Epitopes Of Epidermal Growth Factor Receptor Research Articles

Capsule-like molecular imprinted polymer nanoparticles for targeted and chemophotothermal synergistic cancer therapy

Selective cancer cell targeting, controlled drug release, easy construction and multiple therapeutic modalities are some of the desirable characteristics of drug delivery systems. We designed and built simple capsule-like molecular imprinted polymer (MIP)-based nanoparticles for targeted and chemo-photothermal synergistic cancer therapy. Using dopamine (DA) as functional monomer, cross-linking agent as well as photo-thermal agent, ZIF-8 (zeoliticimidazolate framework-8) as drug carrier, epitope of EGFR (epidermal growth factor receptor) as template molecules, molecular imprinted polymer (MIP) drug carrier was constructed. The ability of MIP layer to bind to EGFR epitope endowed the MD (DOX@MIP) particles to recognize EGFR-overexpressing cancer cells, while the pH-responsiveness and photothermal conversion ability of PDA (polydopamine) achieved chemo-photothermal synergistic effects upon NIR irradiation. Taken together, the MD nanoparticles integrated cancer cell targeting recognition, intelligent drug release, biocompatibility and chemo-photothermal effects, and is therefore a promising tool for targeted cancer therapy with minimal toxicity to normal cells, as well as tumor imaging.

Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.

Targeting and Efficacy of Novel mAb806-Antibody-Drug Conjugates in Malignant Mesothelioma.

Epidermal growth factor receptor (EGFR) is highly overexpressed in malignant mesothelioma (MM). MAb806 is a novel anti-EGFR antibody that selectively targets a tumor-selective epitope. MAb806-derived antibody drug conjugates (ADCs), ABT-414, ABBV-221 and ABBV-322, may represent a novel therapeutic strategy in MM. EGFR and mAb806 epitope expressions in mesothelioma cell lines were evaluated using an array of binding assays, and the in vitro cell effects of ABT-414 and ABBV-322 were determined. In vivo therapy studies were conducted in mesothelioma xenograft and patient-derived xenograft (PDX) tumor models. We also performed biodistribution and imaging studies to allow the quantitative targeting of MM by mAb806 using a 89Zr-labeled immunoconjugate—ch806. A high EGFR expression was present in all mesothelioma cell lines evaluated and mAb806 binding present in all cell lines, except NCIH-2452. ABT-414 and ABBV-322 resulted in significant tumor growth inhibition in MM models with high EGFR and mAb806 epitope expressions. In contrast, in an EGFR-expressing PDX model that was negative for the mAb806 epitope, no growth inhibition was observed. We demonstrated the specific targeting of the mAb806 epitope expressing MM tumors using 89Zr-based PET imaging. Our data suggest that targeting EGFR in MM using specific ADCs is a valid therapeutic strategy and supports further investigation of the mAb806 epitope expression as a predictive biomarker.

Anti-Proliferative Effect of Specific Anti-EGFR Single Chain Antibody on Triple Negative Breast Cancer Cells.

Targeted therapy is an important treatment strategy that is widely used for cancer therapy. Epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of Triple-negative breast cancer (TNBC) patients. Although Cetuximab, which targets EGFR, has shown some inhibitory effects on TNBC cells, Cetuximab resistance cases due to ligand-independent activating mutations in the EGFR gene limit its application. Due to various benefits of single chain antibodies (scFvs), the use of these antibodies in cancer targeted therapy is increasing. In this study, a specific anti-EGFR antibody was isolated and evaluated. Panning procedure was used against an immunodominant epitope of EGFR in its dimerization arm using a diverse phage library. Polymerase Chain Reaction (PCR) and fingerprinting were applied to identify the specific clones. The MTT tetrazolium assay was performed to evaluate the inhibitory effects of selected anti- EGFR scFv phage antibody on MDA-MB-468, a TNBC cell line. After four round of panning, one dominant pattern was observed in DNA fingerprinting with frequency of 85%. The growth of MDA-MB-468 cells was decreased dose-dependently after treatment with anti-EGFR scFv phage antibody. No significant inhibitory effect of M13KO7 helper phage as negative control on the cell growth of MDA-MB-468 was observed (p> 0.05). The selected anti-EGFR scFv with high anti proliferative effect on TNBC cells offers an effective alternative for TNBC targeted therapy. The antibody, which binds to the dimerization arm of EGFR and inhibits EGFR dimerization, could also overcome TNBC cases with Cetuximab resistance due to ligandindependent activating mutations.

