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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 IBC Pharmaceuticals, Inc.; 2 Immunomedics, Inc., Morris Plains, New Jersey; and 3 Garden State Cancer Center, Center for Molecular Medicine and Immunology, Belleville, New Jersey
Requests for reprints: Chien-Hsing Chang, Immunomedics, Inc., 300 American Road, Morris Plains, NJ 07950. Phone: 973-605-1330, ext. 108; Fax: 973-605-1103; E-mail: kchang{at}immunomedics.com or David M. Goldenberg, Garden State Cancer Center, 520 Belleville Avenue, Belleville, NJ 07109. E-mail: dmg.gscancer{at}att.net.
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Abstract |
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Introduction |
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At present, next-generation anti-CD20 mAbs include human and humanized forms, with some claiming enhanced potency by antibody reengineering (1, 3–5). However, it is still unclear what improvements are required to show better efficacy than rituximab. Predicting on the basis of laboratory results has been difficult, because despite 10 years of clinical use of rituximab and an expansive preclinical literature on the use of anti-CD20 mAbs in lymphoma models in vitro and in vivo, how rituximab or other anti-CD20 mAbs kill lymphoma cells is still being debated (5) among the three mechanisms proposed: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and apoptosis or growth inhibition induced by direct signaling.
Dimeric forms of IgG made by genetic engineering (6–10) or chemical conjugation (11, 12) may become more effective antibodies than their monomeric counterparts as a result of increased valency, as well as enhanced effector functions. For example, a mAb for CD19 or CD20 that has little or no signaling activity was shown to induce growth inhibition or apoptosis when each is converted into an IgG-IgG homodimer (11, 12). Similar observations of attaining signaling activity or exhibiting higher cytotoxicity were reported for recombinantly produced fusion proteins comprising three identical anti-HER2 (13) single-chain Fvs (scFv), four identical anti-CD22 (14, 15) scFvs, or four identical anti-CD20 (16) scFvs. In addition, a dextran-rituximab polymer was found to inhibit the growth of Raji xenografts in mice, whereas the homodimer or monomer of rituximab was ineffective (17). Therefore, we propose that making multivalent or multimeric mAbs of a defined composition and proved homogeneity should be promising for developing more potent anti-CD20 agents, which may also help elucidate the most critical mechanism of action in vivo.
Veltuzumab or hA20 is a complementarity-determined region–grafted, anti-CD20 humanized mAb that exhibits CDC and ADCC in vitro, as well as inhibits cell proliferation and induces apoptosis upon cross-linking with a second antibody (18). Initial clinical studies have shown good safety and efficacy results in NHL patients and confirmed that low doses of veltuzumab are effective and that all doses can be given in shorter infusion times than the approved dose of rituximab (19).
The Dock-and-Lock (DNL) method exploits the dimerization and docking domain (DDD) and the anchoring domain (AD) that are involved in the natural binding between cyclic AMP–dependent protein kinase and A-kinase anchoring proteins (20, 21) as a pair of linkers for specifically docking a DDD-containing module into an AD-containing module, with the resulting binary complex further stabilized covalently by locking with disulfide bonds (22, 23). One distinct advantage of the DNL method, as noted previously (23), is to provide a modular approach for generating multivalent antibodies of monospecificity or bispecificity via site-specific conjugation of two types of recombinantly produced fusion proteins that are derived from the same or different antibodies. Here, we describe a veltuzumab-based hexavalent antibody (Hex-hA20) made by DNL to consist of a bivalent IgG linked to four monovalent Fabs, and the evaluation of its antitumor activity against CD20-expressing human lymphoma cells in vitro and in vivo. The versatile DNL method was also applied to generate two other multivalent anti-CD20 antibodies without the Fc region: Tri-hA20, a trivalent construct composed of three hA20 Fabs, and Tetra-hA20, a tetravalent construct composed of four hA20 Fabs. All three multivalent anti-CD20 antibodies were purified to near homogeneity and shown to retain the binding activity of each constituent Fab. In addition, testing of Tri-hA20 allowed us to establish that clustering three CD20 molecules on the cell surface via antibody binding is sufficient to induce growth inhibition. Schematic diagrams for Hex-IgG, Tri-Fab, Tetra-Fab, and the modules used for building them are shown in Fig. 1 .
