Mouse monoclonal antibodies at the red cell surface—I. Generation of EAC4 and interaction with C1

Molecular immunology, Vol. 22, No. 3, pp. 223..227, 1985

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MONOCLINAL ANTIBODIES AT THE RED CELL SURFACE-I. GENERATION OF EAC4 AND INTERACTION WITH Cl *

HERBERTJ. KRATZ, TIBOR BORSOSand HENRI ISLIKER? Department of Biochemistry, University of Lausanne, Switzerland; and Laboratory of Immunobiology, National Cancer Institute, Frederick, MD 21701, U.S.A. (First received 13 June 1984; accepted in revised form 28 August 1984) Abstract-Cl

and CT activity measurements were performed with EA and EAC4 prepared with rabbit anti-Forssman IgG or IgM and were compared to measurements with EA and EAC4 prepared with mouse mon~lonal IgGZb and IgM anti-DNP antibodies on cells coupled with TNP: the amount of TNP per cell was optimal for antibody activity. No differences were found in the ability of EAC4 made with polyor monoclonal IgM to measure Cl activity; in contrast, monoclonal IgM was capable of activating only about 30% of Cl when compared to activation by polyclonal IgM. Monoclonal vs polyclonal IgGs behaved in a similar manner but they were detecting only 50% of Cl or CT activity when compared to IgM of the appropriate class. It was concluded that monoclonal antibodies were capable of generating EAC4 intermediate, and that the ability of monoclonal antibodies in the EAC4 complex to bind Cl and to detect Cl activity is not significantly different from that of polyclonal antibodies but that monocional antibodies are less efficient in activating Cl than polyclonai antibodies.

INTRODUCTION Immunoglobulins acquire complement-fixing properties as a consequence of their interaction with antigens or haptens. Generally, immune complexes generated with IgGf or IgM antibodies are effective in binding and activating the first component of complement and thereby activating the whole sequence, whereas IgA, IgE and IgD do not fix and activate complement (C) by the classical pathway (Ishizaka et al., 1970). Studies with well-defined cell intermediates were indispensable for analyzing the activity of C on a molecular basis. These studies were performed with polyclonal antibodies, composed of a mixture of Ig molecules in which antibodies of different specificities and class and subclass may be present. Monoclonal antibodies may thus behave differently from polyclonal antibodies in generating C fixing and activating sites. _.._ *This study was supported by grant No. 3.267-0.82 of the Swiss National Science Foundation. TCorrespondence should be addressed to: Prof. H. Isliker. Institut de Biochimie. Chemin des Boveresses, CH-1066, Epalinges, Switzerland. 2,4_dinitrophenyl; TNBS, SAbbreviations: DNP, 2,4,6_trinitrobenzene sulfonic acid; E-TNP, E to which TNP was coupled covalently; Ig, immunoglobulin; IgM and IgG, immuno~lobulins of class G and M respectively; IgGl, IgG2, IgG2b and IgG3, subclasses of mouse immunoglobulin G; BSA, bovine serum albumin; NPCB, p-nitrophenyl-p’-guanidinobenzoate; ECDI, SDS, I-ethyl-3(%dimeihyla&opropyl)carbodiimide; sodium dodecvl sulfate: SDS-PAGE, sodium dodecvl sulfate-polyac;ylamide gel electrophoresis; 2, -in (fraction of unlysed cells) = average number of effective lytic sites/cell; NHS, normal human serum. 223

There are no studies on the generation of cell intermediates with monoclonal antibodies and only one study on the effect of hapten density on the fixation of Cl by monoclonal antibodies (Okada et al., 1983). In our first study results are presented on the binding and activation of Cl by EA and EAC4 generated with monoclonal antibodies; in a second investigation the effect of hapten density on C fixation and activation by monoclonal antibodies of different classes was examined.

