- Email: [email protected]
Accessing biomolecular diversity: the challenge
Research Update
TRENDS in Biotechnology Vol.19 No.2 February 2001
37
Meeting Report
Accessing biomolecular diversity: the challenge Andrew Bradbury The IBC Biomolecular Diversity conference was held in Waltham, MA, USA, 11–13 September 2000 and Understanding Phage Display: Structure, Biology and Applications, was held in Vancouver, BC, Canada, 21–23 September 2000.
The recent change in name of the IBC Phage Display meeting to ‘Biomolecular Diversity’ is indicative of the changes that have been occurring in this field. Five years ago, almost all exploration of biomolecular diversity was carried out using phage display, with binding being the universal means of selection. Although phage display is still the most commonly used molecular diversity technique, indeed another congress has been entirely devoted to it, many other methods are now being developed successfully. Much exciting work was discussed at both of these meetings, but this report will concentrate on only a few highlights representing interesting advances in the field. Wet and dry diversity
The concept of creating diversity and selecting from it in a Darwinian fashion remains the concept that unites all the technologies discussed, but the different ways in which this is being done has undergone significant changes. With one exception, the methods to generate diversity have remained similar: accessing natural diversity found in different genes; creating synthetic genes with random oligonucleotides; or subjecting such genes to either targeted, random or recombinatorial mutagenesis. All of these methods are essentially ‘wet’, involving extensive laboratory manipulation of DNA. The one exception is a ‘dry’ in silico method using a program called ‘Protein design automation’ developed by Bassil Dahiyat (Xencor, Pasadena, CA, USA) to identify mutations that are likely to improve protein properties. Starting from either a known or a modeled structure, up to 60 different amino acids are targeted for in silico mutation, with a claimed potential library size of 1080. All members of this vast virtual library are then ranked for stability using energy minimization. By eliminating all those mutations that will be
destabilizing, and thus clearly unsuitable, the top members can then be ‘wet’ tested, by either screening or applying selection. This was successfully applied to the development of a human granulocytecolony stimulating factor (G-CSF), modeled on bovine G-CSF, which was ten times more stable than the clinically used G-CSF, as well as a version of β lactamase that was 30 000 times more active against cefotaxime than the wild-type enzyme. In both cases, novel mutations that had not been encountered in previous mutational selections were identified.
Although phage display is still the most commonly used molecular diversity technique … many other methods are now being developed successfully. Clearly, this technique has great potential but, at least in its present format, it requires knowledge of the structure and function of the protein. Filamentous phage display
Sachdev Sidhu (Genentech, San Francisco, CA) showed that filamentous phages still hold surprises. He demonstrated that p8 can be mutated and selected to increase growth hormone (GH) display levels by ten times, with very few p8 residues being essential; however, whether this p8 mutant will display all proteins equally well, remains to be seen. Furthermore, he showed that both p3 and p8 can be modified to display C-terminal proteins, with C-terminal p3 display being as effective as that at the N terminus. This was especially surprising, given that it was previously thought that the C termini of both proteins were buried within the phage core. The extreme tolerance of the phage coat for nonwild-type proteins was further demonstrated by the design and evolution of completely artificial coat proteins that resemble p8 in reverse and also permit C-terminal protein display in a phagemid system. This C-terminal display was used to
investigate PDZ (PSD-95/Discs-Large/ZO-1) specificity, and will probably find a use in cDNA display. This was also the goal of Duncan McGregor (Rowett Research Institute, Aberdeen, Scotland) who similarly used a non-wild-type protein, in this case the DNA binding domain (DBD) of the estrogen receptor, as a display protein in a phagemid that contained the hormone response element (HRE) sequence; the idea being that the DBD would bind the HRE in the phagemid DNA and so become incorporated into the phage. Although display was not dependent on HRE presence, it was increased by it and antibodies were successfully selected from an scFv library displayed in this way. The rationale of using a C-terminal display protein was also exploited by Alessandra Luzzago (Istituto di Richerche di Biologia Moleculare, Rome, Italy) who described a λ-based cDNA display vector that was used successfully to display several cDNA libraries and select for binding to either protein or DNA targets. In both cases, expected and unexpected targets were identified. cDNA libraries, based on a p6 filamentous phage display vector (Hennie Hoogenboom, TargetQuest, Maastricht, The Netherlands), also have the characteristic that display is C terminal. Libraries derived from colon cancer cDNA were selected with patient sera, in the hope that tumor-specific antisera might identify appropriate therapeutic targets. Selection did occur and when cDNA targets were screened with wider panels of patient sera, ~25% remained tumor specific, with 10–20% of patient sera reactive with at least one of a panel of four individual cDNA clones. As the number of such cDNAs increases, it is hoped that this approach to characterize immune responses to cancer antigens might eventually yield panels of disease-predictive cDNA products. Using phage display to select for function
