Biol. Chem. 2015; 396(8): 917–921

Shenzhi Xiea, Xiaohui Zhua, Yi Li, Li Li, Yimin Si and Na Yang*

Characterization of a new dual-targeting fully human antibody with potent antitumor activity against nasopharyngeal carcinoma Abstract: Despite the effectiveness of the anti-EGFR chimeric antibody (mAb), cetuximab, in treating nasopharyngeal carcinoma (NPC), its efficacy remains variable and often modest. In this study, a full human dual targeted anti-EGFR/HER3 antibody, CA1182, was generated from phage display library. CA1182 was as effective as cetuximab or trastuzumab in inhabiting phosphorylation of EGFR or HER2, but it exhibited as much more potent than cetuximab or trastuzumab. Moreover, our studies showed that CA1182 was significantly more effective than cetuximab in prolonging the survival of severe combined immune deficient mice bearing human NPC, suggesting that it might be a promising therapeutic agent for NPC. Keywords: EGFR; human antibody; nasopharyngeal carcinoma; oncogene. DOI 10.1515/hsz-2014-0236 Received August 8, 2014; accepted November 16, 2014; previously published online November 26, 2014

Introduction Nasopharyngeal carcinoma (NPC) is a rare malignancy in most populations of the world, with incidence rates lower than 1 per 100 000 person-years (Parkin et  al., 2005). However, among populations in the southern parts of a These authors contributed equally to this work. *Corresponding author: Na Yang, South Building No. 2 Division, General Hospital of Chinese People’s Armed Police Forces, Beijing 100039, China, e-mail: [email protected] Shenzhi Xie: Department of Oncology, General Hospital of Chinese People’s Armed Police Forces, Beijing 100039, China Xiaohui Zhu: Department of Gastroenterology, General Hospital of Chinese People’s Armed Police Forces, Beijing 100039, China Yi Li: Department of Oncology, Kunming General Hospital of Chengdu Military Command, Kunming, Yunnan 650032, China Li Li: Department of Nutrition, General Hospital of Armed Police Forces, Beijing 100039, China Yimin Si: South Building No. 2 Division, General Hospital of Chinese People’s Armed Police Forces, Beijing 100039, China

China and Southeast Asia, where NPC is more endemic than any parts of the world, the incidence rates are as high as 20–50 per 100 000 person-years, especially in Cantonese-speaking men residing in Guangdong Province and Hong Kong of Southern China (Jia et al., 2006). Salted-fish consumption (Mimi et al., 1986), Epstein-Barr virus (EBV) infection (Lanier et  al., 1981) and genetic susceptibility (Niedobitek, 2000) are considered to be the major risk factors that contribute to such a distinguished geographic distribution for this cancer. Although it has not been addressed thoroughly, several pieces of evidence suggest that EBV infection is strongly associated with the occurrence of NPC, especially the undifferentiated subtype of non-keratinising carcinoma (Niedobitek et  al., 1991) – the most common histopathological type in southern China according to WHO classification (Liu, 1999). As early as in 1966, elevation of antibodies against EBV antigens in NPC patients was observed (Old et  al., 1966). The therapeutic EGFR antibodies cetuximab and panitumumab antagonize EGFR signaling by blocking ligand binding. These antibodies show marginal objective responses in advanced colorectal cancer patients when used as single agents. Randomized trials of EGFR antibodies combined with chemotherapy showed a statistically significant, albeit modest, increase in progression free survival vs. chemotherapy alone, especially in tumors that are wild-type for K-ras (Grothey, 2010) The most impressive single-agent activity of EGFR antagonists is observed in non-small cell lung cancer (NSCLC) patients whose tumors harbor somatic kinase domain mutations and are treated with erlotinib or gefitinib (Lynch et al., 2004). In contrast, the clinical benefit in patients with wild-type EGFR NSCLC is considerably diminished. Preclinical studies report a strong association between sensitivity to gefitinib in NSCLC cell lines and the inactivation of HER2 (Hu et  al., 2013; Fu et  al., 2014). Moreover, this observation was extended to a panel of colorectal and pancreatic cancer cell lines, where erlotinib anti-proliferative activity was again correlated with inhibition of HER2 phosphorylation. These findings raise the possibility that in the absence of EGFR kinase domain

Brought to you by | University of California Authenticated Download Date | 12/31/15 2:13 AM

918      S. Xie et al.: A new dual-targeting fully human antibody mutations, optimal clinical benefit of anti-EGFR requires that HER2 is also inhibited. In the present study, a full human anti-human EGFR/HER2 monoclonal antibody CA1182 was generated by phage display method. Then we described the construction and characterization of the antibody. Our data indicated that CA1182 was shown to retain the same antigen-binding affinity and specificity as cetuximab and trastuzumab. The in vitro and in vivo antitumor activity of CA1182 was further examined and compared with cetuximab or trastuzumab.

