INVESTIGATION OF THE BINDING SPECIFICITY OF IGF-IR USING MONOCLONAL ANTIBODIES

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1 INVESTIGATION OF THE BINDING SPECIFICITY OF IGF-IR USING MONOCLONAL ANTIBODIES By Mehrnaz Keyhanfar, Pharm.D. A thesis submitted to the University of Adelaide, South Australia in fulfilment of the requirements for the degree of Doctor of Philosophy '" THE UNIVERSITY OF ADELAIDE AUSTRALIA School of Molecular and Biomedical Science The University of Adelaide Adelaide, South Australia, 5005 September 2005

2 Abstract The insulin-like growth factor type one receptor (IGF-IR) plays a critical role in cancer. The receptor is over expressed in many tumours and mediates growth, motility, and survival from apoptosis. Several studies have shown that the inhibition of IGF-lR's expression or its function blocks tumour growth or metastasis, and also enhances sensitivity to cytotoxic drugs and irradiation. To date, IGF-IR is widely considered as a very promising target for cancer treatment. Among different techniques for targeting IGF-IR, blocking the receptor with specific monoclonal antibodies (MAbs) is an attractive way to inhibit the receptor signalling. In addition, antibodies specific for the IGF-IR are potential diagnostic reagents as the IGF-IR is overexpressed in several cancers. A similar approach has been successfully utilized in the treatment of breast cancer using Herceptin, which is a humanised MAb against human epidermal growth factor receptor-2. In this thesis, two high affinity murine MAbs against IGF-IR namely 7C2 and 9El l were isolated and cloned. To obtain these MAbs, s-igf-ir (the soluble extracellular part of the IGF-IR, amino acids 1-906) was used as an mmunogen. These MAbs were IgGIK isotype and did not cross react with either insulin receptor isoform IR-A or IR-B. MAbs 7C2 and 9Ell successfully detected IGF-IR in a number of immunoassays such as in enzyme linked immunosorbent assays (ELISAs), flow cytometry and immunohistochemistry. The MAbs also immunoprecipitated IGF-IR from lysates of cells overexpressing recombinant IGF-IR. Both MAbs 7C2 and 9El l showed high affnity to the s-igf-ir. The binding affnities of the MAbs 7C2 and 9El l to s-igf-ir were 0.5±1.6 nm and 2.1±0.4 nm, respectively. The sequences of the variable regions of these MAbs' heavy and light chains were also described in this study. Comparison of the CDR sequences of the MAbs with those of other MAbs against the IGF-IR showed that MAbs 7C2 and 9El l each had a unique sequence.

3 During this research it was found that MAbs 7C2 and 9El l blocked the binding of IGF-I to the IGF-IR. but did not show an inhibitory effect on the binding of IGF-I1 to this receptor. Moreover, epitope mapping of MAbs 7C2 and 9El l revealed that they both bound to the cysteine-rich domain of the receptor. These fmdings support a previous study, which demonstrated that the cysteine-rich domain oflgf-lr is critical for specific binding oflgf-i but not IGF-I1 to the receptor. In this thesis, fuher epitope mapping studies on MAbs 7C2 and 9El l using alanine mutants of the IGF-IR in the cysteine-rich domain are reported. The results showed that amino acids F241, F251 and F266 were involved in binding to both MAbs 7C2 and 9EII. On the other hand, another study showed that amino acids F241 and F251 were crucial for IGF-I binding to the receptor. Therefore, binding to these two amino acids by MAbs 7C2 and 9EI l is most likely responsible for the selective blocking of IGF-I but not IGF-I1 to the receptor. Competition experiments performed during this research revealed that the chimeric IGF analogues IGF-ICII (IGF-I with IGF-II C domain) and IGF-IICI (IGF-II with IGF-I C domain) behaved like IGF-II and IGF-I. respectively in their ability to inhibit the binding of MAbs 7C2 and 9EIl to s-igf-ir. These findings imply that out of all four domains of IGF-I, " it is the C domain, which binds to residues in IGF-IR that form the epitope for MAbs 7C2 and 9El l. Because in the C domain of IGF-I, amino acids R36 and R37 and also Y31 have been shown to be important for binding to IGF-IR, it could be proposed that the binding of the IGF-I C domain to the IGF-IR cysteine-rich domain is through binding amino acids R36, R37 and Y31 of IGF-I to residues F241 and F251, or to the nearby residues, which are sterically affected by the presence of the MAbs binding to the receptor. In this research, in vitro studies, using MAbs as anti-cancer reagents revealed that both MAbs 7C2 and 9E 11 inhibited the proliferation of colon cancer cell (HT -29) induced by IGF-I and IGF-IL However, the neutralizing effect for IGF-I1 was less potent than for IGF-L Since the MAbs did not inhibit binding of IGF-II to the receptor, their effect on IGF-II induced cell proliferation could be claimed to be due to an indirect effect. This is consistent

