Activation of hemolytic complement by mouse monoclonal IgG1 antibody: Comparison with rabbit IgG

Molecular

Immunology,

Vol. 22, No. 3, pp. 215-221,

1985

c

Printed in Great Britain

ACTIVATION OF HEMOLYTIC COMPLEMENT MOUSE MONOCLONAL IgGl ANTIBODY: COMPARISON WITH RABBIT IgG

0161-5890/85 $3.00 + 0.00 1985 Per8amon Press Ltd

BY

PASQUALE BATTISTA, ANTONELLA CIRCOLO and TIBOR BORSOS* Laboratory

of Immunobiology,

National Cancer Institute, National Frederick, MD 21701, U.S.A.

Institutes

of Health,

FCRF,

(First received 31 July 1984; accepted in revised form 11 September 1984) Abstract-We

investigated the ability of a mouse anti-hapten monoclonal IgGl antibody (Ab) to bind to cell-bound specific hapten and to fix and activate Cl and thus the lytic sequence of complement (C). In a comparative study with polyclonal rabbit anti-hapten IgG Ab, we found that about 6 times more monoclonal Ab molecules than polyclonal were necessary for the generation of 1 hemolytic site/cell: the data were interpreted to mean that a cluster of four cell-bound monoclonal Ab molecules was necessary to bind Cl and activate C-mediated hemolysis. Experiments performed under conditions of low density of cell-bound hapten and excess of antibody showed that both monoclonal and polyclonal IgG Abs were able to react only with 20-30% of the cell-bound hapten and that both Abs recognized the same hapten specificity. We also found that even though monoclonal IgGl Ab was able to bind strongly to a protein A-Sepharose column and could be eluted only by a low-pH buffer, the purified Ab, when bound to cell surface hapten, showed a weak ability to react with free protein A.

INTRODUCTION

The availability of monoclonal antibodies permits the study of the interaction of antibodies with their corresponding epitopes on a molecular basis. From studies on the interaction of small haptens with polyclonal IgG antibodies it was learned that on a cell surface the distribution of the hapten controlled both the generation of Clt-fixing complexes and the ability of the complex to activate Cl and C. It was determined that the two activities were separable and that activation required a relatively high density of hapten distribution. Hapten binding was measured by the use of 3H-labelled hapten and average distances between molecules were calculated based on these measurements (Circolo and Borsos, 1982; Borsos and Circolo, 1983). In this study we compared the binding of antiMTX monoclonal and polyclonal antibodies to sheep

*To

whom correspondence should be addressed at: NCI-FCRF, Bldg 560, Room 1271, Frederick, MD 21701, U.S.A. tAbbreviations: Mab, mouse monoclonal antibody; Ra IgG, rabbit polyclonal IgG immunoglobulin: MTX, methotrexate;-PA, protein A of Staphylococcus aureus; E, sheep red cells: E-MTX. MTX covalentlv bound to guinea EDAC, complement;’ E; pig 1-ethyl-3(3-dimethylaminopropyl)carbodiimide; PBS, phosphate-buffered isotonic saline; VBS, veronalbuffered saline containing 0.00015 M CaCI,, 0.001 M MgC& and 0.1% gelatin; C, complement; CT, active form of the first complement component; EA, sheep red blood cells sensitized with rabbit anti-Forssman IgM antibody; EAC4, EA to which C4 has been bound; EAC14, EA carrying the first and fourth complement components.

&>

215

cells to which MTX was coupled covalently and compared their ability to bind Ci and activate C, and we measured the ratio of the number of MTX molecules bound (as determined by [3H]MTX uptake) to the number of antibody molecules bound at saturating levels of antibody, i.e. the ratio of antibody-reactive to non-reactive MTX molecules on the cell. The results showed that only about 20-30x of the cell-bound MTX molecules were recognized by either antibody, that both antibodies seemed to recognize the same bound MTX molecules and that a tetramer of monoclonal IgGl was necessary to bind Cl and to activate C. It was also found that, while mouse IgGl could bind to and could be eluted from a PA-Sepharose column, the purified IgGl when bound to cell surface hapten was inefficient in binding free PA. Thus, like the ability to binding C, reactivity with PA may also depend on the physical condition of the Ig and possibly of PA.

