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Monoclonal antibodies and cancer: photoimmunotherapy and radioimmunoimagery Mew, Daphne Jung Yen 1984

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M o n o c l o n a l A n t i b o d i e s and C a n c e r : Photo immunotherapy and Radio immunoimagery by Daphne Jung Yen Mew B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1980 A t h e s i s s u b m i t t e d i n p a r t i a l f u l f i l l m e n t o f the r e q u i r e m e n t s f o r the degree o f D o c t o r o f P h i l o s o p h y i n The F a c u l t y o f G radua te S t u d i e s (Department o f M i c r o b i o l o g y , U n i v e r s i t y o f B r i t i s h Co lumbi We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h C o l u m b i a May 1984 © Daphne Jung Yen Mew, 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Uu^RpfeiOLQ(yS{ The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 i i ABSTRACT The advantage o f u s i n g m o n o c l o n a l a n t i b o d i e s i n the t r e a t m e n t o f m a l i g n a n c i e s l i e s i n i t s p o t e n t i a l t o make c a n c e r t h e r a p y o r d e t e c t i o n more tumour s p e c i f i c . Tumour s p e c i f i c a n t i b o d i e s l i n k e d t o c h e m o t h e r a p e u t i c d rugs o r r a d i o n u c l i d e s wou ld a i d i n the t r a n s p o r t a t i o n o f the d r u g s ' c y t o t o x i c a c t i v i t y t o tumour t a r g e t s o r make i t p o s s i b l e t o a c c u r a t e l y map the s p r e a d o f p r i m a r y tumour and m e t a s t a s i s r a d i o a c t i v e l y . The te rm "pho to immuno the rapy " d e s c r i b e s an a n t i - c a n c e r t r e a t m e n t t h a t combines the p h o t o t o x i c e f f e c t s o f c h e m i c a l s such as h a e m a t o p o r p h y r i n and the t a r g e t s e e k i n g a b i l i t y o f a n t i b o d i e s . By homing i n on t h e i r d e s i g n a t e d t a r g e t s , m o n o c l o n a l a n t i b o d i e s c o n j u g a t e d t o h a e m a t o p o r p h y r i n a re a b l e t o l o c a l i z e the photodynamic a c t i v i t y t o the tumour s i t e , m i n i m i z i n g n o n s p e c i f i c d i s p e r s a l t h r o u g h the body and t h e r e f o r e d e c r e a s i n g the p o t e n t i a l f o r norma l t i s s u e t o x i c i t y . Haematoporphy r in c h e m i c a l l y c o u p l e d to m o n o c l o n a l a n t i b o d i e s d i r e c t e d t o DBA/2J rhabdomyosarcoma M-l was a s sayed f o r tumour s p e c i f i c p h o t o t o x i c i t y i j i v i t r o and i n v i v o . When c o n j u g a t e d to m o n o c l o n a l a n t i b o d i e s (CAHAL-1) d i r e c t e d a g a i n s t a human myelogenous l e u k a e m i a - a s s o c i a t e d a n t i g e n (CAMAL), the photoimmuno-t h e r a p e u t i c a c t i v i t y o f haematoporphyr in-CAMAL-1 a n t i b o d i e s was examined as a p o t e n t i a l method f o r s p e c i f i c a l l y p u r g i n g l eukaemic c e l l s f r om a norma l h a e m a t o p o e i t i c c e l l p o p u l a t i o n it i v i t r o . A d m i n i s t r a t i o n o f a n t i - M - l - H a e m a t o p o r p h y r i n c o n j u g a t e s i n t r a v e n o u s l y t o M-l t u m o u r - b e a r i n g an ima l s f o l l o w e d by exposure t o l i g h t r e s u l t e d i n s u p p r e s s i o n o f M-l g rowth . The t ime i n t e r v a l between i n j e c t i o n and l i g h t i i i exposu re was an i m p o r t a n t pa ramete r i n terms o f tumour s u p p r e s s i o n . Tumour-bea r ing an ima l s m a i n t a i n e d i n the d a r k f o r 96 t o 196 h r s a f t e r h a e m a t o p o r p h y r i n - a n t i b o d y i n j e c t i o n f o l l o w e d by 4 h r l i g h t exposu re demons t r a t ed s i g n i f i c a n t l y lower tumour i n c i d e n c e and l o n g e r l a t e n c y p e r i o d s , i n compa r i son t o c o n j u g a t e t r e a t e d an ima l s i n s t a n t l y exposed to l i g h t . The growth i n h i b i t i n g p r o p e r t i e s o f the c o n j u g a t e appea red t o be M-l s p e c i f i c s i n c e i t had no e f f e c t on the growth o f a C5BL/6J lymphoma EL4 . In a d d i t i o n , c o n j u g a t e s made w i t h a n o n - s p e c i f i c m o n o c l o n a l a n t i b o d y d i d not have any s p e c i f i c a n t i - t u m o u r e f f e c t on M-l g rowth . T rea tmen t w i t h e q u i v a l e n t doses o f h a e m a t o p o r p h y r i n o r a n t i b o d y had no s i g n i f i c a n t i n h i b i t i n g e f f e c t on tumour g rowth . C l e a r l y , the homing a b i l i t y o f the s p e c i f i c m o n o c l o n a l a n t i b o d y - h a e m a t o p o r p h y r i n c o n j u g a t e was e s s e n t i a l f o r e f f e c t i v e d rug d e l i v e r y and i n h i b i t i o n o f tumour g rowth . In v i t r o e x p e r i m e n t s w i t h haematoporphyr in-CAMAL-1 d e m o n s t r a t e d t h a t the c o n j u g a t e s p e c i f i c a l l y e l i m i n a t e d CAMAL b e a r i n g c e l l s i n mye logenous l eukaem i c c e l l p o p u l a t i o n s . The c y t o t o x i c i t y o f Hp-CAMAL-1 was shown t o be t a r g e t s p e c i f i c s i n c e i r r e l e v a n t c o n j u g a t e s (Hp-anti-M-1 o r Hp-an t i -L1210 ) had m i n i m a l e f f e c t on the l eukaemic c e l l s a m p l e s . In a d d i t i o n , Hp-CAMAL-1 had no a c t i v i t y on norma l bone marrow c e l l s , norma l p e r i p h e r a l b l o o d l y m p h o c y t e s , CAMAL n e g a t i v e ALL bone marrow o r lymphoma c e l l s . These r e s u l t s i n d i c a t e d t h a t Hp-CAMAL-1 photo immunotherapy may be a p o t e n t i a l method f o r s p e c i f i c a l l y e l i m i n a t i n g tumour c e l l s f rom bone marrow s u s p e n s i o n s p r i o r t o a u t o l o g o u s t r a n s p l a n t a t i o n . In r ad io immuno image ry , l a b e l l e d a n t i b o d i e s a re used t o d e t e c t c a n c e r i n v i v o . By c o m b i n i n g the s p e c i f i c i t y o f an t i - tumour a n t i b o d i e s and the i v s e n s i t i v i t y o f r a d i o i s o t o p e t r a c e r t e c h n o l o g y , a h i g h e r l e v e l o f d e t e c t i o n was c r e a t e d f o r c a n c e r d i a g n o s i s . The p o t e n t i a l o f r ad io immuno imagery was examined i n v i v o mur ine tumour s t u d i e s w i t h " t e c h n e t i u m l a b e l l e d 99m ant i-M-1 m o n o c l o n a l a n t i b o d i e s Tc-ant i-M-1 a n t i b o d i e s were a b l e t o 2 d e t e c t and l o c a l i z e M-l tumours as s m a l l as 0 .3 g ( < 1 cm ) 6 - 18 h r s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n . R a d i o n u c l i d e l o c a l i z a t i o n was M-l tumour s p e c i f i c s i n c e EL4 lymphomas were no t d e t e c t e d w i t h " m T c - a n t i - M - l . S e n s i t i v i t y o f the rad io immuno imag ing approach was d i m i n i s h e d by the h i g h backgrounds o b s e r v e d i n norma l t i s s u e s such as l i v e r , k i d n e y s , and s p l e e n . B i o c h e m i c a l e x a m i n a t i o n o f the l a b e l l e d a n t i b o d y p r e p a r a t i o n s i n d i c a t e d t h a t the background p rob l em c o u l d be due t o the h i g h l y a g g r e g a t e d n a t u r e o f the i n j e c t e d immunot race r . Improvement o f the r ad io immuno imag ing scans migh t be a c h i e v e d i f the l a b e l l e d a n t i b o d i e s c o u l d be a d m i n i s t e r e d i n a nonagg rega ted f o r m . V TABLE OF CONTENTS Page A b s t r a c t i i L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i Acknowledgements x i i A b b r e v i a t i o n s x i i i C h a p t e r I - I n t r o d u c t i o n 1 C h a p t e r I I - Photo immunotherapy : A n i m a l S t u d i e s 15 A . I n t r o d u c t i o n 16 B. M a t e r i a l and Methods 19 C . R e s u l t s 28 D. D i s c u s s i o n 63 Chap t e r I I I - Photo immunotherapy : Human S t u d i e s 67 A . I n t r o d u c t i o n 68 B. M a t e r i a l and Methods 72 C . R e s u l t s 77 D. D i s c u s s i o n 1 ° 4 Chap t e r IV - Radioimmuno imagery . . 109 A . I n t r o d u c t i o n 1 1 ° B. M a t e r i a l and Methods 117 C . R e s u l t s 122 D. D i s c u s s i o n 174 C h a p t e r V - Summary D i s c u s s i o n 179 R e f e r e n c e s I 9 0 v i L IST OF TABLES T a b l e T i t l e Page I E f f e c t o f a n t i - M - l - h a e m a t o p o r p h y r i n c o n j u g a t e on norma l DBA/2J s p l e n o c y t e s and C57BL/6J EL4 lymphoma c e l l s i l l v i t r o 45 I I E f f e c t o f a n t i - M - l - h a e m a t o p o r p h y r i n c o n j u g a t e on M-l tumour growth i n v i v o 55 I I I E f f e c t o f a n t i - M - l - h a e m a t o p o r p h y r i n c o n j u g a t e on the growth o f EL4 tumour i n B6D2 mice 57 IV The i n f l u e n c e o f t i m i n g o f da rk i n c u b a t i o n f o l l o w e d by l i g h t exposu re o f M-l b e a r i n g DBA/2J mice t r e a t e d w i t h haema topo rphy r i n-an t i -M-1 c o n j u g a t e on the t ime o f p a l p a b l e tumour i n d u c t i o n and s u r v i v a l 61 V The i n f l u e n c e o f t i m i n g o f d a r k i n c u b a t i o n f o l l o w e d by l i g h t exposu re o f M-l b e a r i n g DBA/2J mice t r e a t e d w i t h h a e m a t o p o r p h y r i n , ant i-M-1 o r PBS on p a l p a b l e tumour i n d u c t i o n 62 V I E f f e c t o f Hp-an t ibody c o n j u g a t e s on PBL samples f rom CGL p a t i e n t I I 97 v i i L IST OF FIGURES F i g u r e T i t l e Page 1 A c t i v i t y o f s p e c i f i c m o n o c l o n a l ant i-M-1 and n o n s p e c i f i c m o n o c l o n a l an t i -G I P on v a r i o u s a n t i g e n p r e p a r a t i o n s 29 2 C h e m i c a l S t r u c t u r e o f Haematoporphy r in 31 3 H a e m o l y t i c a c t i v i t y o f f r e e h a e m a t o p o r p h y r i n , h a e m a t o p o r p h y r i n d e r i v a t i v e and h a e m a t o p o r p h y r i n -ant i-M-1 c o n j u g a t e s 34 4 T i t r a t i o n o f s p e c i f i c m o n o c l o n a l ant i-M-1 and the Hp-ant i-M-1 c o n j u g a t e on M-l a n t i g e n 36 5 A c t i v i t y o f the Hp-ant i-M-1 c o n j u g a t e on v a r i o u s a n t i g e n p r e p a r a t i o n s 38 6 S t a b i l i t y o f h a e m a t o p o r p h y r i n and a n t i b o d y a c t i v i t y a f t e r l i g h t i r r a d i a t i o n 40 7 In v i t r o e f f e c t o f Hp-ant i-M-1 and c o n t r o l s on M-l tumour c e l l s 42 8 In v i t r o e f f e c t s o f Hp-ant i-M-1 and Hp-an t i -L1210 on M-l and L1210 tumour c e l l s ( ^ C r - r e l e a s e assay ) . . . 46 9 T i t r a t i o n o f h a e m a t o p o r p h y r i n , Hp-ant i-M-1 and Hp-an t i -L1210 a c t i v i t y on M-l tumour c e l l s ( v i t a l dye e x c l u s i o n assay ) 49 v i i i L I ST OF FIGURES, C o n t i n u e d F i g u r e T i t l e Page 10 T i t r a t i o n o f h a e m a t o p o r p h y r i n , Hp-ant i-M-1 and Hp-an t i - L1210 a c t i v i t y on M-l tumour c e l l s 3 ( H- thymid ine i n c o r p o r a t i o n a s say ) 51 11 F low d i ag ram i l l u s t r a t i n g the e f f e c t o f Hp-ant i-M-1 i n v i v o : P r o t o c o l I 53 12 F low d i a g r a m i l l u s t r a t i n g the ijn v i v o e f f e c t o f Hp-ant i-M-1 i n a t i m i n g e x p e r i m e n t : P r o t o c o l I I 59 13 T i t r a t i o n o f s p e c i f i c m o n o c l o n a l a n t i b o d y CAMAL-1 and Hp-CAMAL-1 c o n j u g a t e on CAMAL a n t i g e n 78 14 T i t r a t i o n o f Hp a c t i v i t y on norma l and CGL PBLS 80 15 Hp-an t i body c o n j u g a t e and c o n t r o l a c t i v i t y on norma l p e r i p h e r a l b l o o d l ymphocy tes 83 16 Hp-an t i body c o n j u g a t e and c o n t r o l a c t i v i t y on CGL PBLS 85 17 H p - a n t i b o d y c o n j u g a t e and c o n t r o l a c t i v i t y on ANLL PBLS 87 18 The e f f e c t o f Hp-an t i body c o n j u g a t e s on norma l and ALL PBLS, norma l and lymphoma BM c e l l s 90 19 The e f f e c t o f Hp-an t i body c o n j u g a t e s on CGL and ANLL samples f r om f o u r d i f f e r e n t p a t i e n t s 92 20 The e f f e c t o f Hp-an t i body c o n j u g a t e s on PBL samples f rom CGL p a t i e n t I 95 i x L IST OF FIGURES, C o n t i n u e d F i g u r e T i t l e Page 21 T i t r a t i o n o f Hp-RAMIg a c t i v i t y on norma l and CGL PBLS 100 22 The e f f e c t o f Hp-Ramlg on a n t i b o d y l a b e l l e d c e l l s 102 23 T i t r a t i o n o f s p e c i f i c m o n o c l o n a l ant i-M-1 and 99m Tc-ant i-M-1 on M-l a n t i g e n 123 99m 24 A c t i v i t y o f Tc-ant i-M-1 on v a r i o u s a n t i g e n p r e p a r a t i o n s 125 25 T i s s u e d i s t r i b u t i o n s t u d y : 4-44 h r s a f t e r ^ ^ m T c - a n t i - M - l i n j e c t i o n 128 99m_ 26 R e l a t i v e % r a d i o a c t i v e dose TC pe r gram t i s s u e l o c a t e d i n m u s c l e , b l o o d and M-l tumour 4-44 h r s a f t e r i n t r a v e n o u s i n j e c t i o n o f " m T c - a n t i - M - l 130 27 Scans o f M-l b e a r i n g mice 18 h r a f t e r i n t r a v e n o u s 99m a d m i n i s t r a t i o n o f Tc l a b e l l e d ant i-M-1 132 28 Scans o f EL4 b e a r i n g B6D2 mice i n j e c t e d 99m 18 h r e a r l i e r w i t h Tc-ant i-M-1 135 29 P e r c e n t r a d i o a c t i v e dose p e r gram o f t i s s u e d i s t r i b u t i o n s tudy i n B6D2 mice b e a r i n g bo th M-l and EL4 tumours 138 30 Scans o f an E L 4 , M-l b e a r i n g B6D2 mouse i n j e c t e d 99m 18 h r e a r l i e r w i t h Tc-ant i-M-1 140 X L IST OF FIGURES, C o n t i n u e d F i g u r e T i t l e Page 31 F low d i a g r a m i l l u s t r a t i n g p r e t r e a t m e n t o f DBA/2J m ice w i t h u n l a b e l l e d a n t i b o d i e s o r PBS p r i o r t o 99m Tc-ant i-M-1 i n j e c t i o n 143 32 P e r c e n t r a d i o a c t i v e dose p e r gram o f t i s s u e d i s t r i b u t i o n s t udy i n B6D2 v i a f o l l o w i n g p r e t r e a t m e n t w i t h a n t i - G I P , PBS o r ant i-M-1 145 33 R a t i o s o f M-1/EL4 f rom p r e t r e a t e d B6D2 mice 147 34 Scans o f PBS o r ant i-M-1 p r e t r e a t e d B6D2 mice 150 35 Scans o f an t i -G I P p r e t r e a t e d B6D2 mouse 152 36 A c t i v i t y o f the s p e c i f i c m o n o c l o n a l ant i-M-1 and 99m Tc-an t i -M-1 F ( a b ' ) 2 f r a g m e n t s , on M-l a n t i g e n 155 99m_ 37 Compar i son o f T c - a n t i - M - 1 F ( a b ' ) 2 99m f ragments and whole Tc-ant i-M-1 i n a t i s s u e d i s t r i b u t i o n s t udy 157 99m 99m 38 Compar i son o f Tc-ant i-M-1 and Tc-MAMF ( I r r e l e v a n t ) F ( a b * ) 2 i n a t i s s u e d i s t r i b u t i o n s t udy 159 99m 39 Amicon f i l t e r s e p a r a t i o n o f Tc-ant i-M-1 r e s u l t s on PAGE 162 99m 40 E f f e c t o f c e n t r i f u g a t i o n on Tc-ant i-M-1 i n t i s s u e d i s t r i b u t i o n i n M-l tumour , l i v e r and b l o o d . . . . 165 xi LIST OF FIGURES, Continued Figure T i t l e Page 41 Tissue/blood r a t i o s from the l a b e l l e d antibody ce n t r i f u g a t i o n study 168 99m_ 42 Deaggregation of xc-anti-M-1 as shown on PAGE autoradiography 171 x i i ACKNOWLEDGEMENTS I wou ld l i k e t o e x p r e s s my g r a t i t u d e t o D r . J u l i a Levy f o r he r gu idance and s u p p o r t t h r o u g h o u t t h i s t h e s i s . The o r i g i n a l impetus b e h i n d the two p r o j e c t s , pho to immunotherapy , and rad io immuno imagery was g e n e r a t e d as a r e s u l t o f d i s c u s s i o n s w i t h D r . G . H . N e i l Towers , D r . L i l y C h i - K i t Wat and D r . J . G . Levy on the h a e m a t o p o r p h y r i n s t u d i e s , and d i s c u s s i o n s w i t h 99m D r . Don L y s t e r , L a u r a A l c o r n and D r . J . G . Levy on the Tc s t u d y . I wou ld l i k e t o thank each one o f my a s s o c i a t e s i n t hese d i s c u s s i o n s and acknowledge t h e i r i m p o r t a n t c o n t r i b u t i o n s towards the outcome o f t h i s t h e s i s i n bo th t e c h n i c a l a s s i s t a n c e and a d v i c e . I wou ld a l s o l i k e t o thank Agnes Chan f o r he r h e l p i n computer g r a p h i c s and Susan Heming f o r the t y p i n g o f t h i s t h e s i s . ABBREVIATIONS ADCC - a n t i b o d y dependent c e l l u l a r c y t o t o x i c i t y AFP - a l p h a f e t o p r o t e i n ALL - a cu t e l y m p h o c y t i c l e u k a e m i a ANLL - a cu t e n o n - l y m p h o c y t i c l e u k a e m i a a- o r a n t i - G I P - m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t g a s t r i c i n h i b i t o r y p e p t i d e a- o r anti-L1210 - m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t the mur ine l e u k a e m i a L1210 a- o r anti-M-1 - m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t the mur ine rhabdomyosarcoma M-l B.M. - bone marrow CAMAL - common a n t i g e n i n mye logenous a cu te l e u k a e m i a CAMAL-1 - m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t CAMAL a n t i g e n . CEA - c a r c i n o e m b r y o n i c a n t i g e n CGL - c h r o n i c g r a n u l o c y t i c l e u k a e m i a DME - D u l b e c c o ' s M o d i f i e d E a g l e med ia EDCI - l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) - c a r b o d i i m i d e HCI EL ISA - e n z y m e - l i n k e d immunosorbent a s say EPO - e r y t h r o p o i e t i n FCS - f o e t a l c a l f serum Hp - h a e m a t o p o r p h y r i n HpD - h a e m a t o p o r p h y r i n d e r i v a t i v e HSA - human serum a lbumin 1 XIV 131 1 2 5 T 131 . . 1 2 5 T . . I, I - I o d i n e , I o d i n e Ig - immunog lobu l i n 111 111 . . In - Ind ium LCM - l ymphocy te c o n d i t i o n e d med ia Mab - m o n o c l o n a l a n t i b o d y PBL - p e r i p h e r a l b l o o d l ymphocy tes PBS - p h o s p h a t e - b u f f e r e d s a l i n e PAGE - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s R a CAMAL - r a b b i t ant i-CAMAL a n t i g e n a n t i s e r a R a Hu - r a b b i t a n t i - n o r m a l human p e r i p h e r a l b l o o d l ymphocy te membrane a n t i g e n a n t i s e r a R a i d - i r r e l e v a n t r a b b i t a n t i s e r a R a MIg o r RAMIg - r a b b i t an t i-mouse immunog lobu l i n a n t i s e r a RPMI - 1640 - R o s w e l l P a rk Memor i a l I n s t i t u t e 1640 med ia SDS - sod ium d o d e c y l s u l f a t e 99m m 99m„ . Tc - T e c h n e t i u m 1 CHAPTER I INTRODUCTION 2 Chapter I The idea of using antibodies f o r tre a t i n g neoplastic disease has interested and f r u s t r a t e d researchers f o r many years. S p e c i f i c antibodies have been shown to have a therapeutic e f f e c t in experimental animal systems, p a r t i c u l a r l y i n leukaemia and lymphoma models (1,2). However, serotherapy of human cancers with tumour immune antisera has not been e n t i r e l y successful. For the most part, the anti-tumour antibodies have had at best only a transient action with no l a s t i n g e f f e c t on the c l i n i c a l course of the disease (3,4). The r a t i o n a l e behind using antibodies f o r cancer therapy i s complex. B a s i c a l l y , one hopes to manipulate the host immune system into causing regression of the malignant tumour. There are several modes of action f o r the anti-tumour e f f e c t s of serotherapy. Antibodies di r e c t e d to membrane bound antigens could lead to tumour c e l l l y s i s by complement mediation and/or antibody-dependent c e l l u l a r c y t o t o x i c i t y (ADCC). Binding of antibodies to the surface of tumour c e l l s may enhance c e l l s u s c e p t i b i l i t y for engulfment and destruction by r e t i c u l o e n d o t h e l i a l c e l l s ( i . e . opsonization). An increase in tumour c e l l immunogenicity could occur with binding of heterologous antisera to the c e l l surface. A l t e r n a t e l y , the injected a n t isera may act to modulate the immune response by binding up f r e e l y c i r c u l a t i n g antigen, and in so doing, allow the host c e l l u l a r mechanisms to target in on the tumour c e l l s instead of being blocked by c i r c u l a t i n g antigens. 3 There a re many t h e o r e t i c a l pa ramete r s t h a t must be c o n s i d e r e d when examin ing the u s e f u l n e s s o f tumour t h e r a p y w i t h immune s e r a . The re a re s e v e r a l f a c t o r s t h a t e f f e c t the i n t e r a c t i o n between tumour and a n t i b o d y . The s u c c e s s o f c a n c e r s e r o t h e r a p y may depend on v a r i a b l e s such as i ) a n t i g e n e x p r e s s i o n and d e n s i t y on the tumour c e l l s u r f a c e , i i ) h e t e r o g e n e i t y o f a n t i g e n e x p r e s s i o n i n the whole tumour , i i i ) s p e c i f i c i t y o f the a n t i s e r a f o r tumour a n t i g e n i . e . m i n i m a l c r o s s - r e a c t i v i t y t o norma l t i s s u e a n t i g e n s , i v ) a c c e s s i b i l i t y o f the tumour to the i n j e c t e d a n t i b o d y , v) the s u s c e p t i b i l i t y o f the tumour c e l l s t o a n t i b o d y m e d i a t e d l y s i s ( i . e . i n c o l l a b o r a t i o n w i t h complement o r ADCC) , v i ) the s i z e o f the tumour b u r d e n , and v i i ) a n t i g e n i c m o d u l a t i o n , i . e . l o s s o f a n t i g e n e x p r e s s i o n by s h e d d i n g o r e n d o c y t o s i s a f t e r a n t i b o d y b i n d i n g . B u t , w h i l e t h e r e i s a s i g n i f i c a n t body o f r e s e a r c h i n d i c a t i n g c e l l u l a r immune mechan isms , e . g . c y t o t o x i c T c e l l s , a re c a p a b l e o f d e s t r o y i n g tumour c e l l s i n the h o s t ( 5 ) , e v i d e n c e c l a r i f y i n g the c y t o t o x i c r o l e o f a n t i - t u m o u r a n t i b o d i e s in. v i v o i s not as c o n v i n c i n g . Tumour s e r o t h e r a p y u s i n g c o n v e n t i o n a l complex a n t i s e r a was i n i t i a t e d as e a r l y as the 1 9 4 0 ' s . T h i s a r e a o f r e s e a r c h was e x t e n s i v e l y r e v i e w e d by Rosenberg and T e r r y i n 1977 ( 6 ) . T h e i r g e n e r a l concensus was t h a t the r e s u l t s were d i s a p p o i n t i n g . The most s u c c e s s f u l a n t i - t u m o u r e f f e c t s were l i m i t e d to an ima l s t u d i e s i n v o l v i n g a n t i s e r a a d m i n s t r a t i o n p r i o r t o , o r s h o r t l y a f t e r tumour i n o c u l a t i o n . Fo r examp le , G o r e r and Amos f o u n d t h a t s p e c i f i c p r o t e c t i v e e f f e c t s were demons t r a t ed when C57BL mice were i n j e c t e d i n t r a v e n o u s l y w i t h a n t i - E L 4 serum p r i o r to EL4 lymphoma i n t r a p e r i t o n e a l ( i . p . ) i n o c u l a t i o n ( 1 ) . But t h e r e was no such p r o t e c t i v e 4 e f f e c t i f the a n t i s e r u m was a d m i n i s t e r e d 2 days a f t e r tumour i n j e c t i o n . In ano the r s t u d y , L-51784 l e u k a e m i a i n DBA/2 m i c e , i t was f o u n d t h a t an t i - tumour p r o t e c t i v e e f f e c t s c o u l d be a c h i e v e d when s p e c i f i c a n t i s e r u m was i n j e c t e d as l a t e as 2 days a f t e r tumour i n o c u l a t i o n i . p . ( 7 ) . However, the b e n e f i c i a l e f f e c t s seen i n the mouse l e u k a e m i a and lymphoma tumour models were no t d u p l i c a t e d i n subcu taneous s o l i d mur ine tumour m o d e l s . I t was f ound t h a t w h i l e i n t r a v e n o u s l y i n j e c t e d a n t i s e r u m was c a p a b l e o f c u r i n g mice b e a r i n g tumour i n a s c i t e s f o r m , i t d i d no t e f f e c t subcu taneous tumours ( 8 ) . T h i s c o u l d be due to l e s s e r a c c e s s i b i l i t y o f i n t r a v e n o u s a n t i b o d y t o a subcu taneous tumour s i t e . I t was shown t h a t growth o f d i f f e r e n t subcu taneous m e t h y l c h o l a n t h r e n e i n d u c e d tumours i n m ice c o u l d be i n h i b i t e d by i n t r a p e r i t o n e a l i n j e c t i o n o f s p e c i f i c a n t i s e r u m ( 9 ) . But i n g e n e r a l , i t was f ound t h a t a n t i - t u m o u r immune s e r a had m i n i m a l b e n e f i c i a l a c t i v i t y a g a i n s t s o l i d tumour masses i n a n i m a l s t u d i e s . Human t r i a l s w i t h p o l y c l o n a l a n t i s e r u m p r o v e d e q u a l l y u n s u c c e s s f u l . A t t empts were made w i t h e i t h e r a l l o g e n e i c ( i . e . f r om immunized o r " c u r e d " r e m i s s i o n p a t i e n t s ) o r h e t e r o l o g o u s ( i . e . f rom tumour immunized a n i m a l s ) immune a n t i s e r a ( 1 0 , 1 1 ) . One o f the most d r a m a t i c s e r o t h e r a p e u t i c c u r e s was r e p o r t e d by Summer and T o r a k e r i n 1960 ( 1 2 ) . The " immune" serum s o u r c e was a p a t i e n t t h a t had undergone a spontaneous r e g r e s s i o n o f m a l i g n a n t melanoma 4 y e a r s p r e v i o u s l y . Summer and F o r a k e r i n f u s e d 250 ml o f the c u r e d p a t i e n t ' s b l o o d i n t o a second p a t i e n t w i t h m e t a s t a t i c melanoma. A l l melanoma l e s i o n s d i s a p p e a r e d 6 - 2 4 weeks a f t e r t r e a tmen t and the p a t i e n t r ema ined f r e e o f d i s e a s e f o r a t l e a s t a 6 y ea r m o n i t o r i n g p e r i o d . However when they t r i e d to r e p e a t the r e s u l t s w i t h 5 ano the r m a l i g n a n t melanoma p a t i e n t , no an t i - tumour e f f e c t s were s e e n . I t s h o u l d be n o t e d t h a t s i n c e whole b l o o d was u s e d , bo th c i r c u l a t i n g l ymphocy tes and serum were t r a n s f u s e d . So bo th b l o o d components m igh t have had a f u n c t i o n i n the t h e r a p e u t i c e f f e c t s . In 1968 , L a s z l o e t a l . r e p o r t e d the use o f human an t i -HLA a n t i b o d i e s i n the t r e a tmen t o f c h r o n i c l y m p h o c y t i c l e u k a e m i a ( 4 ) . The a n t i b o d y i n f u s i o n c aused an immediate d e c r e a s e i n the l e u k a e m i c c e l l coun t l a s t i n g f rom s e v e r a l days t o weeks . But the a n t i - H L A serum e f f e c t s p r o v e d t o be t r a n s i e n t . The l e u k a e m i c c e l l coun t s e v e n t u a l l y r e t u r n e d to p r e t r e a t m e n t l e v e l s . A l t h o u g h p o s i t i v e e f f e c t s such as t h o s e d i s c u s s e d have been r e p o r t e d ( 12-14 ) , t hey p r o v e d t o be a n e c d o t a l r e l a t i v e t o the e n t i r e f i e l d o f r e s e a r c h . F o r the most p a r t , t h e r a p e u t i c e f f e c t s were u s u a l l y i n c o n s i s t e n t , r a n g i n g f rom no t o t r a n s i e n t a c t i v i t y , and d i f f i c u l t to c o n f i r m ( 4 , 1 0 , 1 1 ) . The l a c k o f r e p r o d u c i b i l i t y c o u l d be due t o use o f s e r a t h a t c o n t a i n e d i n s u f f i c i e n t q u a n t i t i e s o f tumour s p e c i f i c a n t i b o d i e s . O the r f a c t o r s c o u l d i n c l u d e prob lems i n h e r e n t t o complex p o l y c l o n a l a n t i s e r a , e . g . l i m i t e d a v a i l a b i l i t y o f h i g h - t i t r e a n t i s e r a , i n e f f e c t i v e a n t i b o d y i s o t y p e s , and low a v i d i t y . The advent o f hyb r idoma t e c h n o l o g y i n 1975 o f f e r e d the p o t e n t i a l o f p r o d u c i n g v i r t u a l l y u n l i m i t e d amounts o f m o n o c l o n a l a n t i b o d i e s o f d e f i n e d s p e c i f i c i t y and i s o t y p e ( 1 5 ) . I t was hoped t h a t the use o f m o n o c l o n a l a n t i b o d i e s wou ld a l l o w a more c r i t i c a l assessment o f the t h e r a p e u t i c p o t e n t i a l o f a n t i - t u m o u r s e r o t h e r a p y . T rea tment s t u d i e s i n a n i m a l tumour models were r e v i e w e d by Foon e t a l . i n 1982 ( 1 6 ) . I t was f o u n d t h a t a l t h o u g h m o n o c l o n a l a n t i b o d i e s were more e f f e c t i v e t h e r a p e u t i c a l l y than 6 complex a n t i s e r a , p o t e n t i a l l i m i t a t i o n s i n the c l i n i c a l a p p l i c a t i o n s o f s e r o t h e r a p y were b e i n g made o b v i o u s . In the an ima l tumour s t u d i e s o f B e r n s t e i n e t a l . , i t was f o u n d t h a t , as w i t h the p o l y c l o n a l complex a n t i s e r a , b l o o d borne tumour c e l l s were more s u s c e p t i b l e t o t r e a t m e n t than s o l i d tumours ( 1 7 , 1 8 ) . M o n o c l o n a l a n t i b o d i e s o f the i s o t y p e IgG2a , wh ich are a c t i v e i n an t i body-dependen t c e l l u l a r c y t o t o x i c i t y (ADCC) , were the most e f f e c t i v e i n a n t i - t u m o u r t r e a t m e n t . IgM had no d i s c e r n a b l e e f f e c t on tumour g r o w t h , and the IgG3 e f f e c t was i n t e r m e d i a t e . IgG2a and IgG3 i s o t y p e s w i l l l y s e tumour c e l l t a r g e t s in. v i t r o i n the p r e s e n c e o f complement o r nonimmune e f f e c t o r c e l l s . On the o t h e r h a n d , IgM i s c y t o l y t i c o n l y w i t h complement . These d i f f e r e n c e s s u g g e s t e d t h a t c e l l - d e p e n d e n t and not complement-dependent , c y t o t o x i c i t y p l a y e d a g r e a t e r r o l e i n a n t i b o d y m e d i a t e d an t i - tumour a c t i v i t y iri v i v o . Mur ine m o n o c l o n a l a n t i b o d i e s s p e c i f i c f o r l e u k a e m i a - o r l ymphoma-assoc i a t ed markers has been used i n the t r e a t m e n t o f a number o f l e u k a e m i a o r lymphoma p a t i e n t s (19-21 ) . The most c o n s i s t e n t f i n d i n g i n t h e s e c l i n i c a l t r i a l s was a d r a m a t i c bu t t r a n s i e n t d rop i n the l e v e l o f c i r c u l a t i n g l e u k a e m i a and lymphoma c e l l s . The c i r c u l a t i n g tumour c e l l numbers u s u a l l y r e c o v e r e d i n 24 - 36 h r s . An impo r t an t o b s e r v a t i o n made i n t hese s t u d i e s was the o c c u r e n c e o f a n t i g e n m o d u l a t i o n . R i t z e t a l . f o u n d t h a t 90 m i n . a f t e r anti-common a cu t e l y m p h o c y t i c l e u k a e m i a a n t i g e n (CALLA) m o n o c l o n a l a n t i b o d y t r e a t m e n t , t h e r e was a comp le te l o s s o f CALLA e x p r e s s i o n on the p a t i e n t ' s c i r c u l a t i n g l eukaem i c c e l l s ( 1 9 ) . E i g h t days l a t e r , when the p a t i e n t was no l o n g e r i n a n t i b o d y e x c e s s , the l e u k a e m i c c e l l s began to r e - e x p r e s s the common ALL a n t i g e n . Subsequent i n v i t r o 7 studies confirmed that the rapid changes in CALLA expression a f t e r exposure to anti-CALLA antibody were due to antigenic modulation. Regression in s o l i d lymphoma skin and lymph node deposits was demonstrated in the anti-Leu-1 antibody treatment of cutaneous T c e l l lymphoma patients by M i l l e r and Levy (20). However, complete remission was not achieved since the tumour deposits regenerated within 4 months. One of the most dramatic r e s u l t s was the treatment of a B - c e l l lymphoma patient with monoclonal a n t i - i d i o t y p i c antibodies (21). M i l l e r et a l . reported that treatment with monoclonal antibodies dir e c t e d against i d i o t y p i c determinants on the B - c e l l lymphoma resulted in a complete remission of > 1 year. In general, the use of monoclonal antibodies of known isotype, defined s p e c i f i c i t y and a v i d i t y seems to have improved the e f f i c i e n c y of serotherapy in both human and animal cancers. But for the most part, p o s i t i v e e f f e c t s were only of experimental in t e r e s t since c l i n i c a l l y meaningful regressions were rare. Antigenic modulation appeared to be a common f i n d i n g and i s most l i k e l y a l i m i t i n g obstacle to tumour serotherapy. Other obstacles include the heterogeneity of antigen expression in the ent i r e tumour load and the possible s e l e c t i o n f o r tumour associated antigen marker negative variants by serotherapy. These problems may be overcome by the use of a c a r e f u l l y engineered pool of d i f f e r e n t anti-tumour antigen s p e c i f i c monoclonal antibodies. Another c r i t i c a l drawback to serotherapy i s the p o t e n t i a l f o r a host anti-therapeutic antibody reaction. Such an immune response would act to negate any therapeutic e f f e c t of the antibodies and may be the cause of 8 s i d e e f f e c t s sometimes d i s p l a y e d by a n t i b o d y t r e a t e d p a t i e n t s , i . e . f e v e r , r i g o r s , and r e s p i r a t o r y d i s e a s e ( 2 2 ) . Though some o f t hese s i d e e f f e c t s can be e l i m i n a t e d ( e . g . t h rough the use o f non-aggrega ted a n t i b o d y p r e p a r a t i o n s , s l owe r r a t e o f i n t r a v e n o u s i n f u s i o n , e t c . ) , the r e s e a r c h t o da t e i n d i c a t e s t h a t s e r o t h e r a p y by i t s e l f i s not c o n s i s t e n t l y an e f f e c t i v e mode o f c a n c e r t r e a t m e n t . A more r e a l i s t i c approach may be t o u t i l i z e the t a r g e t i n g a b i l i t y o f s p e c i f i c a n t i b o d i e s r a t h e r than t h e i r h o s t c e l l dependent c y t o t o x i c i t y t o e l i m i n a t e tumour c e l l s . A t the b e g i n n i n g o f the t w e n t i e t h c e n t u r y , P a u l E h r l i c h p r o p o s e d the use o f magic b u l l e t s o r " b o d i e s wh ich p o s s e s s a p a r t i c u l a r a f f i n i t y f o r a c e r t a i n o rgan . . . as a c a r r i e r by wh ich to b r i n g t h e r a p e u t i c a l l y a c t i v e g roups t o the o rgan i n q u e s t i o n " f o r the s p e c i f i c e l i m i n a t i o n o f s y p h i l i s s p i r o c h e t e s w i t h o u t a f f e c t i n g norma l t i s s u e ( 2 3 ) . T h i s t ype o f d i s e a s e t a r g e t i n g c o u l d be e a s i l y a t t a i n a b l e w i t h a n t i g e n s p e c i f i c a n t i b o d i e s . A n t i b o d i e s c o u l d be used as c a r r i e r s t h a t l o c a l i z e c o n j u g a t e d c y t o t o x i c d rugs o r r a d i o i s o t o p e s a t tumour s i t e s . T h u s , the d r u g / i s o t o p e - a n t i b o d y c o n j u g a t e wou ld d e l i v e r i t s t o x i c a c t i v i t y e x c l u s i v e l y t o c e l l s e x p r e s s i n g the t a r g e t a n t i g e n w h i l e m i n i m i z i n g n o n s p e c i f i c e f f e c t s on t a r g e t a n t i g e n n e g a t i v e c e l l s . "Mag i c b u l l e t s " c o u l d be u s e f u l i n two d i f f e r e n t a s p e c t s o f c a n c e r t r e a t m e n t . The f i r s t a s p e c t i s c l e a r l y the m o d i f i c a t i o n o f t o x i n s t o tumour s p e c i f i c l e t h a l weapons. The second a s p e c t i s the use o f r a d i o t r a c e r t agged a n t i b o d i e s f o r the d i a g n o s i s and d e t e c t i o n o f tumour ( i . e . r a d i o i m m u n o d e t e c t i o n o r r ad io immuno image r y ) . P r i o r to the deve lopment o f hyb r idoma t e c h n o l o g y , many a t t empts were made t o d e t e c t o r e l i m i n a t e tumours w i t h i s o t o p e o r d rug c o n j u g a t e d 9 h e t e r o a n t i s e r a . The r e s u l t s were no t e n t i r e l y s u c c e s s f u l ( i . e . h i g h n o n s p e c i f i c b a c k g r o u n d s ) , p r o b a b l y due t o the f a c t t h a t o n l y a m ino r p r o p o r t i o n o f the a n t i b o d y p o p u l a t i o n i n the p o l y c l o n a l complex a n t i s e r a was t r u l y tumour s p e c i f i c ( 2 4 ) . Wi th m o n o c l o n a l a n t i b o d y s t u d i e s , i t was p o s s i b l e t o c l e a r l y demons t r a t e the p o t e n t i a l a b i l i t y o f m o n o c l o n a l a n t i b o d i e s t o a c t as c a r r i e r s to d e s i g n a t e d t a r g e t s . T o x i c agen ts such as p r o t e i n t o x i n s ( e . g . R i c i n - A c h a i n , d i p t h e r i a t o x i n , A b r i n , Pseudomonas a e r u g i n o s a e x o t o x i n A , C l o s t r i d i u m p e r f r i n g e n s p h o s p h o l i p a s e C , b o v i n e p a n c r e a t i c r u b o n u c l e a s e e t c . ) o r c h e m o t h e r a p e u t i c d rugs ( e . g . daunomyc in , m e t h o t r e x a t e , c h l o r a m b a c i l , e t c . ) , when l i n k e d t o a n t i b o d i e s d i s p l a y e d an a p p r e c i a b l e l e v e l o f s p e c i f i c c y t o t o x i c i t y f o r a n t i g e n e x p r e s s i n g t a r g e t c e l l s ( 25-37 ) . A n t i - t u m o u r m o n o c l o n a l a n t i b o d i e s t agged w i t h . . . , . 131 1 2 5 T 111,. 99ni . _ r a d i o i s o t o p e s ( e . g . I, I, I n , TC , e t c . ) were f o u n d t o d i s p l a y t a r g e t p r e f e r e n t i a l l o c a l i z a t i o n iri v i v o , hence d e m o n s t r a t i n g an e x q u i s i t e l e v e l o f s p e c i f i c i t y ( 38-40 ) . In the s t u d i e s r e p o r t e d h e r e , b o t h f a c e t s ( t h e r a p y and d e t e c t i o n ) o f magic b u l l e t s were examined . The te rm "pho to immuno the rapy " was c o i n e d to d e s c r i b e an a n t i - c a n c e r t r e a t m e n t t h a t combines the p h o t o t o x i c e f f e c t s o f c h e m i c a l s such as h a e m a t o p o r p h y r i n w i t h the t a r g e t - s e e k i n g a b i l i t y o f a n t i b o d i e s . Haemotopo rphy r i n was c h e m i c a l l y c o u p l e d to m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t the mur ine DBA/2J rhabdomyosarcoma, M - l , o r a g a i n s t human a c u t e n o n - l y m p h o b l a s t i c l e u k a e m i a c e l l s . In C h a p t e r s I I and I I I , the pho to immuno the r apeu t i c approach w i l l be examined i n the mur ine tumour m o d e l , M - l , and the human m a l i g n a n t c o n d i t i o n , a cu t e n o n - l y m p h o b l a s t i c l e u k a e m i a . In C h a p t e r IV , r ad io immuno imagery was 10 s t u d i e d i n the an ima l model M-l w i t h Tc l a b e l l e d ant i-M-1 m o n o c l o n a l a n t i b o d i e s . S e v e r a l p o r p h y r i n s have been f o u n d t o be p r e f e r e n t i a l l y t aken up and r e t a i n e d by m a l i g n a n t l e s i o n s i n c o n t r a s t t o normal t i s s u e ( 41-44 ) . T h i s c h a r a c t e r i s t i c has been used t o advantage as a d i a g n o s t i c t o o l t o l o c a t e and d e l i n e a t e tumours i n man (45-48 ) . P o r p h y r i n s a re p h o t o t o x i c agents t h a t w i l l s e n s i t i z e c e l l s so t h a t t hey become v u l n e r a b l e t o damage by exposu re t o l i g h t . These photodynamic agen ts r e q u i r e oxygen t o med i a t e t h e i r c y t o t o x i c a c t i v i t y . L e t h a l p h o t o a c t i v a t i o n o f the p o r p h y r i n s o r o t h e r photodynamic a g e n t s , e . g . e o s i n , i n v o l v e s the p r o d u c t i o n o f s i n g l e t oxygen i . e . the m o l e c u l a r oxygen i s a c t i v a t e d to a m e t a s t a b l e , e l e c t r o n i c a l l y e x c i t e d s t a t e (49-51 ) . S i n g l e t oxygen has been i m p l i c a t e d as the major c y t o t o x i c agent f o r the p h o t o a c t i v a t i o n o f h a e m a t o p o r p h y r i n o r haema topo rphy r i n d e r i v a t i v e ( 5 2 ) . Though i t has been shown t h a t s i n g l e t oxygen can have a d e t r i m e n t a l e f f e c t by r e a c t i n g w i t h s u b c e l l u l a r o r g a n e l l e s , p r o t e i n s and n u c l e i c a c i d s ( 53-56 ) , the main t a r g e t s i t e f o r c e l l u l a r i n a c t i v a t i o n i s t hough t to be the c e l l membrane ( 5 0 ) . One o f the e a r l i e s t a t t empts i n the use o f h a e m a t o p o r p h y r i n as an a n t i - c a n c e r agent was r e p o r t e d by Diamond e t a l . i n 1972 ( 5 7 ) . In t h i s s t u d y , Diamond e t a l . showed t h a t r a t g l i o m a c e l l s and subcutaneous g l i o m a s c o u l d be e l i m i n a t e d w i t h a c o m b i n a t i o n o f l i g h t and h a e m a t o p o r p h y r i n . Subsequent s t u d i e s w i t h h a e m a t o p o r p h y r i n i j i v i t r o demons t r a t ed i t s p r e f e r e n t i a l r e t e n t i o n and c y t o t o x i c i t y on tumour ( 5 8 ) . T rea tment w i t h h a e m a t o p o r p h y r i n , d e s t r o y e d g l i o m a c e l l s i n c u l t u r e i n l e s s than 8 min . o f i r r a d i a t i o n and g l i o m a s i n r a t s i n about 40 m i n . o f l i g h t i r r a d i a t i o n . 11 Although most of the p r e l i m i n a r y studies on porphyrin a c t i v i t y u t i l i z e d haematoporphyrin as an anti-cancer p h o t o s e n s i t i z i n g agent, i t i s not the porphyrin of choice f o r c l i n i c a l use. Haematoporphyrin (Hp) was not found to be as e f f e c t i v e a p h o t o s e n s i t i z e r of tumour t i s s u e i_n v i v o (59). Haematoporphyrin d e r i v a t i v e (HpD) (prepared by a c e t y l a t i o n and h y d r o l y s i s of Hp d i h y d r o c h l o r i d e ) has been found to have twice the s e n s i t i z i n g e f f e c t as equivalent concentrations of Hp i_n v i t r o on human NHIK 3025 c e l l s (60). I t has been reported to be a superi o r l o c a l i z e r Jji v i v o than Hp (42,59). In v i t r o s tudies on the d e r i v a t i v e ' s anti-cancer e f f e c t have demonstrated i t s e f f e c t i v e n e s s i n both experimental animal models and human malignancies. Dougherty et a l . reported i n 1975 that low dosages of HpD (2.5 - 5.0 mg/kg body wt i n mice and 10 - 15 mg/kg i n r a t s ) i n t r a p e r i t o n e a l l y , f o l l o w e d 24 - 48 hr l a t e r by exposure to l i g h t (620 -640 nm) r e s u l t e d i n long term cures i n tumour bearing r a t s and mice (61). Dougherty and co-workers reported that i n c l i n i c a l t r i a l s using HpD (2.5 -5.0 mg/kg body weight) i n phototherapy on a v a r i e t y of human cutaneous or subcutaneous metastic malignancies, 81% of the t r e a t e d p a t i e n t s had p o s i t i v e anti-tumour e f f e c t ; i . e . biopsy-proven necrosis or re d u c t i o n i n tumour s i z e by at l e a s t 50% (62). Anti-tumour responses were noted i n 6/7 melanomas, 2/3 adenocarcinoma of the colo n , 2/2 breast cancers, 2/2 b a s a l c e l l tumours, 1/2 mycosis fungoides, 1 chondrosarcoma, 1 p r o s t a t i c carcinoma, 1 squamous c e l l carcinoma, and 1 endometrial carcinoma i n a 21 p a t i e n t study. This c l e a r l y i n d i c a t e d that HpD was e f f e c t i v e over q u i t e a wide range of d i f f e r e n t tumour types. S i m i l a r therapeutic responses with HpD p h o t o r a d i a t i o n treatment on human malignancies has been reported from 12 s e v e r a l o t h e r d i f f e r e n t c l i n i c a l s t u d i e s . Haya ta e t a l . o b s e r v e d t h a t HpD caused s h o r t t e rm r e m i s s i o n i n 12 t r e a t e d cases o f l u n g c a n c e r , and a 70% comp le te r e m i s s i o n r a t e i n t r e a t e d u r i n a r y b l a d d e r c a n c e r s ( 6 3 ) . A r e c e n t s t udy f rom C h i n a r e p o r t e d t h a t a f t e r p h o t o r a d i a t i o n t h e r a p y w i t h HpD (5 mg/kg body w e i g h t ) , 49 o f 81 c ance rous l e s i o n s examined r e g r e s s e d c o m p l e t e l y and 14 p a r t i a l l y ( 6 4 ) . The tumours t r e a t e d i n t h i s s t u d y i n c l u d e d squamous c e l l c a r c i n o m a s , b a s a l c e l l c a r c i n o m a s , adenoca rc inomas o f the b r e a s t and l i v e r , o s t e o g e n i c s a r coma , m y c o s i s f u n g o i d e s , and b l a d d e r c e l l c a r c i n o m a s . A common s i d e e f f e c t no ted i n most o f the c l i n i c a l t r i a l s was an u l t r a s e n s i t i v i t y t o d i r e c t and i n d i r e c t b r i g h t l i g h t . P a t i e n t s were warned t o s t a y ou t o f b r i g h t s u n l i g h t f o r a t l e a s t 30 days and then t o g r a d u a l l y b e g i n l i g h t e x p o s u r e . F a i l u r e t o f o l l o w t h e s e i n s t r u c t i o n s r e s u l t e d i n m i l d t o s e v e r e edema, b l i s t e r i n g and e ry thema o f the e x p o s u r e d a r e a s . HpD a t 25 vg/ml has a l s o been shown t o have a g r o w t h - i n h i b i t i n g e f f e c t on " n o r m a l " e s t a b l i s h e d c e l l l i n e s e . g . r a t kangaroo e p i t h e l i a l k i d n e y , norma l mouse embryon ic f i b r o b l a s t s and d i f f e r e n t i a t e d n e o n a t a l r a t m y o c a r d i a l c e l l s i n the absence o f l i g h t ( 6 5 ) . So i n s p i t e o f HpD's p r e f e r r e d r e t e n t i o n i n tumour t i s s u e , t h e r e i s the p o t e n t i a l f o r d e l e t e r i o u s e f f e c t on norma l t i s s u e . Such s i d e e f f e c t s c o u l d be e l i m i n a t e d i f the p o r p h y r i n p h o t o s e n s i t i z e r was made more tumour s p e c i f i c o r i f l ower d rug dosages made were more e f f e c t i v e . In the s t u d i e s p r e s e n t e d i n C h a p t e r s I I and I I I , the t e s t p o r p h y r i n used was the l e s s e r i j i v i v o p h o t o s e n s i t i z e r , h a e m a t o p o r p h y r i n . In the c o n t e x t as "mag i c b u l l e t s " , s p e c i f i c a n t i - t u m o u r m o n o c l o n a l a n t i b o d i e s were used to i n c r e a s e the s p e c i f i c i t y , and i n t u r n the s e n s i t i z i n g a b i l i t y 13 of haematoporphyrin for tumour t i s s u e . Hence by using antibodies as the active targeting agent, Hp could be transformed to a more e f f e c t i v e anti-tumour drug il l vivo. In addition, a higher l e v e l of tumour s p e c i f i c i t y would allow u t i l i z a t i o n of a lower e f f e c t i v e anti-tumour drug dose thereby hopefully decreasing the p o t e n t i a l for toxic action on normal t i s s u e . In Chapter IV, u t i l i z a t i o n of s p e c i f i c monoclonal antibodies in the in vivo radioimmuno-detection or -imagery of cancer was examined. A s i g n i f i c a n t advance in the cure of cancer may l i e in the a b i l i t y to detect cancerous lesions p r i o r to a stage where the tumour burden i s too large for t r a d i t i o n a l methods; i . e . surgery, radio- and/or chemotherapy, to cope with. Antibodies can be used in two ways in the diagnosis of tumour. Radiolabelled antibodies could be used to detect the presence of serum c i r c u l a t i n g antigen shed from tumour. An example of t h i s would be the detection of diagnostic cancer markers such as carcinoembryonic antigen (CEA) and alphafetoprotein (AFP) in serum using radioimmunoassays (66,67). The second approach i s the i n j e c t i o n of r a d i o l a b e l l e d anti-tumour antibodies into a possible cancer patient. The antibodies would s p e c i f i c a l l y l o c a l i z e an antigen expressing tumour l e s i o n s . The l o c a t i o n of the r a d i o l a b e l e.g. the malignant l e s i o n , could then be detected through external scintigraphy. This approach would o f f e r the diagnostician invaluable information on the l o c a t i o n and the spread of the malignancy, important considerations i f surgery i s contemplated or i f the disease i s being monitored a f t e r radio- and/or chemotherapy. In t h i s 99m 99m study, the radioisotope used for cancer detection was Tc. Tc i s 14 c o n s i d e r e d the r a d i o n u c l i d e o f c h o i c e f o r most n u c l e a r m e d i c a l s t u d i e s . I t ' s mode o f decay (pure p h o t o n , 140 keV) i s i d e a l f o r gamma camera d e t e c t i o n . I t has a r e l a t i v e l y s h o r t h a l f l i f e (6 h r s ) . T h i s i s an impo r t an t pa ramete r f o r a r a d i o t r a c e r s i n c e i t would i n p a r t de t e rm ine the t o t a l r a d i a t i o n dose r e c e i v e d by the p a t i e n t . I d e a l l y , the r a d i o a c t i v i t y has to be i n d e t e c t a b l e q u a n t i t i e s i n the body l o n g enough t o a l l o w l o c a l i z a t i o n o f the tumour masses . Any l o n g e r than t h a t wou ld j u s t mean n e e d l e s s i r r a d i a t i o n f o r the p a t i e n t . In the s tudy r e p o r t e d i n C h a p t e r 99m IV , Tc t agged ant i-M-1 m o n o c l o n a l a n t i b o d i e s were used t o d e t e c t the l o c a t i o n o f M-l myosarcomas i n DBA/2J m i c e . The i i i v i v o s p e c i f i c i t y o f the l a b e l l e d a n t i b o d y and the l i m i t a t i o n s o f c a n c e r rad io immuno imagery were i n v e s t i g a t e d . CHAPTER I I PHOTOIMMUNOTHERAPY: ANIMAL STUDIES 16 INTRODUCTION The use o f p h o t o c h e m i c a l s ( e . g . h aema topo rph r y i n and i t s d e r i v a t i v e s ) as a n t i - c a n c e r agents a re b e i n g e x t e n s i v e l y i n v e s t i g a t e d i n e x p e r i m e n t a l an ima l and human s y s t e m s . Haematoporphy r in (Hp) and h a e m a t o p o r p h y r i n d e r i v a t i v e (HpD) have been used i n p h o t o r a d i a t i o n c a n c e r t r e a t m e n t s w i t h some degree o f s u c c e s s . They a re r e a d i l y t aken up and r e t a i n e d by m a l i g n a n t t i s s u e ( 6 8 , 7 0 ) . The pho tod rugs a re a c t i v a t e d by l i g h t and the c y t o t o x i c e f f e c t i s m e d i a t e d m a i n l y t h r o u g h p r o d u c t i o n o f s i n g l e t oxygen ( 5 2 , 7 1 ) . A major drawback o f t h i s fo rm o f t h e r a p y i s the p o t e n t i a l f o r c a u s i n g norma l t i s s u e damage. The norma l t i s s u e o v e r l a y i n g o r a d j a c e n t t o the tumour may be s e v e r e l y damaged i f c a r e i s not t aken to d e f i n e the t i m i n g between d rug a d m i n i s t r a t i o n and l i g h t e x p o s u r e . T h i s i s due t o the d i f f e r e n t i a l up take and/or c l e a r a n c e o f d rug between norma l and m a l i g n a n t t i s s u e ( 4 2 ) . A n o t h e r adve r se d rug i n d u c e d e f f e c t i s u l t r a s e n s i t i v i t y t o s u n l i g h t . P a t i e n t s r e c e i v i n g h a e m a t o p o r p h y r i n t r e a tmen t a re warned to a v o i d exposu re t o s u n l i g h t f o r a t l e a s t f o u r weeks. L i g h t exposu re p r i o r t o t h a t t ime p e r i o d may r e s u l t i n symptoms r a n g i n g f rom s l i g h t e ry thema and edema to e x t e n s i v e s k i n damage and n e c r o s i s (68-70 ) . P a r t o f t h i s p rob l em i s due t o the dose l e v e l s r e q u i r e d f o r tumour r e s p o n s e . The l e v e l s c u r r e n t l y used f o r chemotherapy a re h i g h enough t o have a d e l e t e r i o u s e f f e c t on norma l t i s s u e upon l i g h t e x p o s u r e . I d e a l l y , the p h o t o s e n s i t i z e r s h o u l d be m o d i f i e d so t h a t i t i s more tumour s p e c i f i c . T h i s would r e s u l t i n m i n i m i z i n g the s i d e e f f e c t s o f h a e m a t o p o r p h y r i n s i n c e the e f f e c t i v e t h e r a p e u t i c dose c o u l d be l o w e r e d . G r e a t e r tumour 17 s p e c i f i c i t y wou ld mean l e s s d rug d i s p e r s a l t h rough the body and f a s t e r l o c a l i z a t i o n i n the m a l i g n a n t t i s s u e . The approach w i t h wh ich we have chosen to add ress t h i s p r o b l e m t akes advantage o f " t h e magic b u l l e t " o r m o n o c l o n a l a n t i b o d y t e c h n i q u e f i r s t d e s c r i b e d by P a u l E h r l i c h i n 1908 ( 2 3 ) . He sugges t ed t h a t m o l e c u l e s w i t h an a f f i n i t y f o r c e r t a i n t i s s u e s migh t be a b l e to s e r ve as c a r r i e r s o f c y t o t o x i c a g e n t s . By homing i n on t h e i r d e s i g n a t e d t a r g e t s , m o n o c l o n a l a n t i b o d i e s c o n j u g a t e d to c e l l - k i l l i n g agents may be a b l e t o d e l i v e r and l o c a l i z e c y t o t o x i c a c t i v i t y to the tumour s i t e . The f e a s i b i l i t y o f t h i s t e c h n i q u e has been demons t r a t ed i n v i t r o by G i l l i l a n d e_t a l . ( 1980 ) . U s i n g d i p h t h e r i a t o x i n A c h a i n - a n t i - c o l o r e c t a l m o n o c l o n a l a n t i b o d y c o n j u g a t e s , G i l l i l a n d r e p o r t e d t h a t , in v i t r o v i r t u a l l y 100% o f the t r e a t e d c o l o r e c t a l c a r c i n o m a c e l l s were k i l l e d . Under the same c o n d i t i o n s , c e l l l i n e s no t b e a r i n g the c o l o r e c t a l c a r c i n o m a a n t i g e n were no t a f f e c t e d ( 7 2 ) . In the p r e s e n t s t u d y , we r e p o r t the use o f m o n o c l o n a l a n t i b o d y -h a e m a t o p o r p h y r i n c o n j u g a t e s as an a n t i - c a n c e r agent ijn v i t r o and In v i v o . The a n t i b o d y used was d i r e c t e d a g a i n s t a tumour s p e c i f i c a n t i g e n on the DBA/2J rhabdomyosarcoma M-l ( 7 3 ) . Under the e x p e r i m e n t a l c o n d i t i o n s u s e d , u s i n g the a n t i b o d y - p h o t o d r u g c o n j u g a t e , we have been a b l e t o demons t r a t e s i g n i f i c a n t a n t i - t u m o u r a c t i v i t y w i t h o u t any v i s i b l e s i d e e f f e c t s i . e . damage to norma l t i s s u e . E q u i v a l e n t c o n c e n t r a t i o n s o f h a e m a t o p o r p h y r i n o r m o n o c l o n a l a n t i b o d y a l o n e had no e f f e c t on tumour g rowth . T h i s i n d i c a t e d t h a t : i ) an t i body-Hp c o n j u g a t e s may have a s u p e r i o r a n t i - c a n c e r e f f e c t o ve r d rug o r a n t i b o d y a l o n e , and i i ) the "mag ic b u l l e t " t e c h n i q u e a l l o w e d 18 the lowering of the minimum e f f e c t i v e dose of haematoporphyrin r e q u i r e d f o r anti-cancer a c t i v i t y , hence minimizing adverse side e f f e c t s . 19 C h a p t e r I I : Photo immunotherapy : A n i m a l Mode l MATERIALS AND METHODS E x p e r i m e n t a l A n i m a l s : Female DBA/2J o r B 6 D 2 F 2 m i c e » 2 - 4 months o l d f rom J a c k s o n L a b o r a t o r y , Bar H a r b o r , M a i n e , were u s e d . Mur ine Tumours : A 3 - m e t h y l c h o l a n t h r e n e - i n d u c e d rhabdomyosarcoma (M-l) and the L1210 l e u k e m i a c e l l l i n e s y n g e n e i c f o r DBA/2J m i c e , as w e l l as the EL4 lymphoma o f C57BL/6 mice were m a i n t a i n e d i n v i v o and j j i v i t r o as p r e v i o u s l y d e s c r i b e d (73-75 ) . A n t i g e n e x t r a c t s and i m m u n i z a t i o n : S o l i d tumours f rom mice were used i n the p r e p a r a t i o n o f membrane a n t i g e n e x t r a c t s . The tumour was f o r c e d th rough a s c r e e n mesh i n o r d e r t o o b t a i n a s i n g l e c e l l s u s p e n s i o n . The c e l l s were washed tw i c e i n p h o s p h a t e - b u f f e r e d s a l i n e (PBS) . R e s i d u a l r e d -2 -1 b l o o d c e l l s were removed by 1.7 x 10 M T r i s : 1.25 x 10 NH^Cl (pH 7.2) t r e a t m e n t . The c e l l s were washed aga i n and r e s u s p e n d e d i n PBS. A n t i g e n e x t r a c t s were o b t a i n e d by two methods : s o n i c a t i o n a t 4 ° C f o r 3 x 45 sec ( s e t t i n g 50) b u r s t s u s i n g a B i o s o n i k s o n i c a t o r ( B r o n w i l l S c i e n t i f i c ) as d e s c r i b e d by Al-Rammahy and Levy (76) o r 3 .0 M KC1 e x t r a c t i o n s (18 h r a t 4 ° C ) . C e l l u l a r d e b r i s i n bo th c a se s was removed f rom the s u p e r n a t a n t by c e n t r i f u g a t i o n a t 100 ,000 x g f o r 90 m i n . The s o l u b l e membrane e x t r a c t was d i a l y z e d e x h a u s t i v e l y a g i n s t PBS and c e n t r i f u g e d a g a i n t o remove any p a r t i c u l a t e m a t t e r . 20 DBA/2J mice were immunized s u b c u t a n e o u s l y w i t h 50 ug o f M-l c e l l membrane e x t r a c t i n a 50% e m u l s i o n o f comp le te F r e u n d ' s a d j u v e n t (CFA, G i b c o ) a t i n t e r v a l s o f two weeks f o r a t o t a l o f f o u r i n j e c t i o n s . F o l l o w i n g the second i n j e c t i o n , the m ice were b l e d and ant i-M-1 t i t r e s were d e t e r m i n e d u s i n g an EL ISA (see b e l o w ) . An ima l s d e m o n s t r a t i n g a s i g n i f i c a n t ant i-M-1 r e s p o n s e were s e t a s i d e and r e s t e d f o r f o u r weeks . Th ree days p r i o r t o f u s i o n , t h e s e m ice were g i v e n a b o o s t i n g dose o f 50 ug M-l i n PBS i n t r a v e n o u s l y , v i a the t a i l v e i n . C e l l f u s i o n and c l o n i n g : C o n d i t i o n s f o r c e l l f u s i o n and c l o n i n g were as d e s c r i b e d by O i and He rzenbe rg (77) and Weaver, S i k o r a and Levy ( 7 8 ) . B r i e f l y , M-l membrane e x t r a c t p r imed DBA/2J s p l e n o c y t e s were f u s e d w i t h NS-1 mur ine myeloma c e l l s i n the p r e s e n c e o f 50% p o l y e t h y l e n e g l y c o l (Baker 1540) i n PBS. The f u s e d c e l l s were r e suspended i n DME + 20% FCS a t a d e n s i t y o f 1 0 7 pe r m l , and d i s p e r s e d i n 100 y l vo lumes i n t o 9 6 - w e l l t i s s u e c u l t u r e p l a t e s ( C o s t a r ) . Sp leen-myeloma f u s i o n p r o d u c t s were i s o l a t e d t h rough HAT s e l e c t i o n . Seven days a f t e r f u s i o n , the s u p e r n a t a n t s f rom t i s s u e c u l t u r e w e l l s were t e s t e d f o r a n t i b o d y p r o d u c t i o n by EL ISA p r o c e d u r e s (see b e l o w ) . P o s i t i v e a n t i b o d y s e c r e t i n g h y b r i d s were then c l o n e d by l i m i t i n g d i l u t i o n i n DME medium + 20% FCS , h y p o x a n t h i n e (2 y g / m l ) , t h y m i d i n e (0 .4 yg/ml) and 1 0 7 thymyocy tes pe r m l . Seven days a f t e r c l o n i n g , the t i s s u e c u l t u r e w e l l s u p e r n a t a n t s were t e s t e d a g a i n f o r ant i-M-1 a c t i v i t y w i t h EL ISA . 21 EL ISA: Ant i-M-1 hybr idomas and b l o o d samples were s c r e e n e d f o r a n t i b o d y p r o d u c t i o n u s i n g an EL ISA ( 7 9 ) . P o l y s t y r e n e s u b s t r a t e p l a t e s (Cooke) were c o a t e d w i t h M-l c e l l e x t r a c t a t 25 ug/ml i n 0.05 M c a r b o n a t e c o a t i n g o b u f f e r pH 9.6 o v e r n i g h t a t 4 C . A f t e r wash ing t h r e e t imes i n PBS-Tween 20 , the p l a t e s were c o a t e d w i t h r a b b i t an t i-mouse Ig (1 ug/ml) f o r 30 min to s a t u r a t e any n o n - s p e c i f i c p r o t e i n b i n d i n g s i t e s . The p l a t e s were washed a g a i n t h r e e t imes i n PBS-Tween 20 and the t e s t a n t i s e r a were added t o the w e l l s i n 100 u l a l i q u o t s . F o l l o w i n g i n c u b a t i o n f o r 1.5 h r a t room t empe ra tu r e and subsequent w a s h i n g , the d e v e l o p i n g a l k a l i n e p h o s p h a t a s e - l a b e l l e d r a b b i t an t i-mouse Ig (1 :5000 d i l u t i o n ) was added a t 100 u l pe r w e l l . A f t e r an a d d i t i o n a l 1.5 h r i n c u b a t i o n and f i n a l PBS-Tween b u f f e r wash , 100 u l o f the enzyme s u b s t r a t e s o l u t i o n (S igma-104-105, p - n i t r o p h e n y l phospha te d i sod ium) was added t o each w e l l . The enzyme s u b s t r a t e r e a c t i o n w i t h subsequent c o l o r deve lopment was a l l o w e d t o p r o c e e d f o r 1 h r . The p l a t e s were then r e a d f o r abso rbance a t 405 nm on a T i t e r t e k M u l t i s k a n (FLow L a b o r a t o r i e s , I ng l ewood , C a l i f o r n i a ) . P l a t e s were b l a n k e d a g a i n s t a row o f w e l l s c o n t a i n i n g a n t i g e n a l o n e and a l l t e s t s were done a t l e a s t tw i c e and i n q u a d r u p l e t on each o c c a s i o n . P r o d u c t i o n and i s o l a t i o n o f m o n o c l o n a l a n t i b o d i e s : The ant i-M-1 h y b r i d c e l l l i n e was m a i n t a i n e d as an a s c i t e s tumour i n i r r a d i a t e d DBA/2J mice (600 r a d s ) . A n t i b o d y was p u r i f i e d f rom the a s c i t e s f l u i d by p r e c i p i t a t i o n w i t h 50% s a t u r a t e d ammonium s u l f a t e o v e r n i g h t a t 4 ° C . The r e s u l t i n g s l u r r y was c e n t r i f u g e d a t 10 ,000 x g f o r 20 m i n . The p r e c i p i t a t e was 22 r e suspended i n a m i n i m a l volume o f PBS and d i a l y s e d e x h a u s t i v e l y a g a i n s t PBS a t 4 ° C , c h a n g i n g the f l u i d f o u r t i m e s . F u r t h e r p u r i f i c a t i n o f the a n t i b o d y p r e p a r a t i o n was a c c o m p l i s h e d w i t h DEAE-Sephace l ( Pha rmac i a F i n e C h e m i c a l s ) ch romatog raphy . One ml o f a s c i t e s ( a p p r o x i m a t e l y 10 mg p r o t e i n i n s t a r t i n g b u f f e r ) pe r ml o f beads was a p p l i e d to a DEAE-Sephace l co lumn p r e v i o u s l y e q u i l i b r a t e d i n s t a r t i n g b u f f e r . The column was washed w i t h s t a r t i n g b u f f e r (0 .025 M H C l ; 0 .032 M T r i s ; 0 .2% I « 3 ; pH 7.4) u n t i l p r o t e i n a b s o r p t i o n r e a d i n g s a t 280 nm were 0 . 