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Materials and Methods |
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Cell lines. Daudi, Raji, and Ramos were purchased from the American Type Culture Collection. Sp/ESF, a variant of Sp2/0-Ag14 engineered to grow in serum-free medium,4 was used as the host cell for transfection.
Generation of Hex-hA20. The pdHL2 vector has been used to mediate the expression of recombinant IgGs in mammalian hosts (24). A plasmid shuttle vector was produced to facilitate the conversion of any IgG-pdHL2 vector into a CH3-AD2-IgG-pdHL2 vector. The expression vector encoding CH1-DDD2-Fab-hA20 was generated from the CH3-AD2-IgG-hA20-pdHL2 by excising the coding sequence for the CH1-Hinge-CH2-CH3 domains with SacII and EagI and replacing it with a 507-bp sequence encoding CH1-DDD2, which was excised from the C-DDD2-hMN-14-pdHL2 expression vector (22) with the same enzymes. The expression vector CH3-AD2-IgG-hA20-pdHL2 or CH1-DDD2-Fab-hA20-pdHL2, each 30 µg, was linearized by digestion with SalI and transfected into Sp/ESF (2.8 x 106 cells) by electroporation (450 V, 25 µF). The pdHL2 vector contains the gene for dihydrofolate reductase, thus allowing clonal selection, as well as gene amplification, with methotrexate (MTX). After transfection, the cells were plated in 96-well plates and selected in media containing 0.2 µmol/L MTX. Clones were screened for CH3-AD2-IgG-hA20 or CH1-DDD2-Fab-hA20 productivity by a sandwich ELISA using 96-well microtiter plates coated with WR2 to capture the fusion protein, which was detected with horseradish peroxidase–conjugated goat anti-human IgG F(ab')2. Wells giving the highest signal were expanded and ultimately used for production.
CH3-AD2-IgG-hA20 and CH1-DDD2-Fab-hA20 were produced in roller bottles, purified by Protein A (MabSelect, Amersham-Biosciences) and Protein L (CBind L, Fluka), respectively, and stored in PBS. To generate Hex-hA20, a mixture of CH1-DDD2-Fab-hA20 (134 mg) and CH3-AD2-IgG-hA20 (100 mg) was treated with 1 mmol/L reduced glutathione at room temperature for 16 h, followed by 2 mmol/L oxidized glutathione for 24 h, from which Hex-hA20 was purified by Protein A. DNL-20/14 was made similarly by reacting CH3-AD2-IgG-hA20 with CH1-DDD2-Fab-hMN-14 (22).
Generation of Tetra-hA20 and Tri-hA20. Tetra-hA20 was obtained by purifying the tetrameric form of CH1-DDD2-Fab-hA20 over a Superdex-200 column. Tri-hA20 was obtained by linking the dimeric form of CH1-DDD2-Fab-hA20 covalently to CH1-AD2-Fab-hA20, which was produced as described for h679-Fab-AD2 (22).
Molecular size. SE-HPLC was performed on a Beckman System Gold Model 116 with a Bio-Sil SEC 250 column (Bio-Rad) and 0.04 mol/L PBS (pH 6.8) and 1 mmol/L EDTA as the mobile phase.
SDS-PAGE. Reducing and nonreducing SDS-PAGE analyses were performed using 4% to 20% gradient Tris-glycine gels (Cambrex Bio Science).
Mass spectrometry. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry was performed in a sinapinic acid matrix by Scripps Center for Mass Spectrometry.
Competition ELISA. Microtiter plates were coated overnight with veltuzumab at 5 µg/mL and blocked with PBS containing 2% bovine serum albumin (BSA) for 1 h. Hex-hA20 and veltuzumab, serially diluted in triplicate, were each mixed with WR2 at 1 nmol/L and added to the coated wells. The bound WR2 was quantified with peroxidase-conjugated goat anti-rat IgG and O-phenylenediamine dihydrochloride.
Flow cytometry. Apoptosis, viable cell counting, cell binding, and off-rate measurements were performed by flow cytometry on a Guava PCA (Guava Technologies, Inc.) using the manufacturer's reagents, protocols, and software.
Scatchard analysis. The maximum number of binding sites per cell and the apparent affinity constants were determined by nonlinear regression analysis of the saturation binding data obtained with radio-iodinated samples and Raji cells using Prism software (GraphPad Software, Inc.). Samples were run in triplicate. Immunoreactivity of each radiolabeled preparation was 90% or greater, as measured by binding to WR2.
