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Targeting adenovirus-mediated gene delivery with recombinant antibodies
Abstracts Phage antibody display technology was used for the analysis of two monoclonal antibodies to HIV-1 envelope glycoproteins gpl20 and gp41. From the data obtained, we were able to demonstrate that the recognition of the PND of different strains of HIV-1 by neutralizing monoclonal antibody M77 is restricted by its heavy and light chains in different ways. Native M77 is able to recognize and neutralize HIV-1 strain IIIb through binding to the gpl20 principal neutralization determinant or V3-1oop. M77 is unable to recognise isolates of HIV-1 that differ from IIIB on either the left side or the right side of the V3-1oop tip. A chain switched Fab fragment containing the M77 Fd fragment with a different light chain was able to recognize HIV-I strains that differ from lllb on the left side, but not the right side of the V3-1oop tip, suggesting that the light chain of M77 restricts regocnition ofthe left side of the V3-1oop tip, while mutations on the right side affect the recognition of the V3-1oop by the heavy chain. Targeting adenovirus-mediated gene delivery with recombinant antibodies. Sarah J. Watkins a, Vadim V. Mesyanzhinova'b, Robert E. Hawkins a, ~CentreJbr Protein Engineering, Hills Road, Cam-
bridge CB2 2HQ, UK, bThe Inanovsky Institute of Birology, 16 Gameleya Str. 123098 Moscow, Russia. Adenovirus has a high efficiency of infection and is a widely used gene delivery system. The virus binds to a cell via a two-step process with cell attachment mediated by the fibre protein and internalization occurring as a result of interactions between the penton base proteins and integrins on the cell surface. However, it infects a wide variety of cells and, for gene therapy, targeted delivery would be desirable. Targeting may be achieved by using bispecific antibodies with one end inhibiting the normal virus binding and the other redirecting the virion to specialised cell surface molecules. To make an antibody which inhibited adenoviral infection, the adenoviral fibre protein was expressed in fusion with the fibritin protein of bacteriophage T4 allowing high yields of functional protein to be obtained from E. coli. Mice immunised with this recombinant Fibre-fibritin produced high titres of antibodies that were able to prevent adenovirus infection. A phage library was then generated from the spleen of one animal containing approximately l07 combinations of Vu and V L and was selected for binding to the fusion protein. After two rounds of panning, ELISA's demonstrated that 72/96 of the VH/V L combinations bound to the fusion protein and 20 of these 72 (27%) bound preferentially to fibre as opposed to the T4 phage fibritin. The specificity of the single chain Fv antibodies for the adenovirus fibre protein itself was assessed by assaying their ability to prevent adenovirus infection of HeLa and A431 cells. Three antibody fragments were isolated (termed SI 1, V40 and VVI6) which bound specifically to the fibre protein and blocked viral infection. Preliminary experiments suggest that when the most effective of these three clones (VVI6) was used to create a fusion protein with Epidermal Growth Factor (EGF), re-infection of A431 cells via the EGF receptor occurred, suggesting that targeting of the virus particle had been achieved. This system
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has potential to efficiently target any adenovirus (type 5) vector to specific cell types and it is likely that other adenoviral vectors (and perhaps other viruses) can be targeted by similar methods. The use of antibody fragments for the sensitive detection and removal of organic pollutants from the environment. [Abst. 337] Stephen Williams, Andrew J. Porter, Dianne Learmonth, Steven D. Grant, Gillian Strachan, William J. Harris, Depart-
ment of Molecular and Cell Biology, University of Aberdeen, Aberdeen, AB9 IAS, Scotland, UK. There is extensive pollution of our environment by organic pesticides and other industrial chemicals. European legislative and safety requirements aim to ensure that clean water contains less that 0.1 /zg per litre (100 ppt) of a particular pollutant and no more that 0.5 pg per litre total organic pollutant. Existing removal methods can reduce pollutants to 5 - l 0 / z g per litre ranges, at least 100 fold above targeted levels in a first level clean up. Current detection methods are expensive, sophisticated and cannot be performed 'in the field'. New cost effective methods of detection and removal are required. Antibodies have binding affinities of l0 9 M and can chelate organics present at ng/1 concentrations, and could be used for detection and second level clean up. Whole antibodies are large, expensive to produce and have poor stability in adverse environments making there use in the future for removal and detection limited. Antibody fragments (Fab, Fv, ScFv), however, form as little as 20% of the intact imunoglobulin molecule and can be made in quantity and cheaply in E. coil The use of antibody fragments could increase the sensitivity of detection methods by 10-100 fold and can be designed with increased stability whilst still retaining high binding affinity. We have designed gene constructs which allow the assembly in E. coli of bivalent antibody fragments against paraquat, atrazine and diuron. These fragments have been immobilised and shown to remove trace pesticides from aqueous environments. Their use in ELISA-based immunoassays will also be descirbed. Stabilization of a single-chain FV antibody by introduction of a disulfide bond. [Abst. 338] N. Martin Young ~,b, C. Roger MacKenzie ~,b, Saran A. Narang a'b, Raymond P. Oomen a,b, R. To a'b, John E. Baenzigera'b, OInstitute for Biological Sciences, National Research
