mAb C225 Research Articles

Growth suppression of colorectal cancer expressing S492R EGFR by monoclonal antibody CH12

Colorectal cancer (CRC) is a common malignant tumor in the digestive tract, and 30%–85% of CRCs express epidermal growth factor receptors (EGFRs). Recently, treatments using cetuximab, also named C225, an anti-EGFR monoclonal antibody, for CRC have been demonstrated to cause an S492R mutation in EGFR. However, little is known about the biological function of S492R EGFR. Therefore, we attempted to elucidate its biological function in CRC cells and explore new treatment strategies for this mutant form. Our study indicated that EGFR and S492R EGFR accelerate the growth of CRC cells in vitro and in vivo and monoclonal antibody CH12, which specifically recognizes an EGFR tumor-specific epitope, can bind efficiently to S492R EGFR. Furthermore, mAb CH12 showed significantly stronger growth suppression activities and induced a more potent antibody-dependent cellular cytotoxicity effect on CRC cells bearing S492R EGFR than mAb C225. mAb CH12 obviously suppressed the growth of CRC xenografts with S492R EGFR mutations in vivo. Thus, mAb CH12 may be a promising therapeutic agent in treating patients with CRC bearing an S492R EGFR mutation.

Development and evaluation of a single domain antibody against human epidermal growth factor receptor (EGFR)

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, multiplication and differentiation. Over expression of EGFR is associated with carcinogenesis and seen in variety of cancers. Anti-EGFR monoclonal antibodies can block EGFR downstream signaling pathway resulting in inhibition of uncontrolled cell proliferation. Antibody fragments have a variety of advantages. In comparison to full length antibodies they have smaller size and therefor exhibit better tumor penetration ability. The aim of this study was to prepare a single domain antibody to target extracellular domain of EGFR. mRNA was extracted from C225 hybridoma cells producing anti-EGFR antibody and subjected to reverse transcription reaction (RT-PCR) to obtain cDNA molecules encoding VH domain of mAb C225. The cDNA encoded VH domain was in frame introduced into the pET-22b(+) vector and expressed in BL21 (DE3) bacterial cells. The resultant antibody was purified via Ni- NTA column and its reactivity was assessed by ELISA and western blot techniques using A431 cell lysate. Analysis by ELISA revealed that this single domain antibody was able to bind EGFR on A431cells. This result was further confirmed by western blotting. In conclusion, the results of this study indicated that single domain antibody can identify and bind to EGFR of A431 carcinoma cells. This recombinant fragment antibody would potentially be used for targeting of cancer cells with high EGFR expression.

A phase I study of doxorubicin-loaded anti-EGFR immunoliposomes in patients with advanced solid tumors.

3029 Background: Immunoliposomes (ILs) combine antibody-mediated tumor recognition with liposomal delivery and, when designed for target cell internalization, provide intracellular drug release in order to increase specificity and efficacy of the encapsulated drug. In animal studies we have shown the ILs approach to be active and promising when targeting the epidermal growth factor receptor (EGFR). Methods: ILs were modularly manufactured under GMP conditions with Fab' fragments from MAb C225 (cetuximab), covalently linked to pegylated liposomes containing doxorubicin (PLD). This first in man single-center phase I clinical trial of anti-EGFR ILs-dox was designed for patients (pts) with various solid tumors, overexpressing EGFR (DAKO EGFR pharmDx-test). ILs-dox was administered i.v. q 4 weeks at a doxorubicin (dox) dose of 5, 10, 20, 30, 40, 50 and 60 mg/m2, 3 pts per dose level, for a maximum of 6 cycles. In addition to weekly safety monitoring, echocardiography was performed q 2 cycles, and pharmacokinet...

