Bispecific antibodies and their use in applied research

Bispecific antibodies (BsAb) can, by virtue of combining two binding specificities, improve the selectivity and efficacy of antibody-based treatment of human disease. Antibodies with two distinct binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo immunodiagnosis, therapy and for improving immunoassays. They have shown great promise for targeting cytotoxic effector cells, delivering radionuclides, toxins or cytotoxic drugs to specific targets, particularly tumour cells. The development of BsAb research goes through three main stages: chemical cross linking of murine-derived monoclonal antibody, hybrid hybridomas and engineered BsAb. This article is providing the potential applications of bispecific antibodies.


Brief History
In the first decade of this century Paul Ehrlich proposed the idea of using 'bodies' which possess a particular affinity for a certain organ [1].The introduction of hybridoma technology for producing monoclonal antibodies [2] has revolutionized almost every field of modern medicine, including tumour targeting.Monoclonal antibodies seem to be the ideal 'magic bullets' for specific targeting of tumours.

What is Antibody?
Antibodies (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as and viruses.They are typically made of basic structural units-each with two large heavy chains and two small light chains -to Bispecific Antibody form a "Y" shaped molecule.This variable region, The potential for using monoclonal antibodies composed of 110-130 amino acids, give the antibody (MAb) to enhance the capacity of the immune system its specificity for binding antigen.The variable region to combat tumors and infectious disease has includes the ends of the light and heavy chains.stimulated imagination for the last 25 years [3].In Applications of bispecific antibodies order to improve the therapeutic utility of antibodies 1) Immunoassay different modifications have been suggested.One of 2) Tumor targeting these concerns the generation of BsAb [4].Harmsen 3) Clinical application in cancer [5] developed an immunotherapy using Bispecific 4) Immunotherapy antibody that confers rapid protection against FMD in 5) Radioimmunotherapy outbreak situations.6) Drug/toxin/cytokine targeting Immunotherapy of non-Hodgkin's B cell lymphoma with bispecific antibody construct has the 1) Immunoassays: The effector binding arm can potential to engage all cytotoxic T cells [6].Bispecific be designed to have specificity for marker enzymes or monoclonal antibody (BsMAb), first introduced by other indicator systems.The anti-target-anti-peroxidase Nisonoff and Rivers into the academia 40 years ago, is bispecific antibodies which have been used in a unique type of MAb with two different binding immunohistochemistry have led to improvements in specificities within a single molecule.Bispecific sensitivity, signal-to-noise ratio, and simplification of antibodies which have two different antigen-specific staining procedures with preservation of fine ultra binding sites, one for tumour-associated antigen structural detail.(target binding arm) and the other for the effector

2)
Tumour targeting: The use of bispecific compounds (effector binding arm), have been antibodies for immunodiagnosis and therapy has developed.They can be used for any application in shown some encouraging results.They have been used which two molecules need to be juxtaposed within a for delivering effector substances such as toxins and distance of a few nM.cytotoxic drugs to tumours, and some are now in BsMAb holds great promise for numerous therapeutic clinical trials.needs in the light of: 1.
Recent breakthroughs in recombinant DNA 3) Clinical application in cancer: Immunotherapy technology, is a powerful anti-cancer treatment modality.Using 2.
The increased number of identified disease BsAbs, it is possible to take advantage of the highly specific binding characteristics of antibodies and targets as the result of the completion of human combine these with the powerful effector functions of genomic map project, and cytotoxic immune effector cells.BsAbs share two 3.
A better understanding of the mechanism of different, monoclonal antibody-derived, antigenhuman immune system.Radioimmunotherapy: Conventional radiobetween pairs of hybridoma cell lines secreting two immunotherapy (RIT) using systemically administered existing monoclonal antibodies [7].
MAb linked to radionuclides is a promising approach to metastatic cancer treatment.However, conventional RIT with MAb conjugates damages critical organs due to exposure to high radiation dose from long circulating radiolabeled antibody and non-specific accumulation of radiolabeled antibody in exposed organs.Pretargeted radioimmunotherapy with BsMAb is a multi-step strategy that allows quick and specific delivery of radioisotope (Y, I, Re and P) to a tumor with minimal radiation exposure, hence lowered toxicity to normal organs.approach allows the control of hematological toxicity, different immunoglobulins, resulted in the cowhich is dose-limiting toxicity with conventional RIT.
dominant expression of both parental immunoglobulin genes and two sets of heavy and light

