Antibodies targeting human IL1RAP (IL1R3) show therapeutic effects in xenograft models of acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell-mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

Keywords: AML; IL1RAP; antibody; immunotherapy; leukemia.

Fig. 1.

Treatment with mAb81.2 has antileukemic effects and prolongs survival in the MA9Ras xenotransplantation model. (A) Expression of IL1RAP on the surface of human MA9Ras AML cells (red, isotype control; blue, anti-IL1RAP) using flow cytometry analysis. (B and C) Frequency of leukemic cells and platelet counts in PB 35 d after transplantation of MA9Ras AML cells into NOD/SCID mice treated with the IL1RAP-targeting antibody mAb81.2 or a mIgG2a isotype control antibody (means ± SD; n = 7 in both groups). (D) Leukemic cell frequency in PB 62 d after transplantation (means ± SD). (E) Overall survival rates with mAb81.2 (median 75 d) compared with isotype control (median 64 d). P value was calculated by using the Mantel Cox log rank test. (F and G) Leukemic cell frequency in BM and spleen (means ± SD). (H) H&E-stained and human CD45-immunostained BM sections from representative isotype control and mAb81.2-treated mice. (Scale bars, 300 μm.) Unless otherwise stated, P values were calculated with Student’s t test.

Fig. S1.

Treatment with mAb81.2 reduces the leukemic burden in the MA9Ras xenotransplantation model. (A) MA9Ras cell engraftment in BM and spleen of unconditioned NOD/SCID mice without antibody treatment (means ± SD; n = 9). (B) Spleen weight in mice engrafted with MA9Ras and treated with mAb81.2 or isotype control (means ± SD; n = 7 in both groups). (C) H&E-stained and human CD45-immunostained spleen sections from representative control and mAb81.2-treated mice. (Scale bars, 300 μm.) P values were calculated with Student’s t test.

Fig. S2.

In vivo results using mAb3F8 and mAb81.2 dose titration. (A and B) PB leukemic cell frequency (A) and platelet counts (B) at 35 d after transplantation in NOD/SCID mice engrafted with MA9Ras cells and treated with mAb81.2 or mAb3F8 or a corresponding isotype control antibody (means ± SD; n = 3–7 per group). (C) Overall survival rates after treatment with mAb81.2 (median 58 d), mAb3F8 (median 57 d), and control (median 51 d) (P < 0.0001; n = 7 for all groups). P values were calculated by using the Mantel Cox log rank test. (D and E) Leukemic cell frequency in BM (D) and spleen (E) (means ± SD). (F and G) Spleen weight (F) and estimated leukemic spleen burden (G) calculated as frequency of leukemic cells multiplied by spleen weight (means ± SD). Unless otherwise stated, P values were calculated with Student’s t test.

Fig. 2.

Antileukemic in vivo effect on MA9Ras cells depends on murine effector cells. (A) Fold expansion of MA9Ras cells in suspension cultures with addition of 10 μg/mL IL1RAP-targeting antibodies mAb81.2 and mAb3F8 or an isotype control antibody (means ± SEM from four experiments with technical duplicates). (B) Relative levels of viable, apoptotic, and necrotic MA9Ras cells after treatment with 10 μg/mL mAb81.2, mAb3F8, or an isotype control antibody. Staurosporine treatment (STP) was used as positive control for apoptosis (means ± SEM from three experiments with technical duplicates). (C and D) NSG mice were engrafted with MA9Ras cells and treated with mAb81.2 or an isotype control antibody (n = 5 in both groups). Graphs show BM (C) and spleen (D) leukemic cell frequency at death 39 d after transplantation (means ± SD). (E) IL-1 activates NF-kβ in the HEKblue IL1R1 reporter cell line. The graph shows the NF-kβ activation measured by absorbance in the presence of IL-1 upon addition of mAb81.2 or mAb3F8 or an isotype control antibody (means ± SEM from technical duplicates; one representative experiment out of three). (F) Absorbance of NF-kβ activation in the HEKblue IL1R1 reporter cell assay in the absence of IL-1 with IL1RA included as a control (one representative experiment out of three). P values were calculated with Student’s t test.

Fig. S3.

MA9Ras cells do not respond to IL-1 in suspension cultures. (A) Fold expansion of MA9Ras cells without IL-1 or with 0.4 or 10 ng/mL IL-1 (means ± SEM from four experiments with technical duplicates). (B) Relative levels of viable, apoptotic, and necrotic MA9Ras cells in the absence or presence of IL-1 (means ± SEM from two experiments with technical duplicates). P values were calculated with Student’s t test.