The role of EGFR MAbs C225 in breast cancer stem cells

e22093 Background: The epithermal growth factor receptor(EGFR) signaling pathways has been implicated in self-renewal of breast cancer stem cells. It has been proved that the EGFR tyrosine kinase inhibitor-gefitinib significantly decreased the mammosphere- forming efficiency(MFE) in ductal carcinoma in situ (DCIS) derived cancer cells. We investigated the response of breast cancer stem cells in breast cancer cell line MCF-7 to EGFR MAbs C225(cetuximab). Methods: Cells were used for nonadherent (i.e.,mammosphere) culture and were divided into four groups according to the different culture medium whether or not including exogenous EGF and MAbs C225. MFE was calculated as the number of mammospheres (≥60um) and was expressed as a percentage. We Compared the percentage of CD44+/CD24- cells from the four groups, and measured the levels of the Wnt and Notch-3 gene expression of the four groups. Results: There was no significant difference of MFE between groups. In the presence of exogenous EGF, MAbs C225 significantly decreased the MFE and the percentage of CD44+/CD24- cells of mammospheres compared with exogenous EGF alone(0.7% versus 1.5%, P < 0.001 and 4% versus 11%P < 0.001;respectively). In the absence of exogenous EGF, MAbs C225 aslo statistically decreased the MFE and the percentage of CD44+/CD24- cells of mammospheres compared with that of lacking exogenous EGF and MAbs C225(0.53% versus 1.3%,P = 0.003 and 9% versus 3%, P < 0.01;respectively). And we observed that the Notch-3 gene expression was compensativly increased in the two groups of addition of MAb C225. No significant differences of Wnt gene expression were observed between groups. Conclusions: EGFR MAbs C225 could disrupt mammoshpere formation and decrease the percentage of CD44+/CD24- cells of mammoshpere cells. The expression of Notch-3 is increased with the EGFR antagonistics in breast cancer stem cells. No significant financial relationships to disclose.

Cetuximab: From Biology To the Care of Colorectal Cancer Patients

Cetuximab (IMC-C225, Erbitux ImClone Systems Inc., NY, USA) is a chimeric human:murine derivative IgG1 monoclonal antibody (mAb) that binds the extracellular domain of the EGF receptor (EGFR) [1–3]. Cetuximab has been approved by the US FDA, in combination with irinotecan, for use in the treatment of metastatic colorectal cancer, for locally/regionally advanced squamous cell carcinoma of the head and neck (SCCHN) in combination with radiation, and as a monotherapy for recurrent/metastatic SCCHN after failing platinum-based chemotherapy. The mechanisms through which cetuximab is thought to express its antitumor activity are numerous and not completely understood, as yet. They include the direct inhibition of EGFR tyrosine kinase activity [4], the inhibition of cell-cycle progression [5,6], angiogenesis, invasion and metastasization [3], the increase and activation of proapoptotic molecules [7,8], and synergic cytotoxicity with chemotherapy and radiotherapy [9]. Another mode of action is antibody-dependent cellular cytotoxicity. Cetuximab mediates cell-cycle arrest in various tumor cell lines, leading, in some cases, to apoptosis. Wu X et al. investigated the relationship between growth factor availability and the activation of the CDK kinases [10]. They demonstrated that blockage of EGFR mediated by cetuximab induces G1-phase cell-cycle arrest in DiFi human colon adenocarcinoma cells. Moreover, in an in vitro assay, immunodepletion of p27KIP1 removes the inhibitory activity of the lysates of mAb-treated cells, so that immunodepleted and heated lysates lose their capacity to inhibit cyclin E/CDK2 activity. The results suggest that G1 arrest in the cell cycle induced by EGFR blockade involves p27KIP1. Petit et al. were the first to describe the antiangiogenic effects of cetuximab [11]. They demonstrated that tumor xenografts of the human A431 squamouscell carcinoma exposed to mAb C225 resulted in a reduction in the release of angiogenic factors by tumor cells themselves. These results were confirmed by numerous studies on other tumor cell lines. Perrote et al. exposed in vitro human transitional cell carcinoma (TCC) of the bladder cell line 253J B-V to mAb C225 [12]. The effect observed was the inhibition of mRNA and protein production of VEGF, IL-8 and β-FGF by the cells. In vivo, mAb C225 therapy of nude mice with established TCCs growing orthotopically resulted in the downregulation of these angiogenic factors and, consequently, in the involution of blood vessels. Another study examined the impact of C225 exposition in human SCCHN using in vitro and in vivo model systems [13]. Regarding angiogenesis, in vitro treatment with C225 reduced cell-to-cell interaction of human umbilical vascular endothelial cells, resulting in disruption of tube formation. Using an in vivo tumor xenograft neovascularization model of angiogenesis, systemic treatment with C225 not only reduced tumor growth and the number of blood capillaries, but also inhibited the growth of established vessels toward the tumor. Taken together, these results provide evidence that C225 can suppress tumor-induced neovascularization in these tumor cell lines.

