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NEOPLASIA
From the Cancer Immunobiology Center, University of
Texas Southwestern Medical Center at Dallas, TX.
In 1997, a chimeric anti-CD20 monoclonal antibody (mAb)
(Rituxan) was approved for the treatment of
low-grade/follicular B-cell lymphoma. Rituxan has a long half-life and
low immunogenicity, and it mediates effector function. Rituxan induces
apoptosis in some tumor cell lines in vitro. Previous studies with mAbs
that react with neoplastic B cells have demonstrated that homodimers of
immunoglobulin G ([IgG]2) often inhibit cell
growth more effectively than their monomeric (IgG)1
counterparts. In this study, the ability of IgG or
F(ab')2 homodimers vs monomers of Rituxan were
compared for their ability to inhibit the growth of several
different B-lymphoma cell lines in vitro. It was found that homodimers
of Rituxan had superior antigrowth activity in vitro and that
F(ab')2 homodimers were the most active.
Homodimers, but not monomers, of Rituxan induced both apoptosis
and necrosis of several B-cell lymphoma lines in vitro; the inhibition
of cell growth was not dependent upon the presence of Fc receptors
or upon 10-fold or greater differences in the density of CD20
on the target cells. Rituxan homodimers, compared with monomers, also
rendered drug-resistant CD20+ B-lymphoma cells more
sensitive to chemotherapeutic agents and synergized with an anti-CD22
immunotoxin in vitro.
(Blood. 2001;97:1392-1398) In a previous study, we demonstrated that
monoclonal antibodies (mAbs) (ie, anti-CD19, anti-CD20, anti-CD21, and
anti-CD22) that have little or no antigrowth activity on
neoplastic B-cells can become potent antitumor agents when they are
converted into homodimers.1 These homodimers exert their
antigrowth activity either by signaling G0/G1
arrest or by inducing apoptosis, depending upon the cell surface
molecule to which they bind. Our results confirmed and extended
previous reports in which a chimeric mAb (Chi BR96) homodimer killed
tumor cells 10-fold more effectively than its monomeric
counterpart.2
The chimeric anti-CD20 mAb (Rituxan/Rituximab; Genentech, San
Francisco, CA) has recently been approved by the Federal Drug Administration in the United States for the treatment of patients with
relapsed or refractory, low-grade or follicular, B-cell non-Hodgkin lymphoma, and in Europe for patients with relapsed stage III/IV follicular lymphoma (reviewed in Grillo-Lopez et al3 and
Gopal and Press4). The overall response was 50% when it
was used as a single agent,4,5 and response rates were
significantly higher when it was used in combination with
chemotherapy.6 It is not yet known whether Rituxan
will improve the long-term survival of patients with low-grade
lymphoma, although early results look encouraging.3,4
In vitro studies, as well as studies in both animals and humans,
suggest that the antitumor activity of Rituxan can be mediated by
antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).3,4,7 It has also been shown
that Rituxan can induce apoptosis7-10 in DHL-4 or
DHL-4-derived cell lines with bcl2 gene
rearrangements.9,10 However, in accord with studies using
other anti-CD20 mAbs such as 1F5 and B1,11 we could induce
only modest levels of apoptosis with Rituxan (even in DHL-4 cells)
unless we hypercrosslinked the bound Rituxan with rabbit antihuman
immunoglobulin (Ig)-G (RAHIg) or goat antimouse immunoglobulin
(GAMIg). In accord with these observations, it has recently
been reported that Rituxan can induce apoptosis in lymphoma cells from
patients only when GAMIg is added.10
Because hypercrosslinking with a secondary antibody would be
impractical in vivo, in this study we evaluated tetravalent
F(ab')2 or IgG homodimers of Rituxan for their ability to
exert antigrowth activity and to induce apoptosis in B-lymphoma cell
lines. We found that homodimers had superior antigrowth activity and
induced apoptosis in several neoplastic B-cell lines in vitro.
