www.elsevier.com/locate/jim

Journal of Immunological Methods 291 (2004) 137–151

Research paper

Isolation of human prostate cancer cell reactive antibodies

using phage display technology

Mikhail Popkov, Christoph Rader, Carlos F. Barbas III*

The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute,

La Jolla, CA 92037, USA

Received 20 February 2004; received in revised form 19 May 2004; accepted 19 May 2004

Available online 6 July 2004

Abstract

Here we describe a phage display strategy for the selection of rabbit monoclonal antibodies that recognize cell surface

tumor-associated antigens expressed in prostate cancer. Two immune rabbit/human chimeric Fab libraries were displayed on

phage and used to search for tumor-associated antigens by panning on DU145 human prostate cancer cells. For this, we

developed a novel whole-cell panning protocol with two negative selection steps designed to remove antibodies reacting with

common antigens. After three rounds of subtractive panning, a majority of clones bound to DU145 cells as detected by flow

cytometry. Among these, we identified several clones that bound selectively to DU145 cells but not to primary human prostate

epithelial cell line PrEC. In summary, our work demonstrates the potential of immune rabbit antibody libraries for target

discovery in general and the identification of cell surface tumor-associated antigens in particular.

D 2004 Published by Elsevier B.V.

Keywords: Prostate; Cancer; Phage display; Negative selection; Antibodies

1. Introduction

Prostate cancer is the most common cancer

observed in men in North America and Europe,

and is second only to lung cancer in numbers of

cancer-related deaths. Neither effective prevention

0022-1759/$ - see front matter D 2004 Published by Elsevier B.V.

doi:10.1016/j.jim.2004.05.004

Abbreviations: FACS, fluorescence activated cell sorting; HA,

influenza hemagglutinin; HUVEC, human umbilical vein endothe-

lial cells; mAb, monoclonal antibody; PSMA, prostate-specific

membrane antigen; RT, room temperature.

* Corresponding author. Department of Molecular Biology,

BCC-550, The Scripps Research Institute, 10550 North Torrey

Pines Road, La Jolla, CA 92037, USA. Tel.: +1-858-784-9098; fax:

+1-858-784-2583.

E-mail address: carlos@scripps.edu (C.F. Barbas).

nor cure is established. In the United States of Ame-

rica, prostate cancer will affect between one and two

in 10 men in their lifetimes (Trump, 2002). Al-

though most prostate cancer patients initially re-

spond to hormonal therapy, eventually the cells

become androgen-insensitive, resulting in disease

progression (Catalona, 1994). Unfortunately, there

is no effective cure that increases survival rate of

patients with hormone-refractory tumors. Therefore,

alternate strategies for treatment of prostate cancer

are needed.

Antibody therapy offers promise for cancer treat-

ment; indeed several therapeutic antibodies have

been approved and many are in clinical trials for

cancer. Antibody therapy depends on the identifica-

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151138

tion of molecular targets, i.e., antigens that are specif-

ically expressed on the cell surface of tumor cells or

tumor-supporting cells. By binding to these antigens,

antibodies can mediate the selective destruction of

tumor cells. In contrast to conventional treatments,

antibody therapy should not harm healthy cells and,

consequently, will cause far fewer side effects. Anti-

gens should be expressed at high levels on the cell

surface of tumor cells or tumor-supporting cells and

should be absent, or expressed at a very low level,

from highly sensitive tissue, including bone marrow,

heart, and the central and peripheral nervous system.

Although the ideal molecular target is expressed in

the context of the tumor only, few, if any, truly

tumor specific antigens have been identified (Hell-

strom and Hellstrom, 1997; Scott and Welt, 1997).

However, even molecular targets with less restricted

expression have proven useful for antibody therapy.

For example, the antigen targeted by the approved

mAb HerceptinR, the EGF receptor family protein

ErbB2, also called HER-2/neu in humans, is over-

expressed in 20–30% of human breast and ovarian

cancers. HER-2/neu is also expressed at low levels in

epithelial cells from a variety of organs (Disis and

Cheever, 1997). Another example is human antigen

A33, which is a promising molecular target for

antibody therapy of colon cancer, yet is expressed

in both normal and malignant colon epithelia (Heath

et al., 1997).

No approved molecular target has yet been iden-

tified for antibody therapy of human prostate cancer.

A potential molecular target is prostate-specific mem-

brane antigen (PSMA), a 100-kD integral membrane

protein, identified by mAb 7E11 derived from mice

immunized with the prostate cancer cell line LNCap

(Horoszewicz et al., 1987; Carter et al., 1996). Initial

immunohistochemical analysis showed that PSMA

expression was highly restricted to normal, benign,

and malignant prostate epithelia (Horoszewicz et al.,

1987; Lopes et al., 1990; Israeli et al., 1994). More

recent reports, however, suggest that the PSMA

expression may not be as prostate-specific as origi-

nally thought (Troyer et al., 1995; Silver et al., 1997).

The detection of PSMA in non-prostate tissue has

raised questions regarding safety of using mAbs

against PSMA for the therapy of prostate cancer.

