Journal of Immunological Methods, 100 (1987) 73-82 73

Elsevier

JIM 04354

Cyclophosphamide treatment used to manipulate the immune

response for the production of monoclonal antibodies
William D. Matthew and Alfred W. Sandrock, Jr.
Harvard Medical School, Department of Neurobiology, Boston, MA, U.S.A.
(Received 16 ~ b e r 1986, accepted 26 January 1987)

After immuniTation with a complex mixture of antigens, a considerable bias toward obtaining
monoclonal antibodies to immunodominant determinants exists. By selectively kilting antigen-stimulated
lymphocytes, the cytotoxic drug cyclophosphamide can be used to manipulate the bias of the normal
immune response. Cyclophosphamide has been used to tolerize mice to one set of antigens followed by
immunization with a similar but slightly different set of antigens. This approach yields an enhanced
frequency of antibodies that distinguish the two sets of antigens. Cyclophosphamide treatment has also
allowed us to produce monoclonal antibodies to weakly immunogenic glycosaminoglycans and to obtain a
high frequency of apparently anti-idiotypic antibodies.

Key words: Cyclophospbamide; Monocional antibody; Anti-idiotyp¢; Glycosaminoglycan; Nervous system

Introduction obtain than others. For example, when mono-

The normal immune system shows considerable clonal antibodies are prepared from mice im-
specificity in its response to antigens with its munized with a membrane preparation from rat
capacity to distinguish 'self' from 'foreign' and an brain, a restricted group of antigens is repeatedly
inherent design which avoids autoimmune reac- identified (for example: Matthew et al., 1981,
tions. Consequendy, when monoclonal antibodies 1985). Furthermore, since many functionally im-
are prepared to tissue extracts, there is a strong portant epitopes are conserved in mammalian
bias in favor of generating antibodies to a few species, the normal bias in the immune response is
immunodominant antigens. Therefore, antibodies a major disadvantage for the production of func-
recogniTing some antigens are more difficult to tion-blocking mouse monoclonal antibodies di-
rected to mammalian antigens.
In order to counteract the bias in the immune
Correspondence to: W.D. Matthew, Department of Neuro- system, we have used a technique designed to
biology, Harvard Medical School, 25 Shattuek Stxtet, Boston, minimize immunodominance and immunosup-
MA 02115, U.S.A.
Abbreviations: CNS, central nervous system; ELISA, en- pression. This approach appears to have been
zyme-linked immtmoadsorbent assay; GAG, glycosaminogly- critical in developing antibodies that block the
can; i.p., intraperitoneally; i.v., intravenously; PNS, peripheral function of a neurite outgrowth-promoting factor
nervous system; RIA, radioimmunoassay; TBS, Tris-buffered (Matthew and Patterson, 1983; Chiu et al., 1986).
saline.
We report here several refinements of the methods
Immunization protocols: DI, developing vs. immature cere-
bellum; EA, embryonic vs. adult spinal cord; TA, transccted
used to manipulate the immune response for the
sciatic nerve vs. adult spinal cord; TN, traxtsccted vs. normal production of monoclonal antibodies. Our strategy
sciatic nerves. is based on the assumption that when a particular

