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Molecular couturiers and designer proteins 'Protein Engineering has come of age', announced Greg Petsko ( Brandeis University, MA, USA) in his introductory presentation at the '93 Miami Biotechnology Winter Symposium on protein engineering*. Petsko went on to explain that sitedirected mutagenesis of proteins has now become a relatively routine tool, used by many biochemists. He predicted that the next Miami Biotechnology Winter Protein Engineering Symposium (in 1995) could be called something else perhaps, finally, 'rational protein design'. This observation seemed to bejustiffed by the posters and papers presented. Many studies were based, not only upon conservative substitution of amino acids, but also upon transposition of protein domains between molecules to produce chimaeras, redesign of antibody molecules for therapy or biocatalysis, development of drugs by phage display of randomized peptide libraries, or design of novel proteins from first principles. Daring breaks with tradition By extrapolating from known protein sequences and three-dimensional (3-D) structures, it is estimated that there are probably only about t 000 naturally occurring folding patterns 1 (combinations of secondary and tertiary structures), although at present we only know about 15% of these. Human beings alone possess 50 000 -100 000 distinct proteins, so that many millions of the different, naturally occurring amino acid sequences must be folded in a limited number of very similar ways. This degree of sequence variability, relative to a few conserved 3-D structures, was not expected, and gives protein engineers, modellers and designers hope that, in the future, more accurate molecular modelling, effective protein redesign, and even invention of protein structures will be possible.

* The '93 MiamiBiotechnologyWinter Symposium 'Advancesin Gene Technology:Prorein Engineering and Beyond' was held in Miami, Florida, USA, 17 22January 1993. © 1993, Elsevier Science Publishers Ltd (UK)

The conservation of 3-D structure, relative to sequence, was evident in many presentations at the meeting. Brian Matthews (Howard Hughes Medical Institute, O R , USA) showed that bacteriophage T4 lysozyme can accommodate multiple amino acid changes. Up to ten contiguous alanine residues can be inserted at points in the protein, without loss of the overall folding pattern, or biological activity. Interestingly, after insertion of alanine residues into ~x-helical structures, the helix often translocates (i.e. the helix does not lengthen, but previously excluded residues are incorporated into the helix and others 'loop-out' of the helix instead). This seems to occur in order to ensure that the existing buried hydrophobic interface on one side of the helix is retained. A broad study revealed that, in over 200 different proteins, 70-80% of amino acid insertion mutants retained their biological activity essentially unchanged. In T4-1ysozyme, it appears that less than 50% of the amino acids are involved in determining the fold of the protein. Deliberately changing buried amino acids in T4 lysozyme - for example, by replacing a leucine with an alanine - led to the formation of a cavity in the protein, as revealed by X-ray crystallography. Although these induced cavities were not connected to the surface of the protein by channels, exogenous hydrophobic molecules, such as benzene, could diffuse rapidly through the protein to occupy the cavity. In addition, to some extent, the protein could adjust its conformation to accept a ligand that was slightly larger than the cavity. Such 'cavity mutants' are very useful for studying the stability and stabilization of proteins. Previous fears that many proteinengineering studies would prove to be inconclusive, by producing molecules that would not fold, appear to have been exaggerated. These concerns arose from the notion that even changing a single amino acid might precipitate catastrophic effects by preventing the formation of a key conformational intermediate on a unique 'folding

pathway'. Earlier evidence from work on the tailspike protein of phage P22 had indicated that this might be the case 2. More recently, evidence for highly localized, fast folding of a polypeptide chain, followed by its collapse to a compact, anhydrous but non-native 'molten globule' state, has become overwhelming (Robert Baldwin, Stanford University, CA, USA). The molten globule appears to be a mobile intermediate that can also be produced by denaturants, and may be determined by N M R measurements (Christopher Dobson, University of Oxford, Oxford, UK; Philip Evans, University o f Cambridge, Cambridge, UK). Sounding a note of caution that folding in vivo may differ from that which occurs in vitro, Georgc Lorimer (DuPont, Wilmington, DE, USA) described the properties of the chaperonin family of proteins, as exemplified by the E. coli protein GroEL. To what extent these proteins actively assist in folding, or passively prevent aggregation, remains unknown, as do the feature(s) recognized by chaperonins which induce their binding to unfolded proteins. The presence of microbial inclusion bodies docs not necessarily indicate that a given polypeptide chain is unable to fold per se. Catherine Schein (Federal Institute o f Technology, Zurich, Switzerland) answered the question shc posed her audience: 'must we live with inclusion bodies?' with the observation that, in many cases, 'probably not unless we want to' (in some cases, the formation of inclusion bodies is useful for aiding purification). She demonstrated that formation of inclusion bodies by 16 different recombinant proteins could be prevented or minimized by simple expedients, such as reducing the growth temperature o f the culture, or the rate of production o f intracellular protein (e.g. by using a weaker promoter to direct expression of the protein). In only a few cases would the amino acid sequence need to be changed to obtain folding.

