Clinical and Biological Characteristics Associated with In Vitro Activity of Anti-CD20 Monoclonal Antibodies, Rituximab and GA101, Against Chronic Lymphocytic Leukemia Cells

The CD20 antibody rituximab (RTX) is an important therapeutic agent in the armamentarium against chronic lymphocytic leukemia (CLL) cells. Several mechanisms have been described that affect low single agent efficacy of CD20 antibodies in B-CLL and relapse after treatment. The novel CD20 antibody GA101 has been developed with the aim of further improving CLL therapy and is currently in phase II/III clinical trials. It is a type II, glyco-engineered CD20 antibody with enhanced ADCC through improved CD16A binding.

To assess in vitro pre-clinical activity of RTX and GA101 against CLL cells in either freshly isolated PBMCs, or after 14 days of culture in vitro, to allow the outgrowth of Nurse-Like Cells (NLC) that may presumably alleviate mAb activity (a process called Environment Mediated-Drug Resistance, or EM-DR).

In 34 CLL patients (all naive for treatment), PBMCs were isolated from blood samples by Ficoll gradient centrifugation. Antibody-mediated B cell depletions was determined by enumerating trypan blue negative, flow cytometrically CD19-positive B lymphocytes after antibody treatment. Three conditions were assessed, all with starting concentrations of 10⁷cells/ml: (i) depletion in freshly isolated PBMCs after 7 days of culture in RPMI+10%FCS and 10μg/ml RTX or GA101 (DEP7), or, after 14 days culture of PBMCs in RPMI+10% FCS, PBMCs were collected and either (ii) washed and cultured in fresh RPMI+10%FCS and antibodies for 7 extra-days (DEP21/RPMI), or (iii) re-suspended in their own conditioned RPMI medium and cultured with NLC and antibodies for 7 extra-days (DEP21/NLC). The Mann-Whitney analysis was used to compare median depletion according to relevant clinical and biological data.

The median B-CLL depletion in freshly isolated PBMC were 56% for GA101 vs 5% for RTX (p=0.000031). As indicated in Table 1, no clinico-biological parameter was significantly associated with antibody response, though unmutated IgVH status seemed to impact on GA101 efficacy. We next sought to study the impact of EM-DR mechanisms afforded by CLL PBMCs+NLC co-cultures in vitro. While RTX activity was virtually abrogated under those conditions, B cell depletion induced by GA101 was significantly reduced to 27% if PBMCs were collected after 14 days of culture with NLC, and incubated with antibodies from days 14–21 either in fresh RPMI (DEP21/RPMI=27% vs 56% for DEP7 GA101, n=18, p=0.024), or to 14% in conditioned RPMI and NLC (DEP21/NLC=14% vs 56% for DEP7 GA101, n=12, p=0.008). This time, clinico-biological parameters linked with active CLL disease like bulky lymph nodes>5cm (p=0.049), presence of at least one NCIWG2008 criteria for initiation of treatment (p=0.01), and unmutated IgVH status (p=0.03) appeared significantly linked to reduced GA101 activity. Interestingly, karyotype abnormalities did not seem to negatively impact on GA101-triggered depletion.

Our results suggest that in vitro EM-DR abrogates the small RTX activity as single agent against CLL cells, whereas GA101 still retains activity under those conditions. It remains to be studied whether this may impact the clinical activity of GA101 in subgroups of patients. Overall, GA101 appeared much more active than rituximab, and should be investigated in combination with strategies aiming at disrupting EM-DR mechanisms.

Off Label Use: GA101 is not currently approved for CLL treatment. Klein: Roche: Employment, Equity Ownership, Patents & Royalties.