In vivo selection of sFv from phage display libraries

Journal of Immunological Methods 239 (2000) 137–151www.elsevier.nl / locate / jim

Recombinant Technology

In vivo selection of sFv from phage display librariesa a a ,*Michael Johns , Andrew J.T. George , Mary A. Ritter

aDepartment of Immunology, Division of Medicine, Commonwealth Building, Imperial College School of Medicine,Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK

Received 11 June 1999; received in revised form 21 December 1999; accepted 5 January 2000

Abstract

The development of phage display technology has facilitated the development of many new and sometimes novel antibodybased reagents for scientific research. However, present methods for selection from phage-sFv display libraries are limited toselection against purified antigens or ex vivo cells of known origin and phenotype. Existing methods therefore preclude theisolation of sFv against unknown molecules in their natural environment, where expression is complex and subject to diversecontrol mechanisms. Since such a complex environment is difficult to mimic in vitro, the development of an in vivo selectionprocedure would greatly enhance the selection from phage display antibody libraries and lead to the development of reagentsagainst cell surface molecules in their natural environment. This would be particularly advantageous for isolation of sFvagainst vascular endothelium which can readily change phenotype when cultured and is believed to express molecules in atissue specific manner and in response to different stimuli. We describe here the development of an in vivo selectionprocedure in the mouse and demonstrate its potential for the selection of sFv from a phage-sFv library. The target antigen forone sFv is expressed solely on the thymic endothelium, while the second, a 165–170 kDa molecule in present on boththymic endothelium and the perivascular epithelium. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Phage display; In vivo selection; Thymus

bacteriophages (Barbas et al., 1991; Hoogenboom1. Introductionand Winter, 1992; Griffiths et al., 1994; Nissim etal., 1994; de Kruif et al., 1995a). In parallel to theThe utilisation of phages as vehicles on which todevelopment of phage display technology, has beenexpress recombinant proteins has led to the develop-the development of a variety of selection methodsment of extensive libraries of antibody fragments, infrom such libraries. Initially, methods were based onparticular Fab or sFv, expressed on the surface ofthe immobilisation of known antigens on a solid

Abbreviations: IPTG, Isopropyl-b-d-thiogalactopyranoside; matrix followed by several rounds of selection byMPBS, Low fat milk powder in phosphate buffered saline; NMS, panning and amplification of selected phage-sFvNormal mouse serum; TMB, 3,39,559-Tetramethylbenzidine; tu, (McCafferty et al., 1990; Marks et al., 1991). ThisTransforming units; sFv, Single chain antibody

method requires a readily available source of purified*Corresponding author. Tel.: 144-208-383-2089; fax: 144-antigen which, in some circumstances, can be a208-383-2786.

E-mail address: [email protected] (M.A. Ritter) problem. The lack of available antigen has led more

0022-1759/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-1759( 00 )00152-6

138 M. Johns et al. / Journal of Immunological Methods 239 (2000) 137 –151

recently to the development of cell based methods specific molecular differences. In vivo methods have,(de Kruif et al., 1995b; Palmer et al., 1997; Siegal et so far, only been described for selection of peptidesal., 1997). Such methods rely either on the existence from phage display libraries (Rajotte et al., 1998;of a cell line or readily available tissues from which Pasqualini and Ruoslahti, 1996) and peptides iso-the cells of interest can be obtained, but do not lated have included those containing integrin bindingrequire purified antigen. motifs (Arap et al., 1998). While this in vivo

Selection of antibodies against the vascular endo- technique has successfully identified organ specificthelium presents a particularly difficult problem. differences in the vascular endothelium (Rajotte etFirstly, it is hard to isolate fresh endothelial cells al., 1998), most peptides (with the exception ofwith intact surface molecules and unchanged pheno- known recognition motifs such as RGD for integrins)type from any tissue. Secondly, in culture, endotheli- will have a relatively low affinity for their target. Weal cells readily change their phenotype thus making have therefore developed an alternative approach,the production of stable cell lines difficult (Borsum taking advantage of the ability of an antibody to bindet al., 1982). Furthermore, the different endothelial its target epitope with high affinity. The immunologi-cell lines that do exist express different levels of cal importance and discrete anatomical structure ofmany common molecules (Mutin et al., 1997) and the thymus provides an excellent model for theare unlikely to be entirely representative of any development of an in vivo phage-sFv display selec-normal in vivo cell counterpart. The above factors, tion procedure. We therefore now describe the de-and the supposition that some endothelial molecules velopment, monitoring and results of a novel methodmay be expressed in a site specific manner (see for the selection of sFv from a phage display librarybelow), make the use of endothelial cell lines a poor against the murine thymus. Selected phage have beencandidate for selection of antibodies from phage shown to bind specifically to the thymic vasculardisplay libraries. endothelium and perivascular epithelium by tissue