5)
Immunotherapy: BsMAb is able to activate chain were found to be secreted.and target the cellular immune system to kill tumor * Fusions have been done between pairs of cells or other pathogens.A number of different effector hybridoma cell lines secreting two existing cells have been studied in this application like CTL monoclonal antibodies.(CD8+) cells, natural killer cells, macrophages and * The main advantage of fusion technique is that polymorphonuclear cells.Two possible mechanisms the resulting bispecific antibodies are have been proposed.First, CTL cells can express death synthesized, assembled, and secreted by the ligands on their surfaces that can bind to the death same process as that of the native immunoreceptors on the target cells, thereby initiating the globulin.target cell's apoptosis.Second, CTL cells can release * Once the 'hybrid hybridoma' cell lines are perforins and granzymes in the narrow intercellular obtained, they will serve as the machines to space between CTLs and target cells.Granzymes can produce endless amounts of antibody in the same enter the target cell to induce apoptosis of the target way as normal hybridomas.through the pores, which are created by perforins on There are some disadvantages: the target cell membrane.
1. Difficulty of fusing hybrid hybridoma cells

6)
Drug/toxin/cytokine targeting: An expanding 2. Stability of the resulting cell lines field in MAb-based therapeutics is the use of MAbs to 3. Low yields direct selective cytotoxic agents.Chemical drugs, 4. Difficulty in purification of the bispecific toxins and cytokines have all been conjugated to molecules.MAbs and are at various stages of development in 5. Cell fusion is labour-intensive, time consuming, clinical trials.BsMAb with intrinsic binding sites to and may not always succeed with the hybridoma any two antigens has the capability to form pairs of choice.homogeneous and reproducible immunoconjugates.6.Not all hybridoma cell lines exhibit good fusion BsMAb has been constructed to deliver various drugs, performance.e.g.doxorubicin, epirubicin, methotrexate or vinca 7. Most parental hybridomas are derived from the alkaloids.In 2001 Ford [9] has successfully fusion of HAT-sensitive myeloma fusion demonstrated a BsMAb targeting of doxorubicin to partners and immune spleen cells.colon cancer cells expressing carcinoembryonic

2.
Chemical linking of antibody molecules antigen (CEA) in vitro and in vivo.BsMAb has been * The technique for producing bispecific expanded to target toxins, e.g.ricin A, saporin and antibodies by chemical manipulation was gelonin to tumors.The advantage of using BsMAb to pioneered by Nisonoff & Rivers.deliver high molecular weight toxins or drugs is that it * It doesn't require cell fusion.avoids the complex chemistry involved in directly * Chemical coupling can be achieved in two ways: linking the antibody to the cytotoxic agent.
direct coupling of the whole antibody molecules or their derivatives, and dissociation and

Production of bispecific antibody (BsAb)
reassociation of heterologous immunoglobulin.BsAb are hybrid proteins that can be produced * The latter requires chemical manipulation to by chemical, genetic or biological methods.Bispecific dissociate immunoglobulins into half molecules antibodies are produced mainly by two methods: without damaging the antigen binding site, then 1.
Fusion of two different hybridoma cell lines to reform the disulfide bonds linking heavy 2.
Chemically linking two antibody molecules chains without allowing any interfering side reactions such as the formation of intrachain or

*
Normal plasma cells secrete only one set of mismatch disulfide bonds.heavy and light chains of immunoglobulin.

