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The preparation of tumour specific antiserum leading to the purification of tumour associated antigen… Al-Rammahy, Abdul Khaliq Abdullah 1979

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THE PREPARATION OF TUMOUR S P E C I F I C ANTISERUM LEADING TO THE PURIFICATION OF TUMOUR ASSOCIATED ANTIGEN AND STUDIES OF ITS ROLE IN THE RECOGNITION OF SYNGENEIC CYTOTOXIC LYMPHOCYTES by ABDUL KHALIQ ABDULLAH AL-RAMMAHY B . V . M . & S . , U n i v e r s i t y o f B a g h d a d , 1969 M . S c , 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 , 1978 A THESIS.' SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY 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 to t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA O c t o b e r , 1979 (c) A b d u l K h a l i q A b d u l l a h Al -Rammahy In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT Considerable evidence i n d i c a t e s that events at the c e l l surface play a c e n t r a l r o l e i n the r e g u l a t i o n of growth of normal and n e o p l a s t i c c e l l s . I t i s p o s s i b l e that m o d i f i c a t i o n of the surface molecules might be, i n p a r t , r e s p o n s i b l e f o r the processes that accompany tumorigenesis. These surface molecules are termed TSTA (tumor s p e c i f i c t r a n s p l a n t a t i o n antigens) and TAA (tumor associated a n t i g e n s ) . The purpose of t h i s study i s to r a i s e xenogeneic antiserum which can recognize TAA. This antiserum, then, can be used as a t o o l to e n r i c h or p u r i f y the antigen i n question. Using the antibody feedback i n h i b i t i o n method which has been developed i n our l a b o r a t o r y , an antiserum d i r e c t e d toward membrane components of DBA/2 mastocytoma P815 c e l l s was r a i s e d . This antiserum was found to be s p e c i f i c f o r tumor c e l l e x t r a c t s and had no r e a c t i v i t y w i t h comparable e x t r a c t s of normal c e l l s when tested by complement f i x a t i o n . This a n t i -serum was a l s o capable of k i l l i n g P815 c e l l s i n the presence of guinea p i g complement but had no r e a c t i v i t y w i t h another DBA/2 tumor or a v a r i e t y of normal DBA/2 c e l l p r e p arations. When mixed w i t h v a r y i n g numbers of tumor c e l l s and i n j e c t e d i n t r a p e r i t o n e a l l y i n t o e i t h e r DBA/2 or B6D2 F l mice, the antiserum demonstrated a p r o t e c t i v e e f f e c t by e i t h e r prolonging s u r v i v a l time o r , apparently f a c i l i t a t i n g complete removal from the body of tumor c e l l s when low numbers of c e l l s were i n j e c t e d . The antiserum was used to monitor the p u r i f i c a t i o n of tumor a s s o c i a t e d antigen (TAA) of P815 c e l l s . Membrane e x t r a c t s of both P815 and normal DBA/2J spleen and p e r i t o n e a l exudate c e l l s were subjected to DEAE i i i f r a c t i o n a t i o n and g e l f i l t r a t i o n , f o l l o w i n g which f r a c t i o n s were test e d f o r r e a c t i v i t y w i t h the anti-P815 antiserum. F r a c t i o n s from P815 e x t r a c t s shown to be enriched f o r the TAA were used to r a i s e a second antiserum s p e c i f i c f o r the tumor. This antiserum was a l s o shown to have s p e c i f i c i t y f o r P815 and none f o r normal DBA/2J c e l l s by "^Cr r e l e a s e assays i n the presence of guinea p i g complement and by surface l a b e l i n g u s i n g peroxidase l a b e l e d sheep a n t i - r a b b i t immunoglobulin a f t e r treatment of e i t h e r P815 or normal c e l l s w i t h the antiserum. This second antiserum (anti-P815-2), 125 when allowed to react w i t h I - l a b e l e d TAA-enriched f r a c t i o n s of the P815 membrane e x t r a c t s and passed over Sepharose-protein A columns, permitted the i s o l a t i o n of a s i n g l e major component, d e t e c t a b l e on autoradiographs of gradient acrylamide g e l s . This component was not present i n equivalent normal DBA/2 t i s s u e e x t r a c t s , nor was i t d e t e c t a b l e when these t e s t s were repeated u s i n g an antiserum r a i s e d against normal DBA/2 membrane prepa-r a t i o n s . I t was thus concluded that t h i s m a t e r i a l c o n s t i t u t e d a TAA of the P815 mastocytoma. Then t h i s major band was c u t , e l u t e d and used i n CFA to immunize a r a b b i t . Three immunizations were needed to get antiserum which was s p e c i f i c to P815 c e l l s and membrane e x t r a c t s . P815 c e l l s t r e a t e d w i t h t h i s l a s t antiserum were r e s i s t a n t to l y s i s by syngeneic c y t o t o x i c c e l l s but they were not when a l l o g e n e i c c y t o t o x i c c e l l s were used. On the other hand, r a b b i t a n t i DBA/2 as w e l l as mouse a n t i H-2^ serum a l s o blocked the syngeneic k i l l i n g as w e l l as having a b l o c k i n g e f f e c t i n the a l l o g e n e i c k i l l i n g . This suggests that t h i s antiserum recognizes membrane molecules which are important f o r r e c o g n i t i o n by syngeneic c y t o t o x i c c e l l s . i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x i i CHAPTER I - LITERATURE REVIEW 1 H i s t o r i c a l Background 1 E t i o l o g y of Tumours 3 Tumour Markers 6 I. The Hormones 7 I I . Enzymes 7 I I I . Chromosomes 8 IV. Antigens 9 A. Antigens of Chemically Induced Tumours 10 1. TSTA and H i s t o c o m p a t i b i l i t y Antigens .... 11 2. Tumour Associated Antigens (TAA) 17 Tumour S p e c i f i c Antigen (TSA) 17 Oncofoetal Antigen (OFA) 19 Alp h a - f o e t o p r o t e i n (aFP) 20 Carcinoembryonic Antigen (CEA) 22 B. Antigens of V i r a l l y Induced Tumours 27 Tumour Antigens: I s o l a t i o n and Assay 32 I. I s o l a t i o n 32 I I . Antigen Assays 35 A. In v i v o C e l l u l a r Immunity 35 B. C e l l u l a r Immunity i n v i t r o .36 C. In v i t r o Antibody Assays 39 I f A Tumour Is A n t i g e n i c , Why Is I t Not Rejected By the Host 43 I . Immunosurveillance (IS) 43 I I . B l o c k i n g Factor and Immune Enhancement 48 III'-. Immune Suppression (Suppressor T C e l l s ) 49 V Page CHAPTER I - LITERATURE REVIEW Objectives of This Study 51 CHAPTER I I - PREPARATION OF XENOANTISERUM SPECIFIC FOR A MURINE MASTOCYTOMA CELL LINE 52 Ab s t r a c t 52 I n t r o d u c t i o n 53 M a t e r i a l s and Methods 55 Experimental Animals 55 Tumour 55 Pre p a r a t i o n of Lymphoid and Tumour C e l l s 55 Antigen E x t r a c t s 56 Immunization 57 Anti-normal DBA/2J 57 Feedback I n h i b i t i o n Anti-P815 57 Hypaque-Ficoll Density C e l l Separation of L1210 A s c i t e s 60 Concanavalin A B l a s t s 61 Antiserum Assays 61 In v i t r o Assays 61 Complement F i x a t i o n 61 51 / Q Cr Release Assay b j In v i v o Assay of Antiserum 63 I n t r a p e r i t o n e a l Growth of P815 Tumour 63 Subcutaneous Growth of P815 Tumour 64 Absorption 64 S t a t i s t i c a l A n a l y s i s • 64 Res u l t s 67 E x t r a c t i o n and Immunization 67 S p e c i f i c i t y of the Anti-P815 Antiserum 67 . In v i v o E f f e c t of Antibody 72 v i Page CHAPTER I I I - ENRICHMENT AND PURIFICATION OF A TUMOUR-ASSOCIATED ANTIGEN OF THE P815 MASTOCYTOMA .81 Abst r a c t . 81 I n t r o d u c t i o n 82 M a t e r i a l s and Methods 85 Pr e p a r a t i o n of Crude Membrane Antigens 85 Pr e p a r a t i o n of A n t i s e r a 88 Immunoglobulin P u r i f i c a t i o n 93 Ammonium S u l f a t e P r e c i p i t a t i o n and Gel Chromatography 93 P u r i f i c a t i o n Procedures 93 DEAE Ion Exchange Chromatography 93 Gel Chromatography 94 R a d i o i o d i n a t i o n 94 A f f i n i t y Chromatography ( P r o t e i n A) 95 Acrylamide Gradient Gel E l e c t r o p h o r e s i s and Autoradiography 95 Staphylococcus aureus Adsorbent Column (SPA) 96 Immunological Assays 97 Complement F i x a t i o n 97 ^Chromium Assay 97 Peroxidase C e l l Surface L a b e l i n g 97 Res u l t s 98 Enrichment of TAA 98 S p e c i f i c i t y of Anti-P815-2 103 P u r i f i c a t i o n of TAA 107 P r o t e i n A Chromatography 107 A n a l y t i c a l E l e c t r o p h o r e s i s and Autoradiography 114 Di s c u s s i o n 115 . v i i Page CHAPTER IV - PRODUCTION OF MONOSPECIFIC RABBIT ANTIBODY TO THE TAA OF P815 AND ITS EFFECT ON SYNGENEIC CYTOTOXICITY TO P815 119 Abst r a c t 119 I n t r o d u c t i o n 120 M a t e r i a l s and Methods 122 Pr e p a r a t i o n of A n t i s e r a 122 Pr e p a r a t i o n of Anti-H-2^ 123 C y t o t o x i c i t y of Assay 124 "^Cr -Release and B l o c k i n g Assays . . 125 Enzyme Linked Immunosorbent Assay 125 Res u l t s 126 Anti-P815-4 S p e c i f i c i t y 126 Bl o c k i n g of the Syngeneic K i l l i n g 128 Bl o c k i n g of the A l l o g e n e i c K i l l i n g 134 D i s c u s s i o n 137 LITERATURE CITED 140 APPENDIX A 159 v i i i LIST OF TABLES TABLE Page I S u r v i v a l times of DBA/2 mice i n j e c t e d i n t r a -p e r i t o n e a l l y w i t h mixtures of P815 and e i t h e r a n t i - P 8 l 5 serum of NRS 74 I I S u r v i v a l times of B6D2 mice i n j e c t e d i n t r a -p e r i t o n e a l l y w i t h mixtures of P815 and e i t h e r anti-P815 serum or NRS 75 I I I The e f f e c t of anti-P815 treatment on tumour recurrence and s u r v i v a l time i n B6D2 F l mice a f t e r r e s e c t i o n of SC implanted tumours that had reacted 1.0 - 2.0 g at the time of r e s e c t i o n 76 IV R e a c t i v i t y of P815 membrane peaks e l u t i n g from DEAE w i t h a n t i - P 8 1 5 - l as assessed by q u a n t i t a t i v e complement f i x a t i o n . Phosphate b u f f e r used was .01, .05, .10 and .20 M 100 V R e a c t i v i t y of P815 membrane peaks e l u t i n g from DEAE w i t h a n t i - P 8 1 5 - l as assessed by q u a n t i t a t i v e complement f i x a t i o n . Phosphate b u f f e r used was .01, .02, .10 M 102 VI R e a c t i v i t y of P815 membrane peaks e l u t i n g from Sephadex G-150 w i t h a n t i - P 8 1 5 - l as assessed by complement f i x a t i o n 105 VII R e l a t i v e l a b e l i n g of P815 or normal DBA/2 spleen c e l l s w i t h e i t h e r anti-P815 or anti-DBA/2 serum at va r i o u s d i l u t i o n s 108 ix LIST OF FIGURES Figure Page 1 Illustration of the steps involved in the process of raising feedback antibody specific for the tumour associated antigen 59 2 Titer of anti-P815 sera of different bleeds as shown by -^Cr release assay. The antisera were absorbed once with either DBA/2 splenocytes or L1210 syngeneic tumour cells 65 3 Titration of anti-P815 serum by the complement fixation assay at a dilution of 1/100 with extracts of P815 membrane and normal tissue extract 66 4 Titration of anti-P815 serum for i t s a b i l i t y to k i l l 51rjr labelled P815 targets in the presence of guinea pig complement before and after absorption 68 5 Titration of various antisera with DBA/2 Con A blasts for 51_r release in the presence of guinea pig complement 70 6 Titration of absorbed anti-P815 with a variety of -*l_r labelled targets in the presence of guinea pig complement 71 7 Neutralization of anti-P815 serum by P815 c e l l membrane extracts 73 8 Flow diagram il l u s t r a t i n g the isolation of membrane fragments of tumour cells and their solubilization with hypertonic salt solution (3 M KC1) 87 9 Elution profiles of anti-P815 serum from Sephadex G-200 . 89 10 Cytotoxicity of IgM and IgG fractions from S-G-200 on 51cr-labelled P815 targets in the presence of guinea pig complement 90 11 Flow diagram il l u s t r a t i n g the enrichment and purification of TAA 92 12 Elution profiles of c e l l membrane extracts of P815 and normal DBA/2 splenocytes and peritoneal exudate cel l s from DEAE, pH 8.0 99 X LIST OF FIGURES (continued) Figure Page 13 E l u t i o n p r o f i l e s of pooled f r a c t i o n s 2 and 3 (.05 and 0.1 M) from DEAE ( F i g . 12) rerun on DEAE .01 M phosphate b u f f e r , pH 8.0 101 14 E l u t i o n p r o f i l e s of pooled DEAE f r a c t i o n s from both P815 and normal DBA/2 membrane preparations from Sephadex-G-150 104 15 C y t o t o x i c i t y of anti-P815-2 i n comparison to anti-DBA/2 serum. Targets were e i t h e r DBA/2 Con A b l a s t s or P815 c e l l s 106 16 Peroxidase l a b e l l i n g of e i t h e r P815 or DBA/2 spleen c e l l s t r e a t e d w i t h anti-P815-2, a n t i -DBA/2 or NRS 110 17 E l u t i o n p r o f i l e s of i o d i n a t e d samples a f t e r treatment w i t h antiserum and passage over p r o t e i n A columns I l l 18 Autoradiographs of m a t e r i a l s eluted from p r o t e i n A columns on gradient g e l e l e c t r o p h o r e s i s 112 19 R e s u l t s of autoradiographs of m a t e r i a l s eluted from p r o t e i n A columns presented as cpm of gradient g e l e l e c t r o p h o r e s i s 113 20 E l u t i o n p r o f i l e of i o d i n a t e d samples a f t e r treatment w i t h antiserum and passage over p r o t e i n A columns 116 21 C y t o t o x i c i t y of anti-P815-4 i n comparison to a n t i -DBA/2 serum. Target c e l l s were e i t h e r DBA/2 Con A b l a s t s or P815 c e l l s both of which had been l a b e l l e d w i t h 51cr 127 22 T i t r a t i o n of P815 enriched antigen, and DBA/2 antigen, w i t h the absorbed (1:300 d i l u t i o n ) anti-P815-4 and anti-DBA/2 serum i n an ELISA assay 129 23 T i t r a t i o n of anti-P815-4 serum w i t h the enriched P815 antigen i n an ELISA assay. The optimum concent-r a t i o n of the antigen was shown to be 375 ng/well 130 24 B l o c k i n g a c t i v i t y of e i t h e r r a b b i t anti-P815 or a n t i -DBA/2 serum on 51-Cr l a b e l l e d t a r g e t c e l l s incubated w i t h CTL r a i s e d to P815 c e l l s 131 x i LIST OF FIGURES (continued) Figure Page 51 25 T i t r a t i o n of v a r i o u s a n t i s e r a w i t h Cr l a b e l l e d P815 t a r g e t s p r i o r to t h e i r a d d i t i o n to assay f o r CTL 132 26 B l o c k i n g a c t i v i t y of e i t h e r r a b b i t anti-P815 or anti-H-2^ antiserum on ^ Cr l a b e l l e d t a r g e t c e l l s incubated w i t h CTL r a i s e d to P815 c e l l s 133 27 B l o c k i n g a c t i v i t y of r a b b i t anti-P815, a n t i - H - 2 d or r a b b i t anti-DBA/2 serum on -'•'-Cr l a b e l e d target c e l l s (L1210) incubated w i t h CTL r a i s e d to L1210 135 28 B l o c k i n g a c t i v i t y of r a b b i t 5 a n t i - P 8 1 5 , a n t i - H - 2 d or r a b b i t anti-DBA/2 serum on Cr l a b e l e d t a r g e t c e l l s (P815) incubated w i t h C57B1/6 CTL r a i s e d to DBA/2 spleen c e l l s 136 x i i ACKNOWLEDGEMENTS I would l i k e to extend my sincere gratitude to my research supervisor, Dr. J u l i a G. Levy for her encouragement, assistance and d i r e c t i o n s throughout t h i s project. Acknowledgement i s also due to Dr. J.J.R. Campbell, Head, Department of Microbiology for h i s continuous support and i n s p i r a t i o n . I am indebted to other members of my committee for t h e i r advice and help i n e d i t i n g t h i s t h e s i s . The consultory contribution of Barbara K e l l y and Dr. Anne Jackson i s f u l l y appreciated. Special thanks to my fellow graduates and the summer students for t h e i r comradeship during the past few years. I am g r a t e f u l to my f r i e n d , Charles Sylvester for h i s excellent t e c h n i c a l assistance and Rosario Bauzon for her s k i l l f u l typing. Last but not the l e a s t , I would l i k e to give recognition of the support given to me both f i n a n c i a l l y and s p i r i t u a l l y by the Minis t r y of High Education, Iraq, through the Directorate of Missions and C u l t u r a l Attaches Section, under the leadership of Baath Party, without t h e i r support the advancement of high education and the development of s c i n e t i f i c i n v e s t i g a t i o n i n Iraq would not be possible. TO MAYSOON AND FURAT 1 CHAPTER I LITERATURE REVIEW H i s t o r i c a l Background: The f i r s t attempts to cure cancer by immunologic means were made more than a century ago (Koldovsky, 1974). The obvious advantages of a c h i e v i n g d i f f e r e n t i a l immunological impairment of cancer t i s s u e growth has l e d to a search f o r some component or f u n c t i o n of the cancer c e l l t hat would d i s t i n g u i s h i t completely from i t s normal counterpart. I n t e r e s t i n tumour immunology has sin c e i n c r e a s e d , but there has been a c e r t a i n degree of pessimism. Tyzzer remarked i n 1916 that reviewing the l i t e r a t u r e on tumour immunology was very d i f f i c u l t because of c o n t r a d i c t o r y and equ i v o c a l f i n d i n g s . In 1929 Woglom included almost 1000 references i n h i s review, and i n 1942, Spencer declared that the l i t e r a t u r e on tumour immunology was voluminous and c o n f l i c t i n g . U n f o r t u n a t e l y , p o s i t i v e and unequivocal r e s u l t s are reported much l e s s f r e q u e n t l y . A p r e l i m i n a r y o b j e c t i v e of the immunologist i n studying tumours has involved the attempt to demonstrate u n e q u i v o c a l l y that tumour c e l l s c o n t a i n q u a l i t a t i v e l y unique c o n s t i t u e n t s , c o l l e c t i v e l y c a l l e d tumour as s o c i a t e d antigens (TAA). Using Japanese w a l t z i n g mice, Loeb, (1901) discovered the s t r a i n s p e c i f i c t y of tumour t i s s u e t r a n s p l a n t a t i o n . A few years l a t e r , Flexner and J o b l i n g (1907) described immunological enhancement of tumour t r a n s -p l a n t s i n r a t s preimmunized w i t h heat i n a c t i v a t e d tumour t i s s u e . Never-t h e l e s s , h a l f a century of inadequately c o n t r o l l e d experiments r e s u l t e d i n a f a i l u r e to prove that the commonly studied t r a n s p l a n t a b l e tumours of 2 rodents contained tumour s p e c i f i c antigens. In 1952, Hauschka expressed s e r i o u s doubt that these c o n s t i t u e n t s would ever be demonstrated. He pointed out two major d e f e c t s prevalent i n the work w i t h animal tumours which had been performed. F i r s t , many i n v e s t i g a t i o n s had been c a r r i e d out w i t h rodent stocks of unproven homogeneity. I t was, t h e r e f o r e , impossible to determine whether normal or t i s s u e s p e c i f i c t r a n s p l a n t a t i o n antigens had been in v o l v e d i n the r e j e c t i o n of c e r t a i n of the t r a n s p l a n t a b l e tumours which had been s t u d i e d . His second point was that many of the tumours employed had been transplanted f o r long periods of time so that t h e i r a n t i g e n i c composition was h i g h l y u n c e r t a i n . In 1953, the experiments of Foley produced the f i r s t c l e a r demonstra-t i o n of s p e c i f i c a n t i g e n i c i t y i n a c l a s s of experimental animal tumours. These s t u d i e s revealed that t r a n s p l a n t a b l e methylcholanthrene-induced sarcomas were a n t i g e n i c i n syngeneic mice. Prehn and Main i n 1957 showed that pretreatment of an inbred mouse w i t h an implant of a methylcholantrene-induced tumour could protect the mouse against challenge w i t h the same tumour c e l l s . Host r e j e c t i o n of a tran s p l a n t e d tumour of syngeneic donor o r i g i n must then be due to the development of new tumour s p e c i f i c t r a n s -p l a n t a t i o n antigen(s) (TSTA) w i t h i n the tumour t i s s u e . Furthermore, c e r t a i n other neoantigens which are not d i r e c t l y i n v o l v e d i n t r a n s p l a n t a t i o n immunity may be detected by examining the host's serum f o r c i r c u l a t i n g a n t i b o d i e s by immunologic techniques. F o l l o w i n g the o r i g i n a l d i s c o v e r y of Sjogren (1961) that polyoma v i r u s induced new TSTA, i t was found that p r a c t i c a l l y a l l oncogenic v i r u s e s d i d the same (Sjogren, 1964). 