Effective in Vivo Targeting of BCP-ALL in a NOD/SCID/huALL Mouse Model By CD70 Directed Immunotherapy

Acute lymphoblastic leukemia (ALL) is the most frequent malignant disorder in children and adolescents. Despite successful treatment, relapse of the disease remains a major problem and is associated with poor prognosis. This emphasizes the need for novel treatment strategies to be applied in addition to established chemotherapy regimens without increasing general toxicity.

Previously, we described a strong association of leukemia cell engraftment of primary patient B cell precursor (BCP) ALL samples transplanted in a NOD/SCID/huALL mouse model and patient outcome. Rapid onset of leukemia related morbidity (time to leukemia, TTL^short) is indicative for patient relapse and characterized by a specific gene expression profile. Among the top differentially regulated genes, the gene coding for CD70 was identified to be significantly up-regulated in TTL^short/high risk ALL. CD70 is a member of the tumor necrosis factor (TNF) family expressed on activated B- and T-lymphocytes and dendritic cells. Binding of CD70 to its receptor CD27 is involved in regulation of T- and B-cells including priming and generation of memory and plasma cells. CD70 has been described to be constitutively expressed on different cancers including hematological malignancies. However, expression and targeting of CD70 in B- cell precursor lymphoblastic leukemia has so far not been investigated.

In this study, we addressed expression of CD70 in patient-derived primograft leukemia samples and primary patient specimens obtained at diagnosis from pediatric patients. Furthermore, we evaluated CD70 as a therapeutic target for directed immunotherapy in vitro and in our BCP-ALL xenograft system in vivo.

Flow cytometric analyses of CD70 surface expression in all together 19 patient-derived xenograft samples (TTL^short n= 7, TTL^long n=12) revealed a higher expression of CD70 on ALL cells with a TTL^short/early relapse phenotype compared to TTL^longsamples. We also investigated expression of the CD70 receptor CD27 and found no significant difference in surface expression between both TTL subgroups.

Moreover, we investigated the transcript expression levels of 198 BCP-ALL specimens obtained at diagnosis. Interestingly, we found a heterogenous expression of CD70 with no association to cytogenetic subgroups, minimal residual disease (MRD) risk classes or patient outcome. Importantly, a significant higher CD70 expression was found in leukemia samples compared to healthy bone marrow controls indicating a general over-expression in BCP-ALL. CD27 however, did not show different transcript expression including healthy bone marrow controls.

To take advantage of increased CD70 expression in BCP-ALL, we addressed CD70 as therapeutic target for immunotherapy. Co-culture in vitro experiments of primograft ALL cells with NK cells in the presence of specific anti-CD70 antibodies revealed five-fold increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the respective isotype control.

To evaluate the efficacy of CD70 directed immunotherapy, we assessed leukemia development in NOD/SCID mice upon transplantation of primograft ALL with high CD70 expression either incubated with anti-CD70 antibody or the respective isotype control. Most importantly, a marked reduction of leukemia load in peripheral blood, bone marrow and spleens of the animals was detected in anti-CD70 treated cases. This indicates, that CD70 provides an immunotherapeutic target on ALL cells inducing ADCC by NK cells present in NOD/SCID mice. Most interestingly, this effect could be abrogated both by NK-cell depletion (pre-treatment with anti-mouse CD122 antibodies) in the recipient animals and by using NK-cell deprived NSG mice as recipients, confirming that decreased in vivo leukemia growth upon anti-CD70 treatment is mediated by NK-cell induced cytotoxicity of anti-CD70 bearing CD70 positive ALL cells.

Taken together, we identified significantly up-regulated CD70 expression in BCP-ALL with varying expression among molecular and prognostic subgroups. BCP-ALL samples with high surface expression of CD70, as detected by flowcytometry, can be targeted by directed immunotherapy with anti-CD70 antibodies leading to efficient NK-cell dependent lysis of leukemia cells in vitro and decreased growth in an in vivo BCP-ALL model. Thus, CD70 provides a novel target for directed immunotherapy of BCP-ALL.