Apoptotic activity did not require the participation of Fc receptors
(FcRs) on target cells and was caspase dependent. Treatment
with homodimers, compared with monomers, also rendered tumor cell lines
more sensitive to chemotherapeutic agents and synergized with an
anti-CD22-immunotoxin (IT) in vitro.
Cells
Antibodies and reagents
Flow cytometric assays Indirect immunofluorescence assays were carried out by means of the panel of mAbs described above and FITC-GAMIg. The mAbs (monomers and homodimers) were labeled with FITC as described elsewhere14 and were used to determine the cell-binding and cell-dissociation rates of mAbs. Cells were suspended at 106 cells per milliliter in CM and were treated with 0.1 to 1.0 µg/mL mAbs for 30 minutes on ice, washed twice with CM containing 0.1% sodium azide, resuspended in 100 µL CM, and treated with 2 to 3 µL FITC-GAMIg. The direct immunofluorescence assay was carried out by means of FITC-mAbs. To determine the dissociation rate of FITC-mAbs (monomers vs homodimers), cells were treated as described above with saturating concentrations of FITC-mAbs. The excess mAbs were washed out, and the cells were incubated at 37°C for 2, 4, 6, 24, 48, and 72 hours in CM. At the indicated intervals, samples were taken, washed, and analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ) to determine the percentage of the remaining cells that were still stained.Preparation of homodimers of mAbs Homodimers of Rituxan and the mouse IgG1 control mAb 3F12 were prepared and purified as previously described.1 Briefly, we used 2 hetero-bifunctional cross-linkers, succinimidyl 4-(maleimidemethyl) cyclohexane-1-carboxylate (SMCC) and N-succinimidyl S-acethylthio-acetate (SATA) (Pierce, Rockford, IL) to bind 2 IgG molecules through a stable thioether bond. The final product was a mixture of polymers, trimers, dimers, and monomers, which were further separated on a preparative 21.5 mm inner diameter (ID) × 60 cm TSKG3000SW (Tosohaas, Montgomeryville, PA) high-performance liquid chromatography (HPLC) column. The protein fractions obtained by HPLC were concentrated to 5 to 10 mg/mL, filter-sterilized, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC. The purity of the homodimer fraction was 80% to 90%.Preparation of F(ab')2 homodimers HPLC-purified homodimers were digested with insoluble pepsin (Sigma) as previously described.15 The F(ab')2 fraction was affinity-purified on a protein G-Sepharose column.Preparation of an immunotoxin using good laboratory practice The mAb, RFB4, which recognizes human CD22, was coupled to deglycosylated (dg) ricin toxin A chain (RTA). RFB4-dgRTA was prepared in our good laboratory practice (GLP) scale-up laboratory as described.16 RFB4-dgRTA, has been evaluated in severe combined immunodeficient (SCID)/Daudi mice (reviewed in Ghetie et al17) and in patients with B-cell lymphoma (reviewed in Sausville and Vitetta18 and Engert et al19).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Proteins were analyzed under both reducing and nonreducing conditions by SDS-PAGE on 4% to 15% gels by means of the Pharmacia PhastSystem Separation Unit.20 Protein bands were visualized by staining the gels with Coomassie blue.Molecular size determination by analytical HPLC The molecular weights of the dimers were determined by analytical HPLC with the use of a 7.5 mm ID × 60 cm TSKG4000SW column (Tosohaas). A standard protein mixture of apoferritin (450 kd), IgA (300 kd), and IgG (150 kd) was fractionated on the same HPLC column to establish the retention time of known molecular weight standards.Measurement of cell growth We incubated CD20+ human cell lines [Ramos, DHL-4, Daudi, Raji] and the CD20 MOLT-4 cells at
2 × 105 cells per milliliter in CM at 37°C for
different time periods in the presence and absence of different
concentrations of anti-CD20 (1, 10, and 100 µg/mL). The irrelevant
IgG1 isotype-matched mAb 3F12 and a human IgG (100 µg/mL) were used as a controls. Cells were stained with trypan blue
and counted. Cell growth rates were then determined.
