HYBRIDOMA Volume 11, Number 3, 1992 Mary Ann Liebert, Inc., Publishers
B-Cell Hybridoma as Intraperitoneal Tumor Model: Correlation Between Tumor Growth and Monoclonal Antibody Production IGNACE VERGÜTE,1 LAURE DE VOS,2 JAN FJELD,2 JAHN NESLAND,3 BENTE KIERULF,2 TERJE MICHAELSEN,4 and KJELL NUSTAD2
'Department of Gynecologic Oncology, 2 Central Laboratory and3 Department of Pathology of the Norwegian Radium Hospital, Montehello, N-0310 Oslo, Norway "The National Institute of Public Health, Geitmyrsveien 75, N-0462 Oslo, Norway
ABSTRACT
Murine B-cell
hybridoma cells producing an immunoglobulin immunoglobulin kappa chains were inoculated intraperitoneally in mice. After intraperitoneal injection of IO6 K13 hybridoma cells, superficial intraperitoneal implants and ascites developed, resulting in death after 10 ± 3 days (mean ± SD) An immunoradiometric assay was developed to GÌ
(K13), specific
for human
.
K13 in murine blood, ascites and culture supernatant. The assay utilized polymer beads coated with human immunoglobulin G. The amount K13 bound to the particles was measured with a 1J5Ilabelled monoclonal rat antibody (LO-MGl-13) specific for mouse IgGl. The assay could be used over a wide working range (2-500 µg/l). Kinetic studies suggested that about 105 secreting cells were required for detection of K13 in blood. After injection of 10' cells, K13 was measurable in blood 1 day later in all animals. Nine of 33 mice injected with 105 or less cells survived, and initially showed rising K13 blood levels followed by decreasing blood levels. In conclusion, a close relationship was established between i.p. growth of the hybridoma K13 cell line and the MAb blood levels. The basic concepts of this assay can readily be adopted for other clones with the limitation that pure antigen is needed for solid phase extraction of the MAb from mouse blood. measure
INTRODUCTION In order to obtain
a monoclonal antibody laboratory practice to inject hybridoma cells intraperitoneally in mice of the same strain from which they are derived. Ascites production is often increased by pretreatment with i.p. injection of a local irritant such as Pristane. It is, however, well recognized that hybridomas show marked differences in growth characteristics and antibody production. In the present experiment, we wanted to establish the correlation between i.p. hybridoma tumor growth and blood levels of the monoclonal antibody. In selecting the hybridoma cells, we searched for the following properties : rapid growth of the clone in the abdominal cavity without any pretreatment of the host
from
ascites, it is
large quantities of
a common
Abbreviations : SD, standard
deviation; MAb, monoclonal PBS, phosphate buffered saline (0.15 M NaCl, 0.01 M NaH2PO,, 0.01% NaN3, pH 7.2); DMEM, Dulbecco's modified Eagle's medium; IRMA, immunoradiometric assay; i.p., intraperitoneal. antibody; IgG, Immunoglobulin G;
323
animal, early detection of the MAb in the murine blood, and the
ability of antigen.
Based
the MAb to be directed on
an
easily
available
criteria,
these
kappa light chains of for measuring the MAb found in blood.
against
a clone producing a MAb to the human IgG was chosen. An assay was developed in the presence of mouse immunoglobulins
MATERIALS AND METHODS
Preparation of the Monoclonal Antibody K13 Female Balb/c mice (Charles River U.K. Ltd., Kent, England) about ten weeks old, were immunized with 10 µg polyclonal human IgG in Freund's complete adjuvant on multiple subcutaneous sites. The animals were boostered with the same dose 3 weeks later. The fusion was performed 5 weeks later after four daily intravenous injections of 50 µg IgG in 0.9% NaCl. The spleen cells were fused with X63.Ag8.653 mouse myeloma cells in a ratio of 4:1 in the presence of polyethylene glycol MW 4000 (Merck, Darmstadt, Germany). The cells, in DMEM (Gibco Ltd., Paisly, Scotland), supplemented with hypoxanthine, aminopterin, thymidine, and 15% of fetal calf serum (Flow Labs., England) were seeded into 96-well tissue culture plates (Nunc, Copenhagen, Denmark), containing 104 mouse peritoneal macrophages per well.
Screening for Anti-Human IgG Antibodies
screening was performed as described for clones antibodies to neuron specific enolase with the exception that 125I-labelled human IgGlK, IgG2K, IgG3X, or IgG4K were used as labelled antigens (1). The clone used in the present study produced an antibody reacting with all Ig's except ^ 3 . Its specificity for the kappa light chain was verified by using 125Ilabelled free kappa chains, free lambda chain and IgMK. The human Ig's were purified from sera, while the free light chains were purified from urine of patients with multiple myelomas with standard Chromatographie techniques and iodinated using Iodogen (Pierce, U.S.A.) as oxidant (2). The
producing
Production and Purification of Monoclonal Antibodies Monoclonal antibodies were produced as ascites and purified Protein- Sepharose (Pharmacia, Uppsala, Sweden) as previously described (1). on
Hybridoma cells and Cell culture 75 cm2 plastic cell Hybridoma cells were cultured in culture flasks at 37°C in a C02 incubator. The medium used was DMEM supplemented with sodium pyruvate (1 mM), L-glutamine (2 Mm), penicillin (100 units/ml), streptomycin (100 µg/ml) and 10% fetal calf serum. For estimating the doubling time the hybridoma cells were incubated in cell culture flasks at a density of about 0.6 10s cells/ml (10 experiments). The cells were counted daily, and cell viability was tested with the trypan blue dye exclusion method.