Carbon dots-embedded epitope imprinted polymer for targeted fluorescence imaging of cervical cancer via recognition of epidermal growth factor receptor.

A carbon dots-embedded epitope imprinted polymer (C-MIP) was fabricated for targeted fluorescence imaging of cervical cancer by specifically recognizing the epidermal growth factor receptor (EGFR). The core-shell C-MIP was prepared by a reverse microemulsion polymerization method. This method used silica nanoparticles embedded with carbon dots as carriers, acrylamide as the main functional monomer, and N-terminal nonapeptides of EGFR modified by palmitic acid as templates. Aseries of characterizations (transmission electron microscope, dynamic light scattering, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, zeta potential,and energy dispersive X-ray spectroscopy) prove the successful synthesis of C-MIP. The fluorescence of C-MIP isquenched by the epitopes of EGFR due to the specific recognition of epitopes of EGFR through their imprinted cavities (analytical excitation/emission wavelengths, 540nm/610nm). The linear range of fluorescence quenching is 2.0 to 15.0μgmL-1 and the determination limit is 0.73μgmL-1. The targeted imaging capabilities of C-MIP are demonstrated through in vitro and in vivo experiments. The laser confocal imaging results indicate that HeLa cells (over-expression EGFR) incubated with C-MIP show stronger fluorescence than that of MCF-7 cells (low-expression EGFR), revealing that C-MIP can target tumor cells overexpressing EGFR. The results of imaging experiments in tumor-bearing mice exhibit that C-MIP has a better imaging effect than C-NIP, which further proves the targeted imaging ability of C-MIP in vivo. Graphical abstract An oriented epitope imprinted polymer embedded with carbon dots was prepared for the determination of the epitopes of epidermal growth factor receptor and targeted fluorescence imaging of cervical cancer.

Abstract B099: Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor

Abstract Dysregulation of the epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of malignancies including glioblastoma and various epithelial cancers. Oncogenesis occurs from overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287-302 loop from the extracellular domain is exposed completely on EGFRvIII (deletion of exons 2-7), partially exposed on some cancers but cryptic on normal cells expressing WT levels of EGFR. We report on the generation of antibodies to this loop. A peptide corresponding to amino acids 286-303 was synthesized and then coupled chemically to Keyhole Limpet Hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V-regions were cloned and sequenced. Receptor binding was characterized using ELISA and flow cytometry. Monoclonal antibodies (n=7) reactive for the 287-302 loop were characterized further. Each one reacted with EGFRvIII but not EGFR WT. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be potently cytotoxic for MDA-468, A431 and F98-EGFRvIII-expressing cells. We conclude that immunization with a peptide corresponding to a cryptic EGFR epitope can produce cancer-binding antibodies. By way of example, the 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be developed as a therapeutic agent for clinical use. Citation Format: Eric Chun Hei Ho, Antonella Antignani, Robert Sarnovsky, David J FitzGerald. Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B099. doi:10.1158/1535-7163.TARG-19-B099

Modulating antibody-dependent cellular cytotoxicity of epidermal growth factor receptor-specific heavy-chain antibodies through hinge engineering.

Human IgG1 and IgG3 antibodies (Abs) can mediate Ab-dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRγIIIa. In the absence of a CH 1 domain, many camelid heavy-chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their VH H and CH 2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge-engineered epidermal growth factor receptor (EGFR)-specific chimeric camelid VH H-human Fc Abs and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR-positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2-hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH 2 fucosylation. Conversely, an EG2-hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab hinge. Although this effect appears to be heavily epitope-dependent, this strategy may be useful to consider during the design of VH H-based therapeutic Abs for cancer.

Specific Drug Delivery to Cancer Cells with Double-Imprinted Nanoparticles against Epidermal Growth Factor Receptor.

Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, is over-expressed in many tumors, including almost half of triple-negative breast cancers. The latter belong to a very-aggressive and drug-resistant form of malignancy. Although humanized anti-EGFR antibodies can work efficiently against these cancers both as monotherapy and in combination with genotoxic drugs, instability and high production costs are some of their known drawbacks in clinical use. In addition, the development of antibodies to target membrane proteins is a very challenging task. Accordingly, the main focus of the present work is the design of supramolecular agents for the targeting of membrane proteins in cancer cells and, hence, more-specific drug delivery. These were produced using a novel double-imprinting approach based on the solid-phase method for preparation of molecularly imprinted polymer nanoparticles (nanoMIPs), which were loaded with doxorubicin and targeted toward a linear epitope of EGFR. Additionally, upon binding, doxorubicin-loaded anti-EGFR nanoMIPs elicited cytotoxicity and apoptosis only in those cells that over-expressed EGFR. Thus, this approach can provide a plausible alternative to conventional antibodies and sets up a new paradigm for the therapeutic application of this class of materials against clinically relevant targets. Furthermore, nanoMIPs can promote the development of cell imaging tools against difficult targets such as membrane proteins.