3 In 1965, evidence f o r the exi s t e n c e of the carcinoembryonic a n t i g e n , CEA, was shown by Gold and Freedman, demonstrating another c l a s s of TAA. Since then, many r e p o r t s have been published proving the concept of tumour a s s o c i a t e d antigens (Watson et a l . , 1975; K e l l y et a l . , 1977; Takashi e_t a l . , 1978), but these antigens have not been i s o l a t e d or p u r i f i e d to an extent that would a l l o w t h e i r p o t e n t i a l use or could v e r i f y t h e i r r o l e i n the immune response to tumour i n tumour bearing animals and human beings. This subject i s one of the main t o p i c s i n t h i s t h e s i s . E t i o l o g y of Tumours: Every time a s c i e n t i s t meets a member of the general p u b l i c , the f i r s t question asked i s "what kind of science do you do?" When a tumour immuno-l o g i s t s t a t e s h i s occupation, the second question asked "what causes cancer?" Since there i s no s i n g l e d e f i n i t e answer when I am asked t h i s question, I am tempted to r e p l y that I am a student i n mic r o b i o l o g y , hoping that I w i l l then be asked the cause of t u b e r c u l o s i s , e t c . There i s a m i n o r i t y group of animals or human beings who are g e n e t i c a l -l y s u s c e p t i b l e to n e o p l a s i a and a general population group who are not. This g e n e r a l i z a t i o n can be made f o r v i r t u a l l y a l l forms of cancer (Knudson, 1977; Kundson et_ a l . , 1973). In both groups, the r i s k of tumour can be increased by environmental agents. The i n i t i a t i o n of malignant tumour growth may i n v o l v e a mutational process, the b a s e l i n e incidence of which would r e f l e c t spontaneous mutation r a t e s i n germ l i n e and somatic c e l l s ; the environmental agents can increase these mutation r a t e s . These mutations are e i t h e r p r e z y g o t i c , i . e . are already present i n germ c e l l s before 4 conception, or p o s t z y g o t i c , i . e . occur a f t e r f e r t i l i z a t i o n . Some dominantly i n h e r i t a b l e mutations are not associated w i t h a p o s i t i v e parent h i s t o r y because they are f r e s h mutations i n the germ l i n e of parents whose somatic c e l l s are normal. How does cancer a r i s e i n normal animals or humans who are not pre-disposed? This question cannot yet be answered w i t h c e r t a i n t y but the model mentioned by Knudson (1973) i n which two mutations are r e q u i r e d i n p o s t z y g o t i c cases, the only d i f f e r e n c e being that both mutations are somatic. Here one can v i s u a l i z e a c e l l which s u s t a i n s one mutation gene-r a t i n g a clone of mutant c e l l s , any one of which can become a malignant c e l l as a r e s u l t of a second mutation. What are the carcinogens that cause cancer? From the work w i t h experimental animals, v i r u s e s e s p e c i a l l y RNA v i r u s e s , may cause t r a n s f o r m a t i o n of c e r t a i n types of t i s s u e s . RNA tumour v i r u s e s have been demonstrated to be present i n c o l d blooded v e r t e b r a t e s (Winguist et_ al_. , 1973) , avian (Temin et a l . , 1974), murine (Aoki et a l . , 1975; F r i e n d , 1957; Moloney, 1960), f e l i n e ( J a r r e t t , 1971; J a r r e t t et a l . , 1964), bovine ( M i l l e r et a l . , 1969; Olson et a l . , 1972), non-human primate leukemias (Prochownik et a l . , 1976; Todaro et a l . , 1975), s a r c o m a s - (Th e i l e n et a l . , 1971) and human beings. As e a r l y as 1965, Dmochowski et^ a l . (1965) reported e l e c t r o n microscopic d e t e c t i o n of p a r t i c l e s resembling RNA tumour v i r u s e s i n human n e o p l a s i a . Soon a f t e r the demonstration of reverse t r a n s c r i p t a s e (RT) i n RNA v i r u s e s , a r e v e r s e t r a n s c r i p t a s e - l i k e a c t i v i t y was detected i n the f r e s h p e r i p h e r a l blood leukocytes of p a t i e n t s w i t h leukemia ( G a l l o et a l . , 1970; G a l l o et a l . , 1974). V i r u s e s which cause murine tumour w i l l be discussed i n d e t a i l under tumour 5 antigens. Of the DNA v i r u s e s , herpesviruses are most l i k e l y to be oncogenic i n man. I t has been shown that the Epstein-Barr v i r u s (EBV) appears to be associated w i t h B u r k i t t ' s lymphoma (BL), and nasopharyngeal carcinoma (Marx et a l . , 1974). L i k e the other herpesviruses, EBV i s widely disseminated i n the human p o p u l a t i o n , e s p e c i a l l y i n A f r i c a where up to 90% of the c h i l d r e n have a n t i b o d i e s to EBV before the age of 2 years. P a t i e n t s w i t h BL have 10 times as much antibody against v i r a l capsid antigen as do normal c o n t r o l s (Marx et_ a l . , 1974). An t i b o d i e s to another EBV-r e l a t e d antigen complex are c a l l e d " e a r l y antigens" because when c u l t u r e d c e l l s are i n f e c t e d w i t h EBV they appear e a r l y i n the v i r u s r e p l i c a t i o n c y c l e . These antigens are r a r e l y found i n healthy people. BL c e l l s a l s o produce an EBV ass o c i a t e d tumour antigen that may be analogous to the T-antigens produced i n c e l l s i n f e c t e d w i t h oncogenic animal v i r u s e s such as SV40. T-antigen, an e a r l y v i r a l gene product, i s present i n the nucleus of transformed c e l l but not i n the v i r u s i t s e l f ( A l l e n e_ a l . , 1972). Evidence i m p l i c a t i n g the herpes simplex v i r u s e s I and I I (HSV I , HSV I I ) i n the e t i o l o g y of human cancer i s s i m i l a r to that o u t l i n e d f o r EBV. These v i r u s e s cause o r a l and cutaneous h e r p e t i c i n f e c t i o n s and g e n i t a l i n f e c t i o n s , r e s p e c t i v e l y . P a t i e n t s w i t h those i n f e c t i o n s have been shown to react w i t h n o n v i r i o n antigens, but antigens" which are v i r a l gene products thought to be ass o c i a t e d w i t h transformation (Sabin e_ a l . , 1973). P h y s i c a l agents l i k e r a d i a t i o n , both u l t r a v i o l e t , i o n i z i n g and X-rays have been shown to be c a r c i n o g e n i c . E p i d e m i o l o g i c a l and l a b o r a t o r y s t u d i e s i n d i c a t e that some chemicals are h i g h l y c a r c i n o g e n i c . Some of these chemicals are N-nitrosamines, 6 p o l y c y c l i c hydrocarbons, methyl-methane sulphonate, e t h y l methyl sulphonate and the wi d e l y used chemical to induce experimental tumours, methylcholan-threne (Magee, 1974). Many of these chemicals are not carc i n o g e n i c i n the n a t i v e form to which man or mouse might be exposed, but they may be metabolized to an a c t i v e form (Knudson, 1977). For example, p o l y c y c l i c hydrocarbons i n tobacco smoke might be transformed e n z y m a t i c a l l y to form epoxides, suggesting the p o s s i b i l i t y that somatic mutation may lead to lung cancer. There i s evidence that N-nitrosamines are a c t i v a t e d by an enzyme i n the l i v e r to form a l k y l a t i n g agents (Magee, 1974). The l i v e r s of some s t r a i n s of mice are b e t t e r able to a c t i v a t e these chemical carcinogens than others. Genetic v a r i a t i o n of t h i s type i s l i k e l y to e x i s t i n man and may determine whether the p o s s i b i l i t y w i l l be low or high that a c i g a r e t t e smoker w i l l acquire the mutations that lead to lung cancer (Kellermann et a l . , 1973). While there i s good reason to b e l i e v e that some cancer i s an inherent b i o l o g i c a l problem a s s o c i a t e d w i t h spontaneous mutation, the incidence of cancer i n many p a r t s of the world, e s p e c i a l l y i n i n d u s t r i a l i z e d n a t i o n s , i s apparently much higher than would r e s u l t from the operation of spontaneous mutation. Chemical agents, e s p e c i a l l y those derived from i n d u s t r i a l a c t i v i t y and from d i e t seem to be p a r t i c u l a r l y important. The high incidence of cancer caused by such agents i s t h e o r e t i c a l l y preventable. Tumour Markers: A tumour marker may be defined as a substance ( g e n e r a l l y a p r o t e i n i n the form of an ant i g e n , hormones or enzyme) which can be i d e n t i f i e d i n the blood and i s r e p r e s e n t a t i v e of the tumour, i n that i t i s produced and 7 secreted by tumour c e l l s or i s part of the c e l l s t r u c t u r e released i n t o the blood. The hypothesis that tumour markers s p e c i f i c a l l y r e f l e c t the n e o p l a s t i c process i s dependent on co n f i r m a t i o n of the f o l l o w i n g p o s t u l a t e s , s i m i l a r to Koch's p o s t u l a t e s f o r e s t a b l i s h i n g and confirming an e t i o l o g i c agent f o r i n f e c t i o u s disease ( L o k i c h , 1978). (a) The c i r c u l a t i n g l e v e l of the tumour marker must be abnormal i n the presence of the n e o p l a s t i c process, (b) The c i r c u l a t i n g l e v e l of the tumour marker must change i n d i r e c t r e l a t i o n s h i p to tumour progression and r e g r e s s i o n . (c) The tumour t i s s u e i t s e l f must c o n t a i n the tumour marker. I. The Hormones Hormones may be associated w i t h endocrine derived t i s s u e ( t h y r o i d , adrenal etc.) i n which case they are immunologically i d e n t i c a l w i t h the p h y s i o l o g i c hormone. In other i n s t a n c e s , non-endocrine neoplasias (e.g. lung) may express abnormal hormone sy n t h e s i s . In these i n s t a n c e s , i t i s not uncommon f o r the hormones secreted to be chemic a l l y d i s t i n c t from t h e i r p h y s i o l o g i c a l counter-p a r t . The best example of high hormone production as a r e f l e c t i o n of neoplasm i s the human c h o r i o n i c gonadotropin (HCG) i n c h o r i o -carcinoma. I I . Enzymes A m u l t i p l i c i t y of serum enzymes have been associated w i t h s o l i d tumours, i n c l u d i n g a l k a l i n e phosphatase (Regan enzyme or . p l a c e n t a l a l k a l i n e phosphatase) (Stolbach et a l . , 1969), histaminase ( B a y l i n e_t a l . , 1972) , amylase (Ammann e_t a l . , 1973) , neuraminidase (Cooper et a l . , 1974) and t e r m i n a l d e o x y n u c l e o t i d y l 8 t r a n s f e r a s e (TdT) which has been suggested to be a marker f o r immature c e l l s of T - c e l l l i n e a g e (McCaffrey et a l . , 1975). I t i s , t h e r e f o r e , of i n t e r e s t that almost a l l acute lymphocytic leukemias and a p r o p o r t i o n of chronic myelogenous b l a s t c r i s e s are TdT p o s i t i v e . I I I . Chromosomes In 1914 the German b i o l o g i s t , Theodore B o v e r i published a t h e o r e t i c a l t r e a t i s e i n which he argued that the fundamental cause of tumours i s an imbalance i n the chromatin content of the c e l l s from which they a r i s e . Boveri's theory i s s t i l l an open question, i n s p i t e of the a s s o c i a t i o n of some malignancies w i t h chromosomal a b n o r m a l i t i e s . Chronic myeloid leukemia (CML) i s always accompanied by a p a r t l y d e l e t e d number of group G ( P h i l a d e l p h i a or Ph') chromosomes (Nowell et a l . , 1960). The c l o s e s t a s s o c i a t i o n to CML was the f i n d i n g that a G-group chromosome i s f r e q u e n t l y l o s t , p a r t i a l l y deleted or involved i n s t r u c t u r a l rearrangements i n meningioma (Mark, 1970). The r e s u l t s from f u s i o n between malignant and non-malignant c e l l s i n v a r i o u s combinations, suggest that the t u m o r i g e n i c i t y of the product depends on the r e t e n t i o n of p a r t i c u l a r chromosomes i n the hy b r i d and the e l i m i n a t i o n of c e r t a i n others ( K l e i n et a l . , 1971). However convincing the evidence, i t can be argued that s i n c e many tumours have, i n t h e i r e a r l y stages, no d e t e c t a b l e chromosome abno r m a l i t i e s , subsequent rearrangement do not c o n t r i b u t e to the 9 malignant p o t e n t i a l , and non random a b e r r a t i o n s merely r e f l e c t the r e s t r i c t e d range of chromosome gains or l o s s e s which can be t o l e r a t e d without i m p a i r i n g the v i t a l i t y of the c e l l and i t s progeny. Many balanced t r a n s l o c a t i o n s , p e r i c e n t r i c i n v e r s i o n s and other c o n s t i t u t i o n a l chromosome ab n o r m a l i t i e s have been detected i n apparently h e a l t h y people (The Lancet, 1977). However, four genetic d i s o r d e r s of man c h a r a c t e r i z e d by chromosomal i n s t a b i l i t y - Fanconi's anaemia, a t a x i a t e l a n g i e c t a s i a , xeroderma pigmentosum and Bloom's syndrome are associated w i t h a g r e a t l y increased r i s k of malignant disease. IV. Antigens Much of the e a r l y work i n tumour immunity was confusing because i t was not appreciated that tumours l i k e other t i s s u e s , e x h i b i t t r a n s p l a n t a t i o n antigens. Only when syngeneic tumours are used can tumour antigens alone be s t u d i e d , and i t was the i n t r o d u c t i o n of inbred mice s t r a i n s which allowed Foley i n 1953 to produce the f i r s t evidence f o r the s p e c i f i c a n t i g e n i c i t y of experimental tumours. In p r e p a r a t i o n f o r the more d e t a i l e d d i s c u s s i o n and r e p r e s e n t a t i v e systems, the f o l l o w i n g d e f i n i t i o n s provide a b a s i s f o r i n i t i a l d i s t i n c t i o n between the v a r i o u s types of tumour neoantigens: Tumour s p e c i f i c t r a n s p l a n t a t i o n antigens (TSTA) - TSTA are capable of inducing r e s i s t a n c e to tumour t r a n s p l a n t a t i o n i n the autochthonous host or i n syngeneic r e c i p i e n t s . They are a l s o 10 c a l l e d tumour r e j e c t i o n antigens. This d e f i n i t i o n i s based s o l e l y on the i n v i v o observations. Tumour as s o c i a t e d antigens (TAA) - The term TAA i s widely used i n the l i t e r a t u r e to designate an antigen which i s tumour s p e c i f i c and a l s o antigens which appear to be tumour s p e c i f i c , i e . , without appropriate s p e c i f i c i t y c o n t r o l s . These may i n c l u d e : Tumour s p e c i f i c antigen (TSA) - Those antigens which are d e t e c t a b l e only on tumour c e l l s and which d i f f e r q u a l i t a t i v e l y from those expressed by the normal counterparts. Embryonic antigens - This i s an antigen whose occurrence i s l i m i t e d to normal embryonic t i s s u e and i n a d u l t s to malignant tumours or i s present to a markedly reduced extent i n normal t i s s u e . Of these, the carcinoembryonic antigen (CEA) and a - f o e t o p r o t e i n (a-FP) ( S i l v e r et_ a l . , 1973; Abelev, 1974) are the best documented. Antigens induced by oncogenic v i r u s e s - They are a l s o d e t e c t a b l e i n m o r p h o l o g i c a l l y normal c e l l s before n e o p l a s t i c transformation (Wahren, 1966). These antigens are absent from non-infected animals. A. Antigens of Chemically Induced Tumours Each tumour induced by any chemical carcinogen has an i n d i v i d u a l p e r s i s t a n t and apparently unique a n t i g e n i c i t y which d i f f e r from other tumours induced i n i d e n t i c a l f a s h i o n (Basom-b r i o et a l . , 1972). 11 TSTA and H i s t o c o m p a t i b i l i t y Antigens Chemically induced neoplasms express TSTA which can cause r e j e c t i o n of otherwise l e t h a l challenge of syngeneic tumour c e l l s by animals that had been immunized w i t h the r e s p e c t i v e tumour. Immunization of animals w i t h d i f f e r e n t tumour of s i m i l a r o r i g i n i s i n e f f e c t i v e proving the uniqueness of TSTA i n these tumours (Basombrio, 1970). The polymorphism of chemi-c a l l y induced TSTA thus resembles that of h i s t o -c o m p a t i b i l i t y antigens and d i f f e r s from the group and t y p e - s p e c i f i c c r o s s r e a c t i v i t y of TSTA on neoplasms induced by DNA and RNA c o n t a i n i n g oncogenic v i r u s e s . The d i v e r s i t y of a n t i g e n i c expression among tumours of chemical e t i o l o g y has been the subject of a comprehensive review (Baldwin, 1973). The possession of p r i v a t e antigens has served to d i s t i n g u i s h tumours of chemical e t i o l o g y from those induced by oncogenic v i r u s e s i n which TSTA are common to tumours induced by the same v i r u s , i r r e s p e c t i v e of tumour morpho-logy or of the s t r a i n or species of o r i g i n ( K l e i n , 1968). S e r o l o g i c a l i n v e s t i g a t i o n of TSTA suggest that t h i s d i s t i n c t i o n may not be absolute i . e . there i s c r o s s - r e a c t i v i t y between 12 s e v e r a l tumours of chemical o r i g i n (Parker et a l . , 1977). The expression of an endogenous v i r a l antigen by some che m i c a l l y induced neoplasms i s an explanation f o r t h i s d i s c r e -pancy between t r a n s p l a n t a t i o n and s e r o l o g i c a l s t u d i e s (Brown et a l . , 1978). This could be true i n MC-sarcoma of guinea pigs i n which case a n t i g e n i c cross r e a c t i v i t y could be a t t r i b u t a b l e to passenger v i r u s , s i n c e v i r u s a s s o c i a t e d cavian leukemias have been detected only i n s t r a i n 2 guinea p i g while sarcomas induced i n s t r a i n 13 guinea pigs have p r i v a t e antigens (Holmes e__ a l . , 1971) . Antigen conversion (Stuck et a l . , 1964) represents a p o t e n t i a l c o m p l i c a t i o n i n the search f o r t r u l y c r o s s -r e a c t i n g antigens on tran s p l a n t e d tumours. Recently, Brown et a l . , 1978) found that i n murine sarcomas of the Balb/C s t r a i n s , primary tumours r a r e l y expressed d e t e c t a b l e murine leukemia v i r u s MuLV antigens whereas tumours that had been repeatedly transplanted i n syngeneic mice commonly produced MuLV and expressed v i r a l antigens at the c e l l s u r f a ce. T h e i r e x p l a n a t i o n was that v i r a l expression r e s u l t e d from a m p l i f i c a t i o n of a v a r i a n t clone 13 owing to some s e l e c t i v e advantage of expression of the MuLV genome. In s p i t e of that the l i t e r a t u r e i s f u l l of references to tumours which are demonstrably and uniform l y a n t i g e n i c i n syngeneic host. The most e x t e n s i v e l y studied are the 3-MC induced tumours and 4-dimethylaminoazobenzene (DMAB) induced hepatomas (Baldwin, 1973; Stolbach et a l . , 1969). The degree of immunogenicity d i f f e r s from one species to another e.g. r a t and guinea p i g (Oettgen et a l . , 1968). V a r i a b i l i t y i n immuno-g e n i c i t y i s encountered among tumours of d i f f e r -ent morphology i n the same species e.g. squamous c e l l carcinoma was shown to be h i g h l y immuno-genic (Old et a l . , 1962), while mammary tumours are weakly immunogenic even though the same chemical carcinogen was used. Why are the TSTAs of each carcinogen induced tumour d i s c r e t e components? One e a r l y e x p l a n a t i o n proposed i s that the a n t i g e n i c i t y might be due to a m u t a t i o n - l i k e event (Koldovsky, 1974) or h e r e d i t a r y a l t e r a t i o n of the c e l l s . (Prehn et a l . , 1957). These p r o p o s i t i o n s are c o n s i s t e n t w i t h the observation that oncogenic hydrocarbons are mutagenic (Vaage et a l . , 1969) 14 and that the r e s u l t i n g a n t i g e n i c determinants are d i v e r s e and p e r s i s t e n t i n the progeny of each. Another explanation proposed by Burnet (1970) was that the development of malignancy i s merely made evident by c l o n a l a m p l i f i c a t i o n of a n t i g e n i c d i f f e r e n c e s which were already present i n the c e l l from which the malignant clone was i n i t i a t e d . The e x p l a n a t i o n that the carcinogen induce genetic a l t e r a t i o n s r a t h e r than c l o n a l a m p l i f i -c a t i o n of p r e - e x i s t i n g a n t i g e n i c v a r i a n t s was shown to be more l i k e l y the mechanism (Basombrio et a l . , 1972; Baldwin et a l . , 1973). Balb/C 3T3 c e l l s were f i r s t cloned i n v i t r o and the progeny c e l l s were then n e o p l a s t i c a l l y t r a n s -formed by MC i n d i f f u s i o n chambers i n the abdomen of mice. The antigens of the tumours induced were s t i l l i n d i v i d u a l l y d i s t i n c t , even though they a l l had been derived from the same o r i g i n a l c e l l (Basombrio et a l . , 1972). TSTA are c e r t a i n l y associated w i t h the c e l l membrane. I t i s d i f f i c u l t to conceive how i n t r a -c e l l u l a r antigens could f u n c t i o n i n immunological r e j e c t i o n , s i n c e the tumour c e l l membrane i s l a r g e l y impermeable to antibody, and c e l l - c e l l i n t e r a c t i o n i s required f o r tumour c e l l 15 k i l l i n g mediated by s e n s i t i z e d lymphocytes (Baldwin, 1973). Normal h i s t o c o m p a t i b i l i t y antigens are known to r e s i d e on the c e l l membrane ( C u l l e n et a l . , 1974). Membrane p r o t e i n s may be p e r i p h e r a l and are bound by weak, non-covalent forces and are r a p i d l y shed o f f the membrane. I n t e g r a l p r o t e i n s (Singer et a l . , 1972) are more c l o s e l y a s s o c i a t e d w i t h membrane l i p i d , and are more d i f f i c u l t to d i s s o c i a t e from membranes. I t has been suggested that H-2 and H-LA a l l o -antigens are i n t e g r a l ( V i t e t t a et a l . , 1974) The biochemical p r o p e r t i e s of some of the H-2 products have been the subject of a review (Nathenson et_ a l . , 1974). B r i e f l y , the molecules i s o l a t e d by detergent s o l u b i l i z a t i o n are g l y c o -p r o t e i n s w i t h an approximate molecular weight of 45,000. Two d i s t i n c t p o l ypeptides, c a r r y i n g s p e c i f i c i t i e s assigned to the K or D regions can be i s o l a t e d and separated from each other. In between these two regions a r e t h e I r . genes and Ss/Slp r e g i o n s . Many t r a i t s h a v e been assigned to each region of the H-2 gene complex. The most s i g n i f i c a n t ones immunologically are the t r a n s p l a n t a t i o n antigens, 16 c o n t r o l of s p e c i f i c , immune responses, g r a f t v s . host r e a c t i o n , and T c e l l - B c e l l i n t e r a c t i o n s . I t has been r e c e n t l y reported that TSTA of c h e m i c a l l y induced tumours i n the mouse represent H-2 modified antigens (Garrido et a l . , 1977; I n v e r n i z z i et _ 1 . , 1975; I n v e r n i z z i e_ a l . , 1977; M a r t i n e t a l . , 1977), but i n the st u d i e s presented by N a t o r i et a l . (1978;1979) and others (Clemetson et a l . , 1976a; 1976b) i t was shown that TSTA of MC induced P815 masto-cytoma are molecules d i s t i n c t from major h i s t o -c o m p a t i b i l i t y antigen. N a t o r i ' s f i n a l products of TSTA were obtained from a m a t e r i a l that d i d not bind to the LcH (Leno c u l i n a r i s ) a f f i n i t y column to which H-2 antigens would have bound. A l s o , H-2 a c t i v i t i e s were not detected i n the isotachophoresis f r a c t i o n c o n t a i n i n g TSTA. Furthermore, immunologically p u r i f i e d TSTA d i d not bind to r a b b i t a n t i H-2 antiserum. TSTA could c o n s t i t u t e a l l o a n t i g e n s other than H-2 i n the mouse, as i n the case of TL antigen ( I n v e r n i z z i et a l . , 1977). P u r i f i c a t i o n of these molecules from tumour c e l l s w i l l shed some l i g h t on the nature of TSTA. 17 2. Tumour Associated Antigens (TAA) Tumour s p e c i f i c antigen (TSA) - As mentioned above, i t i s w e l l e s t a b l i s h e d that many tumours of animals and man possess antigens that are q u a l i t a t i v e l y or q u a n t i t a t i v e l y d i f f e r e n t from normal t i s s u e (Stolbach et a l . , 1969; Baldwin, 1973; D i S c i u l l o et a l . , 1977; K e l l y et a l . , 1979). Humoral a n t i b o d i e s to c e l l surface components of d i f f e r e n t tumours have been shown to be de t e c t a b l e i n sera of hosts immunized w i t h i r r a d i a t e d tumour c e l l s ( E z a k i et_ a l . , 1978) , s o l u b i l i z e d membrane e x t r a c t s ( K e l l y et^ a l . , 1979; Al-Rammahy et a l . , 1979), or i n some cases sera of tumour bearers (Metzgar et a l . , 1975; M i t c h e l l et_ a l . , 1973). Detection of c e l l s urface antigens unique to tumours have been revealed by many immunological techniques, (see below). Even though they y i e l d l i t t l e i n f o r m a t i o n on the b i o l o g i c a l r o l e of these antigens they have proved v a l u a b l e f o r the a n a l y s i s of s p e c i f i c i t y among tumours as opposed to t h e i r normal counterparts. There i s no evidence to show that the antigen(s) detected by tumour r e j e c t i o n t e s t s (TSTA) or s e r o l o g i c a l l y by s p e c i f i c a n t i s e r a (TAA) are n e c e s s a r i l y i d e n t i c a l . 