Rabbit monoclonal antibodies: Generating a fusion partner to ...

Proc. Natl. Acad. Sci. USA
Vol. 92, pp. 9348-9352, September 1995
Immunology
Rabbit monoclonal antibodies: Generating a fusion partner to
produce rabbit-rabbit hybridomas
(myc/abl/transgenic rabbits/plasmacytoma/B cells)
HELGA SPIEKER-POLET, PERIANNAN SETHUPATHI, PI-CHEN YAM, AND KATHERINE L. KNIGHT*
Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
Communicated by Alfred Nisonofft Brandeis University, Waltham, MA, June 16, 1995 (received for review April 10, 1995)
ABSTRACT During the last 15 years several laboratories
have attempted to generate rabbit monoclonal antibodies,
mainly because rabbits recognize antigens and epitopes that
are not immunogenic in mice or rats, two species from which
monoclonal antibodies are usually generated. Monoclonal
antibodies from rabbits could not be generated, however,
because a plasmacytoma fusion partner was not available. To
obtain a rabbit plasmacytoma cell line that could be used as
a fusion partner we generated transgenic rabbits carrying two
transgenes, c-myc and v-abl. These rabbits developed plasmacytomas,
and we obtained several plasmacytoma cell lines
from which we isolated hypoxanthine/aminopterin/thymidine-sensitive
clones. One of these clones, when fused with
spleen cells of immunized rabbits, produced stable hybridomas
that secreted antibodies specific for the immunogen. The
hybridomas can be cloned and propagated in nude mice, and
they can be frozen without change in their ability to secrete
specific monoclonal antibodies. These rabbit-rabbit hybridomas
will be useful not only for production of monoclonal
antibodies but also for studies of immunoglobulin gene rearrangements
and isotype switching.
Monoclonal antibodies (mAbs) from rabbits have not been
available because no rabbit plasmacytomas, from which a
hybridoma fusion partner could be generated, have been
identified. The availability of rabbit mAbs is, however, highly
desirable for several reasons. First, rabbits are known to
produce antibodies to many antigens that are not especially
immunogenic in mice (1-5). For example, Bystryn et al. (2)
directly compared rabbit and mouse antibodies directed
against human melanoma cells and showed that they recognize
different epitopes. Second, rabbit antibodies are generally of
high affinity. Third, because most mAbs are generated in mice
and rats, relatively few mAbs are available that react with
mouse or rat immunogens. Because of this desire for rabbit
mAbs several laboratories developed mouse-rabbit heterohybridomas,
but this technology has had limited success. The
earliest mouse-rabbit heterohybridomas were unstable and/or
secreted only light (L) chain (6-9). Raybould and Takahashi
(5) reportedly overcame this problem by using normal rabbit
serum (NRS) instead of fetal calf serum (FCS) as a supplement
to the culture medium. However, Verbanac et al. (10) described
major problems with this method. For example, they
found that the heterohybridomas were highly unstable and had
to be subcloned every 4-6 weeks to avoid loss of antibody
secretion. In our laboratory, we obtained no more than two to
five hybridomas per fusion when using the method described
by Raybould and Takahashi (5). Further, these heterohybridomas
were difficult to clone, and the clones were generally
unstable and did not secrete antibody over a prolonged period
of time. Thus it became clear that heterohybridomas were not
a satisfying solution and that rabbit-rabbit hybridomas were
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9348
needed to stably produce monoclonal rabbit antibodies. We
have now developed a fusion partner from a myc/abl doubletransgenic
rabbit, and we report the successful production of
stable antigen-specific rabbit-rabbit hybridomas.
MATERIALS AND METHODS
Transgenic Rabbits. Single-cell zygotes were injected with a
murine E,,-abl construct [kindly provided by S. Cory (11); E,,
is the immunoglobulin heavy chain enhancer] at a concentration
of 1 ,tg/ml and implanted into the uterus of pseudopregnant
females (12). Offspring were tested at 3-4 weeks of age
by Southern blot analysis of peripheral blood lymphocyte DNA
for the presence of the E,,-abl transgene. Rabbits carrying the
E,,-abl transgene were mated with EK-myc transgenic rabbits
established previously in our laboratory (EK is the K-chain
enhancer) (13). The offspring were tested for the presence of
both transgenes as described above. In addition, we directly
microinjected zygotes from a transgenic EK-myc rabbit with the
EK-abl transgene.