Production of Specific Anti-EGFR Single Chain Antibodies: A Promising Strategy in the Immunotherapy of EGFR Expressing Tumor Tissues

Background: EGFR (Epidermal growth factor receptor) is overexpressed in a number of cancers and plays an important role in several phenomena such as aggressiveness of tumor, decreased survival of the patient, and resistance to treatments such as hormone therapy, chemotherapy, and also radiation. Objectives: The aim of this study is to produce specific human single-chain antibodies against EGFR and evaluate its specificity against the immunodominant epitope in order to offer a new and efficient way in the treatment of EGFR-expressing tumor tissues. Methods: A phage antibody display library of scFv (single chain fragment variable) was panned against an immunodominant epitope of EGFR. In order to select the specific clones, DNA fingerprinting was performed and the common patterns were differentiated. ELISA (Enzyme linked immunosorbent assay) was done to confirm the panning results and show the specificity of the selected clones. Results: Two specific clones with the frequencies of 55% and 30% were differentiated. The clones showed positive ELISA with the corresponding epitope while no positive reaction was observed for negative controls: unrelated peptide, M13KO7 (helper phage), unrelated scFv and no peptide. Conclusions: Immunotherapy against cancer has been a new treatment strategy in the recent years. Small and high affinity scFvs have had a crucial role in this regard. The specific human anti-EGFR scFvs that were selected in this study and reacted with the corresponding epitopehave the potential to be applied as a blocking antibody for interfering with tumor growth in EGFR-expressing tumors. Further studies are needed to evaluate the effects of these antibodies in vitro and in vivo.

Preclinical efficacy of Sym004, novel anti-EGFR antibody mixture, in esophageal squamous cell carcinoma cell lines.

Epidermal growth factor receptor (EGFR) is a well-validated oncological target molecule for monoclonal antibody therapies and Sym004 is a novel anti-EGFR antibody mixture comprising two recombinant chimeric IgG1 antibodies against non-overlapping epitopes of EGFR. Because EGFR is highly expressed in the majority of esophageal squamous cell carcinomas (ESCCs), we investigated the efficacy of Sym004 in human ESCC cell lines. Forty eight ESCC cell lines were treated with three kinds of anti-EGFR antibodies (Sym004, cetuximab, and panitumumab). Genetic background was investigated by next generation sequencing. The internalization of anti-EGFR antibodies into ESCC cells and inhibition of the EGFR signaling cascade by anti-EGFR antibodies were investigated in vitro. Furthermore, growth inhibition by anti-EGFR antibody treatment was investigated in vitro and in vivo. Sym004 treatments were more effective at inducing EGFR internalization and degradation than the two other anti-EGFR antibodies. Sym004 was more sensitive significantly to cell lines with EGFR gene amplification than those without amplification (P = 0.002). Growth inhibition of Sym004 was greater than in that of cetuximab or panitumumab in vitro and in vivo. These studies showed that Sym004 exhibited antitumor activity in some ESCC cell lines in preclinical settings and warrant a clinical evaluation in patients with ESCC. EGFR amplification is a potential biomarker of response to Sym004.

Abstract 578: Potent complement-dependent cytotoxicity of tumor cells by target bound hexamerization of EGFR antibodies