0 0 . The co lumn-bound a n t i b o d y was e l u t e d by r u n n i n g the column w i t h a s a l t g r a d i e n t o f 0 .0-0 .2M N a C l . The a n t i b o d y was d e t e c t e d by r e a d i n g f r a c t i o n s f o r a b s o r p t i o n a t 280 nm, p o o l e d , and d e s a l t e d on an Amicon PM 10 D i a f l o u l t r a f i l t e r . A l l a n t i b o d y p r e p a r a t i o n s were kep t a t 4 ° C a t one mg p e r ml c o n c e n t r a t i o n . An i r r e l e v a n t m o n o c l o n a l a n t i b o d y o f the same s u b c l a s s as the ant i-M-1 a n t i b o d y was used as a c o n t r o l i n t h e s e s t u d i e s . Drug.: Haematopo rphy r i n d i h y d r o c h l o r i d e (95%) was o b t a i n e d f r om Sigma C h e m i c a l C o . , S t . L o u i s , M o . , and used w i t h o u t f u r t h e r p u r i f i c a t i o n . P r e p a r a t i o n o f a n t i b o d y - haema topo rphy r i n c o n j u g a t e s : The c o n j u g a t i o n p r o c e d u r e was d e v e l o p e d and done by D r . L i l y C h i - K i t Wat, B o t a n y , U . B . C . To 20 mg h a e m a t o p o r p h y r i n 2 HC l (Sigma) i n 1.25 ml wa te r and 0 .8 ml N ,N-d ime thy l f o rmamide , was added 20 mg l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) -c a r b o d i i m i d e HC l i n 0 .6 ml w a t e r . A f t e r 30 m i n , t h i s s o l u t i o n was mixed f o r 5 h r w i t h 15 mg o f the a n t i b o d y i n 5 ml d i s t i l l e d w a t e r . D u r i n g t h i s p e r i o d , the pH o f the s o l u t i o n was m o n i t o r e d and a d j u s t e d t o a range 2 3 between pH 6-7. A f t e r the 5 h r i n c u b a t i o n p e r i o d , 50 u l o f monoethano lamine was added and the s o l u t i o n was l e f t o v e r n i g h t a t room t e m p e r a t u r e . The f o l l o w i n g day , the s o l u t i o n was d i a l y s e d a g a i n s t a 0.001 M phosphate b u f f e r pH 7.A f o r 4 days w i t h 3 changes pe r day and then a g a i n s t PBS o v e r n i g h t . The s o l u t i o n was l y o p h i l i z e d , r e d i s s o l v e d i n PBS and p a s s e d t h rough a Sephadex G-25 column t o remove n o n s p e c i f i c a l l y abso rbed p r o p h y r i n . The amount o f h a e m a t o p o r p h y r i n (Hp) bound t o the a n t i b o d i e s was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y . Haematoporphy r in c o n c e n t r a t i o n was c a l c u l a t e d by t a k i n g the abso rbance a t 505 nm o f 0 .644 f o r 80 ug Hp/ml . P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by t a k i n g the abso rbance a t 280 nm o f 1.4 f o r 1.0 mg p r o t e i n / m l . P o t e n t i a l c r o s s abso rbance was a c c o u n t e d f o r by measu r i ng the abso rbance o f f r e e Hp a t 280 nm and c a l c u l a t i n g the % c o n t r i b u t i o n o f Hp a t 280 nm and the same f o r p r o t e i n abso rbence a t 505 nm. In t h i s s t u d y , the r e l a t i v e q u a l i t i e s o f Hp bound t o the i n d i v i d u a l m o n o c l o n a l a n t i b o d i e s was d e t e r m i n e d t o be as f o l l o w s : A n t i b o d y Hp (mg) bound/mg a n t i b o d y ant i-M-1 0.134 i r r e l e v a n t MAb a L1210 0.172 a G1P 0.103 Assay o f Haematopo rphy r i n a c t i v i t y i n Hp-an t i body c o n j u g a t e s : The h a e m o l y t i c a s say was p e r f o r m e d by D r . Wat, Bo t any , U . B . C . Red b l o o d c e l l s were i s o l a t e d f rom whole b l o o d by c e n t r i f u g a t i o n a t 500 x g f o r 5 m i n . A 1:20 d i l u t i o n (v/v) o f the packed c e l l s were p r e p a r e d and used i n the a s say s y s t e m . 24 The r e d b l o o d c e l l s u s p e n s i o n was d i s p e n s e d i n 200 u l a l i q u o t s t o 13 x 100 nm t e s t t u b e s . 150 u l PBS and 50 y l o f the p r e p a r e d c o n j u g a t e , Hp a l o n e (10 yg) o r PBS was added t o the t u b e s . D u p l i c a t e s o f each t e s t s e t were p r e p a r e d and one s e t was kep t i n the d a r k f o r a backg round c o n t r o l . The tubes were then p l a c e d i n a s l a n t e d p o s i t i o n under a bank o f 4 G e n e r a l E l e c t r i c C o o l Whi te f l u o r e s c e n c e lamps o (F20-T12-CW) i n a P s y c h r o t h e r m i n c u b a t o r a t 30 C . L i g h t i n t e n s i t y was 2 1.5 mW/cm as measured w i t h Y S I - K e t t e r i n g Mode l 65 Rad i ome te r . The shake r speed was s e t a t 100 s t r o k e s pe r m i n . The amount o f h e m o l y s i s r e s u l t i n g was q u a n t i f i c a t e d s p e c t r o p h o t o m e t r i c a l l y . Immed ia te l y a f t e r i r r a d i a t i o n , the samples were c e n t r i f u g e d . To each 100 y l o f the s u p e r n a t a n t , 900 y l D r o b k i n ' s s o l u t i o n was added , and the r e s u l t i n g s o l u t i o n s were r e a d f o r abso rbance a t 540 nm. A s s a y f o r A n t i b o d y a c t i v i t y o f c o n j u g a t e s : Ant i-M-1 m o n o c l o n a l a n t i b o d y , a f t e r c o n j u g a t i o n t o Hp was a s sayed f o r a n t i g e n b i n d i n g and s p e c i f i c i t y u s i n g the EL ISA as d e s c r i b e d above . A f r e e haema topo rphy r i n c o n t r o l was added t o one row o f each p l a t e to m o n i t o r to Hp abso rbance a t 405 nm. S t e r i l i z a t i o n o f t e s t s o l u t i o n s : A n t i b o d y , d r u g - a n t i b o d y c o n j u g a t e s and h a e m a t o p o r p h y r i n s o l u t i o n s were f i l t e r s t e r i l i z e d on Gelman A c r o d i s c 0.45 ym f i l t e r s . A l l s o l u t i o n s were d i l u t e d i n t o medium f rom s t o c k s o l u t i o n s . The d i l u t e d t e s t s o l u t i o n s were made up f r e s h f o r each assay o r used w i t h i n t h r e e days o f d i l u t i o n . A b s o r p t i o n r e a d i n g s a t 280 nm and 505 nm f o r p r o t e i n and h a e m a t o p o r p h y r i n r e s p e c t i v e l y were t aken b e f o r e and 25 a f t e r f i l t r a t i o n t o m o n i t o r f o r l o s s on f i l t e r s . Th roughou t the s t u d y , d rug o r p r o t e i n l o s s due to f i l t r a t i o n was n e g l i g i b l e . In v i t r o A s s a y f o r Hp-an t i body a c t i v i t y : C y t o t o x i c a c t i v i t y o f the ant i-M-1 h a e m a t o p o r p h y r i n c o n j u g a t e s i i i v i t r o were d e t e r m i n e d u s i n g a 0 .2% t r y p a n b l u e dye e x c l u s i o n v i a b i l i t y a s s a y . C e l l s (5 x 10^/ml) were mixed w i t h h a e m a t o p o r p h y r i n - a n t i b o d y c o n j u g a t e s (0 .2 mgHp: 0 .8 mg Ab/10^ c e l l s ) as w e l l as the a p p r o p r i a t e c o n t r o l s , and i n c u b a t e d i n the d a r k f o r two h r i n a h u m i d i f i e d 3 7 ° C 6% CO2 i n c u b a t o r . The c e l l s were washed t h r e e t imes i n RPMI 1640 (Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , New Yo rk ) medium, and r e s u s p e n d e d i n RPMI 1640 medium + 10% h e a t - i n a c t i v a t e d f o e t a l c a l f serum ( FCS ) . A l i q u o t s o f 5 x 10 c e l l s (100 y l vo lume) were p l a t e d i n s e x t e t i n m i c r o t i t r e p l a t e s ( L i n b r o C h e m i c a l s , ISO-96-Te, New Haven, C o n n . ) . Care was taken t o m a i n t a i n da rk c o n d i t i o n s d u r i n g the l a b e l l i n g and wash ing s t eps t o p r e v e n t p remature l i g h t a c t i v a t i o n . The m i c r o t i t r e p l a t e s were exposed t o i n c a n d e s c e n t l i g h t a t room t empe ra tu r e f o r 2 h. The p l a t e s were then i n c u b a t e d o v e r n i g h t ( a p p r o x i m a t e l y 16 h) a t 3 7 ° C , i n a h u m i d i f i e d 6% CO^ i n c u b a t o r . C e l l s were then examined m i c r o s c o p i c a l l y f o r v i a b i l i t y u s i n g 0 . 2 % t r y p a n b l u e . 3 The H-thymid ine i n c o r p o r a t i o n a s say was a l s o used t o a s say the c y t o t o x i c e f f e c t o f the Hp-an t i body c o n j u g a t e s . The assay f o l l o w e d the same p r o t o c o l as the 0 .2% t r y p a n b l u e dye e x c l u s i o n a s say up t o the p o i n t 3 a f t e r l i g h t e x p o s u r e . A t t h i s p o i n t , 2 uC i H-thymid ine (New E n g l a n d N u c l e a r , B o s t o n , M a s s . ) i n RPMI 1640 medium + 10% FCS (50 y l ) was added 26 t o each w e l l . The p l a t e s were i n c u b a t e d f o r 24 h i n a 37 C , 6% CO^ i n c u b a t o r . The c e l l s were then c o l l e c t e d on a MASH h a r v e s t e r as d e s c r i b e d 3 p r e v i o u s l y ( 8 0 ) . I n c o r p o r a t i o n o f H-thymid ine was measured i n coun t s per m inu te (cpm) . The l a s t i n v i t r o a s say used t o t e s t the Hp-an t i body c o n j u g a t e s was the ^ C r - r e l e a s e a s s a y . T a r g e t tumour c e l l s were l a b e l l e d w i t h ^ C r - s o d i u m chromate (New E n g l a n d N u c l e a r , B o s t o n , M a s s . ) as d e s c r i b e d p r e v i o u s l y ( 8 1 ) . T e s t c o n j u g a t e s and the a p p r o p r i a t e c o n t r o l s (2 yg Hp: 10 yg a n t i b o d y / 1 0 7 c e l l s ) were added t o 2 x 1 0 6 c e l l s i n RPMI 1640 medium per t u b e . The f i n a l volume was 200 y l p e r t u b e . The tubes were i n c u b a t e d i n the d a r k f o r 1 h r a t 3 7 ° C i n a 6% CO^ h u m i d i f i e d i n c u b a t o r . A l l a s say tubes were then washed t h r e e t imes i n medium, r e s u s p e n d e d i n RPMI 1640 medium to a f i n a l volume o f 2 ml pe r tube and exposed t o i n c a n d e s c e n t l i g h t a t 3 0 ° C f o r 2 h. A g a i n , a l l s t e p s p r i o r t o l i g h t exposu re were pe r f o rmed under d a r k c o n d i t i o n s . F o l l o w i n g l i g h t a c t i v a t i o n , the tubes were i n c u b a t e d a t 3 7 ° C , 6% CO^ f o r 6 h. The c e l l s were then sed imen ted a t 200 x g f o r 5 m i n u t e s . 0 .3 ml o f the s u p e r n a t a n t s were removed and the r a d i o a c t i v e c o n t e n t was measured on a gamma c o u n t e r (Beckman Biogamma). P e r c e n t a g e o f s p e c i f i c c y t o t o x i c i t y was c a l c u l a t e d as f o l l o w e d : = t e s t r e l e a s e (cpm)-spontaneous r e l e a s e (cpm) % s p e c i f i c c y t o t o x i c i t y X 100 maximum r e l e a s e ( cpm)-spontaneous r e l e a s e (cpm) 27 In v i v o a s say f o r Hp-an t i body a c t i v i t y : Tumour b e a r i n g mice were used t o t e s t the jLn v i v o a n t i - t u m o u r a c t i v i t y o f the a n t i-M- l - h a e m a t o p o r p h y r i n c o n j u g a t e . The an ima l s were shaved a t the tumour i n j e c t i o n s i t e (on the back ) p r i o r t o tumour i n j e c t i o n . T h i s was done t o max imize l i g h t 4 6 p e n e t r a t i o n i n t o the a f f e c t e d a r e a . 1.0 - 5.0 x 10 M-l o r 2 .0 x 10 EL4 c e l l s were i n j e c t e d s u b c u t a n e o u s l y i n the lower back r e g i o n o f DBA/2J o r B , D 0 F . m i c e . The an ima l s were r e s t e d f o r 3 - 2 4 hours and then o i. 1 i n j e c t e d i n t r a v e n o u s l y v i a t a i l v e i n w i t h t e s t c o n j u g a t e (0 .268 mg Hp: 2 .0 mg Ab/kg body we igh t ) o r the a p p r o p r i a t e c o n t r o l s . The an ima l s were e i t h e r immed i a t e l y exposed to i n c a n d e s c e n t l i g h t o v e r n i g h t o r kep t i n the d a r k f o r v a r y i n g t ime i n t e r v a l s f o l l o w e d by 4 h l i g h t e x p o s u r e s . The a n i m a l s were examined d a i l y f o r s i g n s o f tumour g row th . L i g h t sou r ce used i n the i n v i v o and i n v i t r o a s s a y s : The l i g h t i n t e n s i t y 2 used was 22 .5 mW/cm , as measured by a Y S I - K e t t e r i n g model 65 r a d i o m e t e r . The l i g h t s o u r c e was p o s i t i o n e d 24 cm away f rom the e x p e r i m e n t a l a n i m a l s o r 40 cm away f rom assay tubes o r m i c r o t i t r e p l a t e s . A s t a n d a r d 100 W GE 125 V l i g h t b u l b e m i t t i n g wave l eng ths between 200 and 1600 nm was u s e d . RESULTS The hybr idoma c e l l l i n e r e p o r t e d i n t h i s s t udy was the f u s i o n p r o d u c t o f M-l p r imed DBA/2J s p l e n o c y t e s and NS1 myeloma c e l l s . The f u s i o n p r o d u c t s were s c r e e n e d by the EL ISA method f o r ant i-M-1 s e c r e t i o n . P o s i t i v e c e l l c u l t u r e s were c l o n e d a t l e a s t t h r e e t imes and m o n i t o r e d a t each s t age f o r a n t i b o d y p r o d u c t i o n . The hybr idoma l i n e u t i l i z e d i n t h i s s t u d y , d e s i g n a t e d a n t i - M - 1 , was chosen because o f i t s h i g h a n t i b o d y t i t r e and the ease w i t h wh ich i t adap ted t o a s c i t e s g rowth . The m o n o c l o n a l p r o d u c t demons t r a t ed h i g h s p e c i f i c i t y f o r M-l a n t i g e n ( F i g . 1 ) . T h i s was not s u r p r i s i n g s i n c e immune s p l e n o c y t e s used i n the f u s i o n were f rom DBA/2J an ima l s wh i ch a re s yngene i c f o r the M-l tumour . As can be seen i n F i g u r e 1, the m o n o c l o n a l a n t i - M - 1 , when t e s t e d i n the EL ISA w i t h a v a r i e t y o f comparab le membrane p r e p a r a t i o n s f r om norma l DBA/2J t i s s u e s , o n l y showed p o s i t i v e r e a c t i v i t y w i t h M-l a n t i g e n p r e p a r a t i o n s . In c o m p a r i s o n , the i r r e l e v a n t m o n o c l o n a l an t i -G I P demons t r a t ed n e g l i g i b l e a c t i v i t y on the p a n e l o f a n t i g e n s t e s t e d i n c l u d i n g the M-l tumour c e l l a n t i g e n . When t e s t e d a g a i n s t s u b c l a s s - s p e c i f i c a n t i s e r a i n i m m u n o d i f f u s i o n , the ant i-M-1 m o n o c l o n a l a n t i b o d y was shown to be o f the IgG^ s u b c l a s s . The h a e m a t o p o r p h y r i n compound ( F i g . 2) and the ant i-M-1 m o n o c l o n a l a n t i b o d y were c h e m i c a l l y c o n j u g a t e d t h rough a l - e t h y l - 3 ( 3 - d i m e t h y l a m i n o p r o p y l ) - c a r b o d i i m i d e (EDC1) c o u p l i n g r e a c t i o n . The EDCI r e a c t i o n had no d e t r i m e n t a l e f f e c t s on the a n t i g e n b i n d i n g a b i l i t y o f the a n t i b o d y o r the photodynamic e f f e c t o f h a e m a t o p o r p h y r i n . Haematoporphy r in 29 F i g u r e 1. A c t i v i t y o f s p e c i f i c m o n o c l o n a l ant i-M-1 and n o n s p e c i f i c m o n o c l o n a l an t i -G I P on v a r i o u s a n t i g e n p r e p a r a t i o n s . Each b a r r e p r e s e n t s the amount o f a n t i b o d y a c t i v i t y on 25 yg/ml a n t i g e n as measured i n the EL ISA. Each ba r i s the mean ± S .D . f o r s i x w e l l s . 30 z o H «t CC 2. UJ or o. z < 10 pg anti M-l 10 ug anfi GIP 0 .05 .10 .15 20 ABSORBANCE AT 405 nm 31 Figure 2. The chemical structure of Haematoporphyrin. 32 33 a c t i v i t y was a s sa yed by the n o n s p e c i f i c h a e m o l y s i s o f r e d b l o o d c e l l s . E q u i v a l e n t c o n c e n t r a t i o n s o f a n t i b o d y - h a e m a t o p o r p h y r i n c o n j u g a t e , f r e e h a e m a t o p o r p h y r i n , and h a e m a t o p o r p h y r i n d e r i v a t i v e (HpD) were e q u a l l y e f f i c i e n t i n l y s i n g r e d b l o o d c e l l s ( F i g . 3 ) . The Hp i n c o n j u g a t e p r e p a r a t i o n s seem t o r e s u l t i n a s l i g h t l y h i g h e r r a t e o f h a e m o l y s i s i n compar i son t o the f r e e p o r p h y r i n , but no t q u i t e as h i g h as the HpD p r e p a r a t i o n . No h a e m o l y s i s was o b s e r v e d i n th d a r k c o n t r o l s . A n t i g e n r e a c t i v i t y o f the c o n j u g a t e s was r o u t i n e l y t e s t e d a f t e r Hp c o n j u g a t i o n . When t e s t e d on the EL ISA , the an t ibody-Hp c o n j u g a t e had the same l e v e l o f a n t i g e n b i n d i n g as e q u i v a l e n t c o n c e n t r a t i o n s o f u n t r e a t e d m o n o c l o n a l a n t i b o d y ( F i g . 4 ) , and m i n i m a l a c t i v i t y on i r r e l e v a n t a n t i g e n s ( F i g . 5 ) . S t a b i l i t y o f the an t ibody-Hp c o n j u g a t e s a f t e r l i g h t i n r r a d i a t i o n was t e s t e d to e s t a b l i s h the wo rk ing c o n d i t i o n s a t wh ich the e x p e r i m e n t s were t o be p e r f o r m e d . In t h i s e x p e r i m e n t , the c o n j u g a t e s were exposed to l i g h t f o r v a r y i n g p e r i o d s o f t ime and then t e s t e d on r e d b l o o d c e l l s f o r h a e m o l y t i c a c t i v i t y and i n the EL ISA f o r a n t i g e n r e a c t i v i t y . A f t e r 10 min o f l i g h t e x p o s u r e , a p p r o x i m a t e l y 90% o f the h a e m o l y t i c a c t i v i t y o f the Hp-con juga te was a b r o g a t e d . More than 20 min o f l i g h t exposu re b e f o r e t e s t i n g h a e m o l y s i s c aused comp le te l o s s o f Hp a c t i v i t y . A f t e r 60 min o f l i g h t e x p o s u r e , the a n t i g e n b i n d i n g a b i l i t y o f the Hp-ant i-M-1 c o n j u g a t e d e c r e a s e d t o 40% o f i t s o r i g i n a l b i n d i n g c a p a c i t y as a s s a y e d by EL ISA ( F i g . 6 ) . The c y t o t o x i c c a p a b i l i t y o f the an t i body-Hp c o n j u g a t e was t e s t e d i n  v i t r o ( F i g . 7 ) . M-l tumour c e l l s were t r e a t e d w i t h the c o n j u g a t e s , PBS o r medium c o n t r o l , a n t i b o d y a l o n e , Hp a l o n e , o r a n t i b o d y and Hp u n c o u p l e d . 34 F i g u r e 3. The h a e m o l y t i c a c t i v i t y o f f r e e haema topo rphy r i n (Hp) h a e m a t o p o r p h y r i n d e r i v a t i v e (HpD) and haematoporphy r in-an t i -M-1 c o n j u g a t e s . 10 ug o f e q u i v a l e n t p o r p h y r i n c o n c e n t r a t i o n s o f Hp --*— •-, HpD -A and Hp-anti-M-1 S & were a s sayed on human r e d b l o o d c e l l s f o r h a e m o l y t i c a c t i v i t y . Each p o i n t i s the mean o f t r i p l i c a t e s amp le s . The s t a n d a r d d e v i a t i o n s a re < 5%. R e s u l t s c o u r t e s y o f D r . Wat. Time in min. 36 F i g u r e 4. T i t r a t i o n o f s p e c i f i c m o n o c l o n a l ant i-M-1 and the Hp-ant i-M-1 c o n j u g a t e on M-l a n t i g e n . The i m m u n o l o g i c a l a c t i v i t y o f the Hp-ant i-M-1 c o n j u g a t e (o) and ant i-M-1 (•) a n t i b o d y was compared i n the EL ISA on 25 yg/ml o f M-l tumour membrane e x t r a c t s . Each p o i n t i s the average v a l u e o b t a i n e d f rom q u a d r u p l i c a t e s a m p l e s . The s t a n d a r d d e v i a t i o n s a re < 7%. 37 i 100 15 tjS 67 ug ANTIBODY per WELL 38 F i g u r e 5. A c t i v i t y o f the Hp-ant i-M-1 c o n j u g a t e s on v a r i o u s a n t i g e n p r e p a r a t i o n s . The i m m u n o l o g i c a l a c t i v i t y o f the Hp-ant i-M-1 c o n j u g a t e s (10 ug) was t e s t e d on 25 yg/ml o f tumour and norma l t i s s u e membrane e x t r a c t s i n the EL ISA. Each ba r i s the mean ± S .D . f o r s i x w e l l s . The n o n s p e c i f i c backg round c o n t r o l was the a c t i v i t y f o u n d on BSA; 0 .04 OD 39 40 F i g u r e 6. S t a b i l i t y o f h a e m a t o p o r p h y r i n and a n t i b o d y a c t i v i t y a f t e r l i g h t i r r a d i a t i o n . The Hp-ant i-M-1 c o n j u g a t e was exposed to l i g h t f o r v a r y i n g l e n g t h s o f t i m e . The l i g h t exposed p r e p a r a t i o n s were a s sayed f i r s t f o r h a e m o l y t i c a c t i v i t y (A) and then f o r a n t i b o d y a c t i v i t y ( o ) . The 100% v a l u e s a re the h a e m o l y t i c and a n t i b o d y a c t i v i t i e s o b t a i n e d f r om Hp-anti-M-1 c o n j u g a t e p r e p a r a t i o n s t h a t have had no p r i o r exposu re t o l i g h t . Each p o i n t i s the mean v a l u e o b t a i n e d f r om q u a d r u p l i c a t e s a m p l e s . The s t a n d a r d d e v i a t i o n s a re < 8%. The Y a x i s r e p r e s e n t s % a c t i v i t y o f the haema topo rphy r i n (measured by h a e m o l y s i s ) and the a n t i b o d y (measure by EL ISA) a f t e r l i g h t i r r a d i a t i o n . " 4 1 Stability of Hematoporphyrin and Antibody Activity after Light Irradiation 120-1 Period of Light Exposure Prior to Hemolysis Test 42 F i g u r e 7. In v i t r o c y t o t o x i c i t y o f Hp-ant i-M-1 and c o n t r o l s on M-l tumour c e l l s . Each ba r r e p r e s e n t s the mean v i a b l e c e l l number ± S .D . f o r s i x w e l l s a f t e r c o n j u g a t e o r c o n t r o l t r e a t m e n t (10 ug Hp: 40 yg Ab/ 5 . 2 5 x 10 c e l l s ) and l i g h t a c t i v a t i o n (22 .5 mW/cm ) . 43 3! o Q. X LJ P B S anti M - I anti M-l + H P 4) o H P ~ anti M-l - HP | CON JUGATE o 01 X 10 M - l C E L L S 44 The c e l l s were i n c u b a t e d i n a 6% CO h u m i d i f i e d i n c u b a t o r a t 37 C f o r 2 h . The c e l l s were washed t h r e e t imes i n RPMI 1640 medium i n o r d e r t o m i n i m i z e n o n s p e c i f i c p r o t e i n i n t e r a c t i o n s . The c e l l s , up t o t h i s p o i n t , were m a i n t a i n e d i n the d a r k t o p r e v e n t p remature a c t i v a t i o n o f Hp a c t i v i t y . The washed c e l l s were p l a t e d and exposed to l i g h t . When the c e l l s were examined the nex t day , n e i t h e r the ant i-M-1 a n t i b o d y , h a e m a t o p o r p h y r i n , no r a n t i b o d y and Hp t r e a tmen t had any s i g n i f i c a n t e f f e c t on tumour c e l l v i a b i l i t y i n compar i son t o the PBS c o n t r o l . However , t r e a t m e n t w i t h Hp-ant i-M-1 c o n j u g a t e e l i m i n a t e d 95% o f the tumour c e l l s . The c y t o t o x i c a c t i v i t y o f the c o n j u g a t e appea red to be a n t i g e n s p e c i f i c s i n c e when t e s t e d a g a i n s t a d i f f e r e n t tumour c e l l l i n e , E L 4 , o r no rma l DBA/2J s p l e n o c y t e s , the Hp-ant i-M-1 c o n j u g a t e d i d not demons t r a t e a s i g n i f i c a n t l e v e l o f c y t o t o x i c i t y i n compar i son t o o t h e r c o n t r o l s e . g . Hp a l o n e , Ab a l o n e ( T a b l e I ) . S i n c e the s i t e o f h a e m a t o p o r p h y r i n e f f e c t had been p r o p o s e d t o be on the membrane ( i . e . s i n g l e t oxygen a t t a c k l e a d i n g to membrane l e s i o n s ) ( 5 0 ) , 5 1 C r r e l e a s e a s says were i d e a l f o r d e m o n s t r a t i n g the d i r e c t e f f e c t o f h a e m a t o p o r p h y r i n . In t h i s a s s a y , any p e r t u r b a t i o n o f membrane i n t e g r i t y wou ld l e a d t o l e a k a g e o f the ^ " ' "C r- l abe l l ed c y t o p l a s m i c p r o t e i n s w i t h i n the t a r g e t c e l l . The c o n j u g a t e s , Hp-ant i-M-1 and Hp-an t i -L1210 were used i n a r e c i p r o c a l expe r imen t on 5 1 C r l a b e l l e d M-l and L1210 c e l l s . The a n t i b o d i e s , ant i-M-1 and a n t i - L 1 2 1 0 , f r e e h a e m a t o p o r p h y r i n , and the u n c o u p l e d Hp and a n t i b o d y m i x t u r e s d i d no t appear to have any s i g n i f i c a n t e f f e c t on the i n t e g r i t y o f M-l o r L1210 c e l l membranes ( F i g . 8 ) . However, Hp-ant i-M-1 and Hp-an t i - L1210 had a 45 Table I. E f f e c t of anti-M-l-haematoporphyrin conjugate on normal DBA/2J splenocytes and C57BL/6J EL4 lymphoma in. v i t r o *Mean C e l l Numbers (x 10 ) Treatment** Splenocytes EL4 PBS 37 . 7 + 1.5 51, .3 + 3.1 anti-M-1 36, .3 ± 1.5 49, .3 ± 3.2 haematoporphyrin 37, .3 + 1.2 46, .7 + 3.1 anti-M-1 + Hp 36, ,3 + 2.5 50, .7 + 3.0 anti-M-l-Hp conjugate 36, .9 + 2.9 47 .0 + 2.7 * Each c e l l count was the mean r e s u l t of six samples. Drug concentrations used were 10 yg Hp: 40 yg Ab per 5 x 10 2 c e l l s . Light i n t e n s i t y used f or a c t i v a t i o n was 22.5 mW/cm . 46 Figure 8. In v i t r o e f f e c t s of Hp-anti-M-1 and Hp-anti-L1210 on M - l and L1210 tumour c e l l s ( 5 1 C r - r e l e a s e assay). Hp-anti-M-1, Hp-anti-L1210, and controls were assayed on 5 1 C r - l a b e l l e d M-l (•) and L1210 (0) c e l l s . Each bar represents the mean cytotoxic index from quadruplicate samples. The standard deviations were < 7%. The Hp: Ab concentrations used were 20 ug Hp: 80 ug Ab/10 6 c e l l s . The treated c e l l s were 2 thoroughly washed and exposed to white l i g h t (22.5 mW/cm ). 47 4 8 c y t o t o x i c index of 60-657. on t h e i r r e s p e c t i v e s p e c i f i c t a r g e t c e l l s . Again the conjugate c y t o t o x i c i t y proved to be antigen s p e c i f i c since equivalent concentrations of Hp-anti-M-1 had s i g n i f i c a n t l y l e s s e f f e c t on L1210 c e l l s and Hp-anti-L1210 had a n e g l i g i b l e e f f e c t on M - l c e l l s . T i t r a t i o n of the haematoporphyrin bearing conjugates and f r e e Hp f u r t h e r d e l i n e a t e d the t a r g e t s p e c i f i c i t y of the conjugates. The c y t o t o x i c i t y of the Hp-anti-M-1, Hp-anti-L1210 conjugates and f r e e Hp was t e s t e d on M - l c e l l s at Hp concentrations ranging from 2000 to 0.2 ng Hp 5 3 per 10 c e l l s i n a v i t a l dye e x c l u s i o n assay and a H-thymidine i n c o r p o r a t i o n assay ( F i g . 9 and 10). The Hp-anti-M-1 conjugate was the only Hp compound that demonstrated any s i g n i f i c a n t l e v e l of k i l l i n g on the M - l c e l l s . The c y t o t o x i c e f f e c t ranged from 80-100% at 2000 ng Hp/10 5 c e l l s to 0-33% at 0.2 ng Hp/10 5 c e l l s . Hp alone and Hp-anti-L1210 conjugate had minimal c y t o t o x i c i t y i n t h i s range i n comparison to the PBS c o n t r o l . Since the Hp-anti-M-1 conjugate proved e f f e c t i v e and s p e c i f i c i n  v i t r o , f u r t h e r experiments were c a r r i e d out to e s t a b l i s h i t s a c t i v i t y on tumour i_n s i t u . The conjugate (2.0 mg anti - M-1: 0.268 mg Hp per kg body wt) or the appropriate c o n t r o l s were administered i n t r a v e n o u s l y to animals which had been i n j e c t e d subcutaneously w i t h 5 x 10 M - l c e l l s ( F i g . 11). The conjugate demonstrated high s p e c i f i c i t y f o r the in. s i t u M - l tumour (Table I I ) . S i g n i f i c a n t l y longer l a g time (12.2 ± 1.8 days a f t e r tumour i n o c u l a t i o n ) p r i o r to development of palpable tumour was seen i n animals t r e a t e d w i t h l i g h t a c t i v a t e d Hp-anti-M-1 conjugate. The other c o n t r o l t r e a t e d animals developed palpable tumour masses i n a 49 Figure 9. Titration of haematoporphyrin, Hp-anti-M-1 and Hp-anti-L1210 activity on M-l tumour cells ( v i t a l dye exclusion assay). The photocytotoxic activity of different concentrations of haematoporphyrin (•), Hp-anti-M-1 (4) , and Hp-anti-L1210 ( I ) was assayed on M-l tumour cel l s . The v i a b i l i t y of the test cells was determined with 0.2% Eosin Y v i t a l dye. 50 2 0 0 0 2 0 0 2 0 2.0 0.2 S HP CONC. ng / 10 c t l l s 51 F i g u r e 10. T i t r a t i o n o f h a e m a t o p o r p h y r i n , Hp-anti-M-1 and Hp-anti-L1210 3 a c t i v i t y on M-l tumour c e l l s ( H-thymid ine i n c o r p o r a t i o n a s s a y ) . The p h o t o c y t o t o x i c a c t i v i t y o f d i f f e r e n t c o n c e n t r a t i o n s o f h a e m a t o p o r p h y r i n (•), Hp-anti-M-1 (4), and Hp-anti-L1210 (*) was a s sayed on M-l tumour 3 . . c e l l s . S u r v i v a l o f the t e s t c e l l s was measured by H- thymidme i n c o r p o r a t i o n . 53 F i g u r e 1 1 . P r o t o c o l used i n d e m o n s t r a t i n g the e f f e c t o f Hp-ant i-M-1 and c o n t r o l s i n v i v o on tumour b e a r i n g m i c e : P r o t o c o l I. The t r e a t e d mice were exposed f o r 18 h r w i t h wh i t e l i g h t o f i n t e n s i t y 22 .5 mwVcm 2. 54 18 hr. light Dark Control ( maintained in darkness until all animals developed palpable tumours. ) Table II. Effect of anti-M-l-haematoporphyrin conjugate on M-l tumour growth in vivo Mean Tumour Induction Time3 S.P. (days) Treatment Nd) Light Treatment N Dark Treatment0^ haematoporphyrin 10 9.0 ± 1.6 5 7.2 ± 1.1 anti-M-1 5 7.2 ± 1.1 - N.D. drug conjugated to anti-M-1 10 12.1 ± 1.8C> 5 7.2 ± 1.6 drug conjugated to non-specific monoclonal anti-GIP 10 7.5 ± 1.9 5 7.8 ± 1.1 anti-M-1 + haematoporphyrin 10 8.4 ± 1.5 5 8.0 ± 1.2 anti-GIP + haematoporphyrin 10 7.5 ± 1.9 5 7.8 ± 1.1 PBS 5 6.7 ± 1.0 - N.D. a^ Animals were palpated every day. The examinations were carried out blind.Because tumour cells were injected subcutaneously on the back, tumour masses of 2 mm in diameter could be detected relatively easily. These animals were maintained in the dark until they developed palpable tumours. N.D., not determined. c) This time is significantly different from a l l others at P = 0.0005. d) N = number of experimental animals tested. 56 s i g n i f i c a n t l y shorter period of time (6-9 days). Animals not exposed to l i g h t a f t e r Hp treatment developed tumours in 7-8 days. The Hp-anti-M-1 conjugate and appropriate controls had no e f f e c t on the growth of i r r e l e v a n t tumour, EL4 in vivo (Table I I I ) . The r e s u l t s shown in Tables II and III d i r e c t l y correlated with the jji v i t r o r e s u l t s shown in F i g . 6 and Table I. Hp or antibody alone, Hp and antibody uncoupled had no e f f e c t on M - l tumour growth. Light proved to be e s s e n t i a l f o r drug a c t i v i t y i n vivo since conjugate-treated animals kept in the dark developed tumours at the same rate as the control animals e.g. Hp alone, Ab alone, etc. (Table I I ) . S p e c i f i c recognition of the tumour c e l l s by the monoclonal antibody was also e s s e n t i a l f o r maximal drug e f f e c t . Conjugates made with an i r r e l e v a n t monoclonal antibody (anti-GIP) had no e f f e c t on M - l tumour growth. Though a s i g n i f i c a n t anti-tumour e f f e c t was shown with Hp-anti-M-1 conjugate on M - l in. s i t u , t h i s was only on the l e v e l on tumour development. Thus, even i f tumour appearance was s i g n i f i c a n t l y delayed, the fa c t remained that tumour d i d eventually develop r e s u l t i n g in the death of test animals. So the t e s t parameters of the f i r s t iri vivo experiment were re-examined to see i f animal s u r v i v a l could be improved. In the above in vivo experiments, the tumour bearing animals were exposed to l i g h t immediately a f t e r intravenous i n j e c t i o n . These experiments d i d not take into account the time which may be required f o r optimal l o c a l i z a t i o n of the antibody at the tumour s i t e , and much of the conjugate's p o t e n t i a l effectiveness could be l o s t through drug a c t i v a t i o n while i t was f r e e l y c i r c u l a t i n g . In the next experiment, the Table III. Effect of anti-M-l-haematoporphyrin conjugate on the growth of EL4 tumour in B6D2 mice Mean Tumour Mean Survival Treatment3) Nb) Induction Time ± S.D. Time ± S.D. (days) (days) haematoporphyrin 9 6.7 ± 1.9 19.6 ± 1.4 anti-M-1 9 6.8 ± 1.9 19.8 ± 2.0 drug conjugated to anti-M-1 9 7.0 ± 2.3 19.9 ± 1.9 anti-M-1 + haematoporphyrin 9 6.9 ± 1.8 19.9 ± 1.5 V a^ The animals were treated with light (22.5 mW/cm2). b) N = # of experimental animals 0.268 mg Hp: 2.0 in each group. mg Ab/kg body weight, and exposed to 58 conjugate-treated animals were maintained in the dark f o r varying lengths of time, from 0 to 192 hours, and then exposed to l i g h t ( F i g . 