Selection of biologically active peptides by phage display of random peptide libraries

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Selection of biologically active peptides by phage display of random peptide libraries Riccardo Cortese*t, Paolo Monaci*, Alessandra Luzzago*, Claudia Santini*, Fabrizia Bartoli*, Irene Cortese$, Paola Fortugno*, Giovanni Galfr@*, Alfredo Nicosia* and Franco Felici*

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the

identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.

Addresses * Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina km 30.600, 00040 Pomezia (Roma), Italy $ Clinica Neurologica, Universit&di Roma 'Tor Vergata' and Clinica S. Lucia, Via Ardeatina 306, 00179 Roma, Italy t e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:616-621 © Current Biology Ltd ISSN O958-1669 Abbreviations Cryoll EPO

type II mixed cryoglobulinemia cerebrospinalfluid erythropoietin

EPOr

erythropoietinreceptor

HCV MS

hepatitis C virus multiple sclerosis randompeptide library Src homology 3

CSF

RPL SH3

Introduction

Phage M13 and other members of the filamentous phage family have been used as expression vectors in which foreign gene products are fused to the phage coat proteins and are displayed on the surface of the phage particle. Many proteins or peptides have been successfully displayed on phage as have complex random peptide libraries (RPLs). Phage particles displaying the desired ligand can be isolated by affinity chromatography or panning on immobilized selector molecules. Selectors that have been used include antibodies, membrane receptors, enzymes, cultured cells, serum samples, and even the whole animal. Numerous papers have appeared during the past year that describe successful applications of RPLs displayed on phage: we have restricted this review to a small subset of the publications that contain or lead to novel applications and/or novel methods of selection.

Preparation

of libraries

The probability of finding a ligand in an RPL is a function of its affinity for the selector molecule, as well as its frequency in the library. The latter can be maximized by constructing libraries with great sequence diversity and insert flexibility, composed of longer, unconstrained inserts capable of making many contacts with the ligate through induced fit. In contrast, libraries that potentially contain higher affinity ligands are those in which more specific structural information has been introduced in the form of constraints like disulfide bridges [1-5] or molecular scaffolds of predefined structure [6-9]. By using seventeen different monoclonal antibodies for screening eleven libraries with and without the potential to display the foreign peptides as disulfide bridged loops, Bonnycastle et al. [5] have concluded that each antibody has an unpredictable preference for a specific type of constraint or no constraint at all. Therefore, the chance of identifying good ligands increases by screening more libraries. If diversity between the selected sequences is desired, each library needs to be surveyed separately, although best binders can be selected from a pool of different repertoires. Most of the RPLs are constituted by no more than 107-109 independent clones, due to the relatively low efficiency of bacterial transformation. Such complexity theoretically covers all the possible sequences of 5-7 amino acid length peptides, but is incomplete in the case of longer peptides. Despite this, phage-displayed peptide libraries of 107-109 complexity have been successfully used for the selection of functional mimics for many different ligands. This simply means that for most ligand-ligate complexes, few critical amino acid residues at the interface of interaction are enough to provide sufficient stability to the complex. The complexity limit due to DNA transformation efficiency can be overcome by combinatorial approaches. Fisch and co-workers [10] have used a ribozyme+ recombination method (using self-splicing group I intron with inserted lox-Cre site) to assemble a phage-displayed peptide library of more than 1011 members. However, an upper limit to the complexity of phage-displayed libraries is set by the amount of reagents needed in the selection protocols: in practice, libraries with more than 10 le clones would be difficult to use.

Selection of biologically active peptides by phage display of random peptide libraries Cortese et