18 In human beings many tumours have been shown to c o n t a i n unique antigens. K e l l y et a l . (1977) c l e a r l y demonstrated the tumour a s s o c i a t e d components from human squamous c e l l carcinoma of the lung u s i n g a feedback i n h i b i t i o n h e t ero-antiserum r a i s e d i n r a b b i t to monitor the p u r i -f i c a t i o n of TAA. This antigen(s) was shown to be common to a l l squamous c e l l lung carcinoma t e s t e d . A TAA of human melanoma was a l s o detected by a n i t s e r a r a i s e d i n chimpanzee ( S e i g l e r et a l . , 1975) . Immunological response of human leukemia p a t i e n t s to t h e i r own malignant c e l l s (Hersey et a l . , 1973; Metzgar et a l . , 1975) and the apparently s p e c i f i c r e a c t i v i t y of c e r t a i n hete-rologous a n t i s e r a toward leukemia c e l l s (Baker et a l . , 1973; Mann et a l . , 1971) have provided con s i d e r a b l e evidence f o r the presence of leukemia a s s o c i a t e d antigen (LAA). On the other hand, recent work by Tupchong et a l . (1978), i n which they analyzed 95 r a b b i t a n t i s e r a to CML and AML showed that none o f - t h e i r a n t i s e r a were . s p e c i f i c w i t h leukemia b l a s t c e l l s . The few a n t i s e r a which reacted w i t h AML c e l l s were a l s o r e a c t i v e against PHA-induced lymphoblasts even a f t e r extensive absorption w i t h normal spleen 19 c e l l s . This might i n d i c a t e that these a n t i -sera were d e t e c t i n g d i f f e r e n t i a t i o n antigens. However, HL-D locus antigens commonly a s s o c i a t e d w i t h c e l l s ( l a antigen) ( A r b e i t et a l . , 1975) have been reported on the surface of a high p r o p o r t i o n of acute leukemia c e l l s ( B i l l i n g et a l . , 1977; Schlossmann et a l . , 1976). So, the xenoantisera which were reported to be s p e c i f i c f o r leukemia might be d i r e c t e d against these antigens. I t i s not c l e a r whether leukemias express d i f f e r e n t i a t i o n , embryonic or leukemia s p e c i f i c antigens that may be present on leukemia c e l l s and found on c e r t a i n normal c e l l s only during embryogenesis or on a d u l t c e l l s d u r i ng a c e r t a i n stage of d i f f e r e n t i a t i o n . Oncofoetal antigen (OFA) - B i o l o g i s t s have long been c o n s i d e r i n g the s i m i l a r i t i e s between cancer development and processes a s s o c i a t e d w i t h d i f f e r e n t i a t i o n i n c e l l s . Schone (1906) reported that mice i n j e c t e d w i t h f o e t a l t i s s u e acquired the a b i l i t y to r e j e c t t r a n s p l a n t s of tumour t i s s u e which otherwise grew and k i l l e d the host. Adult t i s s u e d i d not show t h i s response. A t t e n t i o n has been refocussed on these f i n d i n g s s t a r t i n g w i t h the d i s c o v e r y of ct-foetoprotein (Abelev e_t a l . , 1963) and 20 carcinoembryonic antigen (CEA) (Gold e_t a l . , 1965). They are c a l l e d OFA because they are f o e t a l substances which are synthesized at c e r t a i n stage of g e s t a t i o n and appear again i n ad u l t s i n a s s o c i a t i o n w i t h c e r t a i n kinds of tumours. However, c e r t a i n f o e t a l substances appear i n a d u l t animals i n c o n d i t i o n s other than cancer ( H i r a i et^ a l . , 1975; J e r r y et a l . , 1976). The immunbgenicity of these substances and t h e i r r e l a t i o n s h i p to t r a n s p l a n t a t i o n antigens i s s t i l l a matter of controversy, but the c l a s s i f i c a t i o n proposed by Alexander (1972) emphasizes that the f o e t a l m a t e r i a l i s immuno-genic i n heterologous animal but may not be autoimmunogenic and may or may not e l i c i t c e l l-mediated or humoral responses. A l p h a f o e t o p r o t e i n (a-FP) - a-FP i s the major plasma p r o t e i n of the e a r l y foetus (Abelev, 1974) c o n s t i t u t i n g almost o n e - t h i r d of the whole serum p r o t e i n and i s about one m i l l i o n times higher than the adu l t l e v e l . Normal human serum contains 2-25 ng per ml, mice about 100 ng per ml and r a b b i t about 170 ng per ml. High serum l e v e l s (1500 ng/ml) as measured by radioimmunoassay (RIA) may occur i n a d u l t s harboring h e p a t o c e l l u l a r carcinoma, 21 ovarian and testicular teratocarcinoma (Silver et_ a l . , 1974; Abelev e t a l . , 1963). a-FP has been identified and purified in a l l species studied using immunochemical techniques. It consists of a single polypeptide chain with about 4% carbohydrate and has a molecular weight of 65,000 - 75,000. a-FP seems to share genetic ancestry with albumin since these two molecules exhibit sequence homology (Ruoslahti et_ a l . , 1976). Cross-reactivity can be demonstrated immunologi-cally when the two molecules are examined as unfolded peptide chains (Ruoslahti et a l . , 1976). No immunological differences have been found between foetal and tumour a-FP which appear to be the products of the same gene (Ruoslahti et_ a l . , 1974) Amounts of up to 1 milligram of protein are needed to generate high t i t e r antisera to a-FP which indicates the striking similarities in amino acid sequence between different species ( Ishi et a l . , 1975). Furthermore, high serum levels of a-FP have been demonstrated during pregnancies and in patients with hepatitis or cirrhosis (Silver e_t a l . , 1974), but this increase i s not followed by detectable anti-a-FP 22 antibody production (Seppala, 1975). Moreover, there are no r e p o r t s of autologous T - c e l l immune response to unmodified a-FP. Since a-FP i s not as s o c i a t e d w i t h the c e l l membrane i t could not be expected to serve as a t r a n s p l a n t -a t i o n antigen ( S e l l et a l . , 1976) . Chemical m o d i f i c a t i o n by haptenation or complexing of a-FP w i t h heterologous antibody can induce production of a potent homologous anti-a-FP. Haptenation or complex formation may all o w homologous a-FP molecules to be recognized by homologous B c e l l s through the c a r r i e r help of T c e l l s r e a c t i n g to heterologous immunoglo-b u l i n or hapten ( R u o s l a h t i et a l . , 1975; N i s h i et a l . , 1972). Carcinoembryonic antigen (CEA) - This tumour a s s o c i a t e d , f o e t a l a s s o ciated antigen was o r i g i -n a l l y described by Gold and Freedman i n 1965. I t was found i n malignant colon cancer and f o e t a l c olon t i s s u e but not i n adu l t t i s s u e . CEA i s a g l y c o p r o t e i n comprising 65% carbohydrate and 35% p r o t e i n w i t h a sedimentation r a t e of 7-8 S and a molecular weight of.approximately 200,000 as determined by g e l f i l t r a t i o n (Gold, 1971). The heterogeneity of CEA preparations may r e s i d e i n the carbohydrate p o r t i o n . V a r i a t i o n s 23 i n the s i a l i c a c i d contents have been shown to play a major r o l e i n e l e c t r o p h o r e t i c hetero-geneity (Coligan e_ a l . , 1973) . The p r o t e i n moeity i s a s i n g l e peptide c h a i n . Glycopeptides of CEA obtained by cleavage at the c y s t e i n e residues and separated by polyacrylamide g e l e l e c t r o p h o r e s i s were capable i n d i v i d u a l l y of 30-50% maximal i n h i b i t i o n i n the radioimmuno-assay, although the pooled glycopeptides were capable of 100% i n h i b i t i o n (Egan et a l . , 1974). Several s t u d i e s have suggested that CEA may comprise a f a m i l y of r e l a t e d g l y c o p r o t e i n s . CEA i s o l a t e d from colon carcinoma has been reported to be d i s t i n c t from that of breast or ovarian cancer i n regard to the a n t i g e n i c determinants and r e a c t i v i t y w i t h l e c t i n (Chism e_ a l . , 1976). U n l i k e a-FP, CEA-like molecules have not been detected i n species other than man and high t i t e r a n t i s e r a are r e a d i l y generated i n many animals. Autoantibody to CEA has been reported i n the serum of colon cancer p a t i e n t s without m e t a s t a t i c disease (Costanza e_ a l . , 1973) , but CEA does not e l i c i t a T c e l l response i n these p a t i e n t s (Gold, 1971). In f a c t , T c e l l t o l e r a n c e i s i n d i c a t e d by f a i l u r e of the lymphocytes from those p a t i e n t s to undergo b l a s t o g e n e s i s when 24 c u l t u r e d w i t h CEA. In addition,' s k i n t e s t i n g w i t h p u r i f i e d CEA was shown to be negative. Since elevated serum CEA l e v e l s occur i n p a t i e n t s w i t h c i r r h o s i s and v a r i o u s inflammatory c o n d i t i o n s and i n i n d i v i d u a l s who are heavy smokers, CEA cannot be considered as a marker of cancer. Do OFA act as t r a n s p l a n t a t i o n antigens?-Immunization w i t h f o e t a l t i s s u e has been shown to have some p r o t e c t i v e e f f e c t i n mice (Le Mevel et a l . , 1973) and guinea pigs (Grant et a l . , 1974) to subsequent tumour c e l l c h a l lenge, but p r o t e c t i o n was dependent on the number of tumour c e l l s which were used f o r the challenge. The higher the tumour dose, the higher the incidence of the tumour. However, animals immunized w i t h tumour c e l l s (TSTA) were protected at the high challenge dose. The p o s s i b i l i t y that embryonic antigens may e l i c i t weak t r a n s p l a n t -a t i o n responses analogous to those of weak h i s t o -c o m p a t i b i l i t y (non H-2) antigens r e q u i r e that an appropriate assay be used (Chism e_t a l . , 1978) . Other workers have observed enhanced tumour growth f o l l o w i n g f o e t a l immunization and the enhancing e f f e c t could be t r a n s f e r r e d to naive 25 animals by 0.2 ml of serum from f o e t a l primed animals (Goldberg et a l . , 1976). The l a c k of p r o t e c t i o n a t t r i b u t e d to OFA could be due to b l o c k i n g f a c t o r (see below), antibody or a n t i g e n -antibody complexes, which i n t e r f e r e w i t h expression of c e l l u l a r immunity (Baldwin et a l . , 1972) . This l a s t hypothesis was supported by the f i n d i n g that s o l u b l e antigen e x t r a c t e d from 10-13 day o l d embryonic t i s s u e abrogated the i n h i b i t o r y e f f e c t of lymph node c e l l s from multiparous pregnant mice on the i n v i t r o colony formation assay (Brawn, 1971). Thompson and Alexander (1973) us i n g immuno-fl u o r e s c e n t techniques, detected four tumour ass o c i a t e d macromolecules us i n g a n t i s e r a prepared against r a t sarcoma c e l l s , and embryonic e x t r a c t s . In a d d i t i o n to the p r i v a t e TSTA, three other antigens were detected. Two of these antigens d i d not e l i c i t humoral immunity i n the r a t . So one could conclude that OFA are d i s s i m i l a r to TSTA. The observations of the s t u d i e s of immunity to tumour as a consequence of pregnancy i s worth mentioning. Apart from the more negative r e s u l t s , there are some r e p o r t s showing the existence of of tumour t r a n s p l a n t a t i o n r e s i s t a n c e induced by 26 f o e t a l antigens (Winchester e_ a l . , 1975). Comparing tumour incidence i n post-partum r e t i r e d breeders r e l a t i v e to v i r g i n age matched female r a t s and guinea p i g s , Le Mevel et a l . (1973) found fewer tumours i n the breeders than i n the c o n t r o l group. The r e s i s t a n c e to tumour growth was shown by others (Chism et^ a l . , 1976) to d i m i n i s h as a f u n c t i o n of time a f t e r p a r t u r i t i o n , w i t h no evidence of secondary immune r e a c t i o n s when challenged at a l a t e r time w i t h tumour. SV-40 transformed c e l l s expressed a surface antigen which reacted w i t h serum obtained from pregnant females (Duff et a l . , 1970). This antibody q u i c k l y disappeared from the serum a f t e r p a r t u r i t i o n . P e r i p h e r a l lymphocytes from multiparous pregnant animals was shown to c o n t a i n c y t o t o x i c c e l l s which can destroy r a t carcinoma and a v a r i e t y of f o e t a l c e l l s but not normal a d u l t colon mucosa c e l l s ( S teele et_ a l . , 1974). In c o n t r a s t , the p e r i p h e r a l blood lymphocytes and lymph node c e l l s of r a t s bearing a colon cancer were c y t o t o x i c only to tumour c e l l s and f o e t a l colon c e l l s but not to other h i s t o l o g i c a l types. The i n v i t r o s t u d i e s of generating c y t o t o x i c c e l l s to syngeneic OFA u s i n g f o e t a l c e l l s have 27 shown promising r e s u l t s i n that a c t i v a t e d T c e l l s were capable of l y s i n g a v a r i e t y of tumour t a r g e t c e l l s ( Chism et a l . , 1975; Chism et a l . , 1976), or when mixed w i t h tumour c e l l s and i n j e c t e d i n t o the animal a c e r t a i n degree of p r o t e c t i o n was observed. In summary, OFAs are d i s t i n c t from TSTA and when i n j e c t e d i n t o an animal they can e l i c i t weak, n o n - s p e c i f i c r e s i s t a n c e . Antigens of V i r a l l y Induced Tumours The appearance of tumour v i r u s e s i n d i f f e r e n t species has already been discussed (see above, e t i o l o g y of tumours). Virus-induced tumours are c h a r a c t e r i z e d by the sharing of common antigens between d i f f e r e n t tumours induced by the same v i r u s , even i n d i f f e r e n t host species ( K l e i n et a l . , 1968). Two antigens are expressed i n the virus-tumour system, the new tumour-associated antigen(s) and the v i r a l antigen which i s c a r r i e d i n a n o n - r e p l i c a t i n g form and r a r e l y produces i n t a c t v i r u s ( A l l e n et_ a l . , 1972) and so both antigens are not present under the same c o n d i t i o n s . The s i t u a t i o n w i t h oncornaviruses i s more complex; the v i r u s production i s u s u a l l y c a r r i e d out contin u o u s l y by the i n f e c t e d and transformed c e l l , so that . v i r a l and tumour antigens are present simultaneously (Green et a l . , 1977). 28 RNA v i r u s e s that cause c e l l t ransformation are more common, but some DNA v i r u s e s are a l s o known to produce tumours. Of the DNA v i r u s e s , the one most thoroughly s t u d i e d i s the papovavirus SV-40 which i s oncogenic i n hamsters. Polyoma v i r u s i s another DNA v i r u s which can produce polyoma i n mice and hamsters among other species. These DNA v i r u s e s can induce neoantigens on the c e l l surface of the transformed c e l l s . These antigens evoke c e l l u l a r and/or humoral responses i n syngeneic hosts which are d e t e c t a b l e by t r a n s p l a n t a t i o n or s e r o l o g i c a l procedures. I n j e c t i o n of mice or hamsters w i t h polyoma v i r u s render these animals r e s i s t a n t to subsequent i n j e c t i o n w i t h polyoma tumour c e l l s (Sjogren et a l . , 1961). The same f i n d i n g s were shown w i t h SV-40. These tumour-specific t r a n s p l a n t a t i o n antigens (TSTA) have no r e l a t i o n s h i p to the V or capsid antigens of the corresponding v i r u s e s , since a n t i -v i r a l a n t i b o d i e s have no adverse e f f e c t on tumours. TSTA s o l u b i l i z e d w i t h 3M KC1 or papain treatment are a l s o immunogenic i n the syngeneic host (Drapkin e_ a l . , 1974). The surface antigen(s) found on SV-40 transformed c e l l s was d e t e c t a b l e n e i t h e r on normal c e l l s nor c e l l s transformed by v i r u s e s other than SV-40. There i s e v i d e n c e that S antigen and TSTA are not the same, si n c e there are SV-40 c e l l l i n e s found to be p o s i t i v e f o r surface antigen but l a c k TSTA (Tevethia et a l . , 1968). This was supported by c y t o t o x i c i t y s t u d i e s u s i n g hyperimmune mouse serum (Wright eit a l . , 1971). 29 Another DNA v i r u s which has been as s o c i a t e d w i t h B u r k i t t ' s lymphoman (BL) i n man i s the Epstein-Barr herpes v i r u s (EBV). Antigens associated w i t h BL are v i r a l capsid antigen (VCA) which i s thought to be i n t r a c e l l u l a r but p o s i t i v e sera were able to coat v i r a l l y i n f e c t e d c e l l s . This antigen appears l a t e i n the v i r u s c y c l e compared w i t h the e a r l y antigen (EA) which was found soon a f t e r i n j e c t i o n (Henle e_t a l . , 1970) . I t i s of i n t e r e s t to note that EBV i s a n t i g e n i t i c a l l y very s i m i l a r , i f not i d e n t i c a l , w i t h the agent that i s r e s p o n s i b l e f o r i n f e c t i o u s mononucleosis (Henle et^ a l . , 1970) . RNA-containing v i r u s e s , on the other hand have been recovered from inbred mice and ass o c i a t e d w i t h tumours. I m p l i c a t i o n s have been made that these v i r u s e s are a l s o associated w i t h some human n e o p l a s i a , eg., breast cancer, acute myelogenous leukemia, t r a n s i t i o n a l c e l l carcinoma of the ur o g e n i c a l t r a c t and sarcomas. Type-C RNA tumour v i r u s e s are the main oncogenic v i r u s e s that have been studied so f a r (Bauer, 1974). In mice, the antigens i n these tumours f a l l i n t o two c l a s s e s : (1) S t r u c t u r a l p r o t e i n s of the v i r u s p a r t i c l e s ; (2) C e l l u l a r a n t i g e n i c m a t e r i a l induced by the v i r u s . Group s p e c i f i c v i r u s envelope antigens (VEA) are common to a l l of the known murine C-type v i r u s r e g a r d l e s s of the type of tumour induced. Type s p e c i f i c (TS) v i r u s antigens are s p e c i f i c f o r c e r t a i n tumours w i t h i n a given species and may show subtypes f o r a given tumour, e.g. murine leukemia v i r u s e s 30 (MuLV) are d i v i s i b l e i n t o at l e a s t two major s e r o l o g i c a l groups, G (Gross) v i r u s and FMR ( F r i e n d , Moloney, Rauscher) (Aoki et a l . , 1974). The i n t e r n a l v i r a l antigen can a l s o be grouped i n t o two main a n t i g e n i c c l a s s e s , the i n t e r s p e c i e s group s p e c i f i c antigens which cross r e a c t among C-type p a r t i c l e s i s o l a t e d from d i f f e r e n t mammalian species (Geering e_t a l . , 1970) , and the group s p e c i f i c antigen ( G i l d e n , 1971). The enzyme reverse t r a n s c r i p t a s e (RT) was shown to be an i n t e r s p e c i e s antigen when checked by a n t i s e r a prepared to the enzyme from d i f f e r e n t species (Aaronson e_ a l . , 1971) Since the v i r u s production and malignant transformation take place i n the same c e l l , the search f o r the c e l l u l a r antigen i s complicated. However, i t has been noted that tumours can be induced w i t h C-type v i r u s of heterologous species w i t h the propagation of v i r u s . No t r u e l y v i r u s induced i n t r a c e l l u l a r antigens of these v i r u s e s have been found i n the mammalian system. That surface of the C-type RNA v i r u s transformed c e l l s c o n t a i n TSTA was shown f i r s t by K l e i n e_t a l . (1962) f o r murine leukemia v i r u s and f o r Rous sarcoma v i r u s (Sjogren e_t a l . , 1963) . Since there i s a continuous budding of the v i r u s , i t i s not. c l e a r whether these are t r u e new c e l l u l a r antigens or v i r a l components. To separate these i d e n t i t i e s , one has to have antiserum which w i l l bind to the transformed c e l l s and have no 31 s p e c i f i c i t y f o r the v i r u s i t s e l f . T his can be achieved through absorption of a n t i s e r a w i t h v i r u s p a r t i c l e s . There have been r e p o r t s about the presence of cytoplasmic group s p e c i f i c antigens of C-type RNA v i r u s i n c h e m i c a l l y induced tumours (Huebner et a l . , 1969; Whitmire et a l . , 1971). Whitmire e_t a l . (1971) have shown that chemical carcinogenesis could be i n h i b i t e d by f o r m a l i n - i n a c t i v a t e d p u r i f i e d C-type RNA v i r u s . Immunization w i t h Friend leukemia v i r u s (FLV) was a l s o shown to have p r o t e c t i v e e f f e c t against chemical carcinogeneis (Gericke et_ a l . , 1976). Whether t h i s p r o t e c t i o n i s n o n - s p e c i f i c l i k e the e f f e c t of BCG on methylcholanthrene carcinogenesis shown by L a r v i n et^ a l . (1973) , or due to the i n d u c t i o n of i n t e r f e r o n or i n t e r f e r o n s t i m u l a t i n g f a c t o r i s not known. However, RT could not be detected i n c h e m i c a l l y induced tumours. The p o s s i b l e o c c u l t involvement of RNA-virus as assessed by the presence of R T - l i k e a c t i v i t y or by e l e c t r o n microscopy i n human n e o p l a s i a e s p e c i a l l y leukemias was reported (Dmochowski et a l . , 1965; G a l l o et a l . , 1970; G a l l o , 1974). F i n a l l y , the common f a c t between ch e m i c a l l y induced or v i r a l l y induced tumours i s that TSTA of the transformed c e l l s are a l l thymus (T) dependant antigens. Resistance to TSTA can be abrogated e i t h e r by thymectomy, treatment w i t h a n t i -thymocyte serum or treatment w i t h a n t i 0 serum and complement (Tevethia et a l . , 1974). 