01991 S. Karger AG. Basel 0042-9007/91/0614-0255 $2.75/0
Vox Sang 1991;61:255-257
A Human Anti-JkbMonoclonal Antibody M . Lecointre-Coatmelec, D . Bowel, J . Ferrette, B. Genetet’ Centre Rkgional de Transfusion Sanguine, Rennes, France
Abstract. Peripheral blood mononuclear cells of an immunized patient were transformed with Epstein-Barr virus and then fused with P3Xs3Ag8mouse myeloma cells by polyethylene glycol. After the cloning, a hybridoma cell line secreting specific anti-Jkb monoclonal antibody was isolated. The antibody was produced in supernatant form and tested for its use as a blood grouping reagent.
Introduction The murine hybridoma technology was unsuitable to produce specific anti-Rhesus monoclonal antibodies (MAbs). Transformations by the Epstein-Barr virus (EBV) of peripheral blood mononuclear cells (PBMCs) from immunized donors or patients were performed in an attempt to produce human blood grouping reagents. Although the efficiency of such a technique was very low, several reagents were produced as anti-D [1-4]. The major problem of this technology was the stability of the production. Several protocols were proposed to stabilize the secretion of MAbs and particularly heterohybridizations of human secreting lymphoblastoid cell lines or human B lymphocytes with mouse myelomas [ 5 , 6 ] . Most of the human monoclonal blood grouping reagents were produced by this heterohybridoma technology: anti-D [7,8], anti-c, anti-E and anti-Kell[g]. By using such procedures a human anti-Jkb MAb was produced; its production and characteristics are reported.
Material and Methods EBV Transformation and Cloning. An immunized patient was selected who had an anti-D alloantibody (titer Y64) without any other detectable antibody in his serum. A blood sample was taken 6 months after the first identification of the anti-D. 50 x lo6PBMCs were isolated I We thank Christine Gouaillier for the preparation of the manuscript.
on Ficoll Hypaque and transformed by the EBV [lo]. Supernatant of B95.8ceU line was added to a suspension of PBMCs and incubated in 96-well microculture plates containing mouse peritoneal cells as feeder layer. Culture medium was fed after 24h with RMPI containing 20% of fetal calf serum (FCS) and lpg/ml of ciclosporin A. Supernatant of growing cells was tested against papain-treated red blood cells (RBCs) of defined phenotypes. One cell line which produce an anti-Jkb antibody was cloned successively at 10, 5 and 1 cell per well with mouse peritoneal cells. Cell Fusion and Hybrid Selection. To preserve antigen-specific immunoglobulin secretion the EBV cell line was fused with a mouse myeloma P&Ags. Cell fusion was performed with PEG 4000 (Serva) at 50% in a buffered solution and hybridomas were grown in a selection medium containing hypoxanthine (13 pg/ml), azaserine (1.7 pg/ml) and ouabain (0.2pg/ml). Microagglutination tests were performed to isolate secreting heterohybridomas: 50p1 of culture supernatant was mixed with an equal volume of 2% papain-treated RBC. The plates were incubated for 35min at 3 7 T , centrifuged at 1,000rpm for lmin and stirred before reading of agglutinations. The production was performed in rolling bottles with a RPMI medium containing Hepes (25mM) and FCS (5%). The stability of the production was controlled by continuous culture over lyear. Every 3months the titer of the supernatant was controlled. Serological Tests. Specificity tests were assessed using a large panel of extensively phenotyped red cells by different tube techniques: (1) Agglutination tests using RBCs at 5% in saline medium: 2 drops of reagent and 2 drops of RBC suspension were mixed in a tube, incubated for 30min at 22°C; agglutination was observed after stirring. (2) Tests with papain-pretreated RBCs at 5%: the protocol was identical except the incubation for 35min at 37°C. (3) Tests with RBCs diluted at 3% in low ionic strengh solution (LISS) without incubation. Absorption elution tests were performed in parallel to commercial human polyclonal anti-Jkb sera. 500 pI of the reagent was incubated with 250 wl of different red cells (Jkb+and Jkb) for 4h at 37°C. After 5 washings the absorbed antibodies were eluted for 15min at 56°C and
256
Lecointre-Coatmelec/Bourel/Ferrette/Genetet
tested against papain-treated RBCs according to the described test. Reactivity was assessed using two spin tube techniques: (1)with papaintreated RBCs; (2) with RBCs diluted in LISS. Titer and score were determined by doubling dilution in RPMI containing 5% of FCS according to the international scoring of titrations. Classing of Immunoglobulin. Classing and quantitation of immunoglobulins in hybridoma supernatants were determined by an enzyme-linked immunoassay.