Immunoradiometric Assay of the Mab K13
Duplicates of 0.1 ml standards, controls or samples were incubated for 30 minutes with 0.1 mg beads (Dynabeads M2 80, Dynal AS, Oslo, Norway) coated with polyclonal human IgG in 0.1 ml assay buffer (0.05 M Tris/Hcl, 0.01% Merthiolat, 0.1 M NaCl, 0.1% casein, 0.1% Tween 20, pH 7.4). Coating of the beads with human IgG
was
performed
as
reported previously (3).
Free
antigen
was
removed by washing three times with 0.7 ml PBS supplemented with 1 ml Tween 2 0 per liter. Separation was performed on magnetic racks (Amersham, England) A 125I-labelled rat MAb to mouse IgGl .
324
(LO-MGl-13) (4), provided by H. Bazin (Experimental Immunology Unit, University of Louvain, Belgium) was added and the mixture was agitated for one hour at room temperature. Iodination of LOMGl-13 was performed according to Fraker and Speck (2). Free radioactivity was removed by washing three times as described
above. Counting was performed with an LKB Wallac 1260 Multigamma II counter and the Riacalc software was used for calculating sample concentration of K13 as well as assay sensitivity and
precision profile.
Production of K13 in vivo Female Balb/c mice were purchased at Charles River U.K. Ltd. (Kent, England). The care of the animals was in accordance to the Guidelines on Care of Laboratory Animals of the Royal Society / UFAW (5), and the UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia (6). The mice were allowed food and water
ad libitum and weighed about 20 g at the start of the experiments. The mice were injected i.p. with varying amounts of hybridoma cells suspended in 0.5 ml DMEM. The hybridoma cells were obtained from the in vitro culture after centrifugation (300 g, room temperature for 10 min), and resuspension of the cell pellet with DMEM. The animals were weighed and blood samples were collected 3 times a week during the first three weeks, and later once a week. Blood was collected from the tail vein (5 µ ) and diluted to 0.5 ml with 0.9% sodium chloride solution, centrifuged (300 g at room temperature for 10 min) and the level of K13 was measured in the supernatant. Further dilutions, when necessary, were made with the assay buffer. Animals were killed by cervical dislocation when showing more than 20% weight increase or deteriorating condition. Samples of ascites were collected for determination of K13, and all mice had a necropsy performed. The abdomen and thorax were opened and the organs inspected. The liver, spleen, kidneys, intestines, lungs, diaphragm, and any tumor present were fixed for histological examination in neutral buffered formalin. K13 Metabolism in vivo
The MAb K13 (100 µg) was diluted in 0.5 ml 0.9% sodium chloride solution and injected i.p. in mice. An extra 0.5 ml was injected to rinse the needle. Blood samples were collected for determination of the K13 level. RESULTS
Immunoradiometric Assay of the MAb K13 More than 85% of the highest K13 standard (500 µg/liter, 0.05 µg/tube) was extracted when 0.1 mg particles (coated with about 1 µg human IgG) were incubated with the K13 standard for 30 min. Increasing the amount of particles reduced the incubation time, but this advantage was offset by an increased non-specific binding and lowered assay sensitivity. The assay sensitivity was 1 µg/liter, with a working range of 2-500 µg/liter, with a coefficient of variation below 10% (Figure 1). The precision profile of the assay is also presented in Figure 1. The nonspecific binding was 0.2% when no mouse serum was added, increasing to 0.3% and 0.5% with the addition of 1 µ and 10 µ mouse serum, respectively. LO-MGl-13 was compared with a polyclonal sheep anti-mouse antibody. The polyclonal antibody gave better assay sensitivity (data not shown), but the working range was only 0.5-50 µg/liter due to a low immunoreactive fraction (2035%). Furthermore, addition of mouse serum gave a larger increase in non-specific binding when the polyclonal antibody was used as tracer. Increasing the amount of labelled antibody augmented the assay's precision and enlarged the working range, using either of the two labelled antibodies. However, this was impractical as we IO5 cpm labelled antibody per assay tube. already used 2 Recovery of K13 from mouse serum was 104 ± 6.5%, from fetal calf serum 97 ± 4.9%, compared with 100 ± 5.3% for the assay = buffer (mean ± SD; 10). Thus the analysis can be applied to
325
100
100
200
300
400
500
(Mg/liter)
K13
1. Standard Curve and Precision Profile of an Immunoradiometric Assay using Beads as Solid Phase and 125Ilabelled Rat MAb LO-MGl-13 to Mouse IgGl to Measure K13 in Murine Blood, Ascites and Culture Supernatant. Open circles: standard curve of LO-MGl-13 (total counts: 176.000 cpm). Solid circles: precision profile (based on 4 assays and 510 duplicates).
Figure
the level of K13 in cell culture and ascites samples from mice.
measure
supernatant
and in blood
Hybridoma Cell growth in vivo Four groups of at least 10 Balb/c mice were injected i.p. with different amounts of hybridoma cells (106, 105, 104, and 103 cells). We observed weight increase due to i.p. tumor spread and ascites production. Necropsy and histology showed i.p. spread with superficial implants on the peritoneum and all intraabdominal organs (Figure 2 and 3) in all mice sacrificed because of clear tumor progression, but no extraabdominal spread was noted. Infiltration by the tumor implants in the parenchyma of liver, spleen or pancreas was observed in 13 animals. Survival and histological findings are summarized in Table 1. TABLE 1
Survival and Histological Injected Cells.
in relation to Number of
Findings
i.p.
Histological findings"
Injected cells
Tumor
progression
daysb
Diffuse tumor
i.p. implants
Solid tumor
i.p implant
10'
12/12
10
±
3
11
11
10s
11/13
19
±
6
11
7
10'
9/10
31
±
9
9
3
10J
4/10
33
7
2
1
l None of the mice sacrificed because of tumor tumor spread. None of the surviving mice histological examination. b: Mean ± SD (surviving mice excluded).
progression had extraabdominal had tumor detected at necropsy and
°: At least 0.1 ml
326
Figure 2. Intraperitoneal Hybridoma Kl3 implants: Superficial Implant (arrows) on the Small Bowel ( 140 hematoxylin and eosin),
showed a uniform clinical short survival. In animals that received less than 106 cells, survival was more variable, ascites was often lacking, and the tumors were larger. Mice that survived for 120 days were sacrificed at that time. We observed neither macroscopic nor microscopic tumor in this group of animals. Mice injected with 10e cells picture with i.p. tumor spread and
Figure
3. Intraperitoneal in the liver hilus (x 140
Hybridoma K13 implants: Implant (arrows) hematoxylin and eosin).
327
Production of Kl3 in vivo The Kl 3 blood levels are presented in Figure 4. Since the of the K13 assay ranged from 2-500 µg/liter, and the were diluted 100 times, the lowest detectable K13 blood level was 0.2 mg/liter. None of the mice had a detectable K13 blood level before the injection of the hybridoma cells. In all mice injected with 106 cells, K13 could be detected in the blood 24 hours after the injection (mean, 2.3 mg/liter; range, 0.4 3.2 mg/liter), and all showed a similar K13 blood level over time. The K13 blood levels were less uniform in mice injected with less than 10e cells. All mice with a K13 blood level above 1000 mg/liter were sacrificed because of tumor progression within 14 days. All mice with tumor at necropsy had a measurable K13 blood level (5160 ± 3340 mg/liter; mean ± SD). The lowest K13 level at death in mice receiving IO6 or 105 cells was 980 mg/liter. Three mice with slow progression showed falling K13 blood levels (two received 104 cells, and 1 received 103 cells). All surviving animals (n 9) showed an initial peak in the K13 blood levels between 16 and 24 days (median, 20 days) after the injection of the hybridoma cells. The K13 blood level then decreased, but was still measurable at 2.6 mg/liter). In day 120 in three of these animals (range, 0.3 none of these mice tumor cells were found at necropsy. In the mice injected with 103 cells, the K13 concentrations in blood reached a detectable level after at least 10 days. The K13 level in ascites (6500 ± 4950 mg/liter; mean± SD) was similar to the K13 level found in blood, and correlated well with the K13 blood level sampled at the day of death (Spearman rank correlation, 0.935; t-value, 11.791; degrees of freedom: 20; < 0.001). In mice with tumor the K13 blood level at the day of sacrifice was similar in mice with and without ascites (47 80 ± 2850 mg/liter and 5770 ± 3920 mg/liter, respectively; mean ± SD). Four mice were injected i.p. with 100 µg of the MAb K13 and the K13 blood levels were monitored (Figure 5). The maximum K13 blood level was found 10 h after the injection, and the half-life of K13 was about 11 days. Accepting that mice weighing about 20 g have a blood volume of 1.2 ml (7), we can calculate that 22% of the i.p. injected K13 was present in the blood 24 hours after the injection (mean K13 blood level, 18.1 mg/liter).
working range blood samples
-
=
-
0
Time
Figure 10ß (
4. Blood K13 Levels in Mice =
20
30
0
50
120
Idays
Injected i.p.
12), , 5 ( 13), C, IO4 ( 10) Hybridoma Cells. Full lines represent mice =
10
=
on
Day 0 with A,
10), and D, IO3 (
dashed lines represent surviving mice. The lowest measurable K13 blood level is 0.2 mg/liter, and K13 blood levels below this limit have been
progression, plotted
as
0.1
mg/liter.
328
=
sacrificed because of tumor
120
Time
(days)
Figure 5. K13 blood levels (mean ± SD) after i.p. injection of 0.1 4) The lowest measurable K13 blood level is 0.2 mg/liter, and K13 mg K13 ( blood levels below this limit have been plotted as 0.1 mg/liter. The first day blood samples were collected 1, 2, 3, 4, 10, 24 hours after the injection. =
.
As described above, we injected 106 hybridoma cells i.p. in mice and found a mean blood level of MAb of 2.3 mg/liter after 24 hours. The mean doubling time in vitro was 29 hours (range, 23 34 h). Assuming that the doubling time is approximately the same in vivo as in vitro, a mean of 1.3 IO6 cells were secreting K13. Accepting that the blood volume is 1.2 ml (7), and that about 22% of the produced K13 is present in the blood after 24 hours, we can calculate that a K13 hybridoma cell produces about 10 pg K13 per 24 hours in vivo. Based on the above mentioned assumptions, we can calculate that about 105 secreting cells are needed to give a measurable (> 0.2 mg/liter) K13 blood level. Indeed, 9 of 13 mice injected with 105 cells had a detectable level 24 hours later, while the first measurable blood levels in animals injected with 104 and 103 cells were observed after 3 and 10 days, respectively. -
DISCUSSION
Many methods have been developed to measure the level of MAbs in hybridoma culture supernatant (8-11). The main problem with measuring a MAb in mouse blood is the high concentration of normal mouse immunoglobulins. Therefore, the antigen used on the solid phase to extract the MAb must be available in large quantities, and only clones producing MAbs against such antigens can be used. In the current report we describe a sandwichimmunoradiometric assay using beads coated with human immunoglobulin as solid phase, and 125I-labelled rat MAb LO-MGl-13 to mouse IgGl. In this way it was possible to establish an assay for the monoclonal antibody K13 with negligible interference of mouse immunoglobulins. Kinetic studies suggested that only 105 secreting cells were needed to give a detectable K13 blood level. All animals that survived after injection of 105 cells or less initially showed rising K13 blood levels with a peak 20 days after the injection of the hybridoma cells. In the mice injected with 103 cells, the K13 blood levels were not measurable during the first 10 days. These findings suggest that the hybridoma cells first proliferated before the host managed to eliminate the hybridoma cells. In three of these animals, the K13 blood level was still measurable at day 120 (range, 0.3 2.6 mg/liter), but no tumor was found at necropsy. This may be due to the fact that these blood levels represent less than 10' cells, or may reflect the half-life of K13 which is about 11 days (Figure 5). In all i.p. growing tumors, early diagnosis of tumor -
329
progression is difficult because they often provide multiple métastases at sites where they are difficult to measure. In the present study, the K13 blood levels reflected tumor growth well (Figure 4). In the current study, only 5 µ blood was sampled from the tail vein, and the K13 blood level was measurable in all mice as early as 24 h after the injection of 10e hybridoma cells. Intraperitoneal injection of 6 K13 hybridoma cells resulted in a standard tumor growth pattern with diffuse superficial i.p. tumor spread and ascites, and a short mean survival (mean ± SD, 10 ± 3 days) (Table 1, Figure 2 and 3). This is considerably shorter than the median survival after i.p. inoculation of human cancer lines in nude mice which is 25 43 days (12-15). When using fresh human tumor, survival ranged from 1-9 months (16). conclusion, a close relationship was established between i.p. growth of the hybridoma K13 cell line and MAb blood levels. The basic concepts of this assay can readily be adopted for other clones with the limitation that pure antigen is needed for solid phase extraction of the MAb from mouse blood.
primary
-
In
ACKNOWLEDGMENT
This work
was
grant 88222.
supported by
the
Norwegian
Cancer
Society by
REFERENCES
1. Paus, E. and Nustad, K. (1989) Immunoradiometric assay for and enolase (neuron-specific enolase), with use of monoclonal antibodies and magnetizable particles. Clin. Chem. 35: 2034-2038.
2. Fraker, P.J. and Speck, J.C.Jr. (1978) Protein and cell membrane iodinations with a sparingly soluble chloramide,
l,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem. Biophys.
Res. Commun.
80: 849-857.
3. Nustad, K., Johansen, L., Ugelstad, J., Ellingsen, T. and Berge, A. (1991) Monodisperse particles as solid-phase material in immunoassays: Comparison of shell and core particles with compact particles. Eur. Surg. Res. 16 (Suppl 2): 80-87. 4. Bazin, H. (1982) Production of rat monoclonal antibodies with the LOU rat nonsecreting IR983F myeloma cell line. In: H. Peeters (Ed.), Protides of the biological fluids. Pergamon Press, Oxford pp. 615-618.
Royal Society / Universities Federation for Animal Welfare (1987) Guidelines on the care of laboratory animals and their use for scientific purposes. Part 1 Housing and care. 5.
(UFAW),
UFAW,
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Kingdom Coordinating Committee on Cancer Research UKCCCR guidelines for the welfare of animals experimental neoplasia. Br. J. Cancer. 58: 109-113. 6. United
(UKCCCR), (1988) 7. Cunliffe
mouse.
In:
Beamer, T.L. and Les,
E.P. (1987) The laboratory (Ed.), The UFAW handbook on the care and laboratory animals. Longman Group UK Limited,
T.B. -
in
Poole
management of Harlow, Essex pp. 278-308.
8. Hunter, K.W. and Bosworth, J.M. (19 86) Measurement of monoclonal immunoglobulin concentrations in hybridoma cultures by competitive inhibition enzyme immunoassay. Methods Enzymol. 121: 541-547. 9. Butler, J.E., Spradling, J.E., Suter, M., Dierks, S.E., Heyermann, H. and Peterman, J.H. (1986) The immunochemistry of
sandwich ELISAs
I. The binding characteristics of to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and assymetrical antibody-enzyme conjugates. Mol. Immunol. 23: 971-982.
immunoglobulins
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10. Fleming, J.O. and Pen, L.B. (1988) Measurement of the concentration of murine IgG monoclonal antibody in hybridoma supernatants and ascites in absolute units by sensitive and reliable enzyme-linked immunosorbent assays (ELISA). J. Immunol. Methods. 110: 11-18.
11. Delaunay, T., Louahed, J. and Bazin, H. (1990) Rat (and mouse) antibodies. Vili. ELISA measurement of Ig production in mouse hybridoma culture supernatants. J. Immunol. Methods. 131: 33-39. 12. Hamilton, T.C., Young, R.C, Louie, K.G., Behrens, B.C., McKoy, W.M., Grotzinger, K.R. and Ozols, R.F. (1984)
Characterization of a xenograft model of human ovarian carcinoma which produces ascites and intraabdominal carcinomatosis in mice. Cancer Res. 44: 5286-5290.
Kondo, H., Tannaka, N. and Orita, K. (1987) Novel models of human cancer metastasis in nude mice: lung metastasis, intraabdominal carcinomatosis with ascites, and liver metastasis. J. Cancer. Res. Clin. Oncol. 113: 544-549. 13. Naomoto, Y.,
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14. Ripamonti, F., Canevari, S., Ménard, S., Mezzanzanica, D., Miotti, S., Orlandi, S. and Rilke, F. (1987) Human carcinoma cell lines xenografted in athymic mice: biological and antigenic
characteristics of an intraabdominal model. Cancer Immunol. Immunother. 24: 13-18.
Reale, F.R., Griffin, T.W., Compton, J.M., Graham, S., Townes, and Bogden, A. (1987) Characterization of a human malignant mesothelioma cell line (H-MESO-1): a biphasic solid and ascitic 15.
P.L.
tumor model. Cancer Res.
47: 3199-3205.
Ward, B.G., Wallace, K., Shepherd, J.H. and Balkwill, F.R. (19 87) Intraperitoneal xenografts of human epithelial ovarian
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Reprint Requests: I.
Vergote,
M.D.
Norwegian Radium Hospital Department of Gynecologic Oncology The
Montebello, N-0310 Oslo Norway Telephone: 02/506050 Facsimile: 02/525559
Received for publication: 10/29/91 Accepted after revision: 1/28/92
331
B-Cell Hybridoma as Intraperitoneal Tumor Model: Correlation Between Tumor Growth and Monoclonal Antibody Production IGNACE VERGÜTE,1 LAURE DE VOS,2 JAN FJELD,2 JAHN NESLAND,3 BENTE KIERULF,2 TERJE MICHAELSEN,4 and KJELL NUSTAD2
'Department of Gynecologic Oncology, 2 Central Laboratory and3 Department of Pathology of the Norwegian Radium Hospital, Montehello, N-0310 Oslo, Norway "The National Institute of Public Health, Geitmyrsveien 75, N-0462 Oslo, Norway
ABSTRACT
Murine B-cell
hybridoma cells producing an immunoglobulin immunoglobulin kappa chains were inoculated intraperitoneally in mice. After intraperitoneal injection of IO6 K13 hybridoma cells, superficial intraperitoneal implants and ascites developed, resulting in death after 10 ± 3 days (mean ± SD) An immunoradiometric assay was developed to GÌ
(K13), specific
for human
.
K13 in murine blood, ascites and culture supernatant. The assay utilized polymer beads coated with human immunoglobulin G. The amount K13 bound to the particles was measured with a 1J5Ilabelled monoclonal rat antibody (LO-MGl-13) specific for mouse IgGl. The assay could be used over a wide working range (2-500 µg/l). Kinetic studies suggested that about 105 secreting cells were required for detection of K13 in blood. After injection of 10' cells, K13 was measurable in blood 1 day later in all animals. Nine of 33 mice injected with 105 or less cells survived, and initially showed rising K13 blood levels followed by decreasing blood levels. In conclusion, a close relationship was established between i.p. growth of the hybridoma K13 cell line and the MAb blood levels. The basic concepts of this assay can readily be adopted for other clones with the limitation that pure antigen is needed for solid phase extraction of the MAb from mouse blood. measure
INTRODUCTION In order to obtain
a monoclonal antibody laboratory practice to inject hybridoma cells intraperitoneally in mice of the same strain from which they are derived. Ascites production is often increased by pretreatment with i.p. injection of a local irritant such as Pristane. It is, however, well recognized that hybridomas show marked differences in growth characteristics and antibody production. In the present experiment, we wanted to establish the correlation between i.p. hybridoma tumor growth and blood levels of the monoclonal antibody. In selecting the hybridoma cells, we searched for the following properties : rapid growth of the clone in the abdominal cavity without any pretreatment of the host
from
ascites, it is
large quantities of
a common
Abbreviations : SD, standard
deviation; MAb, monoclonal PBS, phosphate buffered saline (0.15 M NaCl, 0.01 M NaH2PO,, 0.01% NaN3, pH 7.2); DMEM, Dulbecco's modified Eagle's medium; IRMA, immunoradiometric assay; i.p., intraperitoneal. antibody; IgG, Immunoglobulin G;
323
animal, early detection of the MAb in the murine blood, and the
ability of antigen.
Based
the MAb to be directed on
an
easily
available
criteria,
these
kappa light chains of for measuring the MAb found in blood.
against
a clone producing a MAb to the human IgG was chosen. An assay was developed in the presence of mouse immunoglobulins
MATERIALS AND METHODS
Preparation of the Monoclonal Antibody K13 Female Balb/c mice (Charles River U.K. Ltd., Kent, England) about ten weeks old, were immunized with 10 µg polyclonal human IgG in Freund's complete adjuvant on multiple subcutaneous sites. The animals were boostered with the same dose 3 weeks later. The fusion was performed 5 weeks later after four daily intravenous injections of 50 µg IgG in 0.9% NaCl. The spleen cells were fused with X63.Ag8.653 mouse myeloma cells in a ratio of 4:1 in the presence of polyethylene glycol MW 4000 (Merck, Darmstadt, Germany). The cells, in DMEM (Gibco Ltd., Paisly, Scotland), supplemented with hypoxanthine, aminopterin, thymidine, and 15% of fetal calf serum (Flow Labs., England) were seeded into 96-well tissue culture plates (Nunc, Copenhagen, Denmark), containing 104 mouse peritoneal macrophages per well.
Screening for Anti-Human IgG Antibodies
screening was performed as described for clones antibodies to neuron specific enolase with the exception that 125I-labelled human IgGlK, IgG2K, IgG3X, or IgG4K were used as labelled antigens (1). The clone used in the present study produced an antibody reacting with all Ig's except ^ 3 . Its specificity for the kappa light chain was verified by using 125Ilabelled free kappa chains, free lambda chain and IgMK. The human Ig's were purified from sera, while the free light chains were purified from urine of patients with multiple myelomas with standard Chromatographie techniques and iodinated using Iodogen (Pierce, U.S.A.) as oxidant (2). The
producing
Production and Purification of Monoclonal Antibodies Monoclonal antibodies were produced as ascites and purified Protein- Sepharose (Pharmacia, Uppsala, Sweden) as previously described (1). on
Hybridoma cells and Cell culture 75 cm2 plastic cell Hybridoma cells were cultured in culture flasks at 37°C in a C02 incubator. The medium used was DMEM supplemented with sodium pyruvate (1 mM), L-glutamine (2 Mm), penicillin (100 units/ml), streptomycin (100 µg/ml) and 10% fetal calf serum. For estimating the doubling time the hybridoma cells were incubated in cell culture flasks at a density of about 0.6 10s cells/ml (10 experiments). The cells were counted daily, and cell viability was tested with the trypan blue dye exclusion method.
Immunoradiometric Assay of the Mab K13
Duplicates of 0.1 ml standards, controls or samples were incubated for 30 minutes with 0.1 mg beads (Dynabeads M2 80, Dynal AS, Oslo, Norway) coated with polyclonal human IgG in 0.1 ml assay buffer (0.05 M Tris/Hcl, 0.01% Merthiolat, 0.1 M NaCl, 0.1% casein, 0.1% Tween 20, pH 7.4). Coating of the beads with human IgG
was
performed
as
reported previously (3).
Free
antigen
was
removed by washing three times with 0.7 ml PBS supplemented with 1 ml Tween 2 0 per liter. Separation was performed on magnetic racks (Amersham, England) A 125I-labelled rat MAb to mouse IgGl .
324
(LO-MGl-13) (4), provided by H. Bazin (Experimental Immunology Unit, University of Louvain, Belgium) was added and the mixture was agitated for one hour at room temperature. Iodination of LOMGl-13 was performed according to Fraker and Speck (2). Free radioactivity was removed by washing three times as described
above. Counting was performed with an LKB Wallac 1260 Multigamma II counter and the Riacalc software was used for calculating sample concentration of K13 as well as assay sensitivity and
precision profile.
Production of K13 in vivo Female Balb/c mice were purchased at Charles River U.K. Ltd. (Kent, England). The care of the animals was in accordance to the Guidelines on Care of Laboratory Animals of the Royal Society / UFAW (5), and the UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia (6). The mice were allowed food and water
ad libitum and weighed about 20 g at the start of the experiments. The mice were injected i.p. with varying amounts of hybridoma cells suspended in 0.5 ml DMEM. The hybridoma cells were obtained from the in vitro culture after centrifugation (300 g, room temperature for 10 min), and resuspension of the cell pellet with DMEM. The animals were weighed and blood samples were collected 3 times a week during the first three weeks, and later once a week. Blood was collected from the tail vein (5 µ ) and diluted to 0.5 ml with 0.9% sodium chloride solution, centrifuged (300 g at room temperature for 10 min) and the level of K13 was measured in the supernatant. Further dilutions, when necessary, were made with the assay buffer. Animals were killed by cervical dislocation when showing more than 20% weight increase or deteriorating condition. Samples of ascites were collected for determination of K13, and all mice had a necropsy performed. The abdomen and thorax were opened and the organs inspected. The liver, spleen, kidneys, intestines, lungs, diaphragm, and any tumor present were fixed for histological examination in neutral buffered formalin. K13 Metabolism in vivo
The MAb K13 (100 µg) was diluted in 0.5 ml 0.9% sodium chloride solution and injected i.p. in mice. An extra 0.5 ml was injected to rinse the needle. Blood samples were collected for determination of the K13 level. RESULTS