*
Hybridoma cell lines producing monoclonal antibodies derived from the fusion of the 'non-1.Bispecific antibodies were synthesized by secreting' parental myeloma cell lines with chemically cross linking bovine neutrophil monoclonal immune cells.
antibodies to S. aureus 305 capsule polysaccharide * Fusion of two myeloma clones, each secreting monoclonal antibodies [10].To determine the influence of bispecific antibodies on neutrophil (SARS) a simple, easy to use immunoswab method function, S. aureus 305 was preincubated with various was developed by generating a panel of monoclonal concentrations of bispecific antibodies and antibodies (MAbs), Bispecific MAbs and chicken neutrophils were then added to the opsonized bacteria polyclonal IgY antibody against the SARS-CoV at different bacteria to neutrophil ratios.The results nucleocapsid protein (NP).The key feature of this indicated that bispecific antibodies that recognize both simple immunoswab diagnostic assay is its ability to S. aureus 305 capsular polysaccharide and neutrophil detect the presence of the SARS-CoV antigen within antigens potentiate the bactericidal activity of 45-60 min with the availability of the body fluid neutrophils.samples [15].2.
Bispecific antibodies are manufactured or 7.
In 2011 Schaefer correctly assemble two heavy engineered from two separate antibodies to create a and two light chains, derived from two existing molecule with two different binding specificities.antibodies, to form human bivalent bispecific IgG These are used to link a target to trigger molecules on antibodies without use of artificial linkers.Applying effector cells of the immune system.This shows the three possible "CrossMab" formats, they outlines the preparation of bispecific antibodies, generated bispecific antibodies against angiopoietin-2 describes the components of cellular cytotoxicity in (Ang-2) and vascular endothelial growth factor A the immune system [11].
(VEGF-A) and show that they can be produced by 3.
The use of bispecific antibodies as possible standard techniques, exhibit stabilities comparable to therapeutic agents for cancer treatment was proposed natural antibodies, and bind both targets simultaneously in the mid-1980s [12].The common strategy for with unaltered affinity.Because of its superior side-CH1-CL making bispecific antibodies involves combining the product profile, the CrossMab was selected for in variable domains of the desired mAbs into a single vivo profiling and showed potent antiangiogenic and bispecific structure.Many different formats of antitumoral activity [16].bispecific antibodies have been generated within the 8.
In 2010 Robert describes a new procedure for research field of bispecific immunotherapeutics, forming multivalent and/or multispecific proteins, including the chemical heteroconjugation of two known as the dock-and-lock (DNL) technique.This complete molecules or fragments of MAbs, methodology has the flexibility to create a number of quadromas, F(ab')2, diabodies, tandem diabodies and other biologic agents of therapeutic interest.A variety single-chain antibodies.
Jackman and their co workers [13] had applied a antibodies, have been made, with results showing that technology to generate a bispecific antibody suitable multispecific antibodies have very different properties for development as a human therapeutic.This from the respective parental monospecific antibodies.antibody directly inhibits the activation of the high Thus, DNL technology is a highly flexible platform affinity IgE receptor FcγRI on mast cells and basophils that can be used to prepare many different types of by cross-linking FcγRI with the inhibitory receptor agents that could further improve cancer detection and FcγRIIb, an approach that has strong therapeutic therapy [17].potential for asthma and other allergic diseases.9.
In 2010 Wang and their coworker report that the bispecific antibody derivatives that bind two different natural bispecific antibody can also be generated in epitopes on the HIV coreceptor CCR5.In contrast to New Zealand white rabbits by immunization with monospecific CCR5 antibodies, bispecific antibody synthesized conjugates.These antibodies showed derivatives block two alternative docking sites of bispecificity to the components that were CCR5-tropic HIV strains on the CCR5 coreceptor.simultaneously used to immunize the animals.Wang Consequently, these molecules showed 18-to 57-fold observed a trend in test animals that female rabbits increased antiviral activities compared to the parent exhibited stronger bispecific antibody responses than antibodies.Most importantly, one prototypic tetravalent males.The bispecific antibody was monomeric and CCR5 antibody had antiviral activity against virus primarily belonged to immunoglobulin (Ig) G. strains resistant to the single parental antibodies.In Moreover, bispecific antibodies were demonstrated by summary, physical linkage of two CCR5 antibodies mixing 2 purified monospecific antibodies in vivo and targeting different epitopes on the HIV coreceptor in vitro [14].
In 2010 Kammila and their co workers worked with enhanced antiviral potency against wild-type and for the detection of severe acute respiratory syndrome CCR5 antibody-resistant HIV-1 strains [18].10.In 2011 wang and jinming identified the presence potential therapeutic target antigens from <400 to of natural bispecific antibodies against cyclic <4,000.We anticipate that BsAb will play a significant citrullinated peptide (CCP) and immunoglobulin G role in therapies directed to some of these novel (IgG) in RA patients' sera by means of a double-targets.antigen sandwich enzyme-linked immunosorbent assay recognizing moieties within one molecule.By dual A bsAb promotes the formation of conjugates binding, BsAbs reactive with a trigger molecule on an between effectors and target cells by binding to a immune effector cell on the one hand and a surface surface molecule on each cell type.If total random antigen on a tumor target cell on the other are thus able association of heavy and light chains occurs, ten to functionally focus the lytic activity of the immune different combinations of immunoglobulin molecules effector cell towards the target cell [8].are generated; however, only one has the desired bispecific activity (Fig.-1).Fusions have been done 4)