0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

74

immune response is elicited, resulting in the clonal highly conserved among species and their tissue
proliferation of B and T lymphocytes, the dividing distribution is ubiquitous, GAGs are poor im-
cells can be preferentially killed by the alkylating munogens. Therefore, methods which would allow
agent cyclophosphamide (Schwartz and Dames- the production of antibodies to weak antigens
hek, 1959; Berenbaum and Brown, 1964; review: were sought. Finally, antibodies which recognize
Ahmed and Hombal, 1984). This assumption, and receptors for molecules such as GAGs would be
our results, are consistent with previous work very useful and might be obtained through an
showing that cyclophosphamide will selectively anti-idiotypic response. Although there was no
eliminate proliferating clones of B cells (Turk et rationale for thinking that cyclophosphamide
al., 1972) or T suppressor cells (Lagrange et al., could enhance the anti-idiotypic response, the drug
1974; Askenase et al., 1975; Kipps et al., 1978) may prove to have this effect under some cir-
depending on the recent immunological experi- cumstances.
ence of the animal. Immunosuppressive therapy,
therefore, allows one to manipulate the immune
response by selectively affecting either im- Materials and methods
munodominance or immunosuppression. In ad-
dition, the drug appears to disturb the regulation Immunizations
of the immune system in less predictable ways, DL developing vs. immature - cyclophosphamide
perhaps to enhance the production of apparently treatment used to rigorously suppress the response to
anti-idiotypic immunoglobulins. immature rat cerebellum. Newborn rat cerebella
The purpose of this report is to suggest various were passed through a 26 gauge needle to produce
immunization protocols that will help stimulate a tissue homogenate. Female BALB/c mice
the immune response for the production of inter- (Charles River), 6-8 weeks old, each received a
esting monoclonal antibodies. There are several single cerebellum injected intraperitoneaUy (i.p.).
classes of immunological reagents which have been 10 min later cyclophosphamide (Sigma (100
important in our study of molecules that influence mg/kg)) was given i.p. in saline (2 mg/ml). The
axonal growth during the development and regen- same dose of drug was given at 24 h and 48 h.
eration of the mammalian nervous system. One This tolerizing treatment was repeated every 2
approach has been to search for antigenic dif- weeks and mice were bled from tails on interven-
ferences between tissues that differ in their ability ing weeks. Sera were tested for binding to tissue
to promote axonal growth; for instance, the pe- sections of newborn rat cerebellum. The staining
r/pheral nervous system (PNS) has a high capacity pattern changed until after four treatments no
for axonal regrowth following damage in mature reactivity to newborn cerebellum could be de-
animals whereas the central nervous system (CNS) tected. Following a 3 week rest the mice were
does not support significant regrowth of cut axons. immunized with a homogenate of postnatal day 11
A similar strategy has been to identify antigenic rat cerebellum and then 2 weeks later with day 11
differences between embryonic and adult CNS cerebellum. A fusion of spleen cells with NS1
tissues. The difficulty in this approach is that myeloma cells was completed 3 days later. Control
these tissues are quite similar biochemically and mice were prepared without receiving cyclophos-
share many immunodominant antigenic determi- phamide.
nants. Therefore, a method was needed to improve TN, transected vs. n o r m a l - antibodies prepared
the probability of detecting subtle antigenic dif- to transected sciatic nerves. Membranes were pre-
ferences. pared by suspending the tissues in 5 vols. of
Another interest has been to study extracellular ice-cold Tris-buffered saline (TBS: 0.9% NaCI in
matrix molecules which have been linked to neu- 0.01 M Tris-HCl, pH 7.4), homogenization on ice
rite outgrowth. Proteoglycans and glycosamino- in a hand-held glass tissue grinder (Coming), fol-
glycans (GAGs) may play a role in regulating lowed by sonication on ice for two 10 s intervals
axonal growth (Matthew et al., 1985; Chiu et al., with a Microson ultrasonic cell disruptor (Heat
1986). Because their carbohydrate structures are System Ultrasonics, 50 W total power), and
 75

