Methodology for selection of human antibodies to membrane proteins from a phage-display library

Journal of Immunological Methods 204 Ž1997. 193–203

Methodology for selection of human antibodies to membrane proteins from a phage-display library Carol Sawyer a

a,)

, Jim Embleton b, Christopher Dean

a

Section of Immunology, McElwain Laboratories, Institute of Cancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey, UK b The Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK Received 9 January 1997; accepted 13 March 1997

Abstract We describe a simple antigen capture technique for the selection of a specific human antibody to p185 er bB-2 , a transmembrane glycoprotein, from a library of human Fab genes expressed on the surface of bacteriophage. Magnetic beads coated with the rat antibody ICR55 have been used to capture erbB-2 antigen from Triton X-100 extracts of SKOV3 cells. The antigen-coated beads have then been used to select bacteriophage displaying human Fab with affinity for p185 er bB-2 . After 4 rounds of selection, 65 phage clones were isolated which bound specifically to p185 er bB-2 in a capture assay. Nine of the clones which gave the strongest reaction in an ELISA were selected for further development and the Fab genes were subcloned into the expression vector pUC119his6mycXba and electroporated into E. coli TG1. Colonies were grown, induced and the supernatants tested for the presence of secreted human Fab. Supernatants from two of the 9 clones contained human Fab and one of these bound specifically to erbB-2 in a capture assay, stained the membranes of the erbB-2 overexpressing cell lines BT474 and SKBR3 and immunoprecipitated a protein of molecular weight 185 000 kDa from SKOV3 cells. We conclude that a membrane antigen captured by specific monoclonal antibody can be used successfully to select phage displaying human antibodies specific for the antigen. Keywords: Phage; Recombinant Fab; Human antibody; Membrane protein; erbB-2

. 1. Introduction There are convincing reasons why transmembrane receptors for growth factors expressed by tumours Abbreviations: scFv, single chain Fv; FCS, foetal calf serum; PBS, phosphate-buffered saline; PMSF, phenyl methyl sulphonyl chloride; ELISA, enzyme-linked immunosorbent assay; TMB, X X X X 3 ,3 ,5 ,5 -tetramethyl benzidine ) Corresponding author. Tel.: q1 Ž181. 643-8901; Fax: q1 Ž181. 643-0223.

are good targets for antibody-directed therapy. First, the receptors are expressed at the cell surface and are accessible to antibodies injected intravenously. Second, some receptors are overexpressed by tumours due to amplification of the gene or increased mRNA production. Third, antibodies which block growth factor receptor interaction can inhibit growth of the cells. Inhibition of growth in this way can be very important since some tumours produce their own growth factors and undergo autocrine growth. The product of the c-erbB-2 proto-oncogene is a transmembrane tyrosine kinase found to be overexpressed by certain tumours, whereas it is rarely

0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 0 4 8 - 3

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expressed on adult human tissues. For example, p185 er b B-2 is highly expressed on 20–30% of breast cancers ŽSlamon et al., 1987; Gusterson et al., 1988. and this overexpression has been correlated with a poor prognosis in patients with these tumours ŽSlamon et al., 1987; Tiwaris et al., 1992.. A number of mouse and rat monoclonal antibodies have been prepared against the extracellular domain of p185 er b B-2 Že.g., Hudziak et al., 1989; Styles et al., 1990. some of which bind with high affinity and are stable at the cell surface ŽDean et al., 1993.. Some of these antibodies have been used in clinical studies in patients with breast cancer. However, a drawback with the use of rodent antibodies in the treatment of human disease is their likely immunogenicity and the consequent elicitation of human anti-rodent antibodies following their repeated injection into patients. Clearly, human antibodies with properties similar to their rodent counterparts would be ideal, but it has proved particularly difficult to prepare useful human antibodies using conventional hybridoma technology. Recent developments in recombinant antibody technology have solved a number of the problems involved in the preparation of fully human antibodies and libraries of human variable region genes have been prepared in and displayed on the surface of filamentous bacteriophage ŽMarks et al., 1991; Nissim et al., 1994; Griffiths et al., 1993, 1994.. Phage displaying specific single chain Fv ŽscFv. or Fab are isolated by carrying out several rounds of selection involving binding to specific antigen. Using this technology, it has been possible to select recombinant single-chain antibodies from gene libraries isolated from B-cells of either unimmunised or immune individuals ŽMarks et al., 1991; Burton et al., 1991; Zebedee et al., 1992; Griffiths et al., 1993.. An advantage of this methodology over conventional hybridoma technology is that it should be possible to isolate antibodies to highly conserved regions of proteins which is difficult to achieve by immunisation of rodents because of the inter-species similarity in sequence of the immunogen and the rodent homologue. While this technology works well using purified antigen, it has proved difficult to select phage recognising a predefined antigen by directly binding to mammalian cells or cell membranes. The preparation