4 with the in vitro result for the receptor down-regulation experiment. which showed that 24 h incubation of breast cancer cells (MCF-7) with MAbs 7C2 and 9El l, significantly reduced the IGF-IR expression. In this study it was also shown that MAbs 7C2 and 9El l inhibited MCF-7 cells migration induced by IGF-1. This could indicate a potential anti-metastatic property ofmabs 7C2 and 9El l. Taken together, the in vitro effects of MAbs 7C2 and 9E 11 reported in this thesis revealed that they had inhibiting effects on cancer cell proliferation and migration and they also induced IGF-lR down-regulation. Therefore, they could be potentially valuable prospects for cancer treatment if humanised. Finally, MAbs 7C2 and 9El l could be used as diagnostic reagents to detect IGF-IR in cells or tissue samples in a range of immunoassays as mentioned earlier. Due to the IGF-lR overexpression in several cancer tumours, MAbs 7C2 and 9E II could be utilized for malignant tumour recognition. Furtermore, as another application, these MAbs could also be used to identify tumours overexpressing the IGF-lR, allowing tailored anti-cancer treatment including an IGF-IR blocking agent. "

5 Abstract I Statement of Originality" Acknowledgments V Table of Content... VII List of Figures... XI List of Tables List of Publications IV XIV... XV List of Abbreviations...XVII 1. Literature Review Introduction Introduction to the IGF System IGF Ligands IGF Ligand Binding Characterisation IGFBP Structure and Function The Structure and Function of the Receptors IR Function and Structure IGF-IR Function and Strcture The IGF-IR and IR Ectodomain Structure IGF-IR Signaling IGF-IR Residues Involved in IGF Binding IGF Ligand Residues Involved in IGF-IR Binding The IGF System and Cancer IGF Ligands and Cancer IGFBPs and Cancer The IGF-2R and Cancer The Roles ofir Isofonns and Hybrid Receptors in Cancer The Role ofigf-l R in Cancer Different Strategies for Targeting IGF-IR as an Anti-cancer Therapy Antibodies Antibodies Strcture Antibodies Application '. Monoclonal Antibodies Epitope Mapping Methods Used to Localize Epitopes in Proteins Clinical Applications of Monoclonal Antibodies Monoclonal Antibodies Directed Against the IGF-IR Aims and Experimental Strategy Materials and General Methods Materials General Materials Chemicals Reagents Antibodies Bacterial Strains Tissue Culture Cell Lines Tissue Culture Solutions Media for Growing Cells in vitro Primers Commercial Kits

6 Solutions Computer Applications General Methods Culture of Mammalian Cells Protein Chemistr Techniques ELISA Test for Detection of s-igf-ir ELISA Test for Detection of Antibodies Against IGF-IR Protein Concentration Flow Cytometr SDS-PAGE Electrophoresis and Gel Staining Electroblot and Western Immuno-Detection Immunoprecipitation of Proteins from Cell Lysates Molecular Biology Techniques Agarose Gel Electrophoresis Preparation of DH5-a. Competent Cells Transfonnation of Bacteria by Heat Shock Glycerol Stocks Bacterial Culture Purification of Plasmid DNA DNA Sequencing Production, Characterisation and Sequencing of the Variable Regions of Mouse Monoclonal Antibodies Introduction Methods for General Characterisation ofmabs Animal Ethics Antigen Production and Purification Culture and Purification of s-igf-1r ELISA Test for Detection of Purified s-igf-1r Hybridoma Production Immunisation of the Mice ELISA Test for Detection of Antibodies in Plasma from Immunised Mice Preparation of Lymphocytes from Mouse Spleen Preparation of Myeloma Cells for Fusion Fusion of Myeloma Cells with Spleen Lymphocytes Detection of Antibodies Against s-igf-ir in the Hybridoma Culture Mediums Cloning by Limiting Dilution of Hybridoma Freezing and Thawing of Hybridoma Cells Detennination of Immunoglobulin Isotypes Screening of Positive Hybridomas for Binding to the IGF-1R Cell Surface Using Flow Cytometr Large-Scale Production of the Selected MAbs Purification of Antibody and Detennination of the Antibody Concentration SDS-PAGE Electrophoresis and Gel Staining of Purified MAbs Freeze-Drying of the Purified MAbs Immunoprecipitation and Western Blot Analysis Immunoblotting of the IGF-IR Using the Generated MAbs Using the MAbs in Immunohistochemistry Immunohistochemistry on Frozen Cells Immunohistochemistry on Frozen Skin Sections Immunohistochemistry on Paraffn-Embedded Skin Sections Method for CDR Sequencing ofmabs Heavy and Light Chains...94