centrifugation at 20000 × g for 30 min at 4°C. cleaned with concentrated sulfuric acid, rinsed
The pellet was resuspended in 10 vols. of ice-cold with distilled water and then with 0.1 M NaOH,
TBS containing 2 mM CaCI 2 (Ca-TBS), re-soni- air-dried, and reacted with aminopropyltrietho-
cated, and re-centrifuged. The final pellet was xysilane (Sigma) for 3 min. After extensive wash-
adjusted to a 5 mg protein/ml solution with ice- ing with distilled water and then sterile phosphate
cold Ca-TBS and approximately 1 ml was injected buffer, the beads were incubated with the G A G
into each mouse. Protein concentrations were solution for 10 min and the solution was sus-
estimated by the Bio-Rad protein assay. Mice pended in Freund's complete adjuvant. 24 h after
were immunized with the homogenate of normal immunization with the bead preparation, cyclo-
rat sciatic nerves followed by a sequence of three phosphamide (100 mg/kg) was injected i.p. in
cyclophosphamide (100 mg/kg) injections at 10 saline. After an additional 13 days, 0.5 mg of each
min, 24 and 48 h. 14 days later the mice were G A G preparation was injected intravenously (i.v.)
injected with transected sciatic nerves and spleen in saline and the spleen cells fused to myeloma
cells harvested 3 days later were used for prepar- cells 3 days later. Control mice, prepared using an
ing hybridoma cells. Rat sciatic nerves were tran- identical schedule, did not receive cyclophospha-
sected under general anesthesia and sterile tech- mide. Antibodies were tested for binding to
nique. 5 days later, tissue homogenates were pre- chondroitin sulfate, heparin, hyaluronate and
pared from the distal stump of the transected fucoidan in an ELISA and to tissue sections of rat
nerve and from normal intact nerve. testis, retina and cerebellum.
EA, embryonic vs. adult spinal cord - antibodies
prepared to embryonic CNS antigens. Each mouse Production of putative anti-idiotypes to polysac-
was injected i.p. with 5 mg of membrane protein charides
prepared as described above for sciatic nerve. B A L B / c mice were immunized i.p. with 1 mg
200-500 mg wet weight of tissue were removed of hyaluronate attached to positively charged glass
from freshly killed CD (Charles River) rats. In the beads (as above, without adjuvant). After 24 h an
preparation of spinal cord membranes, care was i.p. injection of cyclophosphamide (150 mg/kg) in
taken to remove all adherent roots during dissec- saline was given. After an additional 13 days, 1
tion, since these are peripheral nerve tissues. Mice mg of hyaluronate was injected i.v. in saline and
were twice tolerized to the insoluble fraction of the spleen cells were fused to myeloma cells 3 days
adult rat spinal cord homogenates, immunized later. Antibodies were tested for binding to mam-
with spinal cord membranes from rat embryos at malian and bacterial hyaluronidases (Sigma) and
18 days of gestation (E18), re-tolerized to adult rat to tissue sections of rat testis, retina and cerebel-
spinal cord membranes, and re-immunized with lum. This same procedure was repeated using the
El8 rat spinal cord membranes (see Fig. 1).. sulfated poly-fucose, fucoidan (Sigma). In this
TA, transected sciatic nerve vs. adult spinal cord case, 100 m g / k g of cyclophosphamide was used.
- antibodies prepared to P N S specific antigens. The antibodies were screened against fucoidan,
Each mouse was injected i.p. with 5 mg of mem- bovine fucosidase (Sigma) and the plant lectins
brane proteins as described above for the TN and Tetragonolobus purpureas and Elex europaeus I
EA fusions. Mice were twice tolerized to adult rat (Sigma) in an ELISA as well as for binding to
spinal cord membranes, immunized with tran- tissue sections.
sected sciatic nerve membrane, re-tolerized to adult
rat spinal cord membrane, and re-immunized with Fusions
transected sciatic nerve membrane (see Fig. 1). 3 days after each final immunization, the mouse
was killed and the spleen was immediately re-
Immunizations with glycosaminoglycans moved and mechanically dissociated in Ca 2+-,
A B A L B / c mouse was immunized i.p. with a Mg 2÷- free Hanks' balanced salt solution (Hanks'
solution containing 1 mg of chondroitin sulfates BSS, Gibco). NS1 myeloma cells that had been
(Sigma) and 1 mg of heparin (Sigma) attached to maintained in logarithmic growth were added to
positively charged glass beads (Sigma). Beads were the spleen cells at a ratio of about 5 × 107 cells per
76