Cell proliferation assay. The in vitro cytotoxicity was determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), which is chemically reduced by metabolically active cells into formazan, and the intensity of the resulting color is proportional to the number of living cells.
CDC. Cells were seeded in black 96-well plates (Nunc) at 5 x 104 cells in 50 µL/well and incubated with serial dilutions (concentration range, 3.33 x 10-8 to 2.6 x 10-10 mol/L) of test and control mAbs in the presence of human complement (1:20 final dilution, Quidel Corp.) for 2 h at 37°C and 5% CO2. Viable cells were then quantified using the Vybrant Cell Metabolic Assay Resazurin kit (Invitrogen). Controls included cells treated with 0.25% Triton X-100 (100% lysis) and cells treated with complement alone (background).
ADCC. Daudi cells were incubated with each test article in triplicate at 5 µg/mL for 30 min at 37°C and 5% CO2. Freshly isolated peripheral blood mononuclear cells obtained from healthy volunteers were then added at a predetermined optimal effector to target ratio of 50:1. After a 4-h incubation, cell lysis was assessed by CytoTox-One (Promega).
Calcium mobilization. Intracellular calcium was measured in Ramos cells loaded with 20 µmol/L Fluo-3 AM (Invitrogen) using a Becton Dickinson FACScan and the FlowJo program (Tree Star, Inc.). For all samples, a baseline was obtained for 60 s before adding each test article, which includes ionomycin and anti-human IgM as positive controls. To evaluate the effect of cross-linking, cells were incubated with 1 µg/mL of veltuzumab, rituximab, or tositumomab for further 15 min and stimulated with an appropriate second antibody (50 µg/mL final).
Homotypical adhesion. Daudi cells (1.5 x 106/mL) were treated with veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at 1 nmol/L for 20 h and then examined with an inverted phase-contrast microscope. The results were scored semiquantitatively according to Polyak and Deans (25).
Animal studies. The pharmacokinetic analysis was performed in naive female Swiss-Webster mice (Taconic Farms) and compared with Hex-hA20 and veltuzumab given either i.v. or s.c. using radio-iodinated samples (Supplementary Materials and Methods online). The in vivo efficacy was evaluated in tumor-bearing SCID mice as described in the figure legends. Depletion of natural killer (NK) cells and neutrophils was performed as described (26), with the following modifications. Briefly, mice received i.p. injections of antimouse Gr-1 ascites (100 µL) and TMβ-1 mAb (100 µg) 1 d before inoculating Raji cells and three more weekly i.p. injections of antimouse Gr-1 ascites on days 6, 13, and 20 to maintain neutrophil depletion, which was confirmed by fluorescence-activated cell sorting analysis of blood samples taken from one treated and one untreated mouse on days 3, 13, and 20. Mice deemed to have succumbed to disease progression, when hind-limb paralysis developed or if they otherwise became moribund, were humanely sacrificed. Additionally, if mice lost >20% of initial body weight, they were sacrificed. Survival curves were analyzed using Kaplan-Meier plots (log-rank analysis) and Prism software. All animal studies were performed after approval of the local animal care and use committee.
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Results |
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The purity of Hex-hA20 was shown by reducing SDS-PAGE, which showed only three bands from the constitutive polypeptides (Fig. 2, lanes 1 and 2 ). In addition, nonreducing SDS-PAGE analysis of Hex-hA20 confirmed its covalent structure, because no bands corresponding to the monomeric form of CH3-AD2-IgG-hA20 were observed (Fig. 2, lanes 5 and 6). The molecular mass of Hex-hA20 was determined to be 368,475 Da by MALDI-TOF mass spectrometry, which agrees well with the calculated molecular weight of 362 kDa for Hex-hA20 from the deduced amino acid sequences of the constituent polypeptides.
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chain) on reducing SDS-PAGE. These results are corroborated by the HPLC data shown in Supplementary Fig. S1A-D.
