Analysis of the antibody repertoire of patients with mantle cell lymphoma directed against mantle cell lymphoma-associated antigens.

Abstract

Treatment results of mantle cell lymphomas (MCL) are not satisfactory and novel therapeutic approaches are warranted. Because "shared" tumor antigens like the group of cancer testis antigens are only rarely expressed in MCL, we applied serological analysis of antigens using recombinant expression cloning (SEREX) to a complementary DNA library derived from five cases of MCL using the sera of eight patients with MCL in order to define MCL-associated antigens that are immunogenic in these patients and might be used as vaccines for patients with MCL. Five antigens were detected by SEREX. Four of the five detected antigens (hypothetical protein FLK10233, recombining binding protein suppressor, a chromosomal sequence, and interleukin-1 receptor associated kinase) are also expressed by a wide spectrum of normal human cells, excluding their use as vaccines. In contrast, the expression of CD52, which was detected by antibodies in the serum of an MCL patient, is restricted to hematopoietic cells. Interestingly, anti-CD52 antibodies were detected in this patient before and >2 years after allogeneic transplantation, indicating that both the autologous as well as the allogeneic immune system recognized CD52. Since the anti-CD52 monoclonal antibody alemtuzumab has shown activity in MCL, a vaccine consisting of recombinant CD52 alone or combined with passive immunotherapy using alemtuzumab warrants furthers clinical and immunological evaluation in MCL.

Full text

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ORIGINAL ARTICLE
Analysis of the antibody repertoire of patients
with mantle cell lymphoma directed against mantle cell
lymphoma-associated antigens
Carsten Zwick & Klaus-Dieter Preuss & Boris Kubuschok & Gerhard Held &
Manfred Ahlgrimm & Joerg Bittenbring & Joerg Schubert & Frank Neumann &
Michael Pfreundschuh
Received: 20 August 2008 /Accepted: 28 January 2009 /Published online: 24 February 2009
# Springer-Verlag 2009
Abstract Treatment results of mantle cell lymphomas
(MCL) are not satisfactory and novel therapeutic approaches
arewarranted.Because“shared”tumorantigenslikethegroup
ofcancertestisantigensare onlyrarelyexpressed inMCL, we
applied serological analysis of antigens using recombinant
expression cloning (SEREX) to a complementary DNA
library derived from five cases of MCL using the sera of
eight patients with MCL in order to define MCL-associated
antigens that are immunogenic in these patients and might be
used as vaccines for patients with MCL. Five antigens were
detected by SEREX. Four of the five detected antigens
(hypothetical protein FLK10233, recombining binding pro-
tein suppressor, a chromosomal sequence, and interleukin-1
receptor associated kinase) are also expressed by a wide
spectrum of normal human cells, excluding their use as
vaccines. In contrast, the expression of CD52, which was
detected by antibodies in the serum of an MCL patient, is
restricted to hematopoietic cells. Interestingly, anti-CD52
antibodies were detected in this patient before and >2 years
after allogeneic transplantation, indicating that both the
autologous as well as the allogeneic immune system recog-
nized CD52. Since the anti-CD52 monoclonal antibody
alemtuzumab has shown activity in MCL, a vaccine consist-
ing of recombinant CD52 alone or combined with passive
immunotherapy using alemtuzumab warrants furthers clinical
and immunological evaluation in MCL.
Keywords Mantlecelllymphoma.Lymphoma-associated
antigens.CD52.Vaccinedevelopment
Introduction
Mantle cell lymphomas (MCL) make up 8% of the
lymphomas and have a poor prognosis because of their
eventual refractoriness to many chemotherapy regimens.
Monoclonal antibodies, particularly rituximab, have activity
in MCL patients [1–3], and chemotherapy combined with
rituximab and followed by high-dose therapy with auto-
transplant may be the best treatment for these patients [3].
For older patients, no standard treatment is available, and
allogeneic transplantation is the only, yet toxic approach, to
which a curative potential is ascribed.
The demonstration of responsiveness to the monoclonal
anti-CD20 antibody rituximab and evidence for a graft-
versus-MCL effect after allogeneic transplantation [4]
support the notion that MCL are amenable to immunother-
apy. Immunotherapy holds promise as an alternative
approach for the prolongation of remissions and reduction
of relapses. Vaccine approaches using dendritic cells fused
with MCL cells met with some success in vitro and in a
pre-clinical model in vivo by inducing T cells with specific
cytotoxicity against MCL cells [5]. However, the identifi-
cation of specific antigenic targets on MCL cells is
cumbersome because cytotoxic T-cell-response-based
approaches to the identification of tumor antigens are
hampered by the inherent problems of establishing and
characterizing [6] specific CTL clones. These problems are
Ann Hematol (2009) 88:999–1003
DOI 10.1007/s00277-009-0711-0
C. Zwick:K.-D. Preuss:B. Kubuschok:G. Held:
M. Ahlgrimm:J. Bittenbring:J. Schubert:F. Neumann:
M. Pfreundschuh
Jose-Carreras-Center, Saarland University Medical School,
Homburg, Saar, Germany
M. Pfreundschuh (*)
Innere Medizin I, Saarland University Medical School,
66421 Homburg, Saar, Germany
e-mail: Michael.pfreundschuh@uniklinikum-saarland.de

Page 2

the main reason why no tumor-specific antigens have been
defined to date that are frequently and specifically
expressed in MCL. While the clonal immunoglobulin
idiotype that is expressed by the malignant MCL cells of
a given patient represents a specific antigen, and idiotype
vaccines have been shown to induce remission in follicular
lymphomas [7], their production is extremely expensive
because they must be individually tailored, which has
prohibited their widespread use [8]. Of the antigens with a
broader expression spectrum, the PRAME antigen has been
reported to be frequently expressed in MCL [9]. However,
PRAME does not seem to be a good vaccine candidate
because it is frequently expressed in normal tissues, albeit
at a lower level than in certain neoplastic tissues [10].
Our group has developed an alternative approach for the
identification and molecular characterization of specific
antigens of human malignancies. The experience with
SEREX, an acronym for “serological analysis of antigens
by recombinant expression cloning” which uses antibodies
in patients’ sera and expression cloning techniques, has
taught us that many, if not all human malignancies elicit
responses in the patient’s own immune system. For a
growing number of these antigens, which were identified
by antibodies, i.e., by the patient’s humoral immune
system, a T-cellular immune response has been demonstrat-
ed. This supports the hypothesis that there is an integrated
immune response against tumors in tumor-bearing patients
which involves both B and T cells. The most promising
vaccine candidates are the so-called cancer testis antigens
(CTA); however, a survey by our group on the expression
of cancer testis antigens in lymphoma revealed that MCL
cells express CTA quite rarely [11].
Therefore, the identification of additional antigens that
are specifically and frequently expressed in MCL is
warranted. To identify antigens which might serve as the
target structures for a broadly applicable vaccine, we
applied a SEREX analysis to MCL.
Materials and methods
Patients, sera, and tissues
The study had been approved of by the local ethical review
board (“Ethikkommission der Ärztekammer des Saar-
landes”). Recombinant DNA work was done with the
official permission and according to the rules of the State
Government of Saarland. Sera and tissues were obtained
during routine diagnostic or therapeutic procedures per-
formed at Saarland University Medical School, Homburg,
Germany and stored at –80°C until use after obtaining
written informed consent by the patient. Normal tissues
were collected from autopsies of tumor-free patients. Tumor
samples used for SEREX and reverse transcription poly-
merase chain reaction (RT-PCR) analysis were checked
microscopically for the presence of neoplastic tissue and
absence of contaminating normal tissues. Normal tissues
were collected from autopsies of tumor-free patients.
Identification of MCL-associated antigens by SEREX
Fresh biopsy material obtained from five patients with MCL
was used to establish complementary DNA (cDNA) libraries
as described by us [12]. Poly(A)+ RNA was prepared with a
mRNA isolation kit (Stratagene, La Jolla, USA) out of total
RNA isolated from fresh tumor biopsies [13]. cDNA
expression libraries were constructed by starting with 5 to
8µg of poly(A) RNA. First-strand synthesis was performed
using an oligo(dT) primer with an internal XhoI site and 5-
methylCTP. cDNA was ligated to EcoRI adaptors and
digested with XhoI. cDNA fragments were cloned in sense
direction with respect to the lacZ promotor into the
bacteriophage expression vector λ-ZAP Express using a
commercially available adaptor ligation system according to
the manufacturer’s instructions (Stratagene). After packaging
into phage particles, these cDNA expression libraries were
used to transfect Escherichia coli. The sera from patients
with untreated MCL which had been preabsorbed with
transformed E. coli lysates were screened for IgG reactivity
against transfectants derived from the cDNA expression
library using the phage assay described by us in detail
previously [12]. XLI MRF′ bacteria transfected with recom-
binant λ-ZAP Express phages were plated onto Luria–
Bertani agar plates. Expression of recombinant proteins in
lytic phage plaques on the bacterial lawn was induced with
IPTG. Plates were incubated at 37°C until plaques were
visible and then blotted onto nitrocellulose membranes. The
membranes were blocked with 5% low-fat milk in Tris-
buffered saline and incubated with a 1:250 dilution of the
patient’s serum, which had been preabsorbed with trans-
fected E. coli lysates. Serum antibodies binding to recombi-
nant proteins expressed in lytic plaques were detected by
incubation with alkaline phosphatase-conjugated goat anti-
human IgG and visualization by staining with 5-bromo-
4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
Positive clones were subcloned to monoclonality and
submitted to in vivo excision of pBK-CMV phagemids.
The nucleotide sequence of cDNA inserts was determined
using Excel cycle sequencing kit (Epicentre Technologies,
Madison, WI, USA) on a LICOR sequencer. Sequencing was
performed according to the manufacturers’ instructions
starting with the vector-specific primers. Insert-specific
primers were designed as the sequencing proceeded. Se-
quence alignments were performed with DNASIS (Pharma-
cia Biotech, Freiburg, Germany) and BLAST software on
EMBL, Genbank, and PROSITE databases.
1000 Ann Hematol (2009) 88:999–1003