of recombinant membrane proteins is possible, but the products are often insoluble and denatured. In this communication, we describe the use of capture methodology ŽSawyer et al., 1995. for the selection and isolation of human Fab antibodies directed against one such transmembrane protein, p185 er b B-2 .

2. Materials and methods 2.1. Cell lines SKBR3 ŽATCC HTB30. and BT474 ŽATCC HTB20., human breast carcinoma and SKOV3 ŽATCC HTB77., human ovarian carcinoma cell lines were obtained from Dr M. O’Hare ŽUniversity College, London.. The product of the c-erbB-2 protooncogene is over-expressed in all three lines and there are approximately 10 6 receptor molecules per cell ŽHarwerth et al., 1992; Shawver et al., 1994.. The cell lines were maintained in Dulbecco’s modified Eagle’s medium ŽDMEM. containing 10% heat-inactivated foetal calf serum ŽFCS. and the antibiotics penicillin, streptomycin and neomycin. 2.2. Isolation of p185 er b B - 2 ‘antigen’ A confluent 80-cm2 flask of SKOV3 cells was washed three times in phosphate-buffered saline ŽPBS., pH 7.4, containing 5 = 10y4 M phenyl methyl sulphonyl fluoride ŽPMSF. and 0.02% sodium azide ŽNaN3 .. Cells were placed on ice and then lysed by addition of 3 ml of PBS containing 1% Triton X-100, 5 = 10y4 M PMSF and 0.02% NaN3 . After 1 h at 48C, the lysate was centrifuged at 15 000 rpm for 15 min at 48C in an IEC Centra-3RS. The supernatant was stored at y708C and used as the source of antigen in all methods described in this paper. 2.3. Phage stocks Phage containing a semi-synthetic human Fab library, representing all the heavy and light chain variable regions ŽGriffiths et al., 1994. were produced from Escherichia coli by PEG precipitation of culture supernatants and stored at y708C.

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2.4. Preparation of magnetic beads for phage selection 15 mg of tosyl-activated magnetic beads ŽDynabeads M-450, Dynal Ltd., UK. were coated by rotating for 1 h at room temperature with 75 mg of ICR55, a rat monoclonal antibody to the extracellular domain of the p185 er b B-2 ŽDean et al., 1992.. The rat antibody had been purified from culture supernatant by salt fractionation followed by ion-exchange chromatography. Free unreacted sites on the beads were blocked by incubation with PBS containing 2% skimmed milk ŽPBSM. for 2 h at room temperature. 2.5. Selection of phage The protocol used is summarised in Fig. 1. For the first round of selection 700 ml of bacteriophage Ž2.8 = 10 13 transducing units. were added to a mixture containing 4 ml of detergent cell extract Žantigen. diluted with 4.5 ml PBS supplemented with 4% milk powder. This was incubated with gentle rotation