More recently, a number of tumor-specific (as op-

posed to tumor-associated) alternative molecular tar-

gets for antibody therapy of prostate cancer have been

identified including prostate stem cell antigen (PSCA;

Reiter et al., 1998), STEAP (Hubert et al., 1999), and

plasma membrane proteins P503S, P504S, and P510S

(Xu et al., 2000). The work on PSMA, however,

illustrates the difficulties that are encountered in

identifying and evaluating molecular targets for anti-

body therapy of cancer in general.

Today, mAbs are generated by either hybridoma

technology or from antibody libraries (Rader, 2001).

Whereas hybridoma technology is, for practical rea-

sons, confined to rodents (mice, rats, and hamsters),

antibody libraries allow the generation of mAbs from

virtually any species whose immunoglobulin genes

are known (Rader and Barbas, 1997). Antibody

libraries have been used to exploit large naıve and

synthetic antibody repertoires, or combinations of

both, for the generation of human mAbs (Barbas et

al., 1992; Barbas, 1995; Hoogenboom and Chames,

2000; Rader and Barbas, 2000). In contrast to anti-

bodies derived from large naive or synthetic reper-

toires, however, antibodies from immune animals are

subjected to in vivo selection and, thus, are more

likely to selectively recognize a given antigen, i.e.,

without cross-reactivity to another antigen. In order

to most effectively use antibody libraries, both pos-

itive and negative selection strategies must be

employed.

Using both positive and negative selection strate-

gies with antibody libraries, a wide range of targets

can be identified by antibodies, e.g. molecules highly

conserved between species, toxic molecules, carbohy-

drate structures, and small haptens (Marks et al.,

1991; Griffiths et al., 1994). It is known that malig-

nant transformation of cells often causes dramatic

changes in the expression of cell surface molecules

(Boon et al., 1994; Scott and Welt, 1997). Truly

tumor-cell specific antibodies would make powerful,

and versatile, diagnostic and therapeutic reagents. To

isolate antibodies with desired specificities, phage

library selections must be performed on tumor-derived

antigen sources. However, panning of antibody library

for cellular targets has proved to be experimentally

challenging, mainly because of the tendency of phage

to bind non-specifically to cells. High antigen com-

plexity and low target antigen concentration may also

dramatically decrease the chance of antibody selec-

tion. Nevertheless, several protocols have been re-

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151 139

ported, allowing the isolation of antibodies against

cell surface antigens (Cai and Garen, 1995; de Kruif et

al., 1995; van Ewijk et al., 1997; Ridgway et al.,

1999; Kupsch et al., 1999).

In this communication, we describe a simple

whole-cell-based panning procedure for isolating

antibodies that react specifically with prostate tumor

cells from an immune, rabbit/human chimeric Fab

antibody library. In addition, we have utilized a

generally applicable positive/negative selection stra-

tegy for cell panning. Using this selection strategy,

a panel of prostate cell-specific antibodies has been

isolated. The potential use of selected antibodies

for diagnosis and therapy of prostate cancer is

discussed.

2. Materials and methods

2.1. Antibodies

High-affinity rat anti-HA antibody (clone 3F10)

and FITC-conjugated affinity-purified goat anti-rat

IgG (H +L) antibody were purchased from Roche

Molecular Biochemicals (Mannheim, Germany).

Mouse mAbs LM609 (anti-human integrin avh3)

and P1F6 (anti-human integrin avh5) were pur-

chased from Chemicon (Temecula, CA). FITC-con-

jugated donkey anti-mouse and donkey anti-rabbit

IgG (H +L) polyclonal antibodies were purchased

from Jackson ImmunoResearch (West Grove, PA).

2.2. Cell lines

Human malignant prostate cell lines DU145, PC-3,

and LNCap (ATCC numbers HTB-81, CRL-1435 and

CRL-1740, respectively), human breast carcinoma

cell lines MDA-MB-231 and MDA-MB 453 (ATCC

numbers HTB-26 and HTB-131, respectively), human

colon carcinoma cell lines HT-29 (ATCC number

HTB-38), human epidermoid carcinoma cell line A-

431 (ATCC number CRL-1555), and human ovarian

carcinoma cell line ES-2 (ATCC number CRL-1978)

were purchased from American Type Culture Collec-

tion (ATCC). Cells were cultured in RPMI 1640

medium supplemented with 10% FCS and antibiotics.

Human fibrosarcoma cell line HT-1080 (ATCC num-

ber CCL-121) was purchased from ATCC and cul-

tured in DMEM supplemented with 10% FCS, 1.5

g/l sodium bicarbonate, 0.1 mM non-essential amino

acids, 1 mM sodium pyruvate, and antibiotics. Hu-

man umbilical vein-derived endothelial cells

(HUVEC) and human prostate epithelial cells (PrEC)

were purchased from BioWhittaker (Walkersville,

MD) and maintained in EGM and PrEGM complete

media according to manufacturer’s instructions (Bio-

Whittaker). Human colon carcinoma LIM1215 and

SW1222 cells were obtained from Dr. Lloyd J. Old

(Ludwig Institute for Cancer Research, New York).

Kaposi sarcoma SLK cells were obtained from Dr.