MATERIALS

AND METHODS

Buffers and sheep red blood cells have been previously described (Rapp and Borsos, 1970). gpC was purchased from JEM Research (Kensington, MD), divided into lo-ml aliquots and stored at -30°C. Before use, 10 ml of gpC were absorbed twice (45 min at 4°C) with packed E (1 x lOlo) to eliminate anti-E antibodies, and with Affi-Gel 701 beads (Bio-Rad, Richmond, CA) to which MTX was coupled, to remove natural anti-MTX antibodies. The coupling procedure for the beads was performed as described previously (Langone, 1980~). E,,, complexes were prepared as follow: 5 parts of E (2 x 109/ml) in PBS

216

PASQUALEBATT~STAei ui

were mixed with 2 parts of MTX (Division of Cancer Treatment. NCI!NIH. Bethesda, MD), 20mgiml in PBS: 5 parts of a solution of EDAC (Bio-Rad), IO mgjml in PBS were added dropwise with constant mixing. After i hr of incLibation at room temp, the cells were washed 3 times with PBS, twice with VBS and resuspended in VBS at a concn of I.5 x lO’/ml. By using different vols of a given MTX solution E,,x with varying amounts of bound MTX could be prepared (Borsos et crl., 1980. 1981). The number of cell-hound MTX molecules was measured by using trace-labelling experiments with I’ HlMTX (Amersham, Arlington Heights, IL) (sp. act. I I .9 Cijmmole), as described previously (Circolo and Borsos, 1982). Rabbit anti-MTX serum was obtained as described previously (Levine and Powers, 1974). The IgG fraction containing anti-MTX antibody was separated from the whole serum by DEAE chromatography (King, 1968) absorbed with packed E (I x lO’/ml) to eliminate anti-E antibody and concentrated by filtration on a Diallo PM IO membrane (Amicon Corp, Lexington, MA). Hybridomas producing anti-MTX monoclonal antibody were made by fusion of a FO myeloma with splenocytes obtained from BALB/c mice immunized with MTX covalently bound to keyhole limpet hemocyanin (Kato ef rrl., 1984). Cloned hybridoma cells were injected intraperitoneally in pristane-primed BALB/c mice; ascitic fluid was recovered lo-12 days later. Anti-MTX monoclon~~l antibody was separated from the ascitic fluid by PA-Sepharose affinity chromatography (Ey et al., 1978). Mouse ascitic fluid was applied to a protein PA-Sepharose column in 0.1 M phosphate buffer, pH 8, containing 0.1’:; NaN,. The column was washed with the same buffer until the absorbance ~28Onm) of the effluent was close to 0. PA-bound IgG was eluted with 0.1 M glycineeHC1, pH 3. Fraction size was 3 ml. The final preparation contained 1.5 mg protein/ml. Only the 0.1 M glycine-HCI eluate contained antibody activity. iZ5!-PA has been prepared as described (Langone ef uf., 1977) and was used to quantify cell-bound IgG antibody (Biberfeld (31~1.. 1975; Dorval et ui., 1975; Langone, 1980h). Rabbit anti-MTX IgG and Mab were labelled with “‘1 by the Bolton-Hunter reagent (Amersham) (sp. act. 2100 Ci!mmole). 0.5 mCi of Bolton-Hunter reagent were evaporated under a streatn of air. 0.3 ml of Ra IgG or Mab (protein content 0.93 or 1.5mgjml respectively) were added to the residue and incubated for 30 min at room temp. 0.1 ml of an amino acid mixture [MEM (non-essential amino acid, 10 mMf] (Gibco, Grand Island, NY) was added to react with the excess of BoltonHunter reagent. After I hr at room temp 2ml of VBS were added and the mixture chro~~to~~ph~d on a Sephadex G 25 column [PD-10 (Pharmacia, Piscataway, NJ)] and eluted with 3 ml of VBS. Over 95% of the recovered radioactivity was TCAprecipitable. Radioactivity was determined in a Packard 5460 Autogamma Scintillation Spectrometer.

Rabbit anti-mouse I&Cl, IgG2a. IgG2b and IgG3 antibodies (Miles Laboratories. Elkhart, IN) were absorbed twice with packed E, TXand were used in hemolytic assays to identify the specific monoclonal antibody subclass. Hemolytic assays were performed as described previously (Rapp and Borsos, 1970): the Cl fixation and transfer test (Rapp and Borsos, 1970) was performed as modified by Brown (1982). C 1 was purchased from Cordis Corp. (Miami. FL). The number of Cl molecules fixed;cell and the number of hemolytic sites/cell was calculated according to the one-hit theory of immune hemolysis with the aid of the Poisson equation (Mayer, 1961; Rapp and Borsos, 1970). RESULTS

In the first experiment we determined the class of IgG to which the monoclonal antibody belonged. We exposed aliquots of EMTX(1.5 x IO’ E in 0. I ml) to low levels of anti-MTX Mab for 45 min at 30°C: the cell preparations were washed and were exposed to dilutions of IgG rabbit anti-mouse IgGl, IgGZa, IgGZb and IgG3. After 45 min incubation at 30-C the cells were washed and exposed to gpC for I hr at 37°C. Lysis was measured at 412nm. The results showed that no lysis was detectable with anti-IgG2a, IgG2b and IgG3, while lysis was obtained even at a high dilution of the mouse antibody with anti-IgGl, demonstrating that the anti-MTX Mab belonged to the IgGI subcfass (data not shown). Since the Mab was purified by ad- and desorption on PA-Sepharose we were interested if the Mab was capable of binding PA when bound to its hapten on the cell surface. In Fig. 1 we show the result of an experiment where binding of “‘I-PA was studied by

Average Number

of i2s1-PA Molecules

Input

(X

N?-*q

Fig. 1. Binding of ‘2JI-PA to EMTx carrying anti-MTX Mab or Ra IgG on their surface. O.I-ml portions of E,,, (1.5 x to* E/ml) sensitized with either antibody were mcubated with varying amounts of ‘25I-PA for dete~ining the ability of the cell-bound antibodies to bind PA.

217

Complement activation by monoclonal IgG 1

0.6 -

cr. p

0.5-

-z i&I !? x

0.4 -

u! c 5

0.3-

0.2 -

0.1

I

I

0’

1 1 12

1

I

I

4

6

16

Relative Ab Concentration (1 = 1

2

4

RelativeAb Concentration (1

11800)

8

= i/400)

Fig. 2. Dose-response curve for anti-hapten Ra IgG antibody (Ab) on EM,,, carrying on their surface high concns of hapten. The number of cell-bound Ra IgG Abs was determined by measuring the cell-bound radioactivity of 0.1 ml of EMYX(1.5 x lO*/ml) exposed to different concns of I25I-labelled Ra IgG for 45 min at 30°C and washed twice (1 cpm = i .7 x lo* Ra IgG molecules). All samples in duplicate; data corrected for background cpm (E without MTX plus lzSI-Ra IgG) of 475 f 71.

cell-bound Mab IgGl anti-MTX and rabbit IgG anti-Mm. It is seen that rabbit IgG was saturable by PA close to an equimolar concn of PA and IgG: in contrast, Mab IgGl was not saturable even at a relatively high PA/Mab ratio. For this reason we could not use PA binding as a measure of antibody uptake. In the next experiments we determined the absolute amount of anti-MTX IgG content of the Mab and polyclonal preparations. This was accomplished by studying the uptake of radiolabelled anti-MTX IgGs. IgGs were labelled with Bolton-Hunter reagent and different concns were offered to portions of E,,, made with 40, 20, 10 and 5 mg MTX, respectively; such cells contained relatively high amounts of MTX. The results (Figs 2 and 3) show the uptake of 1gG rabbit anti-MTX and IgGl anti-MTX Mab, respectively. For the rabbit antibody, only that portion of the curves are shown where uptake was independent of MTX concn (Fig. 2) for at higher levels of antibody the cells were agglutinated. Under the conditions of the experiment all offered antibody was taken up by the cells for the supernatant fluids contained no free antibody. For the Mab we can show a much wider range of antibody uptake (Fig. 3) for, even when saturating levels of antibody were