32 Tumour Antigens: I s o l a t i o n and Assay I . I s o l a t i o n Since s i n g l e antigens may be handled d i f f e r e n t l y by the immune system when presented i n a s o l u b l e form r a t h e r than on i n t a c t c e l l s , i s o l a t i o n of tumour antigen(s) i s important f o r studying the i n v i v o s i g n i f i c a n c e of that antigen or f o r r a i s i n g an antiserum to monitor i t s i s o l a t i o n and s p e c i f i c i t y . Dore et a l . (1967) compared d i f f e r e n t techniques ( c y t o t o x i -c i t y , immunofluorescence, mixed c e l l a g g l u t i n a t i o n and complement f i x a t i o n ) and found evidence f o r a n t i b o d i e s i n the sera of p a t i e n t s w i t h d i f f e r e n t types of leukemias. In most i n s t a n c e s , only one of the four t e s t s y i e l d e d p o s i t i v e r e s u l t s . However, McCabe et a l . (1978;1979) observed comparable r e s u l t s u s i n g the same melanoma antigen which were taken from melanoma c e l l c u l t u r e medium and r a b b i t antiserum f o r the f o l l o w i n g three assays; complement mediated c y t o t o x i c i t y , radioimmunometric assay and delayed cutaneous h y p e r s e n s i t i v i t y r e a c t i o n . whether t h i s con-currence was due t o the same a n t i g e n i c components or due t o co-existence of two separable a n t i g e n i c e n t i t i e s i s not known. The l a c k of s p e c i f i c a n t i s e r a i n a d d i t i o n to the heterogeneity of the antigens are the reasons f o r the slow progress i n the evalua-t i o n of the neoantigens i n tumour systems. I t i s not f e a s i b l e to cover i n d e t a i l the methods which have been used f o r i s o l a t i o n , p u r i f i c a t i o n and assay of tumour ass o c i a t e d antigen i n t h i s t h e s i s . A b r i e f summary i s given here. 33 The methods for i s o l a t i o n of TAA can be placed i n t o three c a t e g o r i e s ; mechanical, chemical and enzymatic. A n t i g e n i c membrane fragments have been prepared by mechanical rupture of the c e l l s by s o n i c a t i o n followed by zonal c e n t r i f u g a -t i o n through sucrose gradient ( P r i c e et a l . , 1974; P r i c e et a l . , 1974) . S o n i c a t i o n s o l u b i l i z e s membrane associated p r o t e i n by depolymerization of the membrane components w i t h a low recovery. Intraplasmic c a v i t a t i o n by n i t r o g e n gas has a l s o been used to prepare membrane fragments of v a r y i n g s i z e s as checked by t h e i r c a p a c i t y to n e u t r a l i z e s p e c i f i c a l l y of antiserum prepared i n syngeneic mice against i n t a c t c e l l s . A major chemical method used f o r s o l u b i l i z a t i o n of TAA i s hypertonic s a l t e x t r a c t i o n . The most widely used i s 3 M KC1 which was used o r i g i n a l l y to s o l u b i l i z e h i s t o c o m p a t i b i l i t y antigens ( R e i s f e l d et a l . , 1971; Lebien et a l . , 1979). I t i s b e l i e v e d that hypertonic s a l t a l t e r s the e l e c t r o s t a t i c i n t e r a c t i o n i n macromolecules, r e a c t s w i t h d i p o l a r groups and e f f e c t the hydro-phobic area of p r o t e i n , thus r e l e a s i n g the antigens from the plasma membrane. I t i s a l s o b e l i e v e d that high s a l t c o n c e n t r a t i o n makes p r o t e o l y t i c enzymes more a c t i v e r e s u l t i n g i n the r e l e a s e of the antigen by an a u t o l y t i c a c t i o n (Mann, 1972). Non-ionic detergents l i k e Nonidet-P40 (NP-40), B r i j 99 and sodium deoxycholate (DOC) have been used (Springer et a l . , 1974; N a t o r i et al_. , 1977; Clemetson, 1976) to s o l u b i l i z e membrane antigens w i t h high y i e l d and i n the undegraded form. The detergent 34 removes the a n t i g e n i c molecules from the plasma membrane's l i p i d m i l i e u . The major problem i n these methods i s the removal of the detergent w h i l e maintaining the antigen i n a water-soluble s t a t e . Soluble membrane molecules can be produced w i t h low recovery by papain enzyme treatment of the i n t a c t c e l l s (Hess e_ a l . , 1974). Since p r o t e o l y t i c d i g e s t i o n fragments the a n t i g e n i c molecules,the s i z e of these molecules i s always lower than that i s o l a t e d by detergent e x t r a c t i o n (Nathenson e_t a l . , 1972) . Enzymes such as neuraminidase has been shown to reduce the t r a n s p l a n t a b i l i t y of tumours l i k e L1210 leukemia (Bekesi e_ a l . , 1972). This enzyme i s thought to remove s i a l i c a c i d which i s considered the major masking component of tumour associated antigens at the c e l l s urface (van Beck e_ a l . , 1973). Several i n v e s t i g a t o r s have attempted to i s o l a t e more uniform products of the membrane antigens from p a t i e n t s ' plasma or from the c u l t u r e f l u i d from c e l l s grown i n v i t r o under the assumption that the c e l l sheds membrane products i n t o the medium ( P e l l i g r i n o et a l . , 1973) . A f t e r a p p l y i n g e i t h e r p h y s i c a l , chemical or enzymatic means of e x t r a c t i o n , f u r t h e r enrichment and p u r i f i c a t i o n can be accomplished by g e l f i l t r a t i o n , i o n exchange chromatography, immunoadsorbent columns, a f f i n i t y chromatography columns l i k e l e c t i n columns, i s o e l e c t r i c f o c u s i n g , immunoprecipitation, p o l y -acrylamide g e l e l e c t r o p h o r e s i s and Staphylococcus aureus p r o t e i n A column (SPA). SPA i s a surface p r o t e i n c o v a l e n t l y l i n k e d to the peptidoglycan of the b a c t e r i a ( S j o q u i s t et a l . , 1972). I t i s known that p r o t e i n A binds the Fc fragment of immunoglobulin (Ig) w i t h 35 the b i n d i n g s i t e being expressed on only c e r t a i n Ig c l a s s e s . On the b a s i s of t h i s property, SPA can be used to detect a n t i -bodies r e a c t i n g w i t h surface antigens of tumour c e l l s (McCabe et a l . , 1979). I I . Antigen Assays These t e s t s may be c a r r i e d out i n v i v o and may measure c e l l u l a r immunity or antibody. A. In v i v o c e l l u l a r immunity Tumour r e j e c t i o n i s the main assay to show that immunized animals are more r e s i s t a n t than the untreated c o n t r o l . This subject has been d e a l t w i t h elsewhere (see page 11). I t i s of i n t e r e s t to note that attempts made to induce tumour r e j e c t i o n i n v i v o w i t h s o l u b l e e x t r a c t s has met w i t h only l i m i t e d success ( P e l l i s et a l . , 1975; Law et a l . , 1975). This i s probably dependant on the antigen p r e p a r a t i o n . Moreover, c e l l u l a r c y t o t o x i c i t y has been d i f f i c u l t to demonstrate i n v i t r o i n many animal models. Since g r a f t r e j e c t i o n i s associated w i t h lymphocyte i n f i l t r a t i o n , c e l l u l a r immune response induced by some antigens i n v i v o can be detected by s k i n r e a c t i o n to intradermal i n j e c t i o n of antigen. Delayed cutaneous h y p e r s e n s i t i v i t y (DCHR) (24-48 h) i s a r e f l e c t i o n of c e l l u l a r immunity. Host c e l l s i n t e r a c t s w i t h the antigen and r e l e a s e s o l u b l e f a c t o r s which cause the inflammatory c e l l s to accumulate i n the r e g i o n . This method i s widely used i n human p a t i e n t s w i t h n e o p l a s i a to assess c e l l u l a r immunity. Some i n v e s t i g a t o r s b e l i e v e that 36 t h i s method i s the most u s e f u l technique i n monitoring antigen p u r i f i c a t i o n (Roth et a l . , 1976). There are some problems associated w i t h t h i s assay, 1) the amount of m a t e r i a l used i s c r i t i c a l s ince the i n j e c t i o n of too much p r o t e i n , even of c o n t r o l m a t e r i a l , may provoke f a l s e p o s i t i v e s ; 2) the general immune c a p a c i t y of tumour bearing animals or p a t i e n t s w i t h advanced cancer i s f r e q u e n t l y n o n s p e c i f i c a l l y impaired. This i s r e f l e c t e d by t h e i r i n a b i l i t y to respond i n s k i n t e s t s not only to tumour antigens but als o to other common s k i n t e s t antigens such as PPD, mumps, and st r e p t o k i n a s e . B. C e l l u l a r Immunity In v i t r o C e l l u l a r c y t o t o x i c i t y - Many methods have been developed to demonstrate c e l l u l a r c y t o t o x i c i t y i n v i t r o . Of these, the "'"'"Chromium r e l e a s e assay and the colony i n h i b i t i o n assay are most f r e q u e n t l y used. B r i e f l y , these assays depend on r a i s i n g c y t o t o x i c c e l l s by exposing spleen or other T c e l l c o n t a i n i n g population to i r r a d i a t e d syngeneic tumour c e l l s c a r r y i n g neoantigen or to a l l o g e n e i c c e l l s . These c e l l s , can then recognize the f o r e i g n membrane antigen on the l a b e l e d target c e l l s r e s u l t i n g i n l y s i s of those c e l l s and the re l e a s e of r a d i o a c t i v e isotope i n t o the medium. The problem w i t h t h i s assay i s that there are n o n - s p e c i f i c c y t o t o x i c c e l l s (non-immune lymphocytes) which are c a l l e d n a t u r a l k i l l e r s (NK). Whether these c e l l s are t r u l y non s p e c i f i c c y t o t o x i c c e l l s 37 or represent some expression of previous exposure to tumour r e l a t e d antigens need to be explored. The nature of the antigen(s) on t a r g e t c e l l s to which NK c e l l s r e a c t s i s a l s o not known. Moreover, some antigens may be incorporated and manifested i n the c e l l membrane upon growth i n heterologous serum as has been demonstrated i n human sarcoma when grown i n FCS ( I r i e et a l . , 1974). Lymphocyte s t i m u l a t i o n (blastogenesis) i s another assay to measure the a b i l i t y of a population of lymphocytes to recognize an antigen and undergo b l a s t o g e n e s i s monitored by t r i t i a t e d thymidine uptake. This method has a l s o been used to measure c i r c u l a t i n g antigen from serum of tumour bearing animals (Burk et^ a l . , 1975). The d i s t i n c t i o n between the response of lymphocytes to n o n - s p e c i f i c mitogen or mixed lymphocyte c u l t u r e s (MLC) and that to s o l u b l e tumour antigen or PPD i s that responsiveness to tumour antigen and PPD re q u i r e s p r e s e n s i t i z a t i o n . Many l i m i t a t i o n s to t h i s assay have been observed which make lymphocyte s t i m u l a t i o n not the best assay to check f o r TAA. With t h i s assay, i t i s d i f f i c u l t to d i s t i n g u i s h between TAA from other neoantigens. B l a s t o -genic f a c t o r i s another problem. The responses evoked by f r e s h autochthonous lymphoid c e l l s r a r e l y achieve the same order of magnitude as those induced by c u l t u r e d c e l l s , which again i n d i c a t e s that a l t e r e d a n t i g e n i c i t y may be an i n v i t r o acquired property of the c e l l s (Gutterman et_ aL., 1972). However, p o s i t i v e r e s u l t s have been reported showing that 38 b l a s t o g e n e s i s can be used to study CMI to tumour. Leukemia c e l l s can s t i m u l a t e lymphocytes derived from the autochthonous host (Friedman et a l . , 1969), and from i d e n t i c a l twins (Bach et a l . , 1969). P o s i t i v e b a l s t o g e n e s i s was shown to c o r r e l a t e w i t h greater immunocompetence and more fav o r a b l e prognosis. F i n a l l y , s t u d i e s on murine sarcomas us i n g b l a s t o g e n e s i s and chromium r e l e a s e assays to measure responsive c e l l s i n the spleens showed that these two assays measure d i f f e r e n t c e l l u l a r subpopulations (Senik et a l . , 1975). I n h i b i t i o n assays - I n t e r a c t i o n between s e n s i t i z e d lymphocytes (T c e l l s ) and the corresponding antigen leads to the r e l e a s e of a b i o l o g i c a l l y s o l u b l e substances known as lymphokines such as m i g r a t i o n i n h i b i t i o n f a c t o r (MIF). These substances have been shown to i n h i b i t m i g r a t i o n of normal macrophages. This phenomenon i s used to measure the r e l e a s e of t h i s s o l u b l e i n h i b i t o r i n an assay c a l l e d m i g r a t i o n i n h i b i t i o n of macrophages. Leukocyte m i g r a t i o n i n h i b i t i o n (LMI) i s a l s o mediated by lymphokines, but i t s r e l a t i o n to macrophage i n h i b i t i o n f a c t o r i s unknown. LMI assay i s widely used i n s t u d i e s of measuring c e l l u l a r immunity i n cancer p a t i e n t s by u s i n g tumour e x t r a c t s . The a b i l i t y of tumour e x t r a c t s to i n h i b i t m i g r a t i o n was shown to d i m i n i s h w i t h advancing d i s e a s e , which means the production of lymphokines by s e n s i t i z e d lymphocytes i s dependant on the stage of the tumour. LMI was a l s o 39 depressed i n p a t i e n t s d u r i n g the immediate post-operative period and c o r r e l a t e d very w e l l w i t h DCHR, mitogen respon-siveness and c y t o t o x i c i t y (Cochran et a l . , 1972). Another assay i s the leukocyte adherence i n h i b i t i o n (LAI). The adherence of normal p e r i t o n e a l leukocytes to glass surface appears to be measurably reduced when the c e l l s are recovered from animals s e n s i t i z e d to tumour antigens and s o l u b l e antigen i s present i n the c u l t u r e (Fujisawa e_t al_. , 1978). L i k e other t e s t s , m i g r a t i o n assays have some drawbacks. Of these i s the non-immunological f a c t o r s i n the crude t i s s u e e x t r a c t s which may cause m i g r a t i o n i n h i b i t i o n and a l s o the cross r e a c t i v i t y among tumours of s i m i l a r h i s t o g e n i c d e r i v a -t i o n e s p e c i a l l y i n melanoma. C. In v i t r o Antibody Assays Immunity to tumour as s o c i a t e d antigen can be checked by the antibody response to those antigens. Antigen-antibody i n t e r a c t i o n can be v i s u a l i z e d by d i r e c t or i n d i r e c t immuno- f l u o r e s c e n t techniques (Minowada e_t a l . , 1978; I r i e et_ a l . , 1979) . The p r i n c i p l e of t h i s method i s the use of f l u o r e s c e i n or rhodamine l a b e l l e d a n t i b o d i e s which would bind to the antigen and then can be seen by the emission of the greenish or r e d d i s h colour under the f l u o r e s c e n t microscope. This method has been used s u c c e s s f u l l y as an i n d i c a t o r f o r the p u r i f i c a t i o n of s o l u b l e antigen from a r a t hepatoma (Baldwin et a l . , 1973). 40 I - l a b e l e d a n t i g l o b u l i n i s a s e n s i t i v e method to q u a n t i t a t e TAA. This can be monitored by counting the d i s -i n t e g r a t i o n of the r a d i o l a b e l e d a n t i g l o b u l i n which has bound to tumour s p e c i f i c antibody. Immunoelectron microscopy has been used to determine the c e l l surface l o c a l i z a t i o n and d i s t r i b u t i o n of antigen (Aoki et a l . , 1970). The radioimmunoassay (RIA) i s a commonly used assay to determine AFP and CEA antigens ( S i l v e r e_ a l . , 1973; Egan et a l . , 1974). In t h i s assay a standard r a d i o l a b e l l e d antigen i s used to compete w i t h the unknown q u a n t i t y of a n t i g e n f o r the l i m i t e d number of antigen b i n d i n g s i t e s present i n the added antiserum. The bound and the unbound antigens are separated and counted to determine the amount of l a b e l l e d bound antigen. The amount of the antigen present i n the unknown can be obtained by i n t e r p o l a t i o n on the standard curve. A very s e n s i t i v e and q u a n t i t a t i v e assay to check the s p e c i f i c i t y of an antiserum to TAA i s the enzyme l i n k e d immunosorbent assay (ELISA) ( E n g v a l l e_ a l . , 1971) (see page 125). This assay i s based on the same p r i n c i p l e s as RIA but an enzyme i s used as a marker f o r the antigen or antibody i n s t e a d of r a d i o a c t i v e i s o t o p e s . The advantage w i t h ELISA i s that i t i s f a s t e r , the enzymes are more s t a b l e than the isotopes used i n RIA, i n a d d i t i o n to the hazard 41 inherent i n the use of r a d i o a c t i v e m a t e r i a l . Recently, a method described by R e i s f e l d and h i s as s o c i a t e s (McCabe e_t a l . , 1979) c a l l e d radioimmunometric  assay based on the a p p l i c a t i o n of Staphylococcus aureus p r o t e i n A (SPA) to evaluate xenoantisera to tumour associated antigens. B r i e f l y , the antiserum was allowed to bind to the tar g e t c e l l s f o r 30 min. at 4°C and washed t w i c e , the p e l l e t 125 125 was incubated w i t h I l a b e l l e d SPA. Free I SPA was removed and the p e l l e t was washed twice and counted. Using appropriate c o n t r o l , s p e c i f i c i t y of the antiserum can be judged. Another s e n s i t i v e assay to measure q u a n t i t a t i v e l y tumour antigens i n a s o l u b l e e x t r a c t p r o v i d i n g the a v a i l a b i l i t y of antigen s p e c i f i c antibody i s the complement f i x a t i o n assay (see page 63 ). Complement mediated antibody dependant c y t o t o x i c i t y has been used e x t e n s i v e l y to determine the humoral immune response to TAA. This method i s based on the bi n d i n g of antibody to the s p e c i f i c antigen on the tumour c e l l s followed by b i n d i n g complement to the complement receptor on the Fc p o r t i o n of the antibody w i t h the subsequent a c t i v a t i o n of complement and l y s i s of the t a r g e t c e l l s . The degree of l y s i s can be checked v i s u a l l y under the microscope with the trypan blue e x c l u s i o n method or through the r e l e a s e of the isotopes when r a d i o -l a b e l l e d , u s u a l l y "^Cr, t a r g e t s are used ( f o r d e t a i l see page ). V i r a l l y induced tumours and tumours grown as a s c i t e s are more 42 s u s c e p t i b l e to the antibody and complement mediated k i l l i n g i n comparison to the more d i f f i c u l t to l y s e , s o l i d tumours ( D i s c i u l l o et a l . , 1977). However, t h i s method has been used to detect antigens on MC-induced sarcomas i n mice (Cleveland et a l . , 1974) . Examination of s e r o l o g i c a l data from s t u d i e s on TAA d i s c l o s e s c o n s i d e r a b l e v a r i a t i o n s . Some of the f a c t o r s are a t t r i b u t e d to methodological d i f f e r e n c e s as w e l l as others. The presence of extraneous m a t e r i a l or the source of comple-ment can i n t e r f e r e w i t h these assays. The a f f i n i t y of a n t i -body i s very important. This could be overcome by manipula-t i n g the time allowed f o r the antigen-antibody i n t e r a c t i o n . Exogeneous antigens expressed on the tumour c e l l s are a d i f f i c u l t problem. This i s tr u e when t i s s u e c u l t u r e conta-minated w i t h mycoplasma i s used to r a i s e anti-tumour antibody. Moreover, s i n c e cancer p a t i e n t s are more prone to i n f e c t i o n s , i t i s o f t e n that a n o n - s p e c i f i c antibody have been found i n the sera of those p a t i e n t s which r e a c t s w i t h the tumour c e l l s (Castro et a l . , 1978). In a d d i t i o n , sera from p a t i e n t s may c o n t a i n autoantibody, a n t i HL-A or anti-ABO blood group due to blood t r a n s f u s i o n . 