meeting report

Designer antibodies The successful design and production of proteins that can bind TIBTECHAPRIL1993 (VOL 11)

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f o r131~l either small molecules, or macromolecules, was illustrated by the range of genetically engineered antibodies on show at the meeting. By taking full advantage o f the natural plasticity o f antibody-combining regions, protein engineers can tailor binding sites for therapeutic, diagnostic, or catalytic purposes. The naturally restricted overall conformations o f five of the six antibody complementarity-determining regions (CDRs) has led Tony Rees (University of Bath, Bath, UK) to devise computer programs that can predict the conformation o f C D R amino acid sequences to within, on average, an angstrom of crystallographically determined structures. The programs (now commercially available) can therefore be used in antibody design. Extending this approach of computer-aided modelling, C D R s and potential antigens of known 3-D structure can be accurately and rapidly 'docked' (as confirmed by a comparison of the predicted structures of complexes with those determined experimentally). A combination of site-directed mutagenesis and model building is now being used to engineer catalytic activity, or metal-binding (metal ions are often required as co-factors in biocatalysis), into antibodies (Steve Benkovic, Penn State University, PA, USA; T o n y Rces, University of Bath, UK). Chimaeric antibodyenzymes, or other bi-functional fusion molecules, were also described in many posters. Bridging the gap between antibodies designed by computer and proteins of practical use is the technology for introducing random mutations into the C D R s and displaying the resulting variants on the surface of recombinant bacteriophage (Greg Winter, MRC, Cambridge, UK). Selection of variant antibodies with appropriate binding properties, using immobilized ligands, provides a powerful and flexible way to identify desired properties of selectivity and binding affinity ('irrational' design). By arranging for recombination to occur between separate pools of recombinant antibody heavy and light chains, this process allows a simple type of 'affinity maturation' of the antibody molecules in vitro, analogous to the in vivo process. Random mutagenesis and screening protocols are also effective for increasing the stability of enzymes and enhancing their activity in TIBTECH APRIL 1993 (VOL 1 I)

organic solvents - a very desirable property for many industrial biocatalysts. Frances Arnold (CalTech, Pasadena, CA, USA) described sequential mutations in the protease subtilisin E, followed by enzymatic screening and selection. Several cycles of mutation and selection resulted in a variant protein with ten amino acid substitutions that was able to hydrolyse a peptide substrate 250-fold more efficiently in 60% dimethylformamide than the wildtype enzyme. The engineered protease could be used with esterified amino acids to carry out peptide synthesis in organic solvents. Although crystallographic analyses of enzymes that are particularly stable can often lead to inferences regarding the basis of increased protein stability, it remains difficult to make successful changes to a given enzyme to stabilize it in a 'rational' manner. At present a 'hybrid' approach of rational design, followed by localized random mutagenesis and selection for desired properties, offers the best approach overall. Functional and fashionable fusions The hybrid design approach described above was also applied to the production of fusion proteins and bifunctionat chimaeras. The creation o f a junction, or linker polypeptide, between two proteins or protein domains is unnatural and requires skill and judgement on the part of the protein designer to produce an amino acid sequence that is compatible with the intended function of the fusion, or chimaeric protein. At one extreme, the linker itself became the focus of experiments. This was seen in the use of surface loops of proteins - such as the coat proteins of bacteriophage, or (as in one poster demonstration by P. Martineau, Institut Pasteur, Paris, France) the E. coli maltose-binding protein - to display short, variable or random polypeptide sequences. In such experiments, either the binding properties of the displayed amino acid sequence, or the effect of the fusion partner on the conformation o f a given sequence was studied. This concept is analogous to the selection of antibodies by bacteriophage display. The technique was used by Jim Wells (Genentcch, S. San Francisco, CA, USA), to optimize the sequence of a linker peptide containing five random amino acids flanked by two