3H-thymidine assay The antiproliferative activity of different mAb monomers and homodimers was determined by means of a 3H-thymidine incorporation assay on Ramos, DHL-4, Daudi, Raji, Namalwa, Namalwa/MDR1, and MOLT-4 cells. Briefly, 5 × 104 cells in 100 µL CM were plated in 96-well plates (Costar, Corning, NY) and incubated for 24 to 48 hours with 100 µL of different concentrations (0.01 to 1.0 mg/mL) of mAbs (monomers and homodimers) diluted in CM. To determine the cytotoxic effect of the immunotoxin (IT), the cells were incubated with 6 serial dilutions (108 to 1013 M) of either IT alone or IT in
combination with a constant amount (20 µg/mL) of Rituxan monomers or
homodimers. Doxorubicin was used in a range of 1 to 70 nM, either alone
or in the presence of Rituxan as described above. After incubating the
cells at 37°C, the plates were pulsed for 4 hours with 1 µCi
3H-thymidine (Amersham Pharmacia Biotech,
Piscataway, NJ), harvested, and counted in a liquid scintillation
spectrometer. The percentage reduction in 3H-thymidine
incorporation, compared with untreated controls, was used to quantitate
the cytotoxic effect.
FITC-Annexin V assay We treated 2 × 105 cells with different concentrations (1 to 100 µg/mL) of Rituxan or irrelevant mAbs (monomers and homodimers); with Rituxan monomers (10 µg/mL) and GAMIg (50 µg/mL) or rabbit antihuman immunoglobulin (RAHIg) (10 µg/mL); or with GAMIg or RAHIg alone for 2, 4, 8, and 24 hours at 37°C. Cells were washed, treated with FITC-Annexin V and propidium iodide (PI), washed again, and analyzed within 1 hour on a FACScan. FL1-positive cells (stained with FITC-Annexin) were considered apoptotic and FL2-positive cells (permeable to PI) were considered necrotic. NaN3 (5 to 10 mg/mL) was used as a positive control for the induction of apoptosis. The caspase inhibitors (Z-VAD-FMK, Ac-YVAD-CHO, and Z-DEVD-FMK) were used at 50 µM.
Characterization of Rituxan multimers Rituxan multimers were separated on an HPLC column and were characterized by SDS-PAGE and by binding to, and dissociation from, cells.HPLC-purification and SDS-PAGE.
The initial Rituxan contained approximately 100% monomers (150 kd).
Homodimers of Rituxan were purified by preparative HPLC (Figure
1). We obtained 4 fractions and analyzed
them by SDS-PAGE. Fraction 1 consisted of high molecular weight
polymers (greater than 450 kd); fraction 2 consisted of approximately
80% trimers (450 kd); fraction 3 consisted of 80% to 90% dimers (300 kd); and fraction 4 consisted of greater than 90% monomer (150 kd).
Binding of Rituxan to cells.
The 6 CD20+ B-cell lines and the CD20
Dissociation of Rituxan from cells. Dissociation experiments were performed by means of a direct immunofluorescence assay in which the DHL-4 or Ramos cells were saturated with FITC-mAbs and the positively stained cells were quantified after different time intervals in culture. As shown in Figure 2C, the monomers dissociated much more rapidly than the homodimers (T1/2 of 2 vs more than 72 hours), and the trimers showed no dissociation even after 72 hours. The increased rate of dissociation as the valency of the mAb increased indicates a stronger and more prolonged interaction with the target cells. These results support the previously reported finding that CD20 does not internalize.21,22 Taken together, these experiments demonstrate that homodimers and trimers have similar binding affinity but a slower rate of dissociation than monomers.Growth inhibition by monomers, homodimers, and trimers Growth index.