Differential expression of vascular addressins on staining.the endothelium is known to be responsible for thehoming of lymphocytes to defined locations aroundthe body including the gut and sites of inflammation 2. Materials and methods(Butcher, 1996; Kraal and Mebius, 1996). Forexample, the expression of the mucosal vascular 2.1. Phage libraries and bacterial strainsaddressin MAdCAM-1 guides lymphocytes express-ing a4b7 to gut-associated lymphoid tissues in a The Nissim semi-synthetic phage-sFv display li-specific manner, while VCAM-1, expressed at sites brary was used in these studies (Nissim et al., 1994).of inflammation, is the ligand for a4b1 on activated The library was constructed by PCR of V regionH

lymphocytes (Berlin et al., 1995). In a similar genes with family specific 59 and degenerate 39

fashion, during development of the immune system, primers used to introduce novel synthetic CDR3stem cells migrate from the blood into the thymus at regions of 4–12 amino acids in length. All V genesH

specific time points between which the organ is have been cloned upstream of the Vl3 light chain inrefractory to stem cell entry (Jotereau et al., 1980; the pHEN1 vector (Hoogenboom et al., 1991). TheFontaine-Perus et al., 1981; Jotereau and Le- light chain gene is separated from an amber stopDouarin, 1982; Coltey et al., 1989). This migration codon by a c-myc tag with the result that all sFv aremust involve interaction of the stem cell with the produced with a c-myc tag attached to the lightthymic vascular endothelium in an as yet unknown chain. The tag can be recognised by the monoclonalmanner (Coltey et al., 1987). antibody 9E10, thus allowing the detection of sFv.

The use of phage display technology in combina- The vector contains both the E. coli and M13 originstion with a novel in vivo selection procedure offers a of replication permitting propagation as either plas-way to directly isolate recombinant antibodies to the mid or phage.vascular endothelium in situ and at specific time The following strains of bacteria were used in thispoints during development, and thus elucidate stage- study:

M. Johns et al. / Journal of Immunological Methods 239 (2000) 137 –151 139

2.4. Radiolabelling phage-sFv• E. coli TG1 suppresser strain (K12, D(lac–pro),supE, thi, hsdD5/F9traD36, proA1B1, lacIq,

12 125Phage (1.8310 ) were labelled with 240 mCi IlacZDM15) (Gibson, 1984)(Amersham, Little Chalfont, UK) for 10 min using• E. coli HB2151 non-suppresser strain (K12, ara,the iodogen method (Pierce, Rockford, IL, USA).D(lac–pro), thi /F9proA1B1, lacIqZDM15) (Car-Excess free nucleotide was removed by extensiveter et al., 1985)dialysis against PBS. After dialysis was complete,

11the labelled phage were diluted to 6310 phage/ml2.2. Growth and purification of phage sFvin PBS.

The phage library was propagated as a phagemidin E. coli TG1 bacteria and the phage-sFv particles 2.5. In vivo selection of phage-sFvrescued by the addition of VCS-M13 helper phage(Stratagene, La Jolla, CA, USA). Phage-sFv were

Mice were injected intravenously with 100 ml ofpurified from the culture supernatant by precipitation 12phage-sFv in PBS (1.8310 transforming units (tu) /with 20% PEG–2.5 M NaCl and were resuspended 11ml for round 3 and 9.9310 tu /ml for round 4).in a final volume of 1.5 ml sterile cold PBS.

The appropriate organs were harvested after 2 hBacterial debris was removed by centrifugation at

(optimal time after injection; discussed later) and the11 500 g for 10 min and the final preparation filter

whole organ washed twice in 1 ml of ice-cold PBS.sterilised using 0.45-mM sterile filters (Sartorius,

The tissue was teased out using 19G needles and¨Gottingen, Germany). Phage-sFv were prepared

washed five times with 1.5 ml ice-cold PBS. Boundfresh for each round of selection.

phage sFv were eluted using 0.5 ml 0.1 M glycine,pH 2.8, for 5 min at room temperature. Elution was

2.3. Phage-sFv detection ELISArepeated three times (rounds 1, 2, 3) or four times(round 4) to ensure recovery of all bound phage-sFv.