MATERIALS AND METHODS Buffers

and

reagents

Sheep blood was obtained from Bio-Merieux (Lyon, France). Isotonic veronal-buffered saline (VBS) and sucrose-containing veronal-buffered saline (SVB) were prepared according to Rapp and Borsos (1970). Both SVB and VBS contain 0.1% gelatin or O.Isi/, BSA, 0.15 mM CaCl, and 1 mM MgCl,. Isotonic VBS-EDTA buffer was prepared by mixing 9 parts isotonic VBS lacking Ca2+ and Mg*+ with 1 part 0.1 M trisodium ethylenediaminetetraacetate, pH 1.5. C-EDTA was prepared by mixing 1 part guinea pig serum, containing at least 170 CH,/ml, with 49 parts isotonic VBS-EDTA buffer. Gelatin and BSA

Gelatin was purchased from Sigma Chemical Co. (St. Louis, MO), lot 29C-0532, and BSA was purchased from Merck (Darmstadt, F.R.G.).

7’4

HEK~FKT.I. KKATT (‘I (11

Seru and C comnponrr2ts Human blood, collected without anticoagulant, was obtained from the Swiss Red Cross, Transfusion Center, Lausanne. The serum was separated after 2-3 hr at 4 C and stored in aliquots at -70 C. Guinea pig serum (GPS) was purchased from Behring AG (Marburg, F.R.G.) and partially purified human and guinea pig CT and C4 from Cordis Corporation (Miami, FL). Purified precursor Cl was prepared according to Medicus and Chapuis (1980). In most experiments, the source of C was fresh or fresh-frozen guinea pig serum. All sera were absorbed with sheep or hapten-labelled cells to reduce lysis of cells in the absence of antibodies. Absorption was performed with packed cells (about 2 x IO’ cells/ml) in the cold, and was repeated at least twice. Separution

of’ C2 ,fiom guineu pig serum

Chemical Co. and modified barbital buffer (MBB) prepared according to Campbell ct al. (1963). (a) With ECDI according to Borsos ct N/. (1980). purchased from Calbiochem Behring Corp. (La Jolla, CA). Briefly, 2 parts of E (2 x 10’ E:‘ml in PBS) were mixed with one part of a solution containing between 0 and I5 mg TNBS/ml in PBS. With constant mixing, 2 parts of ECDI solution (IO mg/ml in PBS) were added. The cells were incubated at room temp for 30 min. washed once with PBS and 3 times with VBS. The cells were diluted to a convenient concn and kept at 4 C. Coupling TNP to E was quantified with [‘HITNBS (sp. act. 208 mCi/mmole) from Amersham, U.K. In order to couple E with [jH]TNBS, radioactive TNBS was mixed with unlabelled hapten at a 11200 ratio and either of the above-described procedures was applied. For counting, E were washed and lysed in distilled water: the stromas were washed free of hemoglobin, dissolved in 0.05% SDS, and the amount of radioactivity was measured in a Beckman LSSOOO liquid scintillation counter.

This was achieved by ion exchange chromatography according to Rapp and Borsos (1970). C2 prepared in this manner contained no detectable Cl, C4 or C3, but a considerable amount of C9 and some of the other late-acting components. In some experiments, partially purified guinea pig or human C2 from Cordis were used. Treatment of human C2 according to the iodine oxidation method was performed as described by Polley and Miiller-Eberhard (1967). CT-inhibitor (500 U/ml) was obtained from Cordis. The assay of C components has been described in detail elsewhere (Rapp and Borsos, 1970). Trypsin from bovine pancreas (40 Ujmg) was purchased from Serva Fein Biochemica (Heidelberg, F.R.G.).

The following mouse monoclonal antibodies were a generous gift of Dr G. Kdhler, Institute for Immunology, Basel: SP6, IgM, (anti-TNP); GK 14-1, IgG2b, (anti-TNP); SP2/HL, IgGZb, (anti-SRBC); HY 2-15, IgGI, (anti-TNP); Hy 1-2, IgG2a, (antiTNP); and SP3/HL, IgGl, (anti-SRBC). Mouse anti-DNP IgG3 and mouse anti-DNP IgM monoclonal antibodies were a gift of N. Klinman (Scripps Research Foundation, La Jolla, CA).

Rabbit antisera

Hybrid cell lines

Rabbit antisera against sheep erythrocyte stromas were prepared according to Rapp and Borsos (I 970) and stored at -20°C. IgM and IgG fractions of the rabbit hemolysin were separated by gel filtration on Sephadex G-200. In some instances the latter was substituted by ACA 22 Ultrogel.

