Epidermal Growth Factor Receptor-targeted Antibodies Research Articles

Locally secreted BiTEs complement CAR T cells by enhancing killing of antigen heterogeneous solid tumors

Bispecific Tcell engagers (BiTEs) are bispecific antibodies that redirect Tcells to target antigen-expressing tumors. We hypothesized that BiTE-secreting Tcells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) Tcells. Glioblastomas represent a good model for solidtumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR Tcells, BiTE Tcells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent Tcells compared with bivalent CAR Tcells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent Tcells have potent anti-tumor activity invitro and invivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

Immune- and Non-Immune-Mediated Adverse Effects of Monoclonal Antibody Therapy: A Survey of 110 Approved Antibodies.

Identification of new disease-associated biomarkers; specific targeting of such markers by monoclonal antibodies (mAbs); and application of advances in recombinant technology, including the production of humanized and fully human antibodies, has enabled many improved treatment outcomes and successful new biological treatments of some diseases previously neglected or with poor prognoses. Of the 110 mAbs preparations currently approved by the FDA and/or EMA, 46 (including 13 antibody–drug conjugates) recognizing 29 different targets are indicated for the treatment of cancers, and 66, recognizing 48 different targets, are indicated for non-cancer disorders. Despite their specific targeting with the expected accompanying reduced collateral damage for normal healthy non-involved cells, mAbs, may cause types I (anaphylaxis, urticaria), II (e.g., hemolytic anemia, possibly early-onset neutropenia), III (serum sickness, pneumonitis), and IV (Stevens–Johnson syndrome, toxic epidermal necrolysis) hypersensitivities as well as other cutaneous, pulmonary, cardiac, and liver adverse events. MAbs can provoke severe infusion reactions that resemble anaphylaxis and induce a number of systemic, potentially life-threatening syndromes with low frequency. A common feature of most of these syndromes is the release of a cascade of cytokines associated with inflammatory and immunological processes. Epidermal growth factor receptor-targeted antibodies may provoke papulopustular and mucocutaneous eruptions that are not immune-mediated.

TLR7/8 Agonist-Loaded Nanoparticles Augment NK Cell-Mediated Antibody-Based Cancer Immunotherapy.

Activated natural killer (NK) cells can kill malignant tumor cells via granule exocytosis and secretion of IFN-γ, a key regulator of the TH1 response. Thus, mobilization of NK cells can augment cancer immunotherapy, particularly when mediated through antibody-dependent cellular cytotoxicity (ADCC). Stimulation of toll-like receptor (TLR)7/8 activity in dendritic cells promotes pro-inflammatory cytokine secretion and costimulatory molecule upregulation, both of which can potentiate NK cell activation. However, currently available TLR7/8 agonists exhibit unfavorable pharmacokinetics, limiting their in vivo efficacy. To enable efficient delivery to antigen-presenting cells, we encapsulated a novel imidazoquinoline-based TLR7/8 agonist in pH-responsive polymeric NPs. Enhanced costimulatory molecule expression on dendritic cells and a stronger pro-inflammatory cytokine response were observed with a NP-encapsulated agonist, compared to that with the soluble form. Treatment with NP-encapsulated agonists resulted in stronger in vivo cytotoxicity and prolonged activation of NK cells compared to that with a soluble agonist. In addition, TLR7/8 agonist-loaded NPs potentiated stronger NK cell degranulation, which resulted in enhanced in vitro and in vivo ADCC mediated by the epidermal growth factor receptor-targeting antibody cetuximab. TLR7/8 agonist-loaded NP treatment significantly enhanced the antitumor efficacy of cetuximab and an anti-HER2/neu antibody in mouse tumor models. Collectively, our data show that a pH-responsive NP-encapsulating TLR7/8 agonist could be used as a potent immunostimulatory adjuvant for antibody-based cancer immunotherapy by promoting NK cell activation.

Co-targeting of EGF receptor and neuropilin-1 overcomes cetuximab resistance in pancreatic ductal adenocarcinoma with integrin β1-driven Src-Akt bypass signaling.