Maxisorb plates (96 well, Nunc, Roskilde, Den-A 150-ml volume of 1 M Tris, pH 8.8, was added

mark) were coated overnight, at 48C, with 10 mg/mlimmediately to each eluate to neutralise it. A 500-ml

9E10 antibody in 100 mM NaHCO buffer, pH 9.6.3 volume of each eluate was infected separately into E.Excess capture antibody was removed by washing.

coli strain TG1 for propagation of the phage-sFv andThe remaining available sites on the plate were

further rounds of selection.blocked with 4% (w/v) low fat milk powder in PBS(MPBS) for 2 h at 378C. The plate was washed and

2.6. Production of soluble sFv fragmentsserially diluted phage-sFv samples were applied tothe plate in 0.5% MPBS and incubated for 2 h at

Selected clones were cultured at 378C in 23TYroom temperature. Bound phage-sFv were detected(supplemented with 100 mg/ml ampicillin and 0.1%using rabbit anti-M13 (5.4 mg/ml) (Sigma, Poole,glucose) until they reached an OD of 1.0.UK) followed, after washing the plate, by peroxi- 600 nm

Production of soluble sFv was induced by thedase-conjugated swine anti-rabbit immunoglobulinaddition of 1 mM isopropyl-b-d-thiogalacto-(0.8 mg/ml) (Dako, High Wycombe, UK). Both

¨pyranoside (IPTG) (Alexis, Laufelfing, Switzerland)were applied in 0.5% MPBS and incubated for 1 h atto the culture and growth overnight at 218C. The sFvroom temperature. Between each stage, the plate waswas collected either directly from the culture super-washed three times with PBS–0.1% Tween 20natant (see later) or from the periplasm followingfollowed by a single wash with PBS except the lastlysis of the outer bacterial membrane resulting in thestage where five washes with PBS–0.1% Tween 20release of periplasmic contents. Periplasmic lysisfollowed by two washes with PBS were used.was performed by osmotic shock for 4 h at 48C using3,39,5,59-tetramethylbenzidine (TMB) (59-39 Bishopsan ice-cold lysis buffer containing 20% sucrose, 5Stortford, UK) was used as the substrate and themM EDTA and 100 mM Tris, pH 8.8, and 1/100reaction was stopped with 25 ml of 0.5 M H SO2 4

volume of protease inhibitor cocktail (4-(2-amino-after 10 min.


ethyl)benzenesulfonyl fluoride, pepstatin A, E64, to separate sections and incubated for 1 h at roombestatin, leupeptin and aprotinin in DMSO; Sigma). temperature. Excess sFv was removed by washing

three times in PBS for 5 min. Sequential incubation,for 1 h at room temperature, with biotinylated 9E10

2.7. Purification of sFv from bacterial culture antibody (5 mg/ml plus 5% normal mouse serum;supernatants NMS) and extra-avidin–FITC (10 mg/ml plus 5%

NMS) were used to detect bound sFv. The longBacterial culture supernatants were cleared by incubation times (60 min rather than the usual 30

centrifugation at 12 800 g for 30 min at 48C. The min) should favour those antibodies with relativelycleared supernatants were incubated with 1.0 ml high affinity for their antigens (low off rate).protein-A Sepharose (50%, w/v; Amersham) for 2 hat room temperature with continuous rotation[protein-A binds a sub set of V 3 domains (Hoogen-H 2.10. Preparation of cytospinsboom and Winter, 1982)]. The protein-A waswashed twice with 20 ml and once with 10 ml of ice Mice, 8–10 weeks old, were sacrificed and thecold PBS. Bound sFv was eluted with 1.0 ml 0.1 M thymus removed. A single cell suspension was madeglycine, pH 2.7, for 5 min at room temperature by pushing the thymus through a cell sieve (Bectonfollowed by centrifugation at 3000 g for 5 min. Dickenson, Franklin Lakes, NJ, USA). To removeSupernatant was removed and the low pH neutralised the majority of lymphoid, dendritic and epithelialwith 1 /10 volume of 1 M Tris, pH 8.8. Elution was cells, the cell suspension was incubated withrepeated three times in total. biotinylated rat anti-mouse NLDC-145 (25 mg/ml)