glycine residues (about 10 6 possible sequences) used to link human growth hormone (hGH) to the gene Ill protein of phage M13. The phage expressing the hGH-linker-protein IlI molecules were captured by immobilized hGH receptor (extracellular domain) and treated with a protease that had been engineered to cleave at very infrequent histidine-containing sequences. Rounds of screening and re-selection o f phage released by protease in this manner selected for hGH-peptide-linker sequences that contained histidine in previously unobserved amino acid contexts, but which were clearly substrates for the protease. From this single example of an important and rapidly developing methodology, one can predict that phage display will prove very valuable in 'designing and refining' polypeptides for other specific biological properties. An interesting possibility, suggested by Wells, might be the application of 'phage pressure' to select amino acid sequences that undergo particular post-translational modifications (such as accepting a phosphate group). Fusion proteins, or chimaeras, are also familiar in the form of recombinant toxins currently being evaluated as selective cytotoxic agents. Ira Pastan (National Cancer Institutc, NIH, Bethesda, MD, USA) reviewed his approaches to the design of anticancer drugs. A chimaeric immunotoxin, based upon the Pseudomonas exotoxin (PE), which arrests protein synthesis through irreversible ADPribosylation of target cell elongation factor 2, is entering clinical trials this year. The domain of PE that interacts with receptors on the surface of many cell types was replaced by a monoclonal antibody, B3, which reacts with many human cancers, apparently by targeting the sialyl Lewis x glycan (a sialyl lactose structure). As the bioactivity of PE is dependent on proteolysis and release of the PE catalytic domain from the binding and translocation domains, the recombinant toxin was expressed in a 'cleaved' form that did not require processing in the cell. The B3 antibody, expressed as a single-chain fusion, was then chemically linked to the modified toxin. Toxic conjugates with other specificities were created by exchanging the B3 antibody for anti-Erb-2 [a truncated form of the epidermal growth factor (EGF) receptor, commonly over-expressed in breast

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cancer]. Transforming growth factor tx (TGF-c¢) was also used as a cellbinding ligand, conjugated with PE to treat bladder cancer. As TGF-ci receptors are present on many cell types, specificity for the tumour was achieved by delivering the TGF-ix-PE recombinant toxin locally, rather than systemically. Interestingly, the cytotoxicity of recombinant PE toxins depends critically upon the presence of five C-terminal amino acids (Arg-GluAsp-Leu-Lys). This is a 'retention' signal sequence that is recognized by receptors in the rough endoplasmic reticulum (ER). The toxic catalytic unit appears to enter the cell cytoplasm through this organelle. By replacing the existing PE terminus by the sequence Lys-Asp-Glu-Leu, Ira Pastan's group achieved a 10- to 20fold increase in specific cytotoxic activity. Transposition of domains in chimaeras normally involves exchanging large segments of polypeptide. Dagmar P, inge (Brandeis University, MA, USA) described the transplantation of a short peptide (ten residues) from the 'active site' of human oq-antitrypsin to the structurally homologous region of the human cytokine interleukin 113 (IL-l[3). IL-113 has no known proteaseinhibitory activity. The chimaeric protein acted as an inhibitor of elastase and was cleaved at the same peptide bond as the native inhibitor. Other chimaeras were produced using inhibitory peptides from ovomucin and soybean trypsin inhibitor. All the chimaeras bound to IL-113 receptors, but not all elicited cellular responses (i.e. they could act as antagonists). Ringe thus raised the intriguing possibility that such chimaeras might be used as 'two headed molecules' to treat inflammatoryjoint disease where both IL-1 activity and proteases are involved. Designs for improved quality o f life The best 'designer proteins', from the point of view of a pharmaceutical company, are not proteins, but protein mimetics. Proteins provide problems when compared with conventional drugs because they cross mucosal membranes poorly, are rapidly metabolized in the body and may be antigenic. A recent successfnl approach has been to identify portions of macromolecules that are involved in an interaction (these are generally relatively small): build a

