Maria L. Jaramillo

A map of human cancer signaling

We conducted a comprehensive analysis of a manually curated human signaling network containing 1634 nodes and 5089 signaling regulatory relations by integrating cancer‐associated genetically and epigenetically altered genes. We find that cancer mutated genes are enriched in positive signaling regulatory loops, whereas the cancer‐associated methylated genes are enriched in negative signaling regulatory loops. We further characterized an overall picture of the cancer‐signaling architectural and functional organization. From the network, we extracted an oncogene‐signaling map, which contains 326 nodes, 892 links and the interconnections of mutated and methylated genes. The map can be decomposed into 12 topological regions or oncogene‐signaling blocks, including a few ‘oncogene‐signaling‐dependent blocks’ in which frequently used oncogene‐signaling events are enriched. One such block, in which the genes are highly mutated and methylated, appears in most tumors and thus plays a central role in cancer signaling. Functional collaborations between two oncogene‐signaling‐dependent blocks occur in most tumors, although breast and lung tumors exhibit more complex collaborative patterns between multiple blocks than other cancer types. Benchmarking two data sets derived from systematic screening of mutations in tumors further reinforced our findings that, although the mutations are tremendously diverse and complex at the gene level, clear patterns of oncogene‐signaling collaborations emerge recurrently at the network level. Finally, the mutated genes in the network could be used to discover novel cancer‐associated genes and biomarkers.

Differential tumor-targeting abilities of three single-domain antibody formats

The large molecular size of antibody drugs is considered one major factor preventing them from becoming more efficient therapeutics. Variable regions of heavy chain antibodies (HCAbs), or single-domain antibodies (sdAbs), are ideal building blocks for smaller antibodies due to their molecular size and enhanced stability. In the search for better antibody formats for in vivo imaging and/or therapy of cancer, three types of sdAb-based molecules directed against epidermal growth factor receptor (EGFR) were constructed, characterized and tested. Eleven sdAbs were isolated from a phage display library constructed from the sdAb repertoire of a llama immunized with a variant of EGFR. A pentameric sdAb, or pentabody, V2C-EG2 was constructed by fusing one of the sdAbs, EG2, to a pentamerization protein domain. A chimeric HCAb (cHCAb), EG2-hFc, was constructed by fusing EG2 to the fragment crystallizable (Fc) of human IgG1. Whereas EG2 and V2C-EG2 localized mainly in the kidneys after i.v. injection, EG2-hFc exhibited excellent tumor accumulation, and this was largely attributed to its long serum half life, which is comparable to that of IgGs. The moderate size (approximately 80 kDa) and intact human Fc make HCAbs a unique antibody format which may outperform whole IgGs as imaging and therapeutic reagents.

Roles for Endothelin Receptor B and BCL2A1 in Spontaneous CNS Metastasis of Melanoma

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.

The crystal structure and dimerization interface of GADD45γ

Gadd45 proteins are recognized as tumor and autoimmune suppressors whose expression can be induced by genotoxic stresses. These proteins are involved in cell cycle control, growth arrest, and apoptosis through interactions with a wide variety of binding partners. We report here the crystal structure of Gadd45γ, which reveals a fold comprising an αβα sandwich with a central five-stranded mixed β-sheet with α-helices packed on either side. Based on crystallographic symmetry we identified the dimer interface of Gadd45γ dimers by generating point mutants that compromised dimerization while leaving the tertiary structure of the monomer intact. The dimer interface comprises a four-helix bundle involving residues that are the most highly conserved among Gadd45 isoforms. Cell-based assays using these point mutants demonstrate that dimerization is essential for growth inhibition. This structural information provides a new context for evaluation of the plethora of protein–protein interactions that govern the many functions of the Gadd45 family of proteins.

Differential sensitivity of A549 non-small lung carcinoma cell responses to epidermal growth factor receptor pathway inhibitors.