7 Extraction of Total Cellular RNA Purification of RNA Samples Electrophoresis of Purified RNA Samples on Agarose Gel Quantification of Purified RNA Synthesis of cdna PCR Amplification of cdna Encoding the Variable Region of Heavy or Light Chain of the MAbs Agarose Gel Electrophoresis ofpcr Products Purification of DNA Fragments from the Positive PCR Products Ligation of Variable Region cdnas ofmabs into pgem-t Easy Vector Transformation of the Ligated Plasmids into E. coli DH5a Small-Scale Production of Plasmid DNA Digestion of Purified Plasmid DNA... I DNA Sequencing Results and Discussion for General Characterisation of MAbs Hybridoma Screening and Isotyping Flow Cytometr Results Large Scale Production and Purification ofmabs The Activity of Freeze-Dried Monoclonal Antibodies I Immunoprecipitation and Immunoblotting of the IGF-IR Immunohistochemistry Results Results for CDR Sequencing RNA Extraction PCR Amplification Ligation Sequencing Results Summary and Final Discussion Binding Analysis and Epitope Mapping of MAbs 7C2 and 9Ell to JGF-IR Introduction Methods IGF-IR Competition Binding Assay BIAcore Analysis ofmabs and s-igf-ir Interactions Surface Preparation Kinetic Assays of Anti-IGF-IR MAbs (7C2, 9El 1 and air-3) Binding to the., s-igf-ir BIAcore Analysis of s-igf-ir and MAbs Interaction in Presence of IGF-I or IGF-II BIAcore Analysis of s-igf-ir and MAbs Interaction in the Presence of IGF Chimeras (IGF-ICII and IGF-IICI) Mass Transfer Control Experiment Epitope Mapping by Flow Cytometry Analysis Using Chimeric Receptors Epitope Mapping by Competition Assay Between Different MAbs to IGF-IR The Fragmentation of IgG to Fab Using Papain Europium Labelling of Fab Competition Binding Analysis Between MAbs Against the IGF-IR Epitope Mapping Using Alanine Mutants of the IGF-IR Transfection Methods ELISA to Determine the Expression of the Recombinant Receptors Europium Binding Assay for Epitope Mapping of the MAbs by Alaine Mutants of the IGF-IR Sequencing the Plasmids Containing cdnas for Alanine Mutants of IGF-IR...138

8 4.3. Results Effect of the MAbs on IGF-IR Competition Binding Assay BIAcore Results Kinetic Analysis of the MAb and s-igf-l R Binding Mass Transfer Control The Effects oflgf-i and IGF-II on Binding s-igf-ir to the MAbs The Effects oflgf Chimeras (IGF-ICII and IGF-IICI) on Binding s-igf-ir to the MAbs Results for Flow Cytometry Analysis on Chimeric Receptors Fragmentation ofmabs and Europium Labelling of the Fab Competition of Eu-7C2 and Eu-9E l 1 with other MAbs IGF-IR Alanine Mutant Expression ELISA on s-igf-ir Identification ofmabs Binding Epitope Sequencing of the Plasmids Containing IGF-IR Mutants Discussion Binding Studies Epitope Mapping with Chimeric Receptors Epitope Mapping with Alanine-Scan Receptor Mutants Epitope Mapping with a Cysteine-Rich Domain Chimera MAb Epitopes and IGF Binding to IGF-IR Structural and Functional Epitope on the IGF-IR Interaction oflgf-ir with IGF-I and IGF-II... I Comparing the Affinity of MAbs 7C2 and 9E 11 to other Antibodies Against the IGF-IR Biological Effects of the MAbs in vitro Introduction Methods Cell Proliferation Assay Cell Migration Assays Analysis of the IGF-IR Down-Regulation Results Effect of the MAbs on HT -29 Cell Proliferation Effect of the MAbs on MCF-7 Cell Migration Effect of the MAbs on IGF-IR Down-Regulation in MCF-7Cells Discussion Final Discussion, Future Applications and Directions Discussion Summary of Findings The Selective Blocking Activity of the MAbs and Epitope Mapping Affnity of the MAbs The Biological Effects of MAbs Future Applications Future Directions Conclusion References