constrained peptide and test this as an antagonist and then synthesize a mimetic compound. This approach was exemplified by Mark Greene (University of Pennsylvania, PA, USA) in the design ofa non-peptide mimic of a constrained peptide analogne of an antibody CDFZ. The C D R inhibited the interaction of reovirus haemagglutinin with its cellular receptor molecule (CD4). The topic o f R N A antisense therapeutics was reviewed by Stanley Crooke (Isis Pharmaceuticals, Carlsbad, CA, USA). The early promise of these agents appears to be sustained in the laboratory, and perhaps soon in the clinic as well. The potency and selectivity of such drugs in vitro was not so surprising; perhaps more remarkable has been their ability to be well tolerated by animals, their excellent stability in t,iuo, and their ability to be transported across biological membranes. An extensive number of modifications to the chemistry of 'antisense' molecules have been reported; perhaps one of the most striking and relevant to this meeting was 'peptide nucleic acids' or PNAs, in which the normal ribose phosphodiester backbone is substituted by a polyamide (i.e. peptide) structure. The sequential positioning of the pendant bases is iso-steric with tCNA, and the duplex and triplex structures (with single- or doublestranded nucleic acids, respectively) are, if anything, more stable than when the corresponding nucleic acid molecules are used. Protein structure determination the key to successful design The detailed solution of the 3-D structures of pathogen-associated proteins can explain the basis of existing and therapeutic activity and sometimes suggest other effective agents. This may be the case for HIV-1 reverse transcriptase (Tom Steitz, Howard Hughes Medical Institute, Yale University, CT, USA), whose structure reveals a complex 'palm and fingers' which may 'grasp' the I K N A - D N A duplex as the D N A is constructed on its R N A template. A 'thumb-like' protrusion of the molecule, which may be induced to change its conformation by DNA, has, at its base, a binding pocket for the reverse transcriptase inhibitor, Nevirapine. Studies using this drug may help to resolve the catalytic mechanism of the reverse transcriptase and suggest further therapeutic approaches.

An analogous tale was told by Michael 1Kossman ( Purdue University, W. Lafayette, IN, USA) concerning the binding of human rhinoviruses to their receptors (the immunoglobulin-like ICAM-1 molecule). The ICAM-1 molecule, which has only a single chain, can enter a deep pocket - the 'canyon' formed in the proteins of the icosahedral virus coat. The conservation of residues in the canyon preserves the receptor-binding site, which is inaccessible to immunoglobulin, whilst amino acids that are accessible to immunoglobulin vary among different rhinoviruses. A number of anti-viral agents can bind in a pocket beneath the 'floor' of the canyon, raising the associated polypeptide chains. This appears to render ICAM-1 unable to bind to the vires, thus preventing infection. There is an intriguing indication that freshly isolated viruses may carry an endogenous ligand in this pocket, leading to speculation that an unknown cellular component may play a role in viral assembly or release. In any event, further study of this interaction may provide insights into the virus life-cycle and possibilities for therapeutic intervention. It was notable that all the three special award lectures at the meeting were delivered by foremost exponents of protein structure determination. Kurt Wtithrich (Federal Institute of Technology, Zurich, Switzerland) showed how nuclear magnetic resonance (NMR) spectroscopy was taking on the burden of producing structures of proteins and protein complexes in solution, and solving structures that still present a severe challenge to the X-ray crystallographer. The limits of protein size that can be tackled successfully by NM1K are being extended by developments in instrumentation and computational technology. David Phillips (Advisory Board of the Research Councils, London, UK), in a talk entitled 'Lysozyme Now', showed how his early conception of the way in which a glycosidic enzyme could catalyse a reaction through introducing bond strain in the bound substrate had been validated by recent structural studies. He also traced the consequences of sitedirected nmtagenesis upon various aspects of the catalytic reaction and, referring to most recent data, how natural mutations of amino acids in human lysozyme may lead to the formation of amyloid plaques. TIBTECH APRIL 1993 (VOL 11)

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forum The organization of the various sessions of the conference still permitted time to appreciate the surroundings and climate of Florida. In his colourful presentation o f the X-ray crystallographic structure of human rhinovirus, Michael Rossman drew attention to the 'Everglades

green' of the viral-protein subunits and the 'Atlantic blue' of the canyon in which ICAM-1 binds. The sessions closed and the delegates, mindful o f the canyon hypothesis, but not in the least fearful of the common cold, headed for the bars and the beach.

References 1 Chothia,C. (1992) Nature 357, 543 -544 2 Hasse-Pettingell, C. and King, J. (1988) J. Biol. Chem. 263, 4977- 4981 Michael J. Geisow Biodigm Consultancy, 64 Langdale Grove, Bingham, Nottingham, UK NG13 8SS.