It has been demonstrated that A549 non-small cell lung cancer (NSCLC) cells are sensitive to epidermal growth factor receptor (EGFR) inhibitors in in vivo xenograft animal models, but are relatively resistant in conventional in vitro monolayer growth assays. Here, we utilized anchorage-independent cell growth/survival assays as well as motility assays and demonstrated that these tests detect the effects of two EGFR inhibitors, the small molecule inhibitor AG1478 and the ligand-blocking antibody 225 mAb, on A549 cells more sensitively than monolayer growth assays. AG1478 was more effective than 225 mAb at inhibiting EGF-stimulated anchorage-independent cell growth, in part due to its pronounced ability to inhibit cell survival, whereas 225 mAb and AG1478 were both able to inhibit cell motility. In order to determine which EGFR signalling pathway components were most strongly associated with these cell responses, we analyzed in parallel the phosphorylation levels of EGFR itself as well as several downstream pathway elements. We found that the limited ability of 225 mAb to inhibit MAPK, PI3K and STAT3 phosphorylation correlated with its inability to promote anchorage independent apoptosis, but did not correlate with its ability to inhibit motility. Based on our results in A549 cells, we propose that EGF stimulates tumour progression of NSCLC largely through effects on anchorage-independent growth and survival, as well as motility.

Computational selection of antibody-drug conjugate targets for breast cancer

The selection of therapeutic targets is a critical aspect of antibody-drug conjugate research and development. In this study, we applied computational methods to select candidate targets overexpressed in three major breast cancer subtypes as compared with a range of vital organs and tissues. Microarray data corresponding to over 8,000 tissue samples were collected from the public domain. Breast cancer samples were classified into molecular subtypes using an iterative ensemble approach combining six classification algorithms and three feature selection techniques, including a novel kernel density-based method. This feature selection method was used in conjunction with differential expression and subcellular localization information to assemble a primary list of targets. A total of 50 cell membrane targets were identified, including one target for which an antibody-drug conjugate is in clinical use, and six targets for which antibody-drug conjugates are in clinical trials for the treatment of breast cancer and other solid tumors. In addition, 50 extracellular proteins were identified as potential targets for non-internalizing strategies and alternative modalities. Candidate targets linked with the epithelial-to-mesenchymal transition were identified by analyzing differential gene expression in epithelial and mesenchymal tumor-derived cell lines. Overall, these results show that mining human gene expression data has the power to select and prioritize breast cancer antibody-drug conjugate targets, and the potential to lead to new and more effective cancer therapeutics.

Abstract 1778: Nimotuzumab, a humanized antiepidermal growth factor receptor antibody, interacts with EGFRvIII

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Glioblastomas frequently overexpress a variant form of EGFR, called variant III (EGFRvIII), which has an in-frame deletion of an 801-bp sequence in the extracellular domain. EGFRvIII does not bind EGF ligand but exhibits constitutive kinase activity that results in enhanced transformation, reduced apoptosis, and resistance to therapy. Nimotuzumab (Nmab) is an EGFR-targeting antibody that has demonstrated encouraging results in early clinical trials treating adult and pediatric brain tumors. Here we present data characterizing the interaction of Nmab with EGFRvIII. Methods: Binding of Nmab to the extracellular domain of wtEGFR and EGFRvIII was first characterized by SPR biosensor analysis: Nmab was captured via its Fc domain and varying concentrations of EGFRvIII and EGFRwt were flowed. Flow cytometry analysis was subsequently performed using a parental U87MG glioblastoma cell line and derivatives which were engineered to overexpress either wtEGFR or EGFRvIII. Results: Nmab bound EGFRvIII and EGFRwt with similar affinity, which was in the 10−8 M range. This KD is consistent with the previously reported affinity constant of Nmab for EGFRwt (Telavera et al, 2009). Binding was further analyzed by flow cytometry. Nmab bound both EGFRwt and EGFRvIII expressed on the surface of parental U87MG, U87MG EGFRvIII and U87MG wtEGFR cells. Additional data concerning effects of Nmab on EGFRvIII expressing cell lines will be presented. Conclusion: Nmab binds to wtEGFR and EGFRvIII with similar affinity, which supports development of the antibody as a therapeutic for glioblastoma. In U87MG glioblastoma cells, synergistic activity of Nmab in combination with radiotherapy has been previously reported (Diaz Miqueli et al, 2009). Nmab is currently being tested in an advanced randomized study in the first line setting for the treatment of adult glioma. Nmab is being tested in combination with radiation plus temozolomide, vs radiation plus temozolomide alone, with preliminary results expected in the second half of 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1778.

Assisted Design of Antibody and Protein Therapeutics (ADAPT)

Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20–30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above.

