Nanobody, New Agent for Combating Against Breast Cancer Cells (2011)

IntroductionBreast cancer (BC) is a major public health problem among women throughout the world.More than 1.1 million cases are diagnosed annually and more than 410,000 patients die of it worldwide (Ferlay et al., 2010).BC is a complex and intrinsically heterogeneous disease with different morphologies, molecular profiles and clinical behavior which require different treatments (Bosch et al., 2010).Cancer treatment has come a long way from surgery alone to combined therapies to control tumour growth.By introducing the new therapeutic agents, combinations of existing therapies and targeted therapies, there would be promising future to improve survival and life span of patients who deal with this disease. Breast cancer and therapeutic approachesBreast cancer remains a threatening health problem in both developed and developing countries.However, its mortality rates have declined in recent years because of the broad advancement in the treatment of BC; including modifications in surgical procedures that reduce the risk of surgical morbidity and improvement in the delivery of radiation using novel imaging techniques that allow enhanced dosing to specified locations with fewer side effects on normal tissues (Moulder and Hortobagyi, 2008).Also, developments in systemic therapies include chemotherapy, hormonal therapy and biological therapy or combinations of these supportive cares, hold great promise for future breast cancer therapy. HER2 targeted therapy by clinically approved drugsReceptor tyrosine kinases (RTKs) play great roles in the transmission of extracellular signals that lead to cancer cell growth, survival and differentiation.These proteins are localized within the cell membrane and contain three parts.An extracellular ligand-binding domain (domains I, II, III, IV), a transmembrane domain that anchors the receptor to the cell membrane and a kinase domain that contains the ATP-binding site that phosphorylated a number of downstream target proteins.Phosphorylation causes the activation of signaling www.intechopen.comBreast Cancer -Current and Alternative Therapeutic Modalities 348 cascades such as the mitogen-activated protein kinase and phosphoinositol 3'OH kinase (PI3K) pathways (Kruser and Wheeler, 2010).The HER family consists of four RTKs with similar homology: HER1 (EGFR/ErbB1), HER2 (neu, C-erbB2), HER3 (ErbB3) and HER4 (ErbB4) (Moulder and Hortobagyi, 2008).HER2 is the preferred dimerization partner for activation of other HER receptors.Receptor activation via ligand binding leads to downstream signaling including mitogen-activated protein kinases (MAPK), PI3K and signal transducers and activators of transcription (Stats).Ultimately, cell proliferation, angiogenesis, invasion and metastasis can be promoted by these cascades (Kruser and Wheeler, 2010;Nielsen et al., 2009).Aberrant expression or activity of two members of this family HER1 and HER2 have been connected with the occurrence of breast cancer.Two key parts of HER1 and HER2 (ligand binding domain and tyrosine kinase domain) have attracted scientists to inhibit their activity.After many endeavors, the dream for generating an antibody that could bind to HER2 and block it became true.Trastuzumab (Herceptin®) (Genentech Inc.San Francisco, CA, USA; Hoffmann-La Roche Ltd.Basel, Switzerland) is the only anti-HER2 antibody that approved by FDA.This antibody is a humanized monoclonal antibody that targets the extracellular juxtamembranal domain of HER2 (Park et al., 2010).Trastuzumab by binding to HER2 reveals therapeutic efficacy in HER2-positive early stage and metastatic breast cancer.(Emde et al., 2010;Nielsen et al., 2009). ImmunotherapyThe clinical purpose of cancer immunotherapy is to enhance immune responses against tumour cells with the lowest side effects on healthy tissue (Guinn et al., 2007;Leen et al., 2007).Two therapeutic strategies have been developed to stimulate anti-tumour immunity in patients with breast cancer; vaccination (active immunization) and antibody/ immune cell therapy (passive immunization) (June, 2007;King et al., 2008).Vaccination of patients with breast cancer has not showed satisfactory results (Stauss et al., 2007).Antibody/T cell therapy possess potential benefits, since two main arms of immune system participate in destroying tumours and prevention of cancer recurrence by developing the immunological memory (Guinn et al., 2007).The advent of monoclonal antibodies (mAbs) revolutionized the treatment of cancer.Monoclonal antibodies have always been encouraging for scientist as if they have developed novel approaches to produce various antibody formations; recombinant mAbs (include antibody fragments), chimeric antibodies and more recently recombinant polyclonal antibodies (Elbakri et al., 2010).Monoclonal antibodies have emerged as a class