Table 1. Specificity of the anti-Jkb MAb according to different techniques Red cells
n
Jk(a+b+) Jk(a-h+) Jk(B+b-)
Saline 22°C
Papain-treated cells
Red cells in LISS
loo
+
44 56 5
-
++++ ++++
+++ +++
Jk‘a-b-)
Results and Discussion An EBV-transformed cell line was selected for its antiJkbreactivity: Nb 143. This cell line was rather unstable and difficult to grow in large-scale culture. For these reasons, heterohybridization was performed with P3X63A& mouse myeloma. One hybridoma cell line PJ43 was selected and cloned. It produced IgM molecules at a concentration of lOpg/ml. All the results presented were carried out with pure culture supernatant. The P3143 Mab agglutinated all the Jk(a+b+) and Jk‘”’+) RBCs without reactions with Jk‘a++b-) and Jk‘a--b-)samples. The agglutinations’were weak with RBCs in saline medium but very strong with Jkb+papain-treated cells without differences between homozygous or heterozygous samples. With RBCs in LISS the reactions were weaker than in papain tests but strong enough to make this technique more suitable for practical use (table 1). Adsorption elution tests showed that the P3 143 MAb was adsorbed on Jk(a+b+) and Jk‘”’) cells; no adsorption was observed with Jk(a+b)samples (table 2). The titer of the anti-Jkb reagent was 1:64 when tested against papain-treated cells and with a score of 70 (table 3); with RBCs in LISS the reagent showed a titer of 1:16 with a score of 49. All the results were confirmed during the Second International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (Workshop No. 168). This reagent presented a good stability: after storage during 2 months at 37°C and 12 months at +4”C its reactivity and its titer remained unchanged. The stability of the producing heterohybrids had to be controlled; after 12 months of continuous culture, the secretion quantitatively decreased as shown in table 4. Cloning experiments showed that after 6 months of continuous culture 40% of the heterohybrids were unproductive although the positive clones secreted the antibody with the same potency. This result pointed out that the decrease of production was due to a loss of secretion of some heterohybrids. This relative instability of production required that before each production the cells must be cloned to assure a better efficiency.
+
-
ND
-
ND
n = Number of samples tested; N D = not determined; +, +++, + + + + = intensity of agglutinations; - = no agglutination. Tests were performed in tubes.
Table 2. Adsorption elution tests of the anti-Jkb MAb Adsorbing cells
Reactivity of eluates against papain-treated cells ~~
Jk(a+h+) Jk‘a-b+) Jk(a+b-)
Jk(a+b)
Jk(a-b+)
-
++++ ++++
Reactivity: - = N o agglutination;
-
++++ = strong agglutination.
Table 3. Reactivity of the anti-Jkh MAb (tube tests) Papain-treated cells
Titer
Score
Jk(a+b+) Jk(a-b+) Jk(a+b-) Jk(a-b-)
70 70 0 0 ~~
International code
Strength of agglutination
Score
4+ 3+ 2+ 1+
12 10 8 5
-
0
Scores were evaluated according to the international code.
The P3143 anti-Jkb MAb was specific and could be considered as a good reagent for blood grouping. Its characteristics were in accordance with the international standards. Pretreatment with enzyme was not needed for hemagglutination if RBCs were diluted in LISS. Agglutinations were
257
A Human Anti-Jkb Monoclonal Antibody
Table4. Stability study of the production of the anti-Jkb by the heterohybridoma P3143 Reactivity
Titer Score
Months 1
3
6
9
12
64 70
64 63
32 54
16 37
4 28
Titers and scores were determined as described.
strong enough and superior to those observed with human polyclonal antisera. This MAb was especially interesting because Kidd phenotyping was of great interest in transfusion therapy [ll] and because it was difficult to obtain good polyclonal anti-Jkbreagents [12].