Binding analysis. As shown by competition ELISA in Fig. 3A
, Hex-hA20 has a 3-fold higher avidity than veltuzumab, suggesting that at least three or more of the six Fab components are capable of binding simultaneously to the WR2 antiidiotype antibody. The binding of Hex-hA20 to CD20 on live cells was compared with that of veltuzumab by flow cytometry. Figure 3B shows that Hex-hA20 resulted in 40% to 50% greater fluorescence intensity than veltuzumab when probed by PE–anti-Fab. In contrast, the signal observed for Hex-hA20 with PE–anti-Fc was lower than that of veltuzumab. These results are consistent with Hex-hA20 having four more Fabs than veltuzumab, but only one Fc like veltuzumab. Additional evidence for the higher valency and avidity of Hex-hA20 is provided by Scatchard analysis of binding to Raji cells (Fig. 3C), which shows that the apparent association constant of Hex-hA20 [
3.9 x 108 (mol/L)–1] is 4-fold higher (P < 0.0001) than that of veltuzumab [1 x 108 (mol/L)–1], whereas an F test determined that the saturation binding curves for rituximab and veltuzumab were similar (P = 0.1859). More importantly, the number of receptors per cell calculated from the maximum number of binding sites (Bmax) obtained for Hex-hA20 was found to be 1/4 of that obtained with veltuzumab (100,000 versus 437,5000), suggesting that all six Fabs of Hex-hA20 are capable of binding to CD20 on the cell surface, because the same number of CD20 molecules would require thrice as many veltuzumab to occupy them. The data obtained from the off-rate measurements (Fig. 3D) also indicate that Hex-hA20 dissociates 3-fold slower than veltuzumab, which remarkably dissociates 2-fold to 3-fold slower than rituximab.5
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The effect of Z-VAD-FMK (a broad-spectrum caspase inhibitor) on apoptosis induced by Hex-hA20 was examined in Ramos, and the results (Supplementary Fig. S2) indicate that Z-VAD-FMK at 100 µmol/L completely prevented the apoptosis induced by antihuman IgM, but not that induced by Hex-hA20, suggesting that both caspase-dependent and caspase-independent pathways occur for Hex-hA20, as reported for rituximab with cross-linking (27). Although CD20 clustering is presumably achievable either indirectly by cross-linking the antigen-bound veltuzumab with a second antibody or directly via multivalent engagement of Hex-hA20, the former, but not the latter, leads to a rapid rise in intracellular calcium levels (Supplementary Fig. S3).
Effector functions. CDC activity was evaluated in vitro using human complement and Daudi cells (Fig. 5B, left). Veltuzumab exhibits potent CDC activity. Surprisingly, Hex-hA20 fails to induce CDC in Daudi cells. Because CH3-AD2-IgG-hA20 induces CDC with similar potency as veltuzumab, modification of the carboxyl termini of veltuzumab by the addition of the small AD2 peptide does not affect CDC, suggesting that the addition of the four Fab-DDD2 groups apparently prevents complement fixation, despite the ability of Hex-hA20 to bind C1q (data not shown). Hex-hA20 and veltuzumab have comparable ADCC (Fig. 5B, right). Thus, the Fc of Hex-hA20 induces ADCC.
Homotypical adhesion. Hex-hA20, Tetra-hA20, and Tri-hA20 induced homotypical adhesion of Daudi cells, resulting in >50% of cells having medium-size to large-size aggregates, whereas under the same conditions, the extent of cell aggregation observed for veltuzumab was similar to that of the untreated control (Supplementary Fig. S4).
Membrane localization of CD20/Hex-hA20. Veltuzumab translocates CD20 into lipid rafts, as shown previously (28) by Western blot analysis of the cell lysates in fractions isolated from sucrose density gradient centrifugation. The distribution of CD20 at the cell surface upon binding to veltuzumab or Hex-hA20 was examined further with immunofluorescence microscopy using cholera toxin subunit B-Alexa Fluor 488 as the reporter for ganglioside GM-1, a common lipid raft marker (29). As shown in Supplementary Fig. S5A-D, incubation of Daudi cells with veltuzumab or Hex-hA20 led to the formation of membrane patches or caps with punctuate spots, which were superbly matched by the images obtained with cholera toxin subunit B, indicating the localization of CD20 in lipid rafts. Although the fluorescent patterns observed were similar, veltuzumab seemed to form larger and fewer patches than Hex-hA20.
Serum stability. Hex-hA20 was found to have the same stability in serum as veltuzumab, maintaining 86% binding activity after 11 days (Supplementary Fig. S6). These results are similar to those of the bispecific Tri-Fab complexes reported previously (22).