Generation of Plasmacytoma Cell Lines and a Hypoxanthine/Aminopterin/Thymidine
(HAT)-Sensitive Fusion
Partner. Rabbits that became ill were sacrificed and cells from
the tumorous tissues were placed in tissue culture in an attempt
to obtain plasmacytoma cell lines. Culture medium used was
RPMI 1640 enriched with the following additions: amino acids,
nonessential amino acids, pyruvate, glutamine, vitamins,
Hepes, gentamicin, penicillin, streptomycin, fungizone (all
components were from GIBCO and were used at concentrations
suggested by the supplier), and 50 ,uM 2-mercaptoethanol.
After 6-8 weeks in culture, stable cell lines were growing
from these tumorous tissues.
To obtain a HAT-sensitive fusion partner, three cell lines
were first x-irradiated with 200 rad (1 rad = 0.01 Gy) and then
cultured in the presence of 8-azaguanine. (The concentration
of 8-azaguanine was initially 0.2 ,ug/ml and was slowly increased
to 20 ,ug/ml over a 10-month period.) We obtained
three 8-azaguanine-resistant clones: 20337-7 after one month
and 240E1-1-1 and 240E1-1-2 after 8 months in culture. Cells
of these three clones were sensitive to medium containing
HAT.
Fusions. Rabbits received a primary immunization by injection
of antigen (a total of 2 mg of protein or 2 x 107 cells
per immunization) in complete Freund's adjuvant subcutaneously,
intramuscularly, and intraperitoneally. The animals
were boosted once or twice in the same manner but with
incomplete Freund's adjuvant. The final boost was given
intraperitoneally and intravenously with saline 4 days before
the fusion. Fusions were performed using conventional methodology
(14): spleen cells (1.5-3 x 108) of immunized rabbits
and the fusion partner 240E 1-1-2 were fused at a ratio of 2:1
Abbreviations: mAb, monoclonal antibody; L chain, light chain; NRS,
normal rabbit serum; FCS, fetal calf serum; HAT, hypoxanthine/
aminopterin/thymidine; FITC, fluorescein isothiocyanate.
*To whom reprint requests should be addressed.

Immunology: Spieker-Polet et aL
with 50% PEG 4000 (EM Science, Cherry Hill, NJ 08304) at
37°C in serum-free medium. The cells were plated in 48-well
microtiter plates, at approximately 2 x 105 spleen cells per
well, in medium with 15% FCS. After 72 h, HAT was added.
Medium was changed every 5-6 days. Clones usually were
observed after 2-5 weeks. Supernatants were tested for the
presence of antibody specific for the immunogen, either by
immunofluorescence with Jurkat cells [using fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit L-chain antibody
as secondary reagent] (fusion 1) or by ELISA (fusions 2
and 3). Hybridomas were cloned by limiting dilution in 48-well
microtiter plates. For feeder cells, we used the fusion partner,
240E1-1-2, at 5 x 104 cells per well. These feeder cells were
killed 5-6 days later by the addition of HAT.
In preliminary fusion experiments we noticed excessive
growth of adherent cells in several wells, which prevented the
hybridomas from establishing themselves. Such growth of
adherent cells, which interferes with the growth of fused cells,
has been reported by other investigators (5, 15). The extent of
this growth varied from one experiment to another, and it
could be partially prevented if FCS was totally or partly
replaced by NRS. However, the hybridomas appeared to grow
more slowly in the absence of FCS. In one experiment, we
attempted to remove the adherent cells by incubating the
spleen cell suspension on plastic dishes for 6 h at 37°C before
fusing them. Although this method did not eliminate adherent
cell growth, it did reduce the number of wells with adherent
cells.
ELISA. ELISA (16) was performed in 96-well microtiter
plates (Falcon 3912, Fisher) that were coated overnight with
purified goat anti-rabbit L-chain antibody, 1,g/ml, or with the
immunogen, 2 Ag/ml. The following solutions were added,
sequentially, for 1-2 h at room temperature: first, the supernatant
to be tested, then biotinylated goat anti-rabbit L chain
or, when assaying for rabbit immunoglobulin isotypes, biotinylated
goat anti-rabbit g-, y-, or a-chain antibodies, 1 jig/ml.