Pancreatic ductal adenocarcinoma (PDAC) cells usually overexpress the epidermal growth factor receptor (EGFR); however, most are resistant to the anti-EGFR monoclonal antibody, cetuximab. In this study, we report that the molecular mechanism of resistance to cetuximab in PDAC cells is mediated by the overexpression of active integrin β1 with downstream Src-Akt activation; this triggers an EGFR ligand-independent proliferation signaling, bypassing EGFR-blocking effect. Knockdown of integrin β1 or inhibition of Src or Akt sensitized cetuximab-resistant (CtxR) PDAC cells to cetuximab. We found that neuropilin-1 (NRP1) physically interacts with active integrin β1, but not inactive one, on the cell surface. To inhibit active integrin β1-driven signaling by targeting NRP1, while suppressing EGFR signaling, we generated an EGFR and NRP1 dual targeting antibody, Ctx-TPP11, by genetic fusion of the NRP1-targeting peptide, TPP11, to the C terminus of the cetuximab heavy chain (Ctx-TPP11). We demonstrate that Ctx-TPP11 efficiently inhibited the growth of CtxR PDAC cells, in vitro and in vivo. The sensitization mechanism involved downregulating active integrin β1 levels through NRP1-coupled internalization mediated by the TPP11 moiety, leading to the inhibition of active integrin β1-driven bypass signaling. Our findings identify aberrant active integrin β1-driven Src-Akt hyperactivation as a primary resistance mechanism to cetuximab in PDAC cells and offer an effective therapeutic strategy to overcome this resistance using an EGFR and NRP1 dual targeting antibody.

Abstract A29: Targeting the EGFR in high-grade glioma

Abstract The epidermal growth factor receptor (EGFR) is altered in 57% of high grade glioma (HGG) by mutation, rearrangement and/or focal amplification, suggesting it should be a target for therapy. However, attempts to therapeutically target EGFR in HGG patients have failed for reasons that remain largely unknown. We have derived a panel of primary cell lines from HGG patients expressing different forms of the EGFR including the most common mutations: the autoactivating deletion EGFRvIII and the extracellular domain point mutation A289V. Using anti-proliferative assays and cell cycle analysis we screened our HGG cell lines for response to Panitumumab (an EGFR targeting antibody) or Dacomitinib (an irreversible TKI targeting EGFR). The anti-proliferative activity of Panitumumab and Dacomitinib showed a similar profile across the HGG cell lines, but the response to EGFR inhibition ranged from resistant to highly sensitive. In order to improve the anti-tumour response to Panitumumab we tested it in combination with either a MEK inhibitor (Trametinib) or a PI3K inhibitor (BYL719), as both signalling pathways are downstream of EGFR. The combination of Panitumumab and Trametinib showed additive anti-tumour activity against HGG cell lines expressing the wtEGFR, whereas the combination of Panitumumab with BYL719 did not display additivity. In contrast, EGFRvIII cell lines showed an additive anti-tumour response to Panitumumab and BYL719 but not Trametinib. More generally, the activation of the ras/MEK/ERK pathway through either ras mutation or integrin engagement mediated resistance to EGFR-targeted therapeutics. Finally, the SB2 HGG cell line, which contains the A289V mutation, was completely resistant to EGFR therapy due to an activating mutation in PI3KCA. We are currently confirming these in vitro observations in orthotopic xenograft models. The epidermal growth factor receptor (EGFR) is altered in 57% of high grade glioma (HGG) by mutation, rearrangement and/or focal amplification, suggesting it should be a target for therapy. However, attempts to therapeutically target EGFR in HGG patients have failed for reasons that remain largely unknown. We have derived a panel of primary cell lines from HGG patients expressing different forms of the EGFR including the most common mutations: the autoactivating deletion EGFRvIII and the extracellular domain point mutation A289V. Using anti-proliferative assays and cell cycle analysis we screened our HGG cell lines for response to Panitumumab (an EGFR targeting antibody) or Dacomitinib (an irreversible TKI targeting EGFR). The anti-proliferative activity of Panitumumab and Dacomitinib showed a similar profile across the HGG cell lines, but the response to EGFR inhibition ranged from resistant to highly sensitive. In order to improve the anti-tumour response to Panitumumab we tested it in combination with either a MEK inhibitor (Trametinib) or a PI3K inhibitor (BYL719), as both signalling pathways are downstream of EGFR. The combination of Panitumumab and Trametinib showed additive anti-tumour activity against HGG cell lines expressing the wtEGFR, whereas the combination of Panitumumab with BYL719 did not display additivity. In contrast, EGFRvIII cell lines showed an additive anti-tumour response to Panitumumab and BYL719 but not Trametinib. More generally, the activation of the ras/MEK/ERK pathway through either ras mutation or integrin engagement mediated resistance to EGFR-targeted therapeutics. Finally, the SB2 HGG cell line, which contains the A289V mutation, was completely resistant to EGFR therapy due to an activating mutation in PI3KCA. We are currently confirming these in vitro observations in orthotopic xenograft models. This work is providing new insights into the mechanism that control response to EGFR-targeted therapeutics and should lead to therapeutic combinations that have efficacy in HGG patients. Note: This abstract was not presented at the conference. Citation Format: Vino Pillay, Sameer Greenall, Terrance G. Johns. Targeting the EGFR in high-grade glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A29.

