JOURNAL OF lMMunOlOElcA1 METHODS

ELSEVIER ,auma, aflmm”nol”gical Meti ,89(1996)73-a

Retrieval of human antibodies from phage-display libraries using enzymatic cleavage

A combinamrial human lgG1.~ gene library of 2 X IO’ clones was constmcted from a pericolic lymph node using Ihe

phagemid vector pComb3H. Fabs with birding activity against tetanus toxoid (‘IT) and keyhole limpet hemocyanin (KLH) WCR isolated from this library. and one such IT binding Fab was used to further evaluate a new phagemid vector for Ihe

disolav of recombinant antibalv fraements (MCOI). This vector was deskned to incorpOrate a cleavage site for the en2yme

Gene&x 1. a myc peptide sg,Yand k amber codon belween the heavy chain cloning site and the mm&d Ml3 phage gene

111.

When MCOt phage displaying an am-TT Fab were bound to ‘R on P solid wbrtrate, elution with Genenare I at

concentrations of 5-10 ,~g/ml proved as effective as acid elution in releasing bound phage. Furthermore. enzymatic elution

with Cenenare I was comparable to acid &lion in tie enrichment of a IT binding Fab from the @colic library subcloned

inm the vector MCOI. Importmtly. the use of enzymatic or acid elutions resulted in tie retrieval of different anti-IT Fabs

from this same library.

We conclude tia pamung of phage-displayed combinatorial antibody libraries can be rucczssfully perfanned using

enzymatic elution. and that dds offers a useful alternative to currently available phage elution techniques.

1. lntroductiun

Human antibody libraries have been constructed

from lymphocytes taken from both immunized

@.cbcdee et rd.. 1992; Williamson et al.. 1993) and

unimmunized individuals (Marks et al., 19911, and

mme recently by the use of entirely synthetic vari-

able region genes (Nissim et al.. 1994). This latter

approach, pa~icularly in combination with the con-

struction of much larger libraries Griffiths et nl.,

1994). has allowed the isolation of antibodies with

greater affinities and novel specificities.

Typically phagc-displayed antibodies from these

libraries have been retrieved by affinity selection on

antigen-coated columns or plates (Clackson et al.,

1991; Marks et al., 1991). The selection of reactive

antibodies is mediated by successive rounds of elu-

tion of the bound phage, using either acid or alkali

(Marks et al., 1991). and subsequent amplification of

eluted phage. The retinement of elution techniques

has the potential to contribute significantly to the

retrieval of antibodies with novel speciticities. In this

regard, other shategies for phage elution have al-

lowed the identification of anti(kdies with novel

specificities not identified using acid elution alone.

These strategies have included the competitive elu-

tion of phage-Fab wirh specific monoclonal antibcd-

ies (Meulemans et al.. 1994). and the use of closely

related antigen in excess (Ames et al.. 1994).

We have sought to use site-specific enzymatic

cleavage of phage-Fab as a means of recovering

bound phage, a” approach which provides the oppor-

tunity for the retrieval of hound phage in a manner

independent of the affinity of the antibody. To

achieve this, we have constructed a phage display

vector which features a site for specific cleavage by

the enzyme Genenase 1, a” engineered variant of

subtilisin BPN’ containing the mutations C24A.

H64A. El56S. Gl69A and Y217L (Carter er al..

1989). These mutations have been shown to increase

the specificity and efticacy with which the enzyme

cleaves its substrate @.Iarter et al.. 1%~). The cleav-

age site PmGlyPheAlaHisTyyr has been engineered

between the heavy chais and gene III protein ele-

ments of the displ:‘yed fusion protein, so as to allow

the gentle and specific elution of bound phage-Feb.

We have further sought to test the efficacy of this

system wirh phage of known specificity retrieved

from a human immunoglobulin library made from

RNA dewed from a pericolic lymph node.

2. Materials and methods

After obtaining informed consent, a 200 pg peri-

colic lymph node was isolated from a hemicolec-

tomy specimen from a 65-yew-old woman. She had

been previously immunized with tetanus toxoid, but

had not received a booster injection for 15 yam.