Several different lymphoma cell lines were cultured with different
amounts of Rituxan monomers or homodimers for 6 days. At different time
intervals, the cells were counted, and their growth indexes and
viabilities were compared with those of untreated cells. As shown in
Figure 3 the growth indexes of cells
treated with either monomers or homodimers (either intact IgG or as
F(ab')2) were lower than those of untreated cells. DHL-4
and Ramos cells treated with homodimers grew more slowly than those
treated with monomers, demonstrating that the antigrowth activity of
Rituxan was increased by using it in homodimeric form. The
F(ab')2 homodimers also had significant antigrowth
activity, suggesting that interaction with cellular Fc
Early apoptotic events induced by homodimers versus monomers.
The FITC-Annexin V assay demonstrated that monomers of Rituxan did not
induce apoptosis unless cell-bound mAbs were cross-linked with a
secondary antibody (GAMIg) (Table 2).
Annexin V positivity was achieved after incubating cells with Rituxan
monomers cross-linked with GAMIg at 37°C for 24 hours. A comparable
effect was observed when Ramos cells were treated for 24 hours with
homodimers or with monomers and GAMIgM (Table 2). The positive
control, sodium azide, induced a very profound apoptotic effect after
24 hours. The apoptotic activity of Rituxan homodimers on different
B-cell lines is shown in Table 3.
Apoptosis was completely inhibited by the irreversible caspase
inhibitors Z-VAD-FMK and Z-DEVD-FMK and partially inhibited by the
reversible inhibitor (Ac-YVAD-CHO) of PARP cleavage (Figure
4).
Inhibition of 3H-thymidine incorporation.
The Rituxan monomers, homodimers, and trimers were incubated for
different intervals of time with Ramos, DHL-4, Daudi, Raji, Namalwa,
and Namalwa/MDR1 cells, and the levels of 3H-thymidine
incorporation were measured and compared with those of untreated cells
(Table 3). CD20 Cytotoxic effect of combinations of mAbs and doxorubicin We used 3 cell lines with different sensitivities to doxorubicin: DHL-4, Namalwa, and Namalwa/MDR1. When treated with doxorubicin alone, the IC50 for drug-sensitive Namalwa cells was 3- to 4-fold lower than that of either drug-resistant Namalwa/MDR1 cells (Figure 5C) or the less drug-sensitive DHL-4 cells (Figure 5B). When Rituxan monomers (20 µg/mL) were added to different concentrations of doxorubicin (1 to 70 nM), their IC50 on DHL-4 (Figure 5A) or Namalwa/MDR1 (Figure 5C) cells decreased in a dose-dependent manner, suggesting that both the drug-resistant Namalwa/MDR1 cells and the DHL-4 cells became more sensitive to the cytotoxic effect of doxorubicin. When added to the same amount of homodimer (at a noncytotoxic concentration), doxorubicin became even more toxic for DHL-4 (Figure 5A) and Namalwa/MDR1 (Figure 5C) cells. In contrast, both homodimers and monomers increased the sensitivity of Namalwa cells to doxorubicin to the same extent (Figure 5B), suggesting that the Rituxan homodimers are most advantageous when cells are insensitive to doxorubicin.
Synergistic effect of Rituxan and RFB4-dgA The IT RFB4-dgRTA kills human B-lymphoma cells in vitro and in vivo.24,25 In the presence of a noncytotoxic concentration of Rituxan monomers or homodimers, the IC50 of the IT decreased by 3-fold and greater than 1000-fold, respectively (Figure 6A). RFB4-mAbs plus either Rituxan monomers or homodimers had no effect on the cells (Figure 6B).