Novel selection strategies involving the internalization of filamentuos phage bearing either antibodies (James Marks, UCSF, San Francisco, CA, USA) or specific ligands such as epidermal growth factor (EGF) (David Larocca, Selective Genetics, San Diego, CA, USA), showed that phage
http://tibtech.trends.com 0167-7799/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01530-4
38
Research Update
could specifically ‘infect’ mammalian cells, leading to the expression of marker genes, with efficiencies as high as 45% with appropriate treatments. These efficiencies now approach those found with human gene therapy vectors based on adeno-, or adeno-associated virus, and might find a role in gene therapy. They would have the clear advantage that phage that usually infect bacteria are unlikely to be pathogenic in humans, beyond the generation of an immune response. This could perhaps be engineered out, using appropriate mutation and negative selection schemes, as was shown by Laurent Jespers (MRC, Cambridge, UK) for staphylokinase. In vivo selection with peptide phage in mice has previously been used to identify what have been termed vascular ‘endothelial addresses’. Renata Pasqualini (MD Andersen Cancer Center, Houston, TX, USA) has carried this technology further, showing that a lung-specific peptide selected in this way was able to purify and identify a single band from cell extracts, as well as inhibit lung metastases in a mouse model. Experiments are currently underway in terminal cancer patients to identify equivalent human ‘endothelial addresses’. Selecting directly for function
Although biomolecular diversity started in filamentous phage, some of the most exciting recent results have been obtained in systems in which libraries are introduced into cells, and specific phenotypes are selected for. This principle was
TRENDS in Biotechnology Vol.19 No.2 February 2001
demonstrated with a known peptide by Gunilla Karlsson (M & E Biotech, Hørsholm, Denmark) and was specifically demonstrated using libraries in the study of HIV by Garry Nolan (Stanford University, Stanford, CA & Rigel Inc. South San Francisco, CA, USA). In both cases, mammalian cells were transduced with retroviruses containing the peptides of interest. One thing that became clear was that after the care of library and selection strategy design, traditional biochemistry and cell biology are still required to identify the targets recognized, interpret their modes of action, and exploit such information in a clinically useful way. However, once this work has been carried out, such cell/target/peptide combinations might be very useful in high-throughput screening of potential antagonists. A similar approach designed to identify peptides that inhibit bacterial growth was described by Jonathan Blum (Harvard University, Boston, MA, USA). He also demonstrated the principle of using bacterial mutants that overcome the effects of aptamers to identify targets recognized by such aptamers: thymidylate synthase overexpressors, for example, overcome the toxicity of peptides that target that enzyme. A combination of such phenotypic screening with specific selection was described by Paul Hamilton (Karo Bio, Durham, NC, USA), who selected peptides on known bacterial targets, such as dihydrofolate reductase, or tyrosyl tRNA synthetase, using phage display and then after identifying consensus binding sequences, showed that such peptides had biological
effects when introduced into bacteria as peptides fused to glutathione S transferase (GST). The real power of the technique comes in the potential of using such peptide–target interactions as high throughput screens for antibacterial compounds. Conclusion
The application of Darwinian selection methods to the study of many different problems can be reduced to four essential requirements: (1) library creation; (2) coupling of phenotype and genotype; (3) the application of selective pressure; and (4) amplification. Although this technology was initially almost exclusively focussed on phage display, the application of these four basic principles has become wider and wider as different organisms (e.g. yeast and bacteria), in vitro systems [e.g. ribosome display and covalent RNA–protein fusion (PROfusion)], and now functional selection systems have been initiated. To paraphrase George Smith (University of Missouri, Columbia, MO, USA) in his closing speech, ‘…this particular technology attracts a certain sort of maverick scientist, and this certain sort of maverick has been remarkably imaginative in the application of this technology’. It is clear that the imagination still flows, and is likely to continue to do so. Andrew Bradbury Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545 USA and SI SSA, Trieste, Italy e-mail: [email protected]
Biomedical microdevices and nanotechnology Adam T. Woolley The Cambridge Healthtech Institute’s BioMEMS and Biomedical Nanotechnology World 2000 conference was held in Columbus, OH, USA, 23–26 September 2000.