R. Pasqualini (University of Texas, M.D. Anderson

Cancer Center). Human ovarian carcinoma UCI107

cells were obtained from Dr. Philip M. Carpenter

(University of California, Irvine Medical Center).

Human melanoma cell line M21 was obtained from

Dr. David A. Cheresh and human melanoma cell line

C8161 was obtained from Dr. Ralph Reisfeld (The

Scripps Research Institute, La Jolla, CA). All human

cell lines were maintained in RPMI 1640 containing

10% FCS and antibiotics.

2.3. Rabbit immunization and antibody library

generation

Two pairs of rabbits from the New Zealand White

strain were immunized and boosted two to three

times with either human prostate cancer cell line

LNCap or DU145. For each shot, 106 cells were

injected subcutaneously. Antisera from immune

rabbits were analyzed for binding to the tumor cells

by flow cytometry using FITC-conjugated donkey

anti-rabbit IgG (H +L) antibody for detection (Jack-

son ImmunoResearch).

Chimeric rabbit/human Fab libraries were generat-

ed as described (Rader et al., 2000a). In brief, total

RNA from spleen and bone marrow of the immune

rabbits was prepared and, after oligo(dT)-primed

reverse transcription, the antibody variable domains

VL and VH were amplified. The rabbit VL and VH

domains were then fused to human constant domains

CL and CH1 of light and heavy chain, respectively.

The combination of the chimeric light chains and

heavy chain fragments was cloned into the phagemid

vector pComb3X and resulted in a rabbit/human Fab

library displayed on phage. The pComb3X vector has

all the features of pComb3H, along with several

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151140

additions. One of the modifications is the insertion of

the influenza hemagglutinin (HA) decapeptide tag,

which facilitates detection of the protein using com-

Fig. 1. Outline of two principal cell panning protocols used to isolate Fab

Protocol 1 shows an example of positive panning against the prostate cance

line (PrEC, HUVEC, or SLK). Protocol 2 outlines an example of positive

negative panning on epitope-masked cell line of the same origin.

mercially available anti-HA mAb. Details of the

pComb3H and pComb3X were previously described

elsewhere (Barbas et al., 2001).

s from immune rabbit libraries with a broad pattern of specificities.

r tumor cell line, DU145 and negative panning against a different cell

panning against the prostate cancer cell line, DU145 or LNCap, and

nological Methods 291 (2004) 137–151 141

2.4. Selection by panning on target cells

Seven independent pannings were done using

Costar 96-well V-bottom Assay plates (Corning,

Acton, MA), each with a different setup (Fig. 1 and

Table 1). Three to four rounds of panning were carried

out, each consisting of two steps of negative selection

followed by one step of positive selection. For the

positive selection step, 107 LNCap or DU145 cells

were used, whereas for negative selection, two steps

using of 5� 106 cells, either epitope masked or not,

were used. An aliquot containing 25 Al of phage (from5� 1010 to 1012 cfu) from the rabbit immune Fab

library was blocked with 225 Al of PBS/BSA 3% to

reduce nonspecific binding to the cell surface. The

blocked phage was added to the cells (already resus-

pended in PBS/BSA 3%) used for the first step of

negative selection and mixed gently for 30 min at

room temperature. Cells were then pelleted by centri-

fugation at 500� g for 5 min. The phage-containing

supernatant was used to repeat the counter selection

step a second time. The resultant phage supernatant

was incubated with the target cells (DU145 or

LNCap) for 1 h at room temperature with gentle

mixing. The cells were pelleted and washed with

M. Popkov et al. / Journal of Immu

Table 1

Strategies applied for selection on target cells

Panning

protocol

Target cells for

negative selection

(epitope masking serum)

Target cells

for positive

selection

Enrichmenta

after 3rd

round

Protocol 1

PD PrEC (none) DU145 141

HD HUVEC (none) DU145 390

SD SLK (none) DU145 2

Protocol 2

1L LNCap (anti-LNCap) LNCap 2

1D DU145 (anti-LNCap) DU145 36

2D DU145 (anti-LIM1215) DU145 17

3D DU145 (anti-DU145) DU145 111

Panning was performed independently using either LNCap (1L) or

DU145 as Fab library. During each round, negative selection

(Protocol 1) or epitope masking (Protocol 2) were performed twice

on 5� 106 target cells followed by one-step positive selection on

107 target cells.a Enrichment was calculated as the total number of phage

recovered after the third round of selection (measured in colony-

forming units, cfu) divided by the number of cfu recovered after the

first round of selection.

PBS (three times in the first round, five in the second

round and seven times in the third and fourth rounds).

E. coli strain ER2537 in mid-logarithmic growth

phase (A550 = 0.5–0.8) was directly infected with the

resulting cell pellet and the phage were propagated as

previously described (Rader et al., 2000a; Barbas et

al., 2001). After the final round of panning, several

clones were selected randomly from each library, and

expression of soluble Fabs was induced by activation

of the LacZ promoter with IPTG as described (Barbas

et al., 2001). After overnight growth at 37jC, bacteriawere pelleted and the resulting supernatant was ana-

lyzed for binding to DU145 cells by flow cytometry

using rat anti-HA mAb for detection as described

below. Clones that bound DU145 cells were further

analyzed by DNA fingerprinting.