Molecular Taxonomy and Tumourigenesis of Colorectal Cancer

Over the last 5 years there has been a surge in interest in the molecular classification of colorectal cancer. The effect of molecular subtyping on current treatment decisions is limited to avoidance of adjuvant 5-fluorouracil chemotherapy in stage II microsatellite unstable-high disease and avoidance of epidermal growth factor receptor-targeted antibodies in extended RAS mutant tumours. The emergence of specific novel combination therapy for the BRAF-mutant cohort and of the microsatellite unstable-high cohort as a responsive group to immune checkpoint inhibition shows the growing importance of a clinically relevant molecular taxonomy. Clinical trials such as the Medical Research Council FOCUS4 trial using biomarkers to select patients for specific therapies are currently open and testing such approaches. The integration of mutation, gene expression and pathological analyses is refining our understanding of the biological subtypes within colorectal cancer. Sharing of data sets of parallel sequencing and gene expression of thousands of cancers among independent groups has allowed the description of disease subsets and the need for a validated consensus classification has become apparent. This biological understanding of the disease is a key step forward in developing a stratified approach to patient management. The discovery of stratifiers that predict a response to existing and emerging therapies will enable better use of these treatments. Improved scientific understanding of the biological characteristics of poorly responsive subgroups will facilitate the design of novel biologically rational combinations. Novel treatment regimens, including the combination of new drugs with radiation, and the discovery and validation of their associated predictive biomarkers will gradually lead to improved outcomes from therapy.

Interlaboratory Comparison of K-ras Testing by Real-time PCR and RFLP in Colorectal Cancer Samples

Activating mutations of K-ras have been described in approximately 40% of patients with colorectal cancer, and are associated with resistance to epidermal growth factor receptor-targeted antibodies, such as cetuximab and panitumumab. Cost-effective and easy methods to determine K-ras mutations are urgently needed. Samples from 31 patients were tested. In laboratory 1, a real-time polymerase chain reaction (PCR)-based technique was used. All samples (n=31) were additionally tested using a restriction fragment length polymorphism (RFLP) analysis in laboratory 2. All results were confirmed by direct sequencing. In the first run, a concordance of real-time PCR and RFLP was observed in 77.4% (24 of 31) of samples. After resampling and reevaluation, a concordance of 93.5% (30 of 31) could be achieved. One of 7 (6.5%) initial discordant cases showed a mutation using real-time PCR and no mutation using RFLP, but the mutation was confirmed by direct sequencing. Real-time PCR and RFLP can be considered as valid K-ras mutation detection techniques. However, in patient probes with lower amounts of tumor cells and wild-type K-ras, reanalysis of further tumor tissue is recommended.

Novel Agents in the Treatment of Metastatic Colorectal Cancer

Colorectal cancer (CRC) is one of the leading causes of cancer death in the United States. Research has led to an explosion of knowledge into the molecular basis of CRC in the past decades. Numerous receptors and intracellular proteins have been identified and implicated in the growth and progression of metastatic CRC, thus creating novel targets for drug development. Many agents are under development and have begun to enter early and even later-stage clinical trials. Results of these agents have demonstrated some encouraging activity but in a small number of patients. Research into predictive biomarkers aims to select the patients who may benefit from these novel agents. This review will address several of these promising new agents, their potential relevance to CRC, results from early clinical studies, and their incorporation into future and ongoing CRC clinical trials. Clearly, there is an urgent need for new agents in this disease, but as we learned from the experience with epidermal growth factor receptor-targeted antibodies, patient selection will be increasingly be required for individualized therapy to become a reality in CRC.