Hybrid cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented to contain 100 U/ml penicillin and streptomycin, 1Oy! heatactivated fetal calf serum and 5 x lOeLM 2-mercaptoethanol. Subsequently, SP2 and HY 2-l 5 cells were injected into BALB/c mice, and the ascites fluid was collected.

Cell intermediate

Separation

used in hemolytic

assays

For the preparation of sensitized sheep erythrocytes (EA), I vol of sheep erythrocytes (IO3 cells/ml) was incubated for 15 min at 30 C, with 1 vol of the IgM fraction of rabbit hemolysin diluted in VBS to lOpgg/ml. The cells were washed once in VBS and once in SVB. Finally, they were suspended in SVB to an appropriate concn. EACi, EACl4 and EAC4 were prepared according to Rapp and Borsos (1970), using either IgM or IgG to sensitize E. T,,,,,-determinations were performed on EAC14, and preparations with a T,,, higher than 8 min were discarded. Preparation

oJ‘ TNP-coupled

sheep erythrocytes

(a) With TNBS in cacodylate according to Rittenberg and Pratt (1969), using TNBS from Sigma

Monoclonal

antibodies

qf’ mouse IgG and IgG subclusses

Separation of mouse IgG and IgG subclasses was carried out by affinity chromatography on protein A-Sepharose according to Ey et a/. (1978). When serum was applied to a protein A-Sepharose column at pH X.0, IgM, IgA and IgE were almost quantitatively recovered in the effluent together with non-immunoglobulin serum components. Essentially all IgG was retained at pH 8.0 and was not eluted by the above buffer. Using 0.1 M citrate buffers of decreasing pH, IgGl, IgG2a and IgG2b were sequentially eluted at pH 6.0, 4.5 and 3.0, respectively. Fractions eluted at pH < 5.0 were neutralized immediately with I M Tris+HCl, pH X.5. Protein concns of purified IgG were calculated from absorbance at 2X0 nm, assuming an extinction coefficient of 14. SP6 anti-TNP monoclonal antibody

Mouse

was purified on a Sephacryl S-300 fine column. The 0.1 M Tris-HCl buffer, pH 7.8, contained 0.5 M NaCl. The 50% ammonium sulfate precipitate from SP6 culture supernatants was taken up in a small vol of gel column buffer, dialyzed for l-2 hr and applied onto a 2.5 x 90 cm column, with a 12 ml/hr flow rate.

SDS gel electrophoresis of purified IgG monoclonal antibodies The purity of protein A-Sepharose isolated IgG monoclonal antibodies was controlled by SDS-PAGE (IgGl anti-TNP, IgG2b anti-SRBC and IgG2b anti-TNP). Non-reduced samples showed one band corresponding to the IgG region. Upon reduction, IgG2b antibodies showed differences in migration between the H-chains: this may be due to a different extent of glycosylation of these chains. IgGl showed only one single band corresponding to the H-chain. The four hybridoma antibodies had light chains of similar apparent mol. wt. The anti-TNP IgG2b antibodies revealed a second line in the light-chain region: this is likely to represent a light chain derived from the parent myeloma with which the spleen cells were fused. Zodination of purtjied antibodies Purified GK 14-1, SP2 and HY 2-15 antibodies (10-20 p g) were radiolabelled with 100-200 p Ci of carrier-free ‘*‘I, using the chloramine-T method. Labelled proteins were diluted in VBS containing 1 mg/ml BSA, and dialyzed extensively against VBS to remove unbound iodine; the labelled antibodies were stored at 4°C. Four hundred micrograms purified SP6 were labelled with 1 mCi carrier-free “‘1 according to the standard procedures described for Iodogen (Pierce Chemical Company, Rockford, IL). Hemolytic assay of monoclonal antibodies To 0.1 ml of erythrocytes or TNP-labelled cells (1.5 x 10’ cells/ml), 0.1 ml of antibody dilution in VBS buffer was added. The mixtures were incubated for 15 min at 37°C washed once in VBS, and 2.0 ml of guinea pig serum (l/250) were added to each tube. After a 1-hr incubation at 37°C the cells were

Table 1. Comparison

ci

sedimented by centrifugation, and the amount of hemoglobin released was determined at 412 nm. The number of hemolytic sites per cell was calculated according to the one-hit theory of immune hemolysis (Rapp and Borsos, 1970). When not otherwise stated, the values presented in Results represent mean values of at least three experiments. Labelling of protein A with ‘25I Protein A (sp. act. > 50 mCi/mg) labelled with “‘1 according to Bolton and Hunter, was purchased from Amersham, U.K. In some experiments, protein A labelled by the chloramine-T method was used (sp. act. 26 mCi/mg). Rabbit and mouse antibodies were to estabquantified with ‘251-protein A according lished procedures (Langone, 1982). Rabbit anti-mouse IgGl, IgG2b and IgM were purchased from Nordic Immunological Reagents (Tilburg, Netherlands).

RESULTS AND DISCUSSION

The efJicacy of Cl hemolytic assay as a function oj monoclonal us polyclonal antibodies Comparative Cl and CT titration of EIgM and EIgMC4: polyclonal us monoclonal antibodies. Assays of Cl activity of EA or EAC4 require different functional capacities of the Cl molecule. Assay of Ci with EAC4 measures the ability of Cf to bind to the cell and to cleave C2. Assay of Ci with EA depends, in addition, on the ability of CT to interact with C4 and to bind a portion of C4 to a reaction site. Table 1 compares Cl and Ci efficiencies, when assayed on EA or EAC4 using IgM anti-Forssman antibody. TNP-labelled E (1.5 x lo6 TNP/E) were first optimally sensitized with excess mouse anti-TNP IgM monoclonal (E-TNP SP6) antibody. Using established procedures for EAC4 preparation, E-TNP SP6C4 were finally obtained. The results in Table 1 differ somewhat from those presented by Opferkuch et al. (1971): whereas, in their hands, Cl (NHS) activity was almost identical on EIgM and EIgMC4, we observed a 30% reduced activity when the assay was done on EA. Similarly when fluid-phase activiated Ci was measured on

of effective Cl and Ci molecules on EA, EAC4, EmTNP SP6 and EmTNP SP6C4 (SP6: IgM, anti-TNP)

EA (anti-Forssman) Cl

225

monoclonal antibodies at the red cell surface-1

1.1 x 10’3 2.6 x IO”

EAC4 (anti-Forssman)

EmTNP SP6

E-TNP SP6C4

1.6 x 10” 6.6 x 10”

0.3 x 10’3 3.4 x 10’1

0.8 x IO” 6.1 x 1O’l

“Data are average values from three experiments and are expressed as effective Cl (or Ci) molecules/ml. 0.1 ml NHS or fluid-phase activated Ci (0.8 mg/ml) dilutions in SVB were incubated for 60min at 30°C with 0.1 ml EA, EAC4, E-TNP SP6 or E-TNP SP6C4 (1.5 x 10’ cells/ml). The TNP-labelled erythrocytes carried 1.4 x IO6 TNP/E. When activity was measured on EA or EmTNP SP6, 0.1 ml of partially purified guinea pig C4 (containing 100 effective molecules per cell) was added, and the mixture incubated for 20 min at 30°C. Lysis was brought to completion by adding C2 and GEDTA (l/SO) and hemoglobin release was determined at 412 nm.

M MM

22’3-t

226

HERBERT J. KRATZ (v u/. Table 2. Effect of lrnmuno~lobuhn class on hemom lytic Cl and ci actwity” Cl

ci

E-TNP lgG2b

E-TNP IgM

0.8 x IO” 3.6 x II.”

0.7 x 10’1 4.6 x 1V

2

I o/o

Inii 2

”

I” ’

:

“Data are average values from three experiments and are expressed as effective Cl (or ci) mo~ecu~esjml.cl (NHS) and ci (a.xmgimt) activities were assayed as described in the legend to Table I. C4. C2 and CmEDTA were added sequentially and lysis was measured by hemoglobin release.

