Synergy between CD52 and CD20 Antibody-Induced Apoptosis Correlates with the Extent of Co-Expression of CD20 and CD52 Antigens on Mononuclear Cells Derived from Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin’s Lymphoma (NHL) Patients: Implications to Antibody-Based Therapy.
Authors: Gazitt Yair , Akay Cagla , Doxey Deborah , Thomas Charles 
Published: April 2020

Abstract
CD20 (Rituximab) and CD52 (Campath 1-H) antibodies (Abs) have been employed in the treatment of NHL and CLL pts, as single agents or in combination with chemotherapy. Rituximab has been effective in NHL pts, with &gt;50% response rate (RR). Campath 1-H has been less effective in CLL pts with RR of ~30% with similar results observed for NHL pts, but with increased toxicity. Combination therapy with the two Abs for the treatment of CLL or NHL pts was not attempted. We hypothesized that responsiveness to Ab-based therapy will depend on the extent of expression of the relevant antigen on cancer cells for each disease category resulting with a synergy when the 2 Abs were combined. Expression of CD20 and CD52 antigens on mononuclear cells (MNCs) were determined by 2-color immunofluorescence staining and analysis was done by flow cytometry. Percent positive cells and mean fluorescence intensities (MFI) were determined. Expression of CD38 antigen has been correlated with disease outcome in CLL pts. The expression of CD38 on MNCs from CLL pts was determined by 3-color taining. Samples were obtained from peripheral blood (PB) MNCs of 17 CLL pts and 10 healthy donors (HD). Bone marrow (BM) MNCs were obtained from 35 NHL pts and 10 HD. Apoptosis was induced by 24 hour culture with 1ug/ml of CD20, CD52, or CD52+CD20 Abs in 1ml of medium containing 1 million MNCs. Apoptosis was determined by the Annexin V method and was correlated with % expression and MFIs of CD38; CD20; CD52; CD20/CD52 and CD38/CD20/CD52 antigens.
Results: MFI varied significantly between the 3 groups. MFIs were 449 ±48 fluorescence units (FU); 70±22 and 158±81 FU, in HD, NHL and CLL pts, respectively. Mean % CD20 expression was 32±4; 51.4±15 and 28±8 in HD, NHL and CLL, respectively and MFIs were 62±14; 19±8 and 35±14 FU, respectively. Mean % co-expression of CD20+CD52 in HD, NHL and CLL pts was 26±3; 31±13; and 48±21, respectively.
Mean apoptosis by CD52, CD20 and CD52+CD20 Abs for HD was 25,000±18,000; 23,000±21,000 and 47,000±23,000 apoptotic cells/ug Ab/24h treatment, respectively. For CLL pts apoptosis by CD52, CD20 and CD52+CD20 Abs was 17,000±16,000; 20,000±20,000 and 25,000±22,000, respectively. For NHL pts it was 11,000±7,200; 16,000±7,000 and 13,000±19,000, respectively. MNCs from CLL pts were significantly more sensitive to CD52, CD20 and CD52+CD20-induced apoptosis than MNCs from NHL pts (p&lt;0.01). Significant correlation was observed between MFI and apoptosis for CD52 in NHL pts (R=0.5; p=0.02), for CLL pts (R =0.65; p=0.003) and for CD20 treatment in NHL pts (R=0.6; p=0.004). The combination of CD20+CD52 significantly increased apoptosis for CLL and NHL pts (R =0.59; p&lt;0.02).
Mean % expression of CD38 was 43±22 and MFI was 57±36 FU. Most importantly, no correlation was found between expression of CD38 or CD38/CD20/CD52 and apoptosis induced by CD20, CD52 or CD20+ CD52 Abs.
Conclusions: 1. The extent of expression (%; MFI) of CD20 and CD52 are important for Ab-induced apoptosis by CD20 and CD52 Abs in NHL and CLL MNCs. 2. The combination of CD20+CD52 Abs increases apoptosis in CLL and NHL MNCs. 3. No correlation was found between the expression of CD38 and apoptosis by CD20, CD52 or CD20+CD52 Abs. 4. Prescreening for the expression of CD52 and CD20 is recommended for optimal response to Ab-based therapy.