Total RNA was extracted from the tissue by a gwn-

dinium isothiwyanate method (Chomczynski and

Sacchi, 1987). and a IO-30 pg sample was reverse

transcribed using the primers CGlz or CKlz for the

human yl and K constant chains. as previously

described (Persscm et al.. 1991). 3 pl of the reverse

transcription prcduct was used as a template in IO0

pl PCR reactions containing 0.3 mM dNTPs. 50 mM

KCI, IO mM Tris-HCI pH 8.3. 3 mM MgCI,. Am-

pliTaq DNA palymemse (0.04 U/p& Perkin-Elmer

Cetus) and 60 pmol of the appropriate 5 and 3

primers (after Pcnson et al., 1991). P.mp:itication

was perfomxd under the following conditions; 94°C

for 3 min. 35 cycles of 94°C for 15 s. 52°C for 50 s,

72°C for 90 s. followed by a final incubation at 72°C

for IO min. The success of each amplification was

x’etermined by agerose gel electmphoresis. and pmc-

UELS of a common 3’ primer were pooled and run on

a 1% low melting point agamse gel. Bands of appro-

priate size (660 bp) we. * excised from the gel and

purified using Magic PCR preps @‘romepa), then

digested with either Sac1 and Xbd (K chain) or

Spel and Xhol (heavy chain), before purification

with DNA Clean-Up System (Prome& prior to

ligation.

K chain PCR products (0.7 pg) were ligated with

2 pg of gel purified. SacI/XbaI linearized pComb3H

(Scripps Research Institute. La Jolla. CA) overnight

at 16°C in IO0 pi caaining 20 U T4 ligase (USB!,

and electropaated into XLI-blue cells (Stratagene).

Library size was determined by plating aliquots of

the transfomution. and Dlasmid DNA from individ-

ual colonies was digested with Sacl/Xbol in order

to determine the insert frequency. The transformation

was grown overright at 37C in 100 ml of Super-

broth (SB) containing carbenicillin (50 pg/mI) and

tetracycline (IO fig/ml). and phagemid DNA con-

taining the light chain library was prepared and

digested with Sped and X/ml. Linearized phagemid

containing the light chain library (4.7 kb) was gel

purified and ligated with the SpeI/Xhol digested

heavy chain PCR product. The ligation products

were transformed into XLI-blue cells and grown for

I h in lo0 ml of SB containing carbpnicillin (50

pg/ml) and tetracycline (IO @g/ml). VCS-Ml3

helper phage (IO” colony faming units (cfu).

Stratagene) were added. the culture grow” For a

further 1 h. and then kanamycin (70 fig/ml) was

added prior to incubation overnight et 37°C. Aliquots

of the culture were again removed for titcring and

detemtination of insert frequency. The overnight cul-

ture was cleared by centrifugation at 1600 x g for 15

min at 4°C. the phage precipitated by incubation on

ice for 60 min with 4% PEG Mxw)/O.S M N&I. and pelleted by centrifogation at 65W g for 20 min at 4°C. Phage were that resuspended in 1 ml PBS/I% BSA. microfuged to remove the bacterial debris, and stored at 4°C as library LNC. Phage were titered by infection of logarithmic phase XL-blue cells with serially diluted aliquots of the phage preparation. and subsequent inoculation onto Luria broth/cxbenicil- lin plates. FM use in panning. phage libraries were amplified by the addition of 20-50 ~1 of phagc stock to 2 ml of XL1 -blue cells at OD,, nm 0.8-I .O in SB containing tetracycline (IO &ml). After incubation ondistorbed for 30 min at 37’C. these cells were grown in 100 ml SB. and then rescued with helper phage, PEG precipitated and titered as described &we.

2.2. Selection of antigen binders by library panning

Wells of an ELISA plate (Nunc; Immunosorp) were coated for 16 i. at 4°C with 100 pl of either keyhole limp1 hemocyanin (KLH, I.5 mgjml ill carbonate buffer pH 9.6). tetanus toxoid (‘IT, ad- sorbed: CSL I:20 v/v in carbonate buffer pH 9.6) or hen egg white lysozyme (HEL, 3.0 mg/ml in PBS pH 7.4, Boehringer Mannheim). After washing three times with PBS, the wells were blocked with 200 pl PBS/2% BSA for 2 h at 3TC. A 100 pl aliquot of the amplitied phage library was added to each well (- 5 x IO” cfu/well), and the plate was incubated for 2 h at room temperature. Following three 5 min washes with each of PBS, PBS/O.]% Twa .t 20 and PBS at mom temperature, the phage were cluted with IM) mM glycine-HCI pH 3.0 for IO min. The elation solution was vigorously pipewd in the well prior to neutralization of the eluted phage with I /IO volume of I M Tris-HCI pH 8.0.