43 I f A Tumour I s A n t i g e n i c , Why Is I t Not Rejected By The Host? I t has been shown that most tumours are a n t i g e n i c and can s t i m u l a t e the immune response, but y e t , tumours normally grown p r o g r e s s i v e l y and f i n a l l y k i l l the host. What happens to the s p e c i f i c immune response such as CTL, humoral antibody and the n o n - s p e c i f i c defence mechanisms l i k e macrophages, polymorphonuclear c e l l s , NK c e l l s and p l a t e l e t s ? One p o s s i b i l i t y i s that the host may be immunologically incompetent, permanently or t e m p o r a r i l y at the time the tumor develops. The host may not recognize i t as being a n t i g e n i c or i t does not have the a b i l i t y to r e j e c t i t because of i t s l a t e a c t i o n as w e l l as many other p o s s i b l e mechanisms which w i l l be discussed below. I . Immunosurveillance (IS) The concept of immunological s u r v e i l l a n c e (IS) has been put i n v a r i e t y of ways, most r e c e n t l y by Burnet (1970) who p o s t u l a t e s that there i s a mechanism, probably mediated by c i r c u l a t i n g lymphoid c e l l s whereby a host can recognize the a n t i g e n i c i t y of the tumour c e l l s and destroy those c e l l s which are presumably a r i s i n g by mutational events at a l l times. A s u c c e s s f u l tumour mass i s thus seen as the mutant c e l l s that escape I S , such as when there i s a def e c t i n the s u r v e r i l l a n c e system. So, the r o l e of IS i s not to mediate the r e g r e s s i o n of e s t a b l i s h e d tumours but r a t h e r to seek out and destroy c l i n i c a l l y unrecognized i n s i t u tumours (Burnet, 1970). The a s s o c i a t i o n of immunologic d e f i c i e n c y w i t h increased r i s k f o r tumour development has been reported (Waldmann et a l . , 1972). 44 The theory of IS was supported by the observation that immuno-suppression of r e c i p i e n t s w i t h kidney t r a n s p l a n t s r e s u l t e d i n a higher than normal incidence of cancer. Furthermore, aged people are r e l a t i v e l y l e s s immunocompetent than young people, p a r t i c u l a r l y w i t h regard to T c e l l s mediated immunity, and by and l a r g e cancer i s a disease of o l d e r people. However, cancers i n these p a t i e n t s i s r e s t r i c t e d to some extent to lymphoid o r i g i n and the frequency i s even higher i n p a t i e n t s who are c o n g e n i t a l l y T c e l l d e f i c i e n t . There i s no increase of other tumours l i k e mammary carcinoma which i s one of the most common human tumours i n immunodeficient p a t i e n t s . A s l i g h t increase i n the incidence of s k i n and c e r v i x cancer has been reported i n immunodeficient p a t i e n t s (Moller et  a l . , 1976). These l a s t tumours are suspected to be of v i r a l o r i g i n and t h i s emphasizes one p o i n t ; namely, that the f u n c t i o n of the T c e l l system i s to counteract v i r u s i n f e c t i o n s . The concept of IS would have suggested that immunologically p r i v i l e g e d s i t e s , a number of anatomical l o c a t i o n s where the T c e l l system cannot e f f e c i e n t l y operate, should have a n a t u r a l l y high incidence of tumours or that i t would be r e l a t i v e l y easy to induce tumours i n such s i t e s . One of these i s the a n t e r i o r pouch of the eye, the cheek pouch of the hamster, e t c . However, spontaneous tumours i n these l o c a t i o n s are r a r e . Another important point to consider regarding IS i s the mono-c l o n a l i t y of tumours. One should p r e d i c t that once the T c e l l system f a i l s m u l t i p l e and p o l y c l o n a l tumours should appear. This 45 i s not the case as shown from s t u d i e s on myelomas i n humans and animals. Fahy and P o t t e r (1959) showed that a l l the immuno-g l o b u l i n s belong to the same c l a s s and they have i d e n t i c a l v a r i a b l e regions i n d i c a t i n g that myelomas were derived from one s i n g l e c e l l (Friedman et a l . , 1976; P o t t e r et a l . , 1973). Monoclonal o r i g i n i s not r e s t r i c t e d to lymphoid neoplasm. The isozyme glucose-6-phosphate dehydrogenase (G-6-PD) e x s i t s i n a l l e l i c forms and i s X - l i n k e d ; t h e r e f o r e , heterozygous women w i l l express one of the two a l l e l e s . Studies on t h i s isozyme to show whether tumours express one or both a l l e l e s have supported the monoclonality of a l l tumours studied (Friedman et a l . , 1976). IS argues that a n t i g e n i c i t y of tumour c e l l s i s the r u l e . Carbone and Parmiani (1975) observed that the presence or absence of antigens i n sarcomas induced by chemical carcinogens w i t h i n d i f f u s i o n chambers was r e l a t e d to c e l l c y c l e stage. Non-immunogenic sarcomas are induced by treatment of r e p l i c a t i n g c e l l populations w i t h 3-MC,while immunogenic tumours a r i s e when c e l l s are t r e a t e d d u r ing the r e s t i n g phase of growth. On the other hand," p r o l i f e r a t i n g c e l l s are more s u s c e p t i b l e to c h e m i c a l l y induced t r a n s f o r m a t i o n than t h e i r r e s t i n g counterparts (Marguardt, 1974). The p o s s i b i l i t y that immunological defenses would be e f f e c t i v e against a very small inoculum of i n s i t u tumour and not e f f e c t i v e a g ainst l a r g e r t r a n s p l a n t s i s not always the case as shown by many i n v e s t i g a t o r s . Bonmassar et a l . (1974) observed that extremely small inoculum of h i g h l y immunogenic tumours may grow upon syngeneic t r a n s p l a n t a t i o n or even i n some a l l o g e n e i c systems. 46 This phenomenon is called "sneaking through" (Potter e_ a l . , 1969). Another phenomenon, "immune stimulation" or counter surveil-lance, which states that weak immune reaction in spontaneous tumours, rather than inhibiting tumour growth, may actually stimulate i t was introduced by Prehn (1971). The existence of this phenomenon has been demonstrated both in vivo and in vitro in a variety of systems in a number of laboratories (Kail et a l . , 1975; Bray et a l . , 1975). The results of different immunosuppressive procedures, using antilymphocyte serum (ALS), thymectomy etc., on tumour develop-ment are contradictory. While some experiments indeed show increased susceptibility to chemical oncogenesis but not to spontaneous tumours (Schwartz, 1975), the majority of the experiments give results that do not f i t with the surveillance interpretation. This could be due to deficiency in suppressor T cells or due to immune stimulation. Viral oncogenesis is markedly potentiated. There i s , however, a strong indication that the T c e l l system specifically recognizes virus induced alterations of histocompatibility antigens (Doherty e_ a l . , 1974). The congenitally athymic (nude) mouse should offer a definite test to the role of IS. Nevertheless, the observations with nude mice indicate that the incidence of tumour development after exposure to chemical carcinogens is comparable with that of normal mice. However, lymphoreticular tumours in old germ-free nude mice have 47 been shown to exceed that of the normal (Outzen e_t a l . , 1975) . No other spontaneous tumours have been seen i n those mice. An a l t e r n a t i v e i n t e r p r e t a t i o n of the r e s u l t s i n nude mice i s that antitumour s u r v e i l l a n c e i s exerted by the thymus independent immune system. This i n t e r p r e t a t i o n i s supported by the demon-s t r a t i o n of n a t u r a l c y t o t o x i c c e l l s against syngeneic and a l l o -geneic tumour c e l l s i n v i t r o i n nude mice ( K i e s s l i n g et_ a l . , 1975) and by the l a t e appearance of immune r e s i s t a n c e against polyoma oncogenesis i n nude mice (Stutman, 1975). N a t u r a l a n t i b o d i e s capable of l y s i n g . tumour c e l l s i n the presence of complement have been detected i n nude mice (Martin e_t al_. , 1974) . I t i s of i n t e r e s t to note that some immunologists proposed macrophages as a mediators of s u r v e i l l a n c e r a t h e r than the more s p e c i f i c a l l y a c t i v e lymphocytes. Alexander (1971) has shown that macrophages can k i l l tumour c e l l s . The c a p a c i t y of macrophages to k i l l can be tumour s p e c i f i c and the s p e c i f i c i t y a r i s i n g from e i t h e r antibody or lymphocytes stimulated by tumour antigens. This i n t e r a c t i o n i s c a l l e d s p e c i f i c macrophage arming f a c t o r . A l s o , p e r s i s t e n t i n f e c t i o n makes l a b o r a t o r y animals r e s i s t a n t to cancer and the macrophages of such animals are t u m o u r i c i d a l i n v i t r o (Hibbs et a l . , 1972). Moreover, Syderman et a l . (1976) found an i n h i b i t o r of macrophage chemotaxis produced by v a r i o u s t r a n s p l a n t e d neoplasm i n the p e r i t o n e a l c a v i t y of mice. From these r e s u l t s i t can be argued that macrophages are a c t i n g as a s e l e c t i v e agent against tumours. 48 B l o c k i n g Factor and Immune Enhancement Another phenomenon which may help to e x p l a i n why a tumour, once i n i t i a t e d , r e g u l a r l y progresses to k i l l the host i s c a l l e d "immunological enhancement" ( I E ) . IE may be mediated by the antibody which would combine w i t h the antigen on the tumour c e l l s and prevent them from f u r t h e r immunizing the host, or b i n d i n g these antigens i n such a way that they are masked and not detected by the host immune T c e l l s , or the antigens might compete i n b i n d i n g w i t h e f f e c t o r lymphocytes and f i n a l l y , immune complexes may exert an i n h i b i t o r y e f f e c t on the c e l l u l a r immune response of the host (Sjogren et a l . , 1971). Enhancement has a l s o been seen i n human ne o p l a s i a . H e l l s t r o m e_ a l . (1974) reported the presence of b l o c k i n g f a c t o r i n serum of human p a t i e n t s which antagonizes lymphocyte mediated c y t o t o x i c i t y i n v i t r o . The evidence that BF i s an antibody i s that the a c t i v i t y of the BF i n a serum taken from mice w i t h progressing tumours-can be n e u t r a l i z e d w i t h tumour c e l l s or t h e i r e x t r a c t s , or w i t h the oncogenic v i r u s used to induce that tumour, or by goat anti-mouse Ig (Hellstro m et a l . , 1969). However, s e v e r a l r e s u l t s i n experimental animals and the human tumours are d i f f i c u l t to i n t e r p r e t on the premise that antibody alone represents the only BF. BF e l u t e d d i r e c t l y from specimens of tumours by a c i d treatment was shown to co n t a i n high and low molecular weight f r a c t i o n s by g e l f i l t r a t i o n . These f r a c t i o n s i n d i v i d u a l l y lacked i n b l o c k i n g a c t i v i t y , but the a c t i v i t y was r e s t o r e d on recombination of i n a c t i v e f r a c t i o n s 49 (Sjogren et a l . , 1972; Tamerius et^ a l _ . , 1976). I t i s f e a s i b l e that a c i d treatment of tumour t i s s u e would l i b e r a t e membrane associated antigens as w e l l as antigen-antibody complexes. Papain s o l u b i l i z e d as w e l l as 3 M KC1 e x t r a c t s of tumour antigens have proved to be e f f e c t i v e i n i n h i b i t i n g c e l l mediated c y t o t o x i c i t y i n v i t r o (Embleton et_ a l _ . , 1975; P l a t a et a l . , 1974). The nature of i n t e r a c t i o n between s o l u b l e tumour antigen and s e n s i t i z e d lymphocyte i s not known. Recently, Levy et a l . (1975) have shown that s o l u b l e tumour e x t r a c t s induce the generation of tumour s p e c i f i c suppressor T c e l l s . The evidence suggesting the presence of BF i s convincing. Tumours can be protected from d e s t r u c t i o n by T lymphocytes and macrophages by feedback i n h i b i t i o n provided by the antibody and tumour antigen. Factors which can abrogate the e f f e c t of BF have been described. This i s c a l l e d t r a n s f e r f a c t o r (TF) or unblocking f a c t o r . I t i s simply a leukocyte l y s a t e from the tumour bearing animal or human p a t i e n t s (Fujisawa et a l . , 1978). I I I . Immune Suppression (Suppressor T C e l l s ) Since the e a r l y experiments showing suppressive e f f e c t s of immune T c e l l s on the helper a c t i v i t y of primed T c e l l s (Gershon et a l . , 1971) , many s t u d i e s have reported s p e c i f i c and n o n - s p e c i f i c suppressive e f f e c t s . Many types of c e l l s have been i n c r i m i n a t e d f o r t a k i n g part i n immune suppression during the course of tumour growth. Gorczynski et a l . (1975) have shown that 50 B lymphocytes were r e s p o n s i b l e f o r the i n h i b i t i o n of anti-tumour c y t o t o x i c i t y . Monocytes and macrophage s e r i e s were al s o found to i n h i b i t DNA synth e s i s of T lymphocytes i n the r e g u l a t i o n of the immune response. Immune suppression has been a t t r i b u t e d to them. Loss of tumour immunity i n the progressive tumour has been shown to be due to the development of tumour s p e c i f i c T suppressor c e l l s (Takei e_ a l . , 1976; Takei et a l . , 1977). In mice, the suppressor c e l l s appear f i r s t i n the thymus a f t e r tumour i n d u c t i o n (Takei et a l . , 1978) . Immune suppression by T suppressor c e l l s has r e c e n t l y been shown to i n t e r f e r e w i t h the e f f e c t o r c y t o t o x i c r e a c t i v i t y i n humans (Yu et_ a l . , 1974). P a t i e n t s w i t h advanced cancer have an extensive a c t i v i t y of T suppressor c e l l s capable of i n h i b i t i n g lymphocyte b l a s t o g e n e s i s (Smith et a l . , 1974). Suppressor lymphokines produced by T c e l l s or other types of c e l l s that i n h i b i t both humoral and c e l l u l a r immune response have been demonstrated. I n h i b i t i o n of B c e l l humoral antibody synt h e s i s e i t h e r d i r e c t l y or through the i n h i b i t i o n of a s p e c i f i c helper mechanism have been reported. A l s o , s o l u b l e supernates of sonicated thymocytes s p e c i f i c a l l y suppress the i n v i t r o c y t o t o x i c response (Takei et a l . , 1978). Moreover, a c t i v a t i o n of T suppressor c e l l s can be induced by s o l u b l e immune complexes (Levy et_ a l . , 1979) and s o l u b l e tumour associated antigens (Gershon et a l . , 1971). I t seems l i k e l y that s i n c e suppressor c e l l s appear during tumour growth they play a r o l e i n the response to tumour. Since 51 t h e l a r g e a n t i g e n i c l o a d o f a r a p i d l y g r o w i n g tumour f a v o u r s s u p p r e s s o r c e l l d e v e l o p m e n t , r e m o v a l o f s u p p r e s s o r c e l l s m i g h t be a u s e f u l t h e r a p e u t i c m e a s u r e . O b j e c t i v e s o f T h i s S t u d y The g o a l o f t h i s r e s e a r c h was t o r a i s e tumour s p e c i f i c a n t i b o d y w h i c h c a n be u s e d t o m o n i t o r t h e p u r i f i c a t i o n o f TAA. S i n c e TAA have been d e m o n s t r a t e d by t r a n s p l a n t a t i o n and s e r o l o g i c a l t e c h n i q u e s , t h e e x t r a c t i o n and p u r i f i c a t i o n o f t h e s e m o l e c u l e s h a v e become o f g e n e r a l i m p o r t a n c e . The r e w a r d s f o r i s o l a t i o n and p u r i f i c a t i o n o f tumour a n t i g e n s may be c o n s i d e r -a b l e . They i n c l u d e ; (1) a b e t t e r u n d e r s t a n d i n g o f how tumour a n t i g e n s a r e a s s o c i a t e d w i t h o t h e r n o r m a l s u r f a c e o r c e l l u l a r c o m p o n e n t s , (2) t h e r e l a t i o n s h i p o f t h e tumour a n t i g e n s t o n o r m a l t r a n s p l a n t a t i o n a n t i g e n s , (3) t h e mechan ism by w h i c h i m m u n i z a t i o n t a k e s p l a c e i n v i v o , (4) t h e s i g n i f i c a n c e o f f r e e a n t i g e n o r a n t i g e n - a n t i b o d y c o m p l e x e s i n t h e c i r c u l a t i o n , (5) t h e u s e o f p u r i f i e d a n t i g e n f o r d e v e l o p m e n t o f s e n s i t i v e a s s a y s t o d i a g n o s e and m o n i t o r t h e g r o w t h o f t u m o u r s , and (6) t h e p o t e n t i a l u s e o f s p e c i f i c a n t i g e n f o r immuno the rapy . These s t u d i e s w i l l c o n t i n u e t o depend h e a v i l y on d e v e l o p m e n t s i n two o t h e r a r e a s : ou r g e n e r a l u n d e r s t a n d i n g o f t h e immune r e s p o n s e and t h e advancement o f t e c h n i q u e s i n immunology and b i o c h e m i s t r y . 52 CHAPTER II PREPARATION OF XENOANTISERUM SPECIFIC FOR A MURINE MASTOCYTOMA CELL LINE Abstract A method called feedback inhibition by which the animals were rendered non-responsive to normal components of the tumour cells is described. Using this method, a xenoantiserum directed toward membrane components of DBA/2 murine mastocytoma P815 cells was raised. This antiserum was found to be specific for tumour c e l l membrane extracts and had no reactivity with comparable extracts of normal cells when tested by complement fixation in the presence of guinea pig complement, and had no reactivity with another DBA/2 syngeneic tumour, L1210 leukemia cells or concanavalin A (Con A) blasts of a variety of normal DBA/2J c e l l preparations. When mixed with varying numbers of tumour cells and injected intraperitoneally into either DBA/2 or B6D2F1 mice, the antiserum demonstrated a protective effect by either prolonging survival time or when low numbers of cells were injected, apparently f a c i l i t a t i n g complete removal from the body of tumour c e l l s . Passive immunization with this antiserum after excision of subcutaneously grown tumour in B6D2F1 mice showed better protection when higher numbers of tumour cells were injected. The possible mechanism by which this anti-body eliminates the tumour c e l l s and the potential role of passive immuni-zation in immunotherapy are discussed. 53 Introduction Standard procedures for the preparation of xenoantisera to TAAs routinely involved immunization of animals with either tumour c e l l s , or extracts thereof, following which the antisera were absorbed exhaustively with normal tissue to remove antibodies reacting with normal tissue (Lebien et a l . , 1979; DiSciullo et a l . , 1977), or by passage through normal hosts (Natori et^ a l . , 1978) . Other approaches have been used in attempts to prepare specific anti-tumour antisera. These involve coating the malignant cells with antibodies against normal tissue antigens prior to immunization (Smith et_ a l . , 1974). Such antisera have usually been of low potency, possibly due to the extensive absorptions required to render them tumour specific or to the overwhelming amount of normal tissue components in the immunizing dose of tumour c e l l preparation. Immunizing an animal with their syngeneic tumours i s theoretically an attractive approach to raise antiserum directed to TAA, but there are some p i t f a l l s in using this pro-cedure. In addition to the small volume of serum which can be obtained from a mouse and the procedure of bleeding being tedious and time consuming, immunizing inbred mice with irradiated, immunogenic, syngeneic tumour cells has not been successful at a l l times. The induction of suppressor cells when soluble tumour membrane extracts are used for immunization poses another problem. However, there are some reports proving the success of this method in raising anti-tiimour antibody (Prager et a l . , 1976). In an attempt to improve on this, various investigators have tried to mani-pulate the immune responsiveness of animals so that they respond specifically to 54 the tumour-specific marker rather than to the normal components. Gold and Freedman (1965) f i r s t demonstrated CEA by f i r s t rendering neonatal rabbits tolerant to extracts of normal human colon tissue, following which the tolerant animals were immunized with colon tumour tissue extracts. These animals produced antibodies almost exclusively to CEA. This pro-cedure has had varied success in the hands of other investigators, one of the major problems being the breakdown of this form of tolerance in animals hyperimmunized with tumour extract. The principle of antibody-feedback (Moller, 1969) has been used more recently in a limited number of studies to raise specific anti-tumour antiserum. In an animal model, Weiner, Hubbard and Hardiney (1972) immunized rabbits with mixtures of anti-normal tissue antiserum and whole tumour cells to obtain a tumour specific antiserum. Recently, Greaves (1975) has reported the successful results of a similar approach to obtain antiserum specific against human leukemia c e l l s . In these instances, he also used a mixture of anti-normal antiserum and whole tumour c e l l s . Working on the hypothesis that antibody feedback as a means of obtaining xenoantiserum to tumour associated or other c e l l surface markers would probably work better i f the antigens administered with antibody were in a soluble form, we undertook a series of experiments to test this. Preliminary studies using KC1 extracts of normal human lung and analogous extracts of squamous c e l l bronchogenic