Immunoradiometric Assay of the MAb K13 More than 85% of the highest K13 standard (500 µg/liter, 0.05 µg/tube) was extracted when 0.1 mg particles (coated with about 1 µg human IgG) were incubated with the K13 standard for 30 min. Increasing the amount of particles reduced the incubation time, but this advantage was offset by an increased non-specific binding and lowered assay sensitivity. The assay sensitivity was 1 µg/liter, with a working range of 2-500 µg/liter, with a coefficient of variation below 10% (Figure 1). The precision profile of the assay is also presented in Figure 1. The nonspecific binding was 0.2% when no mouse serum was added, increasing to 0.3% and 0.5% with the addition of 1 µ and 10 µ mouse serum, respectively. LO-MGl-13 was compared with a polyclonal sheep anti-mouse antibody. The polyclonal antibody gave better assay sensitivity (data not shown), but the working range was only 0.5-50 µg/liter due to a low immunoreactive fraction (2035%). Furthermore, addition of mouse serum gave a larger increase in non-specific binding when the polyclonal antibody was used as tracer. Increasing the amount of labelled antibody augmented the assay's precision and enlarged the working range, using either of the two labelled antibodies. However, this was impractical as we IO5 cpm labelled antibody per assay tube. already used 2 Recovery of K13 from mouse serum was 104 ± 6.5%, from fetal calf serum 97 ± 4.9%, compared with 100 ± 5.3% for the assay = buffer (mean ± SD; 10). Thus the analysis can be applied to
325
100
100
200
300
400
500
(Mg/liter)
K13
1. Standard Curve and Precision Profile of an Immunoradiometric Assay using Beads as Solid Phase and 125Ilabelled Rat MAb LO-MGl-13 to Mouse IgGl to Measure K13 in Murine Blood, Ascites and Culture Supernatant. Open circles: standard curve of LO-MGl-13 (total counts: 176.000 cpm). Solid circles: precision profile (based on 4 assays and 510 duplicates).
Figure
the level of K13 in cell culture and ascites samples from mice.
measure
supernatant
and in blood
Hybridoma Cell growth in vivo Four groups of at least 10 Balb/c mice were injected i.p. with different amounts of hybridoma cells (106, 105, 104, and 103 cells). We observed weight increase due to i.p. tumor spread and ascites production. Necropsy and histology showed i.p. spread with superficial implants on the peritoneum and all intraabdominal organs (Figure 2 and 3) in all mice sacrificed because of clear tumor progression, but no extraabdominal spread was noted. Infiltration by the tumor implants in the parenchyma of liver, spleen or pancreas was observed in 13 animals. Survival and histological findings are summarized in Table 1. TABLE 1
Survival and Histological Injected Cells.
in relation to Number of
Findings
i.p.
Histological findings"
Injected cells
Tumor
progression
daysb
Diffuse tumor
i.p. implants
Solid tumor
i.p implant
10'
12/12
10
±
3
11
11
10s
11/13
19
±
6
11
7
10'
9/10
31
±
9
9
3
10J
4/10
33
7
2
1
l None of the mice sacrificed because of tumor tumor spread. None of the surviving mice histological examination. b: Mean ± SD (surviving mice excluded).
progression had extraabdominal had tumor detected at necropsy and
°: At least 0.1 ml
326
Figure 2. Intraperitoneal Hybridoma Kl3 implants: Superficial Implant (arrows) on the Small Bowel ( 140 hematoxylin and eosin),
showed a uniform clinical short survival. In animals that received less than 106 cells, survival was more variable, ascites was often lacking, and the tumors were larger. Mice that survived for 120 days were sacrificed at that time. We observed neither macroscopic nor microscopic tumor in this group of animals. Mice injected with 10e cells picture with i.p. tumor spread and
Figure
3. Intraperitoneal in the liver hilus (x 140
Hybridoma K13 implants: Implant (arrows) hematoxylin and eosin).
327
Production of Kl3 in vivo The Kl 3 blood levels are presented in Figure 4. Since the of the K13 assay ranged from 2-500 µg/liter, and the were diluted 100 times, the lowest detectable K13 blood level was 0.2 mg/liter. None of the mice had a detectable K13 blood level before the injection of the hybridoma cells. In all mice injected with 106 cells, K13 could be detected in the blood 24 hours after the injection (mean, 2.3 mg/liter; range, 0.4 3.2 mg/liter), and all showed a similar K13 blood level over time. The K13 blood levels were less uniform in mice injected with less than 10e cells. All mice with a K13 blood level above 1000 mg/liter were sacrificed because of tumor progression within 14 days. All mice with tumor at necropsy had a measurable K13 blood level (5160 ± 3340 mg/liter; mean ± SD). The lowest K13 level at death in mice receiving IO6 or 105 cells was 980 mg/liter. Three mice with slow progression showed falling K13 blood levels (two received 104 cells, and 1 received 103 cells). All surviving animals (n 9) showed an initial peak in the K13 blood levels between 16 and 24 days (median, 20 days) after the injection of the hybridoma cells. The K13 blood level then decreased, but was still measurable at 2.6 mg/liter). In day 120 in three of these animals (range, 0.3 none of these mice tumor cells were found at necropsy. In the mice injected with 103 cells, the K13 concentrations in blood reached a detectable level after at least 10 days. The K13 level in ascites (6500 ± 4950 mg/liter; mean± SD) was similar to the K13 level found in blood, and correlated well with the K13 blood level sampled at the day of death (Spearman rank correlation, 0.935; t-value, 11.791; degrees of freedom: 20; < 0.001). In mice with tumor the K13 blood level at the day of sacrifice was similar in mice with and without ascites (47 80 ± 2850 mg/liter and 5770 ± 3920 mg/liter, respectively; mean ± SD). Four mice were injected i.p. with 100 µg of the MAb K13 and the K13 blood levels were monitored (Figure 5). The maximum K13 blood level was found 10 h after the injection, and the half-life of K13 was about 11 days. Accepting that mice weighing about 20 g have a blood volume of 1.2 ml (7), we can calculate that 22% of the i.p. injected K13 was present in the blood 24 hours after the injection (mean K13 blood level, 18.1 mg/liter).