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For the demonstration of antibodies against defined
antigens, monoclonalized phages from clones that were
reactive with the patient serum used for the immunoscreen-
ing were mixed with non-reactive phages of the cDNA
library as internal negative controls at a ratio of 1:10 and
used to transfect bacteria. IgG antibodies in the 1:100
diluted E. coli pre-absorbed sera from other patients and
healthy controls were tested with the phage assay to assess
antibody responses against the respective antigen.
Reverse transcription PCR
Total cellular RNA was extracted from frozen tissues,
primed with a dT(18) oligonucleotide and reverse-
transcribed with Superscript II (Gibco BRL, Eggenstein,
Germany) according to the manufacturer’s instructions.
cDNA thus obtained was tested for integrity by amplifica-
tion of β-actin transcripts in a 30-cycle PCR as described
elsewhere [14]. For PCR analysis of the expression of
individual gene transcripts, 1µg first-strand cDNA was
amplified with transcript-specific oligonucleotides (10 pM)
using 2 U AmpliTaq Gold (Perkin Elmer, Weiterstadt,
Germany), 10 nM of each dNTP (dATP, dTTP, dCTP,
dGTP), and 1.5 mM MgCl2in a 30-µl reaction. The primers
specific for the respective cDNA were designed to amplify
coding sequences of the respective gene and were obtained
commercially (MWG Biotech, Ebersberg, Germany). Pres-
ence of specific transcripts was checked by amplification
with specific primer pairs, followed by electrophoresis.
Results
The patients from whom the sera had been taken had no
obvious other diseases apart from their mantle cell
lymphoma; in particular, the sera were negative for anti-
bodies against common autoantigens such as antinuclear
antibodies or rheumatoid factor.
Antigens detected by SEREX
Approximately 1×106recombinant clones represented in
the MCL-derived cDNA expression library expressed in E.
coli were screened by the SEREX approach using the
1:1,000 diluted sera from eight patients with untreated
MCL. The summary of the results of the SEREX analysis
with these sera is shown in Table 1. Eighteen clones
encoded by five different genes reacted with IgG antibodies
in the 1:1,000 diluted sera from the MCL patients. The
antigens were designated HOM-MCL-1 to HOM-MCL-5.
Except for one of them (HOM-MCL-3), they were
represented by several clones with overlapping sequences.
All antigens detected represented known transcripts.
HOM-MCL-1 is identical to hypothetical protein FLJ
10233 and HOM-MCL-2 to recombining protein suppres-
sor. HOM-MCL-3 is identical to CD52, HOM-MCL-4
represented chromosomal sequences, and HOM-MCL-5 is
identical with interleukin-1 associated kinase 4.
Specificity of the detected antigens
For antigens HOM-MCL-1, HOM-MCL-2, HOM-MCL-4,
and HOM-MCL-5, there was information from the pub-
lished literature or from the GenBank that they are
ubiquitously expressed in human tissues. HOM-MCL-3 is
identical with CD52. CD52 is expressed by B and T
lymphocytes, natural killer cells, monocytes and macro-
phages, and some dendritic cells, but not by granulocytes,
red blood cells, platelets, or hematopoietic progenitor cells
[15, 16].
Survey of antibody reactivity against antigens identified
by screening with sera from MCL patients
To determine whether the immune responses against the
antigens identified in this study were MCL-related, the sera
from eight patients with MCL were tested against the SEREX
antigens expressed in E. coli. Sera from healthy donors
served as controls. As can be seen from Table 2, at a serum
dilution of 1:100, antibodies against HOM-MCL-2 and
HOM-MCL-5 were also detected in the sera of healthy
controls. MCL-1, MCL-3, and MCL-4 were not detected by
antibodies in the sera of healthy controls, but only by 2/8, 1/
8, and 1/8 patients with MCL, respectively. The patient with
antibodies against MCL-3/CD52 was tested for anti-CD52
antibodies 2 years before (during her second relapse) and
after allogeneic transplantation, while in continuous com-
plete posttransplant remission. The anti-CD52 antibody titer
Antigen Length (bp)Encoding gene
HOM-MCL-1
HOM-MCL-2
HOM-MCL-3
HOM-MCL-4
HOM-MCL-5
2,100
1,400
450
1,300
650
Hypothetical protein FLK10233
Recombining binding protein suppressor
CD52
Chromosomal sequence
Interleukin-1 receptor associated kinase
Table 1 MCL-derived antigens
detected by SEREX with IgG
antibodies in the sera of eight
MCL patients
Ann Hematol (2009) 88:999–10031001