0

EPIC4

0

-

I

l-

l------•

E-TWPSP6C4

. I

Fig. 1. Activation of C1 to CT on EAC4 and E-TNP igM SPK4. Source of Cl: human serum diluted 1:lOO.O~~. Temp WC. For experimental details see text.

these cells, EA detected about 40%; of the Ci activity estimated on EAC4. EA and EAC4 prepared with monoclonal antibodies behaved much like those prepared with poiyclonal antibodies: Cl activity was almost 3-fold higher on E-TNP SP6C4 than on E-TNP SP6: this ratio decreased to 2 when activated Ci was used. CT and Ci uttiuities with mouse ~n~i~or~o~a[ antibodies IgM and IgG2h. Cohen et al. (1969) reported that the immunoglobulin class of the antibody used to prepare EAC4 also affected the molecular assay of native and activated Cl. They showed that EAC4 prepared with rabbit IgG hemolysin were unsuitable for the titration of Cl and Cf. since use of these cells led to a two-three-fold underestimate of Cl and to a two-three-fold overestimate of CT. Their conclusions were based on the observation that, although EAIgGC4 or EAIgMC4 are capable of fixing Cl to about the same extent, the rate and extent of activation of Cl to CT were greater on EAIgMC4. Overestimation of Ci, according to those authors, was probably due to site transfer and reutilization of about l&207: of the Ci. This observation was reexamined with purified IgG2b and IgM anti-TNP mouse monoclonal antibodies and by using TNPlabelled cells ( lo6 TNP molecules/E) optimally sensitized with excess IgG2b or IgM antibody. Guinea pig serum CH,, activity was two-fold higher on IgMbearing E-TNP (data now shown). Cl and CT activities on E-TNP IgG2b vs E-TNP IgM are shown in Table 2.

The results show that, in the TNP-labelied erythrocyte system, C 1 content was similar, whether the cells are sensitized with IgG2b or IgM antibodies, whereas CT estimates were two-fold higher on the IgMbearing cells. Uptake of Cl and Ci on E-TNP IgG2b and E-TNP IgM was also measured since differences in CT content estimations could be due to a differential uptake of the activated first component of C by E-TNP IgG2b or E-TNP IgM. Data in Table 3 demonstrate that this is not the case. The results indicate that both types of cells take up Cl equahy well. When activated Ci is offered, binding is slightly more efficient on IgGZb-bearing E-TNP. The two-fold higher eficiency of CT expression of E-TNP IgM therefore cannot be explained by an increased uptake of Ci on cells prepared with IgM. Table 1 compares the ability of two types of cells, EAC4 vs E-TNP SPK4, to fix and activate Cl. Whereas Cf estimates are identical, irrelevant of the type of cells chosen, use of anti-Forssman EAC4 results in a three--four-fold higher Cl activity. Cl uptake studies unequivocally rule out the possibility that tbe low Cl activity on E-TNP SP6C4 is due to reduced uptake of C I. Figure 1 shows that the rate and extent of Cl activation on E-TNP SP6C4 is less when compared to its rate of activation of EAC4. The data demonstrate that mouse monoclonal antibodies are nearly

Table 3. Uprake of CL and Ci by E-TNP IgGtb and E-TNP IgM” Cl or ci!cell remaining in supernatant

after exposure to

Input of Wcell

EmTNP IgCZh”

E~TNP tgM”

0.5 1.3 2.6

0.026 (95) Not detectable (100) 0.017 (99)

Not d&table (100) Not detectable (100) Not detectable (100)

2.1

0.138 (93)

4.3

0.305 (93)

0.405 (81) 0.669 (84)

Input 0r ci:cett

“Dilutmns of NHS (Cl) or fluid-phase activated Ci (0.8 mg;ml) in SVB were added to 1.5 x IO’ E--TNP IgC2b or E-TNP IgM. The mixtures were incubated for 60 min at 30 C and then centnfuged. Cl and Ci activities m the original dilutions and the supernatants were determined in a standard hemolytic aswy on EAC4. “As measured with EAC4 (IgM anti-Forssman). The numbers in parentheses relate to the per cent upiake of Cl or CT.