for 45 min at 48C and then washed three times inice-cold PBS (Kraal et al., 1986). The washed cells2.8. Dot blotswere incubated with streptavidin coated magneticbeads (Dynal, Oslo, Norway) for 45 min at 48C withA 2.5–3.5-ml volume of each sample was appliedcontinuous rotation (a 2:1 ratio of beads to cells wasto a nitrocellulose membrane and allowed to dry forused). The bead-coated cells were removed with a20 min. The membrane was blocked with 2.5%magnet and the beads washed three times with ice-MPBS overnight at 48C or for 2 h at 378C. sFv werecold PBS. The supernatant was kept and the NLDC-detected using 9E10 and peroxidase conjugated145 negative cells washed in PBS–10% FCS, re-rabbit anti-mouse immunoglobulin. 9E10 was ap- 6suspended at 1310 cells /ml and 100 ml applied toplied at 10 mg/ml and the peroxidase-conjugate waseach cytospin. The slides were allowed to dry for aapplied at 1.3 mg/ml. Both were diluted in 0.5%minimum of 12 h and fixed in acetone–methanolMPBS. The membranes were developed with en-(50:50, v /v) for 10 min at room temperature. Thehanced chemiluminescence (ECL; Amersham Phar-fixed slides were transferred immediately to PBS andmacia), and exposed to X-ray film for 1–5 minstained with sFv and antibodies either alone or in(Kodak, Rochester, NY, USA).combination. sFv were applied as protein-A purifiedextracts from bacterial culture supernatant diluted in

2.9. Tissue sectioning and staining PBS to 10 mg/ml. Bound sFv were detected withbiotinylated-9E10 antibody followed, after washing

Murine organs (thymus, liver, lungs, heart, kidney three times with PBS, by TRITC-conjugated extra-and pancreas) were placed in cryovials, covered with avidin both at 10 mg/ml in PBS containing 5%a drop of PBS, capped and snap frozen in liquid NMS. Cytospins were double stained with rat anti-nitrogen (de Maagd et al., 1985). Cryostat sections mouse ICAM-1 (10 mg/ml) which was detected with(6 mm) were cut, air dried for a minimum of 12 h FITC-conjugated rabbit anti-rat immunoglobulin (20and fixed for 10 min in absolute acetone. A 100-ml mg/ml). The latter was applied in PBS supplementedvolume of selected sFv (either periplasmic lysate or with 5% NMS. All reagents were incubated for 1 hculture supernatant dialysed into PBS) was applied at room temperature.


2.11. Preparation of tissue lysates ethanol). The samples were separated using 7.5%non-reducing SDS–PAGE and immunoblotted as

Tissue lysates were prepared from frozen tissue described above.sections to improve efficiency, as previously de-scribed (Mat et al., 1993). The frozen tissue sectionswere lysed at 48C for 30 min using 1 ml of lysis 3. Resultsbuffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1%Triton X, 5 mM EDTA and 5 mM NaN plus 1/1003 3.1. Confirmation of sFv expression on phagevolume of protease inhibitor cocktail (4-(2-amino-

particlesethyl)benzenesulfonyl fluoride, pepstatin A, E64,bestatin, leupeptin and aprotinin in DMSO; Sigma)

In previous studies we had noted that, particularlyper thymus. Cell debris was removed by centrifuga-after storage, the phage lose expression of their sFv.tion at 3000 g at 48C for 30 min. Cleared lysatesIt was therefore important to test the phage-sFvwere stored on ice before use.expression prior to each round of selection. To thatend, we established an ELISA which captured2.12. Immunoblottingphage-sFv via the c-myc tag (using the 9E10 captureantibody) incorporated into the sFv. The phage-sFvProteins were transferred to PVDF membranewere detected by an anti M13 antibody. An exampleaccording to standard protocols. The membranesof such an ELISA is shown in Fig. 1a. Similarwere blocked with 2.5% MPBS either overnight at 48ELISAs were performed before every round ofor 378C for 2 h. For the analysis of tissue lysates, theselection and the preparation discarded if expressionmembranes were then probed with sFv by incubationwas not confirmed.at room temperature on a rotator followed by de-