Abstract Introduction: Activation of complement (C) constitutes an important effector mechanism of monoclonal antibodies in cancer therapy. Unmodified IgG1 antibodies against the epidermal growth factor receptor (EGFR), a well-validated tumor target, lack the capacity to induce complement-dependent cytotoxicity (CDC) as single agents. Recently, significantly improved CDC was demonstrated after antigen-bound IgG molecules formed hexameric structures through intermolecular Fc-Fc contacts. Single point mutations (E345K or E430G) that enhance hexamerization of IgG molecules conditional on antigen binding have been identified to represent a platform for enhanced C activation (Diebolder et al Science 343:1260, 2014, de Jong et al, PLOS Biology, in press). Alternative strategies to enhance CDC have focused on enhancing C1q binding via affinity-maturation of the C1q binding site (S239D/H268F/S324T/I332E; DFTE) or by generating IgG1/IgG3 isotype chimeras (113F). The aim of the present study was to investigate whether EGFR antibodies optimized for each of these approaches differed in terms of CDC activity. Methods: EGFR antibodies comprising engineered Fc domains that carry mutations to enhance hexamerization (E345K or E430G), or increase C1q affinity (DFTE), or that exist of IgG1/IgG3 chimeras (113F) were constructed. CDC activity was assessed by 51Cr release assays in the presence of human serum. Antibody mediated C1q binding or C4b deposition on tumor cells was analyzed by flow cytometry. Apparent C1q binding affinity was determined by C1q specific ELISA. Results: Here, we demonstrate that affinity maturation of the C1q binding site (DFTE & 113F) increased C1q binding in the absence of antigen, whereas mutations that enhance Fc-Fc interactions (E345K & E430G), and so enhance hexamerization after target binding, did not. However, these latter mutations strongly increased C1q avidity and CDC for EGFR antibodies bound to cells. Notably, the latter strategy translated in superior CDC activity, which was particularly evident for tumor cells with low EGFR expression levels. Furthermore, hexamerization enhanced CDC was particularly sensitive to peptides inhibiting Fc-Fc interactions, supporting their different mode of action compared to other CDC-enhancing strategies. Interestingly, also the EGFR epitope recognized by different antibodies impacted on the CDC activity. Conclusion: Enhancement of hexamer formation by EGFR antibodies conditional on antigen binding is a promising novel approach to improve their anti-tumor activity. Strategies that enhanced complement binding independent of antigen engagement by the antibody appeared less effective. Citation Format: Annalina Tammen, Frank J. Beurskens, Stefanie Derer, Ralf Schwanbeck, Paul W.H.I. Parren, Janine Schuurman, Thomas Valerius. Potent complement-dependent cytotoxicity of tumor cells by target bound hexamerization of EGFR antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 578.

ATPS-86MUTATIONS IN THE EGF RECEPTOR EXTRACELLULAR DOMAIN REVEAL AN UNTETHERED TRANSITIONAL STATE WHICH MEDIATES mAb806 BINDING

The epidermal growth factor receptor (EGFR) is both amplified and frequently mutated in glioblastoma. While downstream inhibition of EGFR signaling through small molecule tyrosine kinase inhibitors has shown little clinical efficacy in glioblastoma, monoclonal antibody targeting is proving to be a promising alternative. The monoclonal antibody 806 (mAb806) specifically binds the most common EGFR mutant, EGFRvIII, along with a subset of amplified wild type EGFR, but not EGFR expressed at normal levels. The selectivity of mAb806 is thought to be due to its targeting a tumor-specific epitope of EGFR which is only accessible as the receptor transitions between an inactive tethered conformation to an active ligand-bound dimer. Here we used molecular dynamics simulations coupled with in vitro assays in glioblastoma cells to characterize the transitional conformation of EGFR in mAb806 binding. Focusing on the mutations R108K and G63R, molecular dynamics simulation revealed a spontaneous untethering of EGFR to a compact conformation, which is characterized by a backwards rotation of domain I-II exposing both the dimerization arm and the mAb806-epitope. This configuration is structurally equivalent to EGFRvIII, suggesting that several glioblastoma mutations converge to remove domain I from the dimerization interface. Next, we evaluated the effect of these point mutations on the ability of mAb806 to bind glioblastoma cells in vitro. Using site-directed mutagenesis coupled with lentiviral-transduction, we expressed EGFR-R108K or EGFR-G63R in U87 glioblastoma cells. Investigating mAb806 binding by FACS, we show that mAb806 binds the mutant cells five times greater than control cells, validating our simulation predictions. Future studies will examine the effect of mAb806 in an in vivo tumor model. Significantly, as these point mutations have been found in several GBM patients, our results indicate the possibility of a broader patient spectrum for the use of ABT-806, a humanized version of mAb806 currently in phase I clinical trials.