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for 30 min and then stood for a further 90 min at room temperature. Phage bound to p185 er b B-2 were then isolated by adding 1 ml ICR55-coated Dynabeads and rotated for a further 2 h at room temperature. Phage bound to the Dynabeads were separated from the supernatant using a magnetic particle concentrator ŽDynal MPC-1. and washed 8 times with PBS containing 0.1% Tween 20 ŽPBST. and then 8 times with PBS. Phage were eluted from the Dynabeads by incubation with 1 ml of 100 mM triethylamine, pH 9.5, for 10 min at room temperature and then neutralised by the addition of 0.5 ml of 1 M Tris, pH 7.4. The eluate Ž0.8 ml. was used to infect a 10-ml culture of log phase E. coli TG1 cells which were then allowed to stand for 30 min at 378C before plating onto a Nunc 243 = 243 mm dish of TYE agar Ž15 g bacto-agar, 8 g NaCl, 10 g tryptone, 5 g yeast extract per litre water. containing tetracycline Ž12.5 mgrml.. After overnight incubation at 378C, the cells were scraped off into 200 ml 2 = TY broth Ž5 g NaCl, 16 g tryptone, 10 g yeast extract per litre water. containing tetracycline and the culture was shaken at 308C for 6 h. The cells were pelleted by centrifugation at 5000 rpm ŽSorval RC3B. for 20 min and stored in 2 ml 15% glycerol at y708C. Phage were precipitated from the culture supernatant by the addition of 0.2 vols. of 20% polyethylene glycol ŽPEG. 6000 in 2.5 M NaCl and stored on ice for 1 h. After centrifugation, the pellet was resuspended in approximately 20 ml water and the PEG precipitation repeated. The final pellet was resuspended in 2 ml PBS and 1 ml of this was used for the second round of selection. Four rounds of selection were performed. 2.6. Screening of the selected phage library

Fig. 1. Protocol for the selection of human Fab fragments with affinity for p185 e r b B-2 from a library of human Fab genes in filamentous phage.

Phage clones obtained from the fourth round of selection were assayed for binding to antigen by ELISA. Two 96-well plates ŽImmulon-1, Dynatech, VA, USA. were coated with 10 mgrml rat monoclonal antibody ICR55 ŽDean et al., 1992. and blocked with 2% PBSM. Aliquots Ž50 ml. of the erbB-2-containing extract were added to one of the plates, whilst the other plate was blocked for a further 2 h at 378C with PBSM. After 3 washes with PBST, the supernatant from individual phage clones, grown up from tooth-picked colonies in 96-well

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plates, was then added to wells of both plates and incubated for 1 h at room temperature. The plates were washed 3 times with PBST and 3 times with PBS alone. Sheep antibody to M13 phage Ž5 Prime to 3 Prime Inc., Colorado, USA. diluted 1r2000 in 2% PBSM was added to the wells and the plates were incubated at room temperature for 1 h. Following 4 washes, a 1r1000 dilution of donkey anti-sheep IgG peroxidase conjugate ŽSigma, UK. was added and incubated as previously. Finally, bound phageantibody was detected by incubation with 0.1 M acetate ŽpH 6. containing 0.01% 3X ,3X ,5X ,5X-tetramethyl benzidine ŽTMB. and 0.005% H 2 O 2 . Reactions were stopped by the addition of 1 M H 2 SO4 and the colour developed was determined at 450 nm. 2.7. Sub-cloning selected phage DNAs for expression of soluble Fab fragments Bacteria from positive wells were used as a template in a polymerase chain reaction ŽPCR. and the Fab genes were amplified by cycling 30 times Ž958C for 1 min, 508C for 1 min, 728C for 2.5 min.. The primers used were fdSEQ1 Ž5X-GAA TTT TCT GTA TGA GG-3X . and G3LXbaGTGBack Ž5X-GTC CTC GCA ACT TGC TCT AGA CAA TTT CAC AGT AAG GAG GTT TAA CTT GTG AAA AAA TTA TTA TTC GCA ATT-3X Griffiths et al., 1994.. The products were electrophoresed in a 1.3% agarose gel and purified from the gel using ‘Geneclean’ ŽBio 101 Inc., CA, USA.. Each product was cut with the restriction enzymes NotI and XbaI and ligated into the vector pUC119His6mycXba ŽGriffiths et al., 1994. using a DNA ligation kit ŽAmersham, UK.. The ligation mixture was purified using ‘Geneclean’ Žsee above. and 1 ml of this was used to transform E. coli TG1 by electroporation. The transformation conditions were 2.5 kV, 25 mF and 200 V using 0.2-cm cuvettes and a Gene Pulser, both from BioRad Laboratories ŽHemel Hempstead, UK.. Bacteria were plated onto TYE medium containing 1% glucose and 100 mgrml ampicillin. Colonies were screened initially by PCR using the primers CH1.lib.seq Ž5X-GGT GCT CTT GGA GGA GGG TGC-3X . and pelBback Ž5X-GAA ATA CCT ATT GCC TAC GG-3X . and presoaking at 948C for 10 min and 25 cycles Ž948C for 1 min, 558C for 1 min, 728C for 30 s. which amplified the heavy chain

