Combination immunotherapy with anti-CD20 and anti-HLA-DR monoclonal antibodies induces synergistic anti-lymphoma effects in human lymphoma cell lines.

Rituximab is effective in about one half of patients with indolent lymphoma. Even these patients relapse and develop rituximab resistance. To increase potency and circumvent resistance, the anti-lymphoma effects of rituximab, an anti-CD20 MAb(1), combined with chLym-1(2), an anti-HLA-DR MAb, were assessed in human lymphoma cell lines by examining growth inhibition and cell death, apoptosis induction, ADCC(3) and CDC(4). There were additive effects in all assays and synergism in cell lines, such as B35M, which displayed resistance to either MAb alone. In B35M cells, combined rituximab and chLym-1 induced a 27-fold direct reduction in viable cells, whereas equivalent concentrations of rituximab or chLym-1 alone induced only a 1-fold and 10-fold reduction in viable cells, respectively. Because these results occurred at MAb concentrations readily achievable in patients, they suggest that this combination immunotherapy regimen may increase the potency and range of effectiveness of these MAbs in lymphoma patients.

Direct antilymphoma effects on human lymphoma cells of monotherapy and combination therapy with CD20 and HLA-DR antibodies and 90Y-labeled HLA-DR antibodies.

PURPOSE: Monoclonal antibodies (mAb) in combination and mAbs combined with a radionuclide (radioimmunotherapy) have both been more effective in patients than mAb monotherapy. EXPERIMENTAL DESIGN: Using assays of cell growth and viability, the dose response and temporal characteristics of CD20 (rituximab) and HLA-DR (Lym-1) mAbs, singly and in combination, and of 90Y-conjugated Lym-1 mAb have been characterized in five human lymphoma cell lines (B35M, Raji, SU-DHL-4, SU-DHL-6, and Ramos) spanning Burkitt's to diffuse large cell lymphoma. Although Ramos had a lower HLA-DR density, these cell lines were otherwise selected because of high cell surface CD20 and HLA-DR abundance. Assays of cell growth and death were done using microscopy and trypan blue dye. RESULTS: Lym-1 and rituximab, used singly, showed direct antilymphoma effects; those of Lym-1 were often more potent than those of rituximab. Combinations of these mAbs were more effective, sometimes synergistic, than either mAb singly, even in more resistant SU-DHL-4 cells. Conjugation of 90Y to Lym-1 also augmented potency in all cell lines and overcame resistance to both Lym-1 and rituximab in Ramos cells. CONCLUSIONS: Lym-1 exhibited substantially greater direct antilymphoma effects than rituximab in lymphoma cells in culture. Combination of Lym-1 with rituximab or 90Y increased potency and overcame treatment resistance in lymphoma cells. Greater use of combination therapies of this type to increase potency and range of effectiveness seems likely to improve patient outcome.

Antilymphoma effects of anti-HLA-DR and CD20 monoclonal antibodies (Lym-1 and Rituximab) on human lymphoma cells.

AIM: Anti-HLA-DR and anti-CD20 monoclonal antibodies (MAbs) have been effective for immunotherapy and radioimmunotherapy in non-Hodgkin's lymphoma (NHL). The aim of our study was to compare the antilymphoma effects of Lym-1 and rituximab in human lymphoma cell lines, using assays of viability, apoptosis, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC), under conditions relevant to the clinic. METHODS: To characterize response relationships at varied concentrations of Lym-1 and rituximab, growth inhibition and cell death were assayed over 96 hours in four NHL cell lines derived from Burkitt's or large-cell lymphoma patients. Untreated cells and cells treated with an mLym-1 isotype-matched MAb were used as negative controls for direct assays. Western blot was used to detect apoptosis through the activation of caspase-3 and cleavage of poly (ADP-ribase) polymerase (PARP). The indirect cytotoxicity of Lym-1 and rituximab was assayed at varied concentrations, using ADCC activity in the presence of purified peripheral blood leukocytes and CDC activity in the presence of human donor serum. RESULTS: Lym-1 and rituximab showed significant direct and indirect antilymphoma effects. Lym-1 had a substantial, and statistically greater, effect than rituximab over longer intervals of time. In Raji and B35M cells, Lym-1 induced potent growth inhibition reflected by 90% and 94% reductions in viable cells, respectively, whereas rituximab induced 63% and 56% reductions. Concurrently, Lym-1 increased nonviable cells by 372% and 153% in these cells, respectively, whereas rituximab induced 139% and 43% increases. Lym-1-induced apoptosis was greater than that of rituximab in all cell lines tested. Lym-1, both the chimeric form and the mouse parent, mediate ADCC more effectively, in the presence of a total peripheral blood leukocyte (PBL) population, than does rituximab, although the results for CDC activity were mixed. CONCLUSIONS: In conclusion, Lym-1 had more potent direct and indirect cytotoxic effects than rituximab in lymphoma cells under conditions achievable in patients. Because the HLA-DR target antigen of Lym-1 is enriched on most B-cell lymphomas, these results support its complementary use in patients as an alternative to CD20 for monoimmunotherapy and for combination immunotherapy with rituximab, because the HLA-DR and CD20 antigens are physically and functionally coupled on human B cells.