Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.     

Recombinant IgE antibody engineering to target EGFR

Monoclonal antibodies have become a mainstay for the targeted treatment of cancer today. Some of the most successful targets of monoclonal antibodies are constituted by the epidermal growth factor receptor family spearheaded by the epidermal growth factor receptor (EGFR). Prompted by studies indicating that IgE compared to IgG may harness alternate effector functions to eradicate malignant cells, we addressed the establishment, engineering, and the potential tumoricidal effects of recombinant anti-EGFR IgE. Therefore, two different therapeutic EGFR-specific antibodies, 225 and 425, were chosen for re-cloning into different chimeric IgE and IgG formats and produced in human cells. Simultaneous antibody binding to the sEGFR demonstrated accessibility of both epitopes for recombinant IgE. Proliferation and cytotoxicity assays demonstrated signal blocking and effector mediating capability of IgE isotypes. Pronounced degranulation in the presence of sEGFR upon activation exclusively with two IgE antibodies verified the epitope proximity and provides evidence that tumor-targeting by anti-EGFR IgE is safe with regard to soluble target structures. Degranulation mediated by tumor cells expressing EGFR could be demonstrated for singular and combined IgE antibodies; however, use of two IgE specificities was not superior to use of one IgE alone. The data suggest that the surface distribution of EGFR is optimally suited to mount a robust effector cell trigger and corroborate the potential and specificity of the IgE/IgE receptor network to react to xenobiotic or pathogenic patterns for targeting malignancies.     

Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling

The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the search for robust targeting molecules that can be equipped with new effector functions or show a new mechanism of action. Designed ankyrin repeat proteins (DARPins) may provide the targeting component for such novel reagents. Previously, four DARPins were selected against EGFR with (sub)nanomolar affinity. As any targeting module should preferably be able to inhibit EGFR-mediated signaling, their effect on A431 cells overexpressing EGFR was examined: three of them were shown to inhibit proliferation by inducing G(1) arrest, as seen for the Food and Drug Administration-approved antibody cetuximab. To understand this inhibitory mechanism, we mapped the epitopes of the DARPins using yeast surface display. The epitopes for the biologically active DARPins overlapped with the EGF-binding site, whereas the fourth DARPin bound to a different domain, explaining the lack of a biological effect. To optimize the biological activity of the DARPins, we combined two DARPins binding to different epitopes with a flexible linker or with a leucine zipper, leading to a homodimer. The latter DARPin was able to reduce surface EGFR by inhibiting receptor recycling, leading to a dramatic decrease in cell viability. These results indicate that multispecific EGFR-specific DARPins are superior to cetuximab and may form the basis of new opportunities in tumor targeting and tumor therapy.     

Epidermal growth factor receptor (EGFR) antibody down-regulates mutant receptors and inhibits tumors expressing EGFR mutations

Activating mutations in the kinase domain of the EGF receptor have been reported in non-small cell lung cancer. The majority of tumors expressing these mutants are sensitive to ATP mimetics that inhibit the EGFR tyrosine kinase. The effect of antibodies that bind to the ectodomain of the receptor is less clear. We report herein the effects and mechanisms of action of the antibody cetuximab in lung cancer cells that naturally express receptor mutations and in ErbB-null 32D hematopoietic cells transfected with mutant EGFR. Treatment with cetuximab down-regulated EGFR levels and inhibited cell growth both in vitro and in vivo. This was associated with inhibition of ligand-independent EGFR signaling. These effects were seen in 32D cells arguing the growth inhibitory action was not because of the blockade of autocrine ligand action. Both antibody-induced EGFR down-regulation and inhibition of growth required receptor dimerization as monovalent Fab fragments only eliminated receptor levels or reduced cell proliferation in the presence of antihuman IgG. Finally, cetuximab inhibited growth of H1975 lung cancer cells and xenografts, which expressed L858R/T790M EGFR and were resistant to EGFR tyrosine kinase inhibitors. These data suggest that cetuximab is an effective therapy against mutant EGFR-expressing cancer cells and thus can be considered in combination with other anti-EGFR molecules.     