of the Fab gene. Clones which contained an insert were shaken at 378C in 2 = TY medium plus ampicillin and 0.1% glucose until an OD600 of 0.8 was achieved. Isopropyl b-D-thiogalactoside ŽIPTG. was then added to a final concentration of 1 mM and incubation was continued overnight at 308C to induce soluble Fab expression. 2.8. Detection of recombinant human Fab in culture supernatants Secreted human Fab was detected using 96-well plates coated with 20 mgrml rabbit anti-human FŽabX . 2 Žpurified in house from the serum of rabbits immunised with human FŽabX . 2 .. Duplicate 50-ml aliquots of supernatants were added to the plates and incubated for 1 h. After washing, a rabbit anti-human k- and l-light chain peroxidase conjugate ŽDako, UK. was added Ž1r1000 dilution in 2% PBSM. and incubated for 1 h at room temperature. Finally, colour was developed using TMB, as described above. Wells coated with dilutions of human Ig Žpurified in-house from the serum of a patient with myelomatosis. were used to construct a standard curve. 2.9. Detection of recombinant Fab reactiÕe with c-erbB-2 p185 96-well plates were coated first with rat monoclonal antibody ICR55 and then with antigen containing extract. Pairs of plates, plus or minus antigen, were set up and supernatant from each E. coli clone was added to duplicate wells on both plates. After incubation for 1 h, the plates were washed 3 times with PBST then rabbit anti-human FŽabX . 2 Žpreabsorbed on rat FŽabX . 2 , and diluted 1r500. was added to the wells and the plates were incubated for a further 1 h. After washing, 125 I-sheep anti-rabbit Ig radiolabelled by the Iodogen method ŽFraker and Speck, 1978. was added Ž10 5 cpmrwell. and the plates were incubated for a further 1 h. Bound 125 iodine was determined in an Innotron Hydragamma counter. 2.10. Fluorescence staining Cell lines were grown on 13 mm glass coverslips placed in 24-well plates containing DMEM–10%

