Camel Heavy Chain Antibodies Against Prostate-Specific Membrane Antigen

HYBRIDOMA
Volume 31, Number 6, 2012
ª Mary Ann Liebert, Inc.
DOI: 10.1089/hyb.2012.0048
Camel Heavy Chain Antibodies Against
Prostate-Specific Membrane Antigen
Mehdi Evazalipour,1,2 Bahram Soltani Tehrani,2 Mohsen Abolhassani,1
Hamid Morovvati,3 and Kobra Omidfar 4
Prostate-specific membrane antigen (PSMA), a type II integral membrane glycoprotein, is highly overexpressed
in all forms of prostate cancer tissues. It has also been demonstrated in a wide range of neovasculature of nonprostatic solid tumors, including bladder, pancreas, lung, kidney, colorectal, and gastric cancers. Given the
unique expression of PSMA, it is considered an alluring target for antibody-based imaging and therapy of
cancer. In the present study, the production and characterization of camel heavy chain antibodies (HCAbs)
specific for the external domain of the PSMA are reported. Due to the absence of the CH1 domain, HCAbs are
smaller than their counterparts in conventional antibodies. In this study, camel antibodies were generated
through immunization of Camelus dromedarius with a synthetic 28 amino acid peptide corresponding to the
external surface domain of antigen and PSMA-expressing cell lines. Different binding properties to protein A
and protein G affinity columns were deployed to separate three subclasses of camel IgG. The affinity purified
HCAbs bound selectively to the synthetic peptide in enzyme linked immunosorbent assay (ELISA) and reacted
specifically with PSMA-expressing cell line through imunocytochemistry study. Currently, we are attempting to
develop recombinant variable domain of these heavy chain antibodies (VHH or nanobody) for tumor imaging
and cancer therapy.
Introduction
P
rostate cancer is a major cause of morbidity and
mortality among men. Treatment approaches for patients
with advanced prostate cancer are confined. Hormone therapy by androgen ablation and anti-androgenic agents is the
only standard treatment for advanced prostate cancer, and is
effectual only with hormone responsive disease.
Since there is little effective treatment for metastatic prostate disease, it is critical to recognize genes and/or gene
products that represent diagnostic and prognostic markers, as
well as targets for treatment. Prostate-specific antigen (PSA) is
such a beneficial cancer marker in the clinical diagnosis and
staging of prostate cancer. Since the PSA cannot distinguish
benign prostatic hyperplasia (BPH) from prostatitis or prostate cancer in the range of 4 to 10 ng/mL, a cytologic and/or
histologic assessment to confirm the accurate diagnosis is
essential.(1)
Prostate-specific membrane antigen (PSMA) is a type II
transmembrane glycoprotein with a molecular weight of
about 110 kDa (Fig. 1). This protein represents neurocarboxypeptidase and folate hydrolase activity and is reported to be
involved in the neuroendocrine regulation of prostate growth
1
2
4
and differentiation. It is predominantly expressed by prostatic
epithelial cells and its expression is increased in prostate
cancer, particularly in poorly differentiated, metastatic, and
hormone refractory carcinomas.(2–5)
Moreover, PSMA is also expressed in neovasculature of
extraprostatic solid tumors, including bladder, pancreas,
lung, kidney, colorectal, and gastric cancers.
It is also identified in endothelial cells of capillary vessels in
peritumoral and endotumoral areas of certain malignancies,
such as renal cell carcinomas and colon carcinomas, but not in
blood vessels of normal tissues. Further, PSMA is reported to
be related to tumor angiogenesis.(6–8) Several antibodies
against the extracellular fragment of PSMA have been described.(9–18) More recently, human and humanized antibodies with the capability of binding to PSMA have been
described.(19–21)
Such antibodies have been deployed for imaging of prostate cancer cells.(22,23) In addition, they are considered to be
one of the therapeutic approaches to treat prostate cancer,
typically as a conjugate of a chemotherapeutic agent or radioactive isotope.(14,16,24,25)
Development of effective anti-PSMA antibodies to treat and/
or prevent diseases involving PSMA expression, particularly
Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
Cellular and Molecular Research Center; 3Animal Facility, Guilan University of Medical Sciences, Guilan, Iran.
Endocrine and Metabolism Research Center, Tehran University of Medical Sciences, Tehran, Iran.
424
PRODUCTION AND CHARACTERIZATION OF HCAbs AGAINST PSMA
425
Cell culture
At this stage, prostate cancer cell line overexpressing
human PSMA (LNCaP) and the prostate cancer cell lines,
which do not express the human PSMA (DU145 and PC-3),
were cultured routinely in high glucose DMEM (Gibco, Grand
Island, NY) while 10% fetal bovine serum (Gibco) and penicillin and streptomycin antibiotics were added as a supplementary. All cell lines were gained from the National Cell
Bank of Iran at Pasteur Institute.
Immunization of Camelus dromedarius
with LNCaP cell line and immunogen
FIG. 1. Schematic of PSMA demonstrating extracellular, a
hydrophobic transmembrane, and a short NH2-terminal cytoplasmic domain. Nine potential N-glycosylation sites have
been seen in the extracellular domain of the PSMA(Y).
those with cytotoxic effects and without the need to be conjugated to a chemotherapeutic agent or radioactive isotope, can
be considered a desired advancement of such therapeutic
antibodies.
Typically, monoclonal antibodies (MAbs) are produced in
mice (murine Abs), although they have functional constrictions such as high immunogenicity and low penetration
through solid tumor. Such restrictions have largely been
overcome by the benefits of using the camel antibodies. In
1993, natural heavy chain antibodies (HCAbs) bereft of a light
chain and CH1 domain were discovered in camelids.(26–29)
The HCAbs (i.e., IgG2 and IgG3, 90 kDa), void of CH1 domain, are smaller than their counterparts in conventional
mammalian antibodies (150–160 kDa).
The antigen-binding site of variable domain of camelid
HCAbs has been mentioned as VHH, or nanobody.(30–32) It
consists of the smallest known natural intact antigen-binding
domain. These small-sized fragments (VHH) are wellexpressed and have many particular features such as heat
resistance, small size, very high solubility in aqueous, and are
non-immunogenic in humans.(32,33) In this survey, the production and characterization of novel anti-PSMA specific
polyclonal antibodies in dromedary camels are reported.
Materials and Methods
With the use of recombinant PSMA (R&D Systems, Minneapolis, MN), LNCaP cells, and a 28-amino acid peptide
(NFTEIASKFSERLQDFDKSNPIVLRMMN-COOH), which
were in accordance with the external surface domain of
PSMA, the dromedaries were immunized subcutaneously
and intramuscularly. The peptide was synthesized and conjugated to bovine serum albumin (BSA) by Biomatik (Cambridge, Canada).
Each animal received eight doses of LNCaP cells (*108 cells
in 1 mL PBS per injection) and 1 mg of peptide conjugated to
BSA emulsified in 2 mL complete Freund’s adjuvant for the
first and in 2 mL incomplete Freund’s adjuvant for the
following injections, at weekly intervals. Moreover, animals
received 10 mg of recombinant PSMA at weeks 5 and 6 of
immunization.(28)
Fractionation of IgG subclasses from serum
of preimmunized and immunized camels
Before immunization (day 0) and also after passing 6, 7, and 8
weeks of immunization, pre-immune and immune sera were
collected. Afterward, the high-titer antibodies, which were
collected 6 weeks after the primary immunization, were chosen;
then, applying differential absorption on protein G and A
(Hitrap Pharmacia) that was previously reported by Omidfar
and colleagues,(28,34) IgG subclasses were purified. Separation
of the different immuoglobin (IgG) subclasses obtained from
camel immune serum was carried out with modification. After
collecting the serum, the total immunoglobulin was precipitated with ammonium sulfate and then dissolved in a minimum
amount of phosphate buffer. The precipitate was then dialyzed
all night in an extensive manner against the buffer. The obtained
clear solution was first loaded onto a protein G column, then
IgG3 was eluted with acetate buffer (pH 3.5) and IgG1 was
eluted with glycine HCl (pH 2.7). In order to recover the IgG2
subclass, the flow-through was loaded onto a protein A column,
which was eluted with acetate buffer (pH 3.5). The eluent was
stored at - 20C while it was dialyzed immediately against
bicarbonate buffer (pH 9.5) and treated with sodium azide.
Through the Bradford test, the protein concentration of column
fractions was determined, and by means of SDS-PAGE analysis,
the purity of the isolated serum IgG was confirmed.(28)
Animal farming
Two adult male Camelus dromedarius, purchased locally,
were housed in the Guilan University of Medical Sciences
animal facility, where they were given free access to water and
food resources. During all the stages of these experiments,
guidelines prepared by the Guilan University of Medical
Sciences were precisely followed for the care of the animals.
ELISA assay
Enzyme linked immunosorbent assay (ELISA) was employed to determine the specificity and reactivity of the various IgG subclasses toward the peptide. In order to coat wells
of micro-titer plates, they were subjected to 1 mg/100 mL concentration of PSMA peptide-BSA (for specific binding), BSA
426
EVAZALIPOUR ET AL.
(for non-specific binding), and an extraneous peptide (a 14
amino acid peptide, LEEKKGNYVVTDHC, for non-specific
interaction of antibody with peptides). The plate was incubated overnight at 37C, and the remaining areas on every
well were blocked with BSA and treated with anti-peptide
camel antibodies for 1 h at 37C.
