Investigating the Efficacy of Anti-CD200 Immunotherapy in MRD Low and Risk B Cell Precursor ALL

Despite remission rates of >90% in childhood B cell precursor acute lymphoblastic leukaemia (BCP ALL) using frontline treatment, the incidence of relapse remains high. Immunotherapeutic approaches, including chimeric antigen receptor (CAR)-T cells targeting CD19 have resulted in improved outcomes for refractory cases. However, such therapies do not target CD19^- ALL cells, a population known to have leukaemia initiating ability, and there are several reports of patients relapsing with CD19^- leukaemia after CAR-T cell therapy. CD200 (OX-2) is an immunosuppressive molecule overexpressed in BCP ALL. In low risk minimal residual disease (MRD) ALL samples, only CD200⁺ cells have leukaemia initiating capacity in NOD.Cg- Prkdc^scid Il2r g^tm1Wjl/Sz (NSG) mice, suggesting that CD200 may be a suitable target for therapy in these cases. When CD200 binds to its receptor (CD200R) the immune response is suppressed. Consequently, malignant cells that overexpress CD200 may capitalise on this immune suppression and avoid destruction. Anti-CD200 antibodies (Ab) may be used to prevent such cancer cell evasion by binding to CD200 antigen and blocking its interaction with CD200R. The aim of this study was to investigate the expression of CD200 in MRD low and risk BCP ALL cases and assess the effects of using monoclonal (Mo) TTI-CD200 Ab in vitro and in vivo. Samples from MRD low and risk BCP ALL cases were stained with anti-CD200 Ab to quantify (i) the proportion of cells expressing CD200, (ii) the median fluorescence intensity (MFI) of CD200 and (iii) the number of CD200 antigen binding sites. Results were compared to those obtained using normal bone marrow (NBM) controls. The proportion of CD200⁺ cells was higher in ALL cases (median 40.0%, range 5.4-74.4%) compared to NBM cells (median 0.001%, range 0-0.003%, P =0.0004). There was no significant difference between the MRD low (median 33.8%, range 18.1-74.1%) and risk (median 44.93%, range 36.35-74.4%) ALL cases. However, CD200 MFI levels were significantly higher in the low risk cases (median 593, range 469-1313) compared to those observed in MRD risk cases (median 191, range 138-572, P =0.04). CD200 MFI levels in the low risk group were also significantly higher than in NBM cells (median 0.9, range 0-27, P =0.001). Additionally, the number of CD200 antigen sites on ALL cells was 5-fold higher in MRD low risk cases (median 1372, range 324-2179) than in risk cases (median 264, range 87-1500), while the number of CD200 sites on BM cells was only 17 (range 13-112, P =0.006 vs low risk cases). Monocyte-derived macrophages and CD4⁺ T cells from healthy donors were cultured with low risk BCP ALL cells ± TTI-CD200 in a mixed lymphocyte reaction assay. The production of interleukin (IL)-2 was measured as an indicator of immune system activation using an enzyme-linked immunosorbent assay. Median CD200 expression in these cases was 46.9% (range 0.7-94.2%) and MFI was 249 (34.8-1659). The median IL-2 increase observed on addition of TTI-CD200 was 16-fold (range 1.7-120) and it did not correlate with the proportion of CD200⁺ cells or MFI. In these cases, samples with very low proportions of CD200⁺ cells (0.9±0.4%) demonstrated a 2.5-fold increase in IL-2, whereas samples with higher proportions of CD200⁺ cells (91.1±4.4%) produced a 17.2-fold rise. To assess the effects of TTI-CD200 on ALL cells in vivo, NSG were inoculated with BCP ALL cells from MRD low and risk cases. Once leukaemia levels exceeded 0.5% in peripheral blood, animals received 4 doses of TTI-CD200 (20mg/kg i.v.) over 10 days. Consistent with previous findings, TTI-CD200 treatment of mice engrafted with MRD risk cases had no effect on disease burden. This may be due to the lower proportion of CD200⁺ cells in these cases (34.1±11.7% CD200⁺) and also that CD200^- cells contribute to engraftment in risk cases. In contrast, in low risk ALL cases a 47.5±29% reduction in leukaemia burden was observed after treatment with TTI-CD200. The proportion of inoculated CD200⁺ cells was higher in these low risk cases (74.9±30.3% CD200⁺) and had significantly reduced by 99.4±0.6% ( P =0.04) in ALL cells recovered from the murine BM on termination. The dose and schedule of TTI-CD200 used was much less intensive than standard chemotherapy. It may be possible to improve the in vivo efficacy using another round of therapy. These findings demonstrate the potential of using anti-CD200 MoAb therapy for low risk BCP ALL.