spleen; this mixture was centrifuged at 500 × g for ELISA: After rinsing with Ca-TBS, biotiny-
5 min, resuspended in 45 ml of Hanks' BSS, and lated goat anti-mouse IgG and biotinylated goat
re-centrifuged. Approximately 1 ml of polyethyl- anti-mouse IgM (Vector) were diluted 1:250 in
ene glycol (PEG 1500; Boehringer Mannheim) Ca-TBS-BSA and added to the microtest plates at
was added to the pellet of cells over a 2 min 50 #1 per well, incubated for 1 h at room tempera-
period at 37 ° C. Approximately 30 ml of L15-CO2 ture, and rinsed out with Ca-TBS. The avidin
was added gradually to the cells (over a 20 min D-horseradish peroxidase conjugate was prepared
period) to dilute the PEG 1500 and the suspension according to the instructions supplied with the
was centrifuged at 500 x g for 5 min. The fused Vectastain ABC kit (Vector) in Ca-TBS-BSA, and
cells were then gently resuspended in 20 ml of added to the microtest plates at 50 #1 per well.
L15-CO2 supplemented with 0.6% w / v glucose, 2 After a 1 h incubation at room temperature, the
mM glutarnine, 1130 U / m l penicillin, 100 U / m l wells were rinsed twice with 100 #1 of Ca-TBS
streptomycin, 15% fetal calf serum (Hyclone), 100 followed by two rinses with 100 #1 of 0.1 M
#M hypoxanthine (Sigma), 0.4 #M aminopterin Tris-HCl, pH 7.4. The horseradish peroxidase
(Sigma), and 16 #M thymidine (Sigma) (HAT (HRP) reaction was started with the addition of
medium), and incubated for 1 h at 37°C. The 100 #l of 0.5 mg/ml p-phenylenediamine (Sigma),
hybridoma cells were then triturated and plated at 0.001% H202 (Sigma) in 0.1 M Tris-HCl, pH 7.4
clonal density (typically, one spleen is plated into to each well, and terminated by adding 50 #1 of 1
480 wells) into flat-bottom tissue culture multiwell N HCI to each well. The time that the HRP
plates (Linbro) containing a feeder layer of mouse reaction was allowed to proceed varied from ex-
peritoneal macrophages growing in HAT medium. periment to experiment, but was generally in the
After 7 days, the hybridomas were fed HT medium range of 20-40 rain. Absorbance of 490 nm wave-
(same as HAT, without aminopterin). When a length light was obtained for each well in an
particular well contained a visible clump of cells ELISA reader (Bi0-Tek Instruments).
with medium that had become slightly acidified RIA: After rinsing with Ca-TBS, a mixture of
(typically 10-14 days after fusion), it was suitable iodinated goat anti-mouse IgG and goat anti-
for screening. mouse IgM (New England Nuclear) in Ca-TBS-
BSA was added to the microtest plates at 50
Enzyme-linked immunoadsorbent assay (ELISA) #l/well (approximately 50000 cpm of each anti-
and radioimmunoassay (RIA ) body per well), incubated for 1 h at room temper-
The wells of 96-well microtest plates (ELISA: ature, and rinsed out with Ca-TBS. Individual
Nunc-ImmunoPlate I; RIA: Falcon flexible mi- wells were cut out and counted in a gamma coun-
crotest III plates) were treated with 50 #1 of a 1 ter.
mg/ml poly-o-lysine solution in 0.15 M KCI, 0.15
M borate, pH 8.4 for 12-24 h at room tempera- Immunocytochemistry
ture, then rinsed thoroughly with distilled water, Adult tissue was removed from 250-350 g
and allowed to dry. Crude membrane suspensions Sprague-Dawley rats (Charles River) that had been
were prepared according to the procedures de- anesthetized and perfused through the heart with
scribed above and 50 #1 of a 5 mg protein/ml 2% paraformaldehyde (Fisher) in 0.1 m phosphate
suspension were added to each well. After a 12 h buffer, pH 7.4, at room temperature. Embryonic
incubation at 4 ° C, the wells were rinsed twice for and newborn tissues were removed from Sprague-
15 min with 100 #1 ice-cold Ca-TBS containing 1 Dawley rats and immediately immersed in the
mg/ml BSA (Ca-TBS-BSA). Medium conditioned same fixative for 30 min at room temperature.
by each hybridoma was removed aseptically, ad- After rinsing for 30 min in three changes of 20 ml
ded to the microtest plates at 50 #l/well, and 0.1 M phosphate buffer, pH 7.4, the tissues were
incubated for 2 h at room temperature. Uncondi- placed in the same buffer containing 30% sucrose
tioned medium was added to some wells to obtain at 4°C for 24-48 h. Tissues were frozen onto
background values. The subsequent procedures for cryostat chucks in O.C.T. embedding compound
ELISA and RIA are as follows. (Miles); 10 #m cross-sections of tissues were cut
 77