Elated phage were added to 5 ml of logarithmic XLI-blue and adsorbed without shaking for 30 min at 37oC. and then aliquots were plated to determine the output phage titer. Carbenicillin (20 @g/ml) was added to the 5 mf culture and it was shaken for 60 min at 37T. The culture was then made to a final volume of 25 ml wirh 2YT containing 2% glucose, carbenicillin (50 fig/ml), tetracycline (10 pg/ml) and 1.25 X 10” helper phage (VCS-Ml3) and incu- bated with shaking at 37’C for 2 h. Cells were centrifuged for I.5 min at 2000 X g, resuspended in

50 ml 2YT containing carbenicillin (50 #$/ml). tetracycline (IO gg/ml) and kanamycin (70 pg/tttl) and grown overnight at 37°C. Phage were prepared the next day as described above, and panning was continued for a further four rounds.

2.3. Analysis of phage binding by ELBA

An ELISA was used to determine binding of phage-Fab to antigen. Plates were coated with anti- gen in various concentration:, sod blocked as de- scribed for library panning. Pbage (5 X 10”-10’2 cfu) diluted in 100 pl PBS/I% BSA were incubated in the wells for 2 h at roan temperature, and the plates then washed three times with 200 81 of PBS/O.I% Tween 20 (PBST) and three times with PBS. They were then incubated with 100 aI of biotinylated sheep anti-Ml3 phage antibody (I /I@30 in PBS/I% BSA: S-3’. Boulder. CO) for 1 h at room temperature, and washed as before. Wells were then incubated with IO0 pl of avidin-alkaline phos- phrase (l/800 in PBS/l% BSA: Jackson) for 20 min at room temp-eratore, and wa.hed three times with 200 ~1 of PBS and three times with 200 ~1 of carbonate buffer pH 9.6. Substrate (I mg/ml p- nitmphenyl phosphate in carbonate buffer pH 9.6, 100 ~l/well; Sigma) was ad&d and the plate was incubated at 3pC. The absorbance was determined at 405 nm using a Dynatech MR7000 plate reader (Dynatech. Guernsey, UK). Absorbance greater than four times that seen with an irrelevant control anti- gen was taken to indicate binding of pbage-Fab.

2.4. Analysis of soluble Fab binding by ELISA

Soluble Fab were produced in pComb3H by re- moval of gene 111 with N/xl and Spel, as previously described (Burton et al., 1991). Following electropo- ration into XLI-blue cells. individual colonies were selected for restriction analysis with BsrNI. wd those colonies with unique digestion patterns were gmwn in 2YT containing 0.1% glucose and carbeni- cillin (50 &ml) at 37°C. Expression was induced by the addition of 0.5 mM IPTG (DeBellis and Schwartz. 1990) in late log phase, and cells were grown overnight at 30°C. The culture sopcrnatant was clarified by centrifugation at lOOil X g for I5 min at 4°C. while the periplasmic fraction was pn-

pared by chloroform extraction as previously de- scribed @&e&e et al.. 1992).

For assessment of Fab binding. plates were coated with antigen in vatiour concentmtions. and washed and blocked as described for Ibe phage ELISA. Culture samples (IO0 ~1) were added to the wells for 90 min at man temperature. After four washes with PBS/T. wells were incubated with 100 ul of alka- line ’ pbospbatase-conjugated rabbit Anti-human Ftab’),-speeific(l/IGIHl in PBS/l% BSA; Jackson1 for 60 min at room temperature. They were then washed. devr!qed and xad as described for the phage ELISA.