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FCS. The cells were fixed and permeablised by incubating the coverslips for 5 min in methanol that had been precooled to y208C. After 3 washes in PBS, undiluted, filtered bacterial supernatant was added to coverslips and incubated for 1 h at room temperature. Coverslips were washed 3 times and 1 mgrml rabbit anti-human FŽabX . 2 was added and incubated for a further 1 h. The coverslips were washed in PBS and then fluorescein conjugatedanti-rabbit Ig ŽAmersham International, UK; diluted 1r40 in PBS. was added and the cells were incubated for 1 h at room temperature in the dark. Coverslips were washed and mounted on glass microscope slides using a 1:1 mix of Citifluor ŽUniversity of Kent, UK. and Hydromount ŽNational Diagnostics, UK.. Staining was viewed using a =25 water immersion objective lens on a Zeiss Axiovert fluorescence microscope. 2.11. Immunoprecipitation and immunoblotting 50 ml Sepharose protein-A beads Ž10% slurry in lysis buffer. were coated with 2 mg rabbit anti-human FŽabX . 2 and then incubated with 1 ml E. coli culture supernatant containing human Fab. After washing in lysis buffer, the beads were incubated with 1 ml SKOV3 extract, then rewashed and finally resuspended in 50 ml Laemmli sample buffer. Samples were electrophoresed on a 6% SDS–polyacrylamide gel and blotted on to polyvinylidene fluoride membrane ŽImmobilon-P; Millipore, UK.. Blots were probed with either rabbit anti-human krl-light chains directly conjugated to horseradish peroxidase ŽDako, UK. or rabbit anti-erbB-2 Žsc-284, Santa Cruz Biotechnology, USA. or a mouse anti-myc tag antibody, 9e10 Žpurified in-house., followed by an appropriate horseradish peroxidase-conjugated secondary antibody. Bound protein was detected with a chemiluminescent substrate system ŽECL; Amersham, UK.. 2.12. Fab purification Human Fab was purified from filtered culture supernatant using a Qiagen ŽUK. Ni-NTA spin column kit under non-denaturing conditions according to the manufacturer’s instructions. Protein was anal-

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ysed by SDS–PAGE on a 15% gel which was then immunoblotted.

3. Results 3.1. Phage selection A phage library expressing Fab was incubated with a Triton X-100 extract of SKOV3 cells and then the phage that had bound to specific antigen were captured using Dynabeads precoated with a rat monoclonal antibody ŽICR55. to p185 er b B-2 . After 4 rounds of selection, 95 phage clones were tested and gave various ELISA readings in the presence or absence of antigen. An example of some of the readings obtained is shown in Fig. 2. Clones which gave an A 450 of less than 0.1 were designated ‘non-binders’ and approximately 15% of the clones isolated were in this category. Clones which gave an A 450 of greater than 0.1 were designated ‘binders’ and these were further sub-divided into two groups. One subgroup bound only to captured erbB-2, for example, Well 16 ŽFig. 2., and this group represented 56% of the total population. The other subgroup bound equally well whether or not specific antigen was present, as exemplified by Well 2 ŽFig. 2., which accounted for 24% of the total population. The latter group may have included phage bound to the rat mAb ICR55 which was also used for library selection and non-specific phage which cross-reacted with the ELISA blocking reagent. 3.2. Cloning and expression The Fab genes, from 9 of the phage clones which gave the highest binding in the ELISA, i.e., where the A 450 was greater than 0.35 Žline A, Fig. 2., were amplified by PCR and yielded DNA products of approximately 1400 base pairs. After sub-cloning into the expression vector, pUC119his6mycXba, and electroporation into bacteria, the colonies produced were checked for the presence of insert by PCR amplification of the heavy chain gene. A PCR product of 500 base pairs confirmed that all ampicillinresistant colonies contained plasmid with insert. Protein expression was induced and supernatants were

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Fig. 2. Histogram showing a representative sample of ELISA readings of phage clones which were bound to plates coated either with an anti-erbB-2 rat mAb Žopen bars. or with antigen captured from a detergent cell extract by the same anti-erbB-2 rat mAb Žshaded bars.. Bound phage were detected by incubating with a donkey anti-M13 phage antibody followed by an HRPO-antibody conjugate. The addition of TMB substrate produced a colour change which permitted quantitation of bound phage by measuring the absorbance of plates at 450 nm. Line A Ž0.35 absorbance units. was the cut-off point above which the specific clones, i.e., those likely to bind erbB-2, were selected. Line B Ž0.1 absorbance units. was the cut-off point below which clones were designated ‘non-binders’.