in a cryostat (Bright Instruments) and placed onto 0 2 4 6 II 9 l l WEEKS

gelatin-coated glass slides. DI I I I I I;) [;) FUSION
Tissue sections were incubated with hybridoma xxx xxx xxx xxx
DI ° I I I 1 D D FUSION
conditioned media for 12-20 h at 4 ° C, and rinsed
three times, 10 rain each, with 0.9% NaCI in 0.1 M EA A A _ E A E FUSION
xxx xxx xxx
phosphate buffer, p H 7.4, at room temperature.
TA A A T A T FUSION
Fluorescein-conjugated goat anti-mouse Ig anti- xxx xxx xxx
TN N T FUSION
serum, (Antibodies) diluted 1 : 50 in 15% rat serum, xxx
0.9% NaCI, 0.1 M phosphate buffer, pH 7.4, was 0 2 4 6 8 9 II WEEKS
added to the tissue sections for 1 h at room
temperature. The slides were then rinsed in 0.1 M Fig. 1. Immunization schedules. All tissues were prepared from
phosphate buffer, p H 7.4, coverslipped in a 1 : 1 rats as described in the methods section. The following fusions
correspond to antigens injected on the days indicated: DI - I:
( v / v ) mixture of glycerol: 1 m g / m i p-phenylen- immature, newborn cerebellum homogenate, D: developing,
ediamine (Sigma) in 0.1 M carbonate buffer, p H postnatal day 11 cerebellum homogenate; DI * - same as DI
8.3, and examined in a Zeiss microscope equipped without using cyclophosphamide; EA - A: insoluble fraction
with epifluorescence and a fluorescein filter set. from adult spinal cord, E: insoluble fraction from embryonic
day 18 spinal cord; TA - A: insoluble fraction from adult
spinal cord, T: insoluble fraction from the distal stump of
S D S g e l electrophoresis
transected sciatic nerve; TIC - N: insoluble fraction from
Each of the immunization preparations de- normal adult sciatic nerve, T: insoluble fraction from the distal
scribed above was boiled in SDS sample buffer stump of transected sciatic nerve. Cyclophosphamideinjections
and sized by SDS gel electrophoresis using a 12.5% are indicated by an x (100 mg/kg cyclophosphamide, 2
polyacrylamide gel (Laemmli, 1970). The gel was mg/ml). Control fusions not using cyclophosphamideare noted
with an asterisk.
fixed with methanol/acetic acid and proteins
stained with Coomassie blue.
from mice that received cyclophosphamide bound
to tissue sections of postnatal day 11 cerebellum
R ~ but not to sections of newborn cerebellum, whereas
no selective antibodies were obtained from mice
D I : m i c e t o l e r i z e d to i m m a t u r e c e r e b e l l u m a n d t h e n that did not receive cyclophosphamide (Table I,
i m m u n i z e d with d e v e l o p i n g c e r e b e l l u m fusions DI and DI * ).
Because considerable development and neurite The fact that all the antibodies from mice not
growth in the rodent cerebellum occurs between receiving cyclophosphamide bound to both new-
postnatal days 1 and 21, we were interested in born and postnatal day 11 cerebellum implies the
antigens present in the cerebellum of 11 day post- two tissues share similar antigenic determinants.
natal rats, but absent in newborn rats. We used This similarity would be expected since the im-
homogenates of newborn cerebellum as the nega- munogen was a total tissue homogenate and would
tive immunogen in this case, and l l - d a y - o l d have a high proportion of 'housekeeping' mole-
cerebellar tissue as the positive immunogen. Four cules common to many cell types and develop-
repeated tolerization steps (Fig. 1), at 2 week mental stages; indeed, the protein components of
intervals, were found to suppress antibody pro- each immunogen appear identical when assessed
duction when serum titers to newborn rat cerebel- by one-dimensional SDS electrophoresis (Fig. 2,
lum were evaluated. After two immunizations with lanes 1 and 2). The fact that cyclophosphamide-
11 day cerebellum, cell fusion was performed on treated mice resulted in a high frequency of anti-
spleen cells from these mice as well as others not bodies that distinguish the two similar immuno-
receiving the cyclophosphamide treatment. The gens argues in favor of this approach.
antibodies resulting from these fusions (termed
' D I ' for 'developing vs. immature cerebellum') E A : m i c e t o l e r i z e d to a d u l t s p i n a l c o r d a n d i m -
were screened by immunohistochemical staining m u n i z e d with e m b r y o n i c d a y 18 s p i n a l c o r d
of tissue sections. 85% of the antibodies prepared In effort to raise monoclonal antibodies against
78