Stepwise approaches to increase library complexity seem more promising, generating diversity through iterative mutagenesis and selection, mimicking the natural process of biological evolution [11]. An in vivo protocol for the affinity maturation of phage-displayed antibodies utilizing a bacterial mutator strain has been described [12]. In vitro site-directed mutagenesis [13,14] or error-prone PCR [15] have already been used for the affinity maturation of selected peptides. This kind of 'benchtop evolution' allows the generation of libraries of increasingly large complexity, through cycles of production and selection of millions of mutants, and is clearly a major advantage of phage display technology over any other combinatorial library approach. M a p p i n g critical r e s i d u e s at t h e i n t e r f a c e of protein-protein complexes Phage display technology has recently become of broad use for the identification and characterization of contact sites on either side of a protein-protein interface. The role of conformational constraints is highlighted by the results of Koivunen et al. [16]. For this work, several libraries were used in which the random sequence was flanked by one or two cysteine residues, with the general formula CXsC, CX6C, CX7C or CX9, that were aiming to isolate cyclic peptides binding to integrins, while selecting for the optimal ring size at the same time. Each of the four integrins primarily selected RGD-containing peptides (single letter amino acid code), but favoured different ring sizes and flanking residues around the RGD sequence. Removal of the constraints invariably led to a dramatic decrease in binding affinity. In a reverse strategy, phage-displayed peptides binding to RGD-containing fibronectin fragments [17] were isolated. A large number of clones shared the consensus sequence CWDDG/LWLC. It is worth noting that this sequence is similar to the sequence M T S D D L of the peptide binding to the RGD-site on the adenovirus penton selected by Hong and Boulanger [18]. Both groups suggest that these phage-displayed peptides mimic the sequence K D D L W present in the integrin 133 subunit, and included in a 133-integrin fragment previously isolated by RGD cross-linking [19]. Thus, phage selection led to further refinement in the identification of critical residues at the interface between integrins and RGD-containing proteins, and is likely to provide invaluable information for the design of integrin specific inhibitors. Hong and Boulanger [18] have proposed an interesting use for RPLs in mapping protein-protein interactions occurring during adenovirus type 2 attachment and cellular uptake. Phage clones bearing pill-displayed hexapeptide inserts, were selected with wild-type or mutant penton capsomers. On the basis of sequence similarities between the selected peptides and either viral capsid proteins or cell-surface receptors sequences, the authors suggest a molecular definition of interacting sites on the penton

al,

617

capsomer and on proteins involved in receptor or adhesion functions, as well as mechanisms of ligand exchange occurring between viral entry and release. Further validation of the use of phage-displayed RPLs to map amino acid residues at the interface of protein-protein complexes comes from the study of DeLeo et al. [20], on the assembly of N A D P H oxidase. This multimeric protein is formed by flavocytochrome b, located on the plasma membrane and composed of two subunits, gp91-phox and p22-phox, and at least another subunit, p47-phox, which is normally located in the cytosol, but moves to the membrane following cell activation. The subunit p47-phox was used to select peptides from phage-displayed RPLs. T h e selected peptides can be grouped into four families, according to their amino acid sequence. A comparison of these consensus sequences with the sequences of gp91-phox and p22-phox readily reveals regions of homology, thus allowing the mapping of interacting sites. Some of the synthetic peptides corresponding to these regions proved to be inhibitory to the in vitro assembly of the NADPI-I oxidase complex. A very thorough and profound study is the isolation of small cyclic peptides mimicking the functional properties of erythropoietin (EPO). Wrighton et al. [21 °°] selected an eight amino acid cyclic peptide binding to a soluble recombinant erythropoietin receptor (EPOr) from a pVIlI-displayed RPL. However, the same structure, as a free soluble cyclic peptide, had a rather low affinity for the EPOr. In order to be able to identify peptides with higher affinity, a second library was constructed, containing the mutagenized core of the selected peptide flanked by additional random residues in order to expand the epitope. This library was expressed as a pill fusion, which, by displaying fewer copies of peptide, might provide for a higher affinity threshold for selection. In this way, longer cyclic peptides with 10-50 times higher affinity to EPOr were isolated. Some of these peptides were further studied in a series of biological assays, both in vitro and in vivo, with the conclusion that they are truly EPO mimetics, albeit with lower affinity (nanomolar versus picomolar): they induce receptor dimerization, transmission of the signal, promotion of growth and differentiation, and stimulate reticulocyte counts in vivo. The amino acid sequence of these peptides is unrelated to that of EPO. T h e molecular mechanism responsible for receptor activation is the peptide-induced dimerization of the EPOr. In an accompanying paper [22], the 3D structure of the complex between one of the EPO mimetic peptides and the EPOr was determined. T h e quaternary structure of the complex is composed of two peptides and two receptors that form a T-shaped assembly: the peptide dimer interacts with the receptor, generating a twofold symmetrical arrangement, quite different from the asymmetrical structure of the structurally related growth hormone receptor complex [23]. These papers break new ground, not only validating the approach of phage

