Interspecies hybrid antibody with dual specificity

0161-5890/81/0201-0091 602.00/O

Mokcu/ar Immunology, Vol 18.pp. 91-94 QPergamon PressLtd. 1981. Printedin Great Britain

INTERSPECIES HYBRID ANTIBODY WITH DUAL SPECIFICITY GABRIELA Department

of Immunology, (First

MOTA and V. GHETIE V. Babes Institute,

received 5 May

76201 Bucharest

1980; in revisedfbrm

16 Jr&

35, Romania

1980)

Abstract-The preparation of soluble multivalent hybrid antibody by protein A of ~~u~~~~~c~cc~ uureus (SPA) is limited exclusively to rabbit IgG because other species have SPA-precipitating IgGs. Starting from a soluble complex consisting of one rabbit IgG antibody molecule linked to one SpA molecule (rabbit IgG anti-A/SPA), an interspecies hybrid antibody with dual specificity was prepared using either mouse or human IgG antibody, the molecular formula of this complex being (rabbit IgG anti-A/SpA/mouse or human IaG anti-B),. These complexes are useful tools for the investigation ofceil surface antigens against which no aipropriatgantibody can be raised in rabbits.

INTRODUCTION

MATERNAL

AND METHODS

Proteins

Hybrid antibody complexes with dual specificity were obtained by combining two rabbit IgG antibodies with different specificity by means of protein A of Staphylococcus aureus (SPA*) (Ghefie & Mofa, 1980). By the reaction of rabbit IgG antibody (i.e. anti-A) with an excess of SpA a soluble complex was prepared (IgG antiA/SPA) able to further attach another molecule of rabbit antibody (anti-B) and thus yielding a antibody (IgG antimultivalent hybrid A/SpA/IgG anti-B),. Using this reaction several multivalent hybrid antibodies were prepared and their ability to draw together soluble or particulate antigens was clearly demonstrated (Ghefie & Mota, 1980; Mandache et al., 1980). The preparation of hybrid antibody complexes is restricted only to rabbit IgG since the immunoglobulins of other species do not react with SpA (fowl, horse, etc.) or are precipitated by it (mouse, human, etc.) (Kronvall er al., 1970). In order to extend the use of hybrid antibodies to antigens reacting only with mouse (i.e. Ly antigens) or human (i.e. HLA) alloantibodies, we attempted by means of SpA to link two antibody partners, one deriving from rabbit (nonprecipitating with SpA) and the other originating from mouse or human (precipitating with SPA). Thus we succeeded to obtain a soluble interspecies hybrid antibody complex with dual specificity.

SpA (Pharmacia, Uppsala, Sweden) and rabbit IgG antibody were labelled with Na lzsI (The Radiochemical Centre, Amersham, U.K.) using the lactoperoxidase technique (Biberfeld et af., 1975). Horseradish peroxidase (HRP) (type II Sigma, St. Louis, MO, U.S.A.) was coupled to sheep red blood cells (SRBC) by the CrCl, technique (HRP-SRBC) (Gold & Fudenberg, 1967). Antibodies

Anti-peroxidase sera were obtained from rabbits injected with HRP (MedeSan et al., 1979) Purified antibodies were isolated by acid elution from glutaraldehyde-insolubilized HRP as previously described (Ghetie & Mofa, 1980). IgG was isolated from mouse and human sera by SPA-Sepharose 4B affinity chromatography (Hjelm et al., 1972) using 3 iM NH,SCN for elution of bound IgG. Mouse anti-Thy-l sera, extensively absorbed with mouse red blood cells, hepatocytes and bone marrow cells, was kindly provided by Dr. A. Sulica (Babe? Institute, Bucharest, Romania). Human anti-HLA b-12 serum was obtained from National Institute of Health (Bethesda, MD, U.S.A.). Rabbit IgG antibodies specific for bovine (BRBC) and chicken (CRBC) red blood cells were obtained by immunoadsorption on glutaraldehyde treated red blood cells and elution with 2.5 M NaI. Goat anti-rabbit IgG (Behringwerke, Marburg, F.R.G.), goat antimouse IgG (Nordic, Amsterdam, The Netherlands) and goat anti-

*Abbreviationsused: SPA, protein A of S. uurar.s; HRP, horseradish peroxidase; MSL and MT, mouse spleen lymphocytes and thymocytes: BRBC and CRBC. Bovine and chicken red blood cells. 91

92

GABRIEI_A

human IgG (Cantacuzino Romania) were also used.