TA: mice tolerized to adult spinal cord and im-

I 2 3 4 5 6 munized with the distal stump o f adult sciatic nerve
We exploited the differential distribution of
200 neurite growth-promoting molecules suggested by
the observation that peripheral nerves support ef-
ficient axonal regeneration whereas adult CNS
tissues do not. The method was to fuse spleen cells
from mice which had been rendered tolerant to
adult CNS antigens and stimulated by peripheral
nerve antigens. We immunized with the distal
stump from transected sciatic nerve rather than
normal sciatic nerve because we were more inter-
ested in antigens of the microenvironment which
would be encountered by regrowing axons than in
811. antigens of the axons themselves. Similarly, im-
munizations were carried out with membrane pre-
parations because we were interested in molecules
of the cell surface or the extraceUular matrix,
rather than soluble cytoplasmic proteins.
Of the 147 monoclonal antibodies generated by
this fusion (termed ' T A ' for 'transected sciatic
nerve vs. adult spinal cord'), 28 stained transected
Fig. 2. SDS gel electrophoresis of antigen preparations. Anti- sciatic nerve antigens by immunofluorescence. Of
gen preparations were fractionated on a 12.5% acrylamide gel these 28, only 11 also stained material in adult
and proteins were visualized with Coomassie blue. The pre-
spinal cord; therefore, 17 of 28 (or 61%) were
parations are as follows: lane 1: I, newborn cerebellum homo-
genate; lane 2: D, postnatal day 11 cerebellum homogenate; ' P N S specific' (Table I, fusion TA).
lane 3: A, insoluble fraction from adult spinal cord; lane 4: E,
insoluble fraction from embryonic day 18 spinal cord; lane 5: TN: mice tolerized to normal sciatic nerve and
N, insoluble fraction from adult sciatic nerve; lane 6: T, immunized with transected sciatic nerve
insoluble fraction from the distal stump of transected sciatic Denervated target tissues appear to contain
nerve.
higher levels of neurotrophic factors than their
innervated counterparts (cf., Ebendal et al., 1980).
molecules which underlie the ability of developing Likewise, peripheral nerves appear to react to
CNS to support vigorous axon growth, we tolerized injury by increasing their concentrations of neuro-
mice to adult CNS tissue and immunized the same trophic factors (Richardson and Ebendal, 1982).
mice with developing CNS tissue. Of the 401 The possibility exists, then, that the concentration
monoclonal antibodies generated by this fusion of substrate-bound neurite growth-promoting mol-
(termed ' E A ' for 'embryonic vs. adult'), 43 stained ecules is higher in transected sciatic nerves than in
embryonic spinal cord as well as developing cere- normal sciatic nerves. Accordingly, we sought to
bellum. Of these, 10 (or 23%) did not stain adult produce monoclonal antibodies directed against
spinal cord or cerebellum (Table I, fusion EA). molecules that were induced in the distal stump of
These are, in fact, unusual antibodies since of the sciatic nerve after transection. We subjected mice
442 antibodies that bound developing CNS (DI to a single tolerization with normal sciatic nerve
and D I * ) not one was capable of distinguishing homogenates followed by a single immunization
developing from mature CNS tissues. The protein with transected sciatic nerve membranes. The
compositions of embryonic and adult spinal cord tolerization was intentionally kept to a minimum
membranes are very similar, although some dif- because of the suspicion that normal sciatic nerves
ferences can be detected by one-dimensional SDS would actually contain the putative induced fac-
gel electrophoresis (Fig. 2, lanes 3 and 4). tors, albeit at lower levels. With a less aggressive
 79

TABLE I

(a) Fusion protocol: DI D1 * EA TA TN

(b) no. of spleens (fusions): 4 4 1 1 2
(c) Total no. of hybridomas: 387 965 401 147 764
(d) no. tested by immunohistochemistry: 387 230 123 147 287
(e) no. that bound tissue sections: 286 156 43 28 37
( 0 % of (e) that bound both tissues: 15 100 77 39 100
(g) % of (e) that made definitive
distinction: 85 0 23 61 0
(h) % of (e) more concentrated
in immunizing tissue: n.a. n.a. n.a. n.a.

Key: Fusions are as outlined in Fig. 1: DI = developmental antigens selected using cyclophosphamide; DI * = control mice for
fusion DI did not rec~ve cyclophosphamide; EA = embryonic spinal cord specific antigens selected using cyclophosphamide;
T A = PNS-specific antigens selected using cyclophosphamide; T N = antigens induced in the distal stump selected using
cyclophosphamide, n.a. means not assessed.

tolerization protocol, we hoped to avoid the total It is interesting to note that no antibodies were
elimination of lymphocytes responding to antigens obtained which made an absolute distinction be-
in normal nerve. tween normal and transected nerves. This could be
The resulting monoclonal antibodies were due to the single treatment with normal nerve and
screened by immunohistochemistry on tissue sec- cyclophosphamide, or it may actually reflect the
tions of normal and transected sciatic nerves. In molecular profiles of the tissues. SDS gel electro-
addition to the immunohistochemical screen, all phoresis reveals no unique bands; however, several
antibodies were screened in a radioimmunoassay protein bands show relative differences in staining
for binding to membranes prepared from trans- intensities (Fig. 2, lanes 5 and 6).
ected sciatic nerves. Of the 764 monoclonal anti-
bodies generated by two fusions (termed " I N ' for G: mice injected with heparin and chondroitin fol-
'transected vs. normal sciatic nerves'), only 37 lowed by cyclophosphamide and later boosted with
stained transected sciatic nerve antigens by im- the same GAGs
munofluorescence. Of these, 20 stained structures When initial attempts to generate monoclonal
in normal sciatic nerve with the same intensity antibodies to G A G s using standard immunization
and 17 (or 46%) stained antigens that were in- procedures proved unsuccessful, we attempted to
duced in the distal stump of sciatic nerves after use cyclophosphamide to eliminate stimulated T
transection. suppressor cells. The rationale was that because

