Engineered anti-CD20 antibodies with enhanced complement-activating capacity mediate potent anti-lymphoma activity - PubMed (original) (raw)
Engineered anti-CD20 antibodies with enhanced complement-activating capacity mediate potent anti-lymphoma activity
Akito Natsume et al. Cancer Sci. 2009 Dec.
Abstract
One of the major issues in current antibody therapy is insufficient efficacy. Various biological factors relating to the host's immune system or tumor cells have been suggested to reduce the efficacy of anti-CD20 therapy in B-cell malignancies. In this study, we characterized the in vitro anti-lymphoma activity of anti-CD20 antibodies having a novel engineered heavy chain with enhanced complement-dependent cytotoxicity (CDC). Anti-CD20 antibodies having a variant heavy constant region of mixed IgG1/IgG3 isotype, which have previously been found to enhance CDC, were investigated for their in vitro CDC against lymphoma cells and whole blood B-cell depletion activity. Use of the variant constant region greatly increased the CDC of an anti-CD20 antibody having variable regions identical to those of rituximab to the level shown by an IgG1 antibody of ofatumumab. Although the whole blood assay showed different cytotoxicity patterns among individual blood donors, the CDC-enhancing variant of rituximab showed higher activity than the parent IgG1 and consistently showed maximized activity when further combined with antibody-dependent cellular cytotoxicity (ADCC)-enhancing modification by fucose removal from Fc-linked oligosaccharides. In addition, the rituximab variant showed potent CDC against transfectant cells with lower CD20 expression and chronic lymphocytic leukemia-derived cell lines with higher complement regulatory proteins. These findings suggest that CDC enhancement, both alone and in combination with ADCC enhancement, increases the anti-lymphoma activity of anti-CD20 antibodies irrespective of individual differences in effector functions, and renders current anti-CD20 therapy capable of overcoming the potential resistance mechanisms.
Figures
Figure 1
Anti‐CD20 antibodies with IgG1/3 mixed isotype heavy chain exert enhanced lymphoma cell killing. Complement‐dependent cytotoxicity (CDC) activity against B lymphoma cell line Raji (A) and chronic lymphocytic leukemia (CLL) cell line EHEB (B) are shown as mean ± SD of triplicates. Symbols represent 2B8‐IgG1 (○), 2B8‐113F (•), 2F2‐IgG1 (Δ), and 2F2‐113F ().
Figure 2
Antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC) using effectors from a single blood donor. ADCC (A) and CDC (B) of anti‐CD20 antibody variants against chronic lymphocytic leukemia (CLL) cell line MEC‐1 were measured using PBMCs and serum separated from the same blood sample of a single donor as effectors, respectively. Symbols represent 2B8‐IgG1 (○), 2B8‐IgG1‐nf (•), 2B8‐113F (Δ), 2B8‐113F‐nf (), and 2B8‐IgG4 (×). The mean ± SD of triplicates are shown.
Figure 3
B‐cell depletion activity of anti‐CD20 antibody variants in whole blood assay. Anti‐CD20 antibodies were added to whole blood from three donors (A–C), and remaining CD20+ B cells were measured by flow cytometer after incubation. Percentage of CD2+ CD19− T cells was not changed by anti‐CD20 antibodies (data not shown). Symbols represent 2B8‐IgG1 (○), 2B8‐IgG1‐nf (•), 2B8‐113F (Δ), 2B8‐113F‐nf (), and 2B8‐IgG4 (×). The mean ± SD of triplicates are shown.
Figure 4
Complement‐dependent cytotoxicity (CDC) of anti‐CD20 antibody variants against low CD20‐expressing cells. (A) CDC activity against human CD20 transfectant EL4 cells expressing 1.2 × 104/cell (upper left), 4.1 × 104/cell (lower left), 6.7 × 104/cell (upper right), and 2.1 × 105/cell (lower right). (B) Anti‐CD20 antibodies showed no CDC activity against parent murine EL4 cells. (C) CDC activity at a fixed concentration (1.1 μg/mL) of 2B8‐IgG1 (•), 2B8‐113F (), and 2B8‐IgG4 (×) against transfectant cells expressing variable numbers of CD20 molecules. The mean ± SD of triplicates are shown.
Figure 5
Complement‐dependent cytotoxicity (CDC) of anti‐CD20 antibody variants in chronic lymphocytic leukemia (CLL) cells. CDC activity against CLL cell lines EHEB (A), MEC‐1 (B), MEC‐2 (C), and B lymphoma line Daudi (D) are shown. Symbols represent 2B8‐IgG1 (○), 2B8‐IgG1‐nf (•), 2B8‐113F (Δ), and 2B8‐113F‐nf (). The mean ± SD of triplicates are shown.
Figure 6
Effect of complement regulatory proteins (CRP) neutralization on complement‐dependent cytotoxicity (CDC) sensitivity of chronic lymphocytic leukemia (CLL) cells. CDC activity of anti‐CD20 antibodies against CLL cell line EHEB was measured in the absence or the presence of CRP neutralizing antibodies. Symbols represent 2B8‐IgG1 alone (○) 2B8 plus anti‐CD46 (□), 2B8 plus anti‐CD55 (◊), 2B8 plus anti‐CD59 (Δ), 2B8 plus mixture of 3 anti‐CRPs (×), 2B8‐113F alone (•), 2B8‐113F plus anti‐CD46 (), 2B8‐113F plus anti‐CD55 (◆), 2B8‐113F plus anti‐CD59 (), 2B8‐113F plus mixture of three anti‐CRPs (+). Anti‐CRP antibodies were added at 25 μg/mL, except for the experiments with a mixture of three anti‐CRPs (8.3 μg/mL each). The mean ± SD of triplicates are shown. In the absence of anti‐CD20 antibody, anti‐CRP antibodies did not exhibit any cytotoxicity (data not shown).
References
- McLaughlin P, Grillo‐López AJ, Link BK et al. Rituximab chimeric anti‐CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four‐dose treatment program. J Clin Oncol 1998; 8: 2825–33. - PubMed
- Cheung MC, Haynes AE, Meyer RM, Stevens A, Imrie KR; Members of the Hematology, Disease Site Group of the Cancer Care Ontario Program in Evidence‐Based Care . Rituximab in lymphoma: a systematic review and consensus practice guideline from Cancer Care Ontario. Cancer Treat Rev 2007; 33: 161–76. - PubMed
- Bannerji R, Kitada S, Flinn IW et al. Apoptotic‐regulatory and complement‐protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance. J Clin Oncol 2003; 21: 1466–71. - PubMed
- Di Gaetano N, Cittera E, Nota R et al. Complement activation determines the therapeutic activity of rituximab in vivo. J Immunol 2003; 171: 1581–7. - PubMed
- Kennedy AD, Beum PV, Solga MD et al. Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia. J Immunol 2004; 172: 3280–8. - PubMed
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical