Journal of Immunological Methods, 22 (1978) 37--49 © Elsevier/North-Holland Biomedical Press
37
BINDING PATTERNS OF IMMUNOGLOBULINS FROM TUMOR-BEARING MICE TO THE CORRESPONDING TUMOR CELLS *
NEOMI MOAV, YOSEF HOCHBERG, GERALD COHEN and ISAAC P. WITZ
Department of Microbiology, The George S. Wise Center for Life Sciences, and (Y.H.) Department of Statistics, Tel-Aviv University, Tel-Aviv, Israel (Received 25 January 1978, accepted 2 February 1978) In this study we report on measurements to determine the binding constants of immunoglobulins to tumor cells. IgG from normal and from tumor-bearing mice was studied. It was found that IgG preparations from SEYF-a (a syngeneic polyoma-virusinduced ascites tumor)-bearing mice contain at least two distinct populations of molecules which differ from one another with respect to their binding constants to the tumor cells. One population is characterized by a high binding constant to SEYF-a cells, while the second population reveals a much lower binding constant to these cells. Normal mouse IgG preparations also bind to tumor cells, however, their binding constant is low. Soluble antigens extracted from the corresponding tumor cells were able to inhibit specifically the binding of IgG molecules originating from tumor-bearing mice, which exhibited the high binding constants to the tumor cells. Antigens extracted from nonrelated tissues did not affect the binding patterns of IgG to the tumor cells. Moreover, IgG prepared from SEYF-a-bearing mice exhibited a high binding constant only when interacted with the corresponding tumor cells, but not when interacted with non-corresponding ones. These results suggest that the IgG fraction with the high binding constant is composed mainly of antibodies to surface antigens of tumor cells. Determination of binding patterns of anti-tumor antibodies to the corresponding and to non~orresponding tumor cells could prove useful as an additional method for determinations on the degree of serological relations between various tumors and for further characterization of anti-tumor antibodies. INTRODUCTION A variety of assays are currently used for analyzing and measuring the humoral immune response to membrane antigens of tumor cells (Harder and M c K h a n n , 1 9 6 8 ; S p a r k s e t al., 1 9 6 9 ; B y s t r y n e t al., 1 9 7 4 ; W o o d a n d B a r t h , 1 9 7 4 ; H u a n g e t al., 1 9 7 5 ; B l a i r e t al,, 1 9 7 6 ; S h i k u e t al., 1 9 7 6 ; W i t z e t al., 1 9 7 6 ; R e d d y e t al., 1 9 7 7 ; R o s e n b e r g e t al., 1 9 7 7 ; Z e l t z e r e t al., 1 9 7 7 ) . T h e vast majority of these assays do not differentiate between strong and weak interactions of the tumor cells and the corresponding antibody. * This investigation was supported by Grant Number 1R01 CA20088-01 awarded by the National Cancer Institute DHEW.
38 It is our opinion that studies on the binding affinity of anti-tumor antibodies to the corresponding cells are needed if a comprehensive evaluation of the i m m u n o b i o l o g y of tumors is to be attained. R ecen t studies by Alsted et al. (1974), in a bacterial system, underlined the importance of measuring binding affinities. These investigators provided an experimental p r o o f for the logical expect at i on concerning a positive correlation between the protective capacity of antibodies and their binding affinity. In this study we r e p o r t on at t em pt s to measure the binding constants of antibodies to the t u m o r cell membrane. MATERIALS AND METHODS
Mice A.BY mice (H-2 b) were obtained from Dr. G. Klein (Karolinska Institute, S t o ck h o lm, Sweden) and raised at the Animal Quarters, Tel-Aviv University, Israel.
Tumor cells SEYF-a, a p o l y o m a virus-induced ascites t u m o r (Ran et al., 1976; Witz et al., 1976), was maintained by intraperitoneal passage of 2,000,000 cells per mouse to syngeneic A.BY mice. Cells were harvested between 6 and 30 days after inoculation.
Spleen cell suspensions Mice were sacrificed by cervical dislocation. The spleens were removed and teased with forceps on stainless steel No. 80 mesh screen. The cells were washed with Hank's salt solution and then centrifuged at 350 × g for 10 min at r o o m temperature. In order to remove e r y t h r o c y t e s , cells were treated with 0.85% NH4C1, pH 7.0, and then washed with Hank's salt solution.
Serum and ascitic fluids Serum from normal or tumor-bearing mice was collected by bleeding from the retro-orbital sinus. Ascitic fluid was collected from tumor-bearing mice by puncturing their peritoneal cavity with a 19-gauge needle. In normal mice ascitic fluid was elicited by intraperitoneal injections of 0.5 ml of incomplete Freund's adjuvant containing 0.5% oleic acid. Ascitic fluids from such mice were drawn as described above.
Preparation of IgG IgG was prepared from pooled sera and ascites fluids. The fluids were treated with (NH4)2SO4 (27% final saturation) to remove fibrinogen (Harris and Harris, 1972). The supernatant was brought to 50% saturation with (NH4)zSO4 and kept overnight at 4°C to collect the globulin. The precipitated globulin was washed twice with 50% ice-cold (NH4)2SO4 dissolved in a
39 minimal volume of 0.0175 M phosphate buffer (PB), pH 8.0, and then dialyzed against the same buffer overnight at 4°C. The globulin was further purified by ion-exchange chromatography on DEAE-cellulose columns (0.0175 M PB, pH 8.0).
Preparation o f 12SI-labeled IgG Iodination of IgG with ~2sI (specific activity 250 Ci/mmole; ~2sI was purhcased from The Radiochemical Center, Amersham, U.K.) was carried o u t by the chloramine T method of Greenwood et al. (1963) as described previously (Witz et al., 1968). Free iodine was removed by precipitation with 50% ammonium sulfate followed by dialysis against phosphate-buffered saline (PBS). Titration o f cell-bound IgG (at a constant number of cells) Equal numbers of t u m o r cells or mouse spleen cells in Hank's salt solution containing 0.8% bovine serum albumin (Fr. V, Sigma, St. Louis, MO, U.S.A.) were dispensed in triplicate series into microtiter plates (U-shape Microtiter, Cooke Engineering, Alexandria, VA, U.S.A.). The cells were pelleted by centrifugation for 5 min at 350 × g at room temperature, and the supernatants were completely removed by aspiration. The cells were then resuspended by gently shaking the plate over a vortex-mixer. To each triplicate set of wells were added 50 pl of medium containing a mixture of ~2SI-labeled IgG and cold IgG at various amounts (0.2--50 pg). The cells were incubated for 30 min at 4°C and then washed 3 times with cold medium. After the final wash the supernatant was removed completely and the microplate was dried over a heating plate. It was then cut into individual wells using a chrome-nickle heating wire and the radioactivity b o u n d to the cells in each well was monitored in a Packard Gamma Spectrometer. The specific activity of the IgG mixtures used was determined separately for each experiment by measuring the radioactivity of 50 pl aliquots of the IgG at each concentration used. Cell titration The experiments were carried out in a manner completely analogous to the above, except that the concentration of IgG was kept constant and the cell number was varied. Preparation o f KCl extracts (soluble antigens) The method of Reisfeld et al. (1971) was followed with modification according to Fish and Witz (paper in preparation). The tissue was homogenized with a small volume of cold 3 M KC1 in a Sorvall Omnimixer. Phosphate-buffered (pH 7.5) 3 M KC1 solution was then added to a final tissue concentration of 10% w/v. The suspension was stirred for 18 h at 4°C, filtered through gauze, and dialyzed for 24 h against 3 changes of H20. The precipitate that was formed was removed by centrifugation for 10 min at
40 10,000 X g followed by 2 h centrifugation at 150,000 X g. The particle-free extract was freeze-dried and stored at --20°C. RESULTS
Determination of the binding constants of immunoglobulins to cells The most c o m m o n procedure for determining the binding affinity of a ligand to its protein substrate is to present the experimental data in a Scatchard plot (Scatchard et al., 1957; Karush, 1962). A plot of r/c versus r is made, where r is the moles of ligand bound per moles of protein at a free ligand concentration of c. From such a plot a straight line is obtained with a slope o f - - K , where K is the equilibrium association constant. The above procedure has been adapted by a number of research groups for measurements of interactions between cells and artificially induced antiserum reagents (Boone et al., 1971; Nord and Weissman, 1974). In the present study a modification of this procedure was used for measuring the binding patterns to t u m o r cells of components present in IgG (ligand) preparations originating from normal mice or from mice bearing syngeneic tumors. Since the IgG preparations used probably contained a mixture of heterogeneous populations of IgG molecules with different specificities, and since their relative proportions might vary from one preparation to another,
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Fig. 1. E f f e c t o f cell n u m b e r o n t h e b i n d i n g c a p a c i t y o f n o r m a l m o u s e IgG t o s p l e n o c y t e s or S E Y F - a cells. V a r i o u s c o n c e n t r a t i o n s o f S E Y F - a cells or n o r m a l m o u s e spleen cells were i n c u b a t e d w i t h 7 pg or w i t h 15 pg o f 12SI-labeled n o r m a l m o u s e IgG in final v o l u m e o f 50 pl. A t t h e e n d o f t h e i n c u b a t i o n p e r i o d t h e cells were w a s h e d 3 t i m e s a n d t h e cellb o u n d IgG was d e t e r m i n e d f r o m t h e b o u n d r a d i o a c t i v i t y . T h e results are given in pg o f b o u n d IgG (X) p e r cell n u m b e r (N). All t h e e x p e r i m e n t s were p e r f o r m e d in triplicate. a: S E Y F - a t u m o r cells, p r o p a g a t e d in vivo for 10 days in A.BY mice were i n c u b a t e d w i t h 12SI-labeled n o r m a l m o u s e IgG at cell c o n c e n t r a t i o n s of: 3 X 104, 1.2 X 10 s, 2.4 X l 0 s, 4.8 X 10 s, 9.6 X l 0 s , a n d 3.8 X 106 cells p e r well. e , 7 p g IgG p e r well; o, 15 pg IgG p e r well. I n s e r t i o n : SEYF-a cells i n c u b a t e d w i t h 4 p g o f 12SI-labeled IgG p r e p a r e d f r o m ascitic fluid o f S E Y F - a - b e a r i n g A . B Y mice 1 5 - - 2 0 days a f t e r i n o c u l a t i o n o f t u m o r cells. T h e n u m b e r o f cells in t h e r e a c t i o n m i x t u r e was 1 X 106 , 2 X 106 a n d 3 X 106 cells p e r well, in a final v o l u m e o f 50 pl. b: n o r m a l m o u s e spleen cells were i n c u b a t e d w i t h 12sIlabeled n o r m a l m o u s e IgG a t cell c o n c e n t r a t i o n s of: 0.3 X 106 , 0.6 X 106 , 1.2 X 106 , 2.4 X 106 , 4.8 X 106 , 9 X 106 , 19 X 106 a n d 38 X 106 cells per well. A, 7 p g IgG p e r well; A, 15 ~g IgG p e r well.
the individual concentrations, c, o f t h e f r e e l i g a n d s p e c i e s c o u l d n o t b e obtained. Therefore, we have used in this work the term c to represent the total concentration of free IgG and refer to the values obtained from the
42 Scatchard plots as the binding constant (Kb) of the IgG ligand preparation to the cells. These values c a n n o t and should n o t be compared in a simple fashion with the association constants obtained for antigen--antibody interactions. In mo s t cases in the present work curvilinear plots were obtained (Figs. 3--5). Within the f r a m ew or k of our e x p e r i m e n t a t i o n we have assumed (arbitrarily to some degree) that the data reflect a specified n u m b e r of different interactions between t u m o r cell binding sites and IgG molecules. In order to maintain simplicity, we postulated the existence of two such interactions or relations only. The data were divided arbitrarily into two groups, one with a high-binding cons t a nt (Kul) and a second group with a low-binding constant (Kb2). This procedure is c o m m o n l y used in cases where there are interactions between heterogeneous populations of molecules (Scatchard et al., 1957; Kahn et al., 1974). The ratio between the c o n c e n t r a t i o n of IgG and the n u m b e r of binding sites is an i m p o r t a n t factor in binding experiments (Boone et al., 1971). Preliminary experiments were therefore designed to evaluate the binding capacity o f t u m o r cells as a f unc t i on of cell n u m b e r at a const ant IgG concentration. SEYF-a cells at concent r a t i on s ranging from 5 × 10 s to 6 × 106 cells per well, or mouse spleen cells at concent rat i ons ranging from 5 X l 0 s to 2 X 107 cells per well were incubated with 7 pg and 15 pg of normal mouse IgG, in a final volume of 50 pl. It can be seen (Fig. l a , b) that in the range of 1 X 106 to 6 × 106 t u m o r cells per well, or 5 X 106 to 107 spleen cells per well, there is a linear correlation between the n u m b e r of cells in the reaction mixture and the a m o u n t of IgG b o u n d per cell. However, when the cell n u m b e r per well decreases below 1 × 106 for t u m o r cells or 5 × 106 for spleen cells, the a m o u n t of IgG bound is n o t affected by the cell n u m b e r , and the measured bound radioactivity is due to non-specific binding of the labeled IgG to the p o l y s t y r e n e vessel. Non-specific binding to the plastic had no apparent effect at the higher cell c o n cen tr atio n s as indicated by the linearity of the curves at these cell concentrations. In all the experiments r e p o r t e d in this study we have, therefore, used t u m o r cells at c o n c e n t r a t i o n s ranging from 1 × 106 to 3 × 106 per well, and splenocytes at c onc e nt r at i ons of 107 cells per well. When the binding efficiencies of IgG obtained from tumor-bearing mice to SEYF-a t u m o r cells was measured, a similar correlation between cell num ber and binding efficiency was observed (see insertion in Fig. l a).
Comparison of the binding constants of IgG preparations from normal and from SEYF-a-bearing A.B Y mice to SEYF-a cells SEYF-a cells, propagated in vivo in syngeneic A.BY mice for 6--14 days, were incubated with various c onc e nt r at i ons of 12SI-labeled IgG obtained either f r o m ascitic fluids of normal mice, or from SEYF-a-bearing mice. The IgG preparation from tumor-bearing mice contained c y t o t o x i c antibodies against SEYF-a cells (unpublished data). The radioactivity b o u n d to the cells
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Fig. 2. B i n d i n g p a t t e r n s o f IgG f r o m n o r m a l mice a n d f r o m t u m o r - b e a r i n g mice t o S E Y F a cells. 2.5 X 106 S E Y F - a cells p r o p a g a t e d in vivo for 15 days were i n c u b a t e d in a final v o l u m e o f 5 0 / / l w i t h various c o n c e n t r a t i o n s ( 0 . 6 - - 2 6 . 3 p g / w e l l ) o f 12SI-labeled n o r m a l m o u s e IgG or IgG p r e p a r e d f r o m ascitic fluid c o l l e c t e d f r o m A.BY mice 1 1 - - 1 5 days a f t e r t h e y were i n o c u l a t e d w i t h S E Y F - a cells. A t t h e e n d o f t h e i n c u b a t i o n p e r i o d t h e cells were w a s h e d 3 t i m e s a n d t h e c e l l - b o u n d IgG was d e t e r m i n e d f r o m t h e b o u n d r a d i o a c t i v ity. T h e specific a c t i v i t y o f each IgG p r e p a r a t i o n used was d e t e r m i n e d for each experim e n t . T h e results are p r e s e n t e d in a S c a t c h a r d p l o t (for details see text), o . . . . . . ©, IgG p r e p a r e d f r o m ascitic fluid o f t u m o r - b e a r i n g m i c e ; • e , n o r m a l m o u s e IgG.
at each IgG concentration was determined and the values of r (moles of IgG b o u n d per cell) and c (the concentration of free IgG in moles) were calculated from the specific activity of the IgG preparations used, their concentrations, and the known molecular weight of IgG. Results presented in Fig. 2 TABLE 1 M A T H E M A T I C A L E V A L U A T I O N O F T H E B I N D I N G P A T T E R N S O F N O R M A L IgG A N D O F IgG F R O M T U M O R - B E A R I N G A.BY MICE T O S E Y F - a C E L L S T h e e x p e r i m e n t a l p r o c e d u r e is d e s c r i b e d in legend t o Fig. 2. S o u r c e o f IgG p r e p a r a t i o n
High-binding constant ( K b l ) (1/mole)
Low-binding constant ( K b 2 ) (1/mole)
Ascitic fluid f r o m n o r m a l mice
none
6 x 103
Ascitic fluid f r o m t u m o r b e a r i n g miee
9.01 x 106
4 x 103
44
a n d T a b l e 1 s h o w t h a t IgG p r e p a r a t i o n s f r o m t u m o r - b e a r i n g m i c e c o n t a i n at least t w o p o p u l a t i o n s o f IgG molecules. O n e p o p u l a t i o n is c h a r a c t e r i z e d b y a h i g h - b i n d i n g c o n s t a n t to S E Y F - a cells, while the s e c o n d p o p u l a t i o n has a m u c h l o w e r b i n d i n g c o n s t a n t to these cells. T h e IgG p r e p a r a t i o n s f r o m normal m i c e c o n t a i n o n l y m o l e c u l e s with a l o w - b i n d i n g c o n s t a n t .
Comparison of the binding patterns of normal mouse IgG to S E Y F - a cells and to mouse spleen cells Binding o f IgG s a m p l e s to b o t h S E Y F - a cells and n o r m a l m o u s e spleen cells was e x a m i n e d for 3 p r e p a r a t i o n s o b t a i n e d f r o m n o r m a l m o u s e s e r u m a n d f r o m ascitic fluids. It can be seen (Fig. 3) t h a t these IgG p r e p a r a t i o n s s h o w an e x t r e m e l y l o w - b i n d i n g c o n s t a n t f o r e i t h e r cell t y p e . H o w e v e r , m o r e IgG was b o u n d to t u m o r cells t h a n to spleen cells. Whereas t h e r a t i o o f t h e surface areas o f S E Y F - a cells to spleen cells was c a l c u l a t e d to be 3.8 (the surface area o f a S E Y F - a cell a n d t h a t o f a spleen cell w e r e 770 V2 a n d 2 0 0 ;12, r e s p e c t i v e l y ) , the r a t i o o f the n u m b e r o f IgG m o l e c u l e s b o u n d p e r S E Y F - a cell to t h o s e p e r spleen cell was c a l c u l a t e d t o be 7--15. T h u s it w o u l d a p p e a r
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Fig. 3. Binding p a t t e r n s o f n o r m a l m o u s e IgG to SEYF-a cells and to normal m o u s e spleen cells. T h r e e p r e p a r a t i o n s o f 12SI-labeled n o r m a l m o u s e IgG at c o n c e n t r a t i o n s o f 0 . 3 - - 2 0 . 3 / J g / w e l l were i n c u b a t e d with SEYF-a cells or with n o r m a l m o u s e spleen cells. The e x p e r i m e n t a l p r o c e d u r e is as d e s c r i b e d in the legend to Fig. 2. a: normal m o u s e IgG i n c u b a t e d with SEYF-a cells p r o p a g a t e d in vivo for 10 days. A, 1 X 106 cells per well; o, 2 X 106 cells per well; A, 3 X 106 cells per well. b: normal m o u s e IgG i n c u b a t e d with n o r m a l m o u s e spleen cells. A, 2.1 X 105 spleen cells per well; A, 4.2 X 106 spleen cells per well; o, 8.4 X 106 spleen cells per well.
45
that t u m o r cells are able to bind more normal IgG molecules per surface area than are spleen cells.
Binding specificity o f IgG obtained from tumor-bearing mice (a) Binding specificity to homologous and to heterologous tumor cells. The specificity of IgG preparations prepared from ascitic fluids of SEYF-abearing mice to the hom ol ogous SEYF-a cells was studied by comparing the binding patterns of the IgG preparations to these cells and to S-14 cells (a BALB/c plasmacytoma). It can be seen (Fig. 4) that IgG obtained from SEYF-a bearers binds to SEYF-a cells in a different pattern than to the S-14 cells. As shown above, the binding profile of IgG to SEYF-a cells demonstrated two distinct populations of IgG molecules, one with a high-binding c o n s t a n t (Kbl = 16.8 × l 0 s 1/mole) and a second population with a lowbinding co n s tan t (Kb2 = 0.4 × 10 3 1/mole). The binding constant of the same IgG preparation to S-14 cells was below detection. It can be concluded, therefore, that the IgG preparation from SEYF-abearing A.BY mice contains IgG molecules which bind with a high-binding c o n s tan t to the homologous t u m o r cells, but weakly to heterologous, noncross-reacting cells. In contrast to the differences f ound in binding patterns o f the IgG from I
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Fig. 4. Binding p a t t e r n s o f IgG o b t a i n e d f r o m normal mice and from SEYF-a bearers to SEYF-a cells and S-14 cells. 2.75 × 106 SEYF-a cells or S-14 cells were i n c u b a t e d with various c o n c e n t r a t i o n s ( 0 . 6 - - 2 6 . 3 p g / w e l l ) o f 125I-labeled IgG f r o m normal mice and f r o m ascitic fluids c o l l e c t e d f r o m A.BY mice 11--15 days after they were i n o c u l a t e d with SEYF-a cells in a final v o l u m e o f 50 pl/well. The e x p e r i m e n t a l p r o c e d u r e is as described in t h e legend to Fig. 2. Normal m o u s e IgG i n c u b a t e d w i t h : A, SEYF-a cells; o, S-14 cells. IgG f r o m S E Y F - a bearers i n c u b a t e d with: ~:~,SEYF-a cells; ~, S-14 cells.
46 t u m o r - b e a r i n g m i c e t o t h e t w o cell p o p u l a t i o n s , IgG p r e p a r e d f r o m n o r m a l m i c e b i n d s t o S E Y F - a cells a n d t o S - 1 4 cells i n a s i m i l a r f a s h i o n , i.e., w i t h a l o w - b i n d i n g c o n s t a n t (Ku = 0 . 5 5 × 103 1/mole).
(b) The effect o f soluble antigens on the binding patterns of IgG from SEYF-a-bearing A . B Y mice. I n o r d e r t o d e m o n s t r a t e f u r t h e r t h e s p e c i f i c i t y of b i n d i n g b e t w e e n IgG p r e p a r a t i o n s o b t a i n e d f r o m S E Y F - a - b e a r i n g mice a n d S E Y F - a cells, c o m p e t i t i o n e x p e r i m e n t s w e r e p e r f o r m e d u s i n g s o l u b l e a n t i g e n s p r e p a r e d f r o m S E Y F - a cells o r f r o m n o n - c o r r e s p o n d i n g t i s s u e s s u c h as s p l e e n o r l i v e r f r o m C 3 H m i c e . 5 0 p g o f e a c h o f t h e a n t i g e n p r e p a r a t i o n s w e r e a d d e d t o m i c r o t i t e r wells c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f ~2~I-
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Fig. 5. The effect of soluble antigens on the binding patterns of IgG from tumor bearers to SEYF-a cells. 2.6 × 106 SEYF-a cells propagated in vivo for 15 days in A.BY mice were incubated for 30 min at 4°C in wells containing various concentrations (0.6--26.3 pg/ well) of ~2SI-labeled IgG from aseitie fluids of SEYF-a-bearing A.BY mice, 11--15 days after inoculation of tumor cells, and 50 pg/well of KCI extracts from SEYF-a cells or from spleen or livers of C3H mice. The experimental procedure is as described in the legend to Fig. 2. • . . . . . . $, incubation with IgG alone; © 4), incubation with IgG and KCl-extraeted antigens from SEYF-a cells; • . . . . . . •, incubation with IgG and KCIextracted antigens from C3H spleen cells; /~. . . . . . A, incubation with IgG and KCIextracted antigens from C3H livers.
47
TABLE 2 MATHEMATICAL EVALUATION OF THE EFFECT OF SOLUBLE ANTIGENS ON THE BINDING PATTERNS OF IgG FROM TUMOR-BEARING A.BY MICE TO SEYF-a TUMOR CELLS Experimental procedure as described in legend to Fig. 5. A n t i g e n in r e a c t i o n mixture
High-binding constant (Kbl) (l/mole)
No a n t i g e n C3Hspleen C3Hliver S E Y F - a cells
9.0 x 106 1.25 x 107 5.7 x 106 none
Low-binding constant (KD2) ( l / m o l e ) 1.0 x
37
BINDING PATTERNS OF IMMUNOGLOBULINS FROM TUMOR-BEARING MICE TO THE CORRESPONDING TUMOR CELLS *
NEOMI MOAV, YOSEF HOCHBERG, GERALD COHEN and ISAAC P. WITZ
Department of Microbiology, The George S. Wise Center for Life Sciences, and (Y.H.) Department of Statistics, Tel-Aviv University, Tel-Aviv, Israel (Received 25 January 1978, accepted 2 February 1978) In this study we report on measurements to determine the binding constants of immunoglobulins to tumor cells. IgG from normal and from tumor-bearing mice was studied. It was found that IgG preparations from SEYF-a (a syngeneic polyoma-virusinduced ascites tumor)-bearing mice contain at least two distinct populations of molecules which differ from one another with respect to their binding constants to the tumor cells. One population is characterized by a high binding constant to SEYF-a cells, while the second population reveals a much lower binding constant to these cells. Normal mouse IgG preparations also bind to tumor cells, however, their binding constant is low. Soluble antigens extracted from the corresponding tumor cells were able to inhibit specifically the binding of IgG molecules originating from tumor-bearing mice, which exhibited the high binding constants to the tumor cells. Antigens extracted from nonrelated tissues did not affect the binding patterns of IgG to the tumor cells. Moreover, IgG prepared from SEYF-a-bearing mice exhibited a high binding constant only when interacted with the corresponding tumor cells, but not when interacted with non-corresponding ones. These results suggest that the IgG fraction with the high binding constant is composed mainly of antibodies to surface antigens of tumor cells. Determination of binding patterns of anti-tumor antibodies to the corresponding and to non~orresponding tumor cells could prove useful as an additional method for determinations on the degree of serological relations between various tumors and for further characterization of anti-tumor antibodies. INTRODUCTION A variety of assays are currently used for analyzing and measuring the humoral immune response to membrane antigens of tumor cells (Harder and M c K h a n n , 1 9 6 8 ; S p a r k s e t al., 1 9 6 9 ; B y s t r y n e t al., 1 9 7 4 ; W o o d a n d B a r t h , 1 9 7 4 ; H u a n g e t al., 1 9 7 5 ; B l a i r e t al,, 1 9 7 6 ; S h i k u e t al., 1 9 7 6 ; W i t z e t al., 1 9 7 6 ; R e d d y e t al., 1 9 7 7 ; R o s e n b e r g e t al., 1 9 7 7 ; Z e l t z e r e t al., 1 9 7 7 ) . T h e vast majority of these assays do not differentiate between strong and weak interactions of the tumor cells and the corresponding antibody. * This investigation was supported by Grant Number 1R01 CA20088-01 awarded by the National Cancer Institute DHEW.
38 It is our opinion that studies on the binding affinity of anti-tumor antibodies to the corresponding cells are needed if a comprehensive evaluation of the i m m u n o b i o l o g y of tumors is to be attained. R ecen t studies by Alsted et al. (1974), in a bacterial system, underlined the importance of measuring binding affinities. These investigators provided an experimental p r o o f for the logical expect at i on concerning a positive correlation between the protective capacity of antibodies and their binding affinity. In this study we r e p o r t on at t em pt s to measure the binding constants of antibodies to the t u m o r cell membrane. MATERIALS AND METHODS
Mice A.BY mice (H-2 b) were obtained from Dr. G. Klein (Karolinska Institute, S t o ck h o lm, Sweden) and raised at the Animal Quarters, Tel-Aviv University, Israel.
Tumor cells SEYF-a, a p o l y o m a virus-induced ascites t u m o r (Ran et al., 1976; Witz et al., 1976), was maintained by intraperitoneal passage of 2,000,000 cells per mouse to syngeneic A.BY mice. Cells were harvested between 6 and 30 days after inoculation.
Spleen cell suspensions Mice were sacrificed by cervical dislocation. The spleens were removed and teased with forceps on stainless steel No. 80 mesh screen. The cells were washed with Hank's salt solution and then centrifuged at 350 × g for 10 min at r o o m temperature. In order to remove e r y t h r o c y t e s , cells were treated with 0.85% NH4C1, pH 7.0, and then washed with Hank's salt solution.
Serum and ascitic fluids Serum from normal or tumor-bearing mice was collected by bleeding from the retro-orbital sinus. Ascitic fluid was collected from tumor-bearing mice by puncturing their peritoneal cavity with a 19-gauge needle. In normal mice ascitic fluid was elicited by intraperitoneal injections of 0.5 ml of incomplete Freund's adjuvant containing 0.5% oleic acid. Ascitic fluids from such mice were drawn as described above.
Preparation of IgG IgG was prepared from pooled sera and ascites fluids. The fluids were treated with (NH4)2SO4 (27% final saturation) to remove fibrinogen (Harris and Harris, 1972). The supernatant was brought to 50% saturation with (NH4)zSO4 and kept overnight at 4°C to collect the globulin. The precipitated globulin was washed twice with 50% ice-cold (NH4)2SO4 dissolved in a
39 minimal volume of 0.0175 M phosphate buffer (PB), pH 8.0, and then dialyzed against the same buffer overnight at 4°C. The globulin was further purified by ion-exchange chromatography on DEAE-cellulose columns (0.0175 M PB, pH 8.0).
Preparation o f 12SI-labeled IgG Iodination of IgG with ~2sI (specific activity 250 Ci/mmole; ~2sI was purhcased from The Radiochemical Center, Amersham, U.K.) was carried o u t by the chloramine T method of Greenwood et al. (1963) as described previously (Witz et al., 1968). Free iodine was removed by precipitation with 50% ammonium sulfate followed by dialysis against phosphate-buffered saline (PBS). Titration o f cell-bound IgG (at a constant number of cells) Equal numbers of t u m o r cells or mouse spleen cells in Hank's salt solution containing 0.8% bovine serum albumin (Fr. V, Sigma, St. Louis, MO, U.S.A.) were dispensed in triplicate series into microtiter plates (U-shape Microtiter, Cooke Engineering, Alexandria, VA, U.S.A.). The cells were pelleted by centrifugation for 5 min at 350 × g at room temperature, and the supernatants were completely removed by aspiration. The cells were then resuspended by gently shaking the plate over a vortex-mixer. To each triplicate set of wells were added 50 pl of medium containing a mixture of ~2SI-labeled IgG and cold IgG at various amounts (0.2--50 pg). The cells were incubated for 30 min at 4°C and then washed 3 times with cold medium. After the final wash the supernatant was removed completely and the microplate was dried over a heating plate. It was then cut into individual wells using a chrome-nickle heating wire and the radioactivity b o u n d to the cells in each well was monitored in a Packard Gamma Spectrometer. The specific activity of the IgG mixtures used was determined separately for each experiment by measuring the radioactivity of 50 pl aliquots of the IgG at each concentration used. Cell titration The experiments were carried out in a manner completely analogous to the above, except that the concentration of IgG was kept constant and the cell number was varied. Preparation o f KCl extracts (soluble antigens) The method of Reisfeld et al. (1971) was followed with modification according to Fish and Witz (paper in preparation). The tissue was homogenized with a small volume of cold 3 M KC1 in a Sorvall Omnimixer. Phosphate-buffered (pH 7.5) 3 M KC1 solution was then added to a final tissue concentration of 10% w/v. The suspension was stirred for 18 h at 4°C, filtered through gauze, and dialyzed for 24 h against 3 changes of H20. The precipitate that was formed was removed by centrifugation for 10 min at
40 10,000 X g followed by 2 h centrifugation at 150,000 X g. The particle-free extract was freeze-dried and stored at --20°C. RESULTS
Determination of the binding constants of immunoglobulins to cells The most c o m m o n procedure for determining the binding affinity of a ligand to its protein substrate is to present the experimental data in a Scatchard plot (Scatchard et al., 1957; Karush, 1962). A plot of r/c versus r is made, where r is the moles of ligand bound per moles of protein at a free ligand concentration of c. From such a plot a straight line is obtained with a slope o f - - K , where K is the equilibrium association constant. The above procedure has been adapted by a number of research groups for measurements of interactions between cells and artificially induced antiserum reagents (Boone et al., 1971; Nord and Weissman, 1974). In the present study a modification of this procedure was used for measuring the binding patterns to t u m o r cells of components present in IgG (ligand) preparations originating from normal mice or from mice bearing syngeneic tumors. Since the IgG preparations used probably contained a mixture of heterogeneous populations of IgG molecules with different specificities, and since their relative proportions might vary from one preparation to another,
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Fig. 1. E f f e c t o f cell n u m b e r o n t h e b i n d i n g c a p a c i t y o f n o r m a l m o u s e IgG t o s p l e n o c y t e s or S E Y F - a cells. V a r i o u s c o n c e n t r a t i o n s o f S E Y F - a cells or n o r m a l m o u s e spleen cells were i n c u b a t e d w i t h 7 pg or w i t h 15 pg o f 12SI-labeled n o r m a l m o u s e IgG in final v o l u m e o f 50 pl. A t t h e e n d o f t h e i n c u b a t i o n p e r i o d t h e cells were w a s h e d 3 t i m e s a n d t h e cellb o u n d IgG was d e t e r m i n e d f r o m t h e b o u n d r a d i o a c t i v i t y . T h e results are given in pg o f b o u n d IgG (X) p e r cell n u m b e r (N). All t h e e x p e r i m e n t s were p e r f o r m e d in triplicate. a: S E Y F - a t u m o r cells, p r o p a g a t e d in vivo for 10 days in A.BY mice were i n c u b a t e d w i t h 12SI-labeled n o r m a l m o u s e IgG at cell c o n c e n t r a t i o n s of: 3 X 104, 1.2 X 10 s, 2.4 X l 0 s, 4.8 X 10 s, 9.6 X l 0 s , a n d 3.8 X 106 cells p e r well. e , 7 p g IgG p e r well; o, 15 pg IgG p e r well. I n s e r t i o n : SEYF-a cells i n c u b a t e d w i t h 4 p g o f 12SI-labeled IgG p r e p a r e d f r o m ascitic fluid o f S E Y F - a - b e a r i n g A . B Y mice 1 5 - - 2 0 days a f t e r i n o c u l a t i o n o f t u m o r cells. T h e n u m b e r o f cells in t h e r e a c t i o n m i x t u r e was 1 X 106 , 2 X 106 a n d 3 X 106 cells p e r well, in a final v o l u m e o f 50 pl. b: n o r m a l m o u s e spleen cells were i n c u b a t e d w i t h 12sIlabeled n o r m a l m o u s e IgG a t cell c o n c e n t r a t i o n s of: 0.3 X 106 , 0.6 X 106 , 1.2 X 106 , 2.4 X 106 , 4.8 X 106 , 9 X 106 , 19 X 106 a n d 38 X 106 cells per well. A, 7 p g IgG p e r well; A, 15 ~g IgG p e r well.