References Koskimies S: Human lymphoblastoid cell line producing specific antibody against Rh-antigen D. Scand J Immunol 1980:11:73-77. Bolyston AW, Gardner B, Anderson LR, Hughes-Jones NC: Production of human IgM anti-D in tissue culture by EB - virus transformed lymphocytes. Scand J Immunol 1980:12:355-358. Crawford DH, Harisson JF, Barlow MJ, Winger L, Huens ER: Production of human monoclonal antibody to rhesus D antigen. Lancet 1983;i:386388. Bron D, Feinberg MB, Teng NNH, Kaplan HS: Production of human monoclonal IgG antibodies against Rhesus (D) antigen. Proc Natl Acad Sci USA 1984;81:32143217.
Teng N, Lam K, Calvorieva F,Kaplan H: Construction and testing of mouse human heteromyelomas for human monoclonal antibody production. Proc Natl Acad Sci USA 1983;80:7308-7311. Murphy SM, Webb GC, Earl ME, Russ GR, Churcher A, Tait BD, d’Apice AJF: Human monoclonal antibody production by xenohybridomas. Transplant Proc 1986;18:343-346. Lowe AD, Green SM, Voak D, Gibson T, Lennox ES: A human monoclonal anti-D by direct fusion with a lymphoblastoid line. Vox Sang 1986;51:212-216. Foung SKH, Blunt JA, Wu PS, Ahearn P, Winn LC, Engleman G. Grumet FC: Human monoclonal antibodies to Rho (D). Vox Sang 1987;53:44-47. Goossens D, Champomier F, Rouger P, Salmon C: Human monoclonal antibodies against blood group antigens. Preparation of a series of stable EBV immortalized B clones producing high levels of antibody of different isotypes and specificities. J Immunol Methods 1987;lOl:193-200. lOJames K, Bell GT: Human monoclonal antibody production. Current status and future prospects. J Immunol Methods 1987:100:540. 11 Issitt PD: The Kidd blood group system; in Applied Blood Group Serology. Montgomery, 1985, pp 308-315. 12 Goudemand M, Salmon C: Les systhnes immunogknes de groupes trythrocytaires. 4. Le systkme Kidd; in Goudemand M, Salmon C (ed): Immuno-htmatologie et immunogtnitique. Paris, Flammarion-Mtdecine, 1980, pp 231-233.
Received: November 26,1990 Revised manuscript received: April 30,1991 Accepted: May 1,1991
Dr. D. Bourel CRTS Rue Pierre-Jean-Gineste F-35OOO Rennes (France)
Vox Sang 1991;61:255-257
A Human Anti-JkbMonoclonal Antibody M . Lecointre-Coatmelec, D . Bowel, J . Ferrette, B. Genetet’ Centre Rkgional de Transfusion Sanguine, Rennes, France
Abstract. Peripheral blood mononuclear cells of an immunized patient were transformed with Epstein-Barr virus and then fused with P3Xs3Ag8mouse myeloma cells by polyethylene glycol. After the cloning, a hybridoma cell line secreting specific anti-Jkb monoclonal antibody was isolated. The antibody was produced in supernatant form and tested for its use as a blood grouping reagent.
Introduction The murine hybridoma technology was unsuitable to produce specific anti-Rhesus monoclonal antibodies (MAbs). Transformations by the Epstein-Barr virus (EBV) of peripheral blood mononuclear cells (PBMCs) from immunized donors or patients were performed in an attempt to produce human blood grouping reagents. Although the efficiency of such a technique was very low, several reagents were produced as anti-D [1-4]. The major problem of this technology was the stability of the production. Several protocols were proposed to stabilize the secretion of MAbs and particularly heterohybridizations of human secreting lymphoblastoid cell lines or human B lymphocytes with mouse myelomas [ 5 , 6 ] . Most of the human monoclonal blood grouping reagents were produced by this heterohybridoma technology: anti-D [7,8], anti-c, anti-E and anti-Kell[g]. By using such procedures a human anti-Jkb MAb was produced; its production and characteristics are reported.