Equal amounts (I pg) of a 99mer (sense) and a 9lmer (antisense) synthetic oligonucleotide coding for a myc peptide tag, amber codon and Genenase I cleavage site (Fig. 1) were annealed in Sequenase reaction buffer WSB) by heating at 75°C for 2 mm followed by cooling to WC uver 90 min. 30 pmal of the double-stmnded oligonucleotide were phospho- rylated by incubation in polynucleotide kinase buffer containing 10 mM ATP and IO U polynucleortdc kinase (Boehringer Mannheim) at 2PC for I h. The double stranded oligonuclcotide was then phenol ex- tracted and ethanol precipitated before ligation into

phasphatase-treated, Spel/Bsr XI digested pComb3H vector. The construct was electmporated into XL1 -blue cells, and clones containing the car- rect synthetic oligonucleotide sequence were identi- tied by restriction enzyme analysis and confirmed by nucleotide sequencing.

In order tu assess functionality of the MC01 vector. heavy and light chain genes were excised by .Socl/Spel digestion ftum a clone of the LNC library kxwwn to bind to ‘lT (pCombJH-TT7). The fragment was then subcloned into the MC01 vector to form the co”stmct MC0 I -Tl7. To produce soluble myc- tagged Fab fragments, MCOI-TI-7 was electropo- rated into t!! non-suppressor E. co[i strain MC1061, and grown at 3PC in 15 ml of SB containing carbenicillin (50 @g/ml) and 20 mM MgCI, to an

GDta, nm of 0.8. The culture was then grown overnight in media supplemented with 0.5 mM IPTG, and culture supematant and periplasmic fractions were prepared as described above.

Culture supernatant and periplasmic samples con- taining Fab fragments were subject to 10% SDS- PAGE electmphoresis (Laemmli, 1970). and elec- troblotted to a nitrocellulose membrane. The heavy chain with the myc tag at its C-temaus was de- tected using the 9E10 monuclonal antibody (1 fig/ml) &an et al., 1985) and alkaline phos-

CT AGT GGC. CAG GCC GGC CAG CAA CAA AAA CTC ATC s G 0 * G Q E 0 K L I

.._ . . . . . c_myc,ag .._.

TCA GAA GAG GAT CTG PAT GGG GCC GCA TAG lx S E E DLNGAA F

___......_.._.__....... Amber

phataw-conjugated goat anti-mouse Fc-specific anti- body (1/1000 in PBS/l% BSA, Jackson). A sheep

anti-human K chain antibody (IO pg/ml: The Bind- ing Site) and a peroxidase-conjugated donkey anti-

sheep antibody (l/500 in PBS/l% BSA. Silenus.

Australia) were used to detect human K chains. Alkaline phosphatase-conjugated rabbit anti-human F(ab’),-specific antibody (1/1ooO: Jackson) was used to detect intact Fab fragments. P-nitrobloe tetra-

zolium chloride/5-bromo4chloro-3-indolyl phos-

phate (NBT/BCIP: Bio-Rad) was used as the alka-

line phosphatase substrate. and 3.3’,5.5’-tetramethyl- benzidine (TMB) as the peroxidase substrate (Kirkegaard and perry Labs.).

2.5.2. Genenase I cleacagc of phage-Fab

In preliminary experiments to determine the effect

of Genenase I on phage infectivity, MC01 phage (IO” cfu) were incubated for 15 min at room tem- perature in either 200 pl of Genenase I (IO pg/ml

in 10 mM Tris-HCI pH 8.0; gift of J.A. Wells, Genentech), buffer alone or 100 mM glycine-HCI pH

3.0. Phage were then allowed to infect XLI-blue

cells. and the titre of each phage preparation was determined.

For initial evaluation of Geoenase I cleavage of MCOI. phage were prepared from XLI-blur cells

transfonoed with either MCOI-Tl7, pComb3H-TT7 or helper phage. Duplicate samples (10” cfu in 100

~1 diluted in PBS/2% BSA) wcrc added to the wells of an ELJSA piale coated with Tf aqd blocked as described above. After incubation for 2 h at rwm temperature, wells were washed five times with PBS/T and once with IO mM Tris-HCI pH 8.0. Phage were then eluted from each well by the addi-

tion of 100 gl of either 100 mM glycine-HCI pH 3.0 or Genenase I (I, 5, or 10 &g/ml in IO mM Tris-HCI pH 8.0) for IO min at room temperature. Eluatcr wem transferred to a tube containing 100 pl PBS/l% BSA, and also 10 ~1 of 1.0 M Tris-HCI pH 8.0 in the case of glycine elutions. Phage titers

were determined in each eluate by infection of XLI-

blue cc!& while the number of phage remaining in ear!1 well after eluticn was determined by phage ELISA, as described above. Numbers of phage were

calculated with reference to ? standard curve of serially diluted phage pComb3H-TF7.