2.5. Fingerprint analysis of phage clones

Fab-encoding inserts of phage clones were ampli-

fied by PCR, using the primer GBACK (5VGCC CCC

TTA TTA GCG TTT GCC ATC 3 V) and the primer

OMPSEQ GTG (5 VAAG ACA GCT ATC GCG ATT

GCA GTG 3 V) and amplicons were digested with AluI

(Promega, Madison, WI). The restriction patterns of

the samples were then analyzed in 4% (w/v) agarose

gels.

2.6. Analysis of phage antibody binding by flow

cytometry

Target cells were detached from 100-mm dishes

using 1.5 ml of trypsin solution (0.25%). Cells were

washed once in 10 ml of PBS and were resuspended at

106 cells/ml in FACS buffer (1% BSA, 0.03% NaN3,

25 mM HEPES, pH 7.4 in PBS, sterile filtered).

Aliquots of 100 Al containing 105 cells were distrib-

uted into wells of V-bottom serocluster plates. Sixty

microliters of culture supernatant from IPTG-induced

bacteria cultures was mixed with 40 Al of PBS/BSA3% and incubated for at least 5 min. The entire sample

from each well was then added to the cells and

incubated for 40 min at RT. Cells were washed once

with 200 Al of FACS buffer and incubated with 100

Al of rat anti-HA-antibody, diluted to 1:100 in FACS

buffer for 40 min at room temperature. Cells were

washed once as above and incubated with 100 Al ofFITC-conjugated goat anti-rat antibody, diluted to

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151142

1:200 in FACS buffer, for 30 min at room tempera-

ture. Cells were washed twice, resuspended in 200

Al of FACS buffer, and transferred to FACS-tube for

analysis in a FACS scan flow cytometer (Becton

Dickinson).

3. Results

3.1. Generation of rabbit Fab libraries against

human prostate cancer cell line DU145 and LNCap

Two pairs of rabbits from the New Zealand White

strain were immunized and boosted two to three times

with either human prostate cancer cell line LNCap or

DU145. For each immunization, 106 cells were

injected subcutaneously. Rabbit sera were analyzed

for specific recognition of the human prostate cancer

cell lines by flow cytometry (Fig. 2). Rabbit antibody

Fig. 2. Immune rabbit serum binding to human tumor cells. Flow

cytometry histograms showing the binding of a 1:200 dilution of

pre-immune (dotted line) and immune rabbit sera (bold line) to

corresponding human prostate cancer cell lines DU145 (A) and

LNCap (B). For indirect immunofluorescence staining, cells were

incubated with corresponding serum, except for the control (fine

line), followed by FITC-conjugated donkey anti-rabbit IgG

secondary antibodies. The y-axis gives the number of events in

linear scale, the x-axis the fluorescence intensity in logarithmic

scale.

libraries were generated as has been described in

detail (Rader et al., 2000a; Andris-Widhopf et al.,

2000). Both anti-LNCap and anti-DU145 libraries

were of high complexity as indicated by the number

of independent transformants (7� 108 and 1�109,

respectively) and an extensive analysis of unselected

clones by DNA fingerprinting and sequencing (data

not shown). The number of independent transformants

correlates with the number of different antibodies in

the library (Rader et al., 2000b).

Our rabbit antibody library is based on a chimeric

Fab format (Rader et al., 2000a), i.e., variable

domains from rabbit light and heavy chains are fused

to the corresponding human constant domains. The

use of human constant domains offers several advan-

tages. First, while antigen binding is confined to the

variable domains and, thus, is not expected to be

influenced by constant domain swapping, the human

constant domains allow use of established and stan-

dardized detection and purification methods. Second,

the use of human constant regions was found to

improve the E. coli expression level of Fab (Carter

et al., 1992; Ulrich et al., 1995). Lastly, Fabs with

human constant domains are already partially human-

ized and can be readily channeled into strategies for

complete humanization (Rader et al., 1998).

3.2. Selection of rabbit antibody libraries against

human prostate cancer cell line DU145

Two immune chimeric Fab rabbit/human anti-pros-

tate cancer cells libraries were used to search for novel

tumor-associated antigens by positively selecting for

binding to either the DU145 or LNCap prostate cancer

cell lines followed by one of two negative selection

strategies. Precautions were taken to maintain the

integrity of membrane antigens during panning to

facilitate subsequent identification of antigen by ex-

pression-cloning using isolated Fab fragments. First,

live rather than fixed cells were used for panning in an

attempt to preserve surface antigens in their native

state. Second, target cells with phage bound were used

for bacterial infection, thereby avoiding loss of the

specifically bound phage due to phage internalization

(Becerril et al., 1999). Significant time was spent

establishing the parameters for successful selection

of the phage library on the human cancer cell line. It

was found that a rigorous depletion of phage that

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151 143

bound nonspecifically to the cell surface was essential

for the enrichment of specific binders.

