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Experimental Therapeutics |
Abgenix, Inc., Fremont, California 94555
ABSTRACT
A fully human IgG2
monoclonal antibody (MAb), E7.6.3, specific to the human epidermal growth factor (EGF) receptor (EGFr) was generated from human antibody-producing XenoMouse strains engineered to be deficient in mouse antibody production and to contain the majority of the human antibody gene repertoire on megabase-sized fragments from the human heavy and light chain loci. The E7.6.3 MAb exhibits high affinity (KD = 5 x 10-11M) to the receptor, blocks completely the binding of both EGF and transforming growth factor 
(TGF-) to various EGFr-expressing human carcinoma cell lines, and abolishes EGF-dependent cell activation, including EGFr tyrosine phosphorylation, increased extracellular acidification rate, and cell proliferation. The antibody (0.2 mg i.p. twice a week for 3 weeks) prevents completely the formation of human epidermoid carcinoma A431 xenografts in athymic mice. More importantly, the administration of E7.6.3 without concomitant chemotherapy results in complete eradication of established tumors as large as 1.2 cm3. Tumor eradication of A431 xenografts was achieved in nearly all of the mice treated with total E7.6.3 doses as low as 3 mg, administered over the course of 3 weeks, and a total dose of 0.6 mg led to tumor elimination in 65% of the mice. No tumor recurrence was observed for more than 8 months after the last antibody injection, which further indicated complete tumor cell elimination by the antibody. The potency of E7.6.3 in eradicating well-established tumors without concomitant chemotherapy indicates its potential as a monotherapeutic agent for the treatment of multiple EGFr-expressing human solid tumors, including those for which no effective chemotherapy is available. Being a fully human antibody, E7.6.3 is expected to exhibit minimal immunogenicity and a longer half-life as compared with mouse or mouse-derivatized MAbs, thus allowing repeated antibody administration, including in immunocompetent patients. These results suggest E7.6.3 as a good candidate for assessing the full therapeutic potential of anti-EGFr antibody in the therapy of multiple patient populations with EGFr-expressing solid tumors.
INTRODUCTION
Most applications of MAbs2
in cancer therapy rely on the ability of the antibody to specifically deliver to the cancerous tissues cytotoxic effector functions such as immune-enhancing isotypes, toxins, or drugs. An alternative approach is to use MAbs to directly affect the survival of tumor cells by depriving them of essential extracellular proliferation signals such as those mediated by growth factors through their cell receptors. One of the attractive targets in this approach is EGFr, which binds EGF and TGF- (1, 2, 3, 4)
. The binding of EGF or TGF- to EGFr—a Mr 170,000 transmembrane cell surface glycoprotein—triggers a cascade of cellular biochemical events including EGFr autophosphorylation and internalization, which culminates in cell proliferation (1)
.
Several observations implicate EGFr in supporting the development and progression of human solid tumors. Overexpression of EGFr has been shown to induce transformed properties in recipient cells (5)
. EGFr expression has been found to be up-regulated on many human tumors including lung, colon, breast, prostate, brain, head and neck, ovarian, and renal carcinoma, and the increase in receptor levels has been reported to be associated with a poor clinical prognosis (2
, 3
, 6, 7, 8)
. In many cases, the increased surface EGFr expression was accompanied by the production of TGF- or EGF by the tumor cells, suggesting the involvement of an autocrine growth control in the progression of these tumors (2
, 3
, 6
, 8)
. These observations suggested that blocking the interaction between the growth factors and EGFr could result in the arrest of tumor growth and could possibly affect tumor survival (2, 3, 4
, 6)
.
MAbs specific to the human EGFr, capable of neutralizing the EGF and TGF- binding to tumor cells and of inhibiting ligand-mediated cell proliferation in vitro, have been generated from mice and rats (2
, 4
, 6)
. Some of these antibodies, such as the mouse 108 (9)
, 225, and 528 (2
, 3)
or the rat ICR16, ICR62, and ICR64 (6
, 10
, 11)
MAbs, were evaluated extensively for their ability to affect tumor growth in mouse xenograft models. Most of the anti-EGFr MAbs were efficacious in preventing tumor formation in athymic mice when administered together with the human tumor cells (2
, 11)
. When injected into mice bearing established human tumor xenografts, the mouse MAbs 225 and 528 caused partial tumor regression and required the coadministration of chemotherapeutic agents such as doxorubicin or cisplatin for the eradication of the tumors (2
, 3
, 12
, 13)
. A chimeric version of the 225 MAb (C225), in which the mouse antibody variable regions are linked to human constant regions, exhibited an improved in vivo antitumor activity but only at high doses (14
, 15)
. The rat ICR16, ICR62, and ICR64 antibodies caused regression of established tumors but not their complete eradication (11)
. These results established EGFr as a promising target for antibody therapy against EGFr-expressing solid tumors and led to human clinical trials with the C225 MAb in multiple human solid cancers (2
, 3
, 6)
. However, the limited efficacy of these MAbs as monotherapeutic agents required their assessment in combination with chemotherapy (16
, 17)
. This requirement can limit the utilization of anti-EGFr antibodies in patients for whom chemotherapy is not available. Therefore, the identification of an anti-EGFr antibody capable of eradicating established human tumors by itself can expand the patient populations and cancer indications to which EGFr antibody therapy can be applied successfully. In addition, the MAbs currently pursued in human clinical trials, being murine chimeric antibodies (2
, 4)
, are likely to induce immune or allergic responses to the mouse components in immunocompetent patients, which leads to reduction in the antibody efficacy and safety. Therefore, anti-EGFr antibody therapy can be fully evaluated with the availability of a fully human anti-EGFr antibody that exhibits therapeutic efficacy on EGFr-expressing tumors and that can be administered repeatedly to all appropriate patient populations.