3. Results

3.1. Consrrucrion and penning of .!NC library

Cloning of the yl (Fd part) and K immuno-

globulin genes from cells of a pxicolic lymph node into the vector pComb3H produced a library contain- ing 2 x IO’ clones. of which 95% (38/40 clones) contained both heavy and light chains. After four

rounds of panning against TT or KLH, the ELlSA

procedure showed that phage from rhe enriched li-

braries bound to the antigen they had bees selected against, but not to irrelevant antigens including HEL and bovine serum albumin. No binders against HEL were detected after four rounds of panning. Funher

analysis of these enriched libraries demonstrated three

unique Fabs with specificity for TT, ano one with specificity for KLH (Table I). The two TT clones

shown in Table I as belonging to the same V gene families (IT6 and TT7) did not share identical nu- cleotide sequences of either the V, or V, chains.

One of the TT reactive clones that had been isolated from the LNC library (pComb3H-lT7) was cloned into MC01 (MC01 -TTY) in order to evaluate the functionality of this vector. Soluble Fah frag-

ments were produced when the MCOI-lT7 con- strwt was expressed in the non-suppressor E. co/i

dW NMN M

strain MC1061 (Fig. 2). with approximately 90% of

the Fab found in the sopematant and 10% in the

periplasmic extrx*. The presence of a myc tag on

the heavy chain of the expressed Fab was demon-

strated by immunoreactivity of the 9El0 antibody to

a 32 kDa protein under reduced conditions, and a 50

kDa protein under non-reduced conditions (Rg. 2).

MCOI-II7 and pComb3H-II7 showed similar lev-

els of expression of this Fab. as determined by

Western tnnsfer and immtmodetection with anti-hu-

man I( and anti-human Fab s~ciiic antibodies (data

not shown). Binding of the 9ElO antibody was not

seen when Fab were expressed from pComb3H.TT7

(Fig. 2).

The soluble Fab produced from MCOI-Ti7

showed specific binding activity for IT in an ELISA

(Fig. 3). and was not reactive wtth a variety of other antigens including HEL and KLH (data not shown).

Similariy, phage-displayed Fab produced from

MCOI-‘m in XL-blue cells were also shown to

bind specifically to 7T. in a manner comparable to

that seen with pCombSH-Tf7 (Fig. 4).

Initial experiments indicated that *I-: mfectivity of

MC01 phage was largely unaltered following inco- bation with either IO fig/ml G~eettase I (mean infectivity 99.6% f 4.0) or Ifn) mM glycine. pH 3.0

(mean infectivity 105% f 14) when compared to buffer alone (100%. n = 3). When used at room temperator-, Genenase 1 was effective in eluting MCO!-TT7 phage-Fabs from a TT-coated plate at concentrations from 5-10 pg/ml, but was ineffec- tivc at lower concentrations and was unable to elate

pComb3H-TT7 phage from the plate (Fig. 5). Incu-

01

bation at 37°C was ineffective in eluting pbage at

concentrations less than 20 pg/ml (data not shown). The number of phage elated with either 100 mM

glycine-HCI pH 3.0 or Genenase I (5-10 &ml) were both in the order of IO’ cfu per well, as

determined by titration after infection of XLI-blue cells. This indicate that Genenxe 1 had no signifi-

cant effect on phage infectivity as compared with acid elution.

The effectiveness of enzymatic elution in the

selection of phage was finthx assessed by panning libraries containing a defined number of anti-n phage. The LNC library was digested with Sac1 and Spel, and I +g of the cassette containing the anti-

body genes was subcloned into 1.5 pg of digested

MC01 to generate the library LNM. Tbe Mk4COI-

IT7 clone. was then inoculated into the LNM library at a concentration of 1 per 10’ and I per IO’, and the library was panned against TT using either 100 mM glycine-HCI pH 3.0 or Genenase I (5 @g/ml.

room temperature) to eiote bound phage. The re- trieval of MCOI-Tl7 and other ‘IT binding phase

was monitored by restriction enzyme analysis of

colonies, and by ELBA analysis of soluble Fab. After the first round of panning with either acid or

enzyme elution. there was 250-fold enrichment for MCOI-IT!, and after three round- all colonies rep- resented the spiked IT cms~ct (Table 2). No dele-

tions of heavy or light chain segments wet-e noted in

100 colonies after five rounds of panning. Similar results were obtained using the LNM library spiked

with I MCOI-lT7 phage per IO6 library phage. In this case, after four rounds of panning with either acid or enzymatic elution, the spiked TT constract was represented in 3/B colonies and 6/S colonies :espectivcly.