This was followed by incubation with avidin-biotin-horseradish
peroxidase complex (Vectastain ABC Kit, Vector Laboratories)
and finally with the substrate 2,2'-azinobis(3-ethylbenz
thiazolinesulfonic acid) (ABTS) as suggested by the supplier.
Proc. Natl. Acad. Sci. USA 92 (1995) 9349
Color development was read at 405 nm in an ELISA plate
reader.
RESULTS AND DISCUSSION
Generation of Double-Transgenic Rabbits. Because Rosenbaum
et al. (11) obtained mice with plasmacytomas in myc/abi
double-transgenic mice, we decided to generate myc/abl double-transgenic
rabbits in an effort to obtain rabbits with
plasmacytoma. A family of transgenic rabbits that carried the
c-myc oncogene linked to the K-chain enhancer was developed
previously in our laboratory (13). We now generated a second
family of transgenic rabbits with the v-abl oncogene linked to
the immunoglobulin heavy chain enhancer (E,,) as a transgene.
A total of 665 zygotes were microinjected and implanted in 31
pseudopregnant females. From 11 pregnant females we obtained
19 offspring, of which 2 carried the v-abl transgene. To
obtain double-transgenic rabbits we used two methods. In the
first method, EK-myc transgenic rabbits were mated with the
E,,-abl transgenic rabbits. From four matings 22 offspring were
obtained, and of these, 5 carried both transgenes. The plasmacytomas
81E5-1 and 300F1-2 developed in offspring of
transgenic rabbits developed in our laboratory, whereas plasmacytomas
20337-7, 20337-8, and 0022-3 developed in offspring
for which the E,-abl transgenic parent (obtained with
the same E,IL-abl construct) was kindly provided by Andrew
Kelus and Klaus Karjalainen (Basel Institute of Immunology,
Basel, Switzerland). In the second method EK-myc zygotes
were microinjected with E,,-abl DNA. In this case one,
240E1-1, of five offspring carried both transgenes and developed
plasmacytoma. All offspring that carried both transgenes,
c-myc and v-abl, became ill between the ages of 8 and
19 months. Tumors had developed in these rabbits in various
locations. Histologic analysis of these tumors revealed that the
rabbits had developed immunoblastic lymphoma or early
plasmacytoma.
Development of a Rabbit Fusion Partner. From the tumorous
tissue of five of the six rabbits with plasmacytoma
(300F1-2, 0022-3, 20337-7, 20337-8, and 240E1-1) stable cell
lines were obtained. From these lines, three HAT-sensitive
clones were established by selection with 8-azaguanine and
FIG. 1. Rabbit plasmacytoma fusion partner, 240E1-1-2, stained with Wright-Giemsa stain (Diff-Quick, American Scientific Products, McGaw
Park, IL ). (x1200.)

9350 Immunology: Spieker-Polet et al.
Proc. Natl. Acad. Sci. USA 92 (1995)
Table 1. Frequency and stability of hybridomas obtained in three fusions of the rabbit fusion partner with spleen cells from
hyperimmunized rabbits
Wells with Hybrids secreting Hybrids yielding
hybrids/wells Hybrids per 106 specific mAb/total stable clones/total
Fusion Immunogen plated (%) cells fused hybrids tested (%) hybrids cloned (%)
1 Jurkat cells 200/400(50) 0.7 10/104 (10) ND
2 Ovalbumin 38/980* 0.25 9/36 (25) ND
3 Mouse serum proteins 242/980(25) 1.2 43/187 (23) 7/7 (100)
ND, not determined.
*In many wells adherent cells were growing that prevented the growth of the upcoming hybridomas. In such cases the hybridoma clones had to
be removed from the adherent cells, and this was done only with 38 clones.