T A B L E II

(a) Fusion protocol: G G* H H* F

(b) no. of spleens (fusions): 2 2 1 1 1
(c) Total no. of hybridomas: 69 451 56 187 33
(d) no. binding G A G preparations in ELISA: 32 93 14 26 15
(e) ¢/, of (d) which bound
tissue sections: 66 0 50 4 33
(f) no. binding to G A G receptor: n.a. n.a. 35 0 9
(g) • of (f) that bound rat
tissue sections: n.a. n.a. 83 0 1~

Key: Fusions arc as outlined in Fig. 3: ( 3 - antibodies to chondroitin sulfate and heparin selected using cyclophosphamidc;
G * - control mice for fusion G did not receive cyclophosphamide; H - antibodies to hyaluronate and hyaluronate-binding proteins
selected using cyclophosphamide; H * = control mice for fusion H did not receive cyclophosphamide; F = antibodies to fucoidan and
fucose-binding proteins selected using cyclophosphamide, n.a. means not asses~d.
 80

these carbohydrates are highly conserved between H: mice injected with hyaluronate followed by
species and ubiquitous to many tissues, an im- cyclophosphamide and later boosted with hy-
mune response was actively suppressed to these aluronate
molecules. Cyclophosphamide should selectively Seven antibodies were generated which bound
kill any class of stimulated, proliferating lympho- hyaluronate in an ELISA and stained sections of
cytes and therefore might be used to eliminate rat tissues. In addition, this fusion was screened
suppressor cells and in effect make mice autoim- for the production of immunoglobulins which re-
mune to GAGs. cognize a hyaluronate-binding protein, namely,
When cyclophosphamide was included (Table hyaluronidase. This procedure yielded 29 antibod-
II, fusion G), antibodies were obtained which not ies that recognize hyaluronidase and bind to tissue
only bound to commercial GAG preparations in sections. The evidence that the epitopes on hy-
an ELISA, but also recognized rat tissues in both aluronidase might be associated with the GAG
ELISA and immunohistochemistry. Although 93 binding sites includes: (1) hyaluronate inhibits
antibodies were obtained which bound the GAG antibody binding to hyaluronidase, (2) two anti-
preparations when cyclophosphamide was not used bodies which were tested were both able to inhibit
(Table II, fusion G*), none of these antibodies the enzymatic activity of hyaluronidase, (3) these
was able to recognire rat tissues. It is likely that antibodies recognize both bacterial and mam-
many of these antibodies bound contaminating malian hyaluronidases (data not shown). Since an
molecules in the GAG preparations. anti-GAG response was elicited with this protocol,
Our initial analysis revealed 21 antibodies that these immunoglobulins might have elicited an
reacted with heparin and chondroitin in an ELISA anti-idiotypic response. If the anti-hyalurortidase
and also bound to tissue homogenates and sec- antibodies are anti-idiotypic immunoglobulins,
tions. More rigorous biochemical studies have de- they may be valuable in identifying proteins that
termined that most of these antibodies, although recognize hyaluronate and could be used to inhibit
demonstrating a clear preference for either chon- the binding of such proteins to hyaluronate. When
droitin or heparin, display cross reactivity with cyclophosphamide was not used only one anti-
other GAG types. body to hyaluronate was obtained and no putative
anti-idiotypes were detected (Table II, fusions H
and H*).
0 2 WEEKS
F: mice injected with fucoidan followed by
G G G FUSION cyclophosphamide and later boosted with fucoidan
X This fusion verified our results using hy-
G* G G FUSION aluronate. Nine antibodies were obtained which
bind plant lectins specific for fucose as well as
H H H FUSION mammalian fucosidases (Table II, fusion F). Ap-
Y propriate carbohydrates (fucose and fucoidan) in-
H* H H FUSION hibit antibody binding (data not shown). Such
antibodies, like the anti-hyaluronidase immuno-
F F FUSION globulins, represent potential anti-idiotypes. Five
X antibodies with specificity for fucose were also
i) 2 WEEKS obtained.