Monoclonal antibodies that exert antitumor activity may do so by virtue of their effector function (CDC and ADCC)26-28 and/or of their ability to signal growth arrest or cell death.29 In this study, we have evaluated the negative signaling activity of the mAb Rituxan, a chimeric antihuman CD20 mAb that has excellent activity in humans with lymphoma. Previous in vitro studies have shown that Rituxan arrests cell growth most effectively in vitro following cross-linking with a secondary anti-immunoglobulin antibody or with CD32-expressing cells.11 To take advantage of these observations, we have generated mAb homodimers (tetravalent antibody molecules) of Rituxan in order to optimize its ability to hypercrosslink. In general, homodimers have higher avidity, cross-link their target antigen more efficiently, and dissociate from cells more slowly.1,2 Previously, Wolff et al2 and Caron et al30 reported that homodimers had more potent antitumor activity than monomers, and we reported that mAbs that have little or no negative signaling activity can become potent antitumor agents when they are converted to homodimers.1 In vitro, some homodimers can exert antigrowth activity by signaling Go/G1 arrest and/or apoptosis, depending upon which cell surface molecule they bind.1 In some cases F(ab')2 homodimers work as well as, or better than, IgG homodimers in vitro.1 Rituxan has exhibited in vitro antigrowth activity on Ramos, DHL-4, and Raji cells, but apoptosis has been demonstrated only in Ramos and DHL-4 cells.7-11 It has also been shown that exposure of some, but not all, cell lines to anti-CD20 mAbs induces tyrosine phosphorylation.7 Our studies were designed to determine whether homodimers of Rituxan have better antigrowth activity than monomers. The major findings to emerge are the following: (1) homodimers are far more effective than monomers (and as effective as monomers plus GAMIg) in inducing growth arrest; (2) homodimers but not monomers induce both apoptosis and necrosis, and these effects are not dependent upon the presence of CD32 on the tumor cells; (3) the apoptotic effect of homodimers is inhibited by the caspase inhibitors (Z-VAD-FMK and Z-DEVD-FMK); (4) as compared with monomers, homodimers of Rituxan show decreased dissociation from, CD20+ cells; (5) the inhibitory activity of Rituxan on different cell lines does not correlate with their density of CD20; (6) Rituxan homodimers show marked synergy with an anti-CD22 IT in vitro; (7) Rituxan homodimers chemosensitize doxorubicin-resistant or insensitive tumor cells more effectively than monomers in vitro. Taken together with the results of other studies, the results presented here suggest that Rituxan homodimers might have better antitumor activity in vivo by virtue of enhanced negative signaling. This may, of course, be the case only if homodimers cross-link better in vivo where FcR+ cells are present. With regard to the mechanism of action of Rituxan in vivo, it has been clearly demonstrated that its FC portion and effector functions play major roles in its therapeutic efficacy in genetically engineered mice.30 Whether the effector function of homodimers will be enhanced in vivo remains to be determined. Hence, some mAb homodimers show improved ADCC and CMC30 whereas others do not.2 In an in vivo setting, an improvement may depend upon half-life and the binding of homodimers vs monomers of a particular mAb to effector cells. Regardless of whether homodimers will outperform monomers in vivo by virtue of enhanced negative signaling, effector function, or both, another way to improve the activity of Rituxan is to use it in combination with other antitumor agents. For this reason, we also tested Rituxan in combination with ITs and chemotherapy in vitro. With regard to ITs, we have previously reported that an anti-CD19 mAb (HD37) acts synergistically with the anti-CD22 IT RFB4-dgRTA in SCID mice with human lymphoma.24 In this study, we demonstrate that both Rituxan monomers and homodimers can increase the cytotoxic effect of RFB4-dgRTA but not an RFB4 mAb in vitro and that homodimers were more effective than monomers. This finding suggests that Rituxan homodimers plus ITs should be considered for further in vivo development. Our preliminary data in SCID mice with human lymphomas support this hypothesis. Combination therapy with Rituxan and chemotherapeutic agents (eg, CHOP) has been evaluated in patients with indolent B-cell lymphoma; a 95% overall response (55%, complete remissions; 40%, partial remissions) was achieved6; it is too early to predict what impact this will have on long-term survival. In a previous in vitro study, it was shown that Rituxan sensitized a B-lymphoma cell line to several cytotoxic drugs.8 In the present study, we demonstrate that both monomers and homodimers of Rituxan can potentiate the in vitro effect of doxorubicin on partially resistant or MDR+ tumor cells, but that homodimers are much more effective. The fact that Rituxan can render MDR+ cells sensitive to chemotherapy is in accord with our previous finding that an anti-CD19 mAb rendered MDR+ cells (P-gp+) sensitive to chemotherapeutic drugs in vitro.31 Taken together, these findings suggest exploring a strategy using homodimers followed by chemotherapy. In conclusion, Rituxan homodimers are in all respects superior to monomers in the in vitro killing of the CD20+ cell lines used in these studies, and this superiority can be attributed to enhanced negative signaling by Rituxan. Homodimers might also have improved effector function, but this remains to be tested. Finally, homodimers were also highly effective in synergizing with an IT and in chemosensitizing cells to doxorubicin, suggesting that they might be useful for the treatment of patients with MDR+ lymphomas. What remains to be determined is whether homodimers will also show improved antitumor activity in vivo.