Simultaneous Inhibition of Epidermal Growth Factor Receptor (EGFR) Signaling and Enhanced Activation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor-mediated Apoptosis Induction by an scFv:sTRAIL Fusion Protein with Specificity for Human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

Different classes of EGFR inhibitors may have different potential to improve local tumour control after fractionated irradiation: a study on C225 in FaDu hSCC

Background and purposePrevious experiments reported from this laboratory have shown that simultaneous application of the selective epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor BIBX1382BS during fractionated irradiation significantly prolonged growth delay of FaDu human squamous cell carcinoma but did not improve local tumour control. The present study investigates the effect of the EGFR monoclonal antibody (mAb) C225 on local tumour control of FaDu tumours after combined treatment with single dose and fractionated irradiation to address whether different classes of EGFR inhibitors have different potential to improve the outcome of radiotherapy in the same tumour model. Material and methodsIn unirradiated tumours, C225 was given either once or 4 times i.p. to the nude mice. Irradiation experiments were performed with graded single doses under clamp hypoxic conditions or with 30 fractions in 6 weeks with graded total doses under ambient blood flow. C225 was given 6h before or 6h before and 2, 5 and 7 days after single dose irradiation. During fractionated irradiation C225 was given once per week. Experimental endpoints were tumour growth delay and local tumour control 120 after end of irradiation. ResultsC225 treatment resulted in prolongation of tumour growth delay after drug treatment alone as well as after single dose and fractionated irradiation. TCD50 values were reduced from 56.3Gy [95% CI 50; 62Gy] after single dose irradiation alone to 46.0Gy [41;51] (enhancement ratio [ER]=1.22, P<0.01) after 1 C225 injection and 47.7Gy [44; 51] after 4 injections of the drug (ER=1.18, P=0.06). After fractionated irradiation, tumour control dose 50% (TCD50) was 73.0Gy [64; 82] in control tumours and 63.1Gy [57; 69] after simultaneous C225 treatment, corresponding to an ER of 1.2 (P=0.01). ConclusionTreatment of FaDu hSCC with the anti-EGFR mAb C225 resulted in a significant prolongation of tumour growth delay after single dose and fractionated irradiation. In contrast to previous results on the EGFR-TK inhibitor BIBX1382BS, this prolongation of growth delay translated into a slight but significant improvement of local tumour control. The data indicate that different classes of EGFR inhibitors may have different potential to improve the outcome of radiotherapy in the same tumour model.

THE WILLET F. WHITMORE, JR., LECTURESHIP: BLOCKADE OF EPIDERMAL GROWTH FACTOR RECEPTORS AS ANTICANCER THERAPY

In 1981 Sato and I hypothesized that a monoclonal antibody that binds to EGF receptors may inhibit cell proliferation by blocking activation of receptors by EGF or transforming growth factor (TGF)-a. The compelling rationale for this hypothesis is summarized in Appendix 1. In 1983 and 1984 we published a series of studies demonstrating that blockade of human EGF receptors by murine mAb 225 or mAb 528, produced in our laboratory, competitively inhibits the capacity of EGF to activate receptor tyrosine kinase and can inhibit proliferation of cells bearing EGF receptors in culture and in human tumor xenografts in nude mice.1–3 Subsequently, these antiproliferative effects in culture and against xenografts have been observed with numerous human tumor cell lines.4 A human:murine chimeric mAb C225 was produced with the support of the National Cancer Institute drug evaluation network (Appendix 2). This chimeric antibody has higher binding affinity for the EGF receptor than its murine parent, and its activity against well established human tumor xenografts surpasses that of murine 225.5 It should be stressed that C225 is acting as a drug, a tyrosine kinase inhibitor specific for the EGF receptor. We have identified 6 different mechanisms by which C225 may inhibit tumor cell growth and survival in culture and, more importantly, in nude mouse xenografts (Appendix 3). We review some of the experiments that have elucidated each of these mechanisms.

The epidermal growth factor receptor as a target for cancer therapy.

Epidermal growth factor (EGF) receptors are expressed at high levels in about one third of epithelial cancers, and autocrine activation of EGF receptors appears to be critical for the growth of many tumors. We hypothesized that blockade of the binding sites for EGF and transforming growth factor-alpha on EGF receptors with an antireceptor monoclonal antibody (mAb) might be an effective anti-cancer therapy. We produced murine mAb 225 against EGF receptors and demonstrated blockade of receptor function, as well as inhibition of cell growth in cultures and in nude mouse xenografts. mAb C225 is the human:murine chimeric version of mAb 225. Cell cycle inhibition occurred in G(1) phase, and was due to upregulation of p27(Kip1), resulting in inhibition of cyclin E/cyclin dependent kinase-2 activity and hypophosphorylation of Rb. In addition, the amount and/or activities of a number of proapoptotic molecules were enhanced. The antitumor activity in vivo against xenografts was at least partly attributable to reduced vascularization, resulting from decreased vascular endothelial growth factor and basic fibroblast growth factor production by the tumor cells. Metastasis of xenografts was curtailed with mAB C225 treatment, accompanied by a decrease in tumor production of MMP-9. Further studies showed that mAbs 225 and C225 enhanced the cytotoxicity of chemotherapy against xenografts of a variety of human cancer cell lines. Well established xenografts resistant to either mAb or drug treatment alone were eradicated by the combination therapy. Drugs for which this has been demonstrated include doxorubicin, paclitaxel, cisplatin, and topotecan. Antibody treatment also potentiated the responsiveness of human tumor xenografts to radiation therapy. These findings led to clinical trials of human:murine chimeric mAb C225 in combination with chemotherapy or radiotherapy. Results from phase I and II trials involving more than 500 patients are quite promising, in particular in advanced head and neck cancer treated with C225 plus cisplatin or radiation, in advanced colon cancer treated with C225 plus CPT-11, and in advanced pancreatic cancer treated with C225 plus gemcitabine. Phase III trials are now underway.