Fiwdly, a. (II enzymatic elation methods were

directly compared with respect to their ability to retrieve IT binders from the unspiked LNM library. Three unique TT binding clones were isolated with acid elation, and two were isolated using enzymatic elution. While one of these retrieved clones was common to both elution conditions, the remaining

three clones had unique restriction patterns, indicat-

ing that Gcnenase I allowed the identification of clones not found with acid elution.

4. Discussion

We have constructed and assessed a phage display vector which permits clution of bound phage-Fab

with the enzyme Genenasc 1. This vector also incor- porates a myc pcptide tag to facilitate aftinity purifi-

cation or detection of expressed Fab, and an amber codon for the production of soluble Fab without the

need for subcloning. The elution of bound pbage-Fab by site-specific

proteolytic cleavage has several theoretical ad;an- tagcs over conventional acid and alkali elution meth-

ods in the punning of phagc-Fab libraries. Firstly, since it does not rely on tbc disruption of antibody-

antigen interactions, enzymatic cleavage is indepcn- dent of the affinity of the Fab, and may thus bc more

likely to retrieve phage which encode high affinity Fabs. Secondly, it is more likely to result in the

retrieval of specific binders. since phage which have bound through means other than a specific Fab/anti- gcn interaction arc unlikely to be released by sitc- specific protcolysis.

Enzymatic cleavage of phqe-fusion proteins with

subtilisin and human Factor Xa has previously been described for the elution of human growth hormone from its binding protein (Matthews and Wells, 1993).

More recently, a trypsincleavage site has been intro- duced between light chain and gene III protein of a phage-Fub display vector. in order to facilitate rc-

lease of bound phage during clution (Orurn et al.,

1993). While nypsin elution was shown not to rc- duct infectivity in this system. the uscfulncss of trypsin cleavage in the panning of phage libraics was not dcscribcd.

Although several site-specitic cleavage enzymes

are available, we chose to USC Genenasc I, a mutant H64A form of subtilisin. This enzyme is substrare

specific, and stable over a wide rang= of conditions (Matthews and Wells. 1993). Unlike trypsin. the cleavage site is uncommon in human proteins. This reduces the possibility of clurion. and therefore sclec-

lion. of non-specifically bound Fab which contain an

internal trylsin cleavage site.

Using a TT binding phagc-Fab which we had isolated from a p&colic lymph node library. wc were able to show Gcncnasc I was as effective as acid elution in retrieval of bound phage, and drd not

alter their infectivity following elution. Accordingly,

we sought to determine the utility of enzymatic elution in the panning of a diverse phage-Fab library.

It was not possible lo make direct comparisons bc- twcen the p&colic lymph node library created in

pComb3H (LNC) and that subcloned into MCOI

(LNM), since it was likely that the diversity of this library was reduced in the process of subeloning. Therefore. we added a fined number of TT-bindine phage to ‘the LNM library, in order to provide i

model system for tbc direct comparison of the two

metiiods of clution. Using this system, we found

Genenase I to be as effective as acid elution in terms of retrieving antibodies of known binding activity from the library; a finding that was consistent with results using a single IT-binding clone.

Importantly. enzymatic elution also permitted the

retrieval from tbc library of a lT-binding clone which had not been recovered using acid elution.

This suggests that the characteristics of antibodies recovered from a given library may be affected by the method of elution employed during the panning

process. As has teen described in other vector systems

(Ward et al., 1989; Hwgcnbcam et al., 1991). we

have incorporated the gene for a myc pcptide tag into MCOI in order to facilitate affinity purification and immunodctcction of expressed antibody. In tile

past. soluble Fcb fragments have been produced either through the cc+ of amber codons in non-sup- prcsscr &. coli stmins (Hoogenkaom et al.. 1991). or

by excision of gene III with restriction enzymts (Kane et al.. 1991). We have used both these meth- .y.. ads in the MC01 vector, so as to allow production of Fab as a gne 111 fusion protein. or as B Falubte

protein. either ragged or untagged. We have demonstrated the retrieval of antibodies

specific for ll’ and KLH from a library constructed from a human p&colic lymph node. The individual from whom this library was made had been previ- ously immunized with IT. but had not received a booster injection for I5 years. and had ncvcr been exposed to KLH. Despite this fact, several Fabs

specific for ‘IT. and one reactive with KLH, were

produced from a relatively small (2 X IO’) IgGl library.