To this end, we used our human antibody-producing XenoMouse strains to generate potent fully human anti-EGFr MAbs. As described previously (18)
, these mouse strains were engineered to be deficient in mouse antibody production and to contain integrated megabase-sized fragments from the human heavy and light chain loci with the majority of the human antibody gene repertoire. The human immunoglobulin loci provided the XenoMouse strains with the ability to produce high-affinity human MAbs to a broad spectrum of antigens including human antigens (18
, 19)
. As presented in this report, using XenoMouse strains we generated a panel of anti-EGFr fully human IgG2 MAbs from which we selected the E7.6.3 antibody. This antibody exhibits high affinity (KD = 5 x 10-11M) to the receptor, neutralizes both EGF and TGF- binding to EGFr-expressing human carcinoma cell lines, and inhibits ligand-induced tumor cell proliferation. The antibody not only prevents human tumor formation in athymic mice but, more importantly, effectively eradicates large established human tumor xenografts, independent of chemotherapeutic agents. The exhibited potent antitumor activity of E7.6.3 implies that this antibody may be applied as a monotherapeutic agent for treatment of EGFr-expressing human solid tumors.
MATERIALS AND METHODS
XenoMouse Strains.
Generation and characterization of the Xeno-Mouse-G2 strains, engineered to produce fully human IgG2 antibodies, are described by Mendez et al. (18)
.
Human MAb Production and Purification.
XenoMouse strains (8–10 weeks old) were immunized i.p with 2 x 107 A431 cells (American Type Culture Collection, CRL-7907) in complete Freunds adjuvant. Mice were boosted with the same number of A431 cells in incomplete Freunds adjuvant three times before fusion. The fusion of splenocytes from immunized mice and selection of hybridomas were carried out as described previously(18)
. EGFr-specific hybridomas were identified by ELISA using purified A431 cell membrane-derived EGFr (Sigma). Large quantities of antibodies were purified from ascites that were derived from severe combined immunodeficient mice inoculated with antibody-producing hybridomas, using protein-A affinity chromatography.
Measurement of Antibody Affinity to EGFr.
Affinities of purified anti-EGFr antibodies were determined by plasmon resonance technology using BIAcore 2000 (Pharmacia). On the basis of the general procedures outlined by the manufacturer, kinetic analyses of the antibodies were performed using either purified membrane-derived EGFr or recombinant extracellular domain of EGFr (20)
immobilized onto the sensor surface at a low density (303 RU). The association (kon) and dissociation (koff) rates were determined using the BIAevaluation2.1 software provided by the manufacturer. Affinity measurements of antibody in solution were carried out as described previously (18)
.
EGFr Binding Assays.
EGFr binding assays were conducted using human recombinant 125I-EGF or 125I-TGF- (Amersham Life Science, Arlington Heights, IL) as described previously(18)
. Briefly, human carcinoma cells growing in DMEM containing 10% FCS were detached with trypsin, washed with PBS, and resuspended in binding buffer (PBS containing 0.1% BSA (Sigma) and 0.02% NaN3), and distributed in 96-well Multiscreen filter plates (Millipore) at 4 x 105 cells/well in 50 µl of medium. Fully human anti-EGFr or control anti-keyhole lympet hemocyanin MAbs, control human myeloma IgG2 MAb (Calbiochem, Cambridge, MA), or mouse anti-EGFr 225 or 528 MAbs (Calbiochem) diluted in binding buffer, were added in 50 µl aliquots per well. Plates were incubated for 30 min at 4°C. 125I-EGF or 125I-TGF- (0.1 nM) was added and the plates were further incubated for 90 min at 4°C. After incubation, the plates were washed five times with binding buffer, air-dried, and counted in a scintillation counter. The percentage of specifically bound 125I-EGF or 125I-TGF- was calculated as follows:
 |
In Vitro Tumor Cell Proliferation Assay.