Material and Methods EBV Transformation and Cloning. An immunized patient was selected who had an anti-D alloantibody (titer Y64) without any other detectable antibody in his serum. A blood sample was taken 6 months after the first identification of the anti-D. 50 x lo6PBMCs were isolated I We thank Christine Gouaillier for the preparation of the manuscript.
on Ficoll Hypaque and transformed by the EBV [lo]. Supernatant of B95.8ceU line was added to a suspension of PBMCs and incubated in 96-well microculture plates containing mouse peritoneal cells as feeder layer. Culture medium was fed after 24h with RMPI containing 20% of fetal calf serum (FCS) and lpg/ml of ciclosporin A. Supernatant of growing cells was tested against papain-treated red blood cells (RBCs) of defined phenotypes. One cell line which produce an anti-Jkb antibody was cloned successively at 10, 5 and 1 cell per well with mouse peritoneal cells. Cell Fusion and Hybrid Selection. To preserve antigen-specific immunoglobulin secretion the EBV cell line was fused with a mouse myeloma P&Ags. Cell fusion was performed with PEG 4000 (Serva) at 50% in a buffered solution and hybridomas were grown in a selection medium containing hypoxanthine (13 pg/ml), azaserine (1.7 pg/ml) and ouabain (0.2pg/ml). Microagglutination tests were performed to isolate secreting heterohybridomas: 50p1 of culture supernatant was mixed with an equal volume of 2% papain-treated RBC. The plates were incubated for 35min at 3 7 T , centrifuged at 1,000rpm for lmin and stirred before reading of agglutinations. The production was performed in rolling bottles with a RPMI medium containing Hepes (25mM) and FCS (5%). The stability of the production was controlled by continuous culture over lyear. Every 3months the titer of the supernatant was controlled. Serological Tests. Specificity tests were assessed using a large panel of extensively phenotyped red cells by different tube techniques: (1) Agglutination tests using RBCs at 5% in saline medium: 2 drops of reagent and 2 drops of RBC suspension were mixed in a tube, incubated for 30min at 22°C; agglutination was observed after stirring. (2) Tests with papain-pretreated RBCs at 5%: the protocol was identical except the incubation for 35min at 37°C. (3) Tests with RBCs diluted at 3% in low ionic strengh solution (LISS) without incubation. Absorption elution tests were performed in parallel to commercial human polyclonal anti-Jkb sera. 500 pI of the reagent was incubated with 250 wl of different red cells (Jkb+and Jkb) for 4h at 37°C. After 5 washings the absorbed antibodies were eluted for 15min at 56°C and
256
Lecointre-Coatmelec/Bourel/Ferrette/Genetet
tested against papain-treated RBCs according to the described test. Reactivity was assessed using two spin tube techniques: (1)with papaintreated RBCs; (2) with RBCs diluted in LISS. Titer and score were determined by doubling dilution in RPMI containing 5% of FCS according to the international scoring of titrations. Classing of Immunoglobulin. Classing and quantitation of immunoglobulins in hybridoma supernatants were determined by an enzyme-linked immunoassay.
Table 1. Specificity of the anti-Jkb MAb according to different techniques Red cells
n
Jk(a+b+) Jk(a-h+) Jk(B+b-)
Saline 22°C
Papain-treated cells
Red cells in LISS
loo
+
44 56 5
-
++++ ++++
+++ +++
Jk‘a-b-)
Results and Discussion An EBV-transformed cell line was selected for its antiJkbreactivity: Nb 143. This cell line was rather unstable and difficult to grow in large-scale culture. For these reasons, heterohybridization was performed with P3X63A& mouse myeloma. One hybridoma cell line PJ43 was selected and cloned. It produced IgM molecules at a concentration of lOpg/ml. All the results presented were carried out with pure culture supernatant. The P3143 Mab agglutinated all the Jk(a+b+) and Jk‘”’+) RBCs without reactions with Jk‘a++b-) and Jk‘a--b-)samples. The agglutinations’were weak with RBCs in saline medium but very strong with Jkb+papain-treated cells without differences between homozygous or heterozygous samples. With RBCs in LISS the reactions were weaker than in papain tests but strong enough to make this technique more suitable for practical use (table 1). Adsorption elution tests showed that the P3 143 MAb was adsorbed on Jk(a+b+) and Jk‘”’) cells; no adsorption was observed with Jk(a+b)samples (table 2). The titer of the anti-Jkb reagent was 1:64 when tested against papain-treated cells and with a score of 70 (table 3); with RBCs in LISS the reagent showed a titer of 1:16 with a score of 49. All the results were confirmed during the Second International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (Workshop No. 168). This reagent presented a good stability: after storage during 2 months at 37°C and 12 months at +4”C its reactivity and its titer remained unchanged. The stability of the producing heterohybrids had to be controlled; after 12 months of continuous culture, the secretion quantitatively decreased as shown in table 4. Cloning experiments showed that after 6 months of continuous culture 40% of the heterohybrids were unproductive although the positive clones secreted the antibody with the same potency. This result pointed out that the decrease of production was due to a loss of secretion of some heterohybrids. This relative instability of production required that before each production the cells must be cloned to assure a better efficiency.