Fig. 3.

Targeting of IL1RAP results in reduced frequencies of primary human AML cells in vivo. (A) Frequency of leukemic cells in BM of NOD/SCID mice engrafted with sample AML2 and treated with mAb81.2 (n = 10) or isotype control antibody (n = 8) at death 28 d after transplantation (Left; means ± SD), and flow cytometry plots of representative isotype control (Center) and mAb81.2-treated (Right) mice. (B) Frequency of leukemic cells in BM (Left) and spleen (Right) of NSGS mice engrafted with sample AML4 and treated with mAb81.2 or isotype control antibody (n = 6 in both groups) at death 56 d after transplantation (means ± SD). P values were calculated with Mann–Whitney test.

Fig. S4.

Treatment with mAb81.2 has an antileukemic effect in mice engrafted with primary human AML cells. (A) Flow cytometry pseudocolor plots from analyzed BM of all NOD/SCID mice transplanted with sample AML2 and treated with mAb81.2 or isotype control antibody. CD45⁺CD33⁺ cells were considered human leukemic cells. Two nontransplanted and untreated mice were included in the analysis as specificity controls for the CD45 and CD33 antibodies. (B) Frequency of leukemic cells in BM (Upper) and spleen (Lower) of NSGS mice transplanted with sample AML1 at death 57 d after transplantation. In BM, three of five mAb81.2-treated mice were below the median of the control (n = 5 in both groups), and in spleen, all five were (medians ± interquartile range). (C) Frequency of leukemic cells of NSG mice transplanted with sample AML3 at death 35 d after transplantation. In BM (Upper) and spleen (Lower), all six mAb81.2-treated mice were below the median of the control (n = 6 in both groups; medians ± interquartile range). P values were calculated with the Mann–Whitney test.

Fig. 4.

IL1RAP-targeting antibodies mediate ADCC with human NK cells, and mAb3F8 blocks proliferation of AML cells. (A) Frequency of dead MA9Ras cells with human NK cells in an ADCC assay (means ± SEM from technical duplicates; one representative experiment out of three). (B) Relative amount of human phagocytic macrophages in an ADCP assay with MA9Ras target cells. Data were normalized to 1.0 at conditions without antibody addition (means ± SEM from four experiments with technical duplicates in which the typical frequency of MA9Ras⁺ macrophages were 20–50% without the addition of antibody). (C) Frequency of dead MA9Ras cells in a CDC assay using human normal serum (NS) or heat-inactivated serum (HIS) (means ± SEM from three experiments with technical duplicates). (D) Proliferation rate of three primary human AML samples with IL-1, as measured by incorporation of EdU. Data normalized to proliferation without IL-1 set to 1 (means ± SEM from one experiment with technical duplicates or triplicates). (E) Proliferation rate of samples AML5 and AML6 in the presence of IL-1 and mAb3F8 or isotype control antibody measured by EdU incorporation. Data were normalized to conditions without IL-1 set to 1 as indicated by the dotted line (means ± SEM from one experiment with technical duplicates or triplicates). (F) Total expansion of sample AML6 after 6 d of culture, with the dotted line indicating the number of cells in conditions without IL-1 (means ± SEM from one experiment with technical triplicates). (G) The effect of mAb3F8 or isotype control antibody on the IL-1–induced proliferation of primary human AML cells as measured by expression of the proliferation marker Ki-67. Data were normalized to conditions with IL-1 only set to 1 (means ± SEM from four AML patients with technical triplicates). P values were calculated with Student’s t test.

Fig. S5.

IL1RAP-targeting antibodies mAb81.2 and mAb3F8 mediate ADCC with human NK cells on human AML cell lines. Frequency of dead target cells using MonoMac6 (A), EOL1 (B), and OCI-AML1 cells (C) (means ± SEM from technical duplicates; one representative experiment out of two).

Fig. S6.

Immunostaining with mAb3F8 on 30 normal tissues reveals staining in only five. Displayed are the tissues that showed some degree of staining with 0.1 μg/mL mAb3F8 in at least one individual out of three analyzed. (Scale bars, 100 μm.) A mIgG1 antibody at a concentration of 0.1 μg/mL was used as isotype control. IL1RAP high-expressing SKMEL5 cells were used as positive control, and IL1RAP low-expressing KG1 cells were used as negative control.

Fig. S7.

Weak IL-1 signaling of human cells in response to murine IL-1. The graph shows the NF-kβ activation in the HEKblue IL1R1 reporter cell line measured by absorbance in presence of increasing concentrations of murine or human IL-1.