Institute,

MOTA

Bucharest,

Cells Spleen lymphocytes (MSL) and thymocytes (MT) were isolated from CBA mice. Spleen lymphocytes were subsequently purified by a one-step sodium methrizoate/Ficoll procedure (Boyum. 1968). This procedure was also applied for isolation of human peripheral blood lymphocytes. Chromatography,

electrophoresis

and immuno-

logical analysis

Previously described techniques were used (Ghefie & Mofa, 1980). Briefly, gel filtration was carried out on calibrated Sepharose 6B (Pharmacia) column eluted with 0.1 M Tris-HCl buffer, pH 8 with 0.2 A4 NaCl. CM-Sephadex C-50 chromatography was performed on a column equilibrated with 0.02M acetate buffer, pH 5.5. Electrophoresis in 6”; polyacrylamide dodecylsulphate was gel with O.l”:, sodium performed using a horizontal technique. Immunodiffusion was performed in 1% agarose Microagglutination was assayed in gel. microtiter plates. The rosette technique was carried out with thymocytes or lymphocytes treated with hybrid antibody complex (100 pg/107 cellsjml) and further mixed with indicator red blood cells. Cells binding more than three indicator cells were considered positive. Preparation

of

interspecies

hybrid

antibody

complexes

The rabbit IgG/SpA complex (mol. wt 190,000 daltons) was prepared as previously described (Ghefie & Mofa, 1980). Briefly, rabbit IgG antibody was mixed with SpA at final concentrations of 1 mg IgG/ml and 2 mg SPA/ml. After incubation the mixture was chromatographed on CM-Sephadex equilibrated with 0.02 A4 acetate buffer, pH 5.5, and the adsorbed rabbit IgG/SpA complex was eluted with phosphate buffered 0.3 M NaCl at pH 7.4. The solution was gel filtrated on a calibrated column of Sepharose 6B. The peak with mol. wt 190,000 daltons was collected and mixed with an equal volume of a solution containing the same concentration of mouse or human IgG antibody (24 mg rabbit IgG/ml) and after 1 hr incubation at 37C the mixture was gel filtrated on Sepharose 6B. The peak containing the hybrid antibody complex was concentrated and further used in antigen binding experiments. The

and V GHETIE

attachment of the second nonradioactive mouse or human IgG antibody to the iz51-rabbit IgG anti-HRP/SpA complex was determined on a molar basis according to the decrease of the specific radioactivity of the initial IgG antibody (anti-HRP).

RESULTS

An equal amount of mouse IgG anti-Thy-l was added to the complex containing iz51-rabbit IgG anti-HRP/SpA (mol. wt 190,000 daltons) and the mixture gel filtrated on Sepharose 6B. One single peak was eluted in a volume corresponding to mol. wt 669,000 daltons. The specific radioactivity of the peak.was half that of the iz51-rabbit IgG anti-HRP/SpA (0.54 i 0.08; three experiments), thus indicating that mouse

a

b

c d

e f g h Fig. I. Electrophoresls in polyacrylamide gel of hybrid complexes. (a) Human IgG; (b) rabbit antibody IgG/SpA/human IgG; (c) rabbit IgG; (d) rabbit IgG/SpAjmouse IgG; (e) mouse IgG; (0 rabbit IgG/SpA/rabbit IgG: (g) rabbit IgGjSpA; (h) SpA.