carcinoma as the "normal" and tumour associated antigen preparations, we were able to raise an antiserum which was apparently tumour specific (Kelly and Levy, 1977). The work in this chapter constitutes a report of a further study carried out using a murine mastocytoma c e l l line (P815 in DBA/2 mice) from which membranes have been 55 prepared and extracted to serve as the tumour preparation, and an analogous extract of peritoneal exudate cells as the normal. Materials and Methods Experimental Animals DBA/2J and B6D2 F l mice (Jackson Laboratory, Bar Harbor, Maine) between 2 and 4 months were used. Adult outbred albino rabbits were obtained from the University of British Columbia Animal Unit and used to raise the antisera. Tumour P815 x 2 mastocytoma and L1210 leukemia both in DBA/2J mice were obtained from Dr. J.B. Smith (Institute for Cancer Research, Philadel-phia) . Both tumours have been maintained as an ascites tumour or as frozen stock cultures in this laboratory for the past 3 years. Intra-2 peritoneal injection (IP) of 10 P815 cells or subcutaneous (SC) i n -3 jection of 2 x 10 cells into DBA/2 mice always induced tumours which grew progressively and k i l l e d the animals. Preparation of Lymphoid Cells and Tumour Cells Mice were sacrificed by cervical dislocation, their spleens were teased apart in phosphate buffered saline (PBS) and the c e l l clumps disaggregated by either expulsion through a tuberculin syringe with #26 gauge needle or by passing small pieces through a 60 gauge stain-less steel mesh. Red blood cells were lysed by treating spleen c e l l s with .14 M NH,C1 solution in PBS for 2 min. The cells were then washed 4 twice with PBS. 56 Cells from ascites were collected either by sacrificing the animal or by abdominal puncture and washed three times with PBS. Cell counts were carried out by direct microscopic examination and their v i a b i l i t y assessed by the trypan blue exclusion method. Antigen Extracts Ascites tumour cells to be used in the preparation of membrane extracts were washed three times in phosphate buffered saline (PBS), freeze-thawed three times, and ruptured by sonication at 4°C for 30 seconds (setting 50) using a Biosonik sonicator (Bronwill Scientific). A l l particulate material was pelleted by centrifugation at 100,000 x g for 60 min, and resuspended in PBS. Cell debris was removed by centrifugation of the sonicate at 4000 x g for 20 min. Membranes were pelleted from the supernatant by centrifugation at 100,000 x g for 60 min, and washed once (Billing et a l . , 1976 ). Membranes were extracted by suspension in a 0.5% PBS solution of deoxycholate (DOC) (Difco) for 30 min at 30°C, following which the material was again centrifuged at 100,000 x g for 90 min. The soluble membrane extract was dialysed exhaustively against physiological saline, and centrifuged again to remove insoluble material. Normal tissue extracts were made from a pool of spleen and peritoneal exudate cells from DBA/2J mice, and were prepared as 9 described above. When extracts were being made, approximately 10 cells were used in the preparations. Total protein of extracts was measured by either the Lowry assay or by measurement of absorbance at 280 nm. 57 Immunization Anti-normal DBA/2J (Anti-NDBA/2J) Complete Freund's adjuvant (Difco) was used to hyperimmunize rabbits against normal tissue membrane extracts. Normal extracts were diluted 1:1 with CFA, emulsified and 1.0 ml was injected into the rabbits every other week except for the second immunization which was done 4 weeks after the primary immunization. 500 ug protein was injected intramuscularly in four different places. Two weeks after the secondary immunization the rabbits were bled from the marginalvear vein.. The serum was removed from the blood and the complement was inactivated by heating the serum at 56°C for 30 min, following which i t was stored at -20°C. The antisera were tested either by the complement fixation test and/or complement mediated k i l l i n g of Con A blast cells of DBA/2 normal spleen and peritoneal c e l l s . Feedback Inhibition Anti-P815 In order to determine ratios of anti-normal DBA/2J serum to tumour extract for mixing prior to immunization the anti-normal serum was titrated against the P815 tumour extracts by the classical precipitin test described previously (Gerwing e£ a l . 1968), so that the ratio of antiserum:tumour extract constituting optimal proportions could be established (zone of equivalence and antibody excess). A mixture of anti-normal tissue antiserum and tumour extract was prepared using a ten-fold excess of antiserum over that required for optimal proportions. This mixture was allowed to stand for 48 h at 4°C following which any precipitate Figure 1. Illustration of the steps involved in the process of raising feedback antibody specific for the tumour associated antigen. FEED BACK METHOD FOR RAISING ANT ITUMOUR ANTIBODY ANTI TUNOUR ASSOCIATED AG. 60 was removed by centrifugation. An alum precipitate was prepared from 0.5 ml of this soluble mixture and rabbits were immunized with i t (half intravenously and half intraperitoneally), and l e f t for 5 weeks, after which time they were test bled and boosted with 1.0 ml of the soluble mixture of antiserum plus tumour extract. The whole method is illustrated in Fig. 1. They were test bled two and three weeks after this injection and again boosted as above. Sera obtained from these bleeds were fil t e r e d and stored at 4°C. Hypaque-Ficoll Density Cell Separation of L1210 Ascites HF was prepared from stock solutions of sodium hypaque (Winthrop Laboratories, Aurora, Ontario) and F i c o l l 400 (Pharmacia Fine Chemicals, Uppsala, Sweden) and mixed in a ratio providing a f i n a l specific gravity of 1.07 as described by Pope et a l . (1976). The f i n a l solution was filtered and stored at 4°C. L1210 ascites cells were washed three times with PBS and suspended at a concentration of 4 x 10^ c e l l s per ml. This suspension was carefully layered on 4 ml of HF in a 17 x 100 mm plastic tube (Falcon No. 2001, Oxnard, California). The tubes were centrifuged for 30 min at 400 x G. L1210 cells were found to form a thin layer at the inter-face with fewer c e l l s in the HF layer while the red blood cells were always at the bottom of the tube. L1210 cells were collected by pasteur pipette, washed three times and used for the complement mediated k i l l i n g assay. 61 Concanavalin A Blasts Washed DBA/2J splenocytes were suspended at 1 x 10 c e l l s per ml and incubated with 3 ug per ml con A (Sigma Chemical Co., St. Louis, Mo.), in RPMI 1640 containing 5% foetal calf serum (FCS), in tissue culture flasks (Falcon 3012, Oxnard, California), at 37°C and 5% CO,,. After three days incubation, the cells were centrifuged, washed and counted using the trypan blue exclusion method to check the v i a b i l i t y . Antiserum Assays In vitro assays Complement Fixation I n i t i a l l y , the antisera were tested with both the normal and tumour extracts using the quantitative complement fixation assay as described previously (Kelly et a l . , 1977; Kabat et a l . , 1961). Lyophilized guinea pig complement (Flow Laboratories, Rockville, Md.) was used, veronal buffer was used as diluent throughout. A 2% suspension of washed sheep erythrocytes was sensitized by mixing with an equal volume of 1:100 dilution of the recommended standard solution of haemolysin (Difco-Bacto anti-sheep haemolysin) for at least 15 min. Overnight incubation at 4°C for 10 ml mixtures of complement ( C ) , antigen (Ag) and antibody (Ab) along with appropriate controls was used for the fixation stage. The following day, 5 dilutions from 1:1.67 to 1:3.75 in a total volume of 3.0 ml were prepared. To each dilution tube 1.0 ml of the sensitized erythrocytes suspension was added, and ly s i s was allowed to 62 proceed for 30 min at 37°C. The lowest d i l u t i o n from C 1 c o n t r o l mixture was assumed to correspond to 100% haemolysis, and was used as a basis for c a l c u l a t i n g % haemolysis i n a l l other tubes. For each reaction mixture, the d i l u t i o n of C producing 50% haemolysis (D50) was obtained by the method of probits (Waksman, 1949). A unit of C', C ' H ^ Q was defined as the amount of C producing 50% l y s i s of sheep erythrocytes under the conditions of our t e s t . Due to the anticomplementarity of the antigens and an t i s e r a , the amount of complement fixed by s p e c i f i c antigen-* antibody i n t e r a c t i o n c ' H ^ Q ^.g+Ab "'"S 8 i v e n ^ v , * _ , Vrm , 1 1, _ 1 _ L . C H50 Ag+Ab " D C rm X Vdt X \ + D„_ D D ; 5 U A g D UAb ^"C' D UAg+Ab where -DC'rm = d i l u t i o n of complement i n reaction mixture Vrm = volume of reaction mixture (10.1 ml) Vdt = f i n a l volume i n each d i l u t i o n tube (4.0 ml) D 5 0 A ' P50 ' °50 A ^ ' D 5 0 C = the f i n a l d i l u t i o n of Ag Ab Ag+Ab complement causing 50% l y s i s i n d i l u t i o n tubes from Ag co n t r o l , Ab c o n t r o l , Ag+Ab mixture and C' cont r o l reaction mixtures r e s p e c t i v e l y 63 """"Cr Release Assay Once the antisera became positive and appeared to be tumour specific, by complement fixation they were tested against tumour c e l l s , and a variety of normal c e l l preparations for their a b i l i t y to k i l l *^Cr labeled cells in the presence of guinea pig complement. Tests were carried out in microtitre wells (Linbro Chemical, 1S-FB-96-TC, New Haven, Conn.). Doubling dilutions of both antiserum and normal rabbit serum (NRS) were made in RPMI 1640 culture medium, and 50 ul added to tr i p l i c a t e wells. ~^Cr labeled target cells,prepared as described previously (Takei et a l . (1976),were added to each well at a concentration of 2.0 x 10^ per ml in 50 u l . This mixture was incubated for 30 min at 37°C after which guinea pig complement (Flow Laboratories, Rockville, Md.) at a dilution of 1:10 in RPMI 1640 was added in a volume of 50 u l . Incubation was continued for an additional 60 min after which time an additional 50 ul of PBS was added and the plates were centrifuged, and 100 ul aliquots taken from each well, and counted. Spontaneous release was measured from control wells and never exceeded 10% of total counts, and maximum release was established by lysing cells with 5% Triton X100. The target cells used in this assay include: P815, L1210, normal DBA/2J spleen c e l l s and peritoneal exudate c e l l s , and Concanavalin A (Con A) blast cells from DBA/2J splenocytes. In vivo Assay of Antiserum Intraperitoneal Growth of P815 Tumour In order to determine the effects of the antiserum in vivo, tumour c e l l s , at varying concentrations, were incubated with either 64 antiserum or NRS (final dilution of 1:4) in 0.15 M NaCl for 30 min at 37°, after which aliquots of 0.2 ml (containing the required number of cells) were injected intraperitoneally into groups of either DBA/2 mice or B6D2 F l animals. Cell numbers 4 between 10 and 10 were injected. Each group consisted of between 10 and 15 animals. Animals were observed for survival time after tumour injection. Absorption Antisera were absorbed against normal DBA/2J mouse splenocytes. Absorptions were carried out using a ratio of 10 ml of antiserum to 9 o 10 splenocytes for 120 min at 4 C. Stat i s t i c a l Analyses Student's t-test was used to establish significance in differences i f survival times between different animal groups were significantly different p <0.05 were considered significant. Subcutaneous Growth of P815 Tumour The effect of the antiserum on tumour recurrence after resection 3 was also tested. B6D2 F l animals were injected SC with 2 x 10 P815 cells in the right flank. When tumours reached between 1.0 and 2.0 g they were resected and animals were injected IP with 0.2 ml of the anti-P815 anitserum or NRS. Animals (10 per group) were examined for tumour recurrence and survival time. Animals remaining tumour-free after 6 weeks were considered cured of their tumours. 65 Figure 2. Titer of anti-P815 serum of bleeds after the third boost as shown by -^Cr release assay with antiserum dilution of 1/16 (a), and 1/32 (b). The antisera absorbed once with either DBA/2 splenocytes or L1210 syngeneic tumour ce l l s . 66 Figure 3. Titration of anti-P815 serum at a dilution of 1/100 with extracts of: % % , P815 membrane extract and p Q , normal tissue extract. 67 Results Extraction and Immunization Tumour membrane fragments were prepared and solubilized with 0.5% DOC detergent. This concentration was chosen because higher concentrations were shown to increase the solubilization of H-2 antigen Antiserum raised to P815 antigens by immunizing rabbits with mixtures containing a membrane extract of P815 and antiserum to normal DBA/2J c e l l membrane components demonstrated anti-tumour antigen activity within 8 weeks of the i n i t i a t i o n of the immunization protocol. As can be seen in Fig. 2, the titr e s of antibody of different bleeds were comparable after the fourth bleed with l i t t l e variations. The small differences observed could be due to the test assay or due to the high levels of anti-normal antibodies in the circulation, since we have found that the specificity to P815 increases in bleeds 3 weeks or longer after boosting, probably because of circulating passively acquired anti-normal DBA/2 antibodies used in the immunizing protocol. Specificity of the Anti-P815 Antiserum The specificity of the anti-tumour antibody was detected by complement fixation tests in which the antiserum was tested with both tumour and normal tissue extracts. Representative results are shown in Fig. 3. It can be seen that there were very marked differences in the reactivity of antiserum with tumour extract as opposed to the normal tissue extracts. To determine whether this antiserum would effectively recognize antigen on viable tumour c e l l s , assays were run on ^Cr-labelled P815 68 • » UNABSORBED 0 _ _ 0 ABSORBED 1;'6 1:32 1:64 1:128 1:256 DILUTION OF A N T I S E R U M Figure 4. Titration of anti-P815 serum for i t s a b i l i t y to k i l l Cr labeled P815 targets in the presence of guinea pig complement. Values for % cytotoxicity were obtained by using the NRS control at each dilution as the base line. % % , unabsorbed anti-P815; Q Q , once absorbed anti-P815. 69 cells with both crude and absorbed antiserum. The results are shown in Fig. 4. There is l i t t l e difference between the absorbed and unabsorbed antiserum. To determine the specificity of the antiserum, i t was tested against Con A blasts from DBA/2J mice (Fig. 5). While neither the unabsorbed nor the absorbed anti -P815 antisera k i l l e d the Con A blasts at levels significantly above NRS, the anti-normal c e l l antiserum k i l l e d effectively under the same experimental conditions. These data indicated that very l i t t l e antibody that recognized antigenic components common to DBA/2J cells was present in the anti-P815 serum (either absorbed or unabsorbed). Further tests on the anti-P815 antiserum were carried out on a variety of DBA/2J targets. In each instance, the percentage k i l l i n g was calculated by using an equivalent dilution of NRS to establish a baseline, and each test was run simultaneously with known positive controls. The results are summarized in Fig. 6. It can be seen that only P815 c e l l s undergo l y s i s in the presence of guinea pig complement and once absorbed anti-P815 antiserum. It would thus appear that the antibodies in this serum are directed to components of the P815 membrane not found in normal cells or the L1210 leukemia cells of DBA/2J mice. The a b i l i t y of the anti-P815 antibody to bind the ^"^Cr-labelled P815 target cells can be inhibited by incubating 50 ul of antiserum, which was used in the ly s i s assay with 100 ug (equivalent to 5 x 10 P815 cells) of the crude DOC extracts before adding the target c e l l s , while incubating this antiserum with normal DBA/2J membrane extract 70 Figure 5. T i t r a t i o n of v a r i o u s a n t i s e r a w i t h DBA/2J con A b l a s t s f o r Cr r e l e a s e i n the presence of guinea p i g complement. • • , r a b b i t anti-normal DBA/2 serum; A«——A , NRS; • * , unabsorbed anti-P815; Q .Q , once absorbed anti-P815. 0 71 DILUTION OF A N T I S E R U M Figure 6. T i t r a t i o n of absorbed anti-P815 w i t h a v a r i e t y of Cr l a b e l e d t a r g e t s i n the presence of guinea p i g complement % # , P815 c e l l s ; O O » L1210 c e l l s ; A A , normal DBA/2J p e r i t o n e a l exudate c e l l s ; • • , normal DBA/2 spleen c e l l s . 72 did not neutralize the anti-P815 activity (Fig. 7). In vivo Effect of Antibody Since the specificity data of these antisera were shown to be valid, in vivo tests were run with mixtures of antisera and tumour cells injected IP to either DBA/2J or B6D2 F l mice. Survival times were assessed for mice receiving either NRS or anti-P815 antiserum with tumour c e l l s . The results are shown in Tables 1 and 2. When DBA/2 mice were treated in this way some protection was seen at a l l levels of ce l l s injected in the presence of specific antiserum. However, the antiserum appeared to be considerably more effective when used in B6D2 F l animals. The therapeutic value of the antiserum was tested by treating B6D2 F l mice, following tumour resection with 0.2 ml antiserum administered IP immediately after tumour resection. The results are summarized in Table 3. As can be seen, while a l l the control mice receiving NRS experienced tumour recurrence and limited survival time, 50% of the anti-P815 treated animals were free of tumour after 6 weeks, and those that experienced recurrence survived considerably longer than did the controls. 73 Figure 7. N e u t r a l i z a t i o n of anti-P815 serum by P815 c e l l membrane e x t r a c t s . 100 Ug of KC1 e x t r a c t s were incubated w i t h s e r i a l d i l u t i o n of anti-P815 or NRS before the a d d i t i o n of 5 1Chromium l a b e l l e d P815 c e l l s . • • , anti-P815; p Q anti-P815 plus P815 e x t r a c t s ; • • , NRS. 74 TABLE I. S u r v i v a l times of DBA/2 mice i n j e c t e d i n t r a -p e r i t o n e a l l y w i t h mixtures of P815 and e i t h e r anti-P815 serum or NRS. Each group represents between 10 and 15 i n d i v i d u a l s . Serum Added Anti-P815 Tumour c e l l s i n j e c t e d 10 H 10 3 10 2 10 S u r v i v a l time 17.6 ± 2.3 19.2 ± 1.9 21.4 ± 2.3 % S u r v i v a l 0 0 0 100 p value <0.001 <0.025 <0.01 NRS 10 M 10-10' 10 14.0 ± 0.7 16.6 ± 0.9 18.0 ± 1.0 21.9 ± 1.1 0 0 0 0 S u r v i v a l i n days ± standard d e v i a t i o n . Comparable groups t - t e s t . between anti-P815 and NRS t r e a t e d mice were analysed by Student' 75 TABLE I I . Survival times of B6D2 mice iniected intra-peritoneallv with mixtures of P815 and either anti-P815 serum or NRS. Each group represents between 10 and 15 animals. Serum Added Tumour ce l l s Survival 3 % Survival p valu. injected Time Anti-P815 10 3 26.6 ± 3.78 0 N.D. 10 2 29.73 ± 6.18b 15.4 <.001 NRS 102 20.42 ± 3.42 Survival time in days ± standard deviation. Survival time in this group was based on mice dying in under 45 days. Those survivors beyond 45 days were s t i l l free of disease at 120.days and are not included. Analysis by student's t-test between the anti-P815 and NRS treated mice receivin 10 c e l l s . 76 Table III. The e f f e c t of anti-P815 treatment on tumor recurrence and s u r v i v a l time i n B6D2 F l mice a f t e r resection of subcutaneously implanted tumors which had reached between 1.0 - 2.0 g at the time of resection. a b Treatment Survival Time ± SEM Recurrence Rate (%) NRS 13.2 ± 1.59 100 Anti-P815 22.4 ± 2 . 1 6 50 a S u r v i v a l time a f t e r tumor resection and treatment. bMice remaining free of tumor 35 days following s u r g i c a l resection and treatment were considered cured. Results based on 10 mice in each group. 