The initial panning protocol was based on a

published report using a human naıve scFv library

and negative/positive selection to isolate antibodies

against a lung adenocarcinoma cell line (Ridgway et

al., 1999). Each round of this protocol (Fig. 1,

Protocol 1) began with a two-step negative selection

to remove cross-reactive clones to common antigens.

Following the two-steps of the negative selection,

phage were positively selected for binding to pros-

tate tumor cell line DU145. Overall, three to four

consecutive panning rounds were performed, where

the eluted phage was amplified between each round

and phage particles reintroduced each time. Three

negative selections were performed in parallel: on

PrEC, on HUVEC, and on SLK. We observed a

significant increase in the phage titer in the eluent of

the third round of selection using protocols PD and

HD (Table 1).

After completion of all panning rounds, 24 indi-

vidual clones from the third round of selection from

each protocol were randomly selected and were

screened for binding to DU145 cells by FACS anal-

ysis. Of the 24 clones tested for each protocol, 21

from PD, 16 from HD, and 18 from SD were strongly

positive for binding to DU145 cells (data not shown).

When these clones were analyzed by FACS for

binding to the primary human prostate epithelial cell

line, PrEC, we found that negative selection per se

(i.e., pre-adsorption of the phage libraries on irrele-

vant human cells prior to positive selection on the

human prostate cancer cell line DU145) did not

eliminate nonspecific binders efficiently. Therefore,

four steps of extensive absorption were incorporated

into the negative selection immediately after the

second round of panning. Four fresh aliquots of

5� 106 PrEC cells were used in this negative panning

step. One further positive round of panning was then

performed. Forty phage clones were randomly chosen

for FACS assays and none bound to prostate cancer

cell lines DU145 or PC-3 (data not shown).

Fig. 3. Phage selection on human prostate cancer cell line DU145.

Flow cytometry histograms showing binding of phage pools from

rabbit/human Fab library to DU145 cells after zero to four rounds of

selection on whole cells using Protocol 3D. For indirect immuno-

fluorescence staining, cells, except control, were incubated with

phage. Rat anti-HA secondary mAb and FITC-conjugated goat anti-

rat IgG tertiary antibodies were used for detection. The y-axis gives

the number of events in linear scale, the x-axis gives the fluorescence

intensity in logarithmic scale.

Fig. 4. DNA fingerprints. Representative DNA fingerprints of rabbit/human Fabs selected on human prostate cancer cell lines using Protocols

PD (A), HD (B), SD (C), 1L (D), 1D (E), 2D (F), and 3D (G). The rabbit/human Fab-encoding sequence was amplified by PCR and

subsequently digested with the restriction enzyme AluI. Different patterns indicate individual clones.

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151144

Table 2

Summary of the DNA fingerprinting analysis of rabbit/human Fab

selected on human prostate cancer cell lines using Protocol 1 PD

(A), HD (B), and SD (C)

(A) AluI

fingerprint

type

Number

of tumor

cell

binding

clonesa

Clone

identity

PDX

(B) AluI

fingerprint

type

Number

of tumor

cell

binding

clonesb

Clone

identity

HDX

1 1 01 1 4 06

2 5 02 2 3 02

3 2 03 3 3 08

4 1 04 4 2 05

5 1 05 5 2 10

6 2 06 6 2 12

7 2 08

8 1 10

9 1 12

10 1 13

11 1 14

12 1 15

13 1 16

14 1 17

(C) AluI

fingerprint

type

Number

of tumor

cell

binding

clonesc

Clone

identity

SDX

1 4 01

2 4 02

3 3 04

4 3 11

5 1 05

6 1 09

7 1 10

8 1 20

a DU145 positive clones by single clone FACS. Total number is

21 positives clones out of 24 tested for PD setup.b DU145 positive clones by single clone FACS. Total number is

16 positives clones out of 24 tested for HD setup.c DU145 positive clones by single clone FACS. Total number is

18 positives clones out of 24 tested for HD setup.

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151 145

Based on these results, we developed a new cell

panning strategy in which both negative and positive

selections were performed using the same cell line

(Fig. 1, Protocol 2). For negative selection, the cells

were masked with immune serum that originated from

the same rabbit (Table 1, Protocols 1L and 3D) or

from a different rabbit (Table 1, Protocols 1D and

2D). This negative selection strategy was designed to

eliminate phage that bound nonspecifically to the cell

surface or that bound to nonspecific antigens not

masked by the immune serum, but avoided the loss

of tumor-reactive specificities to over-expressed mol-

ecules also presented on primary human cell lines. In

each of three or four rounds of panning, the antibody

libraries were sequentially subjected to two negative

selections followed by a positive selection on

unmasked cells. Phage bound to unmasked cells were

then rescued by addition of male E. coli and re-

amplification as usual (Rader et al., 2000b). In selec-

tions on whole cells, DU145 cells showed better

enrichment and recovery than LNCap cells and thus

represent a better target for the positive selection step

(Table 1).