A bispecific transmembrane antibody simultaneously targeting intra- and extracellular epitopes of the epidermal growth factor receptor inhibits receptor activation and tumor cell growth

Epidermal growth factor receptor (EGFR) plays an important role in essential cellular processes such as proliferation, survival and migration. Aberrant activation of EGFR is frequently found in human cancers of various origins and has been implicated in cancer pathogenesis. The therapeutic antibody cetuximab (Erbitux) inhibits tumor growth by binding to the extracellular domain of EGFR, thereby preventing ligand binding and receptor activation. This activity is shared by the single chain antibody fragment scFv(225) that contains the same antigen binding domain. The unrelated EGFR-specific antibody fragment scFv(30) binds to the intracellular domain of the receptor and retains antigen binding upon expression as an intrabody in the reducing environment of the cytosol. Here, we used scFv(225) and scFv(30) domains to generate a novel type of bispecific transmembrane antibody termed 225.TM.30, that simultaneously targets intra- and extracellular EGFR epitopes. Bispecific 225.TM.30 and related membrane-anchored monospecific 225.TM and TM.30 proteins carrying extracellular scFv(225) or intracellular scFv(30) antibody fragments linked to a transmembrane domain were expressed in EGFR-overexpressing tumor cells using a doxycycline-inducible retroviral system. Induced expression of 225.TM.30 and 225.TM, but not TM.30 reduced EGFR surface levels and ligand-induced EGFR activation, while all three molecules markedly inhibited tumor cell growth. Co-localization of 225.TM with EGFR was predominantly found on the cell surface, while interaction with 225.TM.30 and TM.30 proteins resulted in the redistribution of EGFR to perinuclear compartments. Our data demonstrate functionality of this novel type of membrane-anchored intrabodies in tumor cells and suggest distinct modes of action of mono- and bispecific variants.

EGFR inhibitors augment antitumour helper T-cell responses of HER family-specific immunotherapy

Background:Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy.Methods:We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition.Results:Among several predicted peptide epitopes, EGFR875–889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR875–889 epitope could be detected in the blood of HNSCC patients. EGFR875–889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR875–889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells.Conclusion:We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR875–889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.

Abstract 870: A clinical study of ABT-806, a humanized recombinant anti-EGFR monoclonal antibody, in patients with recurrent glioblastoma multiforme.

Abstract Background: Glioblastoma (GBM), the most common primary CNS malignancy, has a dismal prognosis with a median survival of under 15 months. Epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in GBM and has been implicated in tumor growth and resistance to therapy. ABT-806 is a humanized recombinant IgG1k monoclonal antibody that binds to a unique EGFR epitope exposed only in the untethered, (activated) state of EGFR or when the de2-7 (EGFRvIII) deletion mutation is present. ABT-806 has shown significant antitumor activity over other EGFR antibodies in both in vitro and in vivo models of GBM. We evaluated the safety, pharmacokinetics (PK), and antitumor activity of ABT-806 in a cohort of recurrent GBM patients treated at the recommended phase 2 dose determined from the dose-escalation portion of this phase 1 study. Methods: Patients with recurrent, EGFR-amplified GBM and measurable disease by RECIST (v1.1) were included. ABT-806 was administered at 24 mg/kg intravenously every other week until disease progression or study withdrawal. Tumor response by MRI or CT was assessed every 8 weeks. Adverse events (AEs) were graded by NCI CTCAE v4.0. Serum samples for determination of ABT-806 concentration and antidrug antibody were collected at multiple time points throughout the study. Archival tumor analysis for EGFR amplification/mutation was also performed. Results: Among 11 patients; median age was 60 years (range 39 to 69) and 9 were male. Median number of prior therapies was 2 (range 1 to 7). Six patients had prior bevacizumab exposure. No patients required dose modification. Hypophosphatemia was the only treatment-related emergent AE that occurred in 2 or more patients. Best responses were n=3 stable disease, n=7 progressive disease. One patient withdrew consent prior to the first post-baseline tumor assessment. Median time to disease progression and median progression free survival [95% CI] were 43 [22,57] and 29 [25,57] days, respectively. PK and EGFRde2-7 data were not available at the time of abstract submission. Conclusions: While ABT-806 related AEs were infrequent, there was little evidence of a clinical benefit in this refractory GBM population. Whether the lack of significant anti-tumor effect was due to poor binding to intracranial tumors, lack of response to the mechanism of action, or the refractory population is to be determined. A study is ongoing to evaluate the biodistribution of 111Indium-labeled ABT-806 in pts with advanced solid tumor types, including GBM. Further preclinical efforts to increase the potency of ABT-806 are also ongoing. Citation Format: David A. Reardon, James M. Cleary, Hao Xiong, Wijith P. Munasinghe, Ya-Hui Hsu, Edward B. Reilly, Jiang Qian, Michelle S. Pedersen, Rod A. Humerickhouse, Kyle D. Holen. A clinical study of ABT-806, a humanized recombinant anti-EGFR monoclonal antibody, in patients with recurrent glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 870. doi:10.1158/1538-7445.AM2013-870