A combination of two antibodies recognizing non‐overlapping epitopes of HER2 induces kinase activity‐dependent internalization of HER2

The human epidermal growth factor receptor 2 (HER2/ErbB2) is overexpressed in a number of human cancers. HER2 is the preferred heterodimerization partner for other epidermal growth factor receptor (EGFR) family members and is considered to be resistant to endocytic down‐regulation, properties which both contribute to the high oncogenic potential of HER2. Antibodies targeting members of the EGFR family are powerful tools in cancer treatment and can function by blocking ligand binding, preventing receptor dimerization, inhibiting receptor activation and/or inducing receptor internalization and degradation. With respect to antibody‐induced endocytosis of HER2, various results are reported, and the effect seems to depend on the HER2 expression level and whether antibodies are given as individual antibodies or as mixtures of two or more. In this study, the effect of a mixture of two monoclonal antibodies against non‐overlapping epitopes of HER2 was investigated with respect to localization and stability of HER2. Individual antibodies had limited effect, but the combination of antibodies induced internalization and degradation of HER2 by multiple endocytic pathways. In addition, HER2 was phosphorylated and ubiquitinated upon incubation with the antibody combination, and the HER2 kinase activity was found to be instrumental in antibody‐induced HER2 down‐regulation.     

Antitumor activity of combinations of antibodies directed against different epitopes on the extracellular domain of the human EGF receptor

The receptor (EGFR) for epidermal growth factor (EGF) and transforming growth alpha (TGFα) is often overexpressed in certain types of human malignancy and high levels of expression of the receptor and/ or coexpression of growth factors. EGF and TGFα have also been correlated with poor prognosis in many patients. We have produced a number of rat monoclonal antibodies (MAbs) against four distinct epitopes on the external domain of the EGF receptor and are currently evaluating their potential for therapeutic use. Nine of these of MAbs were found to inhibit the binding of TGF and EGF to the receptor on tumor cells and these MAbs were able to inhibit the growth in vitro and in vivo of tumor cells that overexpress the EGF receptor. Here, we describe the results of experiments to determine the antitumor activity of combination treatment with two antibodies directed against separate epitopes on the external domain of human EGFR. Our results showed that treatment of human tumor xenografts with a combination of two anti-EGFR MAbs that bind to two distinct epitopes on the external domain of EGF receptor was not as effective as treatment with ICR62 alone, which binds to epitope C on the EGFR and is of IgG2b isotype. A phase I clinical trial with antibody ICR62 is currently underway in Royal Marseden Hospital (UK) in patients with head and neck and lung carcinomas.     

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.     

Anti-EGFR biparatopic-SEED antibody has enhanced combination-activity in a single molecule

Certain combinations of non-competitive anti-EGFR antibodies have been reported to produce new effects on cells compared to either antibody used separately. New and enhanced combination-activity includes increased inhibition of signaling, increased receptor internalization and degradation, reduced proliferation of tumor cell lines and induction of complement-dependent cytotoxicity (CDC) effector function. To test requirements and mechanisms to elicit enhanced combination-activity with different EGFR binding domains, we created an anti-EGFR biparatopic antibody. A biparatopic antibody interacts through two different antigen-binding sites to a single antigen. A heterodimeric antibody with one binding domain derived from the C225 antibody and one binding domain derived from the humanized 425 (hu425) antibody was built on the strand-exchange engineered domain (SEED) scaffold. This anti-EGFR biparatopic-SEED antibody was compared to parental antibodies used alone and in combination, and to the corresponding monovalent anti-EGFR-SEED antibodies used alone or in combination. We found that the anti-EGFR biparatopic-SEED had enhanced activity, similar to the combination of the two parental antibodies. Combinations of monovalent anti-EGFR-SEED antibodies did not produce enhanced effectiveness in cellular assays. Our results show that the anti-EGFR biparatopic antibody created using the SEED scaffold has enhanced combination-activity in a single molecule. Furthermore, these data suggest that the potential to cross-link the two different epitopes is an important requirement in the mechanism of enhanced combination-activity.     

Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking monoclonal antibody and ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine pathway plays an important role in cancer cell growth. Vascular endothelial growth factor A (VEGF-A) is a key regulator of tumor-induced endothelial cell proliferation and vascular permeability. ZD6474 is an orally available, small molecule inhibitor of VEGF receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase activity. We investigated the activity of ZD6474 in combination with cetuximab, an anti-EGFR blocking monoclonal antibody, to determine the anti-tumor activity of EGFR blockade through the combined use of two agents targeting the receptor at different molecular sites in cancer cells and of VEGFR-2 blockade in endothelial cells. EXPERIMENTAL DESIGN: The anti-tumor activity in vitro and in vivo of ZD6474 and/or cetuximab was tested in human cancer cell lines with a functional EGFR autocrine pathway. RESULTS: The combination of ZD6474 and cetuximab determined synergistic growth inhibition in all cancer cell lines tested as assessed by the Chou and Talalay method. In nude mice bearing established human colon carcinoma (GEO) or lung adenocarcinoma (A549) xenografts and treated with ZD6474 and/or cetuximab for 4 weeks, a reversible tumor growth inhibition was caused by each drug. In contrast, a more significant tumor growth delay resulted from the combination of the two agents with an approximately 100-110 days increase in mice median overall survival as compared to single agent treatment. CONCLUSIONS: This study provides a rationale for evaluating in a clinical setting the double blockade of EGFR in combination with inhibition of VEGFR-2 signaling as cancer therapy.     