of novel oncology therapeutics.The unique pharmacokinetic characteristics, high specificity and the ability to engage and activate the immune system have made them valuable therapeutic agents in breast cancer treatment (Yan et al., 2008).Monoclonal antibodies exert their effects through activating antibody dependent cellular cytotoxicity and complement-dependent cytotoxicity, triggering apoptosis and blockade of growth factor receptors (King et al., 2008).Several monoclonal antibodies against tumour-associated antigen (TAA) have been used as fascinating targeting agents in breast cancer therapy.Clinical success has been observed with passively acquired monoclonal antibodies directed against a number of targets including HER2, HER1, MUC1 and vascular endothelial growth factor (King et al., 2008).However, these molecules have shown some side effects, resistance, immunogenicity and toxicity that have limited their uses.Endeavors to find solutions for these drawbacks led to advent several alternatives (Elbakri et al., 2010). www.intechopen.comNanobody, New Agent for Combating Against Breast Cancer Cells 349 Nanobody; an old concept and new tool for immunotargetingThe large molecular size and immunogenicity are some complications that explain why treatment of solid tumours by mAbs is so elusive.These problems in therapeutic efficacy of mAbs have been partly solved with applying novel approaches such as phage display in the development of new therapeutically effective antibody domains.Antibody domains which lack the Fc region such as Fabs, diabodies, single chain variable fragments (scFvs), bispecific antibodies and variable domains (VH) offer many therapeutic advantages for therapeutic applications (Elbakri et al., 2010).By serendipity, in 1993 the nicest substitutes, the new generation of magic bullets that called heavy-chain antibodies (HCAbs) was found in Camelidae (Hamers-Casterman et al., 1993) and opened new window in breast cancer therapy (Rahbarizadeh et al., 2004b).HCAbs have evolved to be fully functional in the absence of a light chain.The smallest antigen-binding fragment, harbouring the full binding capacity of the naturally occurring HCAbs, is called VHH (variable domain of heavy chain antibodies) or single domain antibody.The crystal structure of an isolated single domain antibody is a particle of 2.5 nm in diameter and about 4 nm height, and is termed nanobody because of its size in nm scale and single domain structure. Nanobody; structure and characteristicsSequence analysis and studies on the crystal structure of nanobodies have revealed several structural features of nanobody domains (Hamers-Casterman et al., 1993;Muyldermans et al., 1994).These molecules contain four framework regions (FRs) that form the core structure of the immunoglobulin domain and three CDRs that are involved in antigen binding.When compared to human VH domains, the FRs show sequence homology of more than 80%, and their 3D structures can be superimposed (Desmyter et al., 1996).The genes of gamma 2 and gamma 3 chains of Camelidae HCAb show four amino acid changes at positions 42 (F, Y), 49 (E, Q), 50 (C, R) and 52 (F, G, L, W) according to the IMGT unique numbering, that are involved in forming the hydrophobic interface with VL domains.This co-evolution of the variable region (more hydrophilic) and of the constant region (absence of CH1 due to a mutation in the splicing site) is particularly remarkable.Occasionally, antigen-binding single domain antibody fragments that lack these characteristic FR2 substitutions are isolated from camelids.These fall into two categories:  VHH-like conventional VHs: low-affinity binders isolated from a non-immune library which was originated from conventional antibodies, presumably because of the polymerase chain reaction crossover cloning artifact, as they were linked to the CH1 domain (Tanha et al., 2002)  Conventional-like VHH domains: single-domain antibody fragments with conventional-like FR2 sequences that bind antigens with high affinity, isolated from immune libraries, which might have a hydrophobic residue at position 103 (mostly arginine) (Conrath et al., 2001;Harmsen and De Haard, 2007;Saerens et al., 2004).CDRs of nanobodies are somewhat unique.The first amino acids of CDR1 are highly variable (Harmsen et al., 2000;Nguyen et al., 2000;Vu et al., 1997).VHH libraries generated from immunized camelids retain full functional diversity.High-affinity antigen binding domains can be isolated through screening a limited number of clones from immune libraries (Frenken et al., 2000;Harmsen et al., 2005).This outstanding nature of nanobodies results in several advantages over the classical antibody families.The single domain nature of a nanobody makes molecular manipulation How to referenceIn order to correctly reference this scholarly work, feel free to copy and paste the following: