Journal of Immunological Methods 291 (2004) 137 – 151
www.elsevier.com/locate/jim
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
Abbreviations: FACS, fluorescence activated cell sorting; HA,
influenza hemagglutinin; HUVEC, human umbilical vein endothelial 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).
0022-1759/$ - see front matter D 2004 Published by Elsevier B.V.
doi:10.1016/j.jim.2004.05.004
nor cure is established. In the United States of America, prostate cancer will affect between one and two
in 10 men in their lifetimes (Trump, 2002). Although most prostate cancer patients initially respond 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 treatment; indeed several therapeutic antibodies have
been approved and many are in clinical trials for
cancer. Antibody therapy depends on the identifica-
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M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
tion of molecular targets, i.e., antigens that are specifically 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. Antigens 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 (Hellström and Hellström, 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 overexpressed 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 identified for antibody therapy of human prostate cancer.
A potential molecular target is prostate-specific membrane 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 originally 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 opposed 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 antibody 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 reasons, 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 naı̈ve 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 antibodies derived from large naive or synthetic repertoires, 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 positive and negative selection strategies must be
employed.
Using both positive and negative selection strategies with antibody libraries, a wide range of targets
can be identified by antibodies, e.g. molecules highly
conserved between species, toxic molecules, carbohydrate structures, and small haptens (Marks et al.,
1991; Griffiths et al., 1994). It is known that malignant 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 complexity and low target antigen concentration may also
dramatically decrease the chance of antibody selection. Nevertheless, several protocols have been re-
M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
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 strategy 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 purchased from Chemicon (Temecula, CA). FITC-conjugated 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 A431 (ATCC number CRL-1555), and human ovarian
carcinoma cell line ES-2 (ATCC number CRL-1978)
were purchased from American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640
medium supplemented with 10% FCS and antibiotics.
Human fibrosarcoma cell line HT-1080 (ATCC number CCL-121) was purchased from ATCC and cul-
139
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. Human 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 (BioWhittaker). 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 (Jackson ImmunoResearch).
Chimeric rabbit/human Fab libraries were generated 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
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M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
additions. One of the modifications is the insertion of
the influenza hemagglutinin (HA) decapeptide tag,
which facilitates detection of the protein using com-
mercially available anti-HA mAb. Details of the
pComb3H and pComb3X were previously described
elsewhere (Barbas et al., 2001).
Fig. 1. Outline of two principal cell panning protocols used to isolate Fabs from immune rabbit libraries with a broad pattern of specificities.
Protocol 1 shows an example of positive panning against the prostate cancer tumor cell line, DU145 and negative panning against a different cell
line (PrEC, HUVEC, or SLK). Protocol 2 outlines an example of positive panning against the prostate cancer cell line, DU145 or LNCap, and
negative panning on epitope-masked cell line of the same origin.
M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
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 (from
5 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 resuspended 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 centrifugation 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
Table 1
Strategies applied for selection on target cells
Target cells
for positive
selection
Enrichmenta
after 3rd
round
Protocol 1
PD
PrEC (none)
HD
HUVEC (none)
SD
SLK (none)
DU145
DU145
DU145
141
390
2
Protocol 2
1L
LNCap (anti-LNCap)
1D
DU145 (anti-LNCap)
2D
DU145 (anti-LIM1215)
3D
DU145 (anti-DU145)
LNCap
DU145
DU145
DU145
2
36
17
111
Panning
protocol
Target cells for
negative selection
(epitope masking serum)
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 colonyforming units, cfu) divided by the number of cfu recovered after the
first round of selection.
141
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, bacteria
were pelleted and the resulting supernatant was analyzed 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 amplified 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 3V) 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 distributed into wells of V-bottom serocluster plates. Sixty
microliters of culture supernatant from IPTG-induced
bacteria cultures was mixed with 40 Al of PBS/BSA
3% 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 of
FITC-conjugated goat anti-rat antibody, diluted to
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M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
1:200 in FACS buffer, for 30 min at room temperature. 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
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 advantages. 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 standardized 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 humanized 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
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.
Two immune chimeric Fab rabbit/human anti-prostate 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 expression-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
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 naı̈ve scFv library
143
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 prostate 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 individual clones from the third round of selection from
each protocol were randomly selected and were
screened for binding to DU145 cells by FACS analysis. 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 irrelevant 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 immunofluorescence staining, cells, except control, were incubated with
phage. Rat anti-HA secondary mAb and FITC-conjugated goat antirat 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.
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M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
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) 137–151
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 molecules 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 reamplification as usual (Rader et al., 2000b). In selections 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 Protocol 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-
145
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 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 Clone
(B) AluI
of tumor identity fingerprint
cell
type
PDX
binding
clonesa
Number Clone
of tumor identity
cell
HDX
binding
clonesb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
5
2
1
1
2
2
1
1
1
1
1
1
1
4
3
3
2
2
2
01
02
03
04
05
06
08
10
12
13
14
15
16
17
1
2
3
4
5
6
06
02
08
05
10
12
(C) AluI
Number Clone
fingerprint of tumor identity
type
cell
SDX
binding
clonesc
1
2
3
4
5
6
7
8
4
4
3
3
1
1
1
1
01
02
04
11
05
09
10
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.