3.3. DNA fingerprint analysis of specific binders

Phage populations after each round of panning

were then monitored by flow cytometry as described

(Steinberger et al., 2000). As shown in Fig. 3, the

phage from the third and fourth rounds using Proto-

col 3D bound strongly to DU145 cells, whereas

neither phage from earlier rounds nor unselected

phage bound. This apparent increase in binding to

DU145 cells encouraged us to screen individual

phage from different panning protocols for selective

binding to tumor cells. After completion of all

panning rounds, 24 to 26 individual clones were

randomly selected and tested by FACS for their

ability to bind DU145 cells. Of 24 clones tested,

21 clones from Protocol 1D and 16 clones from

Protocol 2D were strongly positive (data not shown).

Of 26 clones from Protocol 1L, 20 were positive.

Extensive analysis was performed on clones from

Protocol 3D where 106 of 135 analyzed bound to

DU145 cells.

Each of the Fab clones from phage that tested

positive for binding to DU145 cells was analyzed by

DNA fingerprinting using the frequently cutting re-

striction enzyme AluI with the recognition sequence

AGCT. In the course of these studies, we found that

AluI gives a more distinctive DNA fingerprint than the

more generally used restriction enzyme BstOI

(CCWGG, W=A or T; Steinberger et al., 2000).

The DNA fingerprint of selected clones is shown in

Fig. 4. Tables 2 and 3 show the numbers of clones

identified with different AluI patterns from each

Table 3

Summary of the DNA fingerprinting analysis of rabbit/human Fab

selected on human prostate cancer cell lines using Protocol 2 3D

(A), 2D (B), 1D (C), and 1L (D)

(A) AluI

fingerprint

type

Number

of tumor

cell

binding

clonesa

Clone

identity

3DX

(B) AluI

fingerprint

type

Number

of tumor

cell

binding

clonesb

Clone

identity

2DX

1 22 09 1 2 03

2 18 03 2 1 05

3 15 13 3 1 07

4 19 10 4 1 08

5 5 08 5 1 09

6 5 32 6 1 10

7 3 28 7 1 11

8 3 29 8 1 12

9 3 22 9 1 13

10 1 40 10 3 14

11 1 41 11 1 15

12 1 45 12 5 16

13 2 70 13 1 18

14 1 71 14 1 24

15 2 01

16 1 06

17 2 02

18 1 137

19 1 125

(C) AluI

fingerprint

type

Number

of tumor

cell

binding

clonesc

Clone

identity

1DX

(D) AluI

fingerprint

type

Number

of tumor

cell

binding

clonesd

Clone

identity

1LX

1 4 08 1 4 01

2 2 03 2 3 04

3 1 02 3 1 06

4 1 04 4 4 07

5 1 05 5 1 09

6 1 06 6 1 15

7 1 07 7 2 16

8 1 10 8 1 21

9 1 11 9 3 25

10 1 12

11 1 13

12 1 14

a DU145 positive clones by single clone FACS. Total number is

106 positives clones out of 135 tested for 3D setup.b DU145 positive clones by single clone FACS. Total number is

21 positives clones out of 24 tested for 2D setup.c DU145 positive clones by single clone FACS. Total number is

16 positives clones out of 24 tested for 1D setup.d LNCap positive clones by single clone FACS. Total number is

20 positives clones out of 26 tested for 1L setup.

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151146

protocol. For example, for phage selected using Pro-

tocol 3D, 19 different DNA fingerprints were obtained

from the analysis of 106 positive clones (Table 3).

About 75% of the clones were found to be identical to

one of four clones, designated 3D09, 3D03, 3D13,

and 3D10, while the remaining 25% belonged to a

group of clones that was either unique or repeated two

to five times (Table 3).

3.4. FACS analysis of specific binders on target cells

A representative clone based on the best prelimi-

nary expression profile ratio of binding to DU145/

PrEC was selected from the each panning protocol to

test for binding against prostate cancer cell lines. Fig.

5 shows the results. All the clones were found to bind

strongly to DU145 and PC-3 cells, and less efficiently

to PrEC and LNCap cells. One clone, 3D45, whose

DNA fingerprint was found to be unique (Table 3),

demonstrated significant binding to DU145 and PC-3

cells with minimal cross-reactivity to primary human

prostate epithelial cells PrEC and no binding at all to

HUVEC (Fig. 5). Based on the variation in protein

expression profiles, it is likely that the seven selected

Fabs each recognize a different antigen. However, the

identity of the antigens needs to be determined. None

of our selected antibodies recognize the integrins avh3

and avh5, as deduced from the fact that seven of the

clones bound weakly to HUVEC cells, a cell line that

express a high level of avh3, and that the level of avh5

expression was considerably lower on each of the

other cell lines tested (Fig. 5).

Seven representative rabbit/human Fabs for each of

the different panning protocols were further analyzed

for the binding to a panel of 13 human tumor cell

lines. The panel contained cell lines derived from three

colorectal, three breast, two ovarian, two melanoma,

and a Kaposi sarcoma, a fibrosarcoma, and an epider-

moid tumor (Table 4). All clones reacted strongly with

androgen-independent cell lines DU145 and PC-3. The

clones reacted weakly with hormone-dependent

LNCap cells. Three of the clones SD20, 1D06, and

3D45 showed high antigen over-expression on DU145

cells (up to 12.5 times for the 3D45 clone relative to

background as described in Table 4). All clones reacted

with several other tumor lines. However, clone 3D45

demonstrated no reactivity with HUVEC, SW1222,

UCI107, or M21 cell lines. Mouse mAbs LM609 (anti-

Fig. 5. Flow cytometry analysis of rabbit/human Fab binding to human prostate cancer (LNCap, PC-3, and DU145) and human primary (PrEC

and HUVEC) cell lines. Flow cytometry histograms show the binding of rabbit/human Fabs as a bold line. The background of FITC-conjugated

secondary antibodies is shown as a dashed line.

