycin and Golgi-Stop for 4 hours. (G) Percent intracellular granzyme B expressing CD8+ T cells. (H) Percent CD8+AH1-tet+ T cells following ex vivo stimulation of splenocytes with 10 μg/mL AH1 peptide for 4 hours. Results are representative of 1–3 independent experiments of n = 4–6 mice/group. **p<0.01, ***p<0.001 and ****p<0.0001. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
Fig 8.
Combination of ALX148 and anti-PD-1 reduces suppressive tumor microenvironment and activates adaptive immune response in CT26 tumor.
(A) Percent FOXP3+CD25+ in tumor. (B) Percent Ki67+ Tregs in tumor. (C) Percent AH1-tet+CD8+ T cells. Following antibody treatment, ex vivo tumor-derived single cell suspension stimulation with AH1 peptide at 10 μg/mL. (D) Percent intracellular IFNγ expressing CD8+ T cells (left panel) and percent IFNγ+ of AH1-tet+CD8+ T cells (right panel). Following antibody treatment, ex vivo tumor-derived single cell suspension were stimulated in the presence of PMA/ionomycin or AH1 peptide at 10 μg/mL. Results are representative of 1–3 independent experiments of n = 5–6 mice/group.
Fig 9.
Combination of ALX148 and anti-PD-L1 enhances adaptive immune response in MC38 tumor model.
(A) MC38 colon carcinoma cells were implanted subcutaneously in C57BL/6 mice and treatment was initiated four days post-implantation. Treatment consisted of PBS, ALX148, anti-PD-L1 or ALX148 + anti-PD-L1. ALX148 was dosed twice at 30 mg/kg, ten days agart and anti-PD-L1 was dosed twice, one per week. On day 17 post-tumor challenge, tumors were harvested for immune phenotyping. (B-C) Percent tumor infiltrating CD8+ and CD4+ T cells + (D-E) Percent effector memory (CD44+CD62L-) CD8 and CD4 in tumor (F) Percent IFNγ expression in CD8+ T cells (G) Percent Granzyme B expression in CD8+ T cells. All values are reported as percent of CD45 with 5–6 mice/group. ***p<0.001 and ****p<0.0001. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
Fig 10.
Higher doses of ALX148 saturate CD47 antigen sink and drive tumor penetration.
(A) ALX148 binding to the indicated cells from naïve mice. (B) Serum level of ALX148 at the indicated times after administration of 10 mg/kg (grey column) or 30 mg/kg (black column). (C-E) CD47 occupancy in splenic CD4+ T cells (black columns), tumor-infiltrating CD4+ T cells (grey columns), or tumor cells (open columns) (C) one, (D) four, or (E) eight days after administration of the indicated dose. Mean of five mice is shown and error bars represent standard deviation. Results are representative of two independent experiments.