Biomedical micro- and nanodevices, with components from as large as hundreds of microns to as small as a few nanometers, have potential uses that range from the analysis of biomolecules to disease diagnosis, prevention and treatment. Although micro- and nanotechnology have only been developing for a relatively short
time, they are well positioned to have a significant future impact in biology and medicine. The possibility of improved speed, greater sensitivity, reduced cost, and decreased invasiveness, has generated substantial interest in miniaturized devices. Research into the use of biomedical micro- and nanotechnology was discussed in this conference, which covered the general topics of microfluidics, sensors, microelectro-mechanical systems (MEMS) and nanotechnology.
Microfluidics
A key limitation of previous work involving microfluidic systems has been the difficulty in the integration of sample preparation with high-speed analysis; however, at this conference, promising progress was communicated about several aspects of this critical problem. Richard Mathies (University of California, Berkeley, CA, USA) reported complex, integrated, highly parallel devices that take advantage of many of the benefits that micromachining offers to biochemical analysis. These
http://tibtech.trends.com 0167-7799/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01531-6
TRENDS in Biotechnology Vol.19 No.2 February 2001
37
Meeting Report
Accessing biomolecular diversity: the challenge Andrew Bradbury The IBC Biomolecular Diversity conference was held in Waltham, MA, USA, 11–13 September 2000 and Understanding Phage Display: Structure, Biology and Applications, was held in Vancouver, BC, Canada, 21–23 September 2000.
The recent change in name of the IBC Phage Display meeting to ‘Biomolecular Diversity’ is indicative of the changes that have been occurring in this field. Five years ago, almost all exploration of biomolecular diversity was carried out using phage display, with binding being the universal means of selection. Although phage display is still the most commonly used molecular diversity technique, indeed another congress has been entirely devoted to it, many other methods are now being developed successfully. Much exciting work was discussed at both of these meetings, but this report will concentrate on only a few highlights representing interesting advances in the field. Wet and dry diversity
The concept of creating diversity and selecting from it in a Darwinian fashion remains the concept that unites all the technologies discussed, but the different ways in which this is being done has undergone significant changes. With one exception, the methods to generate diversity have remained similar: accessing natural diversity found in different genes; creating synthetic genes with random oligonucleotides; or subjecting such genes to either targeted, random or recombinatorial mutagenesis. All of these methods are essentially ‘wet’, involving extensive laboratory manipulation of DNA. The one exception is a ‘dry’ in silico method using a program called ‘Protein design automation’ developed by Bassil Dahiyat (Xencor, Pasadena, CA, USA) to identify mutations that are likely to improve protein properties. Starting from either a known or a modeled structure, up to 60 different amino acids are targeted for in silico mutation, with a claimed potential library size of 1080. All members of this vast virtual library are then ranked for stability using energy minimization. By eliminating all those mutations that will be
destabilizing, and thus clearly unsuitable, the top members can then be ‘wet’ tested, by either screening or applying selection. This was successfully applied to the development of a human granulocytecolony stimulating factor (G-CSF), modeled on bovine G-CSF, which was ten times more stable than the clinically used G-CSF, as well as a version of β lactamase that was 30 000 times more active against cefotaxime than the wild-type enzyme. In both cases, novel mutations that had not been encountered in previous mutational selections were identified.