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151 147

Table 4

Protein expression on different cell linesa

Cell line Clone identity

PD05 HD02 SD20 1L15 1D06 2D09 3D45

Primary

PrEC 1.0 1.0 1.0 1.0 1.0 1.0 1.0

HUVEC 0.1 0.2 0.1 0.2 0.2 0.2 neg

Prostate carcinomas

DU145 4.8 4.7 9.6 5.1 11.0 5.3 12.5

PC-3 2.9 2.9 4.2 3.1 4.7 3.2 6.5

LNCap 0.1 0.3 0.1 0.2 0.2 0.2 0.1

Colon carcinomas

LIM1215 1.2 1.0 1.0 0.8 0.5 0.8 1.0

HT29 0.4 0.4 0.3 0.3 0.2 0.4 0.3

SW1222 0.1 0.1 0.1 0.1 0.1 0.1 neg

Breast carcinomas

MDA/MB231 2.8 2.6 2.7 2.6 2.6 2.7 2.6

MDA/MB435 0.3 0.5 0.4 0.4 0.4 0.4 0.2

SKBR-3 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Ovarian carcinomas

ES-2 2.8 2.3 2.5 1.9 1.5 2.1 1.9

UCI107 0.1 0.1 0.1 0.2 0.1 0.1 neg

Melanomas

M21 0.1 0.1 0.1 0.1 0.2 0.1 neg

C8161 2.4 2.1 2.5 2.4 2.6 2.5 2.4

Various cancers

SLK 4.0 2.6 3.1 2.4 1.5 2.4 2.4

HT1080 2.0 2.1 2.0 1.9 2.1 2.0 2.0

A431 0.8 0.8 1.0 0.8 0.7 0.9 0.4

a To allow a comparison of cell lines with different protein

expression levels, the MFI signal obtained for binding to different

cell lines was divided by the MFI signal obtained for binding to

PrEC after subtracting the background signal obtained with FITC-

conjugated secondary antibody that was used for detection.

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151148

human integrin avh3) and P1F6 (anti-human integrin

avh5) were used for comparison (Fig. 5).

4. Discussion

Phage antibody library technology has been used

to generate high-affinity antibodies against previously

defined tumor-associated antigens, such as-CEA and

c-erbB-2 (Begent et al., 1996; Osboum et al., 1996;

Schier et al., 1996). Model systems have been used to

demonstrate the feasibility of isolating an antibody

with specificity for a single antigen using a whole-cell

based panning technique, but these protocols are not

applicable to antigens in their natural environment on

cell surfaces or in settings where the target molecule is

unknown (Waiters et al., 1997; Pereira et al., 1997a).

A difficulty in the use of large, non-immune or

synthetic repertoires for selection is that they contain

antibodies to a wide range of epitopes expressed on

the antigenic surface (Marks et al., 1991; Nissim et al.,

1994). Therefore, antibodies with the desired speci-

ficity may be largely obscured by clones that bind to

irrelevant epitopes. To direct the selection of phage-

expressed antibodies towards specific epitopes, sub-

traction and depletion strategies have been devised

and successfully used (Ames et al., 1994; Cai and

Garen, 1995; de Kruif et al., 1995; Kipriyanov et al.,

1996; Palmer et al., 1997). Alternatively, the starting

frequency of antigen-specific clones in the primary

library may be increased by immunization with the

antigen of interest (in those cases, where the antigen is

known: Ames et al., 1994) or by using patient-derived

repertoires (when the antigen is unknown: Cai and

Garen, 1995). Since these repertoires are shaped by

the immune system, they contain a higher starting

frequency of antibodies with affinity for the antigen of

interest, thus increasing the chances of isolating those

with the desired specificity.

Several groups have successfully selected anti-

bodies from libraries using whole cells. Portolano et

al. (1993) used pairs of untransfected and transfected

COS cells for library pre-clearing and selection.