from one of these clones, 240E1-1-2, stable hybridomas could
be obtained. In characterizing this clone, we determined the
doubling time to be 48 h, and by staining with Wright-Giemsa
stain (Diff-Quick) we found that the cells had features characteristic
of early plasma cells-i.e., they are large cells with
abundant cytoplasm, and the nuclei frequently contain
"lumpy" chromatin. The cells have many vacuoles (Fig. 1),
which indicates that they may be proplasmocytes. These cells
do not secrete immunoglobulin (

Immunology: Spieker-Polet et aL
701
Proc. Natl. Acad. Sci. USA 92 (1995) 9351
C
Control and
Ra anti-
Ms IgG2b
I-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Ju'1
100 101 102 100 101 102 103 100 101 102
Fluorescence intensity
FIG. 3. Immunofluorescence labeling of mouse A20 B-lymphoma cells with monoclonal rabbit (Ra) anti-mouse (Ms) IgG2 antibodies. A20 cells
were incubated with the supernatants of IgG-secreting rabbit-rabbit hybridomas (fusion 3) that were shown by ELISA (Table 2) to recognize mouse
IgG2a (clone 53-5) (0.25 ,ug of mAb per ml) (A), IgG2a and IgG2b (clone 80-1) (5 iLg of mAb per ml) (B), or IgG2b (clone 10-6) (0.5 ,ug of mAb
per ml) (C) and none of the other mouse immunoglobulin isotypes. In control samples, A20 cells were incubated with the supernatant of an
IgG-secreting rabbit-rabbit hybridoma that recognizes an irrelevant antigen-i.e., a surface antigen of Jurkat cells (fusion 1). As secondary antibody
we used FITC-conjugated goat anti-rabbit L chain.
efficiency for the three fusions performed was between 0.25
and 1.2 in 106 cells, which is comparable to the efficiency
generally obtained in mouse-mouse fusions. Of the hybridomas
produced in the three fusions, 10%, 23%, and 25%
secreted mAbs that were specific for the immunogens (Table
1). Again, this percentage of hybridomas that secretes specific
mAb is comparable to that obtained in mouse-mouse fusions.
The hybridomas have been subcloned and they were frozen
and thawed without loss in their ability to secrete mAb. These
data indicate that the hybridomas are stable and that frequent
cloning, which had been necessary for the heterohybridomas,
is not needed for the rabbit-rabbit hybridomas.
Production of IgA-Secreting Hybridomas. Most of the
hybridomas from spleen secreted IgG and none were found
that secreted IgA (Table 3). Because IgA-producing hybridomas
would be valuable reagents we decided to perform
fusions with cells from Peyer's patch (PP) and mesenteric
lymph node (MLN) to obtain IgA-secreting hybridomas.
From two separate fusions 34% of the hybridomas from
MLN and 81% of the hybridomas from PP secreted IgA
(Table 3). Similar results were obtained in two additional
experiments-i.e., 35% of the hybridomas from MLN and
38% of the hybridomas from PP secreted IgA. (The other
isotypes were not determined in these experiments.) A high
Table 3. mAbs produced by rabbit hybridomas obtained from
fusions of 240E1-1-2 with mesenteric lymph node (MLN), Peyer's
patch (PP), or spleen cells
No. of clones (% of total clones)
Cells fused Total IgG IgM IgA
Spleen* 25 25 (100) 0 0
MLNt 82 14 (17) 34 (41) 28 (34)
ppI 48 2 (4) 2 (4) 39 (81)
*Data from fusion 3, Table 1: 25 of 43 specific mAbs were analyzed.
tMLN cells of an unimmunized rabbit were activated by murine
CD40-ligand-transfected CHO cells (generously provided by Melanie
Spriggs, Immunex Research and Development Corp., Seattle) for 48
h prior to fusion. The fused cells were plated in 1000 wells.
tPP cells were treated as described above for MLN cells and plated in
400 wells.
percentage of IgA-producing hybridomas from MLN, PP,
and other mucosal tissues had been found by other investigators
for rat (15, 17, 18). In contrast, fusion with spleen cells
does not generally yield IgA-secreting hybridomas in either
mice or rats, as we now report for rabbits.
Concluding Statement. The research described here, detailing
our search for a rabbit fusion partner, began in the
late 1970s, before transgene technology was available. Once
investigators showed that lymphoid tumors developed in
transgenic mice carrying various oncogenes (11, 19), we used
this technology to develop rabbit plasmacytomas. Our breakthrough
came in 1991 when we found that the myc/abl
double-transgenic rabbits developed plasmacytomas. Since
then, we have established plasmacytoma cell lines and were
able to develop one into a usable fusion partner. The
availability of a rabbit fusion partner provides us with the
opportunity to produce mAbs specific for mouse antigens
and also for antigens or epitopes that are not immunogenic
in mice. Such mAbs will be useful in diagnosis of diseases and
treatment of patients.
This work was supported by Public Health Service Grant Al 11234.
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