Fig. 3. Dos.+response curve for anti-hapten Mab on EMTx carrying on their surface high concns of hapten. The number of cell-bound Mab molecules was determined by using 1251-labelledMab (see legend of Fig. 3) (1 cpm = 1.45 x 10’ Mab molecules). All samples in duplicate: data corrected for background cpm (E without MTX plus ‘2SI-Mab) of 3272 + 240.

offered to the cells, no visible agglutination occurred making determinations feasible. From the portions of the curves where uptake was independent of MTX and was dependent on antibody concn we calculated the absolute amount of IgG in both antibodies: for the polyclonal antibody we calculated it as 0.21 mg anti-MTX IgG/ml, and for the Mab as 1.2 mg/ml: thus for Mab at least 80% of the protein as determined by absorption at 280 nm was antibody. In the next experiment we adjusted the antibody concn of each so that they contained the same number of molecules~ml. Uptake of antibody by E,,,, was than determined under conditions when antibody was limiting, i.e. all antibody molecules offered were taken up by the cells. The results, presented in Fig. 4, show that over a wide range of Ab concn, the same number of IgG molecules were taken up for each antibody. In the next experiments we studied the question if the two antibodies reacted with the same epitope. This was accomplished: (1) by measuring the uptake of each antibody on cells with limiting numbers of MTX, and (2) by measuring the simultaneous uptake of the two antibodies on cells with limiting amounts of MTX. The results of the first type of experiments are shown in Table I. It can be seen that, under conditions where the number of MTX molecules/cell were limiting, for each MTX concn there were approx. twice as many rabbit IgG molecules taken up than Mab IgGI. It is possible that this difference is traceable to differences in the bind-

PASQUALE BATTISTA c'tal

Fig. 4. Number of anti-hapten Ra IgG or Mab bound to EMTx4”. The two antibody (Ab) concns were adjusted to contain the same number of molecules/ml. Limiting amount of Ah(s) was offered to the cells (1 cpm = 0.77 x 10’ or 1.35x 10’ Ra IgG or Mab molecules respectively). All determinations in duplicate; data corrected for background cpm of 1655 k 1 for “‘I-Ra IgG and 2590 k 404 for Mab.

ing constants of the antibodies: from uptake and inhibition studies, we found that the polyclonal IgG had an about 10 times higher affinity than the monoclonal IgG (data not shown). A striking finding was that, even at saturating levels of the antibodies, only about 20-30x of the MTX molecules were reactive with the antibodies (Table 1). This finding raised the possibility that the polyclonal and monoclonal antibodies each recognized a different population of bound MTX molecules. This question was resolved by the results of the second type of experiment: the simultaneous uprake of the two antibodies. Portions of E,,,,, (1.5 x 10’ E) were exposed to labelled and unlabelled polyclonal antibodies, and labelled and unlabelled monoclonal antibodies either in solo or in mixtures with unlabelled antibodies of the same or the opposite species. The results presented in Fig. 5 show that: (1) the uptake of radiolabelled rabbit polyclonal antibody was inhibited by either unlabelled antibody to about the same extent, and (2) the uptake of labelled monoclonal antibody was inhibited by either unlabelled antibody to about the same extent. We interpreted this to mean that, within the limits of the experiments, both antibodies reacted with the same epitope.