Pharmacokinetic analysis. Using radio-iodinated preparations, it was found that Hex-hA20 cleared 4.5 times faster than veltuzumab when given i.v. (Supplementary Fig. S7A), resulting in a 3.7-fold lower mean residence time for Hex-hA20 compared with veltuzumab (127 hours versus 472 hours). When given s.c. (Supplementary Fig. S7B), Hex-hA20 cleared at a much faster rate (T1/2 3 days) than veltuzumab (T1/2 >9 days), with both having the same Tmax of 24 hours. However, the Cmax was only half as high for Hex-hA20 compared with veltuzumab (12.9 nmol/L versus 25.7 nmol/L). Pharmacokinetic variables determined for Hex-hA20 and veltuzumab are provided in Supplementary Table S1.
In vivo efficacy. In the multiple-dose study with the Daudi model, mice were treated with Hex-hA20 at 30 µg (q7dx2) and 6 µg (q7dx2). As shown in Fig. 6A , both treatments resulted in significantly improved median survival time (MST) when compared with the saline control (66.5 and 42 versus 21 days; P < 0.0001). Although no significant difference in survival was observed between mice given Hex-hA20 and veltuzumab at the higher doses, it seemed that mice receiving Hex-hA20 at the lower doses had significantly lower MST than those receiving veltuzumab at an equivalent dose (P = 0.0044).
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Discussion |
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Based on their efficacy in certain in vitro assays, anti-CD20 mAbs have been classified by Cragg and colleagues (35) as either type I, represented by rituximab, or type II, represented by tositumomab (36). Type I anti-CD20 mAbs are characterized by potent CDC, rapid calcium mobilization upon the addition of a second antibody (37, 38), weak induction of homotypical adhesion (39), enhanced association of CD20 with lipid rafts (40), and a general requirement of cross-linking for efficient antiproliferation or apoptosis (39). In contrast, type II anti-CD20 mAbs lack CDC, fail to bring CD20 into lipid rafts (3, 4), induce a high level of homotypical adhesion (39), and do not stimulate calcium mobilization upon further cross-linking (37). A further differentiation is that apoptotic cell death mediated by type I mAbs involves both caspase-dependent and caspase-independent pathways, but only caspase-independent pathways seem to be associated with type II mAbs (4). However, both type I and type II anti-CD20 mAbs are effective in ADCC and apparently bind to the same region of the extracelluar loop of CD20 on the plasma membrane (41–43). Interestingly, at saturation, the number by which a type II mAb binds to each CD20-positive B cell only approaches half of that observed with a type I mAb (3, 4), suggesting that a type II mAb may act virtually as a tetravalent antibody by engaging two adjacent extracellular loops of CD20 with each antigen-binding site.
We note that Hex-hA20 exhibits biological properties attributable to both type II (for example, negative for CDC and calcium mobilization; positive for antiproliferation, apoptosis, and homotypical adhesion) and type I (for example, positive for trafficking to lipid rafts). Thus, one effective approach to converting a type I anti-CD20 mAb to a type II can be achieved by making the type I mAb multivalent. Preliminary investigation of the signaling pathway indicates that Hex-hA20 induces caspase-dependent, as well as caspase-independent, apoptosis. Additional studies are in progress to identify the subcellular events associated with the binding of CD20 by Hex-hA20 or Tri-hA20, which may reveal unequivocally the molecular factors that account for the antiproliferative potency of a multivalent anti-CD20 antibody with defined composition.
When tested against human lymphoma xenografts in mice, the therapeutic activity of Hex-hA20 was comparable, but not better, than the parental veltuzumab, except when a higher mole-equivalent dose (such as 465 µg) was given. Because mouse complement was reported to be inactive in mediating CDC of the DHL-4 cell line in vitro by rituximab (26), the shorter half-life of Hex-hA20 rather than the lack of CDC may explain why the in vivo antitumor efficacy of Hex-hA20 was not superior to that of the bivalent veltuzumab, suggesting that more frequent or higher doses of Hex-hA20 may be indicated. It is also noted that cross-linking of veltuzumab may occur in vivo via binding to effector cells (44), thus mimicking the advantage of Hex-hA20 over veltuzumab in vitro. The observation that neither veltuzumab nor Hex-hA20 is effective in mouse models depleted with NK cells and neutrophils emphasizes the importance of ADCC for in vivo killing of tumor cells. Whether a multivalent anti-CD20 construct can be made by DNL to retain CDC and show more potent antitumor effects in vivo is worthy to determine. On the other hand, because CDC has been suggested to be a major factor in infusion-related toxicity for rituximab, it is tempting to speculate that Hex-hA20 may be a viable alternative where reduced side effects are desired.