12). The r e s u l t s (Table IV) c l e a r l y demonstrated that as the period of darkness increased, the mean time for development of palpable tumour also s i g n i f i c a n t l y increased. Conjugate treated animals that were l e f t in the dark f o r 120 to 192 hours p r i o r to l i g h t exposure took s i g n i f i c a n t l y longer to develop tumours. In addition 7-30% of the test animals (depending on the groups) remained tumour-free. They were considered cured since they remained tumour-free f o r 6 months a f t e r treatment. The enhanced anti-tumour a c t i v i t y was not due to nonspecific stress factors caused by the dark conditions or nonspecific a f f i n i t y of Hp for malignant tis s u e , since animals treated with Hp alone, Ab alone or PBS demonstrated no such lag period f or tumour occurrence (Table V) or increased rate of s u r v i v a l (not shown). 59 F i g u r e 12. P r o t o c o l used i n d e m o n s t r a t i n g the i n v i v o e f f e c t o f Hp-ant i-M-1 i n a t i m i n g e x p e r i m e n t : P r o t o c o l I I . T r e a t e d mice were 2 exposed to wh i t e l i g h t (22 .5 mW/cm ) f o r 4 h r . 60 Table IV. The influence of timing of dark incubation followed by light exposure of M-l bearing DBA/2J mice treated with haematoporphyrin-anti-M-1 conjugate on the time of palpable tumour induction and survival. # of hours Mean tumour induction Mean Survival in darkness N time ± Sx (days) P* time ± Sx (days) P* % Survivors 0 14 13.9 + 1.8 31.9 + 3.8 0 12 7 15.0 + 3.3 32.7 + 3.4 0 24 7 15.1 + 3.2 33.0 + 4.5 0 48 7 15.9 + 3.1 34.0 + 3.9 0 96 14 15.9 + 3.2 35.6 + 3.8 0.01 7 120 7 16.3 + 2.6 0.025 34.7 + 4.0 0 144 7 17.3 + 2.3 0.005 33.9 + 4.2 14 168 8 16.3 + 1.6 0.025 36.0 + 4.8 0.025 25 192 14 18.7 + 3.8 0.005 39.7 + 5.9 0.005 30 Values were compared to the 0 time control group by Student's t-test. * wSurvival designates those animals which remain free of tumour during the course of the experiments and for periods up to 6 months thereafter. Drug concentrations used were 0.268 mg Hp: 2.0 mg Ab/kg body weight. Table V. The influence of timing of dark incubation followed by l i g h t exposure of M-l bearing DBA/2J mice treated with haematoporphyrin, anti-M-1 or PBS on palpable tumour induction. Mean Tumour Induction Time Sx (days)  # of hours i n darkness N haematoporphyrin anti-M-1 PBS 0 5 8.0 ± 2.9 8.1 ± 2.0 8.1 ± 2.1 48 5 8.0 ± 2.9 N.D. N.D. 96 10 8.2 ± 1.8 N.D. N.D. 192 10 8.3 ± 1.8 8.2 ± 1.9 8.1 ± 2.0 192 10 7.9 ± 1.8 8.0 ± 2.9 8.2 ± 2.0 Drug concentrations used were 0.268 mg Hp/kg body weight and 2.0 mg Ab/kg body weight. 63 DISCUSSION The purpose of t h i s study was to e s t a b l i s h the p o t e n t i a l use of photoimmunotherapy as a method of cancer treatment. Photochemical therapy has already been investigated i n both human cancers and experimental animal models. I t has been shown to be e f f e c t i v e , within l i m i t s , i n the treatment of a number of carcinomas including: breast, colon, prostate, renal c e l l , squamous c e l l , basal c e l l , endometrium, lung, retinoblastoma, mycosis fungosides, angrosarcoma, chrondrosarcoma and non-pigmented malignant melanoma (62,64,68). Although photochemical agents have been shown to be e f f e c t i v e , the side e f f e c t s ; i . e . t h e i r e f f e c t s on normal tis s u e , are not t r i v i a l . With the present therapeutic dose l e v e l s and protocols used, patients run the r i s k of s u f f e r i n g moderate to severe l i g h t r e l a t e d skin reactions (69). In the murine model, Dougherty found that normal animals treated with 5.0 mg per kg body weight of haematoporphyrin i n t r a p e r i t o n e a l l y followed by 1 h exposure 24.hr l a t e r had a 50% m o r t a l i t y rate (82). Adverse skin reactions such as erythema, edema, and necrosis have been documented in other animal models (42,49). Haematoporphyrin has been shown to have a tendency to accumulate in neoplastic, embryonic, and regenerating t i s s u e s , and in lymph nodes (41). An approach to improving therapeutic r e s u l t s with Hp include attempts to minimize side e f f e c t s by c o n t r o l l i n g the timing and l o c a t i o n of l i g h t exposure ( i . e . , by increasing the time i n t e r v a l between administration of the photochemical and l i g h t exposure) (61). I s o l a t i o n 64 of l i g h t exposure at the tumour s i t e with lasers or optic f i b e r s i s another method being investigated (68,83). Monoclonal antibodies with s p e c i f i c i t y for tumour antigens on t h e i r own, have been shown to have a therapeutic e f f e c t in some cases (20,21,84,85). However, in many cases, anti-tumour antibodies in i s o l a t i o n have not been found to demonstrate any marked tumouricidal e f f e c t . In f a c t , they have been shown to cause an enhancement of tumour growth in some studies (86). I t i s common f e e l i n g that, by and large, antibodies with s p e c i f i c i t y f o r tumour c e l l s , even monoclonal antibodies, are not l i k e l y to have a dramatic b e n e f i c i a l e f f e c t on the course of in vivo tumour growth. This was borne out by the r e s u l t s in Table I I . Anti-M-1 monoclonal antibody at a concentration of 2 mg per kg body weight had no e f f e c t on M-l tumour growth jun vivo. But i t i s possible that the usefulness of anti-tumour monoclonal antibodies may l i e more in t h e i r d i r e c t i o n a l a b i l i t y rather than in t h e i r i n t r i n s i c cytotoxic a c t i v i t y . The p o t e n t i a l of antibodies could be as a device to increase drug-tumour s p e c i f i c i t y . Cytotoxic drug-anti-tumour monoclonal antibody conjugates should permit l o c a l i z a t i o n of the drug at tumour s i t e s and thus minimize generalized cytotoxic e f f e c t s of the drug. The experiments reported here have used a monoclonal antibody with s p e c i f i c i t y f o r the M-l myosarcoma of DBA/2J mice conjugated to haematoporphyrin, a cytotoxic agent which i s active only when i t i s exposed to l i g h t . The monoclonal antibody was shown, using an ELISA, to have s p e c i f i c i t y f o r a membrane antigen of M-l tumour t i s s u e . Experimental procedures showed that both haematoporphyrin and the 65 monoclonal antibodies retained t h e i r b i o l o g i c a l a c t i v i t y and s p e c i f i c i t y , r e s p e c t i v e l y following conjugation with ECDI. In v i t r o experiments with M - l and EL4 tumour c e l l s , and normal DBA/2J spleen c e l l s showed that the conjugate had both s p e c i f i c i t y f o r , and the a b i l i t y to destroy M - l tumour c e l l s in the presence of l i g h t . Subsequent in. vivo experiments demonstrated c o r r e l a t i o n with the preliminary i i i v i t r o studies. In the f i r s t experiments, M - l tumour bearing animals were injec t e d intravenously with the monoclonal antibody-haematoporphyrin conjugate (or the appropriate controls) and immediately exposed to l i g h t (or kept in the dark as dark c o n t r o l s ) . In these experiments, only those animals injected with the s p e c i f i c conjugate and exposed to l i g h t showed a s i g n i f i c a n t delay in the development of palpable tumours. Equivalent animals l e f t i n the dark developed tumours at the same rate as did the controls. In these experiments, animals received a t o t a l dose of 0.268 mg per kg body weight of Hp in antibody conjugate form. In experiments using haematoporphyrin alone for cancer therapy, dosages in the range of 2.5 - 5.0 mg per kg body weight in mice (61), 5.0 mg per kg body weight in cats and dogs (87), and 2.5 to 5.0 mg per body weight in humans (62,68,69) have been used. Thus, we showed that antibody-haematoporphyrin conjugates were e f f e c t i v e i n tumour growth retardation at doses considerably lower than the doses of free Hp found to be e f f e c t i v e . Further experiments were c a r r i e d out to determine whether the haematoporphyrin-antibody conjugates were more e f f e c t i v e , i f animals following i n j e c t i o n were l e f t in the dark for various time i n t e r v a l s p r i o r to l i g h t exposure. The r e s u l t s showed that M-l bearing mice treated with 66 the tumour s p e c i f i c c o n j u g a t e d i d s i g n i f i c a n t l y b e t t e r when they were l e f t i n the da rk 92-196 h p r i o r t o l i g h t e x p o s u r e . An ima l s so t r e a t e d d e v e l o p e d p a l p a b l e tumours more s l o w l y , s u r v i v e d l o n g e r , and demons t r a t ed c u r e r a t e s t h a t were s i g n i f i c a n t l y d i f f e r e n t f rom o t h e r g roups o f e x p e r i m e n t a l a n i m a l s . These r e s u l t s imp l y t h a t c o n s i d e r a b l e t ime i s r e q u i r e d f o r a n t i b o d y t o l o c a t e and c o n c e n t r a t e on s m a l l _in s i t u tumour masses . An ima l s wh i ch rema ined t umour- f r ee a f t e r t r e a tmen t showed no d e l e t e r i o u s s i d e e f f e c t s . The tumour i n j e c t i o n s i t e , c l e a r l y v i s i b l e due to s h a v i n g , and the s u r r o u n d i n g a r e a had no v i s i b l e symptoms o f norma l t i s s u e damage e . g . e ry thema and edema. T h i s i n d i c a t e d t h a t the amounts o f h a e m a t o p o r p h y r i n a d m i n i s t e r e d (0 .268 mg per kg body we igh t ) were be low t o x i c l e v e l s . T h u s , i t wou ld appear t h a t photo immunotherapy may be a r e a l i s t i c app roach t o the t r e a t m e n t o f tumours . The s p e c i f i c i t y c o n t r i b u t e d by the m o n o c l o n a l a n t i b o d y p e r m i t t e d c o n c e n t r a t i o n o f t he d rug a t the tumour s i t e . T h i s p r o p e r t y c l e a r l y made i t p o s s i b l e t o use lower dose l e v e l s o f d r u g , wh ich m i n i m i z e d u n d e s i r e d s i d e e f f e c t s . CHAPTER I I I PHOTOIMMUNOTHERAPY: HUMAN STUDIES 68 C h a p t e r I I I INTRODUCTION Human n o n - l y m p h o b l a s t i c l e u k a e m i a was chosen to t e s t the p o t e n t i a l o f photo immunotherapy i n man. Acu t e n o n - l y m p h o c y t i c l e u k a e m i a (ANLL) i s the most common acu te l e u k a e m i a o c c u r r i n g i n i n f a n t s be low one y e a r o f age and i n the m i d d l e aged and e l d e r l y . I t i s c h a r a c t e r i z e d by h i g h l e v e l s o f c i r c u l a t i n g m y e l o b l a s t s a t c o n c e n t r a t i o n s o f 2 ,000 t o 5 0 , 0 0 0 / u l , anemia , g r a n u l o c y t o p e n i a o r t h r o m b o c y t o p e n i a i n o ve r 90% o f p a t i e n t s ( 8 8 ) . Auer b o d i e s ( f u s e d a z u r o p h i l g r a n u l e s ) a re f ound i n some m y e l o b l a s t s i n 50% o f the p a t i e n t s . P r o g n o s i s i s g e n e r a l l y p o o r . D e s p i t e t r e a t m e n t , one t h i r d o f a l l p a t i e n t s f a i l t o e n t e r comp le t e r e m i s s i o n and o n l y 10 - 15% o f a l l p a t i e n t s a re s t i l l a l i v e 5 y e a r s a f t e r d i a g n o s i s . C h r o n i c g r a n u l o c y t i c l e u k a e m i a (CGL) o c c u r s more commonly i n m i d d l e age , bu t i t has been found i n p a t i e n t s o f a l l a g e s . I t i s c h a r a c t e r i z e d by marked g r a n u l o c y t o s i s i n the b l o o d , u s u a l l y 1 0 0 , 0 0 0 / y l , i n wh ich a l l d i f f e r e n t i a t i o n s t a g e s f rom m y e l o b l a s t s t o p o l y m o r p h o n u c l e a r g r a n u l o c y t e s a re p r e s e n t ( 8 9 , 9 0 ) . The P h i l a d e l p h i a (Ph"S chromosome i s c h a r a c t e r i s t i c o f CGL. I t i s f o u n d i n 90 - 100% o f p r o l i f e r a t i n g marrow c e l l s i n g r e a t e r than 90% o f CGL p a t i e n t s . The d i s e a s e c o u r s e o f CGL i s c h a r a c t e r i z e d by two p h a s e s , an i n i t i a l phase ( c e l l m a t u r a t i o n i s n o r m a l , and the d i s e a s e i s r e s p o n s i v e t o t h e r a p y ) , f o l l o w e d by t r a n s f o r m a t i o n t o an a cu te o r b l a s t i c c r i s i s phase ( m a t u r a t i o n a r r e s t a t m y e l o b l a s t o r l y m p h o b l a s t l e v e l and r e f r a c t o r y t o t h e r a p y ) . The median s u r v i v a l o f CGL p a t i e n t s was 32 months f rom o n s e t o f 69 symptoms and about 20 months f rom d i a g n o s i s . A f t e r the d i s e a s e t r a n s f o r m s t o the a cu te p h a s e , the medium s u r v i v a l i s < 6 months ( 9 1 ) . A p o t e n t i a l t h e r a p e u t i c m o d a l i t y f o r ANLL and CGL p a t i e n t s i s i n t e n s i v e c h e m o r a d i o t h e r a p y f o l l o w e d by bone marrow t r a n s p l a n t a t i o n . Acu t e l e u k a e m i a and the i n i t i a l phase o f CGL e x h i b i t a dose r e l a t e d r e sponse t o d rug o r r a d i a t i o n t h e r a p y ; the g r e a t e r the t h e r a p e u t i c d o s e , the g r e a t e r the c y t o t o x i c e f f e c t ( 9 2 ) . However the dosage used i s l i m i t e d by t o x i c i t y t o norma l t i s s u e . The d o s e - l i m i t i n g e f f e c t i s the s u p p r e s s i o n o f the h a e m a t o p o i e t i c o r bone marrow c e l l s . More e f f e c t i v e , i . e . h i g h e r , i r r a d i a t i o n o r d rug dosages c o u l d be used i f the p a t i e n t ' s h a e m a t o p o e t i c sys tem c o u l d be r e s c u e d by t r a n s p l a n t a t i o n o f normal v i a b l e bone marrow c e l l s a f t e r c h e m o r a d i o t h e r a p y . A t y p i c a l fo rmat f o r t r e a t m e n t wou ld be as f o l l o w s : The l e u k a e m i c p a t i e n t s wou ld r e c e i v e h i g h doses o f chemotherapy and/or i r r a d i a t i o n . The doses wou ld be h i g h enough t o e l i m i n a t e a l l m a l i g n a n t c e l l s and s u b s e q u e n t l y h i g h enough t o d e s t r o y norma l bone marrow. The p a t i e n t would then be t r e a t e d w i t h i n t r a v e n o u s i n f u s i o n s o f bone marrow c e l l s f rom i ) g e n e t i c a l l y i d e n t i c a l tw in i . e . s y n g e n e i c bone marrow; i i ) HLA i d e n t i c a l s i b l i n g donor i . e . a l l o g e n e i c bone marrow o r i i i ) t h e m s e l f i . e . a u t o l o g o u s bone marrow c e l l s . The c e l l s wou ld home i n to the bone marrow and r e - e s t a b l i s h a f u n c t i o n a l h aema topoe t i c s ys tem i n the p a t i e n t . Fo r a u t o l o g o u s t r a n s p l a n t a t i o n , bone marrow c e l l s must be drawn f rom p a t i e n t s p r i o r t o c h e m o r a d i o t h e r a p y . I d e a l l y , t h i s s h o u l d be done a t a t ime when the p a t i e n t ' s bone marrow appears normal and f r e e f rom m a l i g n a n c y ( i . e . , i n comp le te r e m i s s i o n ) . The p r e s e n c e o f c l o n o g e n i c 70 tumour c e l l s i n the bone marrow p r e p a r a t i o n c o u l d be cause o f l eukaem i c r e l a p s e i n t hese c a s e s . S e v e r a l t r e a tmen t c e n t r e s have t e s t e d a u t o l o g o u s bone marrow t r a n s p l a n t a t i o n s i n ANLL p a t i e n t s . The r e s u l t s were no t c o m p l e t e l y s u c c e s s f u l . Many p a t i e n t s s u f f e r e d a r a p i d r e l a p s e o f l e u k a e m i a , the medium d u r a t i o n o f r e m i s s i o n was 3 - 5 months . But a p p r o x i m a t e l y 20% o f the p a t i e n t s s u r v i v e d l o n g e r than 2 y e a r s w i t h o u t any c l i n i c a l s i g n s o f the d i s e a s e ( 9 3 , 9 4 ) . CGL p a t i e n t s t r e a t e d i n the a cu t e phase o f the d i s e a s e have a median s u r v i v a l t ime o f a p p r o x i m a t e l y 6 months . The median d u r a t i o n o f r e m i s s i o n i s l e s s than 4 months and o n l y 10% s u r v i v e more than 2 y e a r s ( 9 5 , 9 6 ) . In a u t o l o g o u s bone marrow t r a n s p l a n t s o f CGL p a t i e n t s , the b e s t e f f e c t t h a t can be a c h i e v e d i s the r e - e s t a b l i s h m e n t o f the c h r o n i c phase o f d i s e a s e s i n c e the t r a n s p l a n t e d bone marrow w i l l c o n t a i n P h ^ - p o s i t i v e l e u k e m i c c e l l s ( c h a r a c t e r i s t i c o f CGL bone marrow) . Shipman e t a l . have r e p o r t e d a p u r i f i e d a n t i g e n , CAMAL, d e r i v e d f rom ANLL c e l l s ( 9 7 ) . When p a r t i a l l y c h a r a c t e r i z e d , i t was shown to be a p o l y p e p t i d e o f m o l e c u l a r w e i g h t , 68 ,000 d a l t o n s w i t h a p i o f 7 . 16 . A m o n o c l o n a l a n t i b o d y , CAMAL—1, was d e v e l o p e d a g a i n s t the a n t i g e n ( 9 8 ) . CAMAL was f ound to be a common t u m o u r - a s s o c i a t e d marker on human n o n - l y m p h o b l a s t i c l e u k a e m i a . Logan e t a l . . demons t r a t ed t h a t CAMAL-1 was a b l e t o p o s i t i v e l y s t a i n a s i g n i f i c a n t number o f c e l l s i n samples f rom CGL and AML p a t i e n t s ( g r e a t e r than 1% o f c e l l s ) i n immunoperoxidase s i n g l e c e l l s l i d e t e s t s (99) . Normal p e r i p h e r a l b l o o d l ymphocy tes o r bone marrow c e l l s demons t r a t ed m i n i m a l s t a i n i n g w i t h CAMAL-1. 71 In t h i s study, CAMAL-1 monoclonal antibody chemically coupled to haematoporphyrin was tested on normal and leukaemic c e l l s f o r s p e c i f i c c y t o t o x i c i t y . In the past, several d i f f e r e n t approaches have been used to eliminate tumour c e l l s from bone marrow preparations. Tumour c e l l s could be removed p h y s i c a l l y by separation on a bovine serum albumin gradient (100), or by d i f f e r e n t i a l agglutination with l e c t i n s (101-103). They could be destroyed with s e l e c t i v e drugs such as 4-hydroperoxycyclopho-sphamide (104). A l t e r n a t e l y , the tumour c e l l s could be eliminated by s p e c i f i c destructive a c t i v i t y of anti-tumour antibodies in the presence of complement (105) or toxin linked antibodies (26,27,106). Hp-CAMAL-1 a c t i v i t y was examined to see whether photoimmunotherapy could be u t i l i z e d as a purging method for cleansing bone marrow suspensions of tumour c e l l s . 72 Chap t e r I I I MATERIAL AND METHODS P a t i e n t Samples : H e p a r i n i z e d bone marrow a s p i r a t e s and p e r i p h e r a l b l o o d samples were o b t a i n e d f rom the Department o f Haemato logy , Vancouve r G e n e r a l H o s p i t a l , o r the Cance r C o n t r o l Agency o f B r i t i s h C o l u m b i a . The g r a n u l o c y t e and mononuc lea r c e l l p o p u l a t i o n s were i s o l a t e d by F i c o l l - H y p a q u e s e p a r a t i o n ( 1 0 7 , 1 0 8 ) . The c e l l s were washed a t l e a s t t h r e e t imes i n serum f r e e D u l b e c c o ' s M o d i f i e d E a g l e (DME) medium (G ib co L a b o r a t o r i e s , Grand I s l a n d , New Yo rk ) and r e suspended i n DME medium t o a g c e l l d e n s i t y o f 0 .5 - 1.0 x 10 pe r m l . P r e p a r a t i o n and i s o l a t i o n o f a n t i b o d i e s : The CAMAL-1 m o n o c l o n a l a n t i b o d y p r o d u c i n g c e l l l i n e was d e v e l o p e d by D r . Andrew Ma l co lm ( 9 8 ) . I t was r a i s e d a g a i n s t a tumour a s s o c i a t e d 68 ,000 d a l t o n a n t i g e n , CAMAL, i s o l a t e d f rom ANLL b l a s t c e l l membrane e x t r a c t s on 7.5% p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s g e l s ( 9 7 ) . CAMAL-1 a n t i b o d y was demons t r a t ed to be a n t i g e n s p e c i f i c f o r most bone marrow and PBL c e l l s o f ANLL and CGL p a t i e n t samples w i t h the immunoperoxidase a ssay ( 9 9 ) . R a b b i t ant i-CAMAL (R a CAMAL), r a b b i t ant i-human PBL membrane e x t r a c t (R a Hu) and r a b b i t an t i-mouse a n t i b o d y (R a MIg) a n t i s e r a were p roduced i n young f ema le a l b i n o r a b b i t s wh ich were immunized a t month l y i n t e r v a l s w i t h a 50% comple te F r e u n d ' s ad j u van t e m u l s i o n o f the v a r i o u s a n t i g e n s a t a c o n c e n t r a t i o n o f 50 ug per m l . They were b l e d f rom the m a r g i n a l ea r v e i n 7 and 10 days a f t e r i m m u n i z a t i o n . The norma l 73 peripheral blood leukocyte (NPBL) membrane extract was produced from a pooled sonicated preparation of normal PBLs (109). The antigen (CAMAL) was produced as described above. Mouse Ig used was a pooled mixture of d i f f e r e n t mouse IgG isotypes (Miles Laboratories Ltd.). These antisera demonstrated s p e c i f i c p o s i t i v e r e a c t i v i t y when treated by ELISA, FACS IV, and immunoperoxidase assays on t h e i r respective antigen or c e l l type (98,99,109,110). An i r r e l e v a n t rabbit antiserum (Raid) and monoclonal antibody (a L1210 or a M-l) were used as negative controls in t h i s study. P u r i f i c a t i o n and i s o l a t i o n of the antibodies was as described in Chapter I I . R a CAMAL antisera were a f f i n i t y p u r i f i e d on an CAMAL immunoabsorbant column (CAMAL coupled to cyanogen bromide activated Sepharose 4B beads) (Pharmacia Fine Chemicals, Uppsala, Sweden). Rabbit antiserum to CAMAL was run over a CAMAL immunoabsorbent column (beads contained 2.0 mg CAMAL per ml). The column was washed with PBS u n t i l e f f l u e n t had no absorbance at 280 nm. The column bound antibodies were then eluted with 0.1 N HCl. The protein containing f r a c t i o n s were detected by absorbance at 280 nm and immediately neutralized to pH 7 with 5% Na^CO^. R a MIg was i s o l a t e d in the same manner over a mouse Ig immunoabsorbant column (78). Preparation of Haematoporphyrin-antibody conjugates: The Hp-antibody conjugates used in the human studies were prepared in the same manner as described in Chapter I I . In t h i s study, the r e l a t i v e quantities of Hp bound to the i n d i v i d u a l monoclonal antibodies and rabbit antisera were as follows: 74 Antibody Hp (ms) bound/ms antibody CAMAL-1 0.260 R a CAMAL 0.064 R a i d 0.046 R a MIg 0.085 R a Hu 0.107 Haematoporphyrin concentration was calculated by taking the absorbance at 505 nm of 0.644 for 80 ug Hp/ml. Protein concentration was determined by taking the absorbance at 280 nm of 1.4 f o r 1.0 mg protein/ml. Testing of antibody a c t i v i t y a f t e r conjugation was done on ELISA as described below. Hp a c t i v i t y was also tested a f t e r conjugation as described in Chapter I I . ELISA: CAMAL-1, R a CAMAL and R a Hu antisera were screened for r e a c t i v i t y using an enzyme-linked immunosorbent assay (ELISA) as described in Chapter I I . In t h i s case, CAMAL or NPBL membrane extract were coated on ELISA m i c r o t i t r e plates (Cooke Engineering, Alexandra, Va.) at 300 ng per ml in 0.05 M carbonate buffer, pH 9.6 (100 u l per well) overnight at 4°C. The plates were washed three times with PBS-Tween 20 buffer and te s t antisera was added. Af t e r 1.5 h incubation at room temperature, the plates were washed three times with PBS-Tween 20 buffer and 100 y l per well of the developing a l k a l i n e phosphatase-labelled rabbit anti-mouse or sheep a n t i - r a b b i t Ig was added (1:5000 and 1:1500 d i l u t i o n s r e s p e c t i v e l y ) . Plates were incubated for another 1.5 h and washed three 75 times with PBS-Tween 20 buffer. The wells were then tested f o r a l k a l i n e phosphatase r e a c t i v i t y with 100 y l of enzyme substrate buffer (Sigma-104-105, p-nitrophenyl phosphate disodium) at room temperature for 1 h. The plates were read f or absorbance at 405 nm in a T i t e r t e k Multiskan (Flow Laboratories, Inglewood, C a l . ) . S t e r i l i z a t i o n of the te s t solutions: A l l test solutions were d i l u t e d to the appropriate concentrations in DME medium from the stock solutions. They were s t e r i l i z e d by f i l t r a t i o n and treated as described i n Chapter I I . In v i t r o assay of Hp-antibody conjugate a c t i v i t y : C y t o t o x i c i t y of the Hp-antibody conjugates on patient samples was determined in an Eosin Y 3 v i t a l dye exclusion assay or by H-thymidine incorporation. Aliquots of 0.5 - 1.0 x 10 7 c e l l s (100 y l ) in serum free medium were added to 17 x 100 nm Falcon t e s t tubes (Becton, Dickinson and Co., Cockeysville, M.D.). S t e r i l e solutions of Hp-antibody conjugates, haematoporphyrin, or antibody (100 y l ) were added at a concentration of 2 yg Hp: 10 yg 7 o o Ab per 10 c e l l s . The c e l l s were incubated at 4 C or 37 C f o r 1 h. The c e l l s were washed three times in serum free DME medium. Up to t h i s point, a f t e r addition of Hp solutions, the experiment was performed in dim l i g h t i n g to prevent premature Hp a c t i v a t i o n . A f t e r the l a s t wash, each c e l l p e l l e t was resuspended to 13 ml DME medium and exposed to l i g h t at 30°C for 1 h. Afterwards, the c e l l s were sedimented and resuspended in 5 ml of 10% FCS + 10% lymphocyte conditioned medium (LCM) +0.5 unit 76 erythropoietin (EPO) per ml Alpha medium (Terry Fox Laboratory, Vancouver, B.C.). An aliquot of 50 u l from each treated c e l l suspension and an add i t i o n a l 150 p i of 10% LCM +0.5 unit EPO per ml ALpha medium was plated in quintuplet in Linbro m i c r o t i t r e wells. The remaining c e l l suspensions and m i c r o t i t r e plates were incubated at 37°C, in a 6% CO^ incubator. The following day (af t e r 16 h incubation), the treated c e l l suspensions were assayed f o r vi a b l e c e l l numbers with 0.2% Eosin Y dye. 3 The m i c r o t i t r e plates were incubated f o r 4 days. H-thymidine incorporation was assayed on day 4. On day 3, 100 u l of the supernatant 3 was removed a s e p t i c a l l y from each w e l l . 2 yCi H-thymidine (New England Nuclear, Boston, Mass.) in 100 u l of 10% FCS + 10% LCM +0.5 unit EPO per ml Alpha medium was added per well. On day 4, the c e l l s were c o l l e c t e d on a MASH harvester as previously described (80). 3 H-thymidine was measured as counts per min (cpm) in a Packard Tri-Carb 4550 s c i n t i l l a t i o n counter (United Technologies). Light Source: The l i g h t source used was an incandescent l i g h t source 2 2 described in Chapter I I . The l i g h t i n t e n s i t y used was 22.5 mW /cm with a spectrum ranging from 200-1600 nm. Media: The lymphocyte conditioned medium (LCM), Alpha medium, and EPO solutions were prepared and obtained from the B r i t i s h Columbia Cancer Research Centre (Terry Fox Laboratory). 77 C h a p t e r I I I RESULTS The hyb r i doma c e l l l i n e u t i l i z e d i n t h i s s tudy was the f u s i o n p r o d u c t o f CAMAL p r i m e d BALB/C s p l e n o c y t e s and NS1 myeloma c e l l s ( 9 8 ) . CAMAL-1 m o n o c l o n a l a n t i b o d y p o s i t i v e l y i d e n t i f i e d c e l l s f rom p a t i e n t s w i t h ANLL o r CGL i n FACS IV a n a l y s e s and i n d i r e c t immunoperoxidase s t a i n i n g . I t was f o u n d t o be s p e c i f i c f o r myelogenous l eukemic c e l l e x t r a c t s i n the EL ISA . CAMAL-1 d i d no t have any a p p r e c i a b l e a c t i v i t y on a cu te l y m p h o c y t i c l e u k e m i a ( A L L ) , norma l bone marrow (BM) o r norma l p e r i p h e r a l b l o o d l ymphyocy te (PBL) c e l l s i n the FACS, i n d i r e c t immunoperoxidase s t a i n i n g , o r on i r r e l e v a n t a n t i g e n p r e p a r a t i o n s i n the ELISA ( 9 8 , 9 9 , 1 0 9 , 1 1 0 ) . As i n the Hp-ant i-M-1 c o n j u g a t e s tudy i n C h a p t e r I I , the EDCI c o u p l i n g r e a c t i o n had no d e l e t e r i o u s a f f e c t on CAMAL-1 o r the photodynamic e f f e c t o f h a e m a t o p o r p h y r i n . The Hp a c t i v i t y o f the Hp-CAMAL-1 c o n j u g a t e was comparab le t o the a c t i v i t y o f e q u i v a l e n t c o n c e n t r a t i o n s o f f r e e Hp i n the h a e m o l y t i c a s s a y . The r e s u l t s were i d e n t i c a l to F i g . 3 i n C h a p t e r I I . A n t i g e n r e a c t i v i t y o f the Hp-CAMAL-1 and f r e e CAMAL-1 were e q u i v a l e n t when t e s t e d i n EL ISA ( F i g . 1 3 ) . F r ee h a e m a t o p o r p h y r i n was t i t r a t e d on norma l and CGL p e r i p h e r a l b l o o d l ymphocy tes to d e f i n e the l e v e l o f p h o t o t o x i c i t y due t o n o n s p e c i f i c a d h e r e n c e . When t r e a t e d w i t h 2000 - 200 ng Hp pe r 1 0 5 c e l l s , b o t h norma l and CGL PBLs were d r a s t i c a l l y a f f e c t e d ( F i g . 1 4 ) . V i a b i l i t y was d e c r e a s e d t o 0 - 4% o f the PBS o r medium c o n t r o l . However, the v i a b i l i t y o f bo th c e l l t y p e s were u n a f f e c t e d when t r e a t e d w i t h 20 ng Hp pe r 1 0 5 c e l l s . 78 F i g u r e 1 3 . T i t r a t i o n o f s p e c i f i c m o n o c l o n a l a n t i b o d y CAMAL-1 and Hp-CAMAL-1 c o n j u g a t e on CAMAL (-) and BSA (- - -) a n t i g e n s . The a n t i b o d y a c t i v i t y o f the Hp-CAMAL-1 c o n j u g a t e (o) and CAMAL-1 (0) was compared i n the EL ISA on 300 ng/ml a n t i g e n . Each p o i n t i s the mean o b t a i n e d f r om s i x w e l l s . The s t a n d a r d d e v i a t i o n s a re < 8%. 79 T i t r a t i o n o f CAMAL-1 a n d Hp-CAMAL-1 a n t i b o d y a c t i v i t i e s 1.0-o^i ' i ' '"""To Too 1000 Antigen Concentration (ug) 80 F i g u r e 14 . T i t r a t i o n o f Hp a c t i v i t y on norma l and CGL PBLs . The p h o t o c y t o t o x i c a c t i v i t y o f h a e m a t o p o r p h y r i n was t i t r a t e d on norma l (A) and CGL (o) p e r i p h e r a l b l o o d l y m p h o c y t e s . The v i a b i l i t y o f the t r e a t e d c e l l s was d e t e r m i n e d w i t h 0 .2% E o s i n Y v i t a l d y e . Each p o i n t r e p r e s e n t s the mean o f q u a d r u p l i c a t e s a m p l e s . The s t a n d a r d d e v i a t i o n a re <S%. 81 TITRATION OF HP ON NORMAL a CGL PBLS HP CONC . ng/ IO5 cells 82 A l t h o u g h f r e e h a e m a t o p o r p h y r i n has been shown t o have an a f f i n i t y f o r tumour t i s s u e , t h e r e d i d no t appear t o be any d i f f e r e n c e between norma l and CGL PBLs i n terms o f t h e i r s e n s i t i v i t y t o f r e e Hp m e d i a t e d c y t o t o x i c i t y . In the f o l l o w i n g e x p e r i m e n t s , the Hp c o n c e n t r a t i o n i n f r e e Hp and c o n j u g a t e s o l u t i o n s was s e t a t 20 ng pe r 1 0 5 c e l l s . In t h e s e s t u d i e s , j j i v i t r o t e s t i n g o f the Hp c o n j u g a t e s was c a r r i e d out on n o r m a l , CGL and ANLL p a t i e n t s a m p l e s . Hp-R a Hu and Hp- a L1210 were the p o s i t i v e and n e g a t i v e s p e c i f i c i t y c o n t r o l s r e s p e c t i v e l y . When t e s t e d on norma l PBLs , o n l y Hp-R a Hu had any e f f e c t on v i a b i l i t y ( F i g . 