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display for the selection of biologically active peptides, but demonstrating the feasibility of the design of relatively small molecular weight analogs of protein hormones, thus opening the way to an entire new class of therapeutics. L-peptides as such are not usually considered to be ideal as drugs, because they have unsatisfactory pharmacokinetic properties, and are usually rather immunogenic. In this respect, D-peptides are a better starting point for the development of drugs, because they are not degraded by cellular enzymes and are not efficiently processed and presented as antigens. The recent synthesis of D-enantiomeric proteins has allowed the validation of the prediction that D-enantiomers should have substrate specificity and structure that mirrors that of naturally occurring l.-protein. From this premise, Schumacher et al. [24] reasoned that a D-protein used as a receptor should be able to isolate L-peptides from phage libraries, whose D-enantiomers should bind the natural protein. Using a synthetic D-amino acid chicken cSrc SH3 domain as selector molecule (SH3, Src homology 3), these authors isolated several phage-displaying cyclic peptides, with no sequence homology to the well-known SH3 domain ligands. As predicted, following the conversion of the cyclic peptide L-amino-acid sequence into the D-amino-acid sequence generated D-amino-acid peptides that bound specifically to the l.-form of the SH3 domain, with an affinity (64~M) comparable to that of SH3 natural ligands. The interaction of the D-peptide with the SH3 domain appears to involve part of the same SH3 site contacted by polyproline natural ligands and immediately st, ggests how to expand the contact points in order to increase affinity. It remains to be established whether the results obtained with the SH3 domain can be generalized. In any case, this is an ingenious strategy that considerably extends the scope of peptide selection from RPL displayed on phage.

Identification and characterization of disease-specific antigen mimics Based on a substantial body of evidence, the likelihood of success in the selection of phage-displayed peptides with purified and homogeneous ligates is rather high and largely depends on the quality of the RPL and, occasionall3, a d hoc modifications of the selection protocol. It has been possible to extend this technology to selection protocols using complex ligate mixtures, such as whole serum or other body fluids. In a set of experiments, we have applied the phage-display technology to select phage peptides that bind to antibodies present in the serum of patients affected by a certain disease but not to antibodies present in the serum of control individuals. The significance of the putative disease-specific phage peptides is established according to various criteria. Prezzi et al. [25] used as selectors a set of serum samples obtained from patients infected with

human hepatitis C virus (HCV), and identified phage that were able to react with several patients' sera but did not react with a set of control sera. In addition, the phage-displayed peptides, used as immunogens, were able to elicit in rabbits antibodies capable of recognizing several HCV proteins. These results suggest that selections using sera from patients who have successfully cleared an infection could be an effective way to identify peptides with the potential to elicit neutralizing antibodies. In general, during the onset of several diseases, there is the concomitant appearance of novel antibodies, reflecting the exposure to novel antigens or the perturbation of the antibody network. An analysis of the disease-specific B-cell response is often difficult and incomplete if the pathological antigen is not known. However, phagedisplayed RPLs can be used as a surrogate source of antigens, in order to identify peptides that react specifically with disease-related antibodies present in the serum of patients. For instance, Mecchia et al. [26] used phage-displayed RPLs to identify autoantigens characteristic of type II mixed cryoglobulinemia, an autoimmune disorder frequently associated with HCV infection (CryoII/HCV). IgM purified from the cryoprecipitate of patients with CryoII/HCV selected a dominant population of clones sharing the consensus sequence HPLAP (single letter amino acid code). Screening of a protein database revealed that HPLAP is present in an already cloned protein, LAG-3 [27]. It was then shown that anti-HPLAP antibodies, as well as HPLAP-immunopurified IgM, react with LAG-3 fragments, strongly suggesting that the natural target of the CryoII/HCV IgM mimicked by the HPLAP peptides is an exposed epitope of the LAG-3 protein. In the case of type I diabetes, the selection of RPLs using sera from pre-diabetic patients identified several clones showing statistically significant reactivity with diabetic sera when compared with control sera [28,29]. When these phage peptides were used as immunogens in rabbits, they elicited antibodies that were able to stain pancreatic islets, thus suggesting that the phage peptides mimicked diabetes-related self antigen. In the case of multiple sclerosis (MS), in which there is extensive demyelinization of white matter, the cerebrospinal fluid (CSF) is characterized by the presence of oligoclonal immunoglobulins, whose origin and pathogenetic relevance is still unclear. Using the procedures outlined above, Cortese et al. [30] isolated several peptides specifically recognized by antibodies enriched in the CSF of MS patients. With these 'specific' reagents, it was possible to establish three things: that antibodies with the same specificity are present both in the CSF and in the serum of the same patient; that these antibodies are directed against rather ubiquitous antigens; and that each MS patient has a different and characteristic set of intrathecally produced antibodies in the CSE

Selection of biologicallyactive peptides by phage display of random peptide libraries Cortese et a/.

S e l e c t i o n of cell- a n d o r g a n - s p e c i f i c p e p t i d e s T h e selection of ligands for cell-surface receptors has been approached by using whole cells as an affinity matrix. This approach has the great advantage that receptors are more likely to be in their native conformations, with all their natural post-translational modifications, and that neither purification nor prior knowledge of a particular target receptor is required. Activated platelets were used to select cell-binding phage from an RPL [31]. A series of peptide motifs, including the already known RGD motif, that inhibit platelet aggregation were identified. More recently, Barry et al. [32] used RPLs to select peptides that bind to and enter several different cell types. Though very preliminary, this paper reports some observations that should be taken into account for a more systematic search of peptides binding to cells. It appears that the binding of phage to cells is extremely strong and that only non-specific phage are recovered with the usual acid elution step. In addition, higher-affinity phage peptides were selected from libraries displaying longer peptides (20 amino acids versus 12 amino acids). In this report, the isolated phage peptides bound to a variety of cell types, suggesting that they are ligands of rather ubiquitous receptors. In this respect, major progress will be achieved when efficient protocols for the depletion of phages binding to these ubiquitous receptors are developed. It has been known for some time that organ-specific molecules on endothelial surfaces direct the homing of lymphocytes and tumor cells. In a recent and innovative paper, Pasqualini and Ruoslahti [33 °] demonstrate that the identification of such specific endothelial markers can be approached by the in vivo screening of random peptide libraries displayed on phage. Phage-displayed cyclic peptide libraries were injected intravenously into mice and, after a few minutes, phage were rescued from individual organs and amplified in vitro. By multiple rounds of such in vivo panning, peptides selectively targeting phage to brain and kidney blood vessels were identified. A selectivity for these organs of up to ninefold was demonstrated. T h e same peptides coupled to the surface of red blood cells mediated selective localization of intravenously injected cells into the brain. Surprisingly, no peptide with an R G D sequence was isolated, despite the facility with which such peptides have been isolated using purified RGD-binding receptors. Peptides selected according to any of the above described procedures that specifically bind to cancer cells, tumors or other diseased tissues could be widely used in diagnostic imaging or exploited for targeted drug delivery. In addition, cell- or organ-specific peptides could be grafted on to the surface molecules of viruses, cells or could simply be conjugated to DNA for use in gene therapy.

N e w v e c t o r s for p e p t i d e / p r o t e i n d i s p l a y In addition to the most widely utilized plII and pVIII proteins, the minor coat protein VI of filamentous

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phage has also been used to display foreign proteins fused at its carboxyl terminus in a monovalent display system [34]. T h e pVl system may be more suitable for cDNA expression and in those cases in which a free carboxy-terminal end is necessary for proper protein folding or activity. Besides the intrinsic limitations of each of these display systems based on filamentous phage, common restrictions include their inability to display proteins toxic to bacterial cells and the feature that the fusion protein must be secreted across the bacterial membrane. An alternative system to alleviate the toxicity problem is represented by the use of bacteriophage ~,, in which the synthesis of any toxic protein can be repressed during the lysogenic state and induced for a very short time just before cell lysis. Furthermore, protein secretion is not required for phage ~. display because this phage assembles intracellularly. Maruyama et al. [35] demonstrated that foreign proteins can be displayed on the ~. capsid through fusion to the carboxyl terminus of a truncated form of pV, a phage tail protein. Protein D, an 11 kDa protein decorating the ~. viral capsid in 405 copies has been demonstrated to tolerate amino-terminal insertions of peptides and protein domains accessible to ligand interaction, thereby making this vector suitable for affinity selection [36]. In this system, the introduction of a loxP site allows the in vivo incorporation of the plasmid expressing the D fusion protein into the viral genome. Another surface-display vector where displayed proteins are not secreted is the bacteriophage T4, where foreign polypeptides can be exposed by fusion with the carboxyl terminus of fibritin, a 487 amino acid residue fibrous protein building the collar-whiskers complex of the bacteriophage neck [37]. By homologous recombination with a plasmid containing the modified gene, it is possible to obtain phage preparations with all fibritin copies exposing the polypeptide. T h e psu protein decorating the mature P4 capsid has also been proposed for peptide presentation, although, in this case, because psu can dissociate from the phage particle and is nonessential for phage propagation, it remains to be established whether this type of fusion protein is efficiently retained on the phage capsid and can be affinity selected [38]. A variety of eukaryotic viruses have also been proposed for the display of foreign peptides. Most of these vectors have been exploited for the expression of peptides of potential interest in vaccine design. Examples of these are the tobacco mosaic virus, in which Plasmodium rnalariae epitopes were displayed both on the surface loop region and at the carboxyl terminus of the coat protein [39] and