After being washed with PBS several times,(28,34) wells
were treated with 100 mL of horseradish peroxidase (HRP)
conjugated goat anti-camel antibodies for 1 h at 37C. Subsequently, after five washes with PBS, 100 mL of tetramethylbenzidine (TMB) and H2O2 solutions as substrate and
50 mL of HCl (0.5 N) as a stop solution were added to determine and stop the enzyme activity, respectively. Finally, the
absorbance was evaluated in an ELISA plate reader at 450 nm.
Immunocytochemistry assay
Prostate cancer cell line overexpressing human PSMA
(LNCaP) and the human PSMA-negative prostate cancer cell
lines (DU145 and PC-3) were applied as the targets. Succinctly
about 2 · 104 cells were seeded into each well of a 96-well culture plate, and the cells were incubated in culture medium at
37C. Once they became roughly confluent, the medium was
discarded in order to incubate the cells with 100 mL of preimmune whole serum (1:1500), the total serum from immunized camels (1:1500), and purified IgG subclass antibodies
(1 mg/mL) for 1 h on ice. Subsequently, the cells that were washed twice with PBS-BSA were incubated with 100 mL of 1:2000
diluted HRP-conjugated goat anti-camel antibodies for 1 h at
4C. Then, after washing the wells five times according to the
previous described method, the binding activity of each preparation was measured in the ELISA plate reader at 450 nm.(28,34)
Affinity determination
Determination of the mean affinity of HCAbs, which was
produced and purified in the present study toward antigen,
was performed by coating the wells of the microtiter plate
with PSMA peptide–BSA conjugate. Then, the whole surface
of the wells was blocked to avoid further non-specific binding
and, using various dilutions of HCAbs, the wells were treated
for 1 h at 37C. After being washed several times and subjected to HRP–anti-camel goat antibodies for 1 h at 37C. The
optical density and affinity were considered according to the
protocol described by our group(31) and others.(35)
Results
Production and separation of camel IgG subclasses
Immunoglobulin, which was produced from two camels,
was precipitated with ammonium sulfate and then separated
into different IgG subclasses that included conventional
(IgG1) and heavy chain (IgG2 and IgG3) antibodies. The
conventional IgG (G1) antibodies were composed of heavy
chains and light chains with a molecular weight of 160–
170 kDa (Fig. 2). The molecular weights related to two other
HCAbs (IgG2 and IgG3) are 90 kDa (Fig. 2).
Reactivity of separated camel antibodies
toward synthetic peptide
Purification of anti-PSMA subclass antibody from the serum of immunized camel was performed employing affinity
FIG. 2. SDS-PAGE electrophoresis of IgG3 (lanes 1 and 2),
IgG2 (lanes 3 and 4), and IgG1 (lanes 5 and 6). Inner numbers
designate the molecular weight markers in kDa.
column. By means of ELISA against peptide, the activity of
IgG subclass antibody eluted in the camels was assessed. The
reactivity of purified IgG1, IgG2, and IgG3 antibodies with the
28 amino acid PSMA conjugated to BSA, BSA alone, and a 14
amino acid extraneous peptide can be seen in Figure 3. A
strong and specific reactivity towards BSA-PSMA peptide
was observed in IgG subclasses.
Peptide affinity for HCAbs was calculated. The measurements were as follows: 1 · 108 M - 1 for IgG2 and 6 · 107 M - 1
for IgG3.
Reactivity of camel antibodies against PSMA
positive and negative cell lines
Reactivity of whole antiserum and protein G and protein A
purified subclasses was tested against PSMA-overexpressing
and PSMA-negative human prostate cancer cell line, LNCaP,
and DU145 and PC-3, respectively. Based on the results provided in Table 1, it can be observed that the whole antiserum
and IgG subclasses only reacted with LNCaP, but not with
DU145 and PC-3.
Discussion
In the present study, the first survey of the production and
characterization of anti-PSMA–specific antibodies in camels
has been presented. The heavy chain antibodies (HCAbs) that
made up variable regions of heavy chain, CH2, and CH3 domains are shown in camelids (camels, llamas, and alpacas).
Compared with other counterparts in conventional mammalian antibodies, HCAbs are smaller due to the absence of the
CH1 domain.(26) Since the antigen-binding domains of these
antibodies are located on the heavy chain of the protein molecule, production of recombinant smaller fragment of such
antibodies was facilitated here. It could be that the provided
antibody is a more appropriate substitution for the present
conventional antibodies regarding the feasibility, effectiveness, and beneficial aspects of camel antibody production.