The retrieval of anti-lT Fab has previously been reported from libraries made fmm the peripheral

blcad mononuclear cells of hypcr-immunized hosts

(Mullinax et al.. 1990; Persson et al., 1991). but such Fab were not retrieved from an immunized individ- ual who had not recently received a booster injection

(Persson et al.. 1991). It has been suggested that

such booster injections may ix necessary to cnswe

tbe presence of antigen specific B cells at an accept- able frequency in the peripheral blood in order to construct a library which contains m binding phage

Fab (Lum et al., 1990; Persson et al.. 1991). The production of human anti-m Fab has not been at-

tempted from individuals who have not ken immu-

n&d for many years. but it is likely that in the years following immunisation. specific anti-IT B cells will tend to localize in sites such as lymph nodes and spleen. Such an ewnt may explain our retrieval of

specilic anti-‘IT Fab from the lymph node library. It

is possible that lymph nodes may prove a useful

source of immttnoglobulin genes for production of human recombinant Fab.

In conclusion, enzymatic elution offers an altema- tive to conventional elution techniques in the selec- tion of phagedisplayed antibodies from immuno-

globulin gene libraries. and brings with it the possi-

bility of retrieval of antibodies with specificities and aftinities not obtainable with current panning meth-

ods.

Acknowledgements

The authors wish to thank Dr. James Wells, Genentech, for helpful discussions regardins enzy- matic elution of phage, and for tbe gift of the enzyme Genenase I, and Dr. 0. Burton. Scripps Rtsearch Institute. for tbe gift of the pComb3H vector. This work was supported by the Cooperative

Research Centre for Biophatmaceutical Research, and by grants from tbe National Health & Medical Re- search Council, Australia, and the Merck Sharp L Dohme Foundation.

R&t%3Xt5

Burton. D.R.. Barbar III. C.. Persron. M.A.. Kanie. S.. Chanoek.

Caner. P.. Nilrron. 8.. Bumicr. J.P.. Burdick. D. and Wells. J.A.

(1989) Enpincerinp subdlisin BPN’ for ske-swbic wxcdy sis, hr. Srme. Fine. Gcnet. 6. 240-246.

Chonx*yrh.kt. P. and Sac&i. N. (1987, Single-step mcdK.3 Of RNA im,vhn by acid gmidi”i”m ddcty”?,lc-pknol-chloro.

form extraction. Anal. Biochem. 162, 156-159.

Clacksag 7.. Hmgenbcom. H.R.. Griffith% A.D. ati Winm. G. (19%) Making antibody fidSrwn$ “sing phase display ii-

brutes. Nwre 352.624-628. DcBc,,ir. D. and Schwartz. 1. (1950) Regulated expression of

foreign gencr f”Xd t” Joe: cOntrot by g,“cosc ,we,s in grow* medium. N”c,cw Acids Res. 18. 13, I.

Evan, 0.1.. Lewis. G.K.. Ramsay. G. and Bishop. J.M (1985) ,so,~don of mowx,ona, antibedicn specific for human e-nzye

proto~onco~nr pmd”FI. MO,. Cal. Bio,. 5. 3610-3616.

GriftiQr. AD., Williams. SC.. Hanky. CL Tomlinsoon, J.M.. Waterhouse. P.. Crosby. WS... Konlcrmann. R.E., Jones. P.T.. Low. NM.. Allison. T.J.. et al. (1994) lroladon of high affinity h”rnan anlibcdica direcdy fxxli kuge synlhetic reper- IOirc”. EMBG 1. 13. 3245-3260.

Hwgenbw!“, H.K.. Grifiths, A.,,., Johnwn. K.S / Chiswell, D.J.. Hudson. P. and Wlntcr. G. (t99,) Multi-subunit aoteina

on the surface of filamcnms phugape: me*odologics for dis playing antibody (F&b) heavy zx.b fight chains. Nucleic Acids Res. 19.4133-4137.