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies.     

Role of targeted therapy in the treatment of squamous cell head and neck cancer

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck cancer (HNC). Since EGFR has a large extracellular ligand binding as well as an intracellular tyrosine kinase domain, anti-EGFR therapy may involve anti-ligand binding domain antibody- or tyrosine kinase inhibitor therapies. Phase II-III studies confirmed the efficacy of anti-EGFR antibody therapy in case of squamous cell HNC. In combination with irradiation, anti-EGFR antibody therapy improved survival of locally advanced HNC patients. In case of recurrent or metastatic HNC, anti-EGFR antibody therapy in combination with chemotherapy significantly increased remission rate without increasing toxicity. Although studies on EGFR kinase inhibitors in HNC are in early phase, preliminary data are encouraging.     

Primary and acquired resistance to anti-EGFR targeted drugs in cancer therapy

In recent years, the epidermal growth factor receptor (EGFR) has been recognized as a central player and regulator of cancer cell proliferation, apoptosis and angiogenesis and, therefore, as a potentially relevant therapeutic target. Several strategies for EGFR targeting have been developed, the most succesful being represented by monoclonal antibodies, that directly interfere with ligand-receptor binding and small molecule tyrosine kinase inhibitors, that interfere with activation/phosphorylation of EGFR. These agents have been authorized in advanced chemorefractory cancers, including colorectal cancer, non-small-cell lung cancer and head and neck cancer. However, evidence of resistance to these drugs has been described and extensive studies have been performed to investigate whether resistance to EGFR-targeted therapy is primary or secondary. Cellular levels of EGFR do not always correlate with response to the EGFR inhibitors. Indeed, in spite of the over expression and efficient inhibition of EGFR, resistance to EGFR inhibitors may occur. Moreover, given the genetic instability of cancer cells, genetic modifications could enable them to acquire a resistant phenotype to anti-EGFR therapies. Taken together, these findings support the importance of understanding the molecular mechanisms affecting cancer cell sensitivity or resistance to such inhibitors. This review will focus on the most relevant mechanisms contributing to the acquisition of sensitivity/resistance to EGFR inhibitors.     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

Non-agonistic bivalent antibodies that promote c-MET degradation and inhibit tumor growth and others specific for tumor related c-MET

The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519-561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed β-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies.     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

T cells are crucial for the anti-metastatic effect of anti-epidermal growth factor receptor antibodies

Experimental evidences supporting the epidermal growth factor receptor (EGFR) as an important molecule for tumor metastasis had been accumulated. Currently, anti-EGFR monoclonal antibodies (mAbs) constitute a promising approach for the treatment of patients with metastatic tumors. However, the mechanisms associated with the potent anti-metastatic effect of these mAbs have not been completely elucidated due to the lack of appropriate syngeneic preclinical models. In this paper, we have investigated the effects of 7A7, an antibody specific to murine EGFR, on the metastatic properties of D122 murine lung carcinoma. 7A7 mAb significantly impaired metastatic spread of D122 cells in C57BL/6 mice by direct anti-proliferative and pro-apoptotic effects on tumor metastasis. 7A7 mAb capacity to inhibit EGFR activation on D122 cells could contribute to its anti-metastatic effect. In addition, 7A7 mAb was able to induce in vitro antibody-dependent cell-mediated cytotoxicity on D122 cells. Interestingly, 7A7 mAb treatment increased the number of natural killer cells, T lymphocytes and dendritic cells infiltrating the metastatic sites. More strikingly, depletion of CD8⁺ and CD4⁺ T cells in vivo completely abrogated the 7A7 mAb anti-metastatic activity whereas function of natural killer cells was irrelevant. This study supports an in vivo role for T cell response in the mechanism of action of anti-EGFR mAbs, suggesting the induction of an adjuvant effect. This work was supported by the Cuban Government.     