In order to investigate the loss of sFv from phage,tection with either 9E10 and peroxidase-conjugatedwe performed immunoblotting using the 9E10 antirabbit anti-mouse immunoglobulin or biotinylatedc-myc antibody (Fig. 1b). Lane 1 shows a fresh9E10 and peroxidase-conjugated extra-avidin. Forpreparation of phage-sFv. Two predominant formsanalysis of sFv and phage preparations, membranesare seen, one corresponds to full length pIII-sFvwere probed with 9E10 and peroxidase-conjugatedfusion protein. The other is the size of pIII alone andrabbit anti-mouse immunoglobulin. The culturepresumably represents cleavage N-terminal to thesupernatants containing sFv were applied neat andc-myc tag. There is an additional minor band proba-the periplasmic lysates containing sFv were appliedbly due to degradation within the sFv. After storeageat 10 mg/ml (total protein) in 0.5% MPBS. 9E10,for 4 days at 48C (Fig. 1b; lane 2) only the lower9E10–biotin and peroxidase-conjugated extra-avidinband was seen — this was very faint indicating thatwere all applied at 10 mg/ml in 0.5% MPBS,cleavage had occurred C-terminal to the c-mycperoxidase-conjugated rabbit anti-mouse immuno-peptide. This correlated with the loss of reactivity inglobulin was applied at 1.3 mg/ml in 0.5% MPBS.the ELISA. Lane 3 (Fig. 1b) shows a fresh prepara-All were incubated for 1 h at room temperature.tion of the control pHEN1 which, since the c-myctag is encoded by the vector, gives a strong band2.13. Immunoprecipitationcorresponding to pIII.

Mouse thymic lysates (prepared as describedabove) were incubated with 10 mg/ml of periplasmic 3.2. In vivo localisation and clearance kinetics ofextract containing E3A1 sFv for 45 min at 48C with a non selected phage-sFv librarycontinuous rotation. A 40 ml volume of protein-ASepharose (50% w/v) was added and incubated for a To determine the localisation of phage particles

125further 45 min on ice. The protein-A Sepharose was irrespective of their associated sFv specificity, Iwashed once with PBS and then boiled for 10 min in labelled phage (from the non selected Nissim li-SDS–PAGE sample buffer (without mercapto- brary) were injected intravenously (through the tail


Fig. 1. Surface expression sFv on bacteriophage pIII. (A) Serially diluted phage-sFv were applied to ELISA plates coated with 9E10antibody (10 mg/ml). Those phage expressing sFv on their surface were captured by their myc tag and detected using rabbit anti-M13followed by peroxidase-conjugated swine anti-rabbit immunoglobulin antibodies. Helper phage represents the negative control. (B)Representative immunoblot of phage-sFv preparations. Phage-sFv have been boiled in loading buffer and separated by SDS–PAGE using a4–12% gradient gel (Novex, Frankfurt, Germany) and electroblotted to PVDF. The membrane was blocked with 2.5% MPBS and probedwith 9E10 followed by peroxidase-conjugated rabbit anti-mouse immunoglobulin antibody. Staining was revealed with enhancedchemiluminescence. Lanes: 15Fresh phage-sFv preparation; 254-day-old phage-sFv preparation; 35fresh pHEN-1 preparation. Lane 1shows the complete sFv expressed on the pIII and also partly degraded sFv on the pIII.

vein) into 8–10-week-old CBA mice. The major for each organ and normalised to the blood. Fig. 2Aorgans were harvested after various time points and shows that many phage become trapped non spe-the percentage of the injected dose /g was calculated cifically in the lungs, presumably owing to the dense


125 10 125Fig. 2. Circulation of I labelled phage-sFv in the mouse. CBA mice (8–10 weeks old) were injected intravenously with 9310 Ilabelled phage-sFv.Various organs were harvested after fixed time intervals, washed and the radioactivity counted. The data are expressed as(percentage injected dose /g tissue) /(percentage injected dose /g blood) for each organ and are the mean of three animals at each time point.(A) Kidney, spleen, lung, thymus and liver; (B) skin, muscle, brain, heart, ileum and urine. At the 2 h time point, the standard error wasbelow 0.07 of the mean for all organs except the spleen, which was 0.627.


