Mechanisms of leukemia resistance to antibody dependent cellular cytotoxicity

Abstract

Specific immunotherapy for acute leukemia remains a great unmet need. Native unmodified monoclonal antibody therapies, while promising, are inadequately effective for these malignancies, and multiple mechanisms for failure have been described. Antibody-dependent cellular cytotoxicity or phagocytosis is the primary modality of mAb-mediated cell killing in vivo, but ultimately leads to relapse of the leukemias, in model systems and in humans. By use of a T-cell receptor mimic mAb ESKM, derived against a WT1 peptide expressed in complex with HLA-A*02:01, whose only mechanism of therapeutic action is ADCC, we evaluated the mechanisms of leukemic relapse from its potent therapeutic action in mouse xenograft models of human leukemia. Leukemia escape was not associated with loss of the antigenic target, downregulation of cell surface HLA, antibody pharmacokinetic or biodistribution issues, or development of leukemia cell-intrinsic resistance to ADCC. Interestingly, the rapidity of leukemic growth determined whether leukemia was able to evade cytotoxicity independent of the presence of sufficient effector cells. By engineering leukemia cells with upregulated p27^Kip1 and slower cell cycling times, we show that relapse was inversely correlated with growth rates resulting in the eventual inadequacy of effector to target ratio. Moreover, lack of migration of effector cells into lymphomatous pockets of ALL also allowed local escape. Successful leukemia therapy with mAb might therefore be improved in similar situations by combination with measures to reduce burden and slow leukemia cell growth.

Keywords: ADCC; Leukemia; T-cell receptor mimicking; antibody; escape mechanisms.

Figure 1.

BV173 engrafted xenograft NSG mouse model. All histology analyses were performed on mice 3 weeks after leukemia injection. For mice receiving ESKM therapy, treatment was started on day 6 after injection, with mice receiving 2 weeks of therapy. (A) Untreated mouse bone marrow. IHC with mac-2 for macrophage staining. Large lymphomatous BV173 cell cluster evident, with no macrophage infiltration. (B) Same section of bone marrow as in (A), with myeloperoxidase IHC for neutrophil (including precursor) evaluation. As in (A), minimal effector cell penetration into lymphomatous BV173 cluster. (C) Mouse liver from non-treated mouse, IHC for mac-2, demonstrating mouse Kupffer cells (macrophages) infiltrating BV173 lymphomatous cluster. (D) BLI, exponential scale, of BV173 growth 34 d after injection (4 weeks of therapy, starting on day 6). (E) IHC for human IgG, 1 d after treatment dose of ESKM. Diffuse penetration of ESKM is seen throughout lymphoma. (F) Control IHC for human IgG in untreated mouse. No human IgG is seen, demonstrating that the IHC in Fig. 1E can only be ESKM. (G) Large BM BV173 lymphoma, IHC mac-2 staining, showing no macrophage infiltration. (H) Same as BM as (E) with myeloperoxidase staining showing no neutrophil infiltration into lymphomatous BV173 tumor.

Figure 2.

SET2 cell line engrafted xenograft NSG mouse model growth patterns. Error bars: 5th to 95th percentile. All BLI imaging and growth curves use exponential scale. (A) IHC for CD33, outlining discreet SET2 cells in mouse BM with no therapy, 3 weeks after injection. (B) CD33 IHC of SET2 cells in mouse BM 3 weeks after injection on 2 weeks of ESKM therapy (started on day 6). (C) In vivo SET2 growth quantification by BLI over time, with 3 × 10⁶ cells injected on day 0, with and without ESKM therapy. (D) In vivo SET2 growth quantification by BLI over time with and without ESKM therapy. Effector to target ratios are increased from (C) with only 5 × 10⁵ cells injected on day 0 and the addition of GM-CSF. (E) SET2 growth by BLI quantification, comparing starting ESKM after leukemic BM engraftment (on day 6) to leukemic growth in ESKM pre-treated mice (ESKM injected 2 h prior to injection of SET2). (F) SET2 growth by BLI quantification comparing 4 weeks of continuous (bi-weekly) ESKM therapy to only 1 week of treatment.

Figure 3.

SET2 cell evaluation status-post harvest from murine BM. (A) Flow cytometry for surface HLA-A*02:01 expression, showing no difference between SET2 cells harvested from control mice and those treated with ESKM. (B) Same cells as in (A), evaluating for ESKM binding, showing no difference between cells extracted from ESKM treated and untreated mice. (C) SET2 cells from ESKM treated and untreated mice were harvested and passaged into a secondary group of mice. Ten initial mice (5 control, 5 treated) had BM harvested, and the SET2 cells extracted by Ficoll from each mouse were injected into two mice, (a control mouse and an ESKM pre-treated mouse). Error bars: 5th to 95th percentile. BLI growth curve utilizing exponential scale.

Figure 4.

Lack of outgrowth of resistant SET2 cells in vitro. Error bars: 5th to 95th percentile. (A) 3 d ADCC: daily cytotoxicity as measured by flow cytometry. Effector to target ratio set at 25:1, with normal donor PBMCs used as effectors. Cytotoxicity measured as ratio of live SET2 cells with:without ESKM in media. (B) Median HLA-A*02:01 cell surface expression of cells in (A) on days 1–3 of ADCC by flow cytometry. HLA-A*02:01 increases in cells exposed to PBMC with ESKM compared to SET2 cells with PBMC without ESKM likely due to inflammatory cytokines. (C) Median surface HLA-A*02:01 expression by flow cytometry of passaged SET2 cells, status-post 2nd round of ADCC.

Figure 5.

SET2-S cell evaluation in vitro and in vivo. Error bars: 5th to 95th percentile. BLI imaging and growth curves use exponential scale. (A) Western blot evaluating the protein expression of p27^Kip1 measured using a FLAG specific antibody. (B) Dose-dependent growth inhibition of SET2-S cells in vitro with increasing doxycycline concentrations. (C) Competition assay demonstrating decrease in population of SET2-S cells when compared to SET2 cells. SET2 and SET2-S cells were mixed 50:50 at t = 0 h and 2 µg/mL doxycycline was added to the media. Expression of tdTomato, marking SET-S cells, was measured over time. (D) BLI at the end of 3 weeks of therapy in NSG mouse model of SET2-S leukemia (day 28 status-post leukemic injection). Controls had injections of SET2-S cells and no therapy. ESKM treated mice received the antibody only on day 6 onwards. The other two groups got doxycycline alone or doxycycline with ESKM. (E) A plot on log scale of the mice treated in panel D over one month. SET2-S in vivo growth, by BLI, evaluating ESKM ADCC with and without p27^Kip1 overexpression, demonstrating clear superiority of ADCC treatment on slower growing SET2-S cells compared to wild type controls. (F) Flow cytometry of SET2-S cells with and without doxycycline exposure, evaluating ESKM binding. (G) ADCC of SET2-S with and without upregulation of p27^Kip1, activated by doxycycline exposure. The overexpression of p27^Kip1 appears to stabilize SET2-S, decreasing the chromium release of both control and ESKM treated cells.

Figure 6.

Therapy in a Rag2 mouse knock out model. Rag2^−/− mice injected with BV173 leukemia xenograft (day 0) after cytoreduction with radiation and anti-NK mAb. Mice were treated with 3 weeks of biweekly ESK1 starting day 28. BLI was measured per mouse in each group (mean +/− SEM). (A) BV173 tumor growth by BLI comparing ESK1 treated mice with their control (untreated) counterparts. (B) BLI of ESK1 and untreated groups on day 28, at the start of therapy, and day 70, 3 weeks after the final week of therapy.