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human rhinovirus 14, on whose surface a library of variants of the V3 loop of HIV type 1 has been displayed. This was efficiently selected with monoclonal antibodies, leading to the discovery of unknown variants with novel antigenic and immunogenic properties [40]. The eukaryotic virus display of complex proteins has been successfully achieved using the baculovirus virion surface glycoprotein gp64 [41]. Glutathione S-transferase and the HIV major surface glycoprotein gpl20 fused to the amino terminus of gp64 have been efficiently displayed on the Baculoviral surface. In addition, displayed gpl20 is properly folded because it is capable of binding to CD4.

4.

Hoess RH, Mack AJ, Walton H, Reilly TM: Identification of a structural epitope by using a peptide library displayed on filamentous bacteriophage. J Immuno/1994, 153:724-729.

5.

Bonnycastle LLC, Mehroke JS, Rashed M, Gong X, Scott JK: Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage. J Mol Biol 1996, 258:747-762.

6.

Martin F, Toniatti C, Salvati AL, Venturini S, Ciliberto G, Cortese R, Sollazzo M: The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6. EMBO J 1994, 13:5303-5309.

7.

McConnell S, Hoess RH: Tendamistat as a scaffold for conformationally constrained phage peptide libraries. J Mo/ Bio11995, 250:460-470.

8.

Bianchi E, Folgori A, Wallace A, Nicotra M, Acali S, Phalipon A, Barbato G, Bazzo R, Cortese R, Felici F, Pessi A: A conformationelly homogenous combinatorial peptide library. J Mo/Bio11995, 247:154-160.

Conclusions The papers that have appeared during the past year convincingly indicate that phage-display technology is becoming a tool for many purposes, ranging from protein engineering to gene therapy. The selection protocols developed in many labs are very simple, requiring only easily acquired familiarity with basic microbiological manipulations. For this reason, their use has become widespread, and has already yielded relevant results in many fields. Currently, the main limitation is still the relatively low complexity of the RPLs, but there are already ingenious ideas for improvement that will be further developed: in particular, we anticipate the further refinement of mutagenesis methods that will have to be incorporated very early in the selection protocols. Furthermore, there is still an unfulfilled need for effective methods of depletion of unspecific or unwanted phage peptides. Finally, the use of new vectors, such as bacteriophage ~., will strongly improve the efficacy of displaying cDNA libraries, thus allowing the incorporation of selection methods into the current strategies of gene cloning.

Acknowledgements

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lO.

Fisch I, Kontermann RE, Finnern R, Hartley O, Soler-Gonzalez AS, Griffiths AD, Winter G: A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage. Proc Nat/Acad Sci USA 1996, 93:7761-7766.

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Stemmer WPC: Searching sequence space. Using recombination to search more efficiently and thoroughly instead of making bigger combinatorial libraries. Bio-Technology 1995, 13:549-553.

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Low NM, Holliger P, Winter G: Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J Mo/Bio/ 1996, 260:359-368.

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Rickles RJ, Boffield MC, Zhou X-M, Henry PA, Brugge JS, Zoller MJ: Phage display selection of ligand residues important for Src homology 3 domain binding specificity. Proc Nat/Acad Sci USA 1995, 92:10909-10913.

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Yu J, Smith GP: Affinity maturation of phage-displayed peptide ligands. Methods Enzymo11996, 267:3-27.

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Koivunen E, Wang B, Ruoslahti E: Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Bio-Technology 1995, 13:265-270.

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Pasqualini R, Koivunen E, Ruoslahti E: A peptide isolation from phage display libraries is a structural end functional mimic of an RGD-binding site on integrins. J Ceil Bio/1995, 130:1189-1196.

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Hong SS, Boulanger P: Protein ligends of the human adenovirus type 2 outer capsid identified by biopanning of a phage-displayed peptide library on separate domains of wild-type and mutant penton capsomers. EMBO J 1995, 14:4714-4727

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21.

Wrighton NC, Farrell FX, Chang R, Kashyap AK, Barbone FP, Mulcahy LS, Johnson DL, Barrett RW, Jolliffe LK, Dower WJ: Small peptides as potent mimetics of the protein hormone erythropoietin. Science 1996, 273:458-463.