77 Discussion The feedback method described here has permitted the development of an antiserum that demonstrates specificity for P815 mastocytoma and does not appear to show reactivity with either soluble extracts of DBA/2J peritoneal exudate c e l l membrane or a variety of lymphoid c e l l populations from DBA/2J mice. The amount of P815-specific antigen injected into the rabbit along with the anti-normal DBA/2J is d i f f i c u l t to assess. However, in most cases 700-1000ug of the P815 crude membrane extract per ml of anti-normal DBA/2J was mixed before absorption. From the work which was done on antigen purification (see Chapter III) one could estimate the amount of P815 TAA injected to be about 2-3 ug per injection. This is in agreement with the finding of Leung, Jones and Feldman (1978) who reported the purification of TAA from rat Moloney sarcoma devoid of v i r a l , foetal or H-2 antigen to be 9 10 ug per 10 c e l l s . Recently, lectin (Lens culinaris) was found to bind more specifically with H-2 molecules (Clemetson et a l . , 1976). This method can be used to reduce the amount of normal tissue antigen in the tumour extract before absorption with anti-N DBA/2J serum. The cytotoxicity- for P815 of the unabsorbed antiserum and serum absorbed once with DBA/2J splenocytes showed negligible differences i n d i -cating that only l i t t l e antibody directed to common DBA/2 membrane antigens had been made by the immunized rabbits. This type of antiserum has been raised in two individual rabbits, and similar specificity of the antiserum has been noted in both cases. The animals began showing the presence of specific antibody after the third immunization (Fig. 2) and continued to produce similar levels after repeated immunizations with the mixture of 78 anti-N DBA/2J antiserum and tumour c e l l membrane extracts. The specificity of antibody directed to P815 TAA is very clear. Conventional methods of immunization with the whole tumour extract followed by massive absorption with normal tissue lack this specificity. Siegler et a l . (1975) showed that the antisera obtained after immunizing monkey and rabbit with tryptic digest of CLL were of different s p e c i f i c i t i e s , in that rabbit antiserum was unable to differentiate between the morphological classes of leukemias while monkey's antiserum was reactive only to CLL. Their argument was that different animals respond to different molecules or react to different determinants of the same molecule of the same xenogenic tumour digest. In addition to the importance of the phylogenetic relation-ship between the species of the immunizing antigen donor and the antibody producer, rabbit antiserum seems to contain antibodies directed to normal antigens even after massive absorption especially to the D locus of the HLA which was shown to reside in large amounts in some leukemia cells (Arbei et a l . , 1975). The in vivo studies with anti-P815 serum were shown to have a protect-ive effect. When antiserum was injected to DBA/2J mice with varying numbers of tumour c e l l s , significant increases in survival time in a l l cases were noted in comparison with equivalent groups of animals injected with NRS. Even though the time differences in survival are not great, they actually represent the differences equal to an order of magnitude in tumour cells injected. This implies that the presence of the antiserum effectively facilitated the removal of approximately 90% of the tumour load. Since this tumour in these mice can k i l l when a single tumour c e l l i s injected IP, 79 i t i s not surprising that only marginal protection was observed and complete protection was only observed at the lowest number of cells injected. When B6D2 F l animals were used, greater success in complete protection was 2 observed, since some animals injected with 10 tumour cells in the presence of antiserum survived in a tumour-free state for longer than 120 days. The therapeutic value of the antiserum was also demonstrated by i t s a b i l i t y to prevent tumour recurrence in B6D2 Fl mice when administered at the time of resection of SC implanted tumours weighing 1.0-2.0 g. The antiserum significantly prolonged the survival time in mice expressing tumour recurrence and also prevented recurrence in half the mice treated with i t . The effector mechanism by which the tumour cells were eliminated by or through the help of the tumour-specific antibody is probably either by (1) complement mediated l y s i s of the tumour c e l l s , which may explain the difference in the degree of protection when DBA/2J and B6D2 F l were used, since DBA/2J mice are deficient in the f i f t h component of complement, or (2) by the action of activated macrophages. The last may attack tumour cel l s in many ways. A macrophage may ingest a tumour target especially when these targets are coated with the immunoglobulin (Bennet et a l . , 1963) or may lyse the tumour cells without f i r s t ingesting them (Evans et_ a l . , 1976) . or the antibodies might allow macrophages to specifically recognize tumour cells (Jolley e_ a l . , 1976). Similar protection studies have been done in other laboratories. EL4 cells pretreated with antiserum did not grow in C57 Bl/6 mice given 500 R 24 hr earlier. E l i c i t i n g an inflammatory response with thioglycolate increased the protective effect of antibody, implicating the involvement of macrophage in the protective process ( Z i g h e l b o i m et a l . , 1974). 80 The results with B6D2 F l after excision of subcutaneously grown tumour indicate that anti-P815 antibody either localized in the site of tumour and bound to the residual tumour cells and/or reacted with tumour cells in the circulation then preventing metastasis, while treatment with NRS did not show any protective effect. Failure of other investigators to achieve antibody localization may be due to the presence of antibody directed to normal tissue components (Izzo et a l . , 1972). 81 CHAPTER III ENRICHMENT AND PURIFICATION OF A TUMOUR-ASSOCIATED ANTIGEN OF THE P815 MASTOCYTOMA Abstract An antiserum, raised in rabbits (Chapter I) and previously shown to have specificity for the P815 mastocytoma of DBA/2 mice, and no cytotoxic reactivity for either normal DBA/2 cells or another syngeneic tumour c e l l line (L1210), was used to monitor the purification of a tumor associated antigen (TAA) of P815 c e l l s . Membrane extracts of both P815 and normal DBA/2J spleen and peritoneal exudate cells were subjected to DEAE fractio-nation and gel f i l t r a t i o n , following which fractions were tested for reactivity with the anti-P815 antiserum. Fractions from P815 extracts shown to be enriched for the TAA were used to raise a second antiserum specific for the tumor. This antiserum was also shown to have specificity for P815 and none for normal DBA/2J cells by ^ C r release assays in the presence of guinea pig complement and by surface labeling using peroxidase labeled sheep anti-rabbit immunoglobulin after treatment of either P815 or normal cells with the antiserum. This second antiserum (anti-P815-2), 125 when allowed to react with I-labeled TAA-enriched fractions of the P815 membrane extracts and passed over Sepharose-protein A columns, permitted the isolation of a single major component, detectable on autoradiographs-of gradient acrylamide gels. This component was not present in equivalent normal DBA/2 tissue extracts, nor was i t detectable when the tests were repeated using an antiserum raised against normal DBA/2 membrane preparations. 82 It was thus concluded that this material constituted a TAA of the P815 mastocytoma. Introduction There i s overwhelming evidence that transplantable murine tumours bear on their surfaces either tumor specific transplantation antigens (TSTA) and/or tumour associated antigens (TAA). TSTA are recognized as those antigenic determinants immunity to which fac i l i t a t e s T c e l l mediated tumour rejection in a manner analogous to allograft rejection. These antigens have been designated as being of v i r a l origin (e.g. in the case of Moloney sarcoma virus) or of unknown origin, as in the case of a number of chemically induced tumors (e.g. methylcholanthrene) (Whitney et a l . , 1975; Roudabeh et a l . , 1978). Presence of TSTA may be demonstrated by the ab i l i t y of previously immunized animals to reject doses of tumour cells known to be tumorigenic in naive animals, or by the Winn assay, in which T cells from immune animals passively protect syngeneic animals from a challenge with tumour cells (Winn, 1959). TAA, on the other hand, constitute those tumour specific components which do not appear to mediate T c e l l k i l l i n g but may be detected serologically (Brystryn, 1978; Parker et a l . , 1977; Chism et a l . , 1976). These are thought to be generally more cross-reactive and may include fetal antigens. It is generally thought that TSTA constitute "strong" antigens while TAA constitute "weak" antigens. To date one cannot precisely define what strong or weak tumor antigens are or indeed, their role in tumour immunity. With the exception of some v i r a l antigens, very few tumour antigens have been f u l l y characterized. However., the properties of some tumour antigens have been described recently 83 Leung et a l . , 1978; Leung et a l . , 1978). The tumour system under study here is the murine mastocytoma P815 of the DBA/2J mice and was originally induced by methylcholanthrene. This tumour c e l l does not express "strong" TSTA since i t is very d i f f i c u l t to raise any significant level of transplantation immunity in syngeneic hosts either by immunization with irradiated cells or by early resection of early tumours. Both of these methods usually work very well in tumours which express strong TSTA. However, P815 does express some form of TSTA, since T c e l l mediated cytotoxicity against P815 can be demonstrated in splenocytes of animals bearing subcutaneous early localized tumours (Takei et a l . , 1976). It was also shown that primary T cell-mediated cytotoxic responses to this tumour could be induced in vitro (Levy et a l . , 1979). However, the nature of complexity of the antigens on this tumour c e l l have not been characterized, although such antigens have been partially characterized by others (Clemetson et a l . , 1976). The detection of TAA on murine tumour cells has been studied, in the main, by serological methods. Antisera, raised either in syngeneic animals immunized with tumour c e l l extracts of inactivated tumour cells have been shown to react with tumour cells by either direct staining or by cyto-toxicity to tumour cells in the presence of complement (Roudabeh et a l . , 1978; Brystryn, 1978; Parker et^ a l . , 1977; Leung et a l . , 1978; Leung et a l . , 1978). Alternately, antisera raised in allogeneic or xenogeneic animals to tumour cells have been found to have tumour specificity following appropriate absorptions. These antisera are usually not of high t i t r e and frequently show tumour reactivity at high dilutions only because of background levels 84 of antibody directed to normal tissue components. Recently, a procedure has been developed by Kelly and Levy (1977) whereby moderately high t i t r e specific antiserum to tumour antigens can be raised in xenogeneic animals. The principle used here was described by Moller (1969) and fu l l y illustrated in Chapter II. This approach to pro-ducing antiserum to tumour antigens has been used by others with limited success, in which animals were passively immunized with antiserum raised to the appropriate normal tissue while being immunized with tumour cells (Greaves, 1975; Weiner et a l . , 1972). This feedback antiserum was used to monitor purification of lung cancer TAA (Kelly and Levy, 1979) and was also used to develop an antiserum in rabbits with apparently total specificity for P815 ce l l s (Al-Rammahy et a l . , 1979). This antiserum was cytotoxic for P815 cells in the presence of complement at a dilution of 1:64, while i t demonstrated no cytotoxicity for normal DBA/2 ce l l s or another tumour line of DBA/2 origin with dilutions as low as 1:4. It also demonstrated tumour specificity by quantitative complement fixation, and was found to have a protective effect in vivo. This section constitutes an extension of this work with P815. The antiserum has been used to monitor the purification of what appears to be a major TAA associated with the P815 tumour. 85 Materials and Methods Preparation of Crude Membrane Antigens The c e l l membrane extracts used throughout this work were prepared either from a mixture of normal spleen and peritoneal exudate cells from DBA/2J (Jackson Laboratories, Bar Harbor, Maine) or from the syngeneic mastocytoma, P815, taken from ascites fluid of tumour bearing mice. The methods of c e l l preparation have been described previously (Al-Rammahy, 1979) with minor alterations. Instead of DOC, 3.0 M KC1 was used to extract membrane components. This was done because we had experienced solubility problems with DOC extracts as well as some interference with some of the biochemical steps used subsequently (eg. acrylamide gel electrophoresis). Membrane extracts from these cells were made as follows. Cells were frozen and thawed three times following which they were subjected to differential centrifugation at 500 x G for 10 min. The supernatant was then centrifuged at 105,000 x G for 120 min. The resulting membrane pellet was resuspended in phosphate buffered saline (PBS), washed, and centrifuged again at 22,000 x G for 60 min to remove mitochondria and aggregated membrane. The small membrane fragments were subsequently pelleted by centrifugation again at 105,000 x G for 120 min. This preparation was extracted for 24 h at 4° with 3.0 M KC1, and dialysed against PBS. The extract was freed of particulate membrane debris by centrifugation again at 105,000 x G for 120 min. The soluble extracts were used for further studies. The starting material constituted the extract from approximately l O 1 1 c e l l s . The extraction procedure is illustrated in Fig. 8. 86 Figure 8. Flow diagram i l l u s t r a t i n g the i s o l a t i o n of membrane fragments of tumour c e l l s and t h e i r s o l u b i l i z a t i o n w i t h h y p e r t o n i c s a l t s o l u t i o n (3 M KC1). 87 KCL EXTRACTION WASHED P815, L 1 2 1 0 OR DBA SPLEEN C E L L S + PERITONEAL C E L L S F R E E Z E , THAW 3 X B R I E F SONICATION S P I N DOWN 1 0 0 0 RPM/10MIN. * — 1 — * S P I N DOWN SUP. AT 30KRPM FOR 2 HRS RESUSPEND P E L L T T WASH WITH T R I S NACL IOMM RECENTRIFUGE AT 1 0 5 K XG RESUSPEND P E L L E T IN T R I S NACL RECENTRIFUGE AT 22 K XG P E L L E T 1 ( P R L R N 2 ) DISCARD P E L L E T DISCARD SUP, DISCARD SUP. RECENTRIFUGE SUP. AT 1 0 5 K XG I P E L L E T 2 ( P 2 , L2. N 2 ) RESUSPEND WITH 3M KCL INCUBATE OVERNIGHT AT 4°C, SLOW SHAKING i S P I N DOWN AT 1 0 5 K XG FOR 2 HRS. SUPERNATANTS ARE THE SO L U B I L I Z E D FRAGMENTS 88 Preparation of Antisera Four different antisera were raised in rabbits. Antisera to normal DBA/2J membrane antigens were prepared by immunizing animals with either the crude normal c e l l extracts or a specific DEAE fraction (see below) of the normal c e l l extracts by intramuscular injections of a 1:1 emulsion of antigen and complete Freund's adjuvant (CFA, Difco) in four sites; one in each shoulder, and one in each thigh. Animals were immunized at 3 week intervals and bled 10-14 days later. The antiserum raised to the crude membrane extract (anti-N DBA/2-1) was highly cytotoxic to a l l DBA/2J cells or c e l l lines in the presence of guinea pig complement. The second antiserum raised to a Sephadex fraction of the crude extract was used exclusively in the preparation (by the feedback method) of specific anti-tumour antibody. Antisera to the tumour antigens of P815 were raised by employing a feedback method which has been described in detail in Chapter II. This antiserum was used to monitor fractions of the P815 extract to determine the presence of tumour associated antigens (TAA). After a series of fractionations, a TAA-enriched fraction from DEAE was identified. The equivalent fraction from normal DBA/2 membranes was used to immunize a rabbit and this antiserum was used in the same manner with the TAA-enriched DEAE fraction (see below) to raise a tumour specific antiserum of higher t i t e r for further studies. The speci-f i c i t i e s of this antiserum (anti-P815-2) are described in this Chapter, and i t was used in the fi n a l stages of purification of a TAA of the P815 mastocytoma. A l l anti-tumour antisera were absorbed routinely on L1210 tumour ce l l s as described previously. This step removed 89 Figure 9. Elution profiles of anti-P815 serum from S-G200. Samples of 5 ml were run on 2.5 x 90 cm Pharmacia columns in borate saline, pH 8.5. Fractions of 5 ml were collected. Fraction 1 and 2 representing IgM and IgG respectively. 90 to 1/8 1/16 1/32 DILUT ION OF ANTISERUM 1/64 Figure 10. Cytotoxicity of IgM, IgG fractions of S-G200. P815 targets were labelled with 51rjr. Fractions were tested in the presence of commercial guinea pig complement. , IgM; o o , IgG. Figure 11. Flow diagram ill u s t r a t i n g the steps involved in the enrichment and purification of tumour associated antigen. 92 1 2 5 I P U R I F I C A T I O N OF TAA S O L U B I L I Z E D MEMB. (P OR N EXTRACTS) 1 DEAE ( E L U T E WITH .01, .05. .10, .20 M ?ou BUFFER PH 8.00) 1 CONCENTRATE ON P M 1 0 CHECK BY CF (.05, .10 PEAKS = P815) DEAE ( E L U T E WITH .01, .02, .03 0.10 M ?ou BUFFER) I CONCENTRATE CHECK BY CF (.05, .09 PEAKS = P815) S.G150 ( E L U T E WITH BORATE S A L I N E PH 8.5) CONCENTRATE PEAKS CHECK BY CF 'EAK #2 (P OR N) + ANT i P815 OR ANT I DBA OR NRS NRS PROT. A COLUMN ( E L U T E WITH 3 M KSCN PH 6.5) i CONCENTRATE (PEG) | TREAT WITH I Z SDS, T R I S GLYCINE PH 8.00 GRADIENT G E L ELECTROPHORESIS (5.307=) X-RAY F I L M (DEVELOP IN - 7 0 ° C ) CU P815 BAND (USED FOR IMMUNIZATION) 93 residual activity to normal tissue components, as tested by complement fixation or "^Cr release assay. Immunoglobulin Purification Ammonium Sulfate Precipitation and Gel Chromatography The Ig from rabbit anti-P815 serum was precipitated with (NH^^SO^ by slowly adding to serum at room temperature, with stir r i n g to make the desired concentration which was 40%. The mixture was then allowed to settle for 3 hr in 4°C. The super-natant was decanted after centrifugation at 5000 x G for 15 min and the pellet was dissolved in a minimum amount of d i s t i l l e d water. The dissolved precipitate was dialysed against Borate saline pH 8.4 and run on Sephadex-G-200 (Pharmacia) in 100 x 2.5 cm column collecting a 5.0 ml fraction. The elution profile is shswn in Fig. 9. The a b i l i t y of the peaks to lyse P815 cells i s shown in Fig. 10. Immunoglobulin class studies were done because Prot. A preferentially binds the IgG class. Purification Procedures The whole purification procedure is illustrated in Fig. 11. DEAE Ion Exchange Chromatography The crude membrane extracts from both DBA/2 and P815 c e l l s were subjected to column chromatography on DEAE cellulose equilibrated with .01 M phosphate buffer at pH 8.0. Extracts containing 100.0 mg protein in 10-15.0 ml were chromatographed by stepwise elution with increasing buffer molarities at a constant pH. Buffer concentrations were 0.01, 0.05, 0.1 and 2.0 M phosphate. Each peak was tested by quantitative complement 94 fixation with anti-P815-l for the presence of TAA. Active material eluted at both 0.05 and 0.1 M phosphate. These fractions and the equivalent fractions from normal membranes were pooled separately, concentrated by u l t r a f i l t r a t i o n on an Amicon PM-10 f i l t e r and re-run on DEAE again using smaller increments of phosphate molarities of 0.01 to 0.1 molar. Two peaks, eluting at 0.05 and 0.09 molar were identified by comple-ment fixation as being enriched for TAA. This procedure was repeated several times in order to obtain more material. A l l active fractions, and the equivalent normal tissue material were pooled separately, concentrated by u l t r a f i l t r a t i o n , and used for further studies. Gel Chromatography The DEAE, TAA-enriched material and the normal equivalent were subsequently fractionated on Sephadex G-150 in borate-saline, pH 8.5. The TAA-containing fractions were identified by complement fixation tests using the anti-P815-l serum. These materials, both normal and tumour, were used to raise a feedback antiserum in rabbits to the enriched TAA. The tumour specific antiserum thus raised was designated anti-P815-2 and was used in further studies. Radioiodination Protein fractions that were obtained from Sephadex-G-200 which showed high specific reactivity with anti-P815-l from P815 extracts and i t s equivalent fraction from DBA/2 were radioiodinated according to the chloramine-T method of Greenwood et^ a l . (1963). Aliquots 125 (200 ug) of those fractions were reacted with 500 uCi of I (New 95 England Nuclear, Dorval, Quebec). The labelled fractions were exhaustively dialysed against PBS u n t i l the radioactivity counts in the dialysate were down to background levels. Af f i n i t y Chromatography (Protein A) 125 I-labelled P815 TAA-enriched fractions, and the equivalent fraction from normal DBA/2 tissue were allowed to react with undiluted preparations of either anti-N-DBA/2-2 antiserum, anti-P815-2 antiserum or NRS at 4° for 48 h. The antigen-antibody mixtures (total volume 350 ul) were passed over 2.5 ml Bio-Rad columns (i.d. 0.7 cm-7371222) of Protein A-tagged Sepharose 4B (Pharmacia). The columns were washed thoroughly with PBS and then the immunoglobulins and/or immune complexes were eluted with 3.0 M potassium thiocyanate at pH 6.5. Radioactive eluted materials were dialysed after concentration by negative dialysis against the SDS-TRIS glycine buffer used as the running buffer for the acrylamide gel studies. Acrylamide Gradient Gel Electrophoresis and Autoradiography 125 I-labelled materials which had been retained on the protein A columns were tested on gradient acrylamide gels (5-30% acrylamide). These running gels were 10 cm x 14 cm x 2 mm, contained 0.1% SDS and were made according to the method of Laemli (1971) with the exception of the use of a gradient maker to alter the acrylamide concentrations. Test materials were boiled for 3 min and added in 75 ul volumes (maximum), containing approximately 1 x 10^ CPM. The gels were run on SDS-Tris glycine buffer (.09 M) at pH 8.4. The samples were applied to the gel in the running buffer, and run for 75 min at 50 mA (150 V) and 4 h 45 min at 300 V. Running time for the electrophoresis was 96 6 h. On termination of the run the gel was wrapped in Saran wrap, quickly frozen at -70°C and developed against a photographic plate (Kodak X-Omat-R film) in the presence of dessicant and wrapped in f o i l . The plates were developed for 4 days. Gels from which slices were made for quantification were run as described above, but immediately after the run they were frozen, and sliced into 1 mm strips using a Mickle gel slicer (Brinkman Instruments). Individual slices were counted in a gamma counter (Biogamma) for 1 min per slice. Staphylococcus aureus Adsorbent Column (SPA) The detailed procedure is described by Kessler (1975). Briefly, Staphylococcus aureus strain Cowan I (protein A producer) was grown in Penassy broth (Difco Laboratories) supplemented with 5 mg/ml Casi-tone, 2.5 mg/ml yeast extract, 4 ug/ml niacin, 2 ug/ml thiamine HC1. The cells were collected, washed with PBS-azide and fixed with 1.5% formalin for 1.5 h followed by washing and k i l l i n g by rapid swirling in an 80°C water bath for 5 min, followed by rapid cooling in an ice 125 bath. The fixed cells were used as an immunoadsorbent column. I labelled antigen-antibody mixture was described under a f f i n i t y chromatography was mixed with the fixed cells and.after 2 h incubation, the bacterial adsorbent was washed twice. Bound radioactivity was eluted with 4% SDS, 6 M urea in a boiling water bath for 3 min. The eluate was carefully collected after centrifugation. The total counts were determined with Beckman gamma counter. 