Although phage display is still the most commonly used molecular diversity technique … many other methods are now being developed successfully. Clearly, this technique has great potential but, at least in its present format, it requires knowledge of the structure and function of the protein. Filamentous phage display
Sachdev Sidhu (Genentech, San Francisco, CA) showed that filamentous phages still hold surprises. He demonstrated that p8 can be mutated and selected to increase growth hormone (GH) display levels by ten times, with very few p8 residues being essential; however, whether this p8 mutant will display all proteins equally well, remains to be seen. Furthermore, he showed that both p3 and p8 can be modified to display C-terminal proteins, with C-terminal p3 display being as effective as that at the N terminus. This was especially surprising, given that it was previously thought that the C termini of both proteins were buried within the phage core. The extreme tolerance of the phage coat for nonwild-type proteins was further demonstrated by the design and evolution of completely artificial coat proteins that resemble p8 in reverse and also permit C-terminal protein display in a phagemid system. This C-terminal display was used to
investigate PDZ (PSD-95/Discs-Large/ZO-1) specificity, and will probably find a use in cDNA display. This was also the goal of Duncan McGregor (Rowett Research Institute, Aberdeen, Scotland) who similarly used a non-wild-type protein, in this case the DNA binding domain (DBD) of the estrogen receptor, as a display protein in a phagemid that contained the hormone response element (HRE) sequence; the idea being that the DBD would bind the HRE in the phagemid DNA and so become incorporated into the phage. Although display was not dependent on HRE presence, it was increased by it and antibodies were successfully selected from an scFv library displayed in this way. The rationale of using a C-terminal display protein was also exploited by Alessandra Luzzago (Istituto di Richerche di Biologia Moleculare, Rome, Italy) who described a λ-based cDNA display vector that was used successfully to display several cDNA libraries and select for binding to either protein or DNA targets. In both cases, expected and unexpected targets were identified. cDNA libraries, based on a p6 filamentous phage display vector (Hennie Hoogenboom, TargetQuest, Maastricht, The Netherlands), also have the characteristic that display is C terminal. Libraries derived from colon cancer cDNA were selected with patient sera, in the hope that tumor-specific antisera might identify appropriate therapeutic targets. Selection did occur and when cDNA targets were screened with wider panels of patient sera, ~25% remained tumor specific, with 10–20% of patient sera reactive with at least one of a panel of four individual cDNA clones. As the number of such cDNAs increases, it is hoped that this approach to characterize immune responses to cancer antigens might eventually yield panels of disease-predictive cDNA products. Using phage display to select for function
Novel selection strategies involving the internalization of filamentuos phage bearing either antibodies (James Marks, UCSF, San Francisco, CA, USA) or specific ligands such as epidermal growth factor (EGF) (David Larocca, Selective Genetics, San Diego, CA, USA), showed that phage
http://tibtech.trends.com 0167-7799/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01530-4
38
Research Update
could specifically ‘infect’ mammalian cells, leading to the expression of marker genes, with efficiencies as high as 45% with appropriate treatments. These efficiencies now approach those found with human gene therapy vectors based on adeno-, or adeno-associated virus, and might find a role in gene therapy. They would have the clear advantage that phage that usually infect bacteria are unlikely to be pathogenic in humans, beyond the generation of an immune response. This could perhaps be engineered out, using appropriate mutation and negative selection schemes, as was shown by Laurent Jespers (MRC, Cambridge, UK) for staphylokinase. In vivo selection with peptide phage in mice has previously been used to identify what have been termed vascular ‘endothelial addresses’. Renata Pasqualini (MD Andersen Cancer Center, Houston, TX, USA) has carried this technology further, showing that a lung-specific peptide selected in this way was able to purify and identify a single band from cell extracts, as well as inhibit lung metastases in a mouse model. Experiments are currently underway in terminal cancer patients to identify equivalent human ‘endothelial addresses’. Selecting directly for function