Combinations of erythrocytes from different blood

groups have been used to select blood group-specific

antibody phages (Marks et al., 1993). Although ef-

fective in principle, these approaches require the

availability of cloned genes or mutant cell lines. More

recently, Noronha et al. (1998) have selected anti-

bodies from a semi-synthetic scFv phage library for

binding to antigens on human melanoma cells. The

authors performed four rounds of selection on mela-

noma cells, followed by extensive post-absorption on

human B lymphoid cell lines, but without amplifica-

tion in bacteria. Ridgway et al. (1999) used pairs of

non-tumor bronchial epithelial and lung adenocarci-

noma cell lines for naive human scFv library pre-

clearing/selection. Topping et al. (2000) used negative

panning against a breast carcinoma cell line to isolate

a panel of colorectal tumor-reactive antibodies from a

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151 149

human synthetic scFv library by positive selection on

a colon carcinoma cell line. Although convenient, a

potential disadvantage of using cell lines for library

clearing is that immortal cells up-regulate the expres-

sion of antigens associated with proliferation. Selec-

tion against antibodies to proliferation-associated

antigens will result in the loss of tumor-reactive clones

during selection. A different approach for negative

selection was used by Kupsch et al. (1999). PBMCs

were used as a source of highly diverse and abundant

normal human cells for the negative selection step and

two antibodies were selected from a human scFv

library that reacted with melanoma cells but not with

normal human tissues were found.

We adopted a different approach to the pre-clearing

problem for isolating phage antibodies that react

specifically with cell surface antigens on prostate

cancer tumor cell lines. Epitope-masking panning

against the same prostate cancer cells of interest was

used to remove antibodies with unwanted specific-

ities. This strategy was compared to negative selection

against primary epithelial cells. When two negative

panning steps against epithelial cells (Protocol 1) were

followed by positive panning steps with prostate

cancer cells, cross-reactive specificities were not

eliminated. This is probably due to the amplification

of the few clones that bind antigens that are highly

expressed on the cell surface. Addition of two more

negative panning steps resulted in complete loss of

antibody specificity. To reduce the negative selection

pressure on rare, specific clones we used negative

panning on epitope-masked prostate cancer cells (Pro-

tocol 2). Use of the same cell line for negative and

positive selections has the advantage that all rare

binders will be preserved from elimination. Moreover,

the primary cell approach is impractical in most cases

because they are difficult to grow in numbers suffi-

cient for pre-clearing. Here we have shown that three

rounds of negative/positive selection were required to

isolate clones with the desired, restricted specificity

for the prostate cancer cells. Obviously increasing the

number of panning rounds allowed the introduction of

minor mutations that may cause an increase in affinity

(Marks et al., 1991). It is likely that the number of

panning rounds required will vary depending on the

antigen and type of cell used. Using the optimized

protocol (Protocol 3D), it was possible to isolate

prostate cancer-specific antibody after screening less

than 150 clones by DNA fingerprinting followed by

FACS. In contrast, Cai and Garen (1995) screened

1700 clones before discovering a melanoma-specific

antibody.

Clones were first tested by FACS for binding to

cultured human prostate epithelial and to endothelial

cells. This allowed us to identify the prostate cancer-

specific antibodies that do not react or react only

weakly with at least two normal cell types. A group

of seven such clones encoding different antibodies

were further tested by FACS on a panel of 13 human

tumor lines. Several interesting antibodies were iden-

tified: (1) a prostate tumor-specific clone 3D45 (iso-

lated using Protocol 2) with binding restricted to two

hormone-independent prostate cancer cell lines and

several of the other tumor lines, but not to normal

prostate epithelial or endothelial cells; (2) tumor-

specific clones SD20 (isolated using Protocol 1) and

1D06 (isolated using Protocol 2) that bound to pros-

tate cancer cells and also reacted with prostate epithe-

lial cells; and (3) four other clones that reacted with

several tumor lines tested and therefore appear to

recognize antigens common to tumor, but not normal

cells.

A clinical problem in treating prostate cancer is the

conversion of androgen-sensitive tumors to a hor-

mone-refractory state after treatment with anti-andro-

gen therapy. At present no specific therapy is available

for androgen-independent prostate cancer. This study

points to the possibility of targeting the epitopes

present on DU145 and PC-3 cells with antibodies.

Fab 3D45 targets such an epitope. The density of this

epitope is approximately 20 times higher on DU145

cells than on LNCap cells. The reactivity with normal

cells, HUVEC and PrEC, was 10 times lower than

with DU145. The antibody 3D45 is of therapeutic

interest for the androgen-independent prostatic cancer.

One of the useful aspects of antibody-phage dis-

play technology, demonstrated by this study, is that a

second cell line is not required. Simple adjustments to

the negative selection step using epitope masked cells

make it possible to isolate either highly specific or

highly cross-reactive antibodies. Most of our clones

reacted with antigens that are also expressed on

tumors from cancers other than prostate. Therefore,

the epitope masking approach could be used for

isolation of antibodies against tumor markers in gen-

eral. A tumor cross-reactive antibody that could be

M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151150

used for the treatment of a range of cancers of similar

tissue type would be highly valuable. Antibodies with

more restricted specificities could identify prognostic

markers or antigens involved in tumor progression.

Most importantly, these results suggest that it is

possible to adapt the epitope-masking strategy out-

lined here to generate a panel of tumor-reactive anti-

bodies from human immune-donor antibody libraries,

provided that the plasma from the same donors is

readily available. This study has implications for the

design of experiments aimed at identifying novel

tumor-associated antigens.

Acknowledgements

This study was supported by NIH grants RO1-

CA094966, RO1-CA027489, and by a Clinical

Investigation Grant from the Cancer Research Insti-

tute. We thank John A. Neves and Lothar Goretzki for

technical assistance.

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