Council of Canada, Ottawa Canada K1A OR6, hDepartment of Biochemistry, University of Ottawa, Ottawa, Canada KIH 8M5. Single-chain forms of antibodies exist as an equilibrium mixture between monomer and dimer forms. This can be an undesirable trait for therapeutic applications since the dimer's greater size may decrease tumor penetration. Dimer formation can also compromise the panning of phage libraries for stronger-binding species. It also affects physical characterization, interfering in crystallization trials and in binding measurements by surface plasmon resonance, where the avidity of the dimer can lead to it dominating the binding profile even
bridge CB2 2HQ, UK, bThe Inanovsky Institute of Birology, 16 Gameleya Str. 123098 Moscow, Russia. Adenovirus has a high efficiency of infection and is a widely used gene delivery system. The virus binds to a cell via a two-step process with cell attachment mediated by the fibre protein and internalization occurring as a result of interactions between the penton base proteins and integrins on the cell surface. However, it infects a wide variety of cells and, for gene therapy, targeted delivery would be desirable. Targeting may be achieved by using bispecific antibodies with one end inhibiting the normal virus binding and the other redirecting the virion to specialised cell surface molecules. To make an antibody which inhibited adenoviral infection, the adenoviral fibre protein was expressed in fusion with the fibritin protein of bacteriophage T4 allowing high yields of functional protein to be obtained from E. coli. Mice immunised with this recombinant Fibre-fibritin produced high titres of antibodies that were able to prevent adenovirus infection. A phage library was then generated from the spleen of one animal containing approximately l07 combinations of Vu and V L and was selected for binding to the fusion protein. After two rounds of panning, ELISA's demonstrated that 72/96 of the VH/V L combinations bound to the fusion protein and 20 of these 72 (27%) bound preferentially to fibre as opposed to the T4 phage fibritin. The specificity of the single chain Fv antibodies for the adenovirus fibre protein itself was assessed by assaying their ability to prevent adenovirus infection of HeLa and A431 cells. Three antibody fragments were isolated (termed SI 1, V40 and VVI6) which bound specifically to the fibre protein and blocked viral infection. Preliminary experiments suggest that when the most effective of these three clones (VVI6) was used to create a fusion protein with Epidermal Growth Factor (EGF), re-infection of A431 cells via the EGF receptor occurred, suggesting that targeting of the virus particle had been achieved. This system
307
has potential to efficiently target any adenovirus (type 5) vector to specific cell types and it is likely that other adenoviral vectors (and perhaps other viruses) can be targeted by similar methods. The use of antibody fragments for the sensitive detection and removal of organic pollutants from the environment. [Abst. 337] Stephen Williams, Andrew J. Porter, Dianne Learmonth, Steven D. Grant, Gillian Strachan, William J. Harris, Depart-
ment of Molecular and Cell Biology, University of Aberdeen, Aberdeen, AB9 IAS, Scotland, UK. There is extensive pollution of our environment by organic pesticides and other industrial chemicals. European legislative and safety requirements aim to ensure that clean water contains less that 0.1 /zg per litre (100 ppt) of a particular pollutant and no more that 0.5 pg per litre total organic pollutant. Existing removal methods can reduce pollutants to 5 - l 0 / z g per litre ranges, at least 100 fold above targeted levels in a first level clean up. Current detection methods are expensive, sophisticated and cannot be performed 'in the field'. New cost effective methods of detection and removal are required. Antibodies have binding affinities of l0 9 M and can chelate organics present at ng/1 concentrations, and could be used for detection and second level clean up. Whole antibodies are large, expensive to produce and have poor stability in adverse environments making there use in the future for removal and detection limited. Antibody fragments (Fab, Fv, ScFv), however, form as little as 20% of the intact imunoglobulin molecule and can be made in quantity and cheaply in E. coil The use of antibody fragments could increase the sensitivity of detection methods by 10-100 fold and can be designed with increased stability whilst still retaining high binding affinity. We have designed gene constructs which allow the assembly in E. coli of bivalent antibody fragments against paraquat, atrazine and diuron. These fragments have been immobilised and shown to remove trace pesticides from aqueous environments. Their use in ELISA-based immunoassays will also be descirbed. Stabilization of a single-chain FV antibody by introduction of a disulfide bond. [Abst. 338] N. Martin Young ~,b, C. Roger MacKenzie ~,b, Saran A. Narang a'b, Raymond P. Oomen a,b, R. To a'b, John E. Baenzigera'b, OInstitute for Biological Sciences, National Research
Council of Canada, Ottawa Canada K1A OR6, hDepartment of Biochemistry, University of Ottawa, Ottawa, Canada KIH 8M5. Single-chain forms of antibodies exist as an equilibrium mixture between monomer and dimer forms. This can be an undesirable trait for therapeutic applications since the dimer's greater size may decrease tumor penetration. Dimer formation can also compromise the panning of phage libraries for stronger-binding species. It also affects physical characterization, interfering in crystallization trials and in binding measurements by surface plasmon resonance, where the avidity of the dimer can lead to it dominating the binding profile even