The effect of antibodies on the growth of human tumor cells was determined using the method described by Ishiyama et al. (21)
. Briefly, 2 x 103 cells in 100 µl of DMEM:F12 growth medium without serum were seeded into each well of a 96-well plate. Aliquots of each diluted antibody (100 µl/well) were added in triplicate to the wells and the cultures were incubated at 37°C for 5 days. The controls consisted of either medium alone or medium containing dilutions of an human myeloma IgG2 control antibody. After incubation, the medium was removed from each well by aspiration. All of the cells were fixed with 0.25% glutaraldehyde, then washed in 0.9% NaCl, air-dried, and stained with Crystal Violet (Fisher Scientific, Pittsburgh, PA) for 15 min at room temperature. After washing with tap water and air-drying, 100 µl of methanol was added to each well and the absorbance at 595 nm (A595) of each well was determined in a Spectra Max spectrophotometer (Molecular Devices, Sunnyvale, CA). The percentage of growth inhibition is calculated as follows:
 |
EGFr Phosphorylation.
Seventy % confluent A431 cells were preincubated overnight with 0.5% of fetal bovine serum at 37°C. The cells were then treated with 16 nM EGF in the absence or presence of different concentrations of E7.6.3 MAb for 30 min at 37°C. After the 30-min incubation, the cells were washed three times with cold PBS and scraped into 0.5 ml of lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1 mg/ml PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate). After 30 min of incubation on ice, the lysates was centrifuged at 10,000 rpm for 5 min in an Eppendorf microcentrifuge at 4°C. The protein concentration in the supernatant was measured using BCA protein assay reagents (Pierce). A small portion of the supernatant was mixed with sample buffer (Novex, San Diego, CA) and boiled for 3 min. The proteins in the supernatant were then separated by 12% SDS-PAGE. Equal amounts of total protein were loaded from each cell lysate. Mouse antiphosphotyrosine (Zymed Laboratories, South San Francisco, CA) was used for the detection of EGFr tyrosine phosphorylation on Western blots. Enhanced chemiluminescence Western blotting detection reagents (Amersham) and the Hyperfilm Enhanced chemiluminescence (Amersham) were used for visualizing the signal. The integrated densities of the bands of interest were analyzed using an AGFA Scanner and the Scanalytics OneDscan software (Hewlett Packard, Mountain View, CA).
Measurement of Cell Activation by Cytosensor Microphysiometry.
To assess the effect of antibody on EGF-mediated signaling, Cytosensor Microphysiometry (Molecular Devices) was used. The Cytosensor detects early biochemical events in cell activation based on increases in the rate of acid release by the cells (22)
. Acid release was measured as described in the user’s manual provided by Molecular Devices, Inc. Briefly, A431 cells (5 x 104) were seeded in 1 ml of medium in a Cytosensor cell capsule and cultured at 37°C for 24 h. After incubation, the cell capsules were assembled and loaded in the Cytosensor sensing chamber maintained at 37°C. The chamber was perfused with low buffer RPMI 1640 containing 1 mM sodium phosphate and 1 mg/ml endotoxin-free BSA. Acid release rates were determined with 30-s potentiometric pH measurements after an 85-s pump cycle and 5-s delay (120-s total cycle time). Basal acid release rates ranged from 60 to 120 mV/s. Percent inhibition is calculated as follows:
 |
Tumor Xenograft Mouse Models.
Male BALB/c-nu/nu mice (6–8 weeks of age) were injected s.c. with 5 x 106 A431 or MDA-MB-468 (American Type Culture Collection, HTB-132) cells in 100 µl of PBS. Tumor sizes were measured in a blind fashion twice a week with a vernier caliper and tumor volume was calculated as the product of length x width x height x 
/6. Mice with established tumors were randomly divided into treatment groups. Animals were treated with antibodies using different regimens. Typically, mice received antibody treatment twice a week over three consecutive weeks either concomitant with the tumor cell injection (prophylactic treatment) or after tumor establishment (therapeutic treatment). The mice were followed for tumor xenograft growth and survival for at least 60 days.
Tumor Histopathological Evaluation.
Biopsies obtained from athymic mice carrying human xenografts were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. The sections were then stained with H&E as described previously (23)
.
RESULTS
High-Affinity Neutralizing Fully Human Anti-EGFr MAbs from XenoMouse Strains.
XenoMouse-G2 strains that produce fully human IgG2 antibodies were immunized with the human vulvar epidermoid carcinoma A431 cells. These cells express approximately 1 x 106 EGFr/cell (Refs. 2
, 3
and data not shown). Fusion of B cells from immunized XenoMouse strains with mouse myeloma cells yielded a panel of 30 hybridomas that secrete human IgG2 MAbs specific to the extracellular domain of human EGFr, as determined by ELISA, BIAcore, Western blots, and flow cytometry analysis (data not shown). The human 
2 was chosen as the preferred isotype with minimal immune-associated cytotoxicity against EGFr-expressing normal tissues.