Update on HER1–3 in Advanced Non–Small-Cell Lung Cancer

Update on HER1–3 in Advanced Non–Small-Cell Lung Cancer

Functional Dissection of the Epidermal Growth Factor Receptor Epitopes Targeted by Panitumumab and Cetuximab

Cetuximab and panitumumab, two antibodies targeting the extracellular domain of the epidermal growth factor receptor (EGFR), are of major clinical importance particularly in the treatment of metastatic colorectal cancer. As patients may acquire resistance-mediating mutations within the extracellular EGFR domain, functional dissection of the exact binding sites of EGFR targeting antibodies may help predict treatment responses. We therefore assessed the epitope recognition of panitumumab by screening phage-displayed random cyclic 7mer and linear 12mer peptide libraries on this antibody. Phage screenings revealed two strong, potentially epitope-mimicking consensus motifs targeted by panitumumab. A computational approach was used to map the sequences back to the potential epitope region on domain III of EGFR. The presumed epitope regions (386)WPEXRT(391) and a biochemically similar though discontinuous region P349-F352-D355 on a neighboring loop of domain III could be confirmed as part of the functionally relevant binding site of panitumumab by site-directed mutational analysis. To more accurately differentiate the panitumumab epitope from the previously characterized cetuximab epitope, binding studies were performed on a broad range of additional mutants. Taken together, this analysis revealed two large, partially overlapping functional epitopes consisting of 17 critical amino acid positions. Four of these positions were selectively targeted by cetuximab (I467, S468, Q408, and H409), whereas another four were selectively recognized by panitumumab (W386, E388, R390, and T391). In view of the clinical significance of extracellular domain mutations, our data may help guide treatment decisions in selected patients receiving EGFR-targeted therapies.

Epidermal growth factor receptor downregulation by small heterodimeric binding proteins

No single engineered protein has been shown previously to robustly downregulate epidermal growth factor receptor (EGFR), a validated cancer target. A panel of fibronectin-based domains was engineered to bind with picomolar to nanomolar affinity to multiple epitopes of EGFR. Monovalent and homo- and hetero-bivalent dimers of these domains were tested for EGFR downregulation. Selected orientations of non-competitive heterodimers decrease EGFR levels by up to 80% in multiple cell types, without activating receptor signaling. These heterodimers inhibit autophosphorylation, proliferation and migration, and are synergistic with the monoclonal antibody cetuximab in these activities. These small (25 kDa) heterodimers represent a novel modality for modulating surface receptor levels.

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6 × 10 17) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5 × 10 9 human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4 × 10 8 and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K D of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose.

Impact of Human IgA Antibodies on Complement-Dependent Cytotoxicity Mediated by Combinations of EGF-R-Directed Antibodies

Dual combinations of non-crossblocking epidermal growth factor receptor (EGF-R)-directed monoclonal antibodies were demonstrated to effectively induce complement-dependent cytotoxicity (CDC) of tumor cells, whereas individual antibodies were ineffective. Here the modulating effects of different antibody isotypes on CDC were studied by adding them as a third antibody. Two different combinations of non-crossblocking EGF-R antibodies of human IgG1 isotype, 018/003 and 425/005, were investigated against the A431 and A1207 cell lines. As a third antibody, human IgG1, IgA1, and IgA2 isotype variants of the therapeutic EGF-R antibody 225 were employed that bind to an EGF-R epitope distinct from the other EGF-R antibodies. In this model, the human IgG1 antibody proved to further enhance CDC, whereas both IgA antibodies significantly blocked CDC. The IgG1 and IgA variants increased target opsonization at similar levels, but the isotypes differed in their effects on C1q fixation. Addition of IgG1 significantly enhanced complement factor binding on the target surface, whereas both IgA antibodies reduced complement binding. Control experiments revealed this blocking effect to be not specific to IgA antibodies, but to antibody constructs incapable of activating the complement system. Interestingly, the effects caused by the IgA2 isotype were consistently stronger than those by IgA1, which may be caused by stronger steric hindrance due to its reduced hinge flexibility. These results demonstrate that monoclonal IgA antibodies inhibit IgG-mediated complement activation in vitro and suggest that the appearance of IgA antibodies within a polyclonal immune response might inhibit complement activation in vivo.