Part of this work was supported by a grant from the Associazione Italiana Sclerosi Muhipla, Genoa, Italy.

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Nord K, Nilsson J, Nilsson B, Uhlen M, Nygren P-A: A combinatorial library of an cx-helical bacterial receptor domain. Protein Eng 1995, 8:601-608.

ee

Selection of biologically active pepUdes by phage display of random peptide libraries Cortese et aL

This paper describes the discovery of a 14-mer peptide capable of mimicking the biological properties of erythropoietin. This demonstrates the feasibility of substituting polypeptide hormones with small molecular weight synthetic compounds, thus opening up a new field of important applications, 22.

Livnah O, Stura EA, Johnson DL, Middleton SA, Mulcahy LS, Wrighton NC, Dower WJ, Jolliffe LK, Wilson IA: Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8A. Science 1996, 273:464-471.

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Schumacher TNM, Mayr LM, Minor DL Jr, Milhollen MA, Burgess MW, Kim PS: Identification of D-peptide ligand through mirrorimage phage display. Science 1996, 271:1854-1857.

25.

Prezzi C, Nuzzo M, Meola A, Delmastro P, Galfre G, Cortese R, Nicosia A, Monaci P: Selection of antigenic and immunogenic mimics of hepatitis C virus using seta from patients. J Immuno/ 1996, 156:4504-4513.

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Barry MA, Dower WJ, Johnston SA: Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat Med 1996, 2:299-305.

33. PasqualiniR, Ruoslahti E: Organ targeting in vivo using phage • display peptide libraries. Nature 1996, 380:364-366. This paper describes a very original method of ligand selection. The RPL is injected as a phage suspension into a living organism and allowed to circulate in the bloodstream. In this way, phage displaying peptides with affinity for certain districts of the body are isolated. With further refinements, this technique might lead to the identification of peptides important for cancer therapy. 34.

Jespers LS, Messens fill, De Keyser A, Eeckhout D, Van den Brande I, Gansemans YG, Lauwereys MJ, Vlasuk GP, Stanssens PE: Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI. Bio-Technology 1995, 13:378-382.

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Mecchia M, Casato M, Taft R, Filocamo G, Bonomo L, Fiorilli M, Cortese R, Migliaccio G, Nicosia A: Non rheumatoid IgM in HCV-associated type II cryoglobulinemia recognise mimotopes of the CD4-1ike LAG-3 protein. J Imrnuno/1996, in press.

Maruyama IN, Maruyama HI, Brenner S: )~foo: a ~. phage vector for the expression of foreign proteins. Proc Nat/Acad Sci USA 1994, 91:8273-8277.

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Triebel F, Jitsukawa S, Baixeras E, Roman-Roman S, Genevee C, Viergas-Pequignot E, Hercend T: LAG-3, a novel lymphocyte activation gene closely related to CD4. J Exp Med 1990, 171:1393-1405.

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Turpen TH, Reinl SJ, Charoenvit Y, Hoffman SL, Fallarme V, Grill LK: Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Bio-Technology 1995, 13:53-57.

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Resnick DA, Smith AD, Geisler SC, Zhang A, Arnold E, Arnold GF: Chimeras from a human rhinovirus 14-human immunodeficiency virus type 1 (HIV-1) V3 loop seroprevalence library induce neutralizing responses against HIV-I. J Viro/ 1995, 69:2406-2411.

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Boublik Y, Di Bonito P, Jones IM: Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface. Bio-Techno/ogy 1995, 13:1079-1084.

Mennuni C, Santini C, Dotta F, Farilla L, DiMario U, Fierabracci A, Bottazzo G, Cortese R, Luzzago A: Selection of phage-displayed peptides mimicking Type 1 diabetes-specific epitopes. J Autoimmun 1996, 9:431-436. Fierabracci A, Biro PA, Yiangou Y, Mennuni C, Luzzago A, Cortese R, Bottazzo GF: A novel approach to the aetiopathogenesis of human autoimmune diseases: the use of random peptide phage libraries in the search for disease-related epitopes. J Immunol 1996, in press. Cortese I, Taft R, Grimaldi LME, Martino G, Nicosia A, Cortese R: Identification of peptides specific for CSF antibodies in multiple sclerosis using phage libraries. Proc Nat/Acad Sci USA 1996, in press. Fong S, Doyle LV, Devlin JJ, Doyle MV: Scanning whole cells with phage-display libraries: identification of peptide ligands that modulate cell function. Drug Dev Res 1994, 33:64-70.