To identify human MAbs with neutralization activity, purified antibodies were evaluated for their ability to block the binding of EGF and TGF- to human tumor cell lines that express low (colon carcinoma SW948, 5 x 104/cell) or high (A431, or breast adenocarcinoma MDA-MB-468, 1 x 106/cell) levels of EGFr. As positive controls, the commercially available murine MAbs, 225 and 528, were tested in parallel. A XenoMouse-derived IgG2 antibody PK16.3.1, specific for keyhole lympet hemocyanin, or a nonspecific human myeloma IgG2 antibody were used as a negative control. Fig. 1A
represents the results obtained with a subset of the fully human anti-EGFr MAbs tested in these assays. Three of the five human anti-EGFr antibodies shown, E7.6.3, E2.5.1, and E2.3.1, and the mouse anti-EGFr 225 and 528 MAbs blocked the binding of 125I-EGF (0.1 nM) to A431 in a concentration-dependent manner. In contrast, E7.5.2 and E7.8.2 did not have any effect on EGF binding. The calculated IC50 values (3.0 nM for E7.6.3, 5.6 nM for E2.5.1, 9.1 nM for E2.3.1, 8.8 nM for 225, and 15.2 nM for 528) suggested E7.6.3 as a potent neutralizing antibody. Furthermore, EGF binding to SW948 cells was also blocked by the human E7.6.3 and E2.3.1 and by the mouse 225 MAbs (Fig. 1B)
. The IC50 values detected in studies with SW948 cells were 0.9 nM for E7.6.3, 0.24 nM for E2.3.1, and 0.17 nM for 225. The efficacy of E7.6.3 in neutralizing ligand binding was also demonstrated in blocking TGF- binding to A431 cells (data not shown). These results indicated that XenoMouse strains are capable of producing fully human anti-EGFr antibodies that recognize different epitopes on the receptor, including those involved in ligand binding.
|
binding, E7.6.3 MAb was selected for further evaluation of its efficacy in affecting tumor cell growth in vitro and in vivo.
E7.6.3 MAb Inhibits EGF-mediated Tumor Cell Activation.
The ability of E7.6.3 to inhibit tumor cell activation was evaluated by examining its effects on EGF-triggered cellular responses such as the tyrosine kinase activity of EGFr, the extracellular acidification rate, and cell proliferation.
One of the first events after EGF binding to its receptor is the induction of EGFr tyrosine kinase activity, which results in the autophosphorylation of the receptor (1)
. As shown in Fig. 2
, incubation of human EGF (16 nM) with A431 cells induced the tyrosine phosphorylation of the Mr 170,000 EGFr. While E7.6.3 did not activate the receptor tyrosine kinase activity, the antibody blocked EGF-triggered EGFr tyrosine phosphorylation in a dose-dependent manner, with a nearly complete inhibition at a concentration of 133 nM (antibody:EGF molar ratio, 8:1; Fig. 2
).
|
, the addition of 1.67 nM EGF to A431 cells induced an immediate increase in the extracellular acidification rate. No effect was observed when the cells were incubated only with E.7.6.3 antibody at concentrations up to 100 nM (not shown). The concurrent addition of E7.6.3 resulted in a dose-dependent inhibition of EGF-mediated extracellular acidification (Fig. 3, A and B)
, whereas no effect was detected with the isotype-matched control antibody PK16.3.1 (Fig. 3B)
.
|
and to be growth-inhibited by the addition of exogenous EGF at nM concentrations (24
, 25)
. Therefore, the experiments using these two cell lines were carried out in the absence of exogenous EGF. E7.6.3 inhibited the growth of A431 cells in a dose-dependent manner, with a maximal inhibition of 60%, a level similar to that obtained with the mouse antibody 225 and higher than that observed for the 528 antibody (Fig. 4A)
. The control antibody did not have any effect on the cell proliferation (Fig. 4A)
. The calculated IC50 values for E7.6.3 (0.125 nM), 225 (0.48 nM), or 528 (0.66 nM) antibodies, indicated E7.6.3 efficacy in inhibiting A431 cell proliferation (Fig. 4A)
. A similar level of growth inhibition by E7.6.3 was observed with MDA-MB-468 cells (Fig. 4B)
. Because no exogenous EGF was added to the culture, these results indicate the ability of the human antibody to block autocrine growth stimulation and thus to inhibit EGF/TGF--induced tumor cell activation. In experiments carried out with SW948 cells, 10 nM E7.6.3 MAb blocked completely the proliferation of the cells (data not shown).
|
, all of the mice treated with either PBS or the control antibody developed tumors by day 10 after inoculation and were killed at day 30 because of the large size of the tumors. In contrast, none of the mice treated with E7.6.3 antibody developed tumors for more than 8 months after the last antibody injection. The data indicated that E7.6.3 prevented the formation of A431 xenografts, probably by exerting its neutralization activity at the initial phase of the tumor cell proliferation.
