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Blood, 15 November 2008, Vol. 112, No. 10, pp. 4355-4356. This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Beers, S. A. Articles by Glennie, M. J. Search for Related Content PubMed PubMed Citation Articles by Beers, S. A. Articles by Glennie, M. J. Related Collections Related Articles in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  CORRESPONDENCE Response: Superior B cell–depleting activity of type II anti-CD20 mAb is not due to activation of complement Our paper focuses on reporting that tositumomab (a type II anti–human CD20) greatly outperforms rituximab and other type I anti-CD20 monoclonal antibody (mAb) in depleting human CD20-expressing B cells in a transgenic mouse. This, despite the fact that the mAb were all of the same mouse IgG2a isotype, selected for maximum effector function, and recognized the same cross-blocking epitope on CD20.1 Previous data in xenograft models has suggested that a type II mAb might have higher efficacy,2 but our paper is the first to demonstrate this comprehensive difference in a fully syngeneic system using unmanipulated target cells in vivo. The main effector difference between type I and II CD20 mAb is that type I, which translocate CD20 to lipid microdomains, activate complement very efficiently, whereas type II do not.3 Extensive investigations in previous work2–4 and the current paper5 show that rituximab is at least 25 times more active at evoking complement-dependent cytotoxicity (CDC) than tositumomab (B1) in the presence of rat or human serum. The relatively low activity of tositumomab is confirmed by the data shown by Beurskens et al against highly sensitive targets under optimal conditions. This low activity prevents type II mAb, such as tositumomab, from mediating effective CDC against B-cell targets that are protected by complement regulatory proteins, such as CD55 and CD59.6,7 Taken together, it is clear that if complement is an important effector mechanism, as we2,3,8 and many others9–13 have shown, it does not explain why tositumomab is so much more effective in the huCD20-transgenic mice. One potential explanation is that, in certain settings, complement is actually deleterious for therapy as recently suggested.14 To test this idea, we transferred huCD20-transgenic B cells into C1q-deficient mice and made the K322A mutant of Ritm2a (and 1F5; a second type I mAb), a substitution which is known to significantly reduce C1q recruitment in mouse IgG2 antibodies.15,16 In answer to the question raised by Beurskens et al regarding the effectiveness of the K322A mutation in ablating CDC, we agree that the case for human IgG1 is unclear.17 In fact, the paper they cite18(p4178) states, "Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1'." Thus, we do not agree that the K322A substitution in mouse mAb is not removing CDC and our results in Figure 4A of our article clearly support this conclusion. Furthermore, this is an ideal modification for our work as it does not interfere with other effector functions, such as IgG binding to FcR and FcRn. Regardless of whether this mutation ablated or partially removed CDC activity, our results show that it made little difference to the efficacy of Ritm2a and did not convert its performance into that of a type II mAb. Therefore, we conclude that in this model, complement neither helps nor hinders efficacy of CD20 mAb and does not explain the outstanding efficacy of the type II reagent. We are currently exploring alternative mechanisms that explain such activity.    Authorship Top Authorship References   Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Prof Martin J. Glennie, Tenovus Research Laboratory, Cancer Sciences Division, University of Southampton, Southampton, United Kingdom SO16 6YD; e-mail: mjg{at}soton.ac.uk. Stephen A. Beers, Mark S. Cragg, and Martin J. Glennie    References Top Authorship References   Teeling JL, Mackus WJ, Wiegman LJ, et al. The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20. J Immunol. 2006;177:362–371.[Abstract/Free Full Text] Cragg MS, Glennie MJ. Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents. Blood. 2004;103:2738–2743.[Abstract/Free Full Text] Cragg MS, Morgan SM, Chan HT, et al. Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood. 2003;101:1045–1052.[Abstract/Free Full Text] Cragg MS, Walshe CA, Ivanov AO, Glennie MJ. The biology of CD20 and its potential as a target for mAb therapy. Curr Dir Autoimmun. 2005;8:140–174.[Medline] [Order article via Infotrieve] Beers S, Chan H, James S, et al. Type II (tositumomab) anti-CD20 monoclonal antibody outperforms Type I (rituximab-like) reagents in B-cell depletion regardless of complement activation. Blood. Prepublished on July 10, 2008, as DOI 10.1182/blood-2008-04–149161. Cardarelli PM, Quinn M, Buckman D, et al. Binding to CD20 by anti-B1 antibody or F(ab')(2) is sufficient for induction of apoptosis in B-cell lines. Cancer Immunol Immunother. 2002;51:15–24.[CrossRef][Medline] [Order article via Infotrieve] Golay J, Zaffaroni L, Vaccari T, et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood. 2000;95:3900–3908.[Abstract/Free Full Text] Teeling JL, French RR, Cragg MS, et al. Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood. 2004;104:1793–1800.[Abstract/Free Full Text] Di Gaetano N, Cittera E, Nota R, et al. Complement activation determines the therapeutic activity of rituximab in vivo. J Immunol. 2003;171:1581–1587.[Abstract/Free Full Text] Golay J, Cittera E, Di Gaetano N, et al. The role of complement in the therapeutic activity of rituximab in a murine B lymphoma model homing in lymph nodes. Haematologica. 2006;91:176–183.[Abstract/Free Full Text] Golay J, Manganini M, Facchinetti V, et al. Rituximab-mediated antibody-dependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interleukin-2. Haematologica. 2003;88:1002–1012.[Abstract/Free Full Text] Golay J, Lazzari M, Facchinetti V, et al. CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59. Blood. 2001;98:3383–3389.[Abstract/Free Full Text] Gong Q, Ou Q, Ye S, et al. Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy. J Immunol. 2005;174:817–826.[Abstract/Free Full Text] Wang SY, Racila E, Taylor RP, Weiner GJ. NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement. Blood. 2008;111:1456–1463.[Abstract/Free Full Text] Duncan AR, Winter G. The binding site for C1q on IgG. Nature. 1988;332:738–740.[CrossRef][Medline] [Order article via Infotrieve] Wawrzynczak EJ, Denham S, Parnell GD, Cumber AJ, Jones PT, Winter G. Recombinant mouse monoclonal antibodies with single amino acid substitutions affecting Clq and high affinity Fc receptor binding have identical serum half-lives in the BALB/c mouse. Mol Immunol. 1992;29:221–227.[CrossRef][Medline] [Order article via Infotrieve] Thommesen JE, Michaelsen TE, Loset GA, Sandlie I, Brekke OH. Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation. Mol Immunol. 2000;37:995–1004.[CrossRef][Medline] [Order article via Infotrieve] Idusogie EE, Presta LG, Gazzano-Santoro H, et al. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol. 2000;164:4178–4184.[Abstract/Free Full Text] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? Related Articles in Blood Online: Complement activation impacts B-cell depletion by both type I and type II CD20 monoclonal antibodies Frank J. Beurskens, Sigrid R. Ruuls, Patrick J. Engelberts, Tom Vink, Wendy J. Mackus, Jan G. J. van de Winkel, and Paul W. H. I. Parren Blood 2008 112: 4354-4355. [Full Text] [PDF] Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation Stephen A. Beers, Claude H. T. Chan, Sonya James, Ruth R. French, Kathrine E. Attfield, Claire M. Brennan, Anupama Ahuja, Mark J. Shlomchik, Mark S. Cragg, and Martin J. Glennie Blood 2008 112: 4170-4177. [Abstract] [Full Text] [PDF] This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Beers, S. A. Articles by Glennie, M. J. Search for Related Content PubMed PubMed Citation Articles by Beers, S. A. Articles by Glennie, M. J. Related Collections Related Articles in Blood Online Social Bookmarking                   What's this?

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