Efficient mucosal delivery of optical contrast agents using imidazole-modified chitosan

The clinical applicability of antibodies and plasmonic nanosensors as topically applied, molecule-specific optical diagnostic agents for noninvasive early detection of cancer and precancer is severely limited by our inability to efficiently deliver macromolecules and nanoparticles through mucosal tissues. We have developed an imidazole-functionalized conjugate of the polysaccharide chitosan (chitosan-IAA) to enhance topical delivery of contrast agents, ranging from small molecules and antibodies to gold nanoparticles up to 44 nm in average diameter. Contrast agent uptake and localization in freshly resected mucosal tissues was monitored using confocal microscopy. Chitosan-IAA was found to reversibly enhance mucosal permeability in a rapid, reproducible manner, facilitating transepithelial delivery of optical contrast agents. Permeation enhancement occurred through an active process, resulting in the delivery of contrast agents via a paracellular or a combined paracellular/transcellular route depending on size. Coadministration of epidermal growth factor receptor-targeted antibodies with chitosan-IAA facilitated specific labeling and discrimination between paired normal and malignant human oral biopsies. Together, these data suggest that chitosan-IAA is a promising topical permeation enhancer for mucosal delivery of optical contrast agents.

Prospective Cost-Effectiveness Analysis of Cetuximab in Metastatic Colorectal Cancer: Evaluation of National Cancer Institute of Canada Clinical Trials Group CO.17 Trial

The National Cancer Institute of Canada Clinical Trials Group CO.17 study showed that patients with advanced colorectal cancer had improved overall survival when cetuximab, an epidermal growth factor receptor-targeting antibody, was given in addition to best supportive care. We conducted a cost-effectiveness analysis using prospectively collected resource utilization and health utility data for patients in the CO.17 study who received cetuximab plus best supportive care (N = 283) or best supportive care alone (N = 274). Direct medical resource utilization data were collected, including medications, physician visits, toxicity management, blood products, emergency department visits, and hospitalizations. Mean survival times for the study arms were calculated for the entire population and for the subset of patients with wild-type KRAS tumors over an 18- to 19-month period. All costs were presented in 2007 Canadian dollars. One-way and probabilistic sensitivity analysis was used to determine the robustness of the results. Cost-effectiveness acceptability curves were determined. The 95% confidence intervals (CIs) for the incremental cost-effectiveness ratios and the incremental cost-utility ratios were estimated by use of a nonparametric bootstrapping method (with 1000 iterations). For the entire study population, the mean improvement in overall and quality-adjusted survival with cetuximab was 0.12 years and 0.08 quality-adjusted life-years (QALYs), respectively. The incremental cost with cetuximab compared with best supportive care was $23,969. The incremental cost-effectiveness ratio was $199,742 per life-year gained (95% CI = $125,973 to $652,492 per life-year gained) and the incremental cost-utility ratio was $299,613 per QALY gained (95% CI = $187,440 to $898,201 per QALY gained). For patients with wild-type KRAS tumors, the incremental cost with cetuximab was $33,617 and mean gains in overall and quality-adjusted survival were 0.28 years and 0.18 QALYs, respectively. The incremental cost-effectiveness ratio was $120,061 per life-year gained (95% CI = $88,679 to $207,075 per life-year gained) and the incremental cost-utility ratio was $186,761 per QALY gained (95% CI = $130,326 to $334,940 per QALY gained). In a sensitivity analysis, cetuximab cost and patient survival were the only variables that influenced cost-effectiveness. The incremental cost-effectiveness ratio of cetuximab over best supportive care alone in unselected advanced colorectal cancer patients is high and sensitive to drug cost. Incremental cost-effectiveness ratios were lower when the analysis was limited to patients with wild-type KRAS tumors.