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M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
protocol. For example, for phage selected using Protocol 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 preliminary 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 epidermoid 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-
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 Clone
(B) AluI
of tumor identity fingerprint
cell
3DX
type
binding
clonesa
Number Clone
of tumor identity
cell
2DX
binding
clonesb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
22
18
15
19
5
5
3
3
3
1
1
1
2
1
2
1
2
1
1
2
1
1
1
1
1
1
1
1
3
1
5
1
1
09
03
13
10
08
32
28
29
22
40
41
45
70
71
01
06
02
137
125
1
2
3
4
5
6
7
8
9
10
11
12
13
14
03
05
07
08
09
10
11
12
13
14
15
16
18
24
(C) AluI
Number Clone
(D) AluI
fingerprint of tumor identity fingerprint
type
cell
1DX
type
binding
clonesc
Number Clone
of tumor identity
cell
1LX
binding
clonesd
1
2
3
4
5
6
7
8
9
10
11
12
4
3
1
4
1
1
2
1
3
4
2
1
1
1
1
1
1
1
1
1
1
08
03
02
04
05
06
07
10
11
12
13
14
1
2
3
4
5
6
7
8
9
01
04
06
07
09
15
16
21
25
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) 137–151
147
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.
148
M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
Table 4
Protein expression on different cell linesa
Cell line
Clone identity
PD05 HD02 SD20 1L15 1D06 2D09 3D45
Primary
PrEC
HUVEC
1.0
0.1
1.0
0.2
1.0
0.1
1.0
0.2
1.0
0.2
1.0
0.2
1.0
neg
Prostate carcinomas
DU145
4.8
PC-3
2.9
LNCap
0.1
4.7
2.9
0.3
9.6
4.2
0.1
5.1
3.1
0.2
11.0
4.7
0.2
5.3
3.2
0.2
12.5
6.5
0.1
Colon carcinomas
LIM1215
1.2
HT29
0.4
SW1222
0.1
1.0
0.4
0.1
1.0
0.3
0.1
0.8
0.3
0.1
0.5
0.2
0.1
0.8
0.4
0.1
1.0
0.3
neg
Breast carcinomas
MDA/MB231 2.8
MDA/MB435 0.3
SKBR-3
0.1
2.6
0.5
0.1
2.7
0.4
0.1
2.6
0.4
0.1
2.6
0.4
0.1
2.7
0.4
0.1
2.6
0.2
0.1
Ovarian carcinomas
ES-2
2.8
UCI107
0.1
2.3
0.1
2.5
0.1
1.9
0.2
1.5
0.1
2.1
0.1
1.9
neg
Melanomas
M21
C8161
0.1
2.4
0.1
2.1
0.1
2.5
0.1
2.4
0.2
2.6
0.1
2.5
neg
2.4
Various cancers
SLK
4.0
HT1080
2.0
A431
0.8
2.6
2.1
0.8
3.1
2.0
1.0
2.4
1.9
0.8
1.5
2.1
0.7
2.4
2.0
0.9
2.4
2.0
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 FITCconjugated secondary antibody that was used for detection.
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 specificity may be largely obscured by clones that bind to
irrelevant epitopes. To direct the selection of phageexpressed antibodies towards specific epitopes, subtraction 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 antibodies 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 effective in principle, these approaches require the
availability of cloned genes or mutant cell lines. More
recently, Noronha et al. (1998) have selected antibodies from a semi-synthetic scFv phage library for
binding to antigens on human melanoma cells. The
authors performed four rounds of selection on melanoma cells, followed by extensive post-absorption on
human B lymphoid cell lines, but without amplification in bacteria. Ridgway et al. (1999) used pairs of
non-tumor bronchial epithelial and lung adenocarcinoma cell lines for naive human scFv library preclearing/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
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 expression of antigens associated with proliferation. Selection 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 specificities. 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 (Protocol 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 sufficient 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
149
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 cancerspecific 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 identified: (1) a prostate tumor-specific clone 3D45 (isolated 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) tumorspecific clones SD20 (isolated using Protocol 1) and
1D06 (isolated using Protocol 2) that bound to prostate cancer cells and also reacted with prostate epithelial 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 hormone-refractory state after treatment with anti-androgen 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 display 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 general. A tumor cross-reactive antibody that could be
150
M. Popkov et al. / Journal of Immunological Methods 291 (2004) 137–151
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 outlined here to generate a panel of tumor-reactive antibodies 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 RO1CA094966, RO1-CA027489, and by a Clinical
Investigation Grant from the Cancer Research Institute. We thank John A. Neves and Lothar Goretzki for
technical assistance.
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