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Disclosure of Potential Conflicts of Interest |
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Meiyu Loo, Diana Delaney, Diane Nordstrom, and John Kopinski for excellent technical assistance, Dr. Xiaochuan Chen for advice on measuring cytoplasmic calcium by flow cytometry, and Dr. Francisco Hernandez-Ilizaliturri for the protocol of depleting NK cells and neutrophils in SCID mice.
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Footnotes |
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4 D. Nordstrom, E.A. Rossi, D.M. Goldenberg, C-H. Chang, unpublished data. 
D.M. Goldenberg, E.A. Rossi, R. Stein, T.M. Cardillo, M.S. Czuczman, F.J. Hernandez-Ilizaliturri, H.J. Hansen, C-H. Chang. Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody (mAb), submitted for publication.
Received 5/29/08. Revised 8/13/08. Accepted 8/14/08.
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fusion protein induces HER2/neu signaling and facilitates repair of injured epithelia. J Pharmacol Exp Ther 2006;316:983–91.This article has been cited by other articles:
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  E. A. Rossi, D. M. Goldenberg, T. M. Cardillo, R. Stein, and C.-H. Chang CD20-targeted tetrameric interferon-{alpha}, a novel and potent immunocytokine for the therapy of B-cell lymphomas Blood, October 29, 2009; 114(18): 3864 - 3871. [Abstract] [Full Text] [PDF]
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E. A. Rossi, D. M. Goldenberg, T. M. Cardillo, R. Stein, and C.-H. Chang Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma Blood, June 11, 2009; 113(24): 6161 - 6171. [Abstract] [Full Text] [PDF]
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) or veltuzumab (
) were incubated at varying concentrations in the presence of WR2 for competition of binding with immobilized veltuzumab. The percentage of inhibition was plotted versus mAb concentration, and EC50 values were generated with Prizm software. B, binding to Daudi cells as determined by flow cytometry using PE-conjugated anti-human Fab (PE–anti-Fab) or PE-conjugated anti-human Fc (PE–anti-Fc). All incubations and washes were performed at 4°C. Daudi cells were suspended at 1 x 106 cells/mL in 1% BSA-PBS and incubated with Hex-hA20, veltuzumab, or labetuzumab for 1 h. The cells were washed with 1% BSA-PBS, incubated with a 1:200 dilution of PE–anti-Fab or PE–anti-Fc for 30 min, washed once more, and analyzed on a Guava PCA using the Guava Express software application. C, Scatchard analysis using radio-iodinated Hex-hA20 (
), or rituximab (
), veltuzumab (
). Briefly, cells were placed in 96-well plates at 5,000 cells per well in complete RPMI 1640. Five-fold serial dilutions of Hex-hA20, veltuzumab, or veltuzumab cross-linked with goat-anti human Fc were added to triplicate wells at final concentrations ranging from 2 x 10–8 to 6.4 x 10–12 mol/L. The plates were incubated for 4 d, after which 20 µL of CellTiter 96 Aqueous One Solution Reagent (Promega Corp.) was added, and the incubation was continued for an additional 4 h before reading the plates at 490 nm. B, in vitro antiproliferation determined by the viable cell counting assay for Ramos (left) or Raji (right). Cells were seeded in T-flasks at 1 x 105 cells/mL and treated with veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at the indicated concentrations. Viable cell densities (VCD) were determined daily over 5 d by flow cytometry using Guava Viacount. On day 3, cultures were split 1:2 to maintain logarithmic growth. Cells were plotted as viable cells per milliliter measured on days 3, 4, and 5 for veltuzumab (gray lines and symbols) at 15 nmol/L (
), 2.5 nmol/L (
), or 0.5 nmol/L (
), Tri-hA20 (
) at 0.5 nmol/L, Tetra-hA20 (
) at 0.5 nmol/L.
) in the presence of human complement. The percentage complement control (number of viable cells in the test sample compared with cells treated with complement only) was plotted versus the log of the nanomolar concentration. ADCC (right) was measured for Hex-hA20, veltuzumab, epratuzumab, or labetuzumab at 5 µg/mL using Daudi as the target cells and freshly isolated peripheral blood mononuclear cells from two donors as the effector cells. A 100% lysis reference was generated by the addition of detergent to wells containing target cells only. The bar graphs show percentage of lysis obtained for each of the two donors.