|
/6). Initially, mice bearing 0.13- to 0.25-cm3-sized tumors were treated i.p. with 1 mg of either E7.6.3 Mab or the human myeloma IgG2 control antibody, twice a week for 3 weeks. As shown in Fig. 5A
and in Table 2
, the tumors in untreated mice or in mice treated with the control antibody continued their aggressive growth to reach the size of 3 cm3 by day 30, at which point the mice were killed. In contrast, treatment with E7.6.3 not only arrested further growth of the tumors but also caused continuous tumor regression that resulted in a complete tumor eradication in all of the mice treated (Fig. 5A
, Table 2
). No recurring tumors were detected for more than 250 days in any of the mice that were monitored, demonstrating a long-lasting effect of the antibody and its ability to completely eliminate all of the tumor cells.
|
|
, E7.6.3 caused a profound tumor regression in all of the treated mice regardless of their initial tumor size, even with tumors as large as 1.2 cm3. Furthermore, this treatment led to a complete disappearance of the tumors (Fig. 5B)
and no tumor recurrence in any of the mouse groups for 210 days after the last antibody injection (data not shown). As summarized in Table 2
, the antibody effect appears to be dose-dependent with a total dose of 3 mg leading nearly to a complete tumor eradication and a total dose of 0.6 mg eliminating 65% of the established A431 xenografts.
To compare the antitumor activity of E7.6.3 to that of the mouse 225 antibody, which was reported to affect the growth of established A431 tumors but not cause their elimination (12
, 13)
, we used suboptimal E7.6.3 doses (0.05 mg and 0.2 mg, given twice a week for 3 weeks) that also caused primarily tumor regression in A431 xenografts. At these antibody doses, there was a significant difference between the ability of E7.6.3 and 225 to arrest the growth of A431 xenografts (Fig. 5C)
.
E7.6.3 was also shown to be efficacious in inhibiting the growth of the breast carcinoma MDA-MB-468 xenografts (Fig. 6A)
. Treatment of 0.2-cm3-tumor-bearing mice with 2 mg of antibody once a week for 2 weeks led to a complete arrest of the tumor growth, with no apparent growing tumors for 140 days after the last antibody administration.
|
. Administration of 0.5 mg of E7.6.3 into mice carrying 0.15-cm3-sized A431 xenografts twice a week for three weeks by i.p., s.c., i.v., or i.m route all caused complete tumor eradication.
The elimination of all of the tumor cells by E7.6.3 was further supported by the histopathological analysis of the small residual nodules observed in some of the A431 xenograft-bearing mice that were treated with the lower antibody doses. Biopsies taken from these nodules at day 79 were shown to contain a thin fibrovascular capsule lined by necrotic cells with its center filled with keratinic and calcified debris (Fig. 7A)
. There was no evidence of neoplastic cells, which were readily detected in tumor taken from mice treated with PBS or control antibody (Fig. 7B)
. A mild inflammatory infiltration of neutrophils, lymphocytes, plasma cells, macrophages, and multinucleated giant cells surrounded the capsule. Taken together with the long-lasting inhibitory effect, the data strongly indicate complete tumor cell eradication by E7.6.3 antibody.
|
Utilization of XenoMouse strains for the production of human antibodies specific to the human EGFr yielded the fully human IgG2 Mab, E7.6.3, characterized by high affinity and strong neutralization activity. Its demonstrated efficacy in eradicating large established human tumor xenografts without concomitant chemotherapy strongly suggests that it is a suitable candidate for antibody monotherapy in patients with EGFr-expressing tumors.
E7.6.3 exhibited strong efficacy in blocking the binding of EGF and TGF- to EGFr on the surface of different human carcinoma cell lines, including those that express high levels of receptors (Fig. 1)
. The complete inhibition of ligand binding to the receptors on A431 and SW948 cells resulted in an abolishment of the signaling events triggered by EGF or TGF-, including EGFr autophosphorylation, increased extracellular acidification rate, and cell proliferation. Our results indicate that E7.6.3 can block ligand-induced cell activation and that E7.6.3 does not act as an agonist to trigger cellular responses in EGFr-expressing tumors (Figs. 2
and 3
).
The antitumor activity of E7.6.3 was examined in multiple xenograft mouse experiments in which the effects of various antibody doses on different sizes of tumors were established (Figs. 5
and 6
). The results obtained from these studies demonstrated the unique antitumor properties of E7.6.3 MAb as compared with the other reported anti-EGFr antibodies. E7.6.3 not only arrested the growth of human tumor xenografts but also completely eradicated established tumors by itself, without the need for concomitant chemotherapy. Tumor eradication of A431 xenografts was achieved in nearly all of the mice treated with total doses as low as 3 mg administered over the course of 3 weeks, and a total dose of 0.6 mg led to tumor elimination in 65% of the mice (Fig. 5
and Fig. 6B
, Table 2
). In comparison, 8 mg of 225 and 10 mg of 528 antibodies, given over 4 and 10 weeks, respectively, had only a limited effect on A431 tumors and required the coadministration of chemotherapeutic drugs to achieve the elimination of the tumors (12
, 13)
. A direct comparison between E7.6.3 and 225 MAbs at low doses demonstrated E7.6.3 as a more potent antibody in regressing established A431 tumors and arresting their growth (Fig. 5C)
. The chimeric C225 MAb, which was reported to acquire higher affinity to EGFr and enhanced in vivo antitumor activities, achieved complete A431 tumor eradication at a total dose of 10 mg given for 5 weeks, whereas total doses of 2.5 and 5 mg led to only 14% and 57% of tumor-free mice (14)
. The potent antitumor activity of E7.6.3 was further validated by its ability at a 6 mg total dose to completely eliminate established tumors as large as 1.2 cm3 in all of the mice treated.