Fig. 3. Immunization schedules. The following fusions corre-

spond to antigens injected on the days indicated: O - mixture Discussion
of the G A G s chondroitin sulphate and heparin; H - hy-
aluronate; F - fucoidan. Cyclophosphamide injections are
indicated by an x (100 m g / k g ) or by a y (150 m g / k g Cyclophosphamide treatment has pronounced
cyclophosphamide, each at 2 m g / m l ) . Control fusions not effects on the immune response and allows one to
using cyclophosphamide are noted with an asterisk. generate monoclonal antibodies to a different set
 81

of epitopes than would be obtained without the that recogniTe determinants that are transiently
use of the drug. The immune system responds to expressed during the development of the central
any particular antigen in a complex manner which nervous system, (c) antibodies that bind epitopes
depends upon the characteristics of each antigen induced in the distal stump of a severed peripheral
and the immunological history of the animal; this nerve, (d) antibodies that bind giycosaminogly-
response involves the proliferation of distinct cans, and (e) putative anti-idiotypic antibodies
populations of lymphocytes. If, following an im- which recognize carbohydrate binding molecules.
munization, the system is perturbed by selectively Since the histological staining patterns for most of
killing the mitotic cells, then the system will have these antibodies are very different, it appears that
a repertoire of lymphocytes capable of a different a variety of molecules has been identified within
immune response following subsequent antigen each fusion. Additionally, cyclophosphamide does
challenge. seem to minimize immunosuppression since virtu-
Admittedly, it is very difficult to control for the ally all our antibodies bind to rabbit, mouse, rat,
effects of drug treatment on the production of chicken and frog tissue sections with the same
monoclonal antibodies. Since monoclonal anti- apparent specificity.
body production is not an exact process there can It will be interesting to determine if the puta-
be considerable variability between mice im- tive anti-idiotypic antibodies are genuine. At this
munized using the same protocol; therefore, the point the argument is indirect. The network theory
results of any one of these immunization protocols of Jerne (1974) predicts that immunization with
could be considered fortuitous. When one consid- antigen elicits antibodies to both the antigen as
ers the experiments collectively, however, there well as anti-idiotypic clones. In a normal immune
arises a good argument that this methodology response anti-idiotypes have been detected at low
provides a useful strategy for the production of levels and anti-idiotypic monoclonal antibodies
monoclonal antibodies. Although the appearance have been isolated from mice injected with an
of any particular monoclonal antibody has a cer- agonist of the acetylcholine receptor (Cleveland et
tain probability, we believe cyclophosphamide can al., 1983). If it is assumed that the proliferation of
be used in a systematic manner to boost the anti-idiotypic lymphocytes is controlled during the
probability of obtaining a desired antibody. normal immune response, and that cyclophos-
As one example of how this approach can be phamide enhances this response, then the drug
exploited, it is useful to compare the results of the may effect the regulation of the anti-idiotypic
TA and TN fusions where the final immunogen in response.
each case was the transected sciatic nerve mem- The specific protocols presented here are in-
brane preparation. The TA immunization, which tended to be used as models for designing im-
was designed specifically for biasing the immune munization protocols. One important considera-
response toward antigens which are present in the tion is that the tolerization generated by cyclo-
PNS but absent from the CNS, generated a much phosphamide is not permanent. It can be main-
higher proportion of such 'PNS-specific' antibod- tained for relatively long periods if the mice are
ies than the TN immunization (61% (Table I, challenged and treated with cyclophosphamide ev-
fusion TA) vs. 8% (data not shown)). Conversely, ery 4 weeks (for example, fusion DI). When mice
the TN immunizations were more efficient than analogous to those tolerized by four treatments
the TA immunizations at generating monoclonal with newborn cerebellum and drug were then im-
antibodies against epitopes that are induced in the munized over the subsequent 6-7 weeks, rather
distal stump of sciatic nerves after injury (46% than over 5 weeks, there was a strong immune
(Table I, fusion TN) vs. 11% (data not shown)). response to shared epitopes (Felix Eckenstein, per-
Using a variety of immunogens and immuniza- sonal communication). This suggests that some
tion schedules, this approach has allowed the pro- aspect of immunological memory, for instance T
duction of interesting and useful antibodies, in- helper lymphocytes, escapes the cyclophospha-
cluding: (a) antibodies that bind peripheral mide treatment but can be maintained in a quies-
nervous system-specific molecules, (b) antibodies cent state as long as cyclophosphamide treatment
82

occurs every 4 weeks, a result consistent with the A W S is a recipient of a Public Health Service,
in vitro studies of Stevenson and Fauci (1980). National Research Service A w a r d no. 2T 32
Using h u m a n lymphocytes it was determined that GM07753-07, from the National Institute of Gen-
cyclophosphamide has its greatest effect on B eral Medical Sciences.
cells, followed secondarily by the T suppressor
cells. T helper cells were reported to be relatively
resistant. Therefore, when designing an immuniza- References
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