Interspecies

Hybrid

IgG anti-Thy-l was fully integrated into the IgG anti-HRPjSpA complex yielding an interspecies hybrid antibody molecule with dual specificity (rabbit IgG anti-HRP/SpA/mouse IgG antiThy-l),. The complex showed a narrow banding in polyacrylamide gel electrophoresis similar to that of the soluble complex formed by reacting rabbit IgG with rabbit IgG/SpA complex; no free SpA or IgG was detected (Fig. 1). By using the above-mentioned procedure we succeeded in preparing some other hybrid complexes such as (rabbit IgG antiCRBC/SpA/mouse IgG anti-Thy-l), and (rabbit IgG anti-BRBC/SpA/human IgG anti-HLA B-12),. The presence in the same molecule of IgG antibodies originating from two different species was tested by immunodiffusion. Double diffusion with (rabbit antiIgG I, Ability of interspecies

hybrid

antibody

Ligands” Rabbit anti-HRP/SpA/mouse anti-Thy-l Rabbit anti-CRBC/SpA/mouse anti-Thy-l Rabbit IgG anti-HRP + mouse IgG anti-Thy-l (without SPA)

complexes

93

ZO-

Fig. 2. lmmunodiffusion analysis of the hybrid antibody complex. (a) Goat anti-rabbit IgG; (b) goat anti-human IgG; (c) rabbit IgG/SpA/human IgG; (d) rabbit IgG/SpA/mouse IgG; (e) goat anti-mouse IgG.

Table

Antibody

20 Peroxidase

i,. 40

60

80

pg/lO’

concentration,

100 cells/ml

Fig. 3. Inhibition of the rosette formation with HRP-SRBC of hybrid treated thymocytes by various concentration of HRP.10’ thymocytes were treated with 75 pg of (rabbit IgG anti-HRP/SpA/mouse IgG anti-Thy-l), complex. The percentage of rosettes formed without prior incubation with HRP was 80%.

HRP/SpA/mouse IgG anti-Thy-l), and (rabbit IgG anti-BRBC/SpA/human IgG anti-HLA B-12), showed a reaction of complete identity between the precipitin lines given by anti-rabbit IgG and anti-mouse IgG or anti-human IgG, respectively (Fig. 2). antiability of (rabbit IgG The HRP/SpA/mouse IgG anti-Thy-l), to react with both HRP and Thy-l was demonstrated by either HRP-SRBC or agglutination of with dual specificity

to draw together

Percentage of HRP-SRBC rosettes formed by MSLb MT’

two distinct

cell types

Percentage of CRBC rosettes formed by MSL MT

28
85




ND’

ND

“Target cells (MSL, MT) were treated with hybrid antibody complexes (100 pg/ml/lO’ cells). After washing, the cell-bound hybrid antibody was able to attach indicator cells (HRP-SRBC, CRBC) to target cells. The percentage of rosettes was calculated according to the following equation: 100 R/R + NR, where R = number of rosetted cells and NR = number of nonrosetted cells. b MSL = mouse spleen lymphocytes. cMT = mouse thymocytes. d When MSL were pretreated with mouse or human IgG (2 mg/ml/lO’ cells), then treated with hybrid antibody, the same percentage (22”/,)was recorded. ‘ND = not done.

94

GABRIELA

MOTA

thymocytes with titers of 0.88 and 0.45 pg hybrid/ml, respectively. The percentages of radioactivity bound to CRBC or to thymocytes when treated with (l 251rabbit IgG anti-CRBC/SpA/mouse IgG antiThy-l), were similar, although only one of the antibody molecules (anti-CRBC) was radiolabelled. Moreover, the same amount of SpA radiolabelled hybrid complex (rabbit IgG antiHRP;“251-SpA/mouse IgG anti-Thy-l), was bound to HRP-SRBC and to thymocytes. Hybrid antibody complexes were also able to draw together two distinct cell types, thus allowing rosette formation. MSL or MT treated with hybrid complexes containing antibodies against both Thy-l antigen and HRP or CRBC formed rosettes with the indicator cells (Table 1). Rosette formation between MT treated with (rabbit IgG anti-HRP/SpA/mouse IgG antiThy-l), complex and HRP-SRBC was inhibited if the cells were previously treated with HRP (Fig. 3). Likewise, human peripheral blood lymphotreated with (rabbit IgG anticytes BRBC/SpA/human IgG anti-HLA B-12), complex were able to form rosettes with BRBC only if lymphocytes from HLA B-12 positive donors were used. No rosettes were formed with CRBC or with lymphocytes from HLA B-12 negative donors. DISCUSSION