1 5 ) . Hp-CAMAL-1, Hp-a L1210 , Hp-R a CAMAL and R a i d had no d i s c e r n a b l e e f f e c t on the v i a b i l i t y o f norma l PBLs i n compa r i son t o the a n t i b o d y , Hp, and PBS c o n t r o l s . Hp-R a Hu t r ea tmen t d e c r e a s e d the number o f v i a b l e c e l l s t o 3 .2% o f the PBS c o n t r o l . The CGL PBLs were a f f e c t e d by Hp-CAMAL-1 and Hp-R a CAMAL i n a d d i t i o n t o the p o s i t i v e c o n t r o l Hp-R a Hu ( F i g . 1 6 ) . The v i a b i l i t y o f Hp-R a Hu t r e a t e d c e l l s d e c r e a s e d to 6 5 . 1 % , Hp-CAMAL-1 t r e a t e d c e l l s down t o 6 8 . 4 % and Hp-R a CAMAL t r e a t e d c e l l s to 4 7 . 4 % o f the v i a b i l i t y o f the PBS c o n t r o l . The i r r e l e v a n t an t i body-Hp c o n j u g a t e s , Hp-R a i d and Hp-a L1210 had no e f f e c t on v i a b l e c e l l numbers i n compa r i son t o the PBS, a n t i b o d y and Hp c o n t r o l s . Not s u r p r i s i n g l y , AML PBLs were a l s o a f f e c t e d by Hp-CAMAL-1 ( F i g . 1 7 ) . On l y 0 .4% o f the t e s t c e l l p o p u l a t i o n s u r v i v e d the s p e c i f i c Hp-CAMAL-1 c o n j u g a t e t r e a t m e n t . Hp-R a Hu c o n j u g a t e t r e a t m e n t e l i m i n a t e d 100% o f the c e l l s and the i r r e l e v a n t Hp-a L1210 c o n j u g a t e d e c r e a s e d the v i a b l e c e l l numbers t o 8 8 . 7 % o f the PBS c o n t r o l . The bo t tom p o r t i o n o f F i g u r e 17 demons t r a t e s Hp r e a c t i v i t y o f the d i f f e r e n t 83 F i g u r e 1 5 . Hp-an t i body c o n j u g a t e and c o n t r o l a c t i v i t y on norma l p e r i p h e r a l b l o o d l y m p h o c y t e s . The p h o t o c y t o t o x i c a c t i v i t y o f d i f f e r e n t Hp-an t i body c o n j u g a t e s and c o n t r o l s were t e s t e d on norma l PBLs . The v i a b i l i t y o f the t r e a t e d c e l l s were a s sa yed w i t h 0 . 2 % E o s i n Y v i t a l d y e . Each ba r r e p r e s e n t s the mean o f d u p l i c a t e s a m p l e s . The s t a n d a r d d e v i a t i o n s a re < 5 % . The PBS c o n t r o l was t aken as the 100% v a l u e . 85 F i g u r e 16 . Hp-an t i body c o n j u g a t e and c o n t r o l a c t i v i t y on CGL PBLs . The p h o t o c y t o t o x i c a c t i v i t y o f d i f f e r e n t Hp-an t i body c o n j u g a t e s and c o n t r o l s were t e s t e d on PBLs f r om a c h r o n i c g r a n u l o c y t i c l eukaemic p a t i e n t sample The v i a b i l i t y o f the t r e a t e d c e l l s were a s sa yed w i t h 0 .2% E o s i n Y v i t a l d ye . Each b a r r e p r e s e n t s the mean o f d u p l i c a t e s amp le s . The s t a n d a r d d e v i a t i o n s a re < 5 % . The PBS c o n t r o l was t aken as the 100% v a l u e . 87 F i g u r e 17 . Hp-an t i body c o n j u g a t e and c o n t r o l a c t i v i t y on ANLL PBLs . The p h o t o c y t o t o x i c a c t i v i t y o f d i f f e r e n t Hp-an t i body c o n j u g a t e s and c o n t r o l s were t e s t e d on PBLs f rom an a cu t e n o n - l y m p h o b l a s t i c l eukaem ia p a t i e n t samp le . The v i a b i l i t y o f the t r e a t e d c e l l s were a s sa yed w i t h 0 . 2 % E o s i n Y v i t a l d y e . Each ba r r e p r e s e n t s the mean o f d u p l i c a t e s amp les . The s t a n d a r d d e v i a t i o n s a re < 5%. The PBS c o n t r o l was t aken as the 100% v a l u e . 89 c o n j u g a t e s . I t was pe r f o rmed to de t e rm ine whether the e q u i v a l e n t c o n c e n t r a t i o n s o f Hp o f the d i f f e r e n t c o n j u g a t e s had e q u a l photodynamic a c t i v i t y i n v i t r o . In t h i s p a r t o f the e x p e r i m e n t , the c o n j u g a t e s were no t added t o the c e l l p e l l e t s u n t i l the f i n a l wash. A f t e r Hp-con juga te a d d i t i o n , the c e l l p e l l e t s were r e s u s p e n d e d to a t o t a l volume o f 1 ml pe r tube and exposed t o l i g h t w i t h the r e s t o f the s a m p l e s . The H p - c o n j u g a t e s : Hp-R a Hu , Hp-CAMAL-1, Hp-a L1210 , and f r e e Hp had an e q u i v a l e n t l e v e l o f c y t o t o x i c i t y on AML PBLs . V i a b i l i t y o f the c e l l s r anged f rom 5.1 t o 7.5% o f the PBS t r e a t e d c e l l p o p u l a t i o n . When a s s a y e d f o r s p e c i f i c i t y , the c o n j u g a t e s o n l y d e m o n s t r a t e d c y t o t o x i c a c t i v i t y a g a i n s t t h e i r d e s i g n a t e d t a r g e t c e l l s . Normal BM, lymphoma BM, norma l PBLs and ALL PBLs were u n a f f e c t e d by the n e g a t i v e c o n t r o l c o n j u g a t e Hp-a L1210 , the t e s t c o n j u g a t e Hp-CAMAL-1 and f r e e Hp ( F i g . 1 8 ) . The p o s i t i v e c o n t r o l c o n j u g a t e Hp-R a Hu d e c r e a s e d the v i a b i l i t y o f the t e s t c e l l s t o 1.2 - 2 3 . 3 % o f the PBS c o n t r o l . CAMAL b e a r i n g CGL and ANLL PBLs p r o v e d t o be s e n s i t i v e t o the Hp-CAMAL-1 c o n j u g a t e ( F i g . 1 9 ) . T rea tment w i t h the t e s t c o n j u g a t e d e c r e a s e d c e l l v i a b i l i t y down t o 0 .4 - 3 7 . 3 % o f the PBS c o n t r o l The CGL and ANLL c e l l s were a l s o s e n s i t i v e t o the p o s i t i v e c o n t r o l c o n j u g a t e Hp-R a Hu , e . g . 0 .0 - 2 7 . 6 % v i a b i l i t y . In a l l e x p e r i m e n t s , Hp-R a Hu d e m o n s t r a t e d a h i g h e r l e v e l o f c y t o t o x i c i t y on the c e l l s , i n compar i son t o the e f f e c t o f the t e s t c o n j u g a t e Hp-CAMAL-1. T h i s was no t unexpec t ed c o n s i d e r i n g the m u l t i s p e c i f i c n a t u r e o f the a n t i s e r a and the r e l a t i v e h i g h e r d e n s i t y o f human a n t i g e n i c d e t e r m i n a n t s on the t a r g e t c e l l s . The f r e e Hp and Hp-a L1210 had m i n i m a l e f f e c t on the v i a b i l i t y o f the t e s t c e l l s . T h i s c l e a r l y 90 F i g u r e 18 . The e f f e c t s o f Hp-an t i body c o n j u g a t e s on norma l and a cu t e l y m p h o c y t i c l e u k a e m i a PBLs , norma l and lymphoma bone marrow (B .M. ) c e l l s . The p h o t o c y t o t o x i c a c t i v i t y o f Hp 0 , Hp-R a Hu 1, Hp-an t i -L1210 0 and Hp-CAMAL-1 S was t e s t e d on f o u r d i f f e r e n t i r r e l e v a n t tumour o r norma l c e l l s amp le s . Each ba r i s the mean o f t r i p l i c a t e s amp le s . C e l l v i a b i l i t y was measured w i t h 0 . 2 % E o s i n Y v i t a l d y e . The v i a b l e c e l l number o f the PBS c o n t r o l was used as the 100% v a l u e . N. BM LYMPH-OMA N.PBL HP HP- RoiHu HP-a U2I0 HP* CAMAL HP HP- RaHu HP- aU2IO HP-CAMAL HP HP- RaHu HP- aU2T0 HP- CAMAL % VIABILITY (EotinY dy« txclusion anay) >Q 80 30 4.0 80 60 70 BP 90 100 2S.3 % ^ \ \ \ \ \ \ \ \ \ \ \ \ \ V a  m__|2:0_%^ >U2I0 ^ ^ ^ ^ ^ ^ ^ ^ ^ 2 % 92 F i g u r e 19 . The e f f e c t o f Hp-an t i body c o n j u g a t e s on c h r o n i c g r a n u l o c y t i c l e u k a e m i a and acu te n o n l y m p h o b l a s t i c l e u k a e m i a samples f rom f o u r d i f f e r e n t p a t i e n t s . The p h o t o c y t o t o x i c a c t i v i t y o f Hp 0 , Hp - R a Hu 8, Hp-an t i -L1210 • , and Hp-CAMAL-1 IS was t e s t e d on PBLs drawn f rom f o u r l e u k a e m i a p a t i e n t s . C e l l v i a b i l i t y was measured w i t h 0 .2% E o s i n Y v i t a l d y e . Each ba r i s the mean o f t r i p l i c a t e s a m p l e s . The v i a b l e c e l l number o f the PBS c o n t r o l was used as the 100% v a l u e . % V I A M U T V l l o a l n Y dy t • i c l u . l o n a w a y ) 10 2 0 3 0 4 0 8 0 6 0 7 0 8 0 9 0 100 CGL H P HP-R H P - a L U l O HP-CAMAL. • HuPHm^2T_6 .12101 37.S % 'CGL HP H P - R a H u HP -aLI2IO HP-CAM AL 1.9 % s r 7 . 5 % A N L L 2 3 . 0 % 3 8 . 3 % ANLL H P H P - R « H u H P - « U 2 I 0 HP-CAMAL V///////////////////////////A 0 . 0 % 0 . 4 % 94 i n d i c a t e d that conjugate r e c o g n i t i o n of the ta r g e t c e l l was e s s e n t i a l (through the antigen bind i n g s i t e of the antibody) f o r d e l i v e r y of the c y t o t o x i c e f f e c t of the p h o t o s e n s i t i z e r haematoporphyrin. Samples from two leukaemic p a t i e n t s were monitored f o r 4 and 6 weeks, drawing PBL samples every two weeks. In both cases, a l l samples proved to be Hp-CAMAL-1 s e n s i t i v e . In F i g . 20, samples from CGL p a t i e n t 1 were examined every two weeks f o r one month. The black bars represent the r e s u l t s from the f i r s t sampling and the white bars from the second sampling. Hp-R a Hu treatment decreased the v i a b i l i t y of the samples to 26.5 - 33% of c o n t r o l s . The c e l l s were s u s c e p t i b l e to Hp-CAMAL-1 treatment at both samplings, 37.7% at day 9.11.83 and 54.0% at day 23.11.83. In Table V I , samples from CGL p a t i e n t 2 were examined every two 3 weeks f o r 6 weeks. V i a b l e c e l l numbers and H-thymidine i n c o r p o r a t i o n was documented. In both assays, the samples demonstrated s e n s i t i v i t y to the p o s i t i v e c o n t r o l Hp-RaHu and the t e s t conjugate Hp-CAMAL-1. In the Eosin Y v i t a l dye e x c l u s i o n assay, the c e l l s were 0 to 34.43% v i a b l e a f t e r Hp-R a Hu treatment and 7.5 to 61.1% v i a b l e a f t e r Hp-CAMAL-1 treatment. The i r r e l e v a n t conjugate c o n t r o l , Hp-a M-l, had a minor e f f e c t on v i a b i l i t y : the v i a b l e c e l l count was 67.5 to 96.3% of the PBS c o n t r o l . 3 The H-thymidine i n c o r p o r a t i o n r e s u l t s were f o r the most p a r t , not as c l e a r . The data from the day 24.9.83 and 24.10.83 demonstrated the major 3 drawback of t e s t i n g PBLs i n H-thymidine assays. In s p i t e of the use of s p e c i a l l y prepared medium f o r human lymphocyte stem c e l l growth, the 3 amount of p r o l i f e r a t i o n i n 4 days as measured by H-thymidine i n c o r p o r a t i o n was poor. At best, i t only gave a plus or minus i n d i c a t i o n 95 F i g u r e 20 . The e f f e c t o f Hp-an t i body c o n j u g a t e s on PBL samples f rom CGL p a t i e n t I. D i f f e r e n t Hp-an t i body c o n j u g a t e s and c o n t r o l s were a s sa yed on PBL samples f rom a CGL p a t i e n t t aken a t 2 s e p a r a t e i n t e r v a l s , 9 . 1 1 . 8 3 13 and 2 3 . 1 1 . 8 3 S. C e l l v i a b i l i t y was measured w i t h 0 .2% E o s i n Y v i t a l d y e . Each ba r i s the mean o f d u p l i c a t e s amp le s . The v i a b l e c e l l number o f the PBS c o n t r o l was used as the 100% v a l u e . 96 T a b l e VI E f f e c t o f H p - a n t i b o d y c o n j u g a t e s 2 4 . 0 9 . 8 3 CELL VIABILITY #'s (x 1 0 ' ) % v i a b l e PBS 266.67 ± 3 0 . 5 5 Hp 2 6 0 . 0 0 ± 20 .00 9 7 . 5 0 Hp-RaHu 0 0 Hp-CAMAL-1 6 0 . 0 0 ± 2 0 . 0 0 2 2 . 5 0 Hp-aM-1 180.00 ± 0 0 . 8 0 6 7 . 5 0 0 8 . 1 0 . 8 3 H's (x 10 ' ) % v i a b l e PBS 2 5 2 . 9 0 ± 2 1 . 1 7 Hp 288.67 ± 1 9 . 0 7 100.00 Hp-RaHu 5.53 ± 1.12 1.92 Hp-CAMAL-1 18.83 ± 0 .15 7.47 Hp-aM-1 242.67 ± 10.69 9 6 . 3 0 2 4 . 1 0 . 8 3 tt's (x 1 0 5 ) % v i a b l e PBS 3 0 . 0 0 ± 1.00 Hp 3 3 . 3 3 + 3 . 2 1 100.00 Hp-RaHu 10.33 ± 2.52 34 .43 Hp-CAMAL-1. 18.33 ± 1.52 61.11 Hp-aM-1 2 9 . 0 0 ± 1.00 96 .67 PBL samples from CGL p a t i e n t II 'H-THYMIDINE INCORPORATION cpm % i n c o r p . 2388 .50 ± 6 7 7 . 8 0 2714.25 ± 906 .39 100.00 1656.00 ± 340.72 6 9 . 3 3 1536.50 ± 678.74 6 9 . 3 3 2200 .50 + 137.92 92. 16 cpm % i n c o r p . 5 2 , 4 7 2 . 5 0 ± 5690.53 4 8 , 3 2 5 . 9 0 + 4601.51 9 2 . 1 0 2 , 4 5 6 . 0 0 ± 1267.69 4 . 6 8 7 , 1 5 6 . 8 3 ± 2439.42 13.64 4 2 . 7 4 5 . 1 7 ± 5665.96 8 1 . 4 6 cpm % i n c o r p . 6943 .95 ± 805.07 6486.62 ± 1050.00 100.00 9376.95 + 728 .09 6 3 . 0 3 5201 . 12 + 1 145.06 74 .90 6496.87 ± 1137.51 9 3 . 5 6 9 8 of the e f f e c t of the conjugates since the background incorporation (e.g. 3 H-thymidme incorporation from wells bearing no c e l l s ) was as high as 1000 cpm. The assay run on 8.10.83 gave a much better set of r e s u l t s due to a higher c e l l concentration in each w e l l . There was a s i g n i f i c a n t 3 difference in the l e v e l of H-thymidine incorporation between the PBS c o n t r o l , and the p o s i t i v e and test conjugate treated c e l l s ; e.g. PBS c o n t r o l : 52,473 ± 5691 cpm in comparison to Hp-R a Hu treated c e l l s : 2,456 ± 1268 cpm and Hp-CAMAL-1 treated c e l l s : 7,157 ± 2,439 cpm. Again, the i r r e l e v a n t conjugate control Hp-aM-1 had a minimal e f f e c t : incorporation went down to 81.46% (42,745 ± 5666 cpm) of the PBS c o n t r o l treated c e l l s . This assay was a s p e c i a l case in that i t was a rare occurrence to get a high enough c e l l concentration from the patient sample to run the assay in that fashion ( i . e . 2 x 10^ c e l l s per tube and 100 u l of 5 ml c e l l suspension in each m i c r o t i t r e w e l l ) . The l a s t two f i g u r e s , F i g . 21 and 22 demonstrate the use of Hp-Ralg in a secondary l a b e l l i n g method. A l l the conjugate preparations used previously varied in the amount of haematoporphyrin bound e.g. 30 - 70 molecules Hp per molecule of antibody. In the assays, the conjugate concentrations were normalized on the basis of haematoporphyrin concentration. Therefore, the actual amount of antibody added in the d i f f e r e n t conjugate treated c e l l s would d i f f e r (between 10 - 20 yg Ab per 10"* c e l l s ) . In the following assay, the c e l l s were l a b e l l e d with Hp-Ramlg following primary treatment with i n d i v i d u a l antibodies. In t h i s way, the amount of antibody added as well as the amount of Hp would be equivalent throughout the experiment. T i t r a t i o n of Hp-RaMIg on CGL 99 o r norma l PB l s i n the c o n c e n t r a t i o n range o f 0 .3 - 6 0 . 0 ng p e r 10 c e l l s i n d i c a t e d t h a t 15 ng pe r 1 0 5 c e l l s had n e g l i g i b l e e f f e c t s on the c e l l s ( F i g . 2 1 ) . When t e s t e d on CGL and ANLL c e l l s , Hp-RaMIg a l o n e a t 15 ng Hp p e r 10** c e l l s had no e f f e c t on v i a b i l i t y ( F i g . 2 2 ) . However as a s e conda r y l a b e l a f t e r a d d i t i o n o f the s p e c i f i c m o n o c l o n a l a n t i b o d y CAMAL-1, the Hp-RaMIg d e c r e a s e d the v i a b i l i t y down t o 50% f o r ANLL c e l l s and 65% f o r CGL c e l l s . The Hp-CAMAL-1 had a s l i g h t l y g r e a t e r e f f e c t on the c e l l s , 36% s u r v i v a l f o r ANLL and 52% s u r v i v a l f o r CGL. Both a L1210 t r e a t m e n t f o l l o w e d by Hp-Ramlg and Hp-aL1210 c o n t r o l s d i s p l a y e d n e g l i g i b l e e f f e c t s on b o t h ANLL and CGL PBLs . The p o s i t i v e c o n t r o l Hp-R a Hu d e c r e a s e d the v i a b i l i t y o f ANLL and CGL t o a p p r o x i m a t e l y 23 - 25% o f the PBS c o n t r o l . 100 F i g u r e 21. T i t r a t i o n o f Hp-R o M Ig a c t i v i t y on norma l and CGL PBLs . The p h o t o c y t o t o x i c a c t i v i t y o f Hp-R a H Ig c o n j u g a t e was a s sa yed on norma l (A) and CGL (o) p e r i p h e r a l b l o o d l y m p h o c y t e s . C e l l v i a b i l i t y was a s sayed w i t h 0.2% E o s i n Y v i t a l d y e . Each p o i n t i s the mean o f t r i p l i c a t e samp les . The s t a n d a r d d e v i a t i o n s were < 7%. 101 102 F i g u r e 22. The e f f e c t o f Hp-R a M Ig on a n t i b o d y l a b e l l e d c e l l s . The p h o t o c y t o t o x i c a c t i v i t y o f Hp-R a M Ig c o n j u g a t e and c o n t r o l s were a s sayed on PBS, CAMAL-1 and a L1210 l a b e l l e d ANLL 0 and CGL 0 c e l l s . The h e a d i n g s , a L1210 -» Hp-RAMIg and CAMAL-1 -* Hp-RAMIg i n d i c a t e the two s t e p l a b e l l i n g p r o c e d u r e i . e . i n i t i a l l a b e l l i n g w i t h a n t i b o d y a l o n e f o l l o w e d by Hp-RAMIg l a b e l l i n g . C e l l v i a b i l i t y o f the t r e a t e d c e l l s were a s s a y e d w i t h 0.2% E o s i n Y v i t a l d ye . Each b a r i s the mean o f d u p l i c a t e s a m p l e s . The v i a b l e c e l l number o f the PBS c o n t r o l was used as the 100% v a l u e . % Viability (Eosin Y exclusion assay I S ? 8*1 i- r- 3 104 Chapter I I I DISCUSSION In the previous chapter, the successful application of photoimmunotherapy i n a experimental animal model was demonstrated. The next l o g i c a l step was to determine whether an analogous therapy might be applied to a human condition. The malignant condition chosen was human nonlymphoblastic leukaemia; a study of which i s currently under in v e s t i g a t i o n in our laboratory. A tumour associated antigen (CAMAL) was i d e n t i f i e d and i s o l a t e d from human acute nonlymphoblastic leukaemia (ANLL) c e l l s on native polyacrylamide gels (97,109). Rabbit antisera d i r e c t e d against t h i s 68 k dalton antigen demonstrated complete s p e c i f i c i t y f o r ANLL c e l l membrane extracts in the ELISA (109), and p o s i t i v e r e a c t i v i t y on nonlymphoblastic leukaemia (ANLL and CGL) c e l l s in FACS IV analysis and in i n d i r e c t immunoperoxidase staining on single c e l l s l i d e preparations (99,110). A monoclonal antibody (CAMAL-1), produced against CAMAL displayed a r e a c t i v i t y s i m i l a r to that of the rabbit antiserum (98,99). Immunoperoxidase studies with CAMAL-1 showed that most myelogenous leukaemic c e l l samples showed s i g n i f i c a n t l y higher numbers (15 - 20%) of CAMAL-expressing c e l l s compared to those from normal, or lymphoid leukaemic c e l l samples ( < 1.0%) (99). In t h i s study, the photodrug haematoporphyrin was conjugated to CAMAL-1 monoclonal antibodies by EDC1 coupling. As in the Hp-anti-M-1 model, both haematoporphyrin and CAMAL-1 antibody retained t h e i r dynamic a c t i v i t y and immunological s p e c i f i c i t y a f t e r chemical coupling. Iri v i t r o 105 expe r imen t s w i t h Hp-CAMAL-1 demons t r a t ed t h a t the a n t i b o d y c o n j u g a t e s p e c i f i c a l l y e l i m i n a t e d CAMAL b e a r i n g c e l l s i n mye logenous l e u k a e m i c c e l l p o p u l a t i o n s ( F i g . 1 6 , 17 and 1 9 ) . The c y t o t o x i c i t y o f the c o n j u g a t e was shown t o be t a r g e t s p e c i f i c s i n c e i r r e l e v a n t c o n j u g a t e s (Hp-ant i-M-1 and Hp-an t i L1210) have m i n i m a l e f f e c t on the human c e l l s a m p l e s . In a d d i t i o n , the Hp-CAMAL-1 had no a c t i v i t y on normal bone marrow c e l l s and p e r i p h e r a l b l o o d l y m p h o c y t e s , o r CAMAL n e g a t i v e ALL bone marrow c e l l s ( F i g . 1 8 ) . E x a m i n a t i o n o f samples f rom two p a t i e n t s o ve r a p e r i o d o f 4 t o 6 weeks i n d i c a t e d t h a t Hp-CAMAL-1 s e n s i t i v i t y was not a t r a n s i t o r y e f f e c t . A l l s a m p l e s , t aken s e q u e n t i a l l y e v e r y two weeks , d i s p l a y e d d i f f e r e n t deg rees o f s e n s i t i v i t y t o the p o s i t i v e c o n t r o l Hp-R a Hu and the t e s t c o n j u g a t e Hp-CAMAL-1 ( F i g . 20 and T a b l e 6 ) . F o r the most p a r t , the n e g a t i v e c o n t r o l s Hp-an t i -L1210 had no e f f e c t on the v i a b i l i t y o f the c e l l s . I t i s d i f f i c u l t t o de t e rm ine whether the d i f f e r i n g deg rees o f t o x i c i t y o f Hp-CAMAL-1 on CGL c e l l s ( e . g . T a b l e 6 , 7.5 t o 6 1 . 1 % o f t r e a t e d c e l l s s u r v i v e d ) was a t r u e r e f l e c t i o n o f the CAMAL d i s t r i b u t i o n i n the c e l l sample on t h a t p a r t i c u l a r day . I t c o u l d be due t o the f r a g i l i t y o f the c e l l s on t h a t day s i n c e the c e l l s samples showed v a r i a b i l i t y i n terms o f the s t a b i l i t y o f the c e l l membranes. The v i a b l e c e l l number r e c o v e r e d f rom PBS t r e a t m e n t v a r i e d f rom sample t o sample i n d i c a t i n g t h a t the i n t e g r i t y o f c e l l s had d i f f e r e n t deg rees o f s e n s i t i v i t y to the m e c h a n i c a l a c t i o n o f c e n t r i f u g a t i o n , wash ing and c u l t u r i n g o v e r n i g h t . T h e r e f o r e i t was c o n c e i v a b l e t h a t the v a r i a t i o n i n Hp-CAMAL-1 s e n s i t i v i t y c o u l d be p a r t i a l l y due t o the v a r i a t i o n i n c e l l i n t e g r i t y i n the samp le . However , t h i s i n no way d i s c o u n t e d the s p e c i f i c i t y o f the Hp-CAMAL-1 c o n j u g a t e f o r 106 AML-1 e x p r e s s i n g c e l l s . I n c r e a s e d s e n s i t i v i t y to Hp-CAMAL-1 c o r r e l a t e d w i t h i n c r e a s e d s e n s i t i v i t y t o the n e g a t i v e c o n t r o l (Hp-ant i-M-1 o r Hp-an t i -L1210 ) and the p o s i t i v e c o n t r o l Hp-R a Hu. Logan e t a l . (99) demons t r a t ed w i t h the immunoperoxidase s l i d e t e s t , R a CAMAL and CAMAL-1 m o n o c l o n a l a n t i b o d y bo th s p e c i f i c a l l y l a b e l l e d CAMAL p o s i t i v e c e l l s . However, d i f f e r e n t a n t i b o d y b i n d i n g p a t t e r n s were n o t e d w i t h the two d i f f e r e n t s e r a . The r a b b i t a n t i s e r a r e a c t e d p r e d o m i n a n t l y w i t h membrane bound CAMAL and l e s s i n t e n s i v e l y w i t h c y t o p l a s m i c a n t i g e n . On the o t h e r h a n d , CAMAL-1 r e a c t e d p r e d o m i n a n t l y w i t h c y t o p l a s m i c , p e r i n u c l e a r , o r n u c l e a r CAMAL. I t d e m o n s t r a t e d f a r l e s s r e a c t i v i t y on membrane bound a n t i g e n . But i n s p i t e o f p o o r e r b i n d i n g t o membrane a n t i g e n , Hp-CAMAL-1 d i s p l a y e d c o n s i d e r a b l e c y t o t o x i c i t y on CAMAL p o s i t i v e c e l l s . T h i s p o s s i b l e d i s c r e p a n c y c o u l d be e x p l a i n e d by t he d i f f e r e n t i a l l e v e l s o f a n t i b o d y r e q u i r e d f o r Hp d e l i v e r y t o ensu re c y t o t o x i c e f f e c t as opposed t o amounts r e q u i r e d f o r v i s i b l e s t a i n i n g i n the immunoperox idase t e s t . Hp-R a CAMAL d i d appear t o have a h i g h e r l e v e l o f c y t o t o x i c i t y i n compa r i son t o the Hp-CAMAL-1 on the CAMAL e x p r e s s i n g CGL c e l l s ( F i g . 1 6 ) . T h i s was not e n t i r e l y s u r p r i s i n g , c o n s i d e r i n g the h i g h e r r e a c t i v i t y o f R a CAMAL f o r membrane bound CAMAL. The a p p l i c a t i o n o f t h i s human s tudy to a c l i n i c a l s i t u a t i o n i n v o l v e s p o s s i b l y t r e a t i n g bone marrow samples f o r f u t u r e a u t o l o g o u s bone marrow t r a n s p l a n t s i n l e u k a e m i c p a t i e n t s . A u t o l o g o u s bone marrow t r a n s p l a n t a t i o n s c o n s i s t s o f d raw ing o f f a p o r t i o n o f the l e u k e m i c p a t i e n t ' s bone marrow w h i l e he i s i n c l i n i c a l r e m i s s i o n , t r e a t i n g the p a t i e n t w i t h i n t e n s i v e chemotherapy and/or r a d i o t h e r a p y when he r e l a p s e s , 1 0 7 and then r e t u r n i n g the bone marrow c e l l t o r e - e s t a b l i s h a v i a b l e h a e m a t o p o i e t i c s ys tem i n the p a t i e n t . A l t h o u g h the use o f a u t o l o g o u s bone marrow e x c l u d e s the p o t e n t i a l o f g r a f t - v e r s u s h o s t d i s e a s e f o u n d i n a l l o g e n i c bone marrow t r a n s p l a n t a t i o n , t h e r e remains a c r i t i c a l drawback t o t h i s t ype o f t r a n p l a n t a t i o n . The bone marrow p r e p a r a t i o n must be d e v o i d o f c l o n o g e n i c m a l i g n a n t c e l l s . A major c o n t r i b u t o r y f a c t o r i n r e l a p s e s a f t e r a u t o l o g o u s bone marrow t r a n s p l a n t s c o u l d be the p r e s e n c e o f m a l i g n a n t c l o n e s p r e s e n t l y u n d e t e c t e d i n the r e m i s s i o n bone marrow p r e p a r a t i o n s . Bone marrow samples f rom myelogenous l e u k a e m i c p a t i e n t s have been shown t o c o n t a i n CAMAL p o s i t i v e c e l l s ( 9 9 , 1 1 0 ) . Photo immunotherapy w i t h Hp-CAMAL-1 r e p r e s e n t s a s p e c i f i c mode o f e l i m i n a t i n g the CAMAL e x p r e s s i n g tumour c e l l s f rom the bone marrow samp le . The norma l ( i . e . CAMAL n e g a t i v e ) bone marrow c e l l s wou ld be u n a f f e c t e d by the c y t o t o x i c a c t i v i t y o f Hp-CAMAL-1, and wou ld be a v a i l a b l e f o r t r a n s p l a n t a t i o n . An i m p o r t a n t q u e s t i o n t o ask i s whether the m a l i g n a n t c l o n o g e n i c c e l l i n the bone marrow e x p r e s s e s s u r f a c e CAMAL. I f the o r i g i n a l m a l i g n a n t c l o n e s do no t e x p r e s s CAMAL, the Hp-CAMAL-1 w i l l not be a b l e t o r e c o g n i z e the c l o n e as the d e s i r e d t a r g e t and the Hp-CAMAL-1 t r e a t e d bone marrow t r a n s p l a n t would s t i l l r e t a i n i t s m a l i g n a n t p o t e n t i a l . So the nex t o b v i o u s avenue o f r e s e a r c h wou ld be the e x a m i n a t i o n o f bone marrow stem c e l l growth f o r CAMAL e x p r e s s i o n a f t e r Hp-CAMAL-1 t r e a t m e n t . T h i s i s p r e s e n t l y b e i n g i n v e s t i g a t e d bu t r e p r e s e n t s a complex s t udy f r om wh ich u n e q u i v o c a l answers may no t r e a d i l y be f o r t h c o m i n g . 108 A n o t h e r a r e a o f i n v e s t i g a t i o n l e f t undone i n t h i s s t udy was t i t r a t i o n o f the s p e c i f i c c y t o t o x i c e f f e c t o f Hp-CAMAL-1 on CAMAL p o s i t i v e c e l l s . The e x p e r i m e n t a l p r o t o c o l was such t h a t h i g h c e l l numbers were r e q u i r e d . Hence an a s say l i m i t a t i o n was the c e l l d e n s i t y o f the p a t i e n t sample r e c e i v e d . In a d d i t i o n , the q u a l i t y o f c e l l s , as d i s c u s s e d p r e v i o u s l y , v a r i e d f rom sample t o samp le . T h e r e f o r e i t was d i f f i c u l t t o t i t r a t e out the Hp-CAMAL-1 e f f e c t and de t e rm ine a " t r u e " d o s e - r e s p o n s e o f Hp-CAMAL c o n c e n t r a t i o n on p a t i e n t c e l l s amp le s . P r e s e n t l y , a CAMAL b e a r i n g mouse-human h y b r i d c e l l l i n e has been d e v e l o p e d r e c e n t l y by V i n c e n t Lum i n our l a b o r a t o r y . P r e l i m i n a r y r e s u l t s show t h a t t h i s c e l l l i n e i s s p e c i f i c a l l y s u s c e p t i b l e t o Hp-CAMAL-1. I t i s hoped t h a t f u t u r e s t u d i e s w i t h t h i s model c e l l l i n e w i l l e s t a b l i s h an a c c u r a t e d o s e - r e s p o n s e r e l a t i o n s h i p between Hp-CAMAL-1 and the t a r g e t c e l l s i n v i t r o . In v i v o s t u d i e s w i t h Hp-CAMAL-1 and the CAMAL e x p r e s s i n g c e l l l i n e i n m ice w i l l de t e rm ine the p o t e n t i a l o f photo immunotherapy i n s p e c i f i c bone marrow p u r g i n g o f m a l i g n a n t c e l l s . CHAPTER IV RADIOIMMUNOIMAGERY 110 C h a p t e r IV INTRODUCTION The s e n s i t i v i t y w i t h wh ich s m a l l tumour masses can be d e t e c t e d i s an a r e a o f keen i n t e r e s t f o r o n c o l o g i s t s . In o r d e r t o improve the t r e a tmen t o f m a l i g n a n t d i s e a s e , methods must be f ound t h a t a re s e n s i t i v e and a c c u r a t e enough t o d e t e c t the p r e s e n c e o f tumour mass and tumour s p r e a d a t a v e r y e a r l y s t age o f the d i s e a s e . A major p rob l em w i t h c a n c e r d i a g n o s i s a t p r e s e n t i s t h a t by the t ime the c a n c e r i s p o s i t i v e l y d i a g n o s e d , i t may be too l a t e f o r the p a t i e n t . O f t e n , the p a t i e n t a l r e a d y i s c a r r y i n g a l a r g e d i s s e m i n a t e d tumour and p r o g n o s i s i s poor w i t h c o n v e n t i o n a l t r e a t m e n t . Hence , a major t h r u s t i n c a n c e r r e s e a r c h d e a l s w i t h f i n d i n g methods by wh i ch c a n c e r s can be d e t e c t e d w h i l e s t i l l a t a c u r a b l e s t a g e . S i n c e the d i s c o v e r y t h a t r a d i o a c t i v e i o d i n e c o u l d be used t o d e t e c t m e t a s t a t i c t h y r o i d t umours , tumour s p e c i f i c r a d i o a c t i v e t r a c e r s have been p r o p o s e d as a p o t e n t i a l t o o l f o r e a r l y d e t e c t i o n o f tumour . The u l t i m a t e g o a l has been to d e v e l o p a s c a n n i n g agent t h a t wou ld be s p e c i f i c f o r a l l t umours . But even a r a d i o t r a c e r t h a t c o u l d d e t e c t a g i v e n h i s t o l o g i c a l t ype o f tumour o r an i n d i v i d u a l tumour wou ld be i n v a l u a b l e i n d i a g n o s i s . R a d i o t r a c e r s i n c o n j u n c t i o n w i t h p a r t i c l e o r gamma imag ing i n s t r u m e n t s c o u l d v i s u a l i z e s m a l l e r and more d e e p - s e a t e d m a l i g n a n t l e s i o n s , i n v i s i b l e by o t h e r d e t e c t i o n methods . In an i d e a l s i t u a t i o n , the r a d i o a c t i v e s c a n n i n g agent wou ld be a d m i n i s t e r e d t o the p a t i e n t , r a d i o a c t i v i t y wou ld s e q u e s t e r i n the m a l i g n a n t l e s i o n s , and t h e i r l o c a t i o n s p i n p o i n t e d by e x t e r n a l s c i n t i g r a p h y , e . g . gamma cameras , r e c t i l i n e a r s c a n n e r s , I l l s i n g l e - p h o t o n o r p o s i t r o n e m i s s i o n computed tomographs . The use o f r a d i o t r a c e r s ex tends f u r t h e r than j u s t the i n i t i a l d i a g n o s i s . Tumour imag ing c o u l d be a p p l i e d to m o n i t o r i n g c a n c e r m e t a s t a s i s and r e c u r r e n c e . I t c o u l d be u t i l i z e d i n d e t e r m i n g the e x t e n t o r s p r e a d o f the d i s e a s e . Assessment o f the e f f i c i e n c y o f a c a n c e r t r ea tmen t o r f o l l o w i n g up on the p r o g r e s s o f r e s i d u a l l e s i o n s l e f t a f t e r t h e r a p y c o u l d be r e a d i l y a c c o m p l i s h e d w i t h s p e c i f i c r a d i o t r a c e r s . However, a major p r o b l e m l i e s i n how t o make the r a d i o a c t i v e s c a n n i n g agents tumour s p e c i f i c . The m a j o r i t y o f the r a d i o p h a r m a c e u t i c a l s i n c u r r e n t c l i n i c a l use a re n o t , by d e f i n i t i o n , tumour s p e c i f i c . The mechanisms by wh i ch they a re c a p a b l e o f imag ing tumour a re s t i l l no t c l e a r . However, the g e n e r a l consensus i s t h a t r a d i o a c t i v e agents l o c a l i z e due t o n o n s p e c i f i c s e conda r y c h a r a c t e r i s t i c s a s s o c i a t e d w i t h tumour growth (111-113 ) . Fo r examp le , a r a p i d growth r a t e i s a d i s t i n g u i s h i n g f e a t u r e o f many m a l i g n a n c i e s . P r e c u r s o r compounds r e q u i r e d to s u p p o r t such a growth r a t e , i f r a d i o a c t i v e l y l a b e l l e d e . g . l a b e l l e d t h y m i d i n e o r amino a c i d s , wou ld be r e a d i l y i n c o r p o r a t e d i n t o a tumour mass above background l e v e l s . The tumour up take o f ^ G a l l i u m , a w i d e l y u s e d s o f t t i s s u e d e t e c t i n g agent ( 1 1 4 ) , i s t hough t to be p a r t i a l l y due t o some f a c t o r c a u s e d by r a p i d c e l l p r o l i f e r a t i o n ( 1 1 5 ) . Secondary v a s c u l a r changes o b s e r v e d i n tumour masses a re a l s o an i m p o r t a n t f a c t o r . Because o f the r a p i d growth f r e q u e n t l y o b s e r v e d i n t umours , n e o a n g i o g e n e s i s o r the p r o d u c t i o n o f new b l o o d v e s s e l s i s a v i t a l f a c t o r i n the ma in tenance o f the d e v e l o p i n g tumour mass . The h i g h e r degree o f v a s c u l a r i t y f o u n d i n many tumours l e a d s t o a r e l a t i v e i n c r e a s e i n b l o o d volume i n the tumour t i s s u e . T h i s r e s u l t s 112 in an apparent increased concentration of the radioactive scanning agent in the tumour r e l a t i v e to the surrounding tissue due a c t u a l l y to an increase i n volume of blood in the malignant t i s s u e . Another important factor i s the increased permeability of the tumour neovasculature. This acts to f a c i l i t a t e the passage of radioactive scanning agents out of the "leaky" tumour blood vessels in to the i n t e r s t i t i a l f l u i d of the tumour. Since the lymphatic drainage of maligant tissue i s often not as well developed as in normal t i s s u e , the scanning agents would be retained longer in the tumour. Other factors include increased permeability of tumour plasma membrane due to a transport misfunction, increased a b i l i t y to take up the imaging agent by pinocytosis, or uptake of radioactive agents by phagocytes r e s i d i n g in the tumour. Unfortunately, a l l the features discussed that lead to tumour l o c a l i z a t i o n also occur in other disease states, e.g. inflammatory processes, myocardial i n f a r c t i o n , etc. 67 . 