capillary network that is present. Clearance of these 3.3. Recovery of viable phagephage-sFv began after approximately 1 h; after 24 h,the level of phage in the lungs had fallen to that of The data presented in Fig. 2 do not reflect theother organs. In agreement with previous work using viability (i.e. the ability of phage to infect bacteria)a peptide display library (Arap et al., 1998), the of the phage-sFv. To address this point further, thespleen and liver also attract a high percentage of the recovery of viable phage from the thymus, liver andcirculating phage initially. However, the majority of heart was assessed (Fig. 3). In the first 10 min afterorgans, in particular the thymus, trap very few injection, few phage enter any of the organs tested.phage, at any time point, from the non selected The recovery of viable phage from the heart andlibrary (Fig. 2A and B). It is interesting to note that thymus was maximal after 1 h. The differences inin contrast to Arap et al., 1998 who observed organ to blood ratios seen in Figs. 2 and 3 presumab-virtually no phage present in the blood after 24 h, we ly reflected differences in the handling of phage. For

125observed 5.6% of the injected dose of phage still example, for I labelled (total) phage in the liver atpresent in the blood after 24 h (compared to 14% 60 min the organ:blood ratio was 0.9 while for viableafter 1 min). This may be attributable to the fact that phage it was 0.08, indicating a greater localisation ofArap et al. used a selected library and therefore non-viable phage to the liver.many phage were absorbed rapidly into the tissue, From the viewpoint of selection from the phage-whereas the data shown here are for a non selected sFv library, we chose the 2 h time point. We believelibrary. the lower levels of viable phage recovered from the

11Fig. 3. Recovery of viable phage from tissues. CBA mice (8–10-weeks-old) were injected intravenously with 1310 phage-sFv. The liver,heart, thymus and blood were harvested at different time points. Bound phage were eluted using 500 ml of 0.1 M glycine, pH 2.7, for 5 minat room temperature and neutralised with 100 ml of Tris, pH 8.8. The eluted phage were serially diluted and used to infect 500 ml of E. coliTG1 bacteria. Each dilution was mixed with 6 ml of top agarose (0.7% agarose w/v in LB medium) and plated out. Plates were incubated at378C overnight. Each time point represents the mean of three animals and has been normalised to the blood as described in Fig. 2. The

10 10 10 10absolute levels in the blood were as follows: 1 min, 1.63310 ; 10 min, 1.77310 ; 60 min, 2.06310 ; 120 min, 1.16310 ; 1440 min,95.75310 phage/ml /g.


tissue after 2 h is advantageous since it reflects the organ should increase. Using our in vivo thymicclearance of non-specifically trapped phage-sFv from selection method, we observed a 15-fold increasethe tissue in question and so should increase the from the third to fourth round of selection (Fig. 4,efficiency of the specific selection procedure. elution 1). U7.6 and pHEN1 represent irrelevant

control phage either with (U7.6) or without (pHEN1)3.4. Monitoring selection of phage-sFv during in sFv on their surface. Less U7.6 and pHEN1, 592-vivo selection and 244-fold, respectively, were recovered from the

thymus.When selection from a phage-sFv display library

is performed against a known antigen, it is easy to 3.5. Characterisation of in vivo selected anti-monitor the selection procedure after each round endothelial sFvusing ELISA. This would be impossible for in vivoselection. However, in theory, as selection proceeds, The data presented in Fig. 4 are highly suggestivethe percentage of phage recovered from the selecting that selection is occurring but do not provide direct

Fig. 4. Monitoring selection: round 3 versus round 4. Mice injected with phage-sFv were sacrificed after 2 h and the thymus removed. Asample of blood was also recovered. The thymus was teased apart and washed 5–10 times with PBS. The phage-sFv bound to the thymuswere eluted by incubating the tissue for 5 min with 500 ml of 0.1 M glycine, pH 2.8. Eluted phage-sFv were neutralised with 100 ml of Tris,pH 8.8. The elution was repeated four times. Phage eluted from the thymus and those present in the blood were serially diluted and used toinfect 500 ml of E. coli TG1 bacteria. Each dilution was mixed with 6 ml of top agarose and plated out. Plates were incubated at 378Covernight. U7.6 and pHEN1 represent the negative controls which expressed either irrelevant (U7.6) or no (pHEN1) sFv on their surface.