Table

I

Bindmg

I No. of MTX lIl&CUleS,Cell”

(x

IO-‘)

DISCUSSION

Several points emerge from the results presented in this paper. One concerns the determination of the

with hmiting amounts of MTX: determination of Maba and polyclonal antibodles by E,,, the fraction of MTX molecules reactive with anti-MTX IgG 2 No. of Mabs:cell” ( x lo-“)

0.25 0.48 0.76 I3

“Determmed “Determined LDetermmed

These results made it possible to compare the Cl-fixing and C-activating properties of the two antibodies. In Fig. 6 we present the results of Cl fixation experiments with target cells carrying a high level of MTX. Two points emerge from the data. It was found that, to generate one Cl-fixing site, about 6 times more IgGl Mab molecules were needed than rabbit IgG molecules. The other finding was that the slope of the dose-response curve for the Mab was about 4 while that of the polyclonal antibody was about 2. Similar results were obtained for hemolytic data (Fig. 7). Again about 6 times more Mab molecules were needed for the generation of one hemolytic site than for polyclonal molecules. Similarly, the slope of the dose-response curve for Mab was about 4 as compared to that of the polyclonal antibody, which was about 2. In Table 2 we show the average slopes of all experiments with three different clones of IgGl and four different rabbit IgG anti-MTX antibodies. We calculated the average slope for the IgG I Mab as 4.31 + 1.02 and for the rabbit IgG as 1.62 f 0.33.

0.43 0.89 1.6 2.x

with ‘H-labelled with ‘Z I-labelled with “iI-labelled

MTX I,$? I. rabbit I&<;.

3 No of polyclom~l antibodieskell ( x IO 4) 0 79 I .4 2.7 42

of

Ratio 2:1

3.1

3.2

0.17 0 19 0.21 0 22

0.32 0.29 0 36 0.32

IY 1.5 1.7 I.5

Av. 0.20

0.37

1.7 * 0.2

Complement activation by monoclonal IgGl 6-

219 Mab

It

0~

Fig. 5. Uptake of 12%Ra IgG or lz51-Mab by Em, z,5: inhibition by cold Ra IgG or Mab. 0.1 ml of E,,x (1.5 x 108/mI) was exposed to “‘I-Ra IgG alone or to a mixture of lZ51-RaIgG + cold Ra IgG or ‘?-Ra IgG + cold Mab (left panel); 0.1 ml of EMox was exposed to “‘1-Mab alone or to a mixture of ‘251-Mab +coid Mab or l*%Mab + cold Ra IgG (right panel) (1 cpm = 0.85 x 1O’or 1.45 x 10’ Ra IgG or Mab molecules respectively). All determinations in duplicate; data corrected for background IgG and 2544i 109 for cpm of 2280 & 3 for ‘*%Ra ,251_Mab.

actual number of body, the second necessary to form plex and the third IgGl with PA.

epitopes that react with its antithe number of IgGl molecules a Cl binding and activating comthe weak interaction of cell-bound

Average Number of IgG Molecules InpuuE ( x

10-q

Fig. 6. Number of CT molecules bound to E,,, sensitized with Ra IgG or Mab: detection of bound CT by Cl fixation and transfer test. E,,, sensitized with antibodies were exposed to excess purified gpCT. To remove unbound CT, EMTXwere layered in silicone fluid and centrifuged, the supernatant discarded and the cells diluted in VBS and tested for their ability to convert EAC4 to EACtii. Log-log plot.

Average Number of IgQ Molecules Input (x lo-‘)

Fig. 7. gpC-mediated hemolytic activity of anti-hapten Ra IgG or Mab bound to EMTXcarrying on their surface different concns of hapten. 0.1 ml of E,,, (1.5 x lO’/ml) sensitized with Ra IgG or Mab were exposed to excess gpC: the extent of lysis was determined after I hr of incubation at 37°C. Log-log plot.

Experiments determining the actual number of bound hapten molecules reacting with antibody were performed with limiting amounts of hapten (MTX) bound to a cell surface. The absolute amount of MTX bound was determined by mixing a small amount of ‘H-labelled MTX with unlabelled MTX and coupling the mixture to the cell. The number of hapten molecules reacting with anti-MTX antibody was then determined by exposing MTX-labelled cells to a large excess of anti-MTX Mab or anti-MTX polyclonal antibody. The levels of MTX were chosen so as to bind only a few thousand antibody mol~ules~cell: these levels of MTX avoided agglutination and permitted the sparse distribution of antibody molecules. The number of antibody molecules bound was determined by using ‘251-labelled antibodies and, in the case of the rabbit IgG, by also using ‘Z51-labelled PA. To ensure saturation we studied uptake as a function of antibody concn and found that uptake was independent of antibody concn over a wide range. We also ensured that antibody molecules were not lost from the surface due to the manipulation by showing that repeated washing, prolonged incubation or changing vols had no effect

Table 2. Av. slopes of hemolytic dose-response curves of Mabs and rabbit anti-MTX IgG antibodies Mabs” Rabbit polyclonal antibodies”

4.31 i 1.02 1.62 f 0.33

“Total of 15 measurements with three different IgCl clones. *Total of 33 measurements with four different rabbit IgGs.

