In Vitro Activity of the Humanized Anti-HLA-DR Antibodies KRN848 and Apolizumab in Non-Hodgkins Lymphoma Cell Lines.

Apolizumab (Hu1D10) and KRN848 are humanized IgG1 anti-HLA-DR monoclonal antibodies (mAb). Apolizumab binds a polymorphic determinant on the HLA-DR-β chain, whereas KRN848 is a pan-HLA-DR mAb. We undertook preclinical studies to assess the properties of these two mAbs with regard to antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC) and their direct effects on target cells. HLA-DR (+) Raji and Daudi lymphoblastic cell lines served as target cells. KRN848 is reactive with HLA-DR expressed on both cell lines, while apolizumab is reactive only with Raji.

ADCC assays were performed using ⁵¹Cr-labeled target cells and normal donor peripheral blood mononuclear cells (PBMCs) as effector cells. At an E:T ratio of 40:1 and a mAb concentration of 2 μg/ml, all mAbs mediated ADCC. Apolizumab mediated greater specific lysis of Raji cells (37%) than did KRN848 (15%) or rituximab (14%). When Daudi cells were used as targets, apolizumab induced <3% lysis, while KRN848 and rituximab both induced 20% lysis. Similar findings were seen when EBV-transformed lymphoblastoid cells served as targets, and autologous healthy PBMCs as effector cells, with the degree of lysis induced by apolizumab varying depending on the degree of reactivity of apolizumab with the lymphoblastoid cells. In CMC assays, apolizumab, KRN848 and rituximab mediated comparable lysis of Raji cells (77%, 80% and 83%) at 2 μg/ml. In Daudi cells, there was <4% specific lysis with apolizumab, 45% lysis with KRN848 and 90% with rituximab.

Evidence for direct killing mediated by mAbs varied based on the assay used. When ⁵¹Cr-labeled Raji cells were incubated with KRN848 or apolizumab for 4–17 hours, and ⁵¹Cr in the supernatant used as a measure of cell death, there was minimal evidence (<10% specific lysis) of direct antibody-induced lysis over a range of concentrations (0.0016–2 μg/ml). In contrast, anti-HLA-DR mAbs had a significant effect on annexin-V/PI staining of target cells. Annexin-V/PI staining of Raji cells was positive after treatment with apolizumab (78%) and KRN848 (67%) at 2 μg/ml at four hours. KRN848 induced annexin-V/PI uptake by Daudi cells (39%) while apolizumab did not, demonstrating this effect was dependent on expression of the target antigen.

We conclude the anti-HLA-DR mAbs apolizumab and KRN848 can induce ADCC and CMC based on expression of target antigen. Anti-HLA-DR mAbs can directly alter the annexin-V/PI positivity of target lymphoma cells but do not induce ⁵¹Cr release, suggesting anti-HLA-DR mAbs alter cell membrane polarization and permeability but may not induce apoptosis directly.