Human gene therapy- progress on all fronts meeting report

Human genetic therapy has progressed from a distant prospect to reality in a very short space of time. Only four years ago, the first protocol for the treatment of adenosine deaminase (ADA) deficiency in humans was approved in the USA, and in 1990, gene therapy for ADA was first performed. Now, there are at least 11 approved and active protocols involving gene therapy in the USA and Europe. Gene therapy has been heralded as a means of treatment for genetic disorders and multi-factorial diseases (such as cancer and heart disease). In addition, much of the technology can also be used in new, targeted drug-delivery systems. A recent conference* covered a wide range of aspects concerning human somaticcell gene therapy, from the design of vectors to deliver the agents of gene therapy, to antisense technology, as well as the treatment of specific diseases such as cystic fibrosis (CF), familial hypercholesterolaemia (FH), and ADA. Efficacy and regulatory issues were also discussed. Bob Williamson (St Mary's Hospital Medical School, London, UK) introduced the concept and therapeutic potential ofgene therapy. He proposed that the real value of gene therapy lay, in the long term, with its application to the treatment of multifactorial disease, in which gene defects play some role in the disease state, but other factors, such as the environment, also play some part. Gene therapy can be described as an enabling technology, with the vectors, technology, and information on gene location and cloning derived *The mccting 'Human Genetic Therapy', organized by IBC Technical ServicesLtd, was held at the National Heart and Lung Institute, London, UK, 10-11 December, 1992. TIBTECHAPRIL1993 (VOL11)

from gene-therapy research programmes having much wider implications and uses than the treatment of the diseases for which the protocols were designed. The prerequisites for effective gene-therapy strategies for singlegene defects are the identification of the defective gene, functional complementation of the gene defect in vitro with a wild-type copy, and in vivo animal models of the disease and its correction or therapeutic amelioration. Using CF, ADA and FH as examples, the various targeting strategies employed in gene therapy were briefly discussed. Williamson gave an optimistic view of the future for gene therapy and predicted that it would rapidly become more widely available for the treatment of more diseases, and hence would benefit a larger proportion of the population. He added a note of caution, however, by reminding the conference that the development of new pharmacological approaches to the treatment of diseases should not be neglected. Some of these would undoubtedly come about as a result of an improved knowledge of gene and protein structure gleaned from research into gene therapy.

Retroviral vectors for targeted gene delivery R.etroviral vectors have been studied as potential vehicles for the delivery of genes to cells. They are suited to this purpose in that they can tolerate small inserts of heterologous DNA, target cells, elicit uptake into the cell and express the heterologous DNA. Hence, three of the major problems that hamper the development of gene-therapy protocols (i.e. cell targeting, entry and expression) may be surmounted in a single step.

Alan Kingsman (University of Oxford, Oxford, UK) gave a comprehensive summary o f the life cycle ofretroviruses and the ways in which they can be modified to deliver D N A to cells. However, as vector systems, retroviruses have limitations, including the requirement that infection of the target cells has to occur ex vivo (i.e. cells must be isolated from the individual, infected with retrovirus containing the correcting gene, and cultured before being returned to the patient). The infection titre of the virus particles is low - improvements in the packaging cell-lines have helped the situation, but this is still an area for further improvement. Longterm expression of heterologous D N A from the viral promoters is also poor, and integration of the virus into the genome of the host cell is random and cannot be controlled, thus raising the prospect of unintentional insertional mutagenesis o f the host-cell DNA. Celt specificity is also a problem as retroviruses only infect rapidly dividing cells; hence strategies involving retroviral vectors are not applicable to correcting defects in terminally differentiated cells. Despite these limitations, retroviral vectors are the most commonly used system for gene-therapy protocols to date, because retroviral biology is relatively simple when compared with other potential viral vectors, such as adenovirus or herpes virus. Walter H. Giinzburg (GSFMunich, Institute of Molecular Virology, Neuherberg, Germany) discussed the targeting of retroviral vectors to particular cell types by the use of regulatory elements and/or associated transcription factors to direct expression of heterologous genes carried by the retroviral vector exclusively to the desired cell type. The promoter of the whey acidic protein (WAP) gene, which is ex-

© 1993, ElsevierSciencePublishers Ltd (UK)