working range blood samples
-
=
-
0
Time
Figure 10ß (
4. Blood K13 Levels in Mice =
20
30
0
50
120
Idays
Injected i.p.
12), , 5 ( 13), C, IO4 ( 10) Hybridoma Cells. Full lines represent mice =
10
=
on
Day 0 with A,
10), and D, IO3 (
dashed lines represent surviving mice. The lowest measurable K13 blood level is 0.2 mg/liter, and K13 blood levels below this limit have been
progression, plotted
as
0.1
mg/liter.
328
=
sacrificed because of tumor
120
Time
(days)
Figure 5. K13 blood levels (mean ± SD) after i.p. injection of 0.1 4) The lowest measurable K13 blood level is 0.2 mg/liter, and K13 mg K13 ( blood levels below this limit have been plotted as 0.1 mg/liter. The first day blood samples were collected 1, 2, 3, 4, 10, 24 hours after the injection. =
.
As described above, we injected 106 hybridoma cells i.p. in mice and found a mean blood level of MAb of 2.3 mg/liter after 24 hours. The mean doubling time in vitro was 29 hours (range, 23 34 h). Assuming that the doubling time is approximately the same in vivo as in vitro, a mean of 1.3 IO6 cells were secreting K13. Accepting that the blood volume is 1.2 ml (7), and that about 22% of the produced K13 is present in the blood after 24 hours, we can calculate that a K13 hybridoma cell produces about 10 pg K13 per 24 hours in vivo. Based on the above mentioned assumptions, we can calculate that about 105 secreting cells are needed to give a measurable (> 0.2 mg/liter) K13 blood level. Indeed, 9 of 13 mice injected with 105 cells had a detectable level 24 hours later, while the first measurable blood levels in animals injected with 104 and 103 cells were observed after 3 and 10 days, respectively. -
DISCUSSION
Many methods have been developed to measure the level of MAbs in hybridoma culture supernatant (8-11). The main problem with measuring a MAb in mouse blood is the high concentration of normal mouse immunoglobulins. Therefore, the antigen used on the solid phase to extract the MAb must be available in large quantities, and only clones producing MAbs against such antigens can be used. In the current report we describe a sandwichimmunoradiometric assay using beads coated with human immunoglobulin as solid phase, and 125I-labelled rat MAb LO-MGl-13 to mouse IgGl. In this way it was possible to establish an assay for the monoclonal antibody K13 with negligible interference of mouse immunoglobulins. Kinetic studies suggested that only 105 secreting cells were needed to give a detectable K13 blood level. All animals that survived after injection of 105 cells or less initially showed rising K13 blood levels with a peak 20 days after the injection of the hybridoma cells. In the mice injected with 103 cells, the K13 blood levels were not measurable during the first 10 days. These findings suggest that the hybridoma cells first proliferated before the host managed to eliminate the hybridoma cells. In three of these animals, the K13 blood level was still measurable at day 120 (range, 0.3 2.6 mg/liter), but no tumor was found at necropsy. This may be due to the fact that these blood levels represent less than 10' cells, or may reflect the half-life of K13 which is about 11 days (Figure 5). In all i.p. growing tumors, early diagnosis of tumor -
329
progression is difficult because they often provide multiple métastases at sites where they are difficult to measure. In the present study, the K13 blood levels reflected tumor growth well (Figure 4). In the current study, only 5 µ blood was sampled from the tail vein, and the K13 blood level was measurable in all mice as early as 24 h after the injection of 10e hybridoma cells. Intraperitoneal injection of 6 K13 hybridoma cells resulted in a standard tumor growth pattern with diffuse superficial i.p. tumor spread and ascites, and a short mean survival (mean ± SD, 10 ± 3 days) (Table 1, Figure 2 and 3). This is considerably shorter than the median survival after i.p. inoculation of human cancer lines in nude mice which is 25 43 days (12-15). When using fresh human tumor, survival ranged from 1-9 months (16). conclusion, a close relationship was established between i.p. growth of the hybridoma K13 cell line and MAb blood levels. The basic concepts of this assay can readily be adopted for other clones with the limitation that pure antigen is needed for solid phase extraction of the MAb from mouse blood.
primary
-
In
ACKNOWLEDGMENT
This work
was
grant 88222.
supported by
the
Norwegian
Cancer
Society by
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Reprint Requests: I.
Vergote,
M.D.
Norwegian Radium Hospital Department of Gynecologic Oncology The
Montebello, N-0310 Oslo Norway Telephone: 02/506050 Facsimile: 02/525559
Received for publication: 10/29/91 Accepted after revision: 1/28/92
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