Although biomolecular diversity started in filamentous phage, some of the most exciting recent results have been obtained in systems in which libraries are introduced into cells, and specific phenotypes are selected for. This principle was
TRENDS in Biotechnology Vol.19 No.2 February 2001
demonstrated with a known peptide by Gunilla Karlsson (M & E Biotech, Hørsholm, Denmark) and was specifically demonstrated using libraries in the study of HIV by Garry Nolan (Stanford University, Stanford, CA & Rigel Inc. South San Francisco, CA, USA). In both cases, mammalian cells were transduced with retroviruses containing the peptides of interest. One thing that became clear was that after the care of library and selection strategy design, traditional biochemistry and cell biology are still required to identify the targets recognized, interpret their modes of action, and exploit such information in a clinically useful way. However, once this work has been carried out, such cell/target/peptide combinations might be very useful in high-throughput screening of potential antagonists. A similar approach designed to identify peptides that inhibit bacterial growth was described by Jonathan Blum (Harvard University, Boston, MA, USA). He also demonstrated the principle of using bacterial mutants that overcome the effects of aptamers to identify targets recognized by such aptamers: thymidylate synthase overexpressors, for example, overcome the toxicity of peptides that target that enzyme. A combination of such phenotypic screening with specific selection was described by Paul Hamilton (Karo Bio, Durham, NC, USA), who selected peptides on known bacterial targets, such as dihydrofolate reductase, or tyrosyl tRNA synthetase, using phage display and then after identifying consensus binding sequences, showed that such peptides had biological
effects when introduced into bacteria as peptides fused to glutathione S transferase (GST). The real power of the technique comes in the potential of using such peptide–target interactions as high throughput screens for antibacterial compounds. Conclusion
The application of Darwinian selection methods to the study of many different problems can be reduced to four essential requirements: (1) library creation; (2) coupling of phenotype and genotype; (3) the application of selective pressure; and (4) amplification. Although this technology was initially almost exclusively focussed on phage display, the application of these four basic principles has become wider and wider as different organisms (e.g. yeast and bacteria), in vitro systems [e.g. ribosome display and covalent RNA–protein fusion (PROfusion)], and now functional selection systems have been initiated. To paraphrase George Smith (University of Missouri, Columbia, MO, USA) in his closing speech, ‘…this particular technology attracts a certain sort of maverick scientist, and this certain sort of maverick has been remarkably imaginative in the application of this technology’. It is clear that the imagination still flows, and is likely to continue to do so. Andrew Bradbury Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545 USA and SI SSA, Trieste, Italy e-mail: [email protected]
Biomedical microdevices and nanotechnology Adam T. Woolley The Cambridge Healthtech Institute’s BioMEMS and Biomedical Nanotechnology World 2000 conference was held in Columbus, OH, USA, 23–26 September 2000.
Biomedical micro- and nanodevices, with components from as large as hundreds of microns to as small as a few nanometers, have potential uses that range from the analysis of biomolecules to disease diagnosis, prevention and treatment. Although micro- and nanotechnology have only been developing for a relatively short
time, they are well positioned to have a significant future impact in biology and medicine. The possibility of improved speed, greater sensitivity, reduced cost, and decreased invasiveness, has generated substantial interest in miniaturized devices. Research into the use of biomedical micro- and nanotechnology was discussed in this conference, which covered the general topics of microfluidics, sensors, microelectro-mechanical systems (MEMS) and nanotechnology.
Microfluidics
A key limitation of previous work involving microfluidic systems has been the difficulty in the integration of sample preparation with high-speed analysis; however, at this conference, promising progress was communicated about several aspects of this critical problem. Richard Mathies (University of California, Berkeley, CA, USA) reported complex, integrated, highly parallel devices that take advantage of many of the benefits that micromachining offers to biochemical analysis. These
http://tibtech.trends.com 0167-7799/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01531-6