This antitumor potency of E7.6.3 is likely to originate primarily from the intrinsic activity of the antibody inasmuch as its human 2 isotype was shown to minimally engage the immune system-derived effector functions such as cell-mediated cytotoxicity or complement-dependent cytolysis. In comparison, the antitumor activities of the rat ICR62, mouse 528, or chimeric C225 antibodies were suggested to reflect the participation of the host immune effector functions recruited by the respective rodent 2b or human 1 isotypes (2
, 4 , 6
, 26)
.
The molecular mechanism(s) underlying the potent antitumor activity of E7.6.3 still remains elusive. Some hypotheses that can be proposed are based on the ability of the antibody to block ligand-triggered growth and survival signals, whereas others emphasize the possible effects that the antibody may exert upon the cell on its interaction with the receptor. The potency of E7.6.3 can be attributed, at least in part, to the high binding affinity (KD = 5 x 10-11M) to human EGFr, higher than the affinity reported for other anti-EGFr MAbs (12 , 14) . With its high affinity, E7.6.3 can inhibit or dissociate the ligand binding to the receptors very effectively, thus depriving the cells completely from receiving essential growth and survival stimuli. Like other anti-EGFr antibodies (2 , 4 , 6) , E7.6.3 MAb does not act as an agonist and does not activate cells on binding to the receptor. The difference in efficacy between E7.6.3 and the other antibodies tested in xenograft mouse models can also be attributed to a unique E7.6.3 binding epitope on EGFr that can mediate a stronger neutralization effect or induce cytotoxicity. The latter hypothesis is supported by the ability of E7.6.3 to eradicate well-established human xenografts as large as 1.2 cm3. The mechanism behind the in vivo cytocidal effects of E7.6.3 is not yet clear and may involve the induction of programmed cell death, differentiation of the tumor cells, or modulation of expression of angiogenesis factors—mechanisms that were shown to be triggered by antibodies in cultured cells (27, 28, 29, 30, 31) . Different mechanisms may account for the antibody effect on different tumors; and in some cases, probably more than one mechanism contributes to the antitumor activity.
The potency of E7.6.3 in eradicating well-established tumors indicates that this antibody can provide effective therapy to tumors that require EGFr activation for their continuous progression and survival. Because E7.6.3 does not require the presence of chemotherapy to exert antitumor activity, the antibody could be applied to various EGFr-expressing human solid tumors. Furthermore, being a fully human antibody, E7.6.3 is expected to have a long half-life and minimal immunogenicity with repeated administration, including in all immunocompetent patients. In addition, bearing a human 2 constant region that interacts poorly with the effector arm of the immune system, E7.6.3 MAb may not induce cytotoxic effects on EGFr-expressing normal tissues such as liver and skin.
Utilization of Mabs directed to growth factor receptors as cancer therapeutics has been validated recently by the tumor responses obtained from clinical trials with Herceptin, the humanized anti-HER2 antibody, in patients with HER2-overexpressing metastatic breast cancer (32 , 33) . The potent in vivo antitumor activity of E7.6.3, as demonstrated in this report, suggests that it is a good candidate for assessing the therapeutic potential of anti-EGFr therapy in EGFr-expressing human tumors.
ACKNOWLEDGMENTS
We thank Donna Louie and Cathy Roth for tumor measurements; Joanna Hales, Helen Chadd, and Chung Lee for antibody production studies; and Drs. Steve Sherwin, Richard Junghans, Ronald Levy, John Lipani, and Larry Green for comments on the article.
FOOTNOTES
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.
1 To whom requests for reprints should be addressed, at Abgenix, Inc., 7601 Dumbarton Circle, Fremont, CA 94555. Phone: (510) 608-6556; Fax: (510) 608-6511; E-mail: yang_xd{at}abgenix.com 
2 The abbreviations used are: Mab, monoclonal antibody; EGF, epidermal growth factor; EGFr, EGF receptor; TGF, transforming growth factor.
Received 9/29/98. Accepted 1/19/99.