The authors thank Drs J. Uhr and V. Ghetie for their critical reviews of this work, and Ms S. Flowers, Ms T. Bowdler, and Ms R. Lewis for secretarial assistance.
Submitted April 12, 2000; accepted October 16, 2000.
Supported by National Institutes of Health grant CA-64679.
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: Ellen S. Vitetta, Cancer Immunobiology Center, UT Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd, NB9.210, Dallas, TX 75390-8576; e-mail: ellen.vitetta{at}utsouthwestern.edu.
1.
Ghetie M-A, Podar EM, Ilgen A, Gordon BE, Uhr JW, Vitetta ES.
Homodimerization of tumor-reactive monoclonal antibodies markedly increases their ability to induce growth arrest or apoptosis of tumor cells.
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1997;94:7509-7514
2.
Wolff EA, Schreiber GJ, Cosand WL, Raff HV.
Monoclonal antibody homodimers: enhanced antitumor activity in nude mice.
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1993;53:2560-2565 3. Grillo-Lopez AJ, White CA, Varns C, et al. Overview of the clinical development of rituximab: first monoclonal antibody approved for the treatment of lymphoma. Semin Oncol. 1999;26:66-73[Medline] [Order article via Infotrieve]. 4. Gopal AK, Press OW. Clinical applications of anti-CD20 antibodies. J Lab Clin Med. 1999;134:445-450[CrossRef][Medline] [Order article via Infotrieve]. 5. McLaughlin P, Grillo-Lopez 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;16:2825-2833[Abstract].
6.
Czuczman MS, Grillo-Lopez AJ, White CA, et al.
Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy.
J Clin Oncol.
1999;17:268-276 7. Maloney DG, Smith B, Appelbaum FR. The anti-tumor effect of monoclonal anti-CD20 antibody (mAb) therapy includes direct anti-proliferative activity and induction of apoptosis in CD20 positive non-Hodgkin's lymphoma (NHL) cell lines. Blood. 1996;88(suppl 1):63. 8. Demidem A, Lam T, Alas S, Hariharan K, Hanna N, Bonavida B. Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs. Cancer Biother Radiopharm. 1997;12:177-186[Medline] [Order article via Infotrieve]. 9. Taji H, Kagami Y, Okada Y, et al. Growth inhibition of CD20-positive B lymphoma cell lines by IDEC-C2B8 anti-CD20 monoclonal antibody. Jpn J Cancer Res. 1998;89:748-756[CrossRef][Medline] [Order article via Infotrieve]. 10. Berchem GJ, Bosseler M, Heintz D, et al. Rituximab induces caspase 3 mediated apoptosis of B-CLL cells despite rapid internationation of the CD20 protein [abstract]. Proc Am Assoc Cancer Res. 2000;41:826.
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© 2001 by The American Society of Hematology.
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  M. Koslowski, U. Sahin, K. Dhaene, C. Huber, and O. Tureci MS4A12 Is a Colon-Selective Store-Operated Calcium Channel Promoting Malignant Cell Processes Cancer Res., May 1, 2008; 68(9): 3458 - 3466. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
); DHL-4 (
); Ramos (
); Raji (
); Namalwa (
); and
Namalwa/MDR1 (
). (B) Binding of Rituxan monomers (
RII (CD32) is
not involved in this growth inhibition (Figure 
