Investigation of the binding specificity of IGF-1R using monoclonal antibodies.

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-1R's expression or its function blocks tumour growth or metastasis, and also enhances sensitivity to cytotoxic drugs and irradiation. To date, IGF-1R is widely considered as a very promising target for cancer treatment. Among different techniques for targeting IGF-1R, blocking the receptor with specific monoclonal antibodies (MAbs) is an attractive way to inhibit the receptor signalling. In addition, antibodies specific for the IGF-1R are potential diagnostic reagents as the IGF-1R 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-1R namely 7C2 and 9E11 were isolated and cloned. To obtain these MAbs, s-IGF-1R (the soluble extracellular part of the IGF-1R, amino acids 1-906) was used as an immunogen. These MAbs were IgG1k isotype and did not cross react with either insulin receptor isoform IR-A or IR-B. MAbs 7C2 and 9E11 successfully detected IGF-1R in a number of immunoassays such as in enzyme linked immunosorbent assays (ELISAs), flow cytometry and immunohistochemistry. The MAbs also immunoprecipitated IGF-1R from lysates of cells overexpressing recombinant IGF-1R. Both MAbs 7C2 and 9E11 showed high affinity to the s-IGF-1R. The binding affinities of the MAbs 7C2 and 9E11 to s-IGF-1R 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-1R showed that MAbs 7C2 and 9E11 each had a unique sequence. During this research it was found that MAbs 7C2 and 9E11 blocked the binding of IGF-I to the IGF-1R, but did not show an inhibitory effect on the binding of IGF-II to this receptor. Moreover, epitope mapping of MAbs 7C2 and 9E11 revealed that they both bound to the cysteine-rich domain of the receptor. These findings support a previous study, which demonstrated that the cysteine-rich domain of IGF-1R is critical for specific binding of IGF-I but not IGF-II to the receptor. In this thesis, further epitope mapping studies on MAbs 7C2 and 9E11 using alanine mutants of the IGF-1R 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 9E11. 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 9E11 is most likely responsible for the selective blocking of IGF-I but not IGF-II 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 9E11 to s-IGF-1R. These findings imply that out of all four domains of IGF-I, it is the C domain, which binds to residues in IGF-1R that form the epitope for MAbs 7C2 and 9E11. 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-1R, it could be proposed that the binding of the IGF-I C domain to the IGF-1R 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 9E11 inhibited the proliferation of colon cancer cell (HT -29) induced by IGF-I and IGF-II. However, the neutralizing effect for IGF-II was less potent than for IGF-I. 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 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 9E11, significantly reduced the IGF-1R expression. In this study it was also shown that MAbs 7C2 and 9E11 inhibited MCF-7 cells migration induced by IGF-I. This could indicate a potential anti-metastatic property of MAbs 7C2 and 9E11. Taken together, the in vitro effects of MAbs 7C2 and 9E11 reported in this thesis revealed that they had inhibiting effects on cancer cell proliferation and migration and they also induced IGF-1R down-regulation. Therefore, they could be potentially valuable prospects for cancer treatment if humanised. Finally, MAbs 7C2 and 9E11 could be used as diagnostic reagents to detect IGF-1R in cells or tissue samples in a range of immunoassays as mentioned earlier. Due to the IGF-1R overexpression in several cancer tumours, MAbs 7C2 and 9E11 could be utilized for malignant tumour recognition. Furthermore, as another application, these MAbs could also be used to identify tumours overexpressing the IGF-1R, allowing tailored anti-cancer treatment including an IGF-1R blocking agent.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Identification of B and T cell epitope peptide vaccines from IGF-1 Receptor in breast cancer

Introduction: The insulin-like growth factor-1 receptor (IGF-1R) plays a key role in proliferation, growth, differentiation, and development of several human malignancies including breast and pancreatic adenocarcinoma. IGF-1R targeted immunotherapeutic approaches are particularly attractive, as they may potentially elicit even stronger antitumor responses than traditional targeted approaches. Cancer peptide vaccines can produce immunologic responses against cancer cells by triggering helper T cell (Th) or cytotoxic T cells (CTL) in association with Major Histocompatibility Complex (MHC) class I or II molecules on the cell surface of antigen presenting cells.  Methods and Results: In our previous study, we set a technique based on molecular docking in order to find the best MHC class I and II binder peptides using GOLD. In the present work, molecular docking analyses on a library consisting of 30 peptides mimicking discontinuous epitopes from IGF-1R extracellular domain identified peptides 249 and 86, as the best MHC binder peptides to both MHC class I and II molecules. The receptors most often targeted by peptide 249 are HLA-DR4, HLA-DR3 and HLA-DR2 and those most often targeted by peptide 86 are HLA-DR4, HLA-DP2 , and HLA-DR3. Conclusions: These findings, based on bioinformatics analyses, can be conducted in further experimental analyses in cancer therapy and vaccine design

Callogenesis in root explants of four species of the family Solanaceae after inducing by Agrobacterium rhizogenes

