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Differences among individual CEA preparations detected by monoclonal antibodies
2A5
MONOCLONAL
ANTIBODIES
DIFFERENCES AMONG INDIVIDUAL CEA PP~PARATIONS DETECTED BY ~DNOCLONAL ANTIBODIES
I
R. Ben Isaac, Z. Eshhar, N. Ariel and R. Arnon Dept. of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. C a r c i n o e m b r y o n i c a n t i g e n (CEA) i s an i m p o r t a n t t u m o r m a r k e r , a s s o c i a t e d w i t h many n e o p l a s m s and m a i n l y w i t h m a l i g n a n c i e s o f t h e human d i g e s t i v e t r a c t . A~ a c o n s e q u e n c e i t s ~ n i t o r i n g s e r v e s as a c r u c i a l means i n t h e f o l l o w - u p o f c a n c e r o u s s t a t e . The a b i l i t y t o d i f f e r e n t i a t e b e t w e e n d i f f e r e n t t y p e s o f CEA may add a n o t h e r d i m e n s i o n t o t h i s d i a g n o s t i c t e s t . We a p p r o a c h e d t h i s i s s u e by a n a l y s i s o f t h e s p e c i f i c i t i e s of different anti-CEA monoclonal a n t i b o d i e s . S t a b l e c l o n e s h a v e b e e n e s t a b l i s h e d from t e n h y b r i d o ~ a s t h a t p r o d u c e d a n t i - C E A a n t i b o d i e s . These ~ n o c l o n a l a n t i b o d i e s (McAb) were found t o d i f f e r i n t h e i r a b i l i t y t o b i n d i n d i v i d u a l CEA p r e p a r a t i o n s : Whereas t h r e e McAb's r e a c t e d i d e n t i c a l l y w i t h f o u r d i f f e r e n t CEA's, t h e o t h e r s e v e n e x h i b i t e d d i f f e r e n t r e a c t i v i t i e s . The p a n e l o f McAb's, i n c l u d i n g t h e t h r e e i d e n t i c a l l y r e a c t i v e o n e s , d i f f e r e d i n t h e i r c a p a c i t y t o s t a i n CEA p o s i t i v e human c e l l l i n e s d e r i v e d from v a r i o u s t u ~ m r s . T h i s f i n d i n g i s a t p r e s e n t b e i n g e v a l u a t e d f o r i t s p o t e n t i a l as a d i a g n o s t i c t o o l . M o r e o v e r , t h e a n t i b o d y d i s p l a y i n g t h e highest degree of cross-reactivity w i t h a l l t e s t e d CEA p r e p a r a t i o n s h a s b e e n u s e d f o r one step affinity purification o f CEA, from e i t h e r m e t a s t a t i c l i v e r o r c u l t u r e f l u i d s o f d i f f e r e n t e s t a b l i s h e d CEA-producing c e l l l i n e s , f o r t h e p u r p o s e o f t h e i r f u r t h e r characterization. THE DETECTION OF VIRAL PARTICLES IN HYBRIDC~ CLONES SECRETING MONOCLONAL ANTIBODIES.
2
Carl Felt, Arle H. Bartal, Robert Erlandson and Yashar Hirshaut Memorial Sloan-Ketterlng Cancer Center, New York, New York 10021, U.S.A. There is a growing effort to utilize monoclonal antibodies for clinical purposes both diagnostic and therapeutic. Recent work in our laboratory has raised a question about the safety of the use of mouse monoclonal antibodies in h~-,-~s. As part of a stmdy to develop monoclonal antibodies to human connective tissue differentiation antigens appearing on h~-,-= sarcomas we have investigated the ultrastructure of hybridoma clones derived by fusing Balb/c mouse spleen cells with P3UI plasmacytQma cells. Electronmlcroscopy of hybridomas revealed that these cells contain large numbers of viral particles which are also seen budding from the cell surfaces. The intracytoplasmlc particles are intracisternal and resemble type A oncornavirus, while the budding and extracellular forms, with a centrally located nucleold, resemble matdre type C oncornaviruses. Cells of the parental PBUI myeloma line as well as NS-I also contained morphologically identical viral structures. The scientific and medical cummunlty engaged in hybridoma research should be alert to the possible presence of viruses in hybridcmas and their products. MONOCLONAL ANTIBODIES TO CONNECTIVE TISSUE D I F F E R ~ I A T I O N ANTIGENS ASSOCIATED WITH SARCOMA. Arle H. Bartal, Carl Felt, and Yashar Hirshaut Memorial Sloan-Kettering Cancer Center, New York, New York 10021, U.S.A. As part of an effort to comprehensively define antigens characteristic of human sarcc~as, monoclonal antibodies (mAb) to a human malignant fibrohlstiocytoma were prepared. Balb/c spleen cells from a mouse immunized with the sarcoma were fused with PBUI plasmacytoma cells. Supernates were then tested against a panel of 20 h,~,n sarcoma cell lines, 7 normal human tissue lines of mesodermal origin and 8 carcinomas. Screening for surface and internal antigens was accomplished using an indirect Immunofluorescence assay (IF) run against acetone-flxed cells. ELISA was used to detect surface antigens on viable cells. Two mAb showed specificity for mesodermal markers. Vi F3 reacted with 18/20 sarcoma cell lines, 4/5 fibroblast lines, and none of 8 carcinoma lines. By IF, this mAb produced diffuse cytoplasmic fluorescence. It was unreactive by ELISA. A second mAb, VI E4 was positive on 17/20 sarcoma cell lines, 4/6 flbroblast lines and platelets but did not react with lymphocytes and 7 carcinoma lines. VI E4 was detectable both by IF and ELISA. Thus 2 mAh, one recognizing an internal and one a cell surface antigen, distinctive for human mesodermal differentiation and expressed by many h ~ , n sarcomas have been produced.
3
MONOCLONAL
ANTIBODIES
DIFFERENCES AMONG INDIVIDUAL CEA PP~PARATIONS DETECTED BY ~DNOCLONAL ANTIBODIES
I
R. Ben Isaac, Z. Eshhar, N. Ariel and R. Arnon Dept. of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. C a r c i n o e m b r y o n i c a n t i g e n (CEA) i s an i m p o r t a n t t u m o r m a r k e r , a s s o c i a t e d w i t h many n e o p l a s m s and m a i n l y w i t h m a l i g n a n c i e s o f t h e human d i g e s t i v e t r a c t . A~ a c o n s e q u e n c e i t s ~ n i t o r i n g s e r v e s as a c r u c i a l means i n t h e f o l l o w - u p o f c a n c e r o u s s t a t e . The a b i l i t y t o d i f f e r e n t i a t e b e t w e e n d i f f e r e n t t y p e s o f CEA may add a n o t h e r d i m e n s i o n t o t h i s d i a g n o s t i c t e s t . We a p p r o a c h e d t h i s i s s u e by a n a l y s i s o f t h e s p e c i f i c i t i e s of different anti-CEA monoclonal a n t i b o d i e s . S t a b l e c l o n e s h a v e b e e n e s t a b l i s h e d from t e n h y b r i d o ~ a s t h a t p r o d u c e d a n t i - C E A a n t i b o d i e s . These ~ n o c l o n a l a n t i b o d i e s (McAb) were found t o d i f f e r i n t h e i r a b i l i t y t o b i n d i n d i v i d u a l CEA p r e p a r a t i o n s : Whereas t h r e e McAb's r e a c t e d i d e n t i c a l l y w i t h f o u r d i f f e r e n t CEA's, t h e o t h e r s e v e n e x h i b i t e d d i f f e r e n t r e a c t i v i t i e s . The p a n e l o f McAb's, i n c l u d i n g t h e t h r e e i d e n t i c a l l y r e a c t i v e o n e s , d i f f e r e d i n t h e i r c a p a c i t y t o s t a i n CEA p o s i t i v e human c e l l l i n e s d e r i v e d from v a r i o u s t u ~ m r s . T h i s f i n d i n g i s a t p r e s e n t b e i n g e v a l u a t e d f o r i t s p o t e n t i a l as a d i a g n o s t i c t o o l . M o r e o v e r , t h e a n t i b o d y d i s p l a y i n g t h e highest degree of cross-reactivity w i t h a l l t e s t e d CEA p r e p a r a t i o n s h a s b e e n u s e d f o r one step affinity purification o f CEA, from e i t h e r m e t a s t a t i c l i v e r o r c u l t u r e f l u i d s o f d i f f e r e n t e s t a b l i s h e d CEA-producing c e l l l i n e s , f o r t h e p u r p o s e o f t h e i r f u r t h e r characterization. THE DETECTION OF VIRAL PARTICLES IN HYBRIDC~ CLONES SECRETING MONOCLONAL ANTIBODIES.