320

PAS~UALE BATTISTA et crl

on the number of antibody molecules associated with the cells. Cross-inhibition experiments showed that Mab and the polyclonal antibody recognized the same epitope. The question remained why only about 20-30”, of the cell-bound MTX was recognized by the antibodies. We considered the following possibilities (among others). MTX contained a mixture of molecules only part of which was reactive with the antibodies. This was considered unlikely for the absorption curves of MTX were indistinguishable from published data. A second possibility was the coupling procedure altered MTX chemically. We performed a competitive-inhibition experiment comparing the ability of free MTX with MTX bound to a protein carrier to inhibit the uptake of anti-MTX antibody by MTX-labelled cells and found that, mole for mole, the carrier-bound MTX was a somewhat better inhibitor than free MTX (Circolo A., unpublished data). A third possibility was that MTX bound not only to the surface of the red cell but also in the membrane, and thus, was not available for reaction with the antibody. We have no information regarding this possibility. A fourth possibility was that even as small a molecule as MTX has more than one epitope. Again we have no information on this point. The fact remains that only a fraction of the MTX molecules on the cell reacted with the antibodies. This finding necessitates a revision of the calculated angle of the form of IgG that can activate Cl. In previous publications from this laboratory for optimal activation we calculated the average distance of bound MTX/cell to be about 6-7 nm, representing an angle for the Fab arms of about 30-40” (Circolo and Borsos, 1982). If only 20-307; of the bound molecules are reactive with antibodies then the average distance becomes about 12.-16 nm, representing an angle of about 70-90 This angle is about the same as that reported by Hyslop rt ul. (1970) for Cl fixation by IgG antibody in a soluble complex. This revision does not change the original conclusion that binding and activation of Cl are separable events and that in addition to aggregation an activating signal must be generated in the antigen-bound IgG (and IgM) molecule. The second point concerns the generation of Cl-binding and C-activating properties of the two kinds of antibodies. From data comparing the number of molecules required to generate one Cl-fixing site or one hemolytic site we concluded that a doublet of Mab IgGIs was not sufficient to fix Cl or to activate the lytic sequence. From slope analyses of doseeresponse curves we concluded that the aggregation by a random process of four molecules of the Mab was necessary for binding Cl and for activating the lytic sequence. At present there is no evidence why certain IgG antibodies require more than two IgG molecules in a complex to “fix” C. Since there seems to be no relation between the binding affinity of an antibody for Clq (Foikerd et al, 1980; Doekes et al: 1982) and activation of Cl, a possible difference

may relate to the binding affinity of the antibody for the hapten (Okada et al; 1983); strict comparative studies have yet to be performed relating binding affinity of antibodies, Cl and C activation. A curious finding was the apparent reduced ability of IgGl to bind soluble PA. While soluble IgG1 bound firmly to immobilized PA, only a portion of cell-bound IgGI could bind PA. The fraction that bound PA could be increased by the addition of higher concns of PA. Notwithstanding the implication of an equilibrium reaction, IgGl-bound PA did not dissociate upon several washings and upon prolonged incubation in the absence of fluid-phase PA. It is conceivable that, with IgGl, PA binds firmly only to IgG molecules in close proximity so that the tetravalent PA could cross-link several IgG molecules. This reasoning is in line with evidence that certain IgGs that bind PA poorly or not at all bind PA with a high affinity when complexed with an antigen (Langone, 1980~). Alternatively, IgG I monoclonal antibodies may be hindered for steric reasons from interacting with PA when bound to cell surfaces.

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