Gain of CD20 Target Cell Surface Receptor Expression During Serum-Free Ex Vivo Culture of CD19+CD5+ B-Cells In a Sub-Population of Early Stage (Rai 0/1) Chronic Lymphocytic Leukemia Patients
Authors: Frank Robin , Jimenez Hugo , Jagan Sucheta , Tan Lydia Luy , Paganessi Laura A , Larson Melissa L , Venugopal Parameswaran , Gregory Stephanie A. , Christopherson Kent W 
Published: October 2019

Abstract
Abstract 2000
Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by the gradual accumulation of CD19+CD5+ monoclonal B-cells. Rutuximab is a monoclonal anti-CD20 antibody that specifically targets B cells expressing the CD20 cell surface glycoprotein, and it is commonly used to treat B-cell malignancies. Because CLL patients express low levels of CD20, the effectiveness of Rutuximab is limited. Therefore, methods to increase CD20 expression on B-cells would benefit CLL patients. We therefore investigated the CD20 expression on untreated cells or cells treated with normal donor serum, autologous CLL serum, or Human Serum Albumin (HSA, flexbumin) during ex-vivo culture. Peripheral blood (PB) was obtained from six Early Stage (Rai 0/1) CLL patients and five normal healthy donors with IRB approval. CLL samples had low CD38 expression, an average WBC count of 55.4±25.7 Th/μL, and Beta-2 Microglobulin (B2M) of 2.3±0.2 mg/L. Mononuclear cells were isolated by density centrifugation and CD19+ cells were isolated by positive magnetic selection. Cells were cultured at 37°C, 5% CO2, 100% humidity for 0, 24, 48, and 96 hours. CD19+ B-cells from six CLL patients were either untreated (AIM-V serum free media) or treated with 5%, 20%, 40% serum. CD20 expression was measured by multivariate flow cytometry. Data was analyzed using the two-tailed Student's t-Test. Initially, CD19+CD5+ CLL cells had an average CD20 expression of 5.1±1.4% (N=6). Flow cytometric analysis of CLL cells cultured for 96 hours without serum treatment revealed that patient samples subdivide into two distinct groups based on changes in CD20 expression: a variable group (N=3) and a stable group (N=3). Untreated CLL cells within the variable group exhibited a significant increase in CD20 expression over the 96 hour culture period. The average CD20 expression of CLL cells from the variable group was initially upregulated from 6.5±1.7% at 0 hours to 55.0±3.2% (p<0.05, N=3) at 96 hours. Within the stable group, the expression of CD20 in untreated CLL cells had little to no increase at 24, 48, and 96 hours of culture. CD20 expression of normal donor cells was initially 97.8±1.2% (N=5) and remained high when cultured. CD20 expression of CLL cells within the stable group was 3.7±2.2% at 0 hours and 12.0±3.9% (N=3) at 96 hours, which was significantly lower than CD20 expression from the variable group (p<0.05). Treatment of CLL cells with 5% normal donor serum or autologous CLL serum from the variable group suppressed CD20 expression to 14.6±3.9% (p<0.05, N=3) and 16.3±4.8% (p<0.05, N=3) respectively at 96 hours. Suppression of CD20 with normal donor serum or autologous CLL serum was not dose-dependent at 5%, 20%, and 40%. In addition, we found that CD20 expression was not suppressed by HSA treatment, 49.2±13.3% (n=3) at 96 hours. Serum treatment of CLL cells from the stable group did not alter CD20 expression. The average CD20 expression was 20.1±8.8%, 21.3±5.6%, and 18.8±6.9% when treated with normal donor serum, autologous CLL serum, or HSA respectively at 96 hours. Our findings show that cells from Early Stage CLL patients categorize into two subgroups based on CD20 expression after serum free culture for 96 hours: variable and stable. Treatment of CLL cells with serum showed that the variable and stable groups respond differently to the environment at 96 hours. Only CLL cells in the variable group that have a significant upregulation of CD20 after 96 hours of culture respond to serum treatment. Regardless of whether cells were treated with autologous CLL serum or normal donor serum, there was a reduction in CD20 expression in CLL cells within the variable group. CLL cells in the stable group have a low CD20 phenotype and are not responsive to the serum. This data suggests that individual CLL patients may have different cell intrinsic properties that allow for or prevent the upregulation of CD20. Some patients may also have a loss of the ability for environmental factors in serum to effect CD20 expression. Further investigation is needed to determine whether our findings correlate with the clinical outcome.

Disclosures:
Gregory: Amgen Inc.: Consultancy; Astellas: Research Funding; Celgene: Research Funding; Cephalon: Research Funding, Speakers Bureau; Genentech (Roche): Consultancy, Research Funding, Speakers Bureau; GlaxoSmithKline: Research Funding; Immunomedics: Research Funding; NCIC CTG: Research Funding; Novartis: Consultancy, Research Funding; Onyx: Research Funding; Spectrum Pharmaceuticals: Consultancy.