proof either of the selection procedure or that the 7.5% SDS–PAGE and immunoblotted, were probedphage-sFv recognise the endothelium. Therefore with protein-A purified E3A1 sFv. Bound sFv wasphage-sFv recovered from round 4 of selection were revealed with either 9E10 and peroxidase-conjugatedinfected into E. coli strain HB2151 and 96 individual rabbit anti-mouse immunoglobulin or biotinylated-clones (chosen at random) were grown up separately 9E10 and peroxidase-conjugated extra-avidin. E3A1and induced to produce sFv by the addition of 1 mM sFv detected a band of 165–170 kDa not detectedIPTG and overnight incubation at 218C. All clones with other sFv. This was confirmed by immuno-were screened for the production of sFv in both the precipitation (Fig. 7).culture supernatant and the periplasmic lysate.Twelve clones secreted sFv into the culture superna-tant and all produced sFv in the periplasm (shown by 4. Discussiondot blot analysis — data not shown). sFv from the 12clones secreting into the supernatant were dialysed The use of phage display technology to derive afrom culture medium into PBS and screened in- source of analytical reagents has yielded many usefuldividually on mouse thymic tissue sections. The sFv antibodies some allowing recognition of singlefrom periplasmic lysates were pooled in groups of amino acid substitutions (Okamoto et al., 1998) andfour, dialysed into PBS, and screened on mouse often capable of recognising conserved epitopes thatthymus as above. sFv A12, which was secreted into had previously proved difficult using hybridomathe culture supernatant, showed clear staining of the techniques (Palmer et al., 1997, 1999). The details ofendothelium as did the unrelated periplasmic extract the techniques used for phage-sFv display selectionfrom pool 21 (Fig. 5A–F). In contrast, clone E3 have been varied but all have been based upon thelabelled the perivascular epithelium. In all cases a availability of a known purified antigen or ex vivominority of small blood vessels with the appearance cells. We have now developed an in vivo selectionof high endothelial venules (HEV) stained positive procedure for use with phage-sFv display libraries inwith the sFv. Large vessels were never seen to stain order to analyse molecular expression in the naturalpositive (Fig. 5G and H). In addition, these sFv were physiological environment. We have chosen thetested on liver, lung, heart, pancreas and kidney from thymus as our model system since it is easilyCBA mice. No other tissues tested showed staining isolated, well defined and it is likely that moleculeswith either A12 or E3 sFv. E3 was also tested on on the vascular endothelium play a crucial role inNLDC-145-negative (epithelium and dendritic cell stem cell recruitment (Coltey et al., 1987).depleted) thymically derived cell cytospins and Initial consideration of the kinetics of in vivoshown to colocalise to ICAM-1 positive cells using phage circulation show the phage-sFv become non-two colour immunostaining (Fig. 6) thus supporting specifically trapped in some organs (liver, spleen andthe anti-endothelial nature of the sFv, in addition to lungs) but that the majority of tissues trap fewits recognition of perivascular epithelium (Boyd et phage-sFv at any time point during the first 24 h.al., 1993; Ritter and Boyd, 1993). Clones E3 and Previous work using a phage-peptide library (Pas-A12 were subcloned, and their ability to produce sFv qualini et al., 1997) has also noted the localisation ofchecked by dot blot (data not shown), to ensure their phage in the liver and has shown this is a property oforigin as single cells, giving rise to the following the whole phage rather than the peptide expressed onnomenclature for each clone: E3A1 and A12A8, the surface; the same is likely to apply to phagerespectively. Clones E3A1 and A12A8 have been expressing sFv on their surface. Furthermore, thesequenced and belong to families V 3 and V 4, role of the liver in the reticuloendothelial system isH H

respectively and have the following CDR3 sequences also likely to be a factor in the accumulation ofHTVRSL (clone E3A1) WDRS (clone A12A8). phage (Geier et al., 1973; Rajotte et al., 1998).

The possible breakdown of labelled phage, either1253.6. Characterisation of the target endothelial by removal of the I label or breakdown of the

molecule recognised by E3A1 sFv phage itself rendering it non-viable, was an obviousproblem to be considered in the development of the

Mouse thymic lysates, separated by non reducing selection procedure. Our data showing localisation of


Fig. 5. Tissue staining. sFv (either from bacterial culture supernatants or from periplasmic lysates) dialysed into PBS were applied to mousethymic tissue sections. Bound sFv was detected with biotinylated-9E10 (10 mg/ml) and FITC-conjugated extra-avidin (10 mg/ml). Anirrelevant sFv was used as the negative control. Slides were viewed under UV illumination. (A) sFv E3A1; (B) control section negativecontrol for A; (C) sFv A12A8; (D) control section negative control for C; (E) sFv Pool 21; (F) control section negative control for E; (Gand H) negative large blood vessels in sections incubated with A12A8 and pool 21, respectively. The autofluorescent spots of staining in thecontrols are likely to be thymic macrophages.

total and non-viable phage (Figs. 2 and 3) suggest to be maximal after 1 h. However, we chose 2 h asthat non-viable phage-sFv are localised in the liver the time point at which to select phage-sFv in orderyet phage recovered from other organs (thymus and to negate any possible problems that may be incurredheart) are predominantly viable. The recovery of by phage-sFv that are non-specifically trapped in theviable phage from the heart and thymus was shown tissue and have yet to be cleared after 1 h.