A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor

Engineered domains of human fibronectin (Adnectins?) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10¹³ Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 μM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC₅₀ values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.     

Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer

Epidermal Growth Factor Receptor (EGFR) is overexpressed on a number of human cancers, and often is indicative of a poor outcome. Treatment of EGFR/HER2 overexpressing cancers includes monoclonal antibody therapy (cetuximab/trastuzumab) either alone or in conjunction with other standard cancer therapies. While monoclonal antibody therapy has been proven to be efficacious in the treatment of EGFR/HER2 overexpressing tumors, drawbacks include the lack of long-lasting immunity and acquired resistance to monoclonal therapy. An alternative approach is to induce a polyclonal anti-EGFR/HER2 tumor antigen response by vaccine therapy. In this phase I/II open-label study, we examined anti-tumor immunity in companion dogs with spontaneous EGFR expressing tumors. Canine cancers represent an outbred population in which the initiation, progression of disease, mutations and growth factors closely resemble that of human cancers. Dogs with EGFR expressing tumors were immunized with a short peptide of the EGFR extracellular domain with sequence homology to HER2. Serial serum analyses demonstrated high titers of EGFR/HER2 binding antibodies with biological activity similar to that of cetuximab and trastuzumab. Canine antibodies bound both canine and human EGFR on tumor cell lines and tumor tissue. CD8 T cells and IgG deposition were evident in tumors from immunized dogs. The antibodies inhibited EGFR intracellular signaling and inhibited tumor growth in vitro. Additionally, we illustrate objective responses in reducing tumors at metastatic sites in host animals. The data support the approach of amplifying anti-tumor immunity that may be relevant in combination with other immune modifying therapies such as checkpoint inhibitors.     

Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

Epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers acquire resistance to EGFR tyrosine kinase inhibitors through multiple mechanisms including c-Met receptor pathway activation. We generated a bispecific antibody targeting EGFR and c-Met (JNJ-61186372) demonstrating anti-tumor activity in wild-type and mutant EGFR settings with c-Met pathway activation. JNJ-61186372 was engineered with low fucosylation (<10 %), resulting in enhanced antibody-dependent cell-mediated cytotoxicity and FcγRIIIa binding. In vitro and in vivo studies with the single-arm EGFR or c-Met versions of JNJ-61186372 identified that the Fc-activity of JNJ-61186372 is mediated by binding of the anti-EGFR arm and required for inhibition of EGFR-driven tumor cells. In a tumor model driven by both EGFR and c-Met, treatment with Fc-silent JNJ-61186372 or with c-Met single-arm antibody reduced tumor growth inhibition compared to treatment with JNJ-61186372, suggesting that the Fc function of JNJ-61186372 is essential for maximal tumor inhibition. Moreover in this same model, downregulation of both EGFR and c-Met receptors was observed upon treatment with Fc-competent JNJ-61186372, suggesting that the Fc interactions are necessary for down-modulation of the receptors in vivo and for efficacy. These Fc-mediated activities, in combination with inhibition of both the EGFR and c-Met signaling pathways, highlight the multiple mechanisms by which JNJ-61186372 combats therapeutic resistance in EGFR mutant patients.     

表皮生长因子受体胞外区在哺乳动物细胞HEK293T中的分泌表达和鉴定

目的:融合表达表皮生长因子受体(EGFR)胞外功能区,为制备针对该分子的特异性抗体提供可用的靶标抗原。方法:通过PCR扩增EGFR胞外区基因,将其克隆入真核表达载体pABhFc中,重组质粒瞬时转染HEK293T细胞,进行EGFR的瞬时分泌表达,纯化获得电泳纯级的分泌蛋白,并通过ELISA、Western印迹、Biacore3000系统对融合蛋白进行鉴定。结果:经测序证实扩增得到了正确的EGFR胞外区基因序列,SDS-PAGE初步确认获得了单、双体的EGFR胞外区,ELISA检测证实双体融合蛋白hFc-EGFR可与商业化的EGF特异性结合,Western印迹检测证实单体融合蛋白His-EGFR可与商业化抗体特异性结合;经Biacore3000蛋白分子相互作用系统测定,双体融合蛋白hFc-EGFR与商业化抗体爱必妥的亲和力达0.5 nmol/L。结论:利用哺乳动物细胞HEK293T分泌表达系统获得了结构正确的单、双体2种类型的EGFR胞外区融合蛋白纯品,将用于抗EGFR特异性抗体的筛选。