75 The commonly used scanning agents e.g. Gallium c i t r a t e , Se l a b e l l e d selenomethianine, 11"'"Indium chl o r i d e , have no s p e c i f i c i t y f o r tumour tis s u e i n that they w i l l j u s t as e a s i l y image inflammatory lesions (116). This lack of tumour s p e c i f i t y prompted a search f o r more discriminating tumour scanning agents. U t i l i z a t i o n of immunological techniques offered a new route of obtaining tumour imaging s p e c i f i c i t y . By combining the s p e c i f i c i t y of anti-tumour antibodies and the s e n s i t i v i t y of radioisotope tracer technology, a greater l e v e l of detection was created f o r tumour diagnosis. Radioimmunoimagery refers to the use of l a b e l l e d antibodies or fragments f or the ijn vivo detection of cancer. Because of t h e i r innate 113 immunospecific nature, anti-tumour antibodies could transport diagnostic quantities of radioisotopes to tumours with minimal deposition in normal ti s s u e . Since the s p e c i f i c i t y of the radiotracer would be s i g n i f i c a n t l y increased, the amount of radioisotope used could be decreased, thereby minimizing both the e f f e c t i v e r a d i a t i o n dosage and p o t e n t i a l damage to normal rad i o s e n s i t i v e t i s s u e . One of the f i r s t pioneering studies of r a d i o n u c l i d e - l a b e l l e d antibodies for s p e c i f i c l o c a l i z a t i o n was reported by 131 Pressman and Keighley in 1948. They found that I - l a b e l l e d rabbit a n t i - r a t kidney antibodies would s p e c i f i c a l l y l o c a l i z e in the r a t kidney 131 in vivo, while an i r r e l e v a n t I-rabbit anti-ovalbumin antibodies demonstrated no such e f f e c t (117). This was one of the f i r s t i ndications that the u t i l i z a t i o n of r a d i o l a b e l l e d anti-tumour antibodies for the i n vivo detection of cancer might be r e a l i s t i c . In rapid succession, more studies were reported of the s p e c i f i c l o c a l i z a t i o n of r a d i o - l a b e l l e d anti-tumour antibodies in tumour t i s s u e . Pressman and Korngold reported in 1953 that r a d i o l a b e l l e d s p e c i f i c antibodies could be l o c a l i z e d in osteogenic sarcoma iji vivo (118). This was confirmed by Bale's studies of the s p e c i f i c in vivo l o c a l i z a t i o n of antibodies to rat kidney and Walker carcinoma (119). As more tumour markers were i s o l a t e d and characterized, r a d i o l a b e l l e d preparations of s p e c i f i c polyclonal antibodies demonstrated the p o t e n t i a l c l i n i c a l usefulness of the radioimmunodetection approach (120-126). With the advent of monoclonal antibody technology, came a new resurgence in the area of radioimmunodetection research. Monoclonal antibody preparations offered the advantages of being a homogeneous population free from contaminating nonspecific antibodies, available at 114 high concentrations through mass production, ease of p u r i f i c a t i o n from supernatants from hybridoma cultures or ascites f l u i d , and could be selected for high antigen s p e c i f i c i t y and a f f i n i t y . The s u p e r i o r i t y of monoclonal antibodies over polyclonal antisera in radioimmunoimagery has been demonstrated in several experiental model systems (127,128). However, t h i s i s a contentious point since some laboratories have found no comparable differences between monoclonal and polyclonal l a b e l l e d antibody l o c a l i z a t i o n in tumour (129-131). There are obviously a considerable number of parameters and problems associated with developing technology by which monoclonal antibodies can be used optimally in tumour imaging. These include: choice of the most appropriate radionuclide with which to l a b e l the antibody, methods f o r tagging the antibody which permit the maintenance of i t s b i o l o g i c a l s p e c i f i t y , and the elimination of background l e v e l s of c i r c u l a t i n g antibody. In terms of the choice of an appropriate radionuclide, i t i s e s s e n t i a l to take into consideration both the physical parameters ( i . e . the type of decay, energy, and h a l f l i f e ) and the optimal time required for maximum uptake by tumour correlated with maximal clearance of antibody from the c i r c u l a t i o n , or from normal t i s s u e . For diagnostic purposes, radionuclides that produce a pure photon decay are desirable. Charged p a r t i c l e s contribute only to the t o t a l r a d i a t i o n dose received by the patient without increasing diagnostic data. I d e a l l y , the physical h a l f - l i f e should be long enough to allow c o l l e c t i o n of the information required for target detection and short enough so that the patient does not receive needless i r r a d i a t i o n . 115 To d a t e , h i g h l e v e l s o f backg round r a d i o a c t i v i t y r e m a i n i n g i n u n a f f e c t e d t i s s u e have been a major p r o b l e m i n t h i s a r e a o f r e s e a r c h . N o n s p e c i f i c a c c u m u l a t i o n i n the l i v e r , s p l e e n and k i d n e y s i s commonly o b s e r v e d ( 1 3 2 ) . J u s t what the causes f o r t h i s k i n d o f backg round a re and how t o m i n i m i z e them are not o b v i o u s . However, b i n d i n g o f a n t i b o d i e s by d e n t r i t i c c e l l s w i t h F r e c e p t o r s and d i f f i c u l t i e s i n c l e a r a n c e o f c r a g g r e g a t e d a n t i b o d i e s may be c o n t r i b u t i n g f a c t o r s . These p rob l ems c o u l d p o s s i b l y be m i n i m i z e d by u s i n g c a r e f u l l y p r e p a r e d nonoagg rega ted F a b ' o r F ( a b ' ) 2 f r agments i n s t e a d o f whole a n t i b o d y m o l e c u l e s . A n t i b o d y f r agments have been shown to g i v e much h i g h e r t a r g e t up take r a t i o s i n compa r i son t o i n t a c t a n t i b o d y (133-136) . T h i s c o u l d be due i n p a r t t o the f a c t t h a t they move more r e a d i l y t h r o u g h c a p i l l a r y po res and i n t e r s t i t i a l f l u i d than the l a r g e r i n t a c t m o l e c u l e s , hence b e i n g more a c c e s s i b l e f o r a n t i g e n b i n d i n g on the tumour s u r f a c e . In a d d i t i o n , a n t i b o d y f r agments a re more r a p i d l y c l e a r e d f rom the body v i a the k i d n e y s when c i r c u l a t i n g u n a g g r e g a t e d ( 1 3 7 ) . 99m In the s t u d y , a T c - l a b e l l e d ant i-M-1 probe was used t o 99m r a d i o i m a g e M-l tumours i n v i v o . Tc has been c o n s i d e r e d one o f t he most i d e a l r a d i o n u c l i d e s f o r imag ing ( 1 3 8 ) . I t has a r e l a t i v e l y s h o r t h a l f - l i f e (6 h r ) , a s i n g l e photon e m i s s i o n o f 140 keV and low t i s s u e d e p o s i t i o n o f i o n i z i n g r a d i a t i o n (an i m p o r t a n t f a c t o r when c o n s i d e r i n g the p o t e n t i a l rad iodamage o f the s c a n n i n g a g e n t ) . The ant i-M-1 m o n o c l o n a l a n t i b o d y was f o u n d t o have an it i v i v o an t i - tumour t a r g e t i n g a b i l i t y as a h a e m a t o p o r p h y r i n c o n j u g a t e . The pu rpose o f t h i s s tudy was t o e s t a b l i s h whether the same m o n o c l o n a l an t i body- tumour model used i n a t h e r a p e u t i c 116 system (as described i n Chapter I I ) , could be u t i l i z e d i n a radioimmunoimaging study. 117 C h a p t e r IV MATERIALS AND METHODS E x p e r i m e n t a l a n i m a l s , t umours , and m o n o c l o n a l a n t i b o d i e s were as d e s c r i b e d i n C h a p t e r 2. Tc l a b e l l i n g o f a n t i b o d y and a n t i b o d y f r agments M o n o c l o n a l a n t i b o d y , e . g . a n t i - M - 1 , i n the fo rm o f a 50% s a t u r a t e d (NH^^SO^ p r e c i p i t a t e ( r e d i s s o l v e d and d i a l y s e d i n PBS) was s en t t o Summa M e d i c a l C o r p o r a t i o n , A l b u q u e r q u e , New Mex i co t o be p r e p a r e d f o r 99m -Techne t i um (Tc ) l a b e l l i n g . In b r i e f , the p r o t e i n s o l u t i o n was r e a c t e d w i t h s tannous c h l o r i d e i n a p r e t i n n i n g method d e v e l o p e d by D r . B .A. Rhodes ( 1 3 9 ) . To p r e p a r e the l a b e l l i n g k i t s , a s o l u t i o n o f K-H p h t h a l a t e , K-Na t a r t r a t e and S n C l 2 was b rough t to pH 5.6 w i t h NaOH. The s o l u t i o n was m ixed w i t h the a n t i b o d y and i n c u b a t e d i n the absence o f oxygen f o r about 21 h a t room t e m p e r a t u r e . The s o l u t i o n was then l y o p h i l i z e d i n 150 yg dose k i t s f o r l a b e l l i n g a t a l a t e r t i m e . The F ( a b ' ) 2 f r agments were p r e p a r e d by s t a n d a r d t e c h n i q u e s w i t h the enzyme p e p s i n ( 1 4 0 ) . The l a b e l l i n g p r o c e d u r e was done i n the Department o f N u c l e a r M e d i c i n e , Vancouve r G e n e r a l H o s p i t a l , i n c o l l a b o r a t i o n w i t h L a u r a A l c o r n . 99m L a b e l l i n g o f the l y o p h i l i z e d a n t i b o d y k i t s c o n s i s t e d o f a d d i n g Tc sodium p e r t e c h n e t a t e and 10% human serum a lbumin ( c a r r i e r p r o t e i n ) , and i n c u b a t i o n f o r 45 min a t room t e m p e r a t u r e . The s o l u t i o n was then f i l t e r e d t h rough a Sephadex m i n i c o l u m n e s p e c i a l l y d e s i g n e d t o remove f r e e 118 pertechnetate and reduced; hydrolyzed technetium ( F i l t e c h -Summa Medical Corp., Albuquerque, New Mexico). For scanning studies, 15-20 mCi of 99m Tc was used to l a b e l each 150 ug l y o p h i l i z e d pretinned antibody k i t . For studies in which only ti s s u e d i s t r i b u t i o n was measured, 3-5 mCi of " m T c was used to l a b e l each k i t . Radioimaging studies: For radioimaging, the mice were injec t e d subcutaneously with e i t h e r 0.5 - 1. x 10"* M-l or 1 x 10 6 EL4 tumour c e l l s i n the lower flank above the thigh muscle. The mice were then rested for 10 - 14 days p r i o r to imaging studies. At t h i s time, the tumour was 1 cm in diameter (0.3 - 0.7 g). Approximately 0 . 5 - 1 mCi 99m Tc tagged to 5 - 10 ug antibody was injected intravenously per animal. The t e s t animals were scanned on a Picker Anger S c i n t i l l a t i o n gamma camera using a pinhole collimator 4 - 48 hr p o s t - i n j e c t i o n . Animals were sedated for imaging with 60 mg/kg body wt Nembutal (Abbott Laboratories, Limited, Montreal, Canada) i n t r a p e r i t o n e a l l y and stretched out in a supine p o s i t i o n . Tissue d i s t r i b u t i o n studies: Tumour bearing mice were prepared in the same manner as described in the radioimaging studies. For d i s t r i b u t i o n 99m studies, 100 - 500 uCi Tc/5 - 10 ug Ab was administered intravenously per tumour bearing animal. Tissue d i s t r i b u t i o n and blood l e v e l s were assayed at 4 - 72 hours p o s t - i n j e c t i o n . The tes t animals were anaesthetized with ether. Blood samples (0.5 - 1 ml) were taken by c l i p p i n g the renal vein and artery and draining the blood from the 119 p e r i t o n e a l c a v i t y w i t h a p a s t e u r p i p e t . O the r t i s s u e s removed f o r a s say were tumour , m u s c l e , k i d n e y s , l i v e r and l u n g . The t a i l was a l s o c o u n t e d t o c o r r e c t f o r p o s s i b l e i n t e r s t i t i a l i n j e c t i o n . The musc l e sample was the e n t i r e t h i g h musc l e f rom the nontumour b e a r i n g l e g . In e x p e r i m e n t s where tumour was grown i n bo th h i n d l e g s , e q u i v a l e n t musc le mass was e x c i s e d f r om the b a c k , and the f r o n t l e g s . A l l t i s s u e samples were t h o r o u g h l y r i n s e d i n PBS and p l a c e d i n p re-we ighed g l a s s tubes (B-D v a c u t a i n e r B rand e v a c u a t e d b l o o d c o l l e c t i o n tube 3 m l ) . A l l tubes were p r o m p t l y s e a l e d t o p r e v e n t l o s s o f we igh t due t o wa te r e v a p o r a t i o n and w e i g h e d . R a d i o a c t i v i t y was c o u n t e d i n a P i c k e r w e l l - c o u n t e r . T i s s u e d i s t r i b u t i o n c a l c u l a t i o n s were done and r e s u l t s e x p r e s s e d as p e r c e n t i n j e c t e d dose pe r gram t i s s u e o r b l o o d . C o n t r o l m ice r e c e i v i n g no i n j e c t i o n were a s s a y e d to c o r r e c t f o r cage l i t t e r c o n t a m i n a t i o n . Background up take o f the r a d i o i s o t o p e due t o consumpt ion o f c o n t a m i n a t e d l i t t e r l e d to an appa ren t i n c r e a s e i n r a d i o a c t i v i t y i n the m e t a b o l i c o rgan s y s t e m s . The re was 5 - 7 t e s t an ima l s i n each g r o u p . S t anda rds were p r e p a r e d i n o r d e r t o de t e rm ine 100% d o s e . R a d i o a c t i v e coun t s i n the t a i l were t aken as c o n t a m i n a t i n g i n t e r s t i t i a l l e a k a g e f rom i n t r a v e n o u s i n j e c t i o n and s u b t r a c t e d f rom the t o t a l i n j e c t e d d o s e . The c a l c u l a t i o n s t e p s used a re o u t l i n e d as f o l l o w s : K = c o n t r o l t i s s u e (cpm) S = s t a n d a r d r a d i o a c t i v e dose (cpm) E = e x p e r i m e n t a l t i s s u e (cpm) W t 1 = we igh t o t tube + sample 2 Wt = we igh t o f tube 120 Bkg = backg round coun t s d i l F = d i l u t i o n f a c t o r R e s u l t 1 = K (cpm) - K bkg (cpm) = K c p m / g K W t ^ g ) - KWt 2 (g) R e s u l t 2 = 100 [ (S(cpm) - S bkg(cpm) x d i l F] - [E t a i l ( c p m ) - K t a i l ( c p m ) % r a d i o a c t i v e dose = E (cpm) - E bkg (cpm) 1 2 I E Wt - E Wt - R e s u l t s 1 x R e s u l t 2 gram t i s s u e Amicon f i l t r a t i o n o f a n t i b o d y s o l u t i o n : L a b e l l e d a n t i b o d y (150 ug i n 1 ml ) was f i l t e r e d t h rough a XM 300 Amicon f i l t e r . A f t e r the 1 ml volume was r e c o v e r e d as the f i l t r a t e , the m a t e r i a l r e t a i n e d on the f i l t e r was r e s u s p e n d e d i n 1 ml o f PBS. Samples were then run on p o l y a c r y l a m i d e g e l s t o l o c a t e the l a b e l l e d p r o t e i n . 99m C e n t r i f u g a t i o n e x p e r i m e n t : A f t e r T c - l a b e l l i n g , the a n t i b o d y s o l u t i o n s ( 1 .8 ml volume) were c e n t r i f u g e d i n a SS34 r o t o r S o r v a l Supe r speed c e n t r i f u g e f o r 5 h r s a t 19 .5 K rpm (45 ,900 g) . 0 .6 ml o f the l a b e l l e d p r o t e i n was s e t a s i d e p r i o r t o c e n t r i f u g a t i o n and was d e s i g n a t e d the unspun f r a c t i o n . A f t e r c e n t r i f u g a t i o n , two 0 .6 ml samples were c a r e f u l l y drawn o f f . The t op and m i d d l e 0 .6 ml p o r t i o n s were d e s i g n a t e d F r a c t i o n s 1 and 2 r e s p e c t i v e l y . The bo t tom 0.6 ml p o r t i o n was v o r t e x e d v i g o r o u s l y t o r e s u s p e n d any sed imented m a t e r i a l and l a b e l l e d F r a c t i o n 3. A l i q u o t s e q u i v a l e n t t o the dose volume t o be g i v e n t o each a n i m a l were 99m t aken and d i l u t e d f o r d e t e r m i n a t i o n o f Tc c o n t e n t . The d i f f e r e n t 121 f r a c t i o n s were then immediately injected intravenously v i a t a i l vein into tumour-bearing mice for tissue d i s t r i b u t i o n studies. Deaggregation pH treatments of l a b e l l e d antibody solutions: The antibody o solutions were incubated at 37 C in a 100% humidified incubator for 1-2 hr or overnight at pH 4 (addition of 0.1 M HCl) or pH 7.2 before or af t e r 99m Tc l a b e l l i n g . The l a b e l l e d antibody solutions were then examined for t h e i r l e v e l of aggregation by non-reducing non-SDS polyacrylamide gel electrophoresis. The gels were 3 - 10% 1.5 cm thick l i n e a r polyacrylamide slabs poured from a HSI Gradient Maker (Hoefer S c i e n t i f i c Instruments, S.F. C a l . ) . 50 y l of each solution was added to 20 y l of thymol blue + g l y c e r o l tracking dye solu t i o n . Approximately 30 y l of these samples were layered on each sample track. The gel was stacked at 50 V f o r 1 hr and run at 150 V for 6 - 7 hr. A l l gel reagents and running buffers (49.5 mM T r i s base: 0.38 M Glycine, pH 8.3) were f i l t e r e d through a 1.2 ym m i l l i p o r e f i l t e r p r i o r to use. Aft e r completion of the run, the gels were radioautographed on 3 M T r i l i t e TM X-ray f i l m overnight and then developed. Af t e r r a d i o a c t i v i t y l e v e l of the gel reached background, the gel was s i l v e r s t a i n e d f o r the po s i t i o n of the molecular weight standards (141). 123 Figure 23. Titration of specific monoclonal anti-M-1 and Tc-anti-M-1 and M-l antigen. The immunological activity of anti-M-1 (o) and 99m Tc-anti-M-1 (0) were compared on 25 ug/ml M-l membrane extract in the ELISA. Each point is the mean of sextuplet c e l l s . There was no st a t i s t i c a l l y significant difference found between the reactivity of labelled and non labelled antibodies. 124 I 0.2 10 1.0 QI 0.01 pg Ab 125 F i g u r e 24 . A c t i v i t y o f Tc-ant i-M-1 on v a r i o u s a n t i g e n p r e p a r a t i o n s . 99m The a n t i b o d y a c t i v i t y o f 10 ug Tc-ant i-M-1 as a s sa yed i n the EL ISA was d e t e r m i n e d on 25 yg/ml o f s p e c i f i c M-l and i r r e l e v a n t a n t i g e n p r e p a r a t i o n s . Each ba r r e p r e s e n t s the mean ± S .D . o f q u a d r u p l i c a t e w e l l s . 1 2 6 127 p a l p a b l e tumours o f 0.5 - 1.0 cm i n d i a m e t e r . A l l t e s t mice were d i a g n o s e d as tumour p o s i t i v e p r i o r t o the e x p e r i m e n t . S c ann ing o f the an ima l s was c a r r i e d out o ve r a 24 h p e r i o d . An ima l s were s a c r i f i c e d a t v a r i o u s t imes and samples o f d i f f e r e n t t i s s u e s were t aken t o a s say f o r 99m Tc d i s t r i b u t i o n . The t i s s u e s sampled were k i d n e y , l i v e r , s p l e e n , l u n g , m u s c l e , b l o o d and tumour . In the t ime p e r i o d o f 4 - 44 h r s p o s t 99m i n j e c t i o n o f l a b e l , the h i g h e s t l e v e l o f Tc as d e t e r m i n e d by % r a d i o a c t i v e dose pe r gram t i s s u e was f o u n d i n the k i d n e y and l i v e r ( F i g . 2 5 ) . C o n s i d e r a b l e amounts o f 9 9 m T c were a l s o f ound i n the s p l e e n , tumour , b l o o d and l u n g ( i n d e c r e a s i n g o r d e r ) . The l e a s t amount 9 9 m T c was f ound i n the norma l t i s s u e c o u n t e r p a r t o f the M-l myosarcoma, m u s c l e . 99m_ Most xc l e v e l s d e c r e a s e d i n the 4 - 44 h r a ssay p e r i o d . The t i s s u e t h a t d e v i a t e d f rom t h i s p a t t e r n were k i d n e y and tumour . From 4 t o 18 h r s , 99m b o t h t i s s u e s d e m o n s t r a t e d an i n c r e a s e i n Tc i n c o r p o r a t i o n ; tumour 99m showing a s h a r p e r i n c r e a s e then k i d n e y . A f t e r 18 h r , Tc l e v e l s began 99m t o d e c r e a s e as i n the o t h e r t i s s u e s . B l o o d l e v e l s o f Tc d e m o n s t r a t e d the f a s t e s t r a t e o f d e c r e a s e . I t can be seen t h a t the most f a v o u r a b l e 99m t i s s u e / b l o o d r a t i o s f o r tumour were o b s e r v e d 44 h r a f t e r Tc-an t i -M-1 99m i n j e c t i o n ( F i g . 2 6 ) . A f t e r 18 h , the Tc b l o o d l e v e l s were d r o p p i n g 99m more r a p i d l y t han were t h o s e i n tumour t i s s u e . However, w i t h T c , coun t s were v e r y low a t 44 h r p o s t i n j e c t i o n , so s c a n n i n g was ou t o f the q u e s t i o n a t t h i s t i m e . S cann ing o f whole an ima l s showed t h a t tumour c o u l d 99m be imaged as soon as 6 h a f t e r Tc i n j e c t i o n . A t 6 h , the tumour mass showed up as a f a i n t shadow. A t 18 h , the tumour was c l e a r l y d i s c e r n a b l e ( F i g . 2 7 ) . Scans o f the tumour a r e a i n c r e a s e d i n i n t e n s i t y w i t h t i m e , 128 F i g u r e 2 5 . T i s s u e d i s t r i b u t i o n s t u d y : 4-44 h r s a f t e r Tc-an t i -M-1 i n j e c t i o n . The % r a d i o a c t i v e dose/g t i s s u e o f v a r i o u s t i s s u e s were a s sayed a t v a r i o u s t imes (4 , 18 and 44 h r ) a f t e r i n t r a v e n o u s i n j e c t i o n o f 99m Tc l a b e l l e d a n t i - M - 1 . T h i s was done i n o r d e r to examine the d i s t r i b u t i o n o f r a d i o l a b e l o ve r t i m e . Each p o i n t r e p r e s e n t s the ave rage r e s u l t s f rom the f i v e m ice p e r g r o u p . 1 2 9 l 130 99m_ F i g u r e 26 . R e l a t i v e % r a d i o a c t i v e dose TC pe r gram t i s s u e l o c a t e d i n m u s c l e , b l o o d and M-l tumour 4 - 4 4 h r s a f t e r i n t r a v e n o u s i n j e c t i o n o f 9 9 i n T c - a n t i - M - 1 . Each d a t a p o i n t r e p r e s e n t s the mean v a l u e o f samples t aken f rom f i v e mice pe r t e s t g r o u p . 131 i 132 Figure 27. Scans of M-l bearing mice 18 hr after intravenous 99m adminstration of Tc labelled anti-M-1. The M-l tumour masses were clearly visible on the right flank of the test animals. No equivalent activity was observed on the l e f t side of the animals. The arrows point towards the tumor masses on the right flank of the animals. 133 134 e . g . a t 24 and 36 h o u r s . However, o t h e r t i s s u e s (ma in l y k i d n e y and l i v e r ) e x h i b i t e d c o n s i d e r a b l y g r e a t e r l e v e l s o f r a d i o a c t i v i t y than d i d the tumour . In a l l scans t aken f rom 6 - 3 6 hours pos t i n j e c t i o n , the k i d n e y s and l i v e r r e p r e s e n t e d the d a r k e s t masses i n the image. The b l a d d e r would 99m p e r i o d i c a l l y scan p o s i t i v e i n some o f the t e s t an ima l s due t o Tc e x c r e t i o n i n the u r i n e ( F i g . 2 7 ) . I t s h o u l d be n o t e d t h a t even though 99m s p l e e n s demons t r a t ed s l i g h t l y h i g h e r l e v e l s o f Tc i n the t i s s u e d i s t r i b u t i o n s t u d i e s , t hey were no t p o s i t i v e l y imaged i n the s c a n s . T h i s was due m a i n l y because o f ove r shadow ing by the l i v e r and the k i d n e y s i n t h a t ang l e o f the s c a n . 99m In o r d e r t o d e t e r m i n e whether the r e t e n t i o n o f Tc i n tumour was a t t r i b u t a b l e t o the s p e c i f i c i t y o f the a n t i b o d y , f u r t h e r e x p e r i m e n t s were r u n . In t hese s t u d i e s , B6D2 mice were used (F^ c r o s s e s between DBA/2J and C57BL/6 p a r e n t s ) . In t hese a n i m a l s , bo th the M-l tumour and a c o m p l e t e l y u n r e l a t e d tumour l i n e (E14 lyphoma) grow w i t h o u t r e j e c t i o n . Bo th tumours were t i t r a t e d i n the B6D2 mice i n o r d e r t o d e t e r m i n e the number o f tumour c e l l s r e q u i r e d to ge t e q u i v a l e n t s i z e tumours i n the same l e n g t h o f t i m e . B6D2 mice were i n j e c t e d s u b c u t a n e o u s l y i n the r i g h t f l a n k w i t h EL4 tumour . Scans o f EL4 tumour b e a r i n g mice were n e g a t i v e i n tumour mass a r e a ( F i g . 2 8 ) . The tumours were a p p r o x i m a t e l y 1 cm i n d i a m e t e r and ave raged 0 .8 gm wet we igh t a t the t ime o f s c a n n i n g . In the nex t e x p e r i m e n t , B6D2 an ima l s were i n j e c t e d s u b c u t a n e o u s l y i n the r i g h t and l e f t f l a n k s w i t h M-l and EL4- tumours , r e s p e c t i v e l y . The t e s t m ice were s c r e e n e d f o r e q u i v a l e n t tumour s i z e s i n b o t h EL4 and M-l tumours p r i o r to the e x p e r i m e n t . When the tumours were p a l p a b l e (0 .5 - 1.0 cm i n 135 F i g u r e 28 . Scans o f EL4 b e a r i n g B6D2 mice i n j e c t e d 18 h r e a r l i e r w i t h 99m T c - a n t i - M - 1 . The EL4 tumour masses (see a r rows ) were not v i s u a l i z e d on the r i g h t f l a n k o f the t e s t an ima l s w i t h the s p e c i f i c l a b e l l e d a n t i b o d y p r o b e . 136 M o o S f . M o o t e r 5 B6D2/FI Mice ! EL4 tumour s.c. lower right abdominal region 137 d i a m e t e r ) , the mice were i n j e c t e d i n t r a v e n o u s l y w i t h T c - l a b e l l e d a n t i - M - 1 . A f t e r 18 h r s , the an ima l s were scanned and s a c r i f i c e d f o r t i s s u e d i s t r i b u t i o n s t u d i e s . S i g n i f i c a n t l y h i g h e r l e v e l s o f a c t i v i t y was f o u n d i n the M-l tumour than i n the E L 4 , m u s c l e s , o r b l o o d s a m p l e s . In E L 4 , tumours coun t s were lower than i n b l o o d , but somewhat h i g h e r than musc l e ( F i g . 2 9 ) . The r e l a t i v e r a t i o o f M-l to musc l e r a d i o a c t i v i t y was about 5 and about 2.5 when M-l to EL4 r a t i o s were measured . Imaging s t u d i e s showed t h a t M-l tumours were v i s i b l e whereas EL4 tumours i n the same an ima l s were not ( F i g . 3 0 ) . A g a i n , as seen i n the p r e v i o u s s c a n n i n g e x p e r i m e n t s , the l i v e r and k i d n e y s were c l e a r l y v i s i b l e . E x a m i n a t i o n o f the tumour t i s s u e a f t e r imag ing showed t h a t the tumours were o f e q u a l s i z e and appea red to have a s i m i l a r degree o f v a s c u l a r i z a t i o n . The imag ing s t u d i e s d i s c u s s e d above demons t r a t ed c l e a r l y the 99m s p e c i f i c i t y o f the Tc-ant i-M-1 i n v i v o f o r the M-l tumour . However , the s t u d i e s showed an e x t r e m e l y h i g h background o f a p p a r e n t l y n o n s p e c i f i c a c t i v i t y seen i n the l i v e r , k i d n e y and s p l e e n i n the t e s t a n i m a l s . The backg round c o u l d be c aused by a number o f f a c t o r s . F r e e c i r c u l a t i n g a n t i g e n shed by the tumour c o u l d be comp l ex i ng w i t h the l a b e l l e d a n t i b o d y . The complexes m igh t then be t aken up by p h a g o c y t i c c e l l s o r be d e p o s i t e d i n o rgans such as the k i d n e y o r l i v e r . A l t e r n a t e l y , the l a b e l l e d a n t i b o d y - a n t i g e n complexes c o u l d have been shed a f t e r b i n d i n g o f the a n t i b o d y t o c e l l membrane bound a n t i g e n . L a b e l l e d a n t i b o d y c o u l d be t aken up n o n s p e c i f i c a l l y v i a F r e c e p t o r s on d e n d r i t i c c e l l s o r c mac rophages . A n o t h e r p o s s i b i l i t y i s t h a t t h e r e c o u l d be p rob lems w i t h the l a b e l l e d a n t i b o d y p r e p a r a t i o n . A g g r e g a t e s o f l a b e l l e d a n t i b o d i e s wou ld 138 F i g u r e 29 . P e r c e n t r a d i o a c t i v e dose pe r gram o f t i s s u e i n B6D2 mice b e a r i n g b o t h M-l and EL4 tumours . An ima l s were s a c r i f i c e d 18 h r s a f t e r 99m Tc-ant i-M-1 a d m i n i s t r a t i o n . V a r i o u s t i s s u e s were removed, we ighed and coun ted f o r r a d i o a c t i v i t y . Each d a t a p o i n t r e p r e s e n t s the r e s u l t s o b t a i n e d f r om an i n d i v i d u a l t e s t a n i m a l . 139 B lood M - l EL4 Muscle 1.00 2 . 0 0 3 . 0 0 4 . 0 0 % R a d i o a c t i v e Dose per gram tissue 140 F i g u r e 30 . Scan o f a B6D2 mouse i n j e c t e d 18 h r e a r l i e r w i t h 99m T c - a n t i - M - 1 . The t e s t an ima l bea r s a M-l tumour on i t s r i g h t f l a n k ( v i s i b l e ) and an EL4 tumour on i t s l e f t f l a n k (not v i s i b l e ) . The tumours were o f e q u i v a l e n t s i z e (M- l : 0 . 6 5 g , E L4 : 0 . 7 2 g ) . 