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was 1:10,000 during relapse and 1:500 while in complete
remission 2 years after transplant. The patient’s stem cell
donor (her sister) was negative for anti-CD52 antibodies.
Discussion
In previous studies [11], it had been shown that a common
group of “shared” antigens, the cancer-testis or cancer-
germline antigens are only rarely expressed in malignant
lymphomas with the exception of T-NHL and diffuse large
B-cell lymphomas. Furthermore, the frequency of expressed
antigens reactive with the sera from NHL patients was low
compared to studies with the sera of patients with solid
tumors, suggesting that humoral immune responses in NHL
patients against antigenic structures expressed by their
tumors are less frequent, even though antinuclear autoanti-
bodies have been quite often found in patients with NHL
[17]. The rare antitumor immune responses in patients with
MCL and other lymphomas might be partially due to the
lack of the expression of costimulatory molecules by
lymphoma cells, hampering the induction of an efficient
immune response and partially to an immunodeficiency
which is often associated with lymphomas.
The results of this study show that mantle cell
lymphomas express several structures that are immunogen-
ic in patients with MCL. However, most immunogenic
structures are widely expressed in normal and malignant
human cells therefore prohibiting their use as specific
vaccines in MCL. In contrast, the expression pattern of
CD52, which can function as a costimulatory molecule for
the induction of CD4+regulatory T cells [18], is more
restricted: CD52 is expressed by B and T lymphocytes,
natural killer cells, monocytes, macrophages, red blood
cells, platelets, hematopoietic progenitor cells [15, 16], and
mast cells [19]. Among hematologic neoplasms, the
expression of CD52 is heterogeneous [20], but MCL cells
express CD52 consistently [20–22] rendering MCL a
promising target for therapy with the monoclonal antibody
alemtuzumab, a humanized monoclonal antibody against
CD52, even though experience with the anti-CD52 anti-
body in MCL is limited [23, 24]. Notably, the MCL patient
with serum anti-CD52 antibodies in our study had never
been treated with alemtuzumab, excluding the possibility
that the immune response in her serum might have had its
origin in an idiotypic anti-alemtuzumab immune response.
She had a very strong (1:10,000) anti-CD52 antibody
response while in her first relapse 2 years before allogeneic
transplantation. After allogeneic stem cell transplantation, a
new immune system derived from the stem cell donor was
established, and the serum taken 2 years after the
transplantation had an anti-CD52 antibody titer of 1:500.
That this antibody response was a neo-response is shown
by the fact, that the stem cell donor (the patient’s HLA
identical sister) was always negative for anti-CD52 anti-
bodies. This indicates that both the patient’s former and her
new immune system recognized CD52 as an immunosti-
mulatory molecule and raised an immune response against
it. Since the donor was negative for anti-CD52 antibodies,
we must assume that CD52-expressing mantle cells
persisting after allogeneic transplantation have induced the
neo-immune response in the transplanted immune system
by presenting the immunogenic CD52 molecule the context
of “danger” [25]. Therefore, passive immunotherapy of
MCL with anti-CD52 antibodies might work not only by a
direct attack of the monoclonal antibody against the MCL
cells, but may be complemented by an additional immune
response against this MCL surface structure induced, i.e.,
by malignant MCL cells. In light of the considerable
toxicity of the humanized anti-CD52 monoclonal antibody
alemtuzumab [26], it is noteworthy that the presence of the
endogenous anti-CD52 antibody in the patient was not
associated with neutropenia and lymphopenia and the
patient did not suffer from opportunistic infections. This
may be due to the fact that the endogenous antibody bound
to an epitope different from the one which is recognized by
the humanized antibody alemtuzumab (data not shown)
and/or due to the possibility that the immune response is
restricted to mantle lymphoma cells that present CD52 in
the context of danger [25].
The finding that CD52 can be immunogenic in MCL
patients supports additional clinical trials using CD52 as a
vaccine alone or together with alemtuzumab. Apart from
measuring the anti-lymphoma effects of alemtuzumab, such
studies should be designed to evaluate if and to which
degree this therapy is associated with or induces host anti-
CD52 responses that could complement the direct effects of
the monoclonal CD52 antibody. If a cellular anti-CD52
immune response induced by a CD52 vaccine would lack
the toxicity of alemtuzumab, it can only be investigated in a
carefully designed prospective study.
Acknowledgments
Cancer Research Institute (New York). We thank Evi Regitz, Justine
Wroclawski, and Natalie Fadle for excellent technical assistance.
This work was supported by a grant from the
Table 2 Antibody reactivities against MCL-derived antigens in the
sera of MCL patients and healthy controls
AntigenMCL seraHealthy controls
HOM-MCL-1
HOM-MCL-2
HOM-MCL-3/CD52
HOM-MCL-4
HOM-MCL-5
2/8
2/8
1/8
1/8
2/8
0/9
1/9
0/9
0/9
3/9
1002Ann Hematol (2009) 88:999–1003

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