Results Generation of a cetuximab-like anti-EGFR monoclonal antibody To explore the therapeutic potential of a dual-action antibody that can block both signaling pathways, we sought to generate an antibody that would recognize and inhibit EGFR as well as HER2. We first isolated an antiEGFR antibody, CA11, from a phage-displayed Fab library based on its ability to block TGF-a binding to EGFR and then improved CA11 to the high-affinity variant CA12 (Kd 0.2 nm) by affinity maturation. CA12 potently inhibited 125I-EGF binding to EGFR. The ability of CA12 to block ligand binding translated into potent inhibition of TGF-astimulated phosphorylation of EGFR, downstream signaling, and cell proliferation of EGFR-NR6 cells. Further, CA12 demonstrated potent antitumor activity in the EGFR amplified A431 xenograft model that is highly sensitive to anti-EGFR therapeutics. The antitumor activity of CA12 was comparable to the activity seen with cetuximab (Kd = 1.1 nm).

Generation of a dual targeted EGFR/HER2 monoclonal antibody After establishing CA12 as an effective EGFR inhibitor, we sought to add anti-HER2 activity. As CA12 was isolated from an antibody library with diversity restricted to the heavy-chain CDRs, we expected its important antiEGFR residues to be in the heavy chain. We constructed a library of CA12 variants with mutations in the light-chain CDRs and were able to identify clones that bound HER2 while maintaining binding to EGFR. These clones, e.g., CA121, not only bound to both receptors but also blocked ligand binding to EGFR and HER3, albeit with much

reduced affinity for EGFR (EC50: 200 nm). To demonstrate anti-proliferative activity of these dual-specific antibodies, we reformatted and expressed several phage clones as human IgG1 proteins. To evaluate HER2 inhibition, we chose a NPC cancer cell line, BT474, which secretes HRG and as a result activates HER2 in an autocrine manner (Schaefer et  al., 1997). We used EGFR-NR6 cells as described above to verify the antagonism of EGFR. As expected, CA12 potently inhibited ligand-induced growth of EGFR-NR6 cells in a dose-dependent manner but had no effect on the growth of BT474 cells. The dual-specific antibodies, however, were able to inhibit EGFR- and HER2-mediated proliferation in the two cell lines. CA1181, which differs from CA12 by only two amino acid substitutions in the light chain, potently inhibited the growth of BT474 cells, demonstrating that binding to HER2 translated into potent in vitro activity. However, the low affinity of CA1181 for EGFR led to inhibition of TGF-a-driven growth of EGFR-NR6 cells only at high concentrations. To improve the dual affinity of CA1181, we first assessed the energetic importance of the CDR residues necessary for binding to EGFR and HER3 using alanine and homolog mutagenesis scanning (see Materials and methods). The analysis revealed that, as expected, heavy-chain CDR residues are dominant in EGFR binding. In contrast, HER3 binding required residues from both light-chain and heavy-chain CDRs. By stringent affinity-based selection from the homolog libraries, we identified many variants with improved affinity. We focused on one Fab, designated CA1182, which exhibited improved binding affinities for both EGFR and HER2 (Kd 0.9 and 0.3 nm, respectively). CA1182 has ten amino acid substitutions compared with CA1181. The enhanced binding affinities of DL11f for EGFR and HER2 translated into increased anti-proliferative activity in EGFR-NR6 and BT474 cells (Figure 1).