141 B6D2 / F I M I C E : M- l sarcoma and E L 4 Lymphoma 142 a l s o be t aken up by p h a g o c y t i c c e l l s o r d e p o s i t e d i n b l o o d f i l t r a t i o n o rgans such as k i d n e y and l i v e r . The f i r s t approach i n a d d r e s s i n g the backg round p rob l em was w i t h a c o l d a n t i b o d y q u e n c h i n g e x p e r i m e n t . In t h i s e x p e r i m e n t , the p o s s i b l e r o l e o f a n t i g e n s h e d d i n g was examined . Tumour b e a r i n g an ima l s were i n j e c t e d i n t r a v e n o u s l y w i t h 5 yg o f a n t i - M - 1 , an i r r e l e v a n t m o n o c l o n a l a n t i b o d y , a n t i - G I P , and PBS 24 h p r i o r to i n j e c t i o n o f the l a b e l l e d a n t i b o d y ( F i g . 3 1 ) . The a n i m a l s were scanned and s a c r i f i c e d f o r t i s s u e d i s t r i b u t i o n s t u d i e s 18 h a f t e r l a b e l l e d a n t i b o d y a d m i n i s t r a t i o n . I f f r e e a n t i g e n was 99m the cause o f T c n o n - s p e c i f i c a c c u m u l a t i o n , c o l d s p e c i f i c a n t i b o d y s h o u l d c l e a r the c i r c u l a t i o n by c o m p l e x i n g w i t h the a n t i g e n . Hence , the scans s h o u l d have a c l e a r e r d e l i n e a t i o n o f the tumour mass s i n c e more l a b e l l e d a n t i b o d y wou ld be a v a i l a b l e f o r b i n d i n g t o the tumour , and not to f r e e c i r c u l a t i n g a n t i g e n . A d m i n i s t r a t i o n o f c o l d an t i -G I P o r PBS wou ld not have any e f f e c t on the scans i n t h i s c a s e . The % r a d i o a c t i v e dose pe r gram t i s s u e r e s u l t s f rom the t h r e e p r e t r e a t e d groups a re shown i n F i g . 32 . The n o n s p e c i f i c t i s s u e s , musc l e and an i r r e l e v a n t tumour EL4 99m d e m o n s t r a t e d e q u i v a l e n t l e v e l s o f Tc r a d i o a c t i v i t y i n a l l t h r e e p r e t r e a t e d g r o u p s . P r e - i n j e c t i o n o f the c o l d a n t i b o d i e s , s p e c i f i c ant i-M-1 and n o n s p e c i f i c an t i -G I P d i d no t s i g n i f i c a n t l y e f f e c t the n o n s p e c i f i c backgrounds i n EL4 and m u s c l e . However, the up take o f l a b e l i n the s p e c i f i c tumour was changed by the p r e t r e a t m e n t s w i t h s p e c i f i c and n o n s p e c i f i c c o l d a n t i b o d i e s . A d m i n i s t r a t i o n o f c o l d ant i-M-1 a n t i b o d y d e c r e a s e d the l e v e l o f " m T c i n M-l tumour i n compa r i son t o the PBS p r e t r e a t e d M-l tumour . On the o t h e r h a n d , a d m i n i s t r a t i o n o f c o l d an t i -G I P 143 F i g u r e 3 1 . F low d i ag ram i l l u s t r a t i n g the p r o t o c o l used i n the p r e t r e a t m e n t o f DBA/2J m ice w i t h u n l a b e l l e d " c o l d " a n t i b o d i e s o r PBS p r i o r t o " m T c - a n t i - M - l i n j e c t i o n . 144 SCAN 18 Hr TUMOUR BEARING MOUSE t 24 Hr. 'RETREATMENT anti -M -1, anti - GIP, or PBS i.v. ( 5 (19) 99m Tc - anti-M-1 i.v. ( 10 vg • I MCi) 24 Hr SACRIFICE AND TISSUE DISTRIBUTION STUDY i 145 F i g u r e 32 : P e r c e n t r a d i o a c t i v e dose pe r gram o f t i s s u e i n B6D2 m i ce f o l l o w i n g p r e t r e a t m e n t w i t h an t i -G I P ( i r r e l e v a n t a n t i b o d y c o n t r o l ) , PBS, o r ant i-M-1 ( s p e c i f i c a n t i b o d y ) . The an ima l s were a s sa yed 24 h r a f t e r " m T c - a n t i - M - l i n t r a v e n o u s i n j e c t i o n . Each d a t a p o i n t r e p r e s e n t s r e s u l t s o b t a i n e d f rom an i n d i v i d u a l t e s t a n i m a l . 146 3.0 • % Radioactive dose g tissue 2.0 cold non - specific anti-GIP PBS control cold specific dnti - M-l M-l E L 4 Muscle M-l EL4 Muscle M-l E L4 Muscle 147 Figure 33. Ratio of percent radioactive dose in M - l : EL4 tumour tissue from B6D2 mice bearing both tumours and treated with unlabelled anti-GIP, 99m PBS, and anti-M-1, 24 hr prior to injection with Tc-anti-M-1. 148 i ! i j | i 3.0 -! I 1 T u m o u r 1 i r a t i 0 2.5 - ! i r | i _ _ i - M ^ L 2 0 • I I E L 4 r 1 , , 1.5 • io • i I I 0.5 • A B C a n t i - G I P PBS ant i — M - l 149 a n t i b o d y seem t o cause a m a r g i n a l i n c r e a s e i n the amount o f Tc i n M-l 99m tumour . The i n c r e a s e o f s p e c i f i c i t y o f the Tc-ant i-M-1 f o r M-l tumour i s more c l e a r l y seen i n F i g . 3 3 . The r a t i o s o f % r a d i o a c t i v e dose pe r gram M-l t o % r a d i o a c t i v e dose pe r gram EL4 f rom each g roup was c a l c u l a t e d . In a l l t h r e e p r e t r e a t m e n t g r o u p s , the r a t i o o f M-1/EL4 was 99m_ g r e a t e r than 1, i n d i c a t i n g t h a t the xc-ant i-M-1 had a g r e a t e r s p e c i f i c i t y f o r M-l t i s s u e than f o r EL4 t i s s u e . Ant i-M-1 p r e t r e a t m e n t d e c r e a s e d the r a t i o t o 1 .6 , the c o n t r o l o r PBS p r e t r e a t m e n t r a t i o was a p p r o x i m a t e l y 2 .19 and the an t i -G I P p r e t r e a t m e n t i n c r e a s e d the r a t i o t o 2 . 4 8 . S t a t i s t i c a l a n a l y s i s demons t r a t ed t h a t t hese d i f f e r e n c e s were s i g n i f i c a n t . However, t h e s e d i f f e r e n c e s were c o m p l e t e l y i r r e l e v a n t when the scans were examined . The o v e r a l l backg round p rob l em o f h i g h l e v e l s o f r a d i o a c t i v i t y i n o t h e r t i s s u e s was not ma rked l y a f f e c t e d by t h e s e q u e n c h i n g s t u d i e s ( F i g . 34 and 3 5 ) . The 18 h scans d i d no t d e m o n s t r a t e any d i s c e r n a b l e d i f f e r e n c e s between the p r e t r e a t e d g r o u p s . The M-l tumour was v i s u a l i z e d i n a l l t h r e e p r e t r e a t e d an ima l g r o u p s . F a i n t shadowing was seen i n the EL4 tumour a r e a but i t was d i f f i c u l t t o de t e rm ine whether i t 99m was due to Tc i n c o r p o r a t i o n i n EL4 o r s i m p l y backg round f rom the s u r r o u n d i n g t i s s u e s . In scans f rom a l l t h r e e p r e t r e a t e d g r o u p s , b o t h l i v e r and k i d n e y were c l e a r l y i d e n t i f i e d as the d a r k e s t masses i n the s c a n s . S i n c e backg round c o u l d be caused by n o n s p e c i f i c up take o f the l a b e l l e d a n t i b o d y v i a F r e c e p t o r s on d e n d r i t i c c e l l s and mac rophages , c an o b v i o u s app roach wou ld be t o use F ( a b ' ) 2 f r a g m e n t s . Ant i-M-1 a n t i b o d i e s were c o n v e r t e d to F ( a b * ) _ f r agments by p e p s i n d i g e s t i o n and 150 F i g u r e 34 . Scans o f PBS o r ant i-M-1 p r e t r e a t e d B6D2 m i c e . The B6D2 tumour b e a r i n g mice were p r e t r e a t e d w i t h PBS o r ant i-M-1 24 h r p r i o r t o 99m i n t r a v e n o u s i n j e c t i o n o f T c - a n t i - M - 1 . A l l an ima l s had p a l p a b l e tumours i n bo th f l a n k s o f e q u i v a l e n t s i z e ; M-l i n the r i g h t f l a n k , EL4 i n the l e f t f l a n k . A n t i b o d y p r e t r e a t m e n t d i d no t appear t o improve the q u a l i t y o f the scan i n compa r i son t o the PBS c o n t r o l . The arrows p o i n t towards the M-l tumours . PRETRFATMCNT PBS a M - l Bear'"ig DBA/2J Mice 152 F i g u r e 35 . Scan o f an t i -G I P p r e t r e a t e d B6D2 mouse. The tumour b e a r i n g mouse was p r e t r e a t e d w i t h an t i -G I P 24 h r p r i o r t o i n t r a v e n o u s i n j e c t i o n o f 99m T c - a n t i - M - 1 . The mouse had p a l p a b l e tumours o f e q u i v a l e n t s i z e i n bo th f l a n k s ; M-l i n the r i g h t f l a n k , EL4 i n the l e f t f l a n k . A n t i b o d y p r e t r e a t m e n t d i d no t appear t o improve the q u a l i t y o f the scan i n compa r i son to the PBS c o n t r o l . 153 M - l tear ing D B A / 2 J M i c e 154 prepared i n t o t i n n e d antibody fragments k i t s by Summa Medical Incorp. The l a b e l l e d anti-M-1 F ( a b ' ) 2 fragments s t i l l maintained immunological s p e c i f i c i t y e q uivalent to that of whole anti-M-1 antibody when assayed i n 99m ELISA ( F i g . 36). An i r r e l e v a n t F(ab> 2 fragment Tc-MAMF had minimal bindi n g on M-l antigen i n ELISA. Anti-M-1 F ( a b ' ) 2 fragments had a d i f f e r e n t t i s s u e d i s t r i b u t i o n i n comparison to whole l a b e l l e d antibody ( F i g . 37). In terms of % r a d i o a c t i v e dose per gram t i s s u e , the M-l , EL4, 99m_ muscle, spleen, and lung t i s s u e s accummulated s i m i l a r amounts of Tc 99m regardless of whether Tc-anti-M-1 whole or fragment was administered. However, the blood, kidney, and l i v e r samples had d i f f e r e n t 99m Tc d i s t r i b u t i o n s depending on whether l a b e l l e d fragment or whole 99m antibody used. The blood l e v e l of Tc was a l i t t l e more than twice as 99m 99m_ high i n Tc-anti-M-1 F ( a b ' ) 2 t r e a t e d mice i n r e l a t i o n to Tc l a b e l l e d whole anti-M-1 t r e a t e d mice. Kidney samples from fragment 99m t r e a t e d mice had at l e a s t a t h i r d more Tc r a d i o a c t i v i t y than analogous samples taken from whole antibody t r e a t e d mice. On the other hand, l i v e r samples from fragment t r e a t e d mice had only h a l f as much 99m Tc as whole antibody t r e a t e d l i v e r . I t should be noted that since 99m the Tc l e v e l s i n the blood was so high at 18 h post i n j e c t i o n , imaging of the tumours and other t i s s u e s was not p o s s i b l e since a t i s s u e / b l o o d r a t i o of 1 i s e s s e n t i a l f o r v i s u a l i z a t i o n (148). S p e c i f i c i t y of the anti-M-1 F ( a b ' ) 2 f o r M-l tumour was demonstrated i n 99m F i g . 38. Both the Tc-anti-M-1 F ( a b ' ) 2 fragment and an i r r e l e v a n t 99m . Tc l a b e l l e d F ( a b ' ) 2 fragment (MAMF) were administered to B6D2 mice bearing M-l and EL4 tumours. The M-l and EL4 tumours had the same l e v e l 155 F i g u r e 36 . A c t i v i t y o f the s p e c i f i c m o n o c l o n a l a n t i - M - 1 , Tc-an t i -M-1 and 9 9 m T c - a n t i - M - l F ( a b ' ) 2 f r agments on M-l (0) and BSA (B) 99m_ a n t i g e n s . The i m m u n o l o g i c a l a c t i v i t y o f a n t i - M - 1 , T c-an t i -M-1 and 99m Tc-ant i-M-1 F ( a b ' ) 2 f r agments as a s sa yed i n the EL ISA was compared on M-l and BSA a n t i g e n s . Each ba r r e p r e s e n t s the mean ± S .D . o f s i x w e l l s . 156 Reactivity of 9 9 m T e - « N M F(ab')2 Fragments on M-1 and BSA Antigens 99m 9 8 m T c - M A M F <<QIP Tc-«M-1 F ( a b ' ) 2  8 m T o - * M - 1 a M- l 99m T c - M A M F <XQ|p "Tc-ocM-1 F ( a b ' ) 2  9 9 m T c - * M - 1 <x M-1 a-/A a-'////. m — • — i - i i i i i BSA Ag i—M-1 Ag 0 . 0 0 0 .02 0.04 0 .06 0 .0S OD 0.10 0.12 0.14 1 0.16 405 157 F i g u r e 3 7 . Compar i son o f Tc-ant i-M-1 F C a b ' ^ f ragments and whole 99m_ T c - a n t i - M - 1 i n a t i s s u e d i s t r i b u t i o n s t u d y . Fragments (0) and 99m whole (1) Tc-an t i-M-1 were a d m i n i s t e r e d i n t r a v e n o u s l y i n t o B6D2 mice b e a r i n g bo th EL4 and M-l tumours . The an ima l s were s a c r i f i c e d and a s sayed 24 h r l a t e r . Each ba r i s the mean o f 6 - 7 d i f f e r e n t t e s t a n i m a l t i s s u e s a m p l e s . Each d a t a p o i n t i s the v a l u e o b t a i n e d f rom an i n d i v i d u a l a n i m a l . N.B . The % r a d i o a c t i v e dose/g t i s s u e s c a l e f o r the top and bo t tom p a r t s o f the f i g u r e d i f f e r i n magn i t ude . 158 159 Figure 38. Comparison of Tc-anti-M-1 F(ab'>2 and Tc-MAMF (irrelevant) F)ab') 2 fragments in a tissue distribution study. " mTc-anti-M-l F(ab'>2 fragments (B) and 99mTc-MAMF fragments (0) were administered intravenously into B6D2 mice bearing both EL4 and M-l tumours. The animals were sacrificed and assayed 24 hr later. Each bar is the mean ± S.D. of samples from 6 test animals. 160 Distribution of 9 9 m Tc - «M-1 and 9 9 m T c - M A M F Fragments in Tumour Bear ing M i ce Blood Muscle EL 4 M-1 m ' Legend "Tc - M-1 'B^ Tc - MAMF % radioactive dose / g tissue 161 o f r a d i o a c t i v i t y when t r e a t e d w i t h the i r r e l e v a n t F ( a b ' ) 2 f r agment 99m 99m Tc-MAMF. When t r e a t e d w i t h the s p e c i f i c Tc-ant i-M-1 F ( a b ' ) 2 > 99m the l e v e l o f Tc i n the M-l tumour was t h r e e t imes h i g h e r than t h a t i n the EL4 tumour . A g a i n , the b l o o d " m T c l e v e l s were h i g h e r than those i n the sample t i s s u e s so they c o u l d not be v i s u a l i z e d . S i n c e n e i t h e r o f the p r e v i o u s expe r imen t s were s u c c e s s f u l i n i m p r o v i n g the backgrounds o f the s c a n s , the l a b e l l e d a n t i b o d y p r e p a r a t i o n were examined f o r a g g r e g a t i o n . L a b e l l e d a n t i b o d y was f i l t e r e d t h rough an Amicon f i l t e r d e s i g n e d t o a l l o w p a r t i c l e s o f 300 ,000 d a l t o n s t h rough (Amicon XM 300 f i l t e r ) . E q u i v a l e n t vo lumes o f f i l t e r e d , p r e f i l t e r e d a n t i b o d y s o l u t i o n s and r e suspended m a t e r i a l r e t a i n e d on the f i l t e r were run on 7% S D S - p o l y a c r y l a m i d e g e l s t o d e t e c t the p r e s e n c e o f l a b e l l e d a n t i b o d y ( F i g . 3 9 ) . Columns A - C were 50 y l , 25 y l and 10 y l a l i q u o t s f rom the f i l t e r e d a n t i b o d y s o l u t i o n . Column D was the m o l e c u l a r we igh t s t a n d a r d s . Columns E - G were 50 y l , 25 y l and 10 y l a l i q u o t s f rom the p r e f i l t e r e d s o l u t i o n . Columns H - J were 50 y l , 25 y l and 10 y l a l i q u o t s f rom the r e s u s p e n d e d m a t e r i a l r e t a i n e d on the f i l t e r . The p r o t e i n c o n c e n t r a t i o n o f the o r i g i n a l s o l u t i o n was 150 yg i n 1.0 ml o f PBS. The s e n s i t i v i t y o f the s i l v e r s t a i n i n g was such t h a t p r o t e i n bands o f 100 pg c o u l d be d e t e c t e d ( 142 ) . The f i l t e r e d s o l u t i o n had a p r o t e i n c o n c e n t r a t i o n t h a t was l e s s than 500 pg/ml s i n c e l i t t l e p r o t e i n was d e t e c t e d i n t r a c k s A - C . The m a j o r i t y o f the p r o t e i n i n the l a b e l l e d a n t i b o d y s o l u t i o n was r e t a i n e d on the f i l t e r e d as seen on t r a c k s H - J . Bands w i t h m o l e c u l a r we igh t s o f a p p r o x i m a t e l y 50 ,000 and 25 ,000 d a l t o n s c o r r e s p o n d e d w i t h heavy and l i g h t c h a i n s o f the a n t i b o d y were d e t e c t e d . 162 F i g u r e 3 9 . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f Amicon XM 300 f i l t e r 99m s e p a r a t e d Tc-ant i-M-1 on 7 .0% . S D S - p o l y a c r y l a m i d e g e l s . Columns A-C were 50 y l , 25 y l , and 10 y l a l i q u o t s f r om the f i l t e r e d a n t i b o d y s o l u t i o n s . Column D was the m o l e c u l a r we igh t s t a n d a r d s . Columns E - G were 50 y l , 25 y l and 10 y l a l i q u o t s f rom the p r e f i l t e r e d s o l u t i o n . Column H- J were 50 y l , 25 y l and 10 y l a l i q u o t s f rom r e s u s p e n d e d m a t e r i a l r e t a i n e d on the f i l t e r . The p r o t e i n bands were d e t e c t e d w i t h s i l v e r s t a i n i n g . 163 164 So i t would appear t h a t the m a j o r i t y o f the l a b e l l e d a n t i b o d y was i n a h i g h l y a g g r e g a t e d f o r m . S i n c e the p r e v i o u s expe r imen t i n d i c a t e d t h a t the l a b e l l e d a n t i b o d y was a g g r e g a t e d , u l t r a c e n t r i f u g a t i o n was an approach used t o examine t h i s p r o b l e m . The a g g r e g a t e s wou ld be sed imen ted and removed by c e n t r i f u g a t i o n . F(.a.b')^ f r agments o f ant i-M-1 a n t i b o d y were used because whole a n t i b o d y was no l o n g e r a v a i l a b l e . L a b e l l e d a n t i b o d y was c e n t r i f u g e d a t 45 ,900 g f o r 5 h. A f t e r c e n t r i f u g a t i o n , d i f f e r e n t f r a c t i o n s o f the s u p e r n a t a n t , r e s u s p e n d e d p e l l e t and p r e - s p i n a n t i b o d y f ragment s o l u t i o n s were i n j e c t e d i n t o tumour b e a r i n g m i c e . F r a c t i o n 1 was d e s i g n a t e d the t op f r a c t i o n o f the s u p e r n a t a n t , f r a c t i o n 2 was the m i d d l e f r a c t i o n and f r a c t i o n 3 was the bot tom r e suspended p e l l e t f r a c t i o n . When a s sayed f o r r a d i o a c t i v e c o n t e n t 24 h l a t e r , the p r e - s p i n f r a c t i o n was 2.8 6 6 6 x 10 cpm, f r a c t i o n 1 was 1.1 x 10 cpm, f r a c t i o n 2 was 1.6 x 10 cpm and f r a c t i o n 3 was 1 2 . 8 x 10^ cpm pe r 60 y l , i . e . the volume u s e d f o r 1 dose p e r a n i m a l . T i s s u e d i s t r i b u t i o n s t u d i e s i n d i c a t e d t h a t c e n t r i f u g a t i o n o f the a n t i b o d y s o l u t i o n d i d have an e f f e c t on the l o c a t i o n 99m o f Tc l a b e l i n the body ( F i g . 4 0 ) . In the M-l tumour , the l e v e l o f 99m Tc was no t s i g n i f i c a n t l y d i f f e r e n t i n the f o u r f r a c t i o n s . F r a c t i o n 99m 1, 2 and 3 t r e a t e d M-l tumour samples had m a r g i n a l l y l ower Tc l e v e l s than the p r e - s p i n f r a c t i o n t r e a t e < i t umours . The d i s t r i b u t i o n i n the b l o o d samples was s i m i l a r t o t h a t i n the tumour s amp le s . B l o o d samples f r o m f r a c t i o n 1, 2 and 3 were m a r g i n a l l y l ower than those f r om the p r e - s p i n t r e a t e d m i c e . The major d i f f e r e n c e i n the d i s t r i b u t i o n s was f o u n d i n the l i v e r . F r a c t i o n 1 t r e a t e d l i v e r samples were r o u g h l y h a l f as r a d i o a c t i v e 165 F i g u r e 40 . E f f e c t o f c e n t r i f u g a t i o n on Tc-ant i-M-1 f r agments i n i n v i v o t i s s u e d i s t r i b u t i o n i n M-l tumour , l i v e r and b l o o d . The 99m Tc-ant i-M-1 p r e p a r a t i o n was c e n t r i f u g e d a t 45 ,900 g f o r 5 h r . The p r e p a r a t i o n was then d i v i d e d i n t o 3 f r a c t i o n s : f r a c t i o n 1 ( the t op f r a c t i o n ) • ; f r a c t i o n 2 ( the m i d d l e f r a c t i o n ) • ; and f r a c t i o n 3 ( the bot tom r e s u s p e n d e d f r a c t i o n ) . A f o u r t h f r a c t i o n , P r e - s p i n f r a c t i o n , was t aken f r om the l a b e l l e d a n t i b o d y p r e p a r a t i o n b e f o r e c e n t r i f u g a t i o n . The d i f f e r e n t f r a c t i o n s were i n j e c t e d i . v . i n t o tumour b e a r i n g DBA/2J m i c e . They were s a c r i f i c e d and a s sayed 24 h r l a t e r . Each ba r i s the mean ± S .D . o f t i s s u e samples t aken f rom 5 t e s t a n i m a l s . 166 TUMOUR LIVER BLOOD 167 as the p r e - s p i n f r a c t i o n t r e a t e d l i v e r s amp le s . F r a c t i o n 2 t r e a t e d l i v e r 99m samples were about two t h i r d s o f the Tc l e v e l s f ound i n p r e - s p i n f r a c t i o n t r e a t e d l i v e r samples w h i l e f r a c t i o n 3 t r e a t e d l i v e r samples were a p p r o x i m a t e l y as h i g h as t h o s e f rom p r e - s p i n f r a c t i o n t r e a t e d s a m p l e s . R a t i o s o f t umour/b l ood and l i v e r / b l o o d were c a l c u l a t e d t o e s t i m a t e the q u a l i t y o f p o t e n t i a l s cans f rom f r a c t i o n t r e a t e d a n i m a l s . Scans were not 99m t a k e n a t t h i s t ime because the l e v e l o f Tc i n the a n i m a l s were too low f o r the imag ing camera t o coun t a c c u r a t e l y . The c r i t i c a l v a l u e d e s i r e d i n the r a t i o s f o r the pu rposes o f imag ing as m e n t i o n e d b e f o r e i s 1. The t i s s u e s have to have a h i g h e r l e v e l o f r a d i o a c t i v i t y t han b l o o d f o r d e l i n e a t i o n o f the t i s s u e i n the s c a n s . In the p r e - s p i n f r a c t i o n t r e a t e d a n i m a l s , the t umour/b l ood r a t i o was be low 1, i n d i c a t i n g t h a t the tumour wou ld be p o o r l y v i s u a l i z e d ( F i g . 4 1 ) . On the o t h e r hand , the l i v e r wou ld be c l e a r l y seen i n the scans s i n c e the l i v e r / b l o o d r a t i o was above 1. In f r a c t i o n 1 and 2 t r e a t e d a n i m a l s , the t umour/b lood r a t i o s were h i g h e r than the l i v e r / b l o o d r a t i o s . In f r a c t i o n 1 t r e a t e d a n i m a l s , the tumour c o u l d be e a s i l y d i s c e r n a b l e w h i l e the l i v e r wou ld be p o o r l y v i s u a l i z e d s i n c e the t umour/b lood r a t i o s were g r e a t e r than 1 and the l i v e r / b l o o d r a t i o s were l e s s than 1. A s i m i l a r s i t u a t i o n was n o t e d i n F r a c t i o n 2 t r e a t e d an ima l s but bo th r a t i o s were lower than 1. T h e r e f o r e poo r imag ing wou ld be e x p e c t e d f o r bo th tumour and l i v e r . In F r a c t i o n 3 (o r the r e s u s p e n d e d agg rega t e p o r t i o n ) t r e a t e d m i c e , as wou ld be p r e d i c t e d , the l i v e r / b l o o d r a t i o s was c o n s i d e r a b l y h i g h e r than t h o s e f rom the o t h e r t r e a t e d g r o u p s . The r a t i o was w e l l above 1, i n d i c a t i n g t h a t the l i v e r wou ld c l e a r l y v i s u a l i z e d . The tumour would a l s o be seen i n f r a c t i o n 168 F i g u r e 4 1 . T i s s u e / b l o o d r a t i o s f rom the l a b e l l e d a n t i b o d y f ragment c e n t r i f u g a t i o n s t u d y . R a t i o s o f t umour/b l ood and l i v e r / b l o o d were compared among the d i f f e r e n t f r a c t i o n t r e a t e d m i c e . The l i v e r / b l o o d r a t i o (•) i s i n d i c a t i v e o f the l e v e l o f r a d i o a c t i v e backg round p r e s e n t i n norma l i r r e l e v a n t t i s s u e s . The t umour/b lood r a t i o (H) i s i n d i c a t i v e o f 99m the l e v e l o f s p e c i f i c up take o f Tc-ant i-M-1 i n the s p e c i f i c tumour t i s s u e . A r a t i o v a l u e > 1 i s r e q u i r e d f o r v i s u a l i z a t i o n o f the t a r g e t t i s s u e . T I S S U E % rodiooctivt do»e per grom RATIO BLOOD % radioactive dose per gram 170 3 t r e a t e d mice but the backgrounds as i n d i c a t e d by the l i v e r / b l o o d r a t i o wou ld be e x t r e m e l y h i g h . These r e s u l t s demons t r a t ed t h a t a g g r e g a t i o n o f the l a b e l l e d a n t i b o d y was a pr ime f a c t o r i n c a u s i n g the h i g h backgrounds seen i n the s c a n s . Though c e n t r i f u g a t i o n was e f f e c t i v e i n d e c r e a s i n g the l e v e l o f a g g r e g a t i o n i n the l a b e l l e d a n t i b o d y p r e p a r a t i o n , the m a j o r i t y o f t he a n t i b o d y was l o s t as the sed imen ted p e l l e t . C o n s e q u e n t l y , a method was sought to d e a g g r e g a t e the l a b e l l e d a n t i b o d y p r e p a r a t i o n s . I t was o b s e r v e d o by Barandum e t a l . , t h a t t r e a tmen t o f a n t i b o d y a t pH 4 a t 37 C f o r 6 h r was s u f f i c i e n t t o deagg rega t e the s o l u t i o n ( 1 4 3 ) . The t r e a t e d a n t i b o d i e s appea red t o be f u n c t i o n a l l y i n t a c t i n t h a t they were s t i l l e f f e c t i v e i n the i n t r a v e n o u s Ig t h e r a p y f o r Ig d e f i c i e n c i e s and a n t i b o d y f r agments were no t o b s e r v e d i n the u r i n e o f t r e a t e d p a t i e n t s . o Ant i-M-1 F ( a b * ) 2 f r agments were i n c u b a t e d a t pH 4 a t 37 C o v e r n i g h t (18 h) b e f o r e o r a f t e r l a b e l l i n g . The l a b e l l e d a n t i b o d y p r e p a r a t i o n s were then a n a l y s e d i n p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) . A f t e r pH 4 o r 7 ( c o n t r o l ) t r e a t m e n t , e q u i v a l e n t amounts o f sample 99m ( n o r m a l i z e d on the b a s i s o f Tc c o n t e n t ) were run on 3 - 10% n o n r e d u c i n g p o l y a c r y l a m i d e g e l s . The g e l s were a u t o r a d i o g r a p h e d t o 99m de t e rm ine the l o c a t i o n o f the Tc l a b e l . In t r a c k s A and B, t he l a b e l l e d a n t i b o d y was i n c u b a t e d a t pH 4 o r pH 7 r e s p e c t i v e l y , o v e r n i g h t ( F i g . 4 2 ) . In t r a c k s C & D, the a n t i b o d i e s were f i r s t i n c u b a t e d a t pH 4 99m ( t r a c k C) o r pH 7 ( t r a c k D) o v e r n i g h t , and then l a b e l l e d w i t h T c . F r e e p r e t e c h n e t a t e was run i n t r a c k E and u n l a b e l l e d c a r r i e r p r o t e i n (HSA) 99m was run i n t r a c k F. In t r a c k G, the Tc l a b e l l e d a n t i b o d y f ragment 171 99m F i g u r e 4 2 . A u t o r a d i o g r a p h i c d e t e c t i o n o f d e a g g r e g a t e d Tc-ant i-M-1 F ( ab '>2 f r agments i s o l a t e d on 3 - 10% p o l y a c r y l a m i d e g e l s . The F ( ab '>2 p r e p a r a t i o n s were t r e a t e d w i t h pH 4 o r pH 7 i n c u b a t i o n s t o t e s t f o r a g g r e g a t i o n . pH 4 t r e a t m e n t have been found to h e l p deagg rega t e a n t i b o d y s o l u t i o n s . T r a c k A : the l a b e l l e d a n t i b o d y f r agments were i n c u b a t e d a t pH 4 o v e r n i g h t a t 3 7 ° C . T r a c k B: the l a b e l l e d a n t i b o d y f r agments were i n c u b a t e d a t pH 7 o v e r n i g h t a t 3 7 ° C . T r a c k C : the o a n t i b o d y f ragment p r e p a r a t i o n was i n c u b a t e d o v e r n i g h t a t pH 4 , 37 C and 99m then l a b e l l e d w i t h T c . T r a c k D: the a n t i b o d y f ragment p r e p a r a t i o n o . 99m was i n c u b a t e d o v e r n i g h t a t pH 7, 37 C and then l a b e l l e d w i t h T c . 99m -T r a c k E: f r e e p e r t e c h n e t r a t e ( Tc 0^) samp le . T r a c k F: u n l a b e l l e d human serum a lbumin ( the c a r r i e r p r o t e i n used i n the l a b e l l i n g 99m p r o c e d u r e ) . T r a c k G: the T c - l a b e l l e d a n t i b o d y f ragment s o l u t i o n was i n c u b a t e d a t pH 4 , 3 7 ° C f o r 1 h r p r i o r to e l e c t r o p h o r e s i s . 172 240 k 66 k 45 k 173 was incubated at pH 4 for 1 h prior to analysis on PAGE. The results clearly indicated that there was a great deal of aggregation in the antibody fragment solutions; The fragments should have a molecular weight of approximately 100 K. However the majority of radioactivity was found at molecular weights above 240 K daltons (see tracks B - D). The pH 4 99m treatment did decrease the amount of aggregation. The major Tc activity was located at bond corresponding to approximately 100 K daltons (track A). One hr incubation at pH 4 was not sufficient to deaggregate 99m the antibody solution. In track G, the major Tc band was located above 240 K daltons and a minor band at 100 K daltons. 174 Chap t e r IV DISCUSSION 99m In t h i s s t u d y , i t has been shown t h a t Tc-ant i-M-1 m o n o c l o n a l 99m a n t i b o d y e x h i b i t e d b i n d i n g s p e c i f i c i t y f o r M-l tumour c e l l s . When Tc l a b e l l e d ant i-M-1 was i n j e c t e d i n t r a v e n o u s l y i n t o mice b e a r i n g M-l tumour , and the an ima l s were s c a n n e d , the tumour mass was c l e a r l y v i s i b l e ( F i g . 2 7 ) . T h i s was f u r t h e r borne out when i n d i v i d u a l t i s s u e samples were a s s e s s e d f o r r e l a t i v e r a d i o a c t i v i t y a f t e r s a c r i f i c e . These r e s u l t s ( F i g . 25 and 26) s u g g e s t e d t h a t r e l a t i v e s p e c i f i c a c t i v i t y ( e . g . t umour/b l ood r a t i o ) may no t become o p t i m a l f o r some t ime a f t e r a d m i n i s t r a t i o n o f the a n t i b o d y ( p o s s i b l y l o n g e r than 4 4 h ) . Even a f t e r 44 h p o s t i n j e c t i o n , t i s s u e s such as l i v e r and k i d n e y had a r e l a t i v e l y h i g h n o n s p e c i f i c _ , . „ 99in , t , accummula t ion o f Tc l a b e l . Tha t the a c c u m u l a t i o n o f l a b e l l e d a n t i b o d y i n tumour t i s s u e was a t t r i b u t a b l e t o the s p e c i f i c i t y o f the a n t i b o d y was e s t a b l i s h e d by u s i n g F^ an ima l s i n t o wh ich bo th M-l and an u n r e l a t e d tumour (EL4) had been i n j e c t e d . Imaging s t u d i e s and e s t i m a t e s o f r e l a t i v e s p e c i f i c a c t i v i t i e s i n the two tumour masses e s t a b l i s h e d t h a t the r e a c t i o n was s p e c i f i c ( F i g . 2 9 ) , s i n c e p e r c e n t r a d i o a c t i v e dose pe r gram o f t i s s u e was s i g n i f i c a n t l y h i g h e r f o r M-l and s i g n i f i c a n t l y l ower i n EL4 tumours . When t h e s e an ima l s were s c a n n e d , the M-l tumours were v i s i b l e whereas the E14 tumours were no t ( F i g . 3 0 ) . Both tumours were a p p r o x i m a t e l y the same s i z e and upon au topsy appea red t o have the same l e v e l o f v a s c u l a r i t y . T h e r e f o r e 99m a c c u m u l a t i o n o f Tc l a b e l i n the M-l tumour was s p e c i f i c ( e . g . b i n d i n g 175 o f Tc-ant i-M-1 t o M-l c e l l ) and not due t o a l a r g e r r e s i d u a l b l o o d p o o l i n the tumour . Wh i l e t h e s e r e s u l t s were e n c o u r a g i n g , a major p r o b l e m was the h i g h l e v e l o f backg round r a d i o a c t i v i t y seen i n the s c a n s . As r e p o r t e d by o t h e r i n v e s t i g a t o r s , h i g h backg round coun t s i n the l i v e r , k i d n e y and s p l e e n have p r e s e n t e d prob lems o f s e n s i t i v i t y . D e t e c t i o n o f tumour masses nea r o r a d j a c e n t to t h e s e o rgans w i t h rad io immunoimagery wou ld be i m p o s s i b l e due t o the n o n s p e c i f i c c o n c e n t r a t i o n o f r a d i o l a b e l i n the o r g a n s . Such h i g h coun t s i n t h e s e o r g a n s , (p resumab ly n o n - s p e c i f i c s i n c e ant i-M-1 had no r e a c t i v i t y on membrane p r e p a r a t i o n s f rom t h e s e t i s s u e s i n the E L I S A ) , may be a t t r i b u t e d t o a number o f f a c t o r s such as : F r e c e p t o r s on c e l l s c ( d e n d r i t i c c e l l s o r macrophages ) wh ich p i c k up f r e e a n t i b o d y m o l e c u l e s , o r the p r e s e n c e o f a g g r e g a t e s i n the i n j e c t e d s o l u t i o n s ( a n t i b o d y wh i ch was a g g r e g a t e d n o n s p e c i f i c a l l y i n the l a b e l l i n g o r p r e t i n n i n g p r o c e s s ) o r agg rega t e s o f a n t i b o d y wh ich fo rm i i i v i v o w i t h c i r c u l a t i n g s o l u b l e tumour a n t i g e n o r shed a f t e r c o m p l e x i n g w i t h membrane bound a n t i g e n . A n t i b o d y agg rega t e s a re r a p i d l y c l e a r e d (24 - 48 h) f rom the b o d y , and wou ld be e x p e c t e d t o s e q u e s t e r i n bo th the l i v e r and k i d n e y p r i o r t o c l e a r a n c e . T h e r e f o r e , under t h e s e c i r c u m s t a n c e s , a h i g h n o n s p e c i f i c ba ckg round i n the l i v e r , k i d n e y and s p l e e n wou ld no t be s u r p r i s i n g . S o l u b l e f r e e l y c i r c u l a t i n g a n t i g e n o r a n t i g e n s h e d d i n g a f t e r a n t i b o d y c o m p l e x i n g w i t h tumour c e l l was no t the cause o f backg round i n t h i s an ima l 99m m o d e l . C o l d ant i-M-1 was a d m i n i s t e r e d 24 h p r i o r t o Tc l a b e l l e d ant i-M-1 i n j e c t i o n . T h i s was done i n hopes o f b l o c k i n g up any c i r c u l a t i n g a n t i g e n t h a t m igh t be c a u s i n g the b a c k g r o u n d . A l t e r n a t i v e l y , i f a n t i g e n 176 was shed a f t e r a n t i b o d y b i n d i n g t o t a r g e t c e l l , a lower b i n d i n g s p e c i f i c i t y f o r tumour ( i . e . p o o r e r imag ing ) would be e x p e c t e d when ho t a n t i b o d y was i n j e c t e d . The r e s u l t s showed t h a t c o l d ant i-M-1 p r e t r e a t m e n t d i d no t improve the q u a l i t y o f scans i n compar i son t o p r e t r e a t m e n t w i t h c o l d i r r e l e v a n t ( an t i -G IP ) a n t i b o d y o r PBS. The ant i-M-1 p r e t r e a t m e n t 99m caused a m a r g i n a l d e c r e a s e i n the l e v e l o f Tc l a b e l i n M-l t i s s u e ( F i g . 