Mouse monoclonal antibodies at the red cell surface-1

as efficient in generating the key cell intermediate, EAC4, as the rabbit antibodies. Although there are quantitative differences between the ability of IgG and IgM to activate Cl, these differences are not more pronounced with the monoclonal than with the polyclonal antibodies. This is important since it has been assumed-based on scant experimental datathat monoclonal antibodies at cell surfaces are less efficient at C fixation than polyclonal antibodies. A second point that emerges from these data is that monoclonal antibodies directed against hapten chemically coupled to cell surfaces are about as effective in allowing the generation of cell intermediates as antibodies to haptens that are naturally part of the cell surface. This has been documented for polyclonal antibodies with a different hapten (Circolo and BorSOS,1982; Borsos and Circolo, 1983). Thus it can be concluded that the mechanism of C fixation and activation and assembly of the attack complex at a cell surface will proceed effectively as long as there are sufficient numbers of C-fixing antibody-hapten complexes in proximity to the cell surface. This is in keeping with reports demonstrating that the entire C sequence occurs on the cell surface (Circolo and Borsos, 1984a, 6). The critical controlling element in generating C fixing and activating Ig-hapten complexes is the density and distribution of hapten molecules on the cell surface. The effect of this variable on the ability of monoclonal antibodies to fix and activate C is the subject of the accompanying study (Kratz et al., 1985). Acknowledgement-We are grateful to Dr G. Kijhler from the Base1 Institute of Immunology for the monoclonal antibodies.

REFERENCES Borsos T. and Circolo A. (1983) Binding and activation of C 1 by cell-bound IgG: activation depends on cell surface hapten density. NO&X. fmmun. 20, 433438. Borsos T., Dunkel V. C. and Langone J. J. (1980) Immunoassay of antigens and haptens by inhibition of passive immune hemolysis. J. immun. Meth. 32, 105-i 14.

227

Campbell D. H., Garvey J. S., Cremer N. E. and Sussdorf D. H. (1963) In Methods in Immunology. Benjamin, New York. Circolo A. and Borsos T. (1982) Lysis of hapten-la~lled cells by anti-hapten IgG and complement: effect of cell surface hapten density. J. Zmmzm. 128, 1118-I121. Circolo A. and Borsos T. (1984~) C4 does not bind to human and rabbit IgM during activation of the classical complement pathway on the red cell. J. Immun. 129, 1485-1488. Circolo A. and Borsos T. (19846) Lack of binding of C3 to IgG antibodies during the activation of the classical complement pathway on the red cell. IMalec. r~un. 21, 191-195. Colten H. R., Borsos T. and Rapp H. J. (1969) Titration of the first component of complement on a molecular basis: suitability of IgM and unsuitability of IgG hemolysis as sensitizer. Immunochemistry 6, 461-467. Ey P. L. Prowse S. J. and Jenkins C. R. (1978). Isolation of pure IgGl, IgG2a and IgG2b immuno~obulins from mouse serum using protein A-Sepharose. Immunochemistry 15, 429436.

Ishizaka K.. Ishizaka T. and Lee J. M. (1970) Biologic function of the Fc fragments of E myeloma protein. Immunochemistry 7, 687-702.

Kratz H. J., Borsos T. and Ishker H. (1984) Mouse monoclonal antibodies at the red cell surface--II. Effect of hapten density on complement fixation and activation. M&c. Immun. 22, 229-235. Langone J. L. (1982) Use of labelled protein A in quantitative immunochemical analysis of antigens and antibodies. J. immun. Meth. 51, 3-22, Medicus R. G. and Chapuis R. M. (1980) Purification and properties of native Cl. J. Immun. 125, 39&395. Okada M., Takahashi K. and Utsumi S. (1983) Conditions which favor the Cl-fixation by mouse IgG1. Mo/ec. fmmun. 20, 219-285.

Opferkuch W., Rapp H. J., Colten H. R. and Borsos T. (1971) The first component of guinea pig complement: hemolytic assays with EA, EAC4 and with rat and guinea pig late acting components. immunochemistry 8, 511-523. Polley M. J. and Miller-~~rhard J. J. (1967) Enh~~ment of the hemolytic activity of the second component of human complement by oxidation. J. exp. Med. 126, 1013-1025.

Rapp H. J. and Borsos T. (1970) In Molecular BUSTSof Complement Action, Chap. 7, pp. 75-108. Meredith Corporation, New York. Rittenberg H. B. and Pratt K. L. (1969) Antitrinitrophenyl (TNP) plaque assay. Primary response of BALB!c mice to soluble and particulate immunogen. Proc. Sot. exp. Biol. Med. 132, 575-58 1.