Competitive binding assay The human antibody CA1182 was expressed in CHO cells and then purified from the CHO cell serum-free culture supernatant by Protein A affinity chromatography. In the competitive binding assay, CA1182 effectively competed with cetuximab or trastuzumab for binding to EGFR or HER2 (Figure 1). The avidity (mean IC50 ± SD) of CA1182 to EGFR (1.11 ± 0.32 μg/ml, 1.76 ± 0.51 μg/ml) was similar to that of cetuximab (1.25 ± 0.35 μg/ml) and trastuzumab (1.66 ± 0.13 μg/ml), suggesting that this humanized antibody possessed affinity and specificity similar to that of cetuximab or trastuzumab.

Brought to you by | University of California Authenticated Download Date | 12/31/15 2:13 AM

S. Xie et al.: A new dual-targeting fully human antibody      919

Figure 1 Competitive binding assay. Cetuximab, trastuzumab, and CA1182 were evaluated for their ability to compete with cetuximab-FITC or trastuzumab-FITC for binding.

CA1182 has the best inhibition rate in cancer cell growth in vitro and in vivo We compared the ability of trastuzumab, cetuximab, and CA1182 to inhibit the NPC cell lines CNE2 and C666-1. Previous studies have suggested that EGFR plays little or no role in driving the biology of EGFR-overexpressing NPC. Consistent with this, our data indicated that trastuzumab effectively suppressed the growth of CNE2 and C666-1 NPC cancer cells, whereas cetuximab had only a minor effect (Figure 2). The addition of cetuximab to trastuzumab did not result in a significant increase in growth inhibition compared with trastuzumab alone (Figure 2). Remarkably, the anti-EGFR/HER2 antibody, CA1182, was significantly more potent in inhibiting

the proliferation of NPC cancer cell lines than cetuximab and trastuzumab, either alone or in combination (Figure 2). To further investigate the effect of CA1182 treatment on NPC tumor growth in vivo, nude mice bearing established CNE2 and C666-1 xenograft tumors were treated twice weekly with control human IgG, trastuzumab, cetuximab, trastuzumab plus cetuximab, or CA1182 for 3 weeks. As shown in Table 1, trastuzumab, but not cetuximab, was effective in delaying the CNE2 and C666-1 xenograft tumor progression. Combinatorial treatment with these two mAbs did not result in a significant benefit over single-agent trastuzumab treatment in the CNE2 and C666-1 xenograft mouse model. It is particularly noteworthy that CA1182 was significantly more efficient in inhibition of the CNE2 and C666-1

Figure 2 Dual-action antibodies effectively inhibits cancer cell proliferation. Results are shown as the percentage of control cell proliferation.

Brought to you by | University of California Authenticated Download Date | 12/31/15 2:13 AM

920      S. Xie et al.: A new dual-targeting fully human antibody

Materials and methods

Table 1 Tumor inhibition rate of antibodies in day 25. Antibody

Cetuximab   Trastuzumab  Cetu+Tras   CA1182   Cetuximab   Trastuzumab  Cetu+Tras   CA1182  

Xenografts   CNE2 CNE2 CNE2 CNE2 C666-1 C666-1 C666-1 C666-1


Tumor growth inhibition (%CTRL) 45 15 49 67 65 31 58 91

tumors than trastuzumab plus cetuximab. The survival curves of tumor-bearing mice were plotted according to the Kaplan-Meier method and compared using the logrank test. Treatment with CA1182 appeared to be significantly more effective in prolonging the survival of CNE2 and C666-1 tumor-bearing nude mice than all the other treatments (Table 1).

Phage display libraries and selections All enzymes used were purchased from Fermentas Life Science (St Leon-Rot, Germany) unless otherwise specified. The predator library was constructed in the pHEN1 vector by insertion of a synthesized dAb gene between the unique SapI and Mva1269I restriction sites. This means that the library includes ampicillin resistance gene, the f1 phage origin of replication, most of the phage gene III and the bacterial origin of replication from the original pHen1 vector. The dAb scaffold gene was based on the HEL4 sequence (GenBank: CQ761108.1) with silent mutations introduced to create unique restriction sites around the CDR regions. The dAb gene along with the flanking sequences were synthesized at Mr. Gene (Regensburg, Germany) according to our design. The synthesized dAb gene was digested with SapI and Mva1269I and ligated into pHEN1 to create the preliminary predator vector. Finally an isoleucine in the framework region just prior to CDR1 was changed to an aspartic acid by site directed mutagenesis PCR.