tested by ELISA for the presence of secreted human Fab. Supernatants from two of the cultures ŽB7 and D7. contained Fab and from a standard curve it was estimated that the concentration of human Fab in the bacterial supernatants was approximately 2 mgrml. The supernatants from clones B7 and D7 were assayed for binding to erbB-2 in a radioimmunoassay together with a supernatant from clone A11 which was found not to be producing human Fab and so was used as a negative control. The assay was conducted both in the presence and absence of antigen and clone B7 showed elevated binding when antigen was included in the assay. Although clone D7 was found to produce soluble human Fab, no binding to erbB-2 was detected in the radioimmunoassay. Supernatant, indirectly bound to Sepharose protein-A, was used to immunoprecipitate p185 er b B-2

from SKOV3 cell lysate and protein was then immunoblotted. Clone B7 produced Fab which precipitated a protein of 185 000 kDa detected with a rabbit anti-erbB-2 antibody Žsee Fig. 5.. 3.3. Fluorescence staining To investigate whether the recombinant human Fab bound to the receptor expressed on cell surfaces, cells grown on glass cover slips were fixed and permeablised in methanol at y208C and examined by indirect immunofluorescence. The rat monoclonal antibody ICR12, which binds to the extracellular domain of the c-erbB2 p185, was used as a positive control. Supernatant from clone B7 gave strong membrane staining of BT474 cells ŽFig. 3a,b., similar to that observed with ICR12 ŽFig. 3c,d., but BT474 cells were not stained with either the D7 or

Fig. 3. Immunofluorescent staining of methanol fixed BT474 cells viewed using a =25 water immersion objective showing cells under phase contrast Ž a,c,e, g . and the same cells viewed with epifluorescent illumination Ž b,d, f,h.. Cells in a and b were labelled with human Fab B7 and in c and d with a rat monoclonal ICR12. No staining was observed with human Fab D7 Ž e, f . or A11 supernatant Ž g,h. which did not contain human Fab.

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Fig. 4. Immunofluorescent staining of SKBR3 cells viewed under phase contrast Ž a,c,e, g . and the same cells viewed with epifluorescent illumination Ž b,d, f,h.. Cells in a–d were fixed and permeablised with methanol and in e–h were fixed with paraformaldehyde. Staining was with human Fab B7 Ž a,b,e, f . or rat monoclonal ICR12 Ž c,d, g,h..

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ŽFig. 4e,f. indicating that the human antibody fragment binds to an intracellular epitope. 3.4. Purification

Fig. 5. Immunoblot of a 6% SDS–PAGE probed with a polyclonal antibody to erbB-2, sc-284. Protein was immunoprecipitated with human Fab, B7, from a lysate of SKOV3 cells.

A11 supernatant ŽFig. 3e–h.. SKBR3 cells were also stained strongly with B7 supernatant ŽFig. 4a,b. and in a similar pattern to ICR12 ŽFig. 4c,d.. If the treatment with the B7 supernatant was omitted, no staining was observed when the cells were subsequently incubated with the rabbit anti-human antibody and the anti-rabbit-FITC conjugate. SKBR3 cells fixed with 0.2% paraformaldehyde which does not permeablise the cells were stained with the control antibody ICR12 ŽFig. 4g,h. which binds an extracellular epitope of erbB-2, but not with B7 Fab

Fig. 6. Immunoblot of a 15% SDS–PAGE showing Fab B7 purification fractions from a Ni-NTA spin column probed with Ž a. anti-human k-light chain and Ž b . 9e10, anti-myc tag antibodies. Track 1 contains crude bacterial supernatant; track 2, non-binding material; track 3, washes; track 4, eluted protein and track 5, control Fab produced by tryptic digest of a purified rat monoclonal antibody.

Fab B7 was purified on a small-scale using metal ion chelation chromatography. Protein eluted from a Ni-NTA column was analysed by SDS–PAGE and transferred to a membrane. Probing with the appropriate antibodies revealed the Fab contained a human k-light chain ŽFig. 6a. and a myc-tagged heavy chain ŽFig. 6b. of approximate Mr 30 000 and 38 000, respectively. No reactivity was observed when the membrane was probed with an anti-human l-light chain antibody.