The rabbit IgG/SpA complex had the ability to bind mouse or human IgG molecules, thus determining the formation of a soluble complex with the same molecular formula (rabbit IgG/SpA/mouse IgG or human IgG), and mol. wt (669,000 daltons) as with rabbit IgG (Ghelie & Mota, 1980). The attachment of mouse or human IgG to rabbit/SPA complex did not lead to the precipitation of both components, thus allowing the preparation of soluble interspecies hybrid antibody complexes. Mouse or human IgG could not be used for the preparation of the initial IgG/SpA complex (mol. wt 190,000 daltons) since their reaction even with an excess of SpA led to the precipitation of both proteins. Therefore hybrid antibody complexes composed IgGs (i.e. of SPA-precipitating only mouse/human, human/human, etc.) might be prepared starting from moderately carbamylated human and mouse IgG which react but do not precipitate with SpA (unpublished results). The interspecies hybrid antibody complexes were able to react by agglutination with both

and V. GHETIE

specific antigens (red blood cells and lymphoid cells) and to allow cell to cell contact between two unrelated cells, as proved by rosette formation. The binding of hybrid antibody to the lymphoid cell surface was mediated through their specific combining sites without in any way involving attachment of the complex to the MSL Fc receptors, as demonstrated by the inability of IgG to inhibit the binding of hybrid complexes to the cell surface (Table I&. The interspecies hybrid antibody complex (rabbit IgG anti-A/SpA,/mouse or human IgG anti-B), has eight combining sites. four belonging to rabbit IgG antibodies and four to mouse or human IgG antibodies. By its multivalency a hybrid antibody complex has an increased functional affinity for the corresponding antigens and can be used for the study of cell to cell interaction (Table 1). These hybrid antibody complexes can be easily obtained starting from purified antibodies (i.e. rabbit antiHRP, anti-BRBC) or even from the IgG fraction of the antisera (i.e. mouse anti-Thy-l, human anti-HLA). Therefore it was possible to build up a complex with two antibody partners of different origins, thus extending the use of multivalent hybrid antibody with dual specificity to antigens against which no antibodies can be raised in rabbits. AcknoMledgements~This investigation was sponsored by the Academy of Medical Sciences. The technical assistance of Mrs. Mariana Caralicea is greatly appreciated. REFERENCES Biberfeld P., Ghefie V. & Sjaquist J. (1975) Demonstration and assaying of IgG antibodies in tissues and on cells by labeled staphylococcal protein A. J. immunol. M&. 6, 249-259. Bayurn A. (1968) Separation of leucocytes from blood and bone marrow. &and. J. clin. Lab. Inrev. 21, Suppl. 97. Ghetie V. & Mota G. (1980) Multivalent hybrid antibody. M&c. Immun. 17, 395-40 I. Gold E. R. & Fudenberg H. H. (1967) Chromic chloride: A coupling reagent for passive hemagglutination reaction. J. Immun. 99, 859-862. Hjelm H., Hjelm K. & Sj(iquist J. (1972) PI-otein A from Staphylococcus aweus. Its isolation by affinity chromatography and its use as an immunoadsorbent for isolation of immunoglobulins. Fe&. Eur. hiochem. SW. Lett. 28, 73-76. Kronvall G., Seal C!. S., Finstad J. & Williams R. C., Jr. (1970) Phylogenetic insight into evolution of mammalian Fc fragment of gamma globulin using staphylococcal protein A. J. Zmmun. 104, 140-147. Mandache E., Moldoveanu E.. Moia G., Moraru I. & Ghetie V. (1980) Multivalent hybrid antibody with double specificity as a tool for locating cell surface antigens by electron microscopy. J. irnr~mnol. Meth.. 35, 33-41. Medegan C., Gherman M., Onica D.. Ghetie V.. SjOqui\t J. & Sulica A. (1979) Binding properties of various IgG ligands to Fc receptors of macrophage cells. Rrr roum. Biochim 16, 31-47.