2
Carl Felt, Arle H. Bartal, Robert Erlandson and Yashar Hirshaut Memorial Sloan-Ketterlng Cancer Center, New York, New York 10021, U.S.A. There is a growing effort to utilize monoclonal antibodies for clinical purposes both diagnostic and therapeutic. Recent work in our laboratory has raised a question about the safety of the use of mouse monoclonal antibodies in h~-,-~s. As part of a stmdy to develop monoclonal antibodies to human connective tissue differentiation antigens appearing on h~-,-= sarcomas we have investigated the ultrastructure of hybridoma clones derived by fusing Balb/c mouse spleen cells with P3UI plasmacytQma cells. Electronmlcroscopy of hybridomas revealed that these cells contain large numbers of viral particles which are also seen budding from the cell surfaces. The intracytoplasmlc particles are intracisternal and resemble type A oncornavirus, while the budding and extracellular forms, with a centrally located nucleold, resemble matdre type C oncornaviruses. Cells of the parental PBUI myeloma line as well as NS-I also contained morphologically identical viral structures. The scientific and medical cummunlty engaged in hybridoma research should be alert to the possible presence of viruses in hybridcmas and their products. MONOCLONAL ANTIBODIES TO CONNECTIVE TISSUE D I F F E R ~ I A T I O N ANTIGENS ASSOCIATED WITH SARCOMA. Arle H. Bartal, Carl Felt, and Yashar Hirshaut Memorial Sloan-Kettering Cancer Center, New York, New York 10021, U.S.A. As part of an effort to comprehensively define antigens characteristic of human sarcc~as, monoclonal antibodies (mAb) to a human malignant fibrohlstiocytoma were prepared. Balb/c spleen cells from a mouse immunized with the sarcoma were fused with PBUI plasmacytoma cells. Supernates were then tested against a panel of 20 h,~,n sarcoma cell lines, 7 normal human tissue lines of mesodermal origin and 8 carcinomas. Screening for surface and internal antigens was accomplished using an indirect Immunofluorescence assay (IF) run against acetone-flxed cells. ELISA was used to detect surface antigens on viable cells. Two mAb showed specificity for mesodermal markers. Vi F3 reacted with 18/20 sarcoma cell lines, 4/5 fibroblast lines, and none of 8 carcinoma lines. By IF, this mAb produced diffuse cytoplasmic fluorescence. It was unreactive by ELISA. A second mAb, VI E4 was positive on 17/20 sarcoma cell lines, 4/6 flbroblast lines and platelets but did not react with lymphocytes and 7 carcinoma lines. VI E4 was detectable both by IF and ELISA. Thus 2 mAh, one recognizing an internal and one a cell surface antigen, distinctive for human mesodermal differentiation and expressed by many h ~ , n sarcomas have been produced.
3