Anaplastic Large Cell Lymphoma (ALCL) is a T-cell lymphoma driven by the constitutive activation of the Anaplastic Lymphoma Kinase (ALK) due to a chromosomal translocation of the gene, resulting in the formation of the oncogenic chimeric protein NPM-ALK. Many patients with ALK+ ALCL tumors that undergo remission for years quickly relapse upon TKI cessation due to a re-activation of drug-tolerant persister cells (DTPC). We have demonstrated a role for the tumor microenvironment (TME) as a key element in supporting the survival of DTPCs. We identified a role of the CCL19/21-CCR7 survival pathway in DTPCs resistant to ALK TKI. In this work, we investigate whether blocking CCR7 with a newly developed IgG human antibody (CAP-100), combined with ALK TKI crizotinib can prevent relapses by eradicating DTPCs.

CCR7 expression and CAP-100 binding were tested by flow cytometry with a commercial CCR7 antibody and CAP-100 in ALK+ ALCL cell lines and PDX-derived cell line. Patient-Derived Xenograft (PDXs) mouse models were made by subcutaneously injecting ALK+ALCL cells in NSG mice (PDX-DN and PDX-MTK). For in vivo experiments mice were split into four cohorts of treatment: vehicle, CAP-100, crizotinib, and a combination of both drugs. Crizotinib was given orally at 50mg/kg/day in PDX-DN and at 100mg/kg/day in PDX-MTK; CAP-100 was given by peritoneal injection every 3 days at a dosage of 10mg/kg in both PDX models. Tumor growth was tracked with a caliper every three days. Mice were euthanized at a human endpoint. Tumors were resected and snap-frozen or formalin-fixed for further analysis. Immunohistochemistry (IHC) analysis was performed to assess ALK positivity on tumor tissues and organs

CCR7 expression was assessed in ALK+ALCL cell lines and both PDX models, showing positivity in all lines and PDXs with some heterogeneity. In therapeutic experiments, after two weeks of treatment, mice treated with crizotinib alone or with the combo showed decreased tumor mass in both PDX models. In PDX-DN model, after treatment suspension mice that received crizotinib alone all relapsed. In contrast, mice treated with the combo crizotinib+CAP100 had significantly extended survival, and only one out of eight mice had a late relapse. In the PDX-MTK model, mice treated with crizotinib alone developed brain dissemination; mice treated with the crizotinib+CAP100 showed significantly less brain involvement, also confirmed by immunohistochemistry for ALK

CAP-100 boosts crizotinib activity in ALK+ ALCL PDX models by aiding the killing of DTPC in a subset of mice. The addition of CAP-100 to crizotinib markedly reduced the dissemination of lymphoma cells to the brain confirming our previous data that showed that genetic knock-out of CCR7 impaired lymphoma dissemination to the brain. Overall, these data show that the addition of CAP-100 to an ALK TKI could improve the cure rate and prevent lymphoma spread to critical sites like the brain.