Synergistic growth inhibition and induction of apoptosis by a novel mixed backbone antisense oligonucleotide targeting CRIPTO in combination with C225 anti-EGFR monoclonal antibody and 8-Cl-cAMP in human GEO colon cancer cells.

We have evaluated the antiproliferative effect of a novel mixed backbone antisense oligonucleotide generated against the 5'-coding region of the human CRIPTO mRNA in GEO human colon cancer cells. We have also evaluated the effects of this anti-CRIPTO antisense oligonucleotide in combination with a chimeric anti-human epidermal growth factor receptor (EGFR) monoclonal antibody (MAb C225) and with 8-Cl-cAMP, a cAMP analog that specifically inhibits type I protein kinase A (PKAI), since a functional EGFR-driven autocrine pathway is operative and PKAI is overexpessed in GEO colon cancer cells. Treatment with a single agent at low doses determined a 15-35% growth inhibition. A synergistic antiproliferative effect was observed when combinations of two agents were used with a co-operativity quotient ranging between 1.5 and 2.2. Furthermore, the combined treatment with all three drugs caused an almost complete suppression of the ability of GEO cells to form colonies in soft agar. We next evaluated whether any combination of 8-Cl-cAMP, the anti-CRIPTO antisense oligonucleotide and MAb C225 could induce programmed cell death in GEO cells. Treatment with each agent alone at all doses tested did not cause DNA fragmentation. The treatment with any combination of two agents was not able to induce apoptosis. In contrast, treatment with all three compounds determined an approximately three-fold increase in DNA fragmentation. In conclusion, the combination of selective antineoplastic agents directed against different but related key signal tranduction pathways efficiently inhibits cell growth and causes apoptosis in human colorectal cancer cells.

Augmentation of a humanized anti-HER2 mAb 4D5 induced growth inhibition by a human-mouse chimeric anti-EGF receptor mAb C225.

Overexpression of epidermal growth factor (EGF) receptor and HER2 (p185neu) may both contribute to the growth of human cancers. A humanized anti-HER2 monoclonal antibody (mAb) 4D5 and a human-mouse chimeric anti-EGF receptor mAb C225 are currently being investigated in clinical trials for their anti-tumor activities. In the present study, we have examined the effect of concurrent treatment of OVCA 420 human ovarian cancer cells with mAb C225 and mAb 4D5. Exposure of OVCA420 cells to saturating concentrations of C225 (20 nM) for 7 days resulted in 40-50% growth inhibition, and exposure to 20 nM mAb 4D5 also resulted in 30-40% growth inhibition. The growth inhibition of OVCA420 cells by mAb C225 or 4D5 was associated with an increased G1 cell population; an increased level of a cyclin-dependent kinase (CDK) inhibitor p27Kip1 with increased association of p27kip1 with CDK2, CDK4 and CDK6; and decreased activities of these CDKs. Combination treatment with concurrent exposure to mAbs C225 and 4D5 resulted in additive anti-proliferative effects on these cells, which was accompanied by enhanced G1 cell distribution, a greater increase in the levels of p27Kip1 and a greater decrease in the activities of CDK kinases. The anti-proliferative effects and related changes in cell cycle regulators induced by mAb 4D5, mAb C225 or the combination of the two mAbs could be reversed by concurrent exposure to exogenous EGF. Our data suggest the potential fruitful cooperation of anti-EGF receptor mAb and anti-HER2 mAb in the treatment of human cancers stimulated by EGF receptor and HER2 signals.

Cooperative inhibition of renal cancer growth by anti-epidermal growth factor receptor antibody and protein kinase A antisense oligonucleotide.