+
-
ND
-
ND
n = Number of samples tested; N D = not determined; +, +++, + + + + = intensity of agglutinations; - = no agglutination. Tests were performed in tubes.
Table 2. Adsorption elution tests of the anti-Jkb MAb Adsorbing cells
Reactivity of eluates against papain-treated cells ~~
Jk(a+h+) Jk‘a-b+) Jk(a+b-)
Jk(a+b)
Jk(a-b+)
-
++++ ++++
Reactivity: - = N o agglutination;
-
++++ = strong agglutination.
Table 3. Reactivity of the anti-Jkh MAb (tube tests) Papain-treated cells
Titer
Score
Jk(a+b+) Jk(a-b+) Jk(a+b-) Jk(a-b-)
70 70 0 0 ~~
International code
Strength of agglutination
Score
4+ 3+ 2+ 1+
12 10 8 5
-
0
Scores were evaluated according to the international code.
The P3143 anti-Jkb MAb was specific and could be considered as a good reagent for blood grouping. Its characteristics were in accordance with the international standards. Pretreatment with enzyme was not needed for hemagglutination if RBCs were diluted in LISS. Agglutinations were
257
A Human Anti-Jkb Monoclonal Antibody
Table4. Stability study of the production of the anti-Jkb by the heterohybridoma P3143 Reactivity
Titer Score
Months 1
3
6
9
12
64 70
64 63
32 54
16 37
4 28
Titers and scores were determined as described.
strong enough and superior to those observed with human polyclonal antisera. This MAb was especially interesting because Kidd phenotyping was of great interest in transfusion therapy [ll] and because it was difficult to obtain good polyclonal anti-Jkbreagents [12].
References Koskimies S: Human lymphoblastoid cell line producing specific antibody against Rh-antigen D. Scand J Immunol 1980:11:73-77. Bolyston AW, Gardner B, Anderson LR, Hughes-Jones NC: Production of human IgM anti-D in tissue culture by EB - virus transformed lymphocytes. Scand J Immunol 1980:12:355-358. Crawford DH, Harisson JF, Barlow MJ, Winger L, Huens ER: Production of human monoclonal antibody to rhesus D antigen. Lancet 1983;i:386388. Bron D, Feinberg MB, Teng NNH, Kaplan HS: Production of human monoclonal IgG antibodies against Rhesus (D) antigen. Proc Natl Acad Sci USA 1984;81:32143217.
Teng N, Lam K, Calvorieva F,Kaplan H: Construction and testing of mouse human heteromyelomas for human monoclonal antibody production. Proc Natl Acad Sci USA 1983;80:7308-7311. Murphy SM, Webb GC, Earl ME, Russ GR, Churcher A, Tait BD, d’Apice AJF: Human monoclonal antibody production by xenohybridomas. Transplant Proc 1986;18:343-346. Lowe AD, Green SM, Voak D, Gibson T, Lennox ES: A human monoclonal anti-D by direct fusion with a lymphoblastoid line. Vox Sang 1986;51:212-216. Foung SKH, Blunt JA, Wu PS, Ahearn P, Winn LC, Engleman G. Grumet FC: Human monoclonal antibodies to Rho (D). Vox Sang 1987;53:44-47. Goossens D, Champomier F, Rouger P, Salmon C: Human monoclonal antibodies against blood group antigens. Preparation of a series of stable EBV immortalized B clones producing high levels of antibody of different isotypes and specificities. J Immunol Methods 1987;lOl:193-200. lOJames K, Bell GT: Human monoclonal antibody production. Current status and future prospects. J Immunol Methods 1987:100:540. 11 Issitt PD: The Kidd blood group system; in Applied Blood Group Serology. Montgomery, 1985, pp 308-315. 12 Goudemand M, Salmon C: Les systhnes immunogknes de groupes trythrocytaires. 4. Le systkme Kidd; in Goudemand M, Salmon C (ed): Immuno-htmatologie et immunogtnitique. Paris, Flammarion-Mtdecine, 1980, pp 231-233.
Received: November 26,1990 Revised manuscript received: April 30,1991 Accepted: May 1,1991
Dr. D. Bourel CRTS Rue Pierre-Jean-Gineste F-35OOO Rennes (France)