PO-033 Identification and functional evaluation of monoclonal antibodies specifically targeting human carbonic anhydrase IX

Introduction Poor vascularisation of solid tumours leads to inadequate nutrient and oxygen supplies which forces tumour cells to reprogram their metabolism. As a consequence the tumour cell’s environment becomes acidic and hypoxic. This, in turn, triggers signalling cascades involving for example heterodimeric hypoxia-inducible factor (HIF). Activation of this hypoxia-induced transcriptional program is crucial for the survival of tumour cells in their hostile microenvironment but also their ability to metastasize. One of the genes upregulated through the HIF pathway is carbonic anhydrase (CA)-IX (CAIX, gene G250/MN-encoded transmembrane protein). CA-IX catalyses carbon dioxide (CO2) thereby generating a proton (H+) and bicarbonate (HCO3-), the latter of which is transported back into the cell and utilised to help safeguard intracellular pH (pHi) stability. Except for the stomach and the gallbladder, CA-IX expression is negligible in normal tissues. In contrast, a broad range of tumours express high levels of CA-IX, where the protein can serve as a biomarker for the early stages of tumour development but also as tumour marker of hypoxia associated with resistance to chemotherapy and radiotherapy. Material and methods Preclinical and clinical studies have shown that CA-IX is a promising therapeutic target for detection and therapy for several cancer types. To date only a limited number of ant-CAIX monoclonal antibodies (mAbs) have been available for clinical testing as therapeutic and imaging agents. In the current study, we generated and functionally categorised a panel of 51 mouse mAbs that specifically bind to human CA-IX. Results and discussions Characterisation of the mAbs revealed that of the mAbs with the best biophysical characteristics, three3 mAbs are suitable as an antibody-drug conjugate (ADC), two2 mAbs inhibit the CA-IX enzyme activity, and one1 mAb that is suitable for CA-IX imaging purposes. Conclusion These preliminary data presented here could thus form the basis for the development of novel CA-IX targeted immunotherapies and diagnostic tools for the treatment of cancer.

Abstract 3669: Screening platform for development of antibody-drug conjugates against novel targets at the National Research Council of Canada

One of the most promising of the next generation of biologic-based cancer therapeutics builds on the molecular targeting abilities of antibodies by combining them with drugs to generate highly specific antibody-drug conjugates (ADCs). However, the development of ADCs requires time-consuming selection of the antibody for every target and cancer type. High-throughput screening technologies based on the use of conjugated secondary antibodies provide a fast and efficient surrogate assay from which to identify which antibodies are best internalized and suitable for immunoconjugate development into ADCs. As part of its integrated antibody development initiative, NRC has isolated and characterized anti- mouse Fc and anti-human Fc monoclonal antibodies to serve as very selective detective reagents for various IgG isotypes. We have shown that these secondary antibodies are species specific, selective and of high affinity. When conjugated to pH sensitive fluorophores, we have used them to specifically identify internalizing antibodies against tumor targets, which were later validated as ADCs. Furthermore, these secondary conjugates exhibit high specific potency and low background toxicity once conjugated to linkered drugs. This approach allow us to optimize the selection of an antibody for a particular target, tumor type, linker and drug for ADC development. NRC will present results of a screen of 285 mouse antibodies against 20 different targets in 7 different cancer cell lines, using either MCC-DM1 or vc-MMAE-conjugated secondary antibodies. The NRC ADC discovery platform is combining this methodology with our proprietary mRNA and DNA expression database for the selection of appropriate ADC targets. NRC Biologics and Biomanufacturing program is in the process of screening thousands of NRC antibodies generated against a variety of cancer-associated cell surface targets to deliver a steady pipeline of ADCS as part of its drug discovery efforts. This functional screening platform further promotes the integration and advancement of NRC’s capabilities and strengths in the area of biologic-based therapeutics lead candidate selection, including quality attributes and characterization and biomanufacturing. The combined expertise in cell biology, high throughput screening, antibody generation and selection, bioinformatics and expression analysis forms the foundation by which NRC can establish strategic collaborations with other Canadian or international partners to develop antibodies into novel ADC biologics. Citation Format: Maria Luz Jaramillo, Luc Meury, Patrice Bouchard, Allan Matte, Anne Marcil, Mauro Acchione, Jennifer Hill, Francois Fauteux. Screening platform for development of antibody-drug conjugates against novel targets at the National Research Council of Canada [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3669. doi:10.1158/1538-7445.AM2017-3669