Activity of 8F4, a T-cell receptor-like anti-PR1/HLA-A2 antibody, against primary human AML in vivo

Abstract

The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a high-affinity T-cell receptor-like murine monoclonal antibody, 8F4, that binds to the PR1/HLA-A2 complex, mediates lysis of AML and inhibits leukemia colony formation. Here, we explored whether 8F4 was active in vivo against chemotherapy-resistant AML, including secondary AML. In a screening model, coincubation of AML with 8F4 ex vivo prevented engraftment of all tested AML subtypes in immunodeficient NSG (NOD scid IL-2 receptor γ-chain knockout) mice. In a treatment model of established human AML, administration of 8F4 significantly reduced or eliminated AML xenografts and extended survival compared with isotype antibody-treated mice. Moreover, in secondary transfer experiments, mice inoculated with bone marrow from 8F4-treated mice showed no evidence of AML engraftment, supporting the possible activity of 8F4 against the subset of AML with self-renewing potential. Our data provide evidence that 8F4 antibody is highly active in AML, including chemotherapy-resistant disease, supporting its potential use as a therapeutic agent in patients with AML.

Figure 1. 8F4 Ex vivo treatment prevents engraftment of primary AML in NSG mice

AML cells were incubated with 20μg 8F4 or isotype control mouse IgG2a monoclonal antibody (IgG) and intravenously transplanted in sublethally-irradiated NSG recipient mice. At indicated time points tissues were analyzed for the presence of leukemia cells. (A) Representative flow cytometry dot plots depict leukemia cells in tissues of IgG-treated mice transplanted with four HLA-A2+ (UPN1–4) and one HLA-A2- (UPN9) patient samples. The percentages of human cells (hu CD45⁺/mo CD45⁻) are shown within the gate in each plot. (B) Representative H&E stained bone marrow (upper panels) and liver (lower panels) slides from mice that were transplanted with UPN1 patient sample. Left panels - AML infiltration is seen in tissues of control mice following injection of AML cells treated with control IgG isotype antibody. Right panels - no AML infiltration is seen in corresponding tissues of mice injected with AML cells that were treated with 8F4.

Figure 2. 8F4 treatment reduces established leukemia in NSG mice

(A) The schedule of 8F4 treatment in mice transplanted with primary AML cell from patients. (B) Leukemia engraftment in mice, transplanted with four HLA-A2+ patient samples (UPN1, UPN2, UPN7, and UPN8) and one HLA-A2- patient sample (UPN10) was monitored by flow cytometry analysis of human cells in the peripheral blood (left panels). Increasing percentage of human AML cells in the PB of mice pre-treatment shows established and growing leukemia. Mice with established leukemia were treated with 8F4 or control antibody (IgG). Horizontal capped line in each graph in the left panel indicates the treatment period and schedule. Right panels show percent bone marrow engraftment at time of sacrifice. To confirm the BM engraftment of each sample, mice from each leukemia group were sacrificed and analyzed before treatment (Pre Rx). The percentages of human cells are shown. Data shown as mean percent human cells ± SEM of the chimerism for each treatment group (n=2–5); * p< .05; ** p< .005. We were unable to perform statistical testing on UPN 2 and 8 samples because of sample numbers in the group.

Figure 3. 8F4 treatment prolongs survival of NSG mice with established leukemia

AML from UPN8 was expended in vivo by serial subsequent transplants of spleen cells in NSG recipients. Transplanted mice were treated with 8F4 or control IgG starting on day 7. (A) Kaplan-Meier survival curves show longer median survival in 8F4 treated mice (median 50 days, n=10) in comparison with IgG treated mice (34 days, n=9). (B) AML was present in the peripheral blood only in IgG-treated animals at week 4. (C) Spleens were enlarged only in IgG-treated mice. (B-C) Each data point represents one mouse; mean ± SEM for each group is also presented; **** p< .0001. (D) Representative H&E-stained sections of mice, treated with 8F4 or IgG. Spleen: the extramedullary hematopoiesis and all other tissue structures present in 8F4-treated mice are completely effaced by infiltration of AML in the control mice. Lung: AML cells are diffusely infiltrated into the alveolar walls of IgG treated mice, but not in the 8F4-treated mice. Stomach: AML infiltrated diffusely between the glands of gastric mucosa and in the sub-mucosa of glandular stomach of IgG-treated mice, but not in the stomach of 8F4-treated mice. Liver: the AML cells infiltrated severely into the portal triads of liver (black arrows) of IgG-treated mice, and there was minimal or no infiltration of AML in the liver of 8F4-treated mice.

Figure 4. 8F4 treatment depletes leukemia-initiating cells in vivo

(A) Mice with established leukemia UPN1, and UPN7 were treated with 8F4 or control antibody (IgG) as indicated in Figure 2. LSC were defined as Lin⁻CD34⁺CD38⁻. Zebra plots (left panel) show a representative staining of Lin- human cells in mouse BM before and after treatment with 8F4 or IgG, gated on LSC. Numbers show LSC percentage within human Lin- cells. Right panels show LSC percent (mean± SEM) of all live cells (including mouse and human) in bone marrow for each treatment group. (B) Secondary transfer model was used to test the effect of treatment on LSC as measured by leukemia self-renewing capacity. Mice with established leukemia (UPN2) were treated with 8F4 three times per week starting weeks 7. At week 10, residual leukemia in bone marrow from 8F4-treated mice were transplanted into secondary recipient mice. (C) Representative zebra flow cytometry plots of tissues from the transfer recipient mice at week 16 show no detectable leukemia in the bone marrow, spleen and liver of mice that received the AML transplant from 8F4-treated animals, indicating elimination of LSC populations (n=2–4 mice per treatment group).