Because of recent advances in the existing knowledge about
molecular and tumor immunology, new immunotherapies
have been achieved to treat human cancers including humoral
and/or cellular approaches. Antibody therapy has been
PRODUCTION AND CHARACTERIZATION OF HCAbs AGAINST PSMA
427
FIG. 3. Binding of anti-peptide antibody of C. dromedarius with BSA-peptide, BSA, and extraneous peptide. All assays were
performed in duplicate.
studied for various types of human cancers. In fact numerous
studies have recognized cell surface antigens, which are tumor
or lineage specific (e.g., CD20), receptors mutated in cancer
cells (e.g., mutated EGFR), overexpressed receptors, and/or
antigens on cancer cells compared with normal cells (e.g.,
EGFR, HER-2, and MUC1).(2,5,36,37)
PSMA (folate hydrolase 1, glutamate carboxypeptidase II)
is a 750 amino acid type II cell surface membrane protein that
was identified in prostate cancer epithelium for the first time.
Subsequently, it was discovered that PSMA is in fact expressed by the neovascular endothelium of almost all solid
tumor types but without being expressed by the tumor cells or
normal vascular endothelium. PSMA is upregulated 10-fold
in prostate cancer, especially in hormone refractory diseases;
therefore, it can be considered an attractive target for immunotherapy and imaging of prostate cancer. It can also be regarded as an ideal sentinel molecule for use in targeting
prostatic cancer cells. Moreover, PSMA has been exploited as
a diagnostic target to detect prostatic cancer.(2,4,5)
In the present study, two camels were immunized with a
synthetic peptide corresponding to the PSMA antigen and
PSMA-expressing cell line. It was mainly aimed to develop
high-titer HCAbs, which is particular for PSMA and the final
objective was to develop the recombinant camelid single domain antibody. The production of the heavy chain camel antibodies (i.e., IgG2 and IgG3) specific for the human PSMA has
been shown in this study (Fig. 2).
Table 1. Reactivity of Antibodies Toward
Human Prostate Cancer Cell Lines
Camel antiserum
IgG1
IgG2
IgG3
Normal serum
LNCaP
DU145
PC-3
1.23 – 0.03
1.62 – 0.02
1.45 – 0.02
1.86 – 0.04
0.35 – 0.03
0.29 – 0.03
0.32 – 0.05
0.35 – 0.01
0.27 – 0.02
0.27 – 0.01
0.32 – 0.04
0.30 – 0.02
0.23 – 0.05
0.33 – 0.03
0.32 – 0.04
All data are illustrated as OD at 450 nm.
According to the performed analysis, the serum of camelid
species contained approximately 68% HCAbs. As a result of
some techniques, it was made evident that the camelid
HCAbs are specific for the PSMA.
At the first stage, conventional and heavy chain (HC) affinity purified antibody from the serum of immunized camels
was bound specifically to the PSMA peptide conjugate to BSA
but did not bind to BSA alone or an impertinent peptide (Fig.
3). The whole antiserum, conventional, and HC affinity purified antibodies with human prostate cancer cell line that
express high level of PSMA (LNCaP) showed significant
positive reactions.
In contrast these compounds do not interact with the
PSMA-negative prostate cancer cell lines (DU145 and PC-3) in
an apparent way. However, it might be that antibodies of
affinities in the range of 108 to 1010 M - 1 are obtained according to the affinities of anti-PSMA antibodies being observed for more than 90% of the previous reports.
In the present research, the heavy chain anti-PSMA antibodies with comparably good affinity were produced in the
range of 1 · 108 M - 1 and 6 · 107 M - 1 for IgG2 and IgG3, respectively.
Some previously performed reports about the use of
HCAbs against tumor-associated cell-surface markers implied that camel HCAbs are more sensitive compared with the
normal antibody to detect the antigen.(34) Perhaps, this is due
to the presence of the longer complementarily determining
regions (CDR) domain of the HCAbs, which allows better
contact with the antigens and a higher capability to identify
them.(32,38) Therefore, respect to the more feasible, effectual,
and lucrative aspects in the production of this antibody, the
current conventional double-chained antibodies may be replaced with camel antibodies if such an experimentation can
be applied in a wide range.
Acknowledgment
This research was supported by the Endocrinology Metabolism Research Center of Tehran University of Medical
428
Sciences, and the Pasteur Institute, Tehran, and I.R. Iran. The
authors wish to express their gratitude to all who provided
support during the course of this research.
Author Disclosure Statement
The authors have no financial interests to disclose.
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Address correspondence to:
Dr. Kobra Omidfar
Endocrinology and Metabolism Research Center
Tehran University of Medical Sciences
P.O. Box 14395/1179
Tehran
Islamic Republic of Iran
E-mail: [email protected]
Received: June 6, 2012
Accepted: August 6, 2012