Fig. 5. (continued)

No direct method exists to monitor in vivo selec- specificity of the selection procedure, however,tion against unknown molecules. Therefore the best comes from the tissue staining. Two clones (E3A1indication comes from the number of recovered and A12A8 belonging to V 3 and V 4 families,H H

phage-sFv after each round of selection; if phage-sFv respectively) have been isolated and clearly stain theare being specifically selected, the percentage of thymus. Clone A12A8 stains some thymic endo-injected dose per gram of the organ recovered after thelium with the appearance of HEV. However,each round of selection will increase. Our data E3A1 whilst selected against the endothelium ap-clearly demonstrate that this is the case. Proof of the pears to stain the thymus perivascular epithelium


Fig. 6. Cytospins of NLDC-145 depleted thymic cells. Cytospins of NLDC-145 depleted mouse thymic cells were incubated with sFv(either anti-NIP or E3A1) and double stained with rat anti-mouse ICAM-1. sFv were detected with biotinylated 9E10 (10 mg/ml) andTRITC-conjugated extra-avidin (10 mg/ml). The rat anti-mouse ICAM-1 was detected with FITC-conjugated rabbit anti-rat immuno-globulin. All second and third layer reagents were applied in PBS–5% normal mouse serum. (A and B) Stained with anti-NIP sFv plusICAM-1; (C and D) stained with E3A1 sFv and ICAM-1. The small double positive spots are residual magnetic beads remaining afterremoval of NLDC-145 positive cells. This rat monoclonal antibody was bound to the beads via an avidin–biotin bridge and therefore crossreacts with both fluorochromes. The specific staining is marked with arrows.


this work is preliminary, initial data show that theantigen has a molecular weight of 165–170 kDa.This has been demonstrated by immunoblotting andimmunoprecipitation. No information has been ob-tained regarding the identity of the endothelialmolecule recognised by E3A1. A molecular weightof 165–170 kDa rules out Vanin-1, ICAM-1, ICAM-2, VLA-3, LFA-3, VCAM-1, L-selectin, E-selectin,CD31, CD34, CD51, CD61, as possible antigens(Aurrand-Lions et al., 1996; Barclay et al., 1997).However, it is possible, based on the approximatesize of the molecule, that the sFv recognises VLA-2,P-selectin a -integrin or LFA-1 although the dis-E

tribution of the antigen would argue against these(Reza and Ritter, 1994; Barclay et al., 1997). There-fore, it seems likely that sFv, E3A1, recognises apreviously unknown endothelial molecule which isthymus-restricted and hence is a candidate vascularaddressin that may control stem cell entry into thethymus. Our data therefore confirm the efficacy of invivo selection using antibody phage display in athymic model. Moreover, the technique should bereadily applicable to other situations.

Fig. 7. Immunoblot with E3A1 sFv following immunoprecipita-tion. Mouse thymic lysates have been incubated with either E3A1or anti-NIP sFv (10 mg/ml) and immunoprecipitated with protein-A Sepharose. Samples (10 ml) were separated on a non-reducing References7.5% SDS–PAGE gel and transferred by electroblotting to PVDF.The membranes have been probed with E3A1 which has been

Arap, W., Pasqualini, R., Ruoslahti, E., 1998. Cancer treatment bydetected with 9E10 and peroxidase conjugated rabbit anti-mousetargeted drug delivery to tumour vasculature in a mouseimmunoglobulin. Staining was revealed by enhanced chemilumin-model. Science 279, 377–380.escence.

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most strongly (Boyd et al., 1993). Double staining of perivascular molecule involved in thymus homing. Immunity 5(5), 391–405.cytospins with E3A1 and ICAM-1 on NLDC-145

Barclay, A.N., Brown, M.H., Law, S.K.A., McKnight, A.J.,depleted thymic cells shows that the E3A1 antigen isTomlinson, M.G., van der Merwe, P.A., 1997. The Leukocyte

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In vivo selection of sFv from phage display libraries