The expression of epidermal growth factor receptor (EGFR) and type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is associated with neoplastic transformation. By use of human renal cancer cell lines (i.e., 769-P, ACHN, A498, and SW839), we investigated the antiproliferative activity and the antitumor activity of an anti-EGFR humanized chimeric mouse monoclonal antibody, MAb C225, and a novel mixed backbone 18-mer antisense oligonucleotide, HYB 190, that targets expression of the RIalpha regulatory subunit of PKAI. The antiproliferative activity of MAb C225 and oligonucleotide HYB 190, alone or in combination, on different renal cancer cell lines was determined by monitoring cell growth in soft agar. In addition, the induction of apoptosis by treatment with the anti-EGFR antibody and/or antisense PKAI oligonucleotides was evaluated by flow cytometric analysis of fragmented DNA. The antitumor activity of MAb C225 and oligonucleotide HYB 190 was determined in athymic mice bearing established ACHN tumor xenografts. Cell proliferation and tumor growth data were evaluated for statistical significance using Student's t test; reported P values are two-sided. MAb C225 and oligonucleotide HYB 190 inhibited colony formation in soft agar in a dose-dependent manner for all renal cancer cell lines tested. We observed a potentiation of growth inhibition and induction of apoptosis when 769-P cells and ACHN cells were treated with both agents. Combination treatment with MAb C225 and oligonucleotide HYB 190 caused regression of ACHN tumor xenografts, whereas single-agent treatment only delayed tumor growth. The combination of anti-EGFR MAb C225 and ited cooperative antiproliferative effects and cooperative antitumor effects on EGFR and PKAI-expressing human renal cancer cell lines.

Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A.

Epidermal growth factor (EGF)-related proteins, such as transforming growth factor-alpha (TGF-alpha), control cancer cell growth through hormonal pathways (i.e., autocrine [hormone acts on cell that produces it] and paracrine [hormone acts on nearby cells] pathways). Overexpression of TGF-alpha and/or its receptor (EGFR) has been detected in human cancers. The blockade of EGFR activation by the use of anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is generally overexpressed in human cancer cells and is involved in neoplastic transformation. Inhibition of PKAI by selective cAMP analogues, such as 8-chloro-cAMP (8-CI-cAMP), induces growth inhibition in various human cancer cell lines. On the basis of our previous observations of a cooperative anti-proliferative effect of anti-EGFR Mab 528 and 8-Cl-cAMP in human cancer cell lines in vitro, we evaluated the anticancer activity in vivo of the combination of an anti-EGFR MAb (MAb C225) and 8-Cl-cAMP. Athymic mice were injected subcutaneously with 10(7) human colon carcinoma GEO cells. After 7 days, when established tumor xenografts of 0.30-0.35 cm3 were detectable, 10-15 mice per group were treated intraperitoneally twice weekly with different doses of 8-Cl-cAMP and/or MAb C225. Cancer cell expression of various growth factors was evaluated by immunohistochemical analysis in tumors obtained from control and treated mice. Data were evaluated for statistical significance using the Student's t test and the Mantel-Cox logrank test. All P values represent two-sided tests of statistical significance. A 5-week treatment with low doses of 8-Cl-cAMP (0.5 mg/dose) and MAb C225 (0.25 mg/dose) blocked GEO tumor growth (compared with that in control mice; P < .00001) and suppressed cancer cell production of autocrine growth factors, such as TGF-alpha, amphiregulin, and CRIPTO, and of angiogenic (promotes new blood vessel formation) factors, such as vascular endothelial growth factor and basic fibroblast growth factor, with no signs of toxicity. Control and 8-Cl-cAMP (0.5 mg/dose)-treated mice died within 9-10 weeks after tumor cell injection. In MAb C225 (0.25 mg/dose)-treated mice, GEO tumors resumed a growth rate comparable to that in control animals within 3 weeks following the end of treatment and the mice died between 11 and 20 weeks after tumor cell injection. GEO tumor growth was significantly delayed in the MAb C225 plus 8-Cl-cAMP treatment group (P < .00001) and was accompanied by a prolonged survival of mice (P < .00001) as compared with the control group. Long-term treatment with a combination of agents that selectively inhibit two intracellular signal-transduction enzymes, such as the PKAI serine-threonine kinase and the EGFR tyrosine kinase, has anticancer activity in vivo, reflected by suppression of tumor proliferation and angiogenesis, with no signs of toxicity. Since these inhibitors of intracellular mitogenic (growth-stimulating) signaling have a different mechanism(s) of action and do not antagonize the effects of cytotoxic therapy, a combination of anti-EGFR MAb C225 and 8-Cl-cAMP should be investigated as a nontoxic, long-term treatment for cancer patients following chemotherapy.