the individual concentrations, c, o f t h e f r e e l i g a n d s p e c i e s c o u l d n o t b e obtained. Therefore, we have used in this work the term c to represent the total concentration of free IgG and refer to the values obtained from the
42 Scatchard plots as the binding constant (Kb) of the IgG ligand preparation to the cells. These values c a n n o t and should n o t be compared in a simple fashion with the association constants obtained for antigen--antibody interactions. In mo s t cases in the present work curvilinear plots were obtained (Figs. 3--5). Within the f r a m ew or k of our e x p e r i m e n t a t i o n we have assumed (arbitrarily to some degree) that the data reflect a specified n u m b e r of different interactions between t u m o r cell binding sites and IgG molecules. In order to maintain simplicity, we postulated the existence of two such interactions or relations only. The data were divided arbitrarily into two groups, one with a high-binding cons t a nt (Kul) and a second group with a low-binding constant (Kb2). This procedure is c o m m o n l y used in cases where there are interactions between heterogeneous populations of molecules (Scatchard et al., 1957; Kahn et al., 1974). The ratio between the c o n c e n t r a t i o n of IgG and the n u m b e r of binding sites is an i m p o r t a n t factor in binding experiments (Boone et al., 1971). Preliminary experiments were therefore designed to evaluate the binding capacity o f t u m o r cells as a f unc t i on of cell n u m b e r at a const ant IgG concentration. SEYF-a cells at concent r a t i on s ranging from 5 × 10 s to 6 × 106 cells per well, or mouse spleen cells at concent rat i ons ranging from 5 X l 0 s to 2 X 107 cells per well were incubated with 7 pg and 15 pg of normal mouse IgG, in a final volume of 50 pl. It can be seen (Fig. l a , b) that in the range of 1 X 106 to 6 × 106 t u m o r cells per well, or 5 X 106 to 107 spleen cells per well, there is a linear correlation between the n u m b e r of cells in the reaction mixture and the a m o u n t of IgG b o u n d per cell. However, when the cell n u m b e r per well decreases below 1 × 106 for t u m o r cells or 5 × 106 for spleen cells, the a m o u n t of IgG bound is n o t affected by the cell n u m b e r , and the measured bound radioactivity is due to non-specific binding of the labeled IgG to the p o l y s t y r e n e vessel. Non-specific binding to the plastic had no apparent effect at the higher cell c o n cen tr atio n s as indicated by the linearity of the curves at these cell concentrations. In all the experiments r e p o r t e d in this study we have, therefore, used t u m o r cells at c o n c e n t r a t i o n s ranging from 1 × 106 to 3 × 106 per well, and splenocytes at c onc e nt r at i ons of 107 cells per well. When the binding efficiencies of IgG obtained from tumor-bearing mice to SEYF-a t u m o r cells was measured, a similar correlation between cell num ber and binding efficiency was observed (see insertion in Fig. l a).
Comparison of the binding constants of IgG preparations from normal and from SEYF-a-bearing A.B Y mice to SEYF-a cells SEYF-a cells, propagated in vivo in syngeneic A.BY mice for 6--14 days, were incubated with various c onc e nt r at i ons of 12SI-labeled IgG obtained either f r o m ascitic fluids of normal mice, or from SEYF-a-bearing mice. The IgG preparation from tumor-bearing mice contained c y t o t o x i c antibodies against SEYF-a cells (unpublished data). The radioactivity b o u n d to the cells
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Fig. 2. B i n d i n g p a t t e r n s o f IgG f r o m n o r m a l mice a n d f r o m t u m o r - b e a r i n g mice t o S E Y F a cells. 2.5 X 106 S E Y F - a cells p r o p a g a t e d in vivo for 15 days were i n c u b a t e d in a final v o l u m e o f 5 0 / / l w i t h various c o n c e n t r a t i o n s ( 0 . 6 - - 2 6 . 3 p g / w e l l ) o f 12SI-labeled n o r m a l m o u s e IgG or IgG p r e p a r e d f r o m ascitic fluid c o l l e c t e d f r o m A.BY mice 1 1 - - 1 5 days a f t e r t h e y were i n o c u l a t e d w i t h S E Y F - a cells. A t t h e e n d o f t h e i n c u b a t i o n p e r i o d t h e cells were w a s h e d 3 t i m e s a n d t h e c e l l - b o u n d IgG was d e t e r m i n e d f r o m t h e b o u n d r a d i o a c t i v ity. T h e specific a c t i v i t y o f each IgG p r e p a r a t i o n used was d e t e r m i n e d for each experim e n t . T h e results are p r e s e n t e d in a S c a t c h a r d p l o t (for details see text), o . . . . . . ©, IgG p r e p a r e d f r o m ascitic fluid o f t u m o r - b e a r i n g m i c e ; • e , n o r m a l m o u s e IgG.
at each IgG concentration was determined and the values of r (moles of IgG b o u n d per cell) and c (the concentration of free IgG in moles) were calculated from the specific activity of the IgG preparations used, their concentrations, and the known molecular weight of IgG. Results presented in Fig. 2 TABLE 1 M A T H E M A T I C A L E V A L U A T I O N O F T H E B I N D I N G P A T T E R N S O F N O R M A L IgG A N D O F IgG F R O M T U M O R - B E A R I N G A.BY MICE T O S E Y F - a C E L L S T h e e x p e r i m e n t a l p r o c e d u r e is d e s c r i b e d in legend t o Fig. 2. S o u r c e o f IgG p r e p a r a t i o n
High-binding constant ( K b l ) (1/mole)
Low-binding constant ( K b 2 ) (1/mole)
Ascitic fluid f r o m n o r m a l mice
none
6 x 103
Ascitic fluid f r o m t u m o r b e a r i n g miee
9.01 x 106
4 x 103
44
a n d T a b l e 1 s h o w t h a t IgG p r e p a r a t i o n s f r o m t u m o r - b e a r i n g m i c e c o n t a i n at least t w o p o p u l a t i o n s o f IgG molecules. O n e p o p u l a t i o n is c h a r a c t e r i z e d b y a h i g h - b i n d i n g c o n s t a n t to S E Y F - a cells, while the s e c o n d p o p u l a t i o n has a m u c h l o w e r b i n d i n g c o n s t a n t to these cells. T h e IgG p r e p a r a t i o n s f r o m normal m i c e c o n t a i n o n l y m o l e c u l e s with a l o w - b i n d i n g c o n s t a n t .
Comparison of the binding patterns of normal mouse IgG to S E Y F - a cells and to mouse spleen cells Binding o f IgG s a m p l e s to b o t h S E Y F - a cells and n o r m a l m o u s e spleen cells was e x a m i n e d for 3 p r e p a r a t i o n s o b t a i n e d f r o m n o r m a l m o u s e s e r u m a n d f r o m ascitic fluids. It can be seen (Fig. 3) t h a t these IgG p r e p a r a t i o n s s h o w an e x t r e m e l y l o w - b i n d i n g c o n s t a n t f o r e i t h e r cell t y p e . H o w e v e r , m o r e IgG was b o u n d to t u m o r cells t h a n to spleen cells. Whereas t h e r a t i o o f t h e surface areas o f S E Y F - a cells to spleen cells was c a l c u l a t e d to be 3.8 (the surface area o f a S E Y F - a cell a n d t h a t o f a spleen cell w e r e 770 V2 a n d 2 0 0 ;12, r e s p e c t i v e l y ) , the r a t i o o f the n u m b e r o f IgG m o l e c u l e s b o u n d p e r S E Y F - a cell to t h o s e p e r spleen cell was c a l c u l a t e d t o be 7--15. T h u s it w o u l d a p p e a r
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Fig. 3. Binding p a t t e r n s o f n o r m a l m o u s e IgG to SEYF-a cells and to normal m o u s e spleen cells. T h r e e p r e p a r a t i o n s o f 12SI-labeled n o r m a l m o u s e IgG at c o n c e n t r a t i o n s o f 0 . 3 - - 2 0 . 3 / J g / w e l l were i n c u b a t e d with SEYF-a cells or with n o r m a l m o u s e spleen cells. The e x p e r i m e n t a l p r o c e d u r e is as d e s c r i b e d in the legend to Fig. 2. a: normal m o u s e IgG i n c u b a t e d with SEYF-a cells p r o p a g a t e d in vivo for 10 days. A, 1 X 106 cells per well; o, 2 X 106 cells per well; A, 3 X 106 cells per well. b: normal m o u s e IgG i n c u b a t e d with n o r m a l m o u s e spleen cells. A, 2.1 X 105 spleen cells per well; A, 4.2 X 106 spleen cells per well; o, 8.4 X 106 spleen cells per well.
45
that t u m o r cells are able to bind more normal IgG molecules per surface area than are spleen cells.
Binding specificity o f IgG obtained from tumor-bearing mice (a) Binding specificity to homologous and to heterologous tumor cells. The specificity of IgG preparations prepared from ascitic fluids of SEYF-abearing mice to the hom ol ogous SEYF-a cells was studied by comparing the binding patterns of the IgG preparations to these cells and to S-14 cells (a BALB/c plasmacytoma). It can be seen (Fig. 4) that IgG obtained from SEYF-a bearers binds to SEYF-a cells in a different pattern than to the S-14 cells. As shown above, the binding profile of IgG to SEYF-a cells demonstrated two distinct populations of IgG molecules, one with a high-binding c o n s t a n t (Kbl = 16.8 × l 0 s 1/mole) and a second population with a lowbinding co n s tan t (Kb2 = 0.4 × 10 3 1/mole). The binding constant of the same IgG preparation to S-14 cells was below detection. It can be concluded, therefore, that the IgG preparation from SEYF-abearing A.BY mice contains IgG molecules which bind with a high-binding c o n s tan t to the homologous t u m o r cells, but weakly to heterologous, noncross-reacting cells. In contrast to the differences f ound in binding patterns o f the IgG from I
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Fig. 4. Binding p a t t e r n s o f IgG o b t a i n e d f r o m normal mice and from SEYF-a bearers to SEYF-a cells and S-14 cells. 2.75 × 106 SEYF-a cells or S-14 cells were i n c u b a t e d with various c o n c e n t r a t i o n s ( 0 . 6 - - 2 6 . 3 p g / w e l l ) o f 125I-labeled IgG f r o m normal mice and f r o m ascitic fluids c o l l e c t e d f r o m A.BY mice 11--15 days after they were i n o c u l a t e d with SEYF-a cells in a final v o l u m e o f 50 pl/well. The e x p e r i m e n t a l p r o c e d u r e is as described in t h e legend to Fig. 2. Normal m o u s e IgG i n c u b a t e d w i t h : A, SEYF-a cells; o, S-14 cells. IgG f r o m S E Y F - a bearers i n c u b a t e d with: ~:~,SEYF-a cells; ~, S-14 cells.
46 t u m o r - b e a r i n g m i c e t o t h e t w o cell p o p u l a t i o n s , IgG p r e p a r e d f r o m n o r m a l m i c e b i n d s t o S E Y F - a cells a n d t o S - 1 4 cells i n a s i m i l a r f a s h i o n , i.e., w i t h a l o w - b i n d i n g c o n s t a n t (Ku = 0 . 5 5 × 103 1/mole).
(b) The effect o f soluble antigens on the binding patterns of IgG from SEYF-a-bearing A . B Y mice. I n o r d e r t o d e m o n s t r a t e f u r t h e r t h e s p e c i f i c i t y of b i n d i n g b e t w e e n IgG p r e p a r a t i o n s o b t a i n e d f r o m S E Y F - a - b e a r i n g mice a n d S E Y F - a cells, c o m p e t i t i o n e x p e r i m e n t s w e r e p e r f o r m e d u s i n g s o l u b l e a n t i g e n s p r e p a r e d f r o m S E Y F - a cells o r f r o m n o n - c o r r e s p o n d i n g t i s s u e s s u c h as s p l e e n o r l i v e r f r o m C 3 H m i c e . 5 0 p g o f e a c h o f t h e a n t i g e n p r e p a r a t i o n s w e r e a d d e d t o m i c r o t i t e r wells c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f ~2~I-
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Fig. 5. The effect of soluble antigens on the binding patterns of IgG from tumor bearers to SEYF-a cells. 2.6 × 106 SEYF-a cells propagated in vivo for 15 days in A.BY mice were incubated for 30 min at 4°C in wells containing various concentrations (0.6--26.3 pg/ well) of ~2SI-labeled IgG from aseitie fluids of SEYF-a-bearing A.BY mice, 11--15 days after inoculation of tumor cells, and 50 pg/well of KCI extracts from SEYF-a cells or from spleen or livers of C3H mice. The experimental procedure is as described in the legend to Fig. 2. • . . . . . . $, incubation with IgG alone; © 4), incubation with IgG and KCl-extraeted antigens from SEYF-a cells; • . . . . . . •, incubation with IgG and KCIextracted antigens from C3H spleen cells; /~. . . . . . A, incubation with IgG and KCIextracted antigens from C3H livers.
47
TABLE 2 MATHEMATICAL EVALUATION OF THE EFFECT OF SOLUBLE ANTIGENS ON THE BINDING PATTERNS OF IgG FROM TUMOR-BEARING A.BY MICE TO SEYF-a TUMOR CELLS Experimental procedure as described in legend to Fig. 5. A n t i g e n in r e a c t i o n mixture
High-binding constant (Kbl) (l/mole)
No a n t i g e n C3Hspleen C3Hliver S E Y F - a cells
9.0 x 106 1.25 x 107 5.7 x 106 none
Low-binding constant (KD2) ( l / m o l e ) 1.0 x