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L. M. Weiner, A. S. Belldegrun, J. Crawford, A. W. Tolcher, P. Lockbaum, R. H. Arends, L. Navale, R. G. Amado, G. Schwab, and R. A. Figlin Dose and Schedule Study of Panitumumab Monotherapy in Patients with Advanced Solid Malignancies Clin. Cancer Res., January 15, 2008; 14(2): 502 - 508. [Abstract] [Full Text] [PDF]
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W. A. Messersmith and M. Hidalgo Panitumumab, a Monoclonal Anti Epidermal Growth Factor Receptor Antibody in Colorectal Cancer: Another One or the One? Clin. Cancer Res., August 15, 2007; 13(16): 4664 - 4666. [Full Text] [PDF]
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J. A. Lofgren, S. Dhandapani, J. J. Pennucci, C. M. Abbott, D. T. Mytych, A. Kaliyaperumal, S. J. Swanson, and M. C. Mullenix Comparing ELISA and Surface Plasmon Resonance for Assessing Clinical Immunogenicity of Panitumumab J. Immunol., June 1, 2007; 178(11): 7467 - 7472. [Abstract] [Full Text] [PDF]
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K. A. Rogers, F. Scinicariello, and R. Attanasio IgG Fc Receptor III Homologues in Nonhuman Primate Species: Genetic Characterization and Ligand Interactions J. Immunol., September 15, 2006; 177(6): 3848 - 3856. [Abstract] [Full Text] [PDF]
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K. F. Tse, M. Jeffers, V. A. Pollack, D. A. McCabe, M. L. Shadish, N. V. Khramtsov, C. S. Hackett, S. G. Shenoy, B. Kuang, F. L. Boldog, et al. CR011, a Fully Human Monoclonal Antibody-Auristatin E Conjugate, for the Treatment of Melanoma Clin. Cancer Res., February 15, 2006; 12(4): 1373 - 1382. [Abstract] [Full Text] [PDF]
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N. J. Meropol Epidermal Growth Factor Receptor Inhibitors in Colorectal Cancer: It's Time to Get Back on Target J. Clin. Oncol., March 20, 2005; 23(9): 1791 - 1793. [Full Text] [PDF]
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B. D. Cohen, D. A. Baker, C. Soderstrom, G. Tkalcevic, A. M. Rossi, P. E. Miller, M. W. Tengowski, F. Wang, A. Gualberto, J. S. Beebe, et al. Combination Therapy Enhances the Inhibition of Tumor Growth with the Fully Human Anti-Type 1 Insulin-Like Growth Factor Receptor Monoclonal Antibody CP-751,871 Clin. Cancer Res., March 1, 2005; 11(5): 2063 - 2073. [Abstract] [Full Text] [PDF]
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J. Christiansen and A. K. Rajasekaran Biological impediments to monoclonal antibody-based cancer immunotherapy Mol. Cancer Ther., November 1, 2004; 3(11): 1493 - 1501. [Abstract] [Full Text] [PDF]
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W. K. Bleeker, J. J. Lammerts van Bueren, H. H. van Ojik, A. F. Gerritsen, M. Pluyter, M. Houtkamp, E. Halk, J. Goldstein, J. Schuurman, M. A. van Dijk, et al. Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer Therapy J. Immunol., October 1, 2004; 173(7): 4699 - 4707. [Abstract] [Full Text] [PDF]
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E. K. Rowinsky, G. H. Schwartz, J. A. Gollob, J. A. Thompson, N. J. Vogelzang, R. Figlin, R. Bukowski, N. Haas, P. Lockbaum, Y.-P. Li, et al. Safety, Pharmacokinetics, and Activity of ABX-EGF, a Fully Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Patients With Metastatic Renal Cell Cancer J. Clin. Oncol., August 1, 2004; 22(15): 3003 - 3015. [Abstract] [Full Text] [PDF]
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J. L. Dominguez-Escrig, J. D. Kelly, D. E. Neal, S. M. King, and B. R. Davies Evaluation of the Therapeutic Potential of the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Gefitinib in Preclinical Models of Bladder Cancer Clin. Cancer Res., July 15, 2004; 10(14): 4874 - 4884. [Abstract] [Full Text] [PDF]
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M. Nunes, C. Shi, and L. M. Greenberger Phosphorylation of extracellular signal-regulated kinase 1 and 2, protein kinase B, and signal transducer and activator of transcription 3 are differently inhibited by an epidermal growth factor receptor inhibitor, EKB-569, in tumor cells and normal human keratinocytes Mol. Cancer Ther., January 1, 2004; 3(1): 21 - 27. [Abstract] [Full Text]
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J. Mendelsohn and J. Baselga Status of Epidermal Growth Factor Receptor Antagonists in the Biology and Treatment of Cancer J. Clin. Oncol., July 15, 2003; 21(14): 2787 - 2799. [Abstract] [Full Text] [PDF]
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S. Welt, G. Ritter, C. Williams Jr., L. S. Cohen, M. John, A. Jungbluth, E. A. Richards, L. J. Old, and N. E. Kemeny Phase I Study of Anticolon Cancer Humanized Antibody A33 Clin. Cancer Res., April 1, 2003; 9(4): 1338 - 1346. [Abstract] [Full Text] [PDF]