Studying explants affected by Agrobacterium rhizogenes shows that in addition to possible formation of hairy roots, it is likely that callogenesis can be induced in these tissues. The T-DNA region of A. rhizogenes codes enzymes that participate in biosynthesis of plants growth hormones. These hormones also affect callogenesis, hence, the formation of various calluses with different morphological properties are possible. It is very likely that the level of biosynthetic growth hormone, the plasmid carried by each bacteria strain, the position of T-DNA, and the level of gene expression contribute to this morphologic variation. In this study, the root explants of four species of the family Solanaceae namely Atropa belladonna, Datura metel, D. stramonium and Hyoscyamus niger were induced by using different strains of A. rhizogenes (A4, A7, AR15834, AR318, AR9402 and AR9543). Some of these explants entered callus phase and formed various calluses with different colors and shapes. Moreover, in some callus samples hairy roots were also appeared. These variations were probably caused by variations in the levels and ratios of auxin and cytokinine hormons after the induction. As shown in previous studies, the amount of secondary metabolites is reduced due to undifferentiated tissue produced in the callogenesis process

Evaluation of Antibacterial Activities of Some Medicinal Plants, Traditionally Used in Iran: Antibacterial activities of some medicinal plants

The aim of this study was to assess the antibacterial activities of some medicinal plants extracts traditionally used in Iran. Hydroalcoholic extracts obtained from different parts of five plants including Rosmarinus officinalis L. (rosemary), Syzygium aromaticum L. (Clove)., Arctium lappa L. (Burdock) , Coriandrum sativum, Myrtus communis with traditional medicinal use were examined for theirantibacterial activities against some gram-negative strains including Pseudomonas aeruginosa, Salmonella typhi, Proteus mirabilis, Klebsiella oxytoca and Shigella dysentriae. The disc diffusion method was applied to screen the antibacterial efficacy of the extracts. Gentamicin was used as control. This study showed that the extracts obtained from Syzygium aromaticum L., Arctium lappa L. and Myrtuscommunis had antibacterial activity against Proteus mirabilis. In addition, only the Myrtus communis extract had some inhibiting effect on the growth of Pseudomonas aeruginosa. The result of the current study revealed that some of the studied plants could be considered as potential source of antimicrobial agents and supports the traditional applications of a number of the tested plants as antibacterial reagents

The effect of carbon and nitrogen sources on the fatty acids profile of Mortierella vinacea

Introduction: Microbial lipids attract attention of many researchers due to their therapeutic effects. The goal of this study is the production and optimization of lipids and fatty acids in Mortierella vinaceaby applying different media to achieve invaluable fatty acids in pharmaceutical and food industry. Materials and methods: Mortierella vinacea was cultured on potato dextrose agar. Then the spores were inoculated to the production medium. After 72 hours, the lipids were extracted and they were analyzedby gas chromatography. To optimize lipid and important fatty acids production in medium, various carbon and nitrogen sources were substituted with glucose and yeast extract respectively. Results: The effect of some carbon and nitrogen sources on biomass, lipid and fatty acids production were assayed. The highest level of lipid production was in a medium which contains lactose and yeast extract (26.66%). Linoleic acid was only produced in presence of lactose and yeast extract (25.7%). While, M. vinacea yielded the highest level of linoleic acid (52.76%) in a medium containing peptone, linolenic acid was achieved only in presence of lactose and triptone. Discussion and conclusion: In this study, lactose as a carbon source was the most effective one in the production of lipids. In addition, linoleic acid was produced in presence of lactose, so lactose was selected as the best carbon source. Peptone and triptone as a nitrogen source were chosen for the production of linoleic acid and linolenic acid in M. vinacea respectively. All of these findings reveal that Mortierella strain is a potential candidate for enhancement of linoleic acid and linolenic acid production. Furthermore, this simple media can be used in production of linoleic acid and linolenic acid for industrial goals in large scales

Precise mapping of an IGF-I-binding site on the IGF-1R

The IGF-1R [type 1 IGF (insulin-like growth factor) receptor] is activated upon binding to IGF-I and IGF-II leading to cell growth, survival and migration of both normal and cancerous cells. We have characterized the binding interaction between the IGF-1R and its ligands using two high-affinity mouse anti-IGF-1R mAbs (monoclonal antibodies), 7C2 and 9E11. These mAbs both block IGF-I binding to the IGF-1R but have no effect on IGF-II binding. Epitope mapping using chimaeras of the IGF-1R and insulin receptor revealed that the mAbs bind to the CR (cysteine-rich) domain of IGF-1R. The epitope was finely mapped using single point mutations in the IGF-1R. Mutation of Phe(241), Phe(251) or Phe(266) completely abolished 7C2 and 9E11 binding. The three-dimensional structure showed that these residues cluster on the surface of the CR-domain. BIAcore analyses revealed that IGF-I and a chimaeric IGF-II with the IGF-I C-domain competed for the binding of both mAbs with the IGF-1R, whereas neither IGF-II nor a chimaeric IGF-I with the IGF-II C-domain affected antibody binding. We therefore conclude the IGF-I C-domain interacts with the CR (cysteine-rich) domain of the receptor at the cluster of residues Phe(241), Phe(251) and Phe(266). These results allow precise orientation of IGF-I within the IGF-I–IGF-1R complex involving the IGF-I C-domain binding to the IGF-1R CR domain. In addition, mAbs 7C2 and 9E11 inhibited both IGF-I- and IGF-II-induced cancer cell proliferation, migration and IGF-1R down-regulation, demonstrating that targeting the IGF-1R is an effective strategy for inhibition of cancer cell growth