4. Discussion Phage display libraries of recombinant human immunoglobulin genes offer the exciting possibility of generating fully human antibodies for diagnostic and therapeutic use in the clinic. Most of the antibodies that have been generated from these libraries have been selected by panning the phage on purified antigen and, in general, this has limited their targets to soluble molecules of predefined specificity. In malignant disease, most of the accessible targets for antibody-directed therapy of tumours are cell surface molecules, such as transmembrane glycoproteins which may be mutated or over-expressed. While it is relatively easy to select monoclonal antibodies using cell binding assays, at present, such assays have proved to be unsuitable for the selection of phage expressing scFv or Fab antibodies. Also, most of these target molecules are soluble only in non-ionic detergent because of the hydrophobic nature of the transmembrane region and the presence of detergent rendering the isolated protein difficult to handle using standard procedures. We have presented here a simple method for selecting a specific human antibody from a phagedisplay library, which by-passes the need for purified antigen, by capturing the molecule of interest from a mixture of cellular proteins with a monoclonal antibody bound to magnetic beads. The latter facilitates preferential selection of Fabs recognising a different epitope from that bound by the capture antibody. To

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obtain Fabs against the widest variety of epitopes it may be useful to carry out two independent rounds of phage selection using antigen captured by two non-competing antibodies. Currently, there are a large number of monoclonal and polyclonal antibodies available which have been generated against many constituents of the surfaces of human cells, both normal and malignant, and these could be used for capturing antigen. Using such a capture assay, we were able to select a large number of phage clones displaying Fabs with affinity for p185 er b B-2 . Clone B7, one of 9 selected and sub-cloned for bacterial expression, produced soluble human Fab which bound antigen in a radioimmunoassay. Fluorescence staining of fixed, permeablised cells showed that the B7 Fab binds to the plasma membrane of BT474 and SKBR3 cells which express large numbers of erbB-2 molecules on the cell surface. This staining was not observed with supernatants from other clones which did not secrete human Fab and therefore cannot be due to nonspecific binding of secreted bacterial proteins. Also, the absence of staining when Fab secreted by clone D7 was used to stain BT474 cells suggests that the B7 staining was specific. Using immunoprecipitation, we have shown that B7 specifically binds native erbB-2 in a detergent extract of SKOV3 cells which was detected by immunoblotting using a commercially available anti-erbB-2 antibody. The ELISA results also suggest that phage were present in the library which expressed Fab with specificity for the rat antibody ICR55 used to capture antigen. This result was expected and it may be helpful, therefore, to preadsorb the phage library before use with beads coated with the capture antibody to be employed. In our experience, the use of the magnetic Dynabeads greatly simplifies the extensive washing that is needed to reduce non-specific interactions of the phage. Whilst only one specific antibody fragment has been isolated so far, this is an 11% success rate since, in initial studies, only 9 of 95 tested phage clones were selected for sub-cloning into bacterial expression vectors. Now that library selection and screening assays have been developed it is possible to return to the remaining 40 phage clones that showed affinity for p185 er b B-2 in an ELISA and sub-clone the Fab genes for expression. It is likely

that several more phage clones contain Fab genes which when expressed in bacteria will produce further anti-erbB-2 antibodies. Clearly, the affinities of these phage antibodies for p185 er b B-2 may differ widely, although the washing procedure employed would be expected to select preferentially clones expressing higher affinity Fabs. It is interesting that 7r9 bacterial clones did not produce soluble human Fab when assayed by ELISA. Even though the Fab gene insert is present in the pUC119 expression vector, no soluble human Fab was detectable in the supernatant. Experiments where phage have been selected for binding to different antigens have produced similar results ŽMJE, unpublished data.. The reason for apparent non-secreting clones is unclear, although it could be that the protein produced is incorrectly folded and, therefore, not released into the culture supernatant in a soluble form. In conclusion, we have shown that a Fab fragment isolated from a phage library of human antibody genes binds to the intracellular domain of native p185 er b B-2 expressed in the membrane of breast and ovarian cancer cell lines.

Acknowledgements We would like to thank Dr. Andrew Griffiths and Dr. Greg Winter for providing the phage library. This work was supported by grants from the Cancer Research Campaign, London and the Medical Research Council.

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