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R. Nahta, G. N. Hortobagyi, and F. J. Esteva Growth Factor Receptors in Breast Cancer: Potential for Therapeutic Intervention Oncologist, February 1, 2003; 8(1): 5 - 17. [Abstract] [Full Text] [PDF]
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J. Mendelsohn Targeting the Epidermal Growth Factor Receptor for Cancer Therapy J. Clin. Oncol., September 15, 2002; 20(90001): 1s - 13. [Full Text] [PDF]
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L. Mills, C. Tellez, S. Huang, C. Baker, M. McCarty, L. Green, J. M. Gudas, X. Feng, and M. Bar-Eli Fully Human Antibodies to MCAM/MUC18 Inhibit Tumor Growth and Metastasis of Human Melanoma Cancer Res., September 1, 2002; 62(17): 5106 - 5114. [Abstract] [Full Text] [PDF]
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Y. He, W. J. Honnen, C. P. Krachmarov, M. Burkhart, S. C. Kayman, J. Corvalan, and A. Pinter Efficient Isolation of Novel Human Monoclonal Antibodies with Neutralizing Activity Against HIV-1 from Transgenic Mice Expressing Human Ig Loci J. Immunol., July 1, 2002; 169(1): 595 - 605. [Abstract] [Full Text] [PDF]
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S. J. Miknyoczki, W. Wan, H. Chang, P. Dobrzanski, B. A. Ruggeri, C. A. Dionne, and K. Buchkovich The Neurotrophin-Trk Receptor Axes Are Critical for the Growth and Progression of Human Prostatic Carcinoma and Pancreatic Ductal Adenocarcinoma Xenografts in Nude Mice Clin. Cancer Res., June 1, 2002; 8(6): 1924 - 1931. [Abstract] [Full Text] [PDF]
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I. Renard, D. Mezzanzanica, S. Canevari, S. Ferrini, J. Boniver, P. Delvenne, and N. Jacobs Anti-CD3/Anti-Epidermal Growth Factor Receptor-Bispecific Antibody Retargeting of Lymphocytes against Human Neoplastic Keratinocytes in an Autologous Organotypic Culture Model Am. J. Pathol., January 1, 2002; 160(1): 113 - 122. [Abstract] [Full Text] [PDF]
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A. Viloria-Petit, T. Crombet, S. Jothy, D. Hicklin, P. Bohlen, J. M. Schlaeppi, J. Rak, and R. S. Kerbel Acquired Resistance to the Antitumor Effect of Epidermal Growth Factor Receptor-blocking Antibodies in Vivo: A Role for Altered Tumor Angiogenesis Cancer Res., July 1, 2001; 61(13): 5090 - 5101. [Abstract] [Full Text] [PDF]
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O. Christ, S. Seiter, S. Matzku, C. Burger, and M. Zöller Efficacy of Local versus Systemic Application of Antibody-Cytokine Fusion Proteins in Tumor Therapy Clin. Cancer Res., April 1, 2001; 7(4): 985 - 998. [Abstract] [Full Text]
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M. Hambek, C. Solbach, H.-G. Schnuerch, M. Roller, M. Stegmueller, A. Sterner-Kock, J. Kiefer, and R. Knecht Tumor Necrosis Factor {{alpha}} Sensitizes Low Epidermal Growth Factor Receptor (EGFR)-expressing Carcinomas for Anti-EGFR Therapy Cancer Res., February 1, 2001; 61(3): 1045 - 1049. [Abstract] [Full Text]
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V. Guillemard and H. U. Saragovi Taxane-Antibody Conjugates Afford Potent Cytotoxicity, Enhanced Solubility, and Tumor Target Selectivity Cancer Res., January 1, 2001; 61(2): 694 - 699. [Abstract] [Full Text]
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C. D. Woodworth, D. Gaiotti, E. Michael, L. Hansen, and M. Nees Targeted Disruption of the Epidermal Growth Factor Receptor Inhibits Development of Papillomas and Carcinomas from Human Papillomavirus-immortalized Keratinocytes Cancer Res., August 1, 2000; 60(16): 4397 - 4402. [Abstract] [Full Text]
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 V. A. Pollack, D. M. Savage, D. A. Baker, K. E. Tsaparikos, D. E. Sloan, J. D. Moyer, E. G. Barbacci, L. R. Pustilnik, T. A. Smolarek, J. A. Davis, et al. Inhibition of Epidermal Growth Factor Receptor-Associated Tyrosine Phosphorylation in Human Carcinomas with CP-358,774: Dynamics of Receptor Inhibition In Situ and Antitumor Effects in Athymic Mice J. Pharmacol. Exp. Ther., November 1, 1999; 291(2): 739 - 748. [Abstract] [Full Text]
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 A. M. Pilling The Role of the Toxicologic Pathologist in the Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals Toxicol Pathol, November 1, 1999; 27(6): 678 - 688. [Abstract] [PDF]
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, E7.6.3; •, E2.5.1; 
, E2.3.1; 
), E7.5.2; 
, E7.8.2) or murine (
, 225; 
, 528) anti-EGFr antibodies, or in the presence of the human IgG2
, hIgG2
) doses of 225 MAbs, twice a week for 3 weeks. Control mice (
