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Blood, 15 October 2004, Vol. 104, No. 8, pp. 2540-2542. Prepublished online as a Blood First Edition Paper on June 22, 2004; DOI 10.1182/blood-2004-05-1733.
NEOPLASIA A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cellsFrom the Tenovus Research Laboratory, Southampton, United Kingdom; Cancer Research UK (CRUK) Oncology, Cancer Sciences Division, School of Medicine, University General Hospital, Southampton, United Kingdom; and Genmab, Utrecht, The Netherlands.
The chimeric anti-CD20 monoclonal antibody (mAb), rituximab, is an established part of the management of many non-Hodgkin lymphomas. The in vivo action of rituximab remains elusive, and this partially reflects a lack of highly specific reagents to detect rituximab binding at the cell surface. Here we report a new high-affinity mAb (MB2A4) with fine specificity for the idiotype of rituximab. It is able to detect rituximab in vitro, in the presence of high levels of human immunoglobulin G (IgG), in the serum of patients receiving rituximab therapy, and, surprisingly, when rituximab is bound to CD20 on the cell surface. We propose that the anti–idiotype (Id) binds to rituximab molecules bound univalently at the cell surface, facilitated by the relatively high off-rate of rituximab. This reagent provides new insights into the binding of rituximab at the cell surface and demonstrates a mode of binding that could be exploited for the surface detection of other mAbs with clinical and biologic applications.
The chimeric anti-CD20 monoclonal antibody (mAb), rituximab, has now been used in more than 300 000 patients and has become integrated into the treatment of many B-cell lymphomas.1-5 The use of rituximab is likely to increase even further with the recent observations that it may be of clinical benefit to patients with autoimmune diseases.5-8 Remarkably, given its widespread use, we still have a relatively poor understanding of the nature of rituximab binding to B cells and how this mAb actually mediates its therapeutic effects in vivo.3,9-15 There are currently few well-characterized, highly specific reagents to detect rituximab. Previous reagents have been polyclonal and directed either to the rituximab idiotype (Id)16,17 or the remaining mouse regions in rituximab.18 Clearly, these may suffer from batch variation, lack of specificity, and/or cross-reactivity to normal human immunoglobulin G (IgG). For this reason, we raised a highly specific mAb MB2A4 directed to the rituximab Id and have used this tool to probe the nature of rituximab binding to B-cell tumors.
Cell lines and antibodies Cells were obtained from the European collection of cell cultures (ECACC) and maintained in supplemented RPMI. mAbs and their fragments were produced using standard methods.19 Generation of anti-Id mAbs Anti-Id mAbs were generated by immunizing Lou rats with purified rituximab F(ab')2 fragments and fusing their splenocytes with NS-1 cells to generate hybridomas. The hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and assessed for binding to rituximab-coated plates. ELISA for rituximab To detect rituximab in patient serum, MB2A4 was coated onto ELISA plates, blocked, and washed. Subsequently, appropriately diluted rituximab-containing samples were added to the plate for 90 minutes, washed, and incubated with anti–human Fc–horseradish peroxidase (Hu Fc-HRP) mAb (SB2H2) for 60 minutes, before further washing and addition of the ELISA substrate. Flow cytometry for the detection of cell-bound rituximab To detect rituximab bound to the surface of CD20+ cells, standard flow cytometry techniques were used.20 Briefly, rituximab was coated to CD20+ cells for 15 minutes at room temperature (RT), and the cells were washed twice and incubated with fluorescein isothiocyanate (FITC)–labeled MB2A4. Labeled cells were assessed using a FACScan flow cytometer with CellQuest software (BD Biosciences, San Jose, CA).
Anti-rituximab idiotype (MB2A4) detects rituximab in sera Following immunization of Lou rats and subsequent fusions, a number of mAbs were raised that detected rituximab by ELISA. MB2A4 was chosen for further study, as it failed to bind to normal IgG in serum or another chimeric anti-CD20 mAb, chAT80 (Figure 1A). Importantly, MB2A4 did bind to 2B8, the nonchimeric parent mAb of rituximab (data not shown) and thus appears specific for the idiotype region of rituximab. Next, we addressed whether MB2A4 bound rituximab in patients' sera. The linear region of the standard curve occurred at less than 100 ng/mL, so patients' sera was first diluted accordingly. Figure 1B illustrates the variation in rituximab concentration over time for a representative patient during and following weekly administration of rituximab and demonstrates that MB2A4 is able to detect serum rituximab down to 5 to 10 ng/mL.
MB2A4 binds rituximab on the surface of B cells We then assessed whether MB2A4 could detect rituximab bound to CD20 on the surface of B-cell tumor lines, using FITC-labeled MB2A4. To our initial surprise, these experiments indicated that MB2A4 bound to rituximab-coated cells (Figure 1C-D). Binding was specific for the idiotypic region of rituximab and not other chimeric or fully human anti-CD20 mAbs, or chimeric mAbs against other B-cell markers (Figure 1C), and was dependent upon the amount of idiotype-containing mAbs (rituximab or 2B8) bound to the cells (data not shown). Further specificity was demonstrated when rituximab was incubated with whole blood where the anti-Id detected only the CD19+ B cells (Figure 1D). MB2A4 binds to rituximab through univalent binding
Because the specificity of anti-Id mAbs is highly dependent on the epitope structure of the complementarity determining regions (CDRs), it is expected that binding the target antigen will block binding of the anti-Id. Therefore, we wanted to determine the mechanism through which MB2A4 was binding to rituximab-coated cells. The first possibility addressed was whether rituximab was binding to the cells via Fc
In summary, we have developed a new high-affinity anti-Id mAb (MB2A4) that is equally capable of binding rituximab in the serum of patients or when bound to CD20 at the cell surface. This reagent represents a significant advance in our ability to accurately probe the pharmacokinetics, tissue distribution, and modulation of rituximab in vivo. We believe that this novel mode of mAb cell surface detection provides new insights into the mode of rituximab binding to B cells and this type of approach could be exploited for the detection of other mAbs with clinical and biologic applications.
The authors would like to acknowledge the assistance and input of Prof Jan van de Winkel and Carin EG Havenith (Genmab) and the members of the CD20 team both in Southampton and Utrecht.
Submitted May 6, 2004; accepted June 1, 2004.
Prepublished online as Blood First Edition Paper, June 22, 2004; DOI 10.1182/blood-2004-05-1733.
Supported by Tenovus of Cardiff, the Leukemia Research Fund, and Cancer Research UK.
M.S.C. and M.B.B. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mark Cragg, Tenovus Research Laboratory, Cancer Sciences Division, School of Medicine, University General Hospital, Tremona Road, Southampton, SO16 6YD United Kingdom; e-mail: msc{at}soton.ac.uk.
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Abstract
Introduction
), or serum spiked with 100 µg/mL rituximab (
), chAT13/5 (X), or chAT80 (
). The amount of human Fc captured to the plate was then detected using anti–human Fc coupled to HRP. (B) The amount of rituximab present in a patients' serum was detected over a period of time during which they had received rituximab. After collection of serum, a number of dilutions of the sample were made and then measured in an ELISA, such as detailed in panel A, along with a standard curve of known rituximab concentrations. Subsequently, reference to the standard curve allowed calculation of the serum rituximab level in the original sample. 
receptor IIb (Fc
) before washing and detection of FITC-mAb binding. (D) Schematic of MB2A4 binding to rituximab bound to the surface of CD20+ cells.