Cells and culture condition Tumor cell lines HEK293, CHO, and CNE2 and C666-1 were obtained from American Type Culture Collection (ATCC. Manassas, VA) and were grown with either 0.4 mg/ml genecitin or 0.05 mg/ml hygromycin.

Discussion Here we have described the full human monoclonal antibody, CA1182. Antigen-binding activity assays indicated that this version lost the binding activity to EGFR-positive cells. To date, HER2 is not reported to undergo oncogenic activation as a result of mutation or amplification. However, during the last decade unique structural aspects of HER2 emerged that produced insights and questions regarding the receptor’s function in normal tissue and hyperproliferative conditions such as cancer. In the absence of an appropriate coreceptor, HRG binds to HER3 with relatively low affinity. In the presence of another HER family member, the low-affinity HRG binding site is converted to a high-affinity site by the formation of a heterodimeric complex. Additionally, the EGFR extracellular domain exists in a closed conformation and undergoes an impressive conformational change in the presence of ligand that allows for the formation of a two domain ligand binding site and the exposure of a receptor dimerization arm. All in all, there is accumulating evidence to support the hypothesis that simultaneously targeting more than one proliferation and survival signal, or targeting a resistance mechanism, will provide better clinical outcomes. The unique potential of CA1182 to both treat EGFR/HER2driven disease and delay warrants its consideration as a promising anticancer therapy in the clinic.

Coimmunoprecipitation assays Protein interation was evaluated in a coimmunoprecipitation assay, as describe (Muthuswamy et  al., 1999; Agus et  al., 2002; Franklin et  al., 2004). Transfected cells were lysed in 1  ml RPMI lysis buffer [1% v/v DevelopTriton X-100, 1% w/v CHAPS, 10 mm HEPES (pH 7.2), in RPMI medium containing 0.2 mm PMSF, 10 mg/ml leupeptin, 10 U/ml aprotinin, and 1 mm Na3VO4]. Targeted proteins were immunoprecipitated from 500 μl of lysate using mouse anti-V5 (AbD Serotec, Oxford, UK) antibody, covalently coupled to agarose (Pierce ultralink, Rockford, Illinois, USA) at 4°C for 2 h. Complexes were washed twice in lysis buffer and resuspended in SDS sample buffer and boiled. Samples were separated on a 4–12% polyacrylamide gel (Invitrogen, CA, USA) and electro-blotted onto nitrocellulose membranes. Blots were blocked in 10% BSA/TBST and probed with different antibodies. To verify and normalize for expression of transfected constructs between experimental conditions, 50 μg of cell lysate was checked by Western blotting with anti-V5 antibody (AbD Serotec, Oxford, UK) or anti-Flag antibody (Sigma-Aldrich, Germany). Anti-mouse HRP-conjugated secondary antibody was used for visualization by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Uppsala, Sweden).

Western blot analysis For Western blot analysis, 40 μg of whole cell extracts were fractioned by SDS-PAGE and transferred onto Hybond nitrocellulose membranes (GE Healthcare, Pittsburgh, PA, USA). Filters were blocked in PBS-Tween 20/5% skim milk and probed with antibodies against respective target proteins (EGFR Ab., Santa Cruz #sc-20093;

Brought to you by | University of California Authenticated Download Date | 12/31/15 2:13 AM

S. Xie et al.: A new dual-targeting fully human antibody      921 HER2 Ab., Santa Cruz #sc-20093; HER33 Ab., Santa Cruz # sc-17342) at a dilution of 1:50 and 1:100 or probed with anti-β-catenin antibodies, which were visualized by SuperSignal West PICO chemiluminescent detection system (Invitrogen, CA, USA). β-Actin was used as equal protein loading control.

Statistical analysis All in vitro assay results represent the arithmetic mean ± SE of triplicate determinations of three independent experiments done under the same conditions. Student’s t test was used to determine the differences between groups and p 

Characterization of a new dual-targeting fully human antibody with potent antitumor activity against nasopharyngeal carcinoma.

Despite the effectiveness of the anti-EGFR chimeric antibody (mAb), cetuximab, in treating nasopharyngeal carcinoma (NPC), its efficacy remains variab...
537KB Sizes 0 Downloads 11 Views