3 2 ) , however i t was not enough t o e f f e c t the imag ing o f the tumours . F £ r e c e p t o r b i n d i n g o f l a b e l l e d a n t i b o d y was a l s o no t a major f a c t o r i n c a u s i n g b a c k g r o u n d . Though scans were not t a k e n , F ( a b ' ) 2 t i s s u e d i s t r i b u t i o n s t u d i e s i n d i c a t e d t h a t the l i v e r and k i d n e y t i s s u e s s t i l l d i s p l a y e d a h i g h l e v e l o f r a d i o a c t i v i t y ( F i g . 3 7 ) . F ( a b ' ) 2 f r agments have been shown t o have an a c c e l e r a t e d r a t e o f d i s a p p e a r a n c e f rom the b l o o d f o l l o w i n g i n t r a v e n o u s i n j e c t i o n i n c o m p a r i s o n t o whole i n t a c t a n t i b o d y ( 1 3 7 ) . So v i s u a l i z a t i o n o f t a r g e t s wou ld be f a s t e r because the t ime i n t e r v a l r e q u i r e d t o ge t s u f f i c i e n t t a r g e t t o backg round 99m r a t i o s wou ld be s h o r t e r . The p r e p a r a t i o n o f Tc l a b e l l e d ant i-M-1 f r agments used i n t h i s s t udy d i d not improve the q u a l i t y o f the r a d i o l a b e l s cans i n compar i son t o the i n t a c t a n t i b o d y p r e p a r a t i o n . However , t h i s was r e l a t e d t o the a g g r e g a t e d n a t u r e o f the p r e p a r a t i o n and not t o l o s s i n i m m u n o s p e c i f i c i t y . P h y s i c a l e x a m i n a t i o n o f the l a b e l l e d a n t i b o d y p r e p a r a t i o n s gave a c l u e as t o what m igh t be c a u s i n g the h i g h backgrounds i n the s c a n s . F i l t r a t i o n , d e a g g r e g a t i o n , and c e n t r i f u g a t i o n s t u d i e s i n d i c a t e d t h a t the F ( a b * ) 2 f r agment and whole a n t i b o d y p r e p a r a t i o n s were h i g h l y a g g r e g a t e d ( F i g . 39 and 4 0 ) . F ( a b ' ) „ f ragment p r e p a r a t i o n s were c e n t r i f u g e d i n a 1 7 7 SS34 r o t o r a t 19 ,500 rpm (45 ,900 g) f o r 5 h. Under t hese c o n d i t i o n s , a monomeric IgG m o l e c u l e (150 ,000 d a l t o n s ) would t ake 62 h o f c e n t r i f u g a t i o n t o sed iment down ( 1 4 4 ) . A f t e r s e d i m e n t a t i o n o f the l a b e l l e d p r e p a r a t i o n , 99m the s u p e r n a t a n t ( e . g . F r a c t i o n 1 and 2) c aused a l ower l e v e l o f Tc d e p o s i t i o n i n the l i v e r i n compar i son t o the agg rega te f r a c t i o n ( e . g . F r a c t i o n 3) o r the p r e - s p i n p r e p a r a t i o n ( F i g . 4 0 ) . T h i s i n d i c a t e d t h a t i f 99m the l e v e l o f a g g r e g a t i o n i n the T c - a n t i b o d y p r e p a r a t i o n c o u l d be d e c r e a s e d , the q u a l i t y o f the scans i n terms o f backg round wou ld p r o b a b l y be improved ( F i g . 4 1 ) . Ano the r v a r i a b l e t h a t s h o u l d be a d d r e s s e d a t t h i s p o i n t i s t h a t o f the t i m i n g f o r imag ing s t u d i e s . In C h a p t e r I I , i t was shown t h a t o p t i m a l t ime f o r Hp-an t i body tumour l o c a l i z a t i o n ( i n terms o f maximum t h e r a p e u t i c e f f e c t s ) i j i v i v o was i n the r e g i o n o f 5 - 6 days a f t e r a d m i n i s t r a t i o n o f Hp-ant i-M-1 a n t i b o d y . O t h e r l a b o r a t o r i e s have demons t r a t ed t h a t maximum d e f i n i t i o n o f tumour ( i . e . w i t h n e g l i g i b l e backg round r a d i o a c t i v i t y ) t o o k 131 6 - 1 1 days f o r I - l a b e l l e d whole a n t i b o d y and 3 - 4 days f o r 131 I - l a b e l l e d F ( a b ' ) 2 f r agments ( 1 3 5 , 1 4 5 ) . E a r l i e r expe r imen t s i n t h i s s t udy i n d i c a t e d t h a t more f a v o u r a b l e r a t i o s between tumour and o t h e r t i s s u e s ( e . g . l i v e r and k i d n e y ) c o u l d be o b t a i n e d a f t e r 44 h ( F i g . 2 5 ) . However, w i t h such a s h o r t l i v e d r a d i o i s o t o p e as " m T c (T 1/2 = 6 h ) , r a d i o i m a g i n g would be out o f the q u e s t i o n a f t e r t h a t l e n g t h o f t i m e . I t i s , however , c o n c e i v a b l e t h a t i f the l a b e l l e d a n t i b o d y p r e p a r a t i o n was m a i n l y monomer i c , i n t a c t a n t i b o d y o r F ( a b ' ) 2 f r a g m e n t s , the backg round due t o n o n s p e c i f i c up take wou ld be d e c r e a s e d and a c l e a r e r v i s u a l i z a t i o n o f tumour a c h i e v e d i n a s h o r t e r p e r i o d o f t i m e . A l e s s a g g r e g a t e d 178 99m preparation could be obtained by u l t r a c e n t r i f u g a t i o n a f t e r Tc l a b e l l i n g or deaggregation by low pH treatment. CHAPTER V DISCUSSION 180 C h a p t e r V The p r i n c i p l e o f "mag i c b u l l e t s " i s the p r o v i s i o n o f a s p e c i f i c l o c a l i z i n g d e v i c e t h a t w i l l f e r r y c y t o t o x i c o r r a d i o a c t i v e s u b s t a n c e s t o a d e f i n e d t a r g e t f o r the pu rposes o f d e s t r o y i n g o r d e t e c t i n g t h a t t a r g e t . By u s i n g a t a r g e t i n g a n t i b o d y to ensu re t o x i n o r r a d i o i s o t o p e d e l i v e r y , n o n s p e c i f i c i n t e r a c t i o n between e f f e c t o r agents and n o n t a r g e t norma l t i s s u e would be m i n i m i z e d , w h i l e m a x i m i z i n g i n t e r a c t i o n w i t h s p e c i f i c a n t i g e n - e x p r e s s i n g t a r g e t t i s s u e . The a c t i v i t y o f the e f f e c t o r agents wou ld be l i m i t e d t o s p e c i f i e d t a r g e t s i t e s , thus i n c r e a s i n g the e f f i c i e n c y o f the d rug o r r a d i o l a b e l and a l l o w i n g the use o f lower dosages s i n c e l e s s wou ld be u s e l e s s l y d i s p e r s e d t h roughou t the body . The re a re many i m p o r t a n t b i o l o g i c a l pa ramete rs t o be c o n s i d e r e d f o r a n t i b o d y l o c a l i z a t i o n i n v i v o . P rob lems i n d e l i v e r y o f the a n t i b o d y "mag i c b u l l e t " t o the a n t i g e n i c s i t e s on the t a r g e t m a l i g n a n t t i s s u e c o u l d p rove t o be the l i m i t i n g f a c t o r d e t e r m i n i n g the s u c c e s s o r f a i l u r e o f t r e a t m e n t o r d e t e c t i o n . The s p e c i f i c t a r g e t a n t i g e n s must be a c c e s s i b l e t o the a n t i b o d i e s . They have to be e x p r e s s e d on the tumour c e l l membrane i n such a way t h a t p e r m i t s a n t i b o d y r e c o g n i t i o n and b i n d i n g . The a n t i g e n d e n s i t y has t o be s u f f i c i e n t to b i n d up a l l o r most o f the i n j e c t e d a n t i b o d i e s . A n o t h e r p rob l em may l i e w i t h i n the n a t u r e o f the a n t i g e n i t s e l f . The q u e s t i o n as t o whether a n t i g e n s un ique t o a m a l i g n a n t c o n d i t i o n do e x i s t has l o n g been c o n t e s t e d ( 146 ) . What i s no t a rgued i s t h a t tumour c e l l s may e x h i b i t a n t i g e n i c m a r k e r s , e . g . d i f f e r e n t i a t i o n , f e t a l , v i r a l , wh ich can be used t o d i s t i n g u i s h them i m m u n o l o g i c a l l y f rom 181 t h e i r norma l c o u n t e r p a r t s . But such markers cannot be c o n s i d e r e d tumour un ique s i n c e they may have been e x p r e s s e d on normal c e l l s a t some s t age o f deve l opmen t , o r be p r e s e n t a t l e v e l s t oo low t o be d e t e c t e d w i t h p r e s e n t day methods on some o r a l l norma l c e l l s . Normal t i s s u e t o x i c i t y i n t r e a t m e n t o r backg round up take i n rad io immuno imagery c o u l d be p a r t i a l l y due t o a low l e v e l o f " tumour a s s o c i a t e d " a n t i g e n e x p r e s s i o n on no rma l t i s s u e o r t r a n s i e n t e x p r e s s i o n on norma l c e l l s d u r i n g the c o u r s e o f c e l l u l a r m a t u r a t i o n . Fo r a n t i b o d y t o ge t to an a n t i g e n i c s i t e in v i v o , i t must be t r a n s f e r r e d v i a the b l o o d s u p p l y to the tumour , and l e a k i n t o the i n t e r s t i t i a l f l u i d s u r r o u n d i n g the tumour t o f i n a l l y r e a c h the a n t i g e n . The s p e c i f i c i t y o f the i m m u n o l o g i c a l approach b e h i n d "mag i c b u l l e t s " would be d e c r e a s e d by r e t e n t i o n o f t o x i n - o r i s o t o p e - a n t i b o d y c o n j u g a t e s i n the c i r c u l a t i n g b l o o d p o o l o r i n the i n t e r s t i t i a l f l u i d s o f norma l t i s s u e s , i f t h e y d o n ' t r e a d i l y move t h rough c a p i l l a r y p o r e s i n the b l o o d v e s s e l w a l l s . There must be an adequate b l o o d s u p p l y t o the tumour. Many c a nce ro us l e s i o n s due i n p a r t to t h e i r r a p i d growth r a t e , have i n s u f f i c i e n t v a s c u l a r sys tem to s u p p o r t homogeneous g r o w t h , r e s u l t i n g i n a n o x i c c e l l s and n e c r o s i s . I t has been demons t r a t ed t h a t s u c c e s s f u l b i n d i n g o f a n t i b o d y t o t a r g e t c e l l s i n v i v o i s dependent on an adequate b l o o d s u p p l y t o the tumour ( 128 ) . Moshak i s e t a l . r e p o r t e d t h a t i n t r a v e n o u s l y i n j e c t e d m o n o c l o n a l a n t i b o d i e s b i n d p r e f e r e n t i a l l y t o v i a b l e c e l l s s i t u a t e d nea r b l o o d v e s s e l s . N e c r o t i c o r f i b r o t i c a r e a s , d i s t a n t f r om the b l o o d v e s s e l s , d i d not show any a n t i b o d y b i n d i n g . In c o n t r a s t , i f whole t i s s u e s e c t i o n s o f tumour were l a b e l l e d i j i v i t r o , the e n t i r e 182 s e c t i o n wou ld show u n i f o r m d i s t r i b u t i o n o f a n t i b o d y b i n d i n g . Hence the l a c k o f a n t i b o d y b i n d i n g i n v i v o d i s t a n t f rom the b l o o d v e s s e l s was due t o i n s u f f i c i e n t a c c e s s o f a n t i b o d y t h rough the tumour t i s s u e . In C h a p t e r s I I and I I I , the p o t e n t i a l o f photo immunotherapy was examined . The p o r p h y r i n h a e m a t o p o r p h y r i n , was c h e m i c a l l y c o u p l e d t o s p e c i f i c a n t i - t u m o u r a n t i b o d i e s w i t h o u t s i g n i f i c a n t l o s s o f photodynamic o r immunolog ic a c t i v i t y . These "mag i c b u l l e t s " p r o v e d t o be s p e c i f i c a l l y t o x i c f o r t h e i r r e l e v a n t tumour t a r g e t s . The p o r p h y r i n - a n t i b o d y c o n j u g a t e s r e p r e s e n t a r a t h e r un ique fo rm o f "mag ic b u l l e t " t h e r a p y . Photo immunotherapy can be v i ewed as a p r o c e s s w i t h two d i s c r e t e s t a g e s . The f i r s t s t age r e q u i r e s the a n t i b o d y - p o r p h y r i n c o n j u g a t e t o l o c a t e and b i n d t o the tumour c e l l s . A t t h i s s t e p , the c o n j u g a t e i s m a i n t a i n e d i n a t o x i c a l l y i n e r t f o rm ( e . g . i n the absence o f l i g h t ) and the f u n c t i o n a l a c t i v i t y o f the c o n j u g a t e d e a l s o n l y w i t h i m m u n o l o g i c a l l y b r i n g i n g the u n a c t i v a t e d p h o t o c h e m i c a l i n t o the v i c i n i t y o f the t a r g e t c e l l s . S i n c e the p r o p o s e d mode o f c y t o t o x i c a c t i o n i s c r o s s l i n k i n g o f membrane p r o t e i n s l e a d i n g t o c e l l l y s i s ( 5 0 , 5 3 , 5 6 ) , e n d o c y t o s i s o f the H p - a n t i b o d y i s no t e s s e n t i a l f o r e f f e c t i v e c y t o t o x i c i t y . T h i s d i f f e r s f rom the p r o t e i n t o x i n a n t i b o d y c o n j u g a t e models such as r i c i n , a b r i n o r d i p h t h e r i a A c h a i n s wh i ch r e q u i r e i n t e r n a l i z a t i o n o f the t o x i c p e p t i d e s f o r i n a c t i v a t i o n o f t a r g e t c e l l s . In the absence o f l i g h t , the haema topo rphy r i n compound has no d e t e c t a b l e t o x i c a c t i v i t y i n v i v o . Hence Hp-an t i body "mag ic b u l l e t s " d i f f e r f rom o t h e r "mag i c b u l l e t " models i n a c r u c i a l p o i n t . They a re no t t o x i c u n t i l a c t i v a t e d by l i g h t . Most "mag i c b u l l e t " systems use t o x i n s t h a t a re p o t e n t i a l l y c y t o t o x i c to any s u s c e p t i b l e nea rby c e l l a t any t ime 183 w h i l e i t i s c i r c u l a t i n g i n the b l o o d p o o l . So t h e r e wou ld be an u b i q u i t o u s low l e v e l o f n o n s p e c i f i c i n v i v o c y t o t o x i c i t y i n h e r e n t t o the o t h e r t o x i n - a n t i b o d y s y s t ems . The Hp-an t i body c o n j u g a t e s , on the o t h e r h a n d , can be m a n i p u l a t e d so t h a t the t o x i c a c t i v i t y can be c o n t r o l l e d u n t i l the m a j o r i t y o f the Hp c o n j u g a t e i s e i t h e r l o c a l i z e d a t the d e s i r e d t a r g e t tumour s i t e o r e x c r e t e d f rom the body . The second and f i n a l s t e p i s l i g h t a c t i v a t i o n o f the h a e m a t o p o r p h y r i n s e q u e s t u r e d i m m u n o l o g i c a l l y a t the tumour s i t e , w i t h subsequent d e s t r u c t i o n o f the tumour c e l l s i n the v i c i n i t y o f the a c t i v a t e d p o r p h y r i n s . O b v i o u s l y , adequate l i g h t p e n e t r a t i o n i s e s s e n t i a l f o r t r i g g e r i n g the p h o t o c y t o t o x i c i t y o f h a e m a t o p o r p h y r i n i j i v i v o . Wave lengths g r e a t e r than 690 nm, e . g . r e d l i g h t , a re used f o r photochemotherapy because they had been d e m o n s t r a t e d t o have the h i g h e s t l e v e l o f photon t r a n s m i s s i o n t h rough t i s s u e ( 1 4 7 ) . I t has been f o u n d t h a t as much as 0 . 1 % o f the i n c i d e n t l i g h t i n t e n s i t y (600 -700 nm) can p e n e t r a t e t o a dep th o f 7.0 cm o f s o l i d t i s s u e ( 6 1 ) . Deep s e a t e d tumours can be r e a c h e d w i t h dye l a s e r s c o u p l e d t o f i b e r o p t i c s . S u c c e s s f u l t r e a t m e n t o f deep s e a t e d tumours w i t h HpD and l a s e r p h o t o r a d i a t i o n has been demons t r a t ed i n e x p e r i m e n t a l a n i m a l mode ls (87) and i n human m a l i g n a n c y c l i n i c a l t r i a l s ( 6 3 , 6 4 , 6 8 , 8 3 ) . The i m m u n o l o g i c a l s p e c i f i c i t y o f Hp-an t i - tumour a n t i b o d i e s was demons t r a t ed jun v i t r o and iri v i v o i n the DBA/2J M-l tumour mode l i n Chap t e r I I and in. v i t r o w i t h Hp-CAMAL-1 a n t i b o d i e s on human ANLL and CGL c e l l s i n C h a p t e r I I I . The in. v i t r o s t u d i e s i n bo th c h a p t e r s c l e a r l y showed t h a t the photodynamic a c t i v i t y was d e l i v e r e d t o the c e l l s o n l y i f the c a r r i e r a n t i b o d i e s r e c o g n i z e d and bound t o the t a r g e t s . N o n - t a r g e t 184 a n t i g e n - e x p r e s s i n g c e l l s were u n a f f e c t e d by the c o n j u g a t e s iri v i t r o . The i n v i v o s t u d i e s d i s c u s s e d i n C h a p t e r I I d emons t r a t ed the l o c a l i z i n g a b i l i t y o f the Hp-ant i-M-1 c o n j u g a t e s . The i m m u n o l o g i c a l s p e c i f i c i t y o f the Hp c o n j u g a t e d e t e r m i n e d whether o r not the chosen tumour would be e f f e c t e d in. v i v o . EL4 tumour ( i r r e l e v a n t tumour c o n t r o l ) growth i n v i v o , was u n a f f e c t e d by i n t r a v e n o u s a d m i n i s t r a t i o n o f Hp-ant i-M-1 ( T a b l e I I I ) . However , the t a r g e t i n g a b i l i t y o f the Hp-ant i-M-1 c o n j u g a t e a l l o w e d Hp l o c a l i z a t i o n o f M-l tumour i n v i v o and i n h i b i t i o n o f tumour g row th . The a n t i b o d y ' s s p e c i f i c l o c a l i z a t i o n on the t a r g e t was e s s e n t i a l f o r Hp d e l i v e r y t o the tumour s i t e s i n c e f r e e haema topo rphy r i n d i d no t e f f e c t M-l tumour growth a t the dosages used (0 .268 mg Hp/kg body w e i g h t ) . So c o n j u g a t i o n o f a n t i - t u m o u r a n t i b o d y to haema topo rphy r i n p r o v i d e d an i m m u n o l o g i c a l mechanism t h a t would a l l o w the p o r p h y r i n t o be b r o u g h t i n c l o s e p h y s i c a l c o n t a c t w i t h the s p e c i f i c tumour . In t h i s manner , h a e m a t o p o r p h y r i n , a r e l a t i v e l y poor m a l i g n a n t l o c a l i z e r , was c o n v e r t e d t o a s p e c i f i c a n t i - t u m o u r agent w i t h a h i g h e r a f f i n i t y f o r tumour t i s s u e . S i n c e tumour a f f i n i t y was i n c r e a s e d , H p ' s r e l a t i v e c y t o t o x i c i t y f o r tumour was i n c r e a s e d , a l l o w i n g the use o f lower tumour e f f e c t i v e d o s e s . The dose l e v e l u sed t o t r e a t M-l b e a r i n g mice i n C h a p t e r I I was a p p r o x i m a t e l y 1/10 - 1/20 o f the HpD dosage used by Dougher t y and a s s o c i a t e s (2 .5 - 5.0 mg/kg body wt) i n e x p e r i m e n t a l mur ine tumour s t u d i e s o r 1/40 o f the Hp dosage used by Diamond and a s s o c i a t e s (10 mg/kg body wt) i n r a t tumour s t u d i e s ( 5 7 , 6 1 ) . A l l an ima l s used i n the ij i v i v o s t u d i e s r e p o r t e d i n C h a p t e r I I d i s p l a y e d no d i s c e r n a b l e s i g n s o f norma l t i s s u e t o x i c i t y ( i . e . e r y t hema , edema o r b l i s t e r i n g o f the ba re exposed tumour s i t e on the backs 185 o f the mice ) even i f the an ima l s were exposed to l i g h t i m m e d i a t e l y a f t e r Hp a d m i n i s t r a t i o n . The dosages used (0 .268 mg Hp/Kg body wt) appea red t o be too low to cause n o n s p e c i f i c p h o t o t o x i c s i d e e f f e c t s . On l y one s i n g l e dose o f Hp c o n j u g a t e was used i n t r e a t m e n t o f the M-l t umour s , i n compar i son t o m u l t i p l e dose t r e a t m e n t used i n D o u g h e r t y ' s s t udy ( 6 1 ) . Though s i g n i f i c a n t c u r e r a t e s were demons t r a t ed ( T a b l e I V ) , a h i g h e r c u r e r a t e migh t be o b t a i n e d w i t h m u l t i p l e d o s e s . In C h a p t e r I I I , the use o f photo immunotherapy f o r e l i m i n a t i n g l e u k a e m i c c e l l s in. v i t r o was examined . The c l i n i c a l a p p l i c a t i o n o f t h i s t e c h n i q u e c o u l d e n t a i l p u r g i n g a u t o l o g o u s bone marrow p r e p a r a t i o n s o f l e u k a e m i c c e l l s o r c l o n o g e n i c tumour stem c e l l s p r i o r to t r a n s p l a n t a t i o n . T h i s would e l i m i n a t e a p o t e n t i a l cause f o r the o c c u r r e n c e o f l e u k a e m i c r e l a p s e s a f t e r bone marrow t r a n s p l a n t a t i o n . Hp-CAMAL-1 t r e a t m e n t o f CGL o r ANLL p a t i e n t samples r e s u l t e d i n s p e c i f i c e l i m i n a t i o n o f CAMAL a n t i g e n e x p r e s s i n g l e u k a e m i c c e l l s . The p h o t o t o x i c i t y o f the c o n j u g a t e p r o v e d t o be CAMAL a n t i g e n s p e c i f i c s i n c e Hp-CAMAL-1 demons t r a t ed n e g l i g i b l e c y t o t o x i c i t y w i t h CAMAL-negat ive c e l l s ( e . g . norma l PBL o r BM c e l l s , ALL o r lymphoma c e l l s ( F i g . 1 8 ) . These r e s u l t s c l e a r l y i n d i c a t e d t h a t photo immunotherapy w i t h Hp-CAMAL-1 c o u l d be a p r o m i s i n g method f o r bone marrow p u r g i n g . I t s i m m u n o l o g i c a l s p e c i f i c i t y f o r the CAMAL p o s i t i v e l eukaemic c e l l s wou ld ensu re e l i m i n a t i o n o f the c a n c e r c e l l s w i t h o u t a f f e c t i n g the no rma l h a e m a p o i e t i c c e l l s i n the bone marrow samp le . Radio immunoimagery o r d e t e c t i o n w i t h m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t M-l myosarcoma was examined i n C h a p t e r IV . " m T c t a g g e d ant i-M-1 a n t i b o d i e s were a b l e t o l o c a l i z e a t the M-l tumour s i t e i j i v i v o 186 and a l l o w imagery o f t h a t tumour w i t h e x t e r n a l s c i n t i g r a p h y . The imag ing p r o c e d u r e p r o v e d t o be i m m u n o s p e c i f i c s i n c e i r r e l e v a n t EL4 tumours were 99m_ no t d e t e c t e d w i t h the T c-an t i -M-1 p r o b e . A l t h o u g h s p e c i f i c tumours were d e t e c t e d w i t h r ad io immuno image ry , c r i t i c a l p rob lems a f f e c t i n g the s e n s i t i v i t y o f the p r o c e d u r e were e v i d e n t . H igh n o n - s p e c i f i c backg round up take i n the l i v e r , k i d n e y s , and s p l e e n p r e s e n t major l i m i t i n g drawbacks t o d e t e c t i o n o f s m a l l tumours e s p e c i a l l y i f they a re l o c a t e d i n the v i c i n i t y o f t h o s e o r g a n s . In a d d i t i o n , the s e q u e s t r a t i o n o f r a d i o n u c l i d e n o n s p e c i f i c a l l y i n t h o s e o rgans may i n c r e a s e the p o t e n t i a l f o r r a d i a t i o n damage i n norma l t i s s u e . P h y s i c a l e x a m i n a t i o n o f the l a b e l l e d a n t i b o d y i n d i c a t e d t h a t the h i g h n o n s p e c i f i c backg round was p r o b a b l y due to the h i g h l y a g g r e g a t e d n a t u r e o f the i n j e c t e d a n t i b o d y p r e p a r a t i o n and no t to a b i o l o g i c a l l i m i t a t i o n to the p r o c e d u r e e . g . a n t i b o d y - a n t i g e n s h e d d i n g f rom the tumour . What i s a l i m i t a t i o n t o the imag ing method i s the s h o r t h a l f 99m l i f e o f the r a d i o i s o t o p e T c . S i n c e a h a l f o f the r a d i o a c t i v i t y i s l o s t e v e r y 6 h o u r s , c l e a r d e l i n e a t i o n o f tumour s i t e s w i t h the t o t a l 99m absence o f backg round may be an u n r e a l i s t i c g o a l w i t h T c - l a b e l l e d a n t i b o d i e s . However , i f monomeric nonagg rega ted p r e p a r a t i o n s o f l a b e l l e d a n t i b o d i e s were u s e d , the n o n s p e c i f i c backg round c o u l d be l o w e r e d . The monomeric a n t i b o d i e s wou ld t r a v e l more r e a d i l y t h rough the c a p i l l a r y po res t o r e a c h the t a r g e t t i s s u e s and t h e r e f o r e c o u l d be c l e a r e d ou t o f the b l o o d c i r c u l a t i o n f a s t e r . T h i s wou ld a i d i n a q u i c k e r v i s u a l i z a t i o n o f the tumour masses . The prob lems e n c o u n t e r e d i n rad io immuno imagery a l s o i n d i c a t e i m p o r t a n t f a c t o r s t o c o n s i d e r i n immunotherapy. The n o n s p e c i f i c 187 s e q u e s t r a t i o n o f t o x i n - a n t i b o d y i n norma l o rgans e . g . l i v e r , k i d n e y s e t c . c o u l d l e a d t o norma l t i s s u e t o x i c i t y i . e . s i d e e f f e c t s . Nonaggrega ted p r e p a r a t i o n s o f t o x i n - a n t i b o d i e s c o u l d h e l p t o m i n i m i z e t h i s p r o b l e m . The s t u d i e s p r e s e n t e d here c l e a r l y e s t a b l i s h the f e a s i b i l i t y o f the "mag i c b u l l e t " t h e r a p y e n v i s u a l i z e d by E h r l i c h i n 1908. The i m m u n o l o g i c a l advantage o f a n t i b o d i e s adds a h i g h e r l e v e l o f s p e c i f i c i t y t o c a n c e r t r e a t m e n t and d e t e c t i o n . I t can p o t e n t i a l l y e l i m i n a t e the s i d e e f f e c t s due t o n o n s p e c i f i c i t y t h a t i s i n h e r e n t to most c o n v e n t i o n a l c a n c e r t h e r a p i e s . But "mag i c b u l l e t s " a re no t a panacea t r e a t m e n t f o r a l l c a n c e r s . I t i s h e a v i l y dependent on h a v i n g m o n o c l o n a l a n t i b o d i e s w i t h a p p r o p r i a t e s p e c i f i c i t y f o r the c a n c e r under c o n s i d e r a t i o n . A l t h o u g h many a n t i - c a n c e r a s s o c i a t e d marker m o n o c l o n a l a n t i b o d i e s a re p r e s e n t l y a v a i l a b l e e . g . an t i -me lanoma , a n t i - C E A , a n t i - A F P , a n t i - l e u k a e m i a a n t i b o d i e s ( 1 4 6 ) , i t i s c o n c e i v a b l e t h a t m o n o c l o n a l a n t i b o d i e s may have t o be d e v e l o p e d f o r d i f f e r e n t c a se s t o s u i t the s i t u a t i o n . A n o t h e r drawback t o t h i s t ype o f t h e r a p y i s the p o s s i b i l i t y o f the p a t i e n t mount ing an immune r e s p o n s e a g a i n s t the p a s s i v e l y a d m i n i s t e r e d a n t i b o d y c o n j u g a t e s . Even the use o f human m o n o c l o n a l a n t i b o d i e s would not c o m p l e t e l y p r e c l u d e t h i s p o s s i b i l i t y s i n c e an a n t i - i d i o t y p i c r e s p o n s e c o u l d o c c u r . The h o s t r e s p o n s e wou ld a c t t o r a p i d l y e l i m i n a t e the c o n j u g a t e s f rom the c i r c u l a t i o n w i t h o u t a l l o w i n g them to a c t t h e r a p e u t i c a l l y . T h i s p r o b l e m c o u l d be b y p a s s e d by u s i n g d i f f e r e n t m o n o c l o n a l a n t i b o d i e s w i t h the same i m m u n o l o g i c a l s p e c i f i c i t y . They c o u l d be used i n s u c c e s s i o n t o m i n i m i z e a h o s t a n t i - " m a g i c b u l l e t " r e s p o n s e t h a t wou ld a c t t o a b r o g a t e t h e i r t h e r a p e u t i c e f f e c t i v e n e s s . The a n t i g e n h e t e r o g e n e i t y i n tumour t i s s u e 188 poses ano the r p r o b l e m . The use o f a m o n o c l o n a l a n t i b o d y p r e p a r a t i o n s w i t h a s i n g u l a r s p e c i f i c i t y wou ld a l l o w the escape o f any tumour c e l l s t h a t do no t bea r t h a t s p e c i f i c a n t i g e n . A s p e c i a l l y t a i l o r e d p r e p a r a t i o n o f d i f f e r e n t a n t i - t u m o u r m o n o c l o n a l a n t i b o d i e s ( e . g . an a r t i f i c i a l l y p r e p a r e d p o l y c l o n a l a n t i s e r a ) may be more s u c c e s s f u l f o r t r e a t m e n t . F u t u r e c l i n i c a l a p p l i c a t i o n o f "mag i c b u l l e t s " i s dependent on an i n t i m a t e knowledge o f the h e t e r o g e n e i t y and e x p r e s s i o n o f tumour a s s o c i a t e d a n t i g e n s i n m a l i g n a n c i e s . A g r e a t e r u n d e r s t a n d i n g o f the a n t i g e n i c make up o f the tumour , the i d e n t i f i c a t i o n o f the c l o n o g e n i c tumour p r o g e n i t o r s c e l l s , and the d i s t r i b u t i o n o f tumour a s s o c i a t e d a n t i g e n s i n the m a l i g n a n t t i s s u e w i l l a i d i n the e f f e c t i v e c l i n i c a l u t i l i z a t i o n o f "mag i c b u l l e t " t e c h n o l o g y . A l t h o u g h prob lems do e x i s t (as d i s c u s s e d above) i n t he c l i n i c a l a p p l i c a t i o n o f d rug o r r a d i o n u c l i d e a n t i b o d y c o n j u g a t e s , t h e y a re no t u n s u r m o u n t a b l e , e s p e c i a l l y when one c o n s i d e r s the p o t e n t i a l a d v a n t a g e s . D e t e c t i o n o f s m a l l tumour masses w h i l e s t i l l a t a c u r a b l e s t age ( e . g . by s u r g i c a l r emova l o r chemotherapy ) c o u l d be made p o s s i b l e w i t h e x t e r n a l s c i n t i g r a p h y w i t h r a d i o n u c l i d e l a b e l l e d a n t i b o d i e s . Tumour s p e c i f i c t h e r a p y , f r e e o f any s i d e e f f e c t s , c o u l d be a c h i e v e d w i t h t o x i n - a n t i b o d y c o n j u g a t e s . An a d d i t i o n a l degree o f c o n t r o l t h a t wou ld gua rd a g a i n s t norma l t i s s u e t o x i c i t y m igh t be o b t a i n e d i f t he k i l l i n g a c t i o n o f the t o x i n c o u l d be kep t s e p a r a t e f rom the b i n d i n g i n t e r a c t i o n o f the a n t i b o d y . T o x i n s such as h a e m a t o p o r p h y r i n wh ich r e q u i r e a c t i v a t i o n f o r c y t o t o x i c a c t i v i t y p r o v i d e an e x t r a degree o f s p e c i f i c i t y i n a n t i - c a n c e r c o n j u g a t e s s i n c e the d e s t r u c t i v e a c t i o n o f the t o x i n c o u l d be c o n t r o l l e d u n t i l t hey a re l o c a l i z e d a t s p e c i f i c tumour s i t e s . Clearly, the utilization of "magic bullet" technology w i l l prove to be potent tool in the detection and treatment of cancers. 190 R e f e r e n c e s 1. G o r e r , P .A . and D.B. Ames (1956) P a s s i v e immunity i n m ice a g a i n s t C57BL l e u k o s i s EL4 by means o f iso-immune serum. C a n c e r Res . 1 6 : 3 3 8 - 3 4 3 . 2. R e i f , A . E . , and C H . Kim (1971) Leukaemia L1210 t h e r a p y t r i a l s w i t h a n t i - l e u k a e m i a serum and B a c i l l u s C a l m e t t e - G u e r i n . Cance r Res . 3 1 : 1 6 0 6 . 3. M u r r a y , G. 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