IOne. AS.. Jones. TM. and Bunon. D.R. (199,) Andbodv nderi8” by chain sh”f”i”S fro!” random c‘““bi”“,orial i, m”“oglob”bn libraries. Pm. Nat,. &ad. sci. US.4 88. ,,,20-1,123.

Laemmli. U.K. (1970) Ckavnne of sbyct”ra, “roteins durine *e

Lam, L.C.. Bums E.. Jaw”. M.M. and L.,,. (1990) ISO anti- tel?aur toxoid andbody synlhcris by hvman tnnc IlQlnow. 1. Two disdnct pop”,ad~~s of nwrow B cells and functional dtffennces bclwcen marrow and ped~bcra, blood 6 cells. J. Clin. Immune,. 10. 255-264

Marks, J.D., HooScnboom. H.R.. Bmnc”. T.P., MeC”ffcny. 1..

Grif,ithr. AD. and Winlcr. 0. (199,) By-pnssing immunize don. H”UD antibodies fron Y-.gc”e libties displayed on phage. 1. Ma,. Bio,. 222. 581-597.

Ma,,bcwr, D.J. and Wells. LA. (1993) S”b%ra,c ~,?a@: selccdon of proleare subsrrales by monovalent phagc display. Science 260. ,,,3-,111.

Meukmanr. E.“., Slobbe. R.. Wanerva,. I’.. Ramaekea. F. and

Vamys. G. (1994) Sekclion of phagc.display& andbodies spufic for a cy,oskclewt ;mtigcn by comperirive eluion with a manoc,ona, mc,baly. J. Ma,. Biol. 24.4. 353-360.

M”,,inar. R.L.. Cross, EA.. Amberk, J.R.. Hay. B.N., Hogrefc. H.H.. Kubiu. MM.. Greener. A.. Abing-Men, M.. Ardoure,. D.. Shnt. J.M.. Serge. J.A. and Shops. 8. (1990) Idcn,i!ica- lion of b”lnan alabody +aSnlcnt Chm”. a,wbic for a”nur

82

toxoid in a baeterinphage I immunaexprrrsion library. Pmt. Natl. Acad. Sri. USA 87, 8095-8099.

Nbrim. A.. Hwgcnbmm. H.R.. Tomlinson. I.M.. Fiynn. C.. Midgley. C.. Law. D. and Winkr. G. (1994) Antibody frag-

monks horn a ‘single pa’ phase display library as immune

cbcmicrl rcagcnu. lI&tBC ;, 13.692-698.

&urn. H.. Andcrwn. P.S.. 0s~. A.. Johanxn. LX., Rik IX.. Bjomvd. M.. Swcdscn. I. and Engbng. J. (1993) Efticirnt method for constmcting comprchcnsivc murk hb antibody

librafiies displayed ~1 phage. Nucleic Acids RCE. 21. 4491- 4498.

Ward. ES.. Cusww. D.. Griffi+s. A.D.. Jmw. P.T. ;nd Winwr. G. (1989) Binding aFliviOcs of a repnoire of single immuno- globulin variable domsns scatted from Eschtrichio COIL Nature 341.5~544.

PC-. MA,. Caothkn. RH. and Burton. D.R. Il991) Gcncra-

don of diverse high-affmiry human monwlonal andbodies by repnomz cloning. Pnx. Narl. Acal. Sfi. USA 88, 2432-2436.

Williamson, R.A.. Bmioni, R.. Sanna. PI’., Pamidge. LJ., Barbas 111. C. and Bunon, D.R. (1993) Human monwlonal antibodies

against a plcthma of viral pahogcns from single combinatorial

Ikuics. F’mc. Nad. Acad. Sci. USA 90.4141-4145. 2ebed.x. S.L., Barbas Ill. C.. Han. Y.L.. Caotbicn. R.H.. Graff,

R.. DeGraw, J.. Pyati. 1.. LaPolla. R.. Burton. D.R.. Lcmer. R.A.. et al. ( ~9921 Humm combinawial amibcdy libraries to hepatitis B ru?fze ardigcn. Rot. Nat Acad. ki. USA 89.

31753179.