9 7 Immunological Assays Complement fixation A quantitative complement fixation assay was used routinely to identify fractions containing TAA. This method has been described in detail in Chapter II (Kelly et a l . , 1977). "^Chromium Assay The specificity of the various antisera used here, and their a b i l i t y to k i l l target cells in the presence of guinea pig complement was assessed by the standard "^Chromium release assay. This has been described in detail in Chapter II and published elsewhere (Al-Rammahy, 1979). Peroxidase Cell Surface Labeling In order to determine whether the test antisera reacted with c e l l surface components, cells were examined microscopically by the immunoperoxidase method of Stanislawski et a l . (1976) with minor modifications. P815 tumor cells were reacted with antisera at various dilutions or with normal rabbit serum (NRS), washed, and air dried on slides overnight. The slides were then treated with sheep anti-rabbit Ig which had been labeled with horseradish peroxidase. Visualization of the enzyme was effected with 3-amino-9-ethylcarbazole (AEC). 98 Results Enrichment of TAA Crude membrane extracts from both P815 and normal DBA/2 cells were subjected to a variety of fractionation procedures in an attempt to enrich for the TAA of the P815 membrane. The antiserum used to monitor the resulting fractions (anti-P815-l) was raised in a rabbit by immunizing i t with a mixture of the P815 extract and antiserum (anti-DBA-1) raised in another animal to normal DBA/2 membrane components. The characterization of this antiserum has been f u l l y described in Chapter II. I n i t i a l l y , the membrane extracts were subjected to stepwise elution on DEAE cellulose in phosphate buffer at pH 8.0 in a molarity range between 0.01 and 2.0. Typical elution profiles are shown in Figure 12. Each peak was pooled, concentrated by u l t r a f i l t r a t i o n , and tested by complement fixation for positive reactivity with anti-P815-1. The results of these tests are shown in Table 4. Since none of the normal fractions showed any reactivity with the anti-P815-l serum, the data are not presented. It can be seen that only the material eluting at 0.05 and 0.10 M contained material that reacted strongly with anti-P815-l. This procedure was repeated several times in order to accumulate reactive material as well as equivalent normal material. This DEAE fractionated material was also rerun on. DEAE using a narrower stepwise elution. The results of the DEAE fractionation of this material is shown in Figure 13. Complement fixation data on these individual peaks are shown in Table 5. It can 99 200 TUBE NO. Figure 12. E l u t i o n p r o f i l e s of c e l l membrane e x t r a c t s of P815 and normal DBA/2 splenocytes and p e r i t o n e a l wash c e l l s from DEAE at pH 8.0. Stepwise e l u t i o n was c a r r i e d out as i n d i c a t e d w i t h i n c r e a s i n g m o l a r i t i e s of b u f f e r from 0.01 to 2.0 M. P815 e x t r a c t ; _ • , normal DBA/2 e x t r a c t . Table IV. R e a c t i v i t y of P815 membrane peaks e l u t i n g from DEAE with anti-P815-l as assessed by quantitative complement f i x a t i o n . Peak % s t a r t i n g ug/ml C' H*50 number material antigen tested (Ag + Ab) 1 79.6 50 0 (0.01M) 25 0 2 6.8 50 1.60 (0.05M) 25 1.75 3 7.6 50 2.0 (0.10M) 25 1.87 4 6.0 50 0 (0.50M) 25 0 101 Figure 13. Elution profiles of pooled fractions 2 and 3 (.05 and 0.1 M) from DEAE (Fig. 1) rerun on DEAE in 0.01 M phosphate buffer, pH 8.0. Stepwise elution was carried out as indicated with increasing molarities of buffer from 0.01 to 0.10 M. * » , P815 e x t r a c t s ; Q Q , normal DBA/2 extract. 102 Table V. R e a c t i v i t y of P815 membrane peaks e l u t i n g from DEAE w i t h a n t i - P 8 1 5 - l as assessed by q u a n t i t a t i v e complement f i x a t i o n . Peak number Ug/ml antigen C'H*,.^ (Ag + Ab) 1 25 0 (0.01M) 12.5 0 2 25 0.25 (0.02M) 12.5 0.125 3 25 0.125 (0.03M) 12.5 0 4 25 0.375 (0.04M) 12.5 0.125 5 25 2.000 (0.05M) 12.5 2.375 6 25 0.375 (0.06M) 12.5 0.375 7 25 0 (0.07M) 12.5 0 8 25 0.125 (0.08M) 12.5 0.375 9 25 2.375 (0.09M) 12.5 2.000 10 25 0.625 (0.10M) 12.5 0.125 103 be seen that the only fractions containing significant amounts of reactivity with the antibody eluted at 0.05 and 0.09 M phosphate. They also constituted the two major peaks coming from the column, both with the P815 and the normal tissue extracts. It was not clear whether the two fractions each contained a distinct TAA or whether they constituted the same TAA in slightly different forms. These two peaks were pooled, concentrated by u l t r a f i l t r a t i o n and passed over a Sephadex G-150 column. The elution profiles are shown in Figure 14. The peaks were pooled and tested again by complement fixation. The results are shown in Table 6. While a l l three peaks reacted positively in the complement fixation tests, indicating that the TAA was distributed over a variety of molecular weights, the major a c t i v i t y was detected in peak 2. This material was used for further studies and was used to raise the second feed-back anti-P815 antiserum (anti-P815-2). A rabbit was i n i t i a l l y immunized with the normal tissue fractions, and this antiserum was used in combination with the active P815 material to immunize a second animal. Specificity of Anti-P815-2 In order to establish that this second antiserum (anti-P815-2) was directed toward surface antigens of the P815 tumor c e l l , a series of tests were carried out. After a single absorption of the anti-serum with L1210 cells was performed, i t s cytotoxicity to "*^ Cr labeled P815 and normal DBA/2 Con A blasts in the presence of guinea pig complement was assessed. The results are shown in Figure 15. It Figure 14. E l u t i o n p r o f i l e s of pooled DEAE f r a c t i o n s from both P815 and normal DBA/2 membrane preparations from Sephadex G-150. Samples of 4.5 ml were run on 2.5 x 90 cm Pharmacia columns of G-150 i n borate s a l i n e , pH 8.5. F r a c t i o n s of 4.5 ml were c o l l e c t e d . A A , P815 e x t r a c t ; ± ^ , normal DBA/2 e x t r a c t . Table VI. R e a c t i v i t y of P8Jr> membrane peaks e l u t i n g from Sephadex G-150 wi t h a n t i - P 8 1 5 - l as assessed by q u a n t i t a t i v e complement f i x a t i o n . Peak number ug/ml antigen C'H* 5 U ( A g + Ab) 1 (f r a c t i o n s 30-35) 10 2.625 5 1.875 2 (f r a c t i o n s 36-40) 10 3.625 5 3.125 3 (f r a c t i o n s 57-60) 10 2.125 5 1.625 106 Figure 15. Cytotoxicity of anti-P815-2 in comparison to anti-DBA/2 serum. Target c e l l s were either DBA/2 Con A blasts or P815 cells both of which had been labelled with -^Cr. Antisera were tested in the presence of commercial guinea pig complement (1:10). 107 can be seen that this antiserum, like the previously raised one showed high specificity for P815 cells and essentially no cytotoxicity to normal c e l l s , whereas the anti-DBA/2 antiserum effectively k i l l e d both P815 and normal DBA/2 ce l l s . In order to establish that this antiserum was reacting with surface antigens on the P815 c e l l s , a study was performed in which peroxidase-labeled sheep-anti-rabbit Ig was used to examine cells previously treated with either anti-P815-2, anti-DBA/2 serum or normal rabbit serum. The results of these studies are summarized in Table 7. Representative pictures of the labeling studies are shown in Figure 16. Microscopic fields were chosen so that both positively and negatively staining cells could be seen. Thus, both the cytotoxicity tests and the surface labeling studies indicated that this new antiserum was reacting with a surface component present on the P815 tumor cells but not on normal DBA/2 ce l l s . Purification of TAA Protein A Chromatography It was clear that conventional chromatographic procedures could only enrich for the P815 TAA but would not permit i t s purification. Further attempts to purify the TAA involved the use of protein A columns. The TAA enriched material isolated from DEAE chromatography and gel f i l t r a t i o n s , as well as the equivalent normal DBA/2 tissue fraction were iodinated and allowed to react with anti-P815-2, anti-DBA/2 or NRS for 48 h at 4°. The mixtures were then passed over protein A columns. The material adhering to the columns was eluted with 3.0 M 108 Table V I I . R e l a t i v e l a b e l i n g of: P815 or normal DBA/2 spleen c e l l s w i t h e i t h e r a n t i - P 8 l 5 or anti-DBA/2 serum at v a r i o u s d i l u t i o n s i : C e l l s Serum D i l u t i o n i — •— , ~ j P815 Normal Spleen c e l l s counted percent p o s i t i v e c e l l s counted percent p o s i t i v e Anti-P815-2 1 10 293 65 326 5.2 1 20 133 22 355 3.1 Anti-DBA/2 1 20 398 93 431 84 1 40 243 76 263 24 Normal Rabbit serum 1 10 200 0 200 0 / 109 Figure 16. Peroxidase l a b e l l i n g of either P815 or DBA/2 spleen c e l l s treated with anti-P815-2, anti-DBA/2 or NRS. (a) P815 c e l l s pre-treated with 1:20 anti-P815-2 (magnification 400 x ). (b) DBA/2 spleen c e l l s pre-treated with 1:20 anti-P815-2 (magnification 1000 x). (c) P815 c e l l s pre-treat-d with 1:20 anti-DBA/2 (magnification 400 x). (d) . P815 c e l l s pre-treated with 1:20 NRS (magnification 1000 x). 110 I l l 8 0 » 1 T FRACTION NO. Figure 17. Elution profiles of iodinated samples after treatment with antiserum and passage over protein A columns. The peak in material eluting with 3.0 M potassium thiocyanate. (a) P815 material treated with anti-P815-2 serum. (b) Normal DBA/2 material treated with anti-P815-2 serum. (c) P815 material treated with anti-DBA/2 serum. (d) Normal material treated with anti-DBA/2 serum. 112 Figure 18. Autoradiographs of materials eluted from protein A columns, dialysed and boiled in SDS-TRIS-glycine buffer. (a) P815 material treated with anti-P815-2 serum. (b) P815 material treated with anti-DBA/2 serum. (c) Normal DBA/2 material treated with anti-P815-2 serum. (d) Normal DBA/2 material treated with anti-DBA/2 serum. (e) P815 material without treatment. (f) Normal DBA/2 material without treatment. 113 Figure 19. Results presented in CPM of gradient gels of protein A eluted materials. (a) Normal DBA/2 material treated with anti-DBA/2 serum. (b) Normal DBA/2 material treated with anti-P815 serum. (c) P815 material treated with anti-DBA/2 serum. (d) P815 material treated with anti-P815-2 serum. 114 potassium thiocyanate. The elution profiles are shown in Figure 17. It was clear that the anti-P8l5-2 serum contained antibodies which reacted with and bound material present in the P815 fraction but not in the normal tissue equivalent fraction, whereas the anti-DBA/2 serum bound material present in both P815 and normal tissue fractions. Analytical Electrophoresis and Autoradiography The eluted fractions were subjected to gradient acrylamide electro-phoresis. The autoradiographs of these runs are shown in Figure 18. It can be seen that the material eluted with anti-P815-2 and the tumor fraction appears as a single component on the acrylamide, and that this component is not present in the material eluted from the column containing the P815 material and the anti-DBA/2 serum. Individual gels of these material were run, sliced and counted. The results of these runs are shown in Figure 19. Again, i t was clear that a unique component was separated by the anti-P815-2 serum after reaction with the P815 TAA-enriched fraction. These results indicate that these procedures may be successfully employed for the isolation of tumour-unique' components. 115 Discussion The specific antiserum raised against P815 membrane extracts (anti-P815-1) made i t possible to identify, by complement fixation, those peaks eluting from DEAE columns which contained antigenically active material. A l l tests run on equivalent fractions from normal DBA/2 lymphoid c e l l membrane extracts did not react in the complement fixation assay used here, so i t was assumed that the antiserum was detecting an antigen or antigens unique for the P815 c e l l . After pooling and concentration of antigenically active fractions, a narrow stepwise elution was carried out on DEAE. It was of interest to note that two peaks, those eluting at 0.05 M and 0.09 M phosphate were both strongly antigenic (Figuru 13, Table 5). The possibi-l i t y was recognized that the antiserum might be detecting two distinct antigenic entities or that, i f a single major antigen were involved, i t was present in the membrane extracts in two distinct forms. Sephadex gel f i l t r a t i o n of the combined 0.05 and 0.09 M eluting material indicated that at least two different sized molecules were associated with the antigen-i c i t y of the extract (Figure 14, Table 6). While the f i r s t two fractions taken from the Sephadex column were not clearly separated, the third fraction was well retained on the column, and antigenic reactivity was apparent in a l l three fractions. Because the second fraction (that eluting just behind the void volume) contained the strongest reactivity in the complement fixation test, i t was used in further purification studies. We have not yet ascertained whether the third Sephadex fraction constitutes a distinct antigen from that isolated from the second fraction, although the mobility of the isolated antigen in gradient SDS acrylamide gels would 116 Figure 20. Elution profile of iodinated samples after treatment with antiserum and passage over fixed Staph, aureus CoWI column. Each column is material eluting with Tris (.05M) pH 8.4, 6.6 M urea and 4% SDS according to Kessler, S. (1975). (1) P815 material treated with anti-P815 serum. (2) Normal DBA/2 material treated with anti-P815 serum. (3) P815 material treated with anti-DBA/2 serum. (4) Normal DBA/2 material treated with anti-DBA/2 serum. (5) P815 material treated with NRS. (6) Normal DBA/2 material treated with NRS. 117 indicate that in i t s deaggregated form, the antigen has a considerably lower molecular weight (65,000-75,000 when compared to standards) than would material eluting near the void volume of a Sephadex G-150 column. This indicates that the material in the Sephadex G-150 fraction 2 may contain an aggregated form of the antigen. Using the feedback technique described earlier, a second antiserum to P815 was raised with the TAA-enriched fractions from DEAE. This antiserum (anti-P815-2) had a higher t i t r e than the original one (anti-P815-l). In order to test the specificity of this antiserum and to determine i f i t , like the original antiserum was reacting with surface antigens on the P815 c e l l , cytotoxicity tests were run. It was clear that this antiserum, lik e anti-P815-l was capapble of e l i c i t i n g complement-mediated k i l l i n g of P815 cells but not of normal c e l l s , whereas the equivalent antiserum raised to normal DBA/2 membrane antigens could ef f i c i e n t l y cause k i l l i n g of both normal and P815 cells (Figure 15). The earlier antiserum had not been sufficiently strong to permit surface labeling of P815 cells when a sandwich technique using fluorescein-labeled sheep anti-rabbit Ig (unpublished data) was employed. The second antiserum, when tested with P815 and normal spleen c e l l s , followed by development with peroxidase-labeled sheep anti-rabbit Ig showed good levels of labeling of P815 c e l l s , at 1:10, whereas there was essentially no labeling of normal c e l l s at this dilution. These observations indicated that this antiserum was reacting with a sufficiently large number of antigen molecules on the surface of the P815 c e l l to permit visualization using the peroxidase staining procedure. 118 The material eluting in fraction 2 from Sephadex G-150 (both P815 and normal) was iodinated, reacted with either anti-P815-2 or anti-normal DBA/2 antiserum and passed over protein A columns, following which the material eluting with KSCN was dialysed against starting buffer and run on gradient SDS acrylamides after which the gels were developed by autoradiography at -70°. The results of these studies were unambiguous. The anti-P815-2 serum bound to an apparently unique major component in the P815 fraction, and did not bind to any identifiable components in the equivalent fraction from normal DBA/2 ce l l s . Alternately, the anti-normal DBA/2 serum bound materials present in both the P815 and the normal DBA/2 fractions. When preparative gels using the same materials were run, sliced, and counted, the same general picture emerged. It would thus appear that these techniques are applicable to the ultimate isolation of TAA from the very complex array of antigens found in crude membrane extracts. It should be emphasized that the starting materials in these studies constituted approximately 10^ tumour and normal c e l l s , and that the actual yield of antigen at the culmination of these steps less than 100 ug. Recognizing that each step used here involves some loss of product, i t is s t i l l apparent that the TAA described here constitutes a very small proportion of the membrane components of the P815 c e l l . The relationship of this TAA with the cell-mediated cytotoxicity seen in syngeneic animal cells to P815 remains to be c l a r i f i e d . This i s dealt with in Chapter IV. 119 CHAPTER IV PRODUCTION OF MONOSPECIFIC RABBIT ANTIBODY TO THE TAA OF P815, AND ITS EFFECT ON SYNGENEIC CYTOTOXICITY TO P815 Abstract Tumour specific antigen associated with membranes of the P815 masto-cytoma of DBA/2J mice was purified (Chapter III). Antiserum raised in rabbits to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement mediated target c e l l l y s i s or by ELISA. This antiserum was tested for i t s a b i l i t y to block k i l l i n g by in vitro raised syngeneic lymphocytes cytotoxic for P815 c e l l s . It was found that this antiserum, as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^ anti-serum (raised in congenic mice) were a l l able to block k i l l i n g when ^ C r -labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^ serum were capable of slightly blocking syngeneic k i l l i n g of L1210 c e l l s . Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pre-treatment of "^Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^ serum but not by the anti-P815 serum. The implications of these observations are discussed. 0 120 Introduction There is an overwhelming evidence that chemically induced tumours possess tumour specific antigens (Al-Rammahy et a l . , 1979a; Natori et a l . , 1978; Clemetson et a l . , 1976). The evidence came mainly from transplantation studies in inbred mice (Natori et a l . , 1977), or by the demonstration that these tumours are immunogenic by stimulating a humoral immune reaction in syngeneic mice (Ezaki e_t a l . , 1978) or also by raising tumour specific xenoantibody (Al-Rammahy et a l . , 1979a; Bertschmann et a l . , 1976). Although these antigens have been known to exist for a long time, l i t t l e is known about the nature of these antigens and their relationship to c e l l surface molecules, especially histocompatibility antigens. Serological means could be used to characterize these molecules, but this has been d i f f i c u l t , mainly because of the unavailability of clean, high t i t r e , specific antisera because of the heterogeneity of the antigenic preparations. Immunogenicity of chemically induced tumours as shown by the humoral response in syngeneic mice is rarely demonstrated. It has been suggested that these neoantigens are altered histocompatibility antigens (Garrido e_t a l . , 1976) which may only induce cellular immunity but not humoral immune reaction (Klein, 1978), or that they are in close association with H-2 antigens which would make purification of these tumour antigens very d i f f i c u l t (Callahan et a l . , 1979). If tumour antigens constitute such cross-reactive moieties, then the reactivity of an antibody to these molecules would be absorbed by normal tissue. Only i f TAA constitute separate entities on the c e l l surface can they be isolated using biochemical and immunological methods. 121 Few tumour antigens of chemically induced tumours have been characte-rized (Clemetson e_ a l . , 1976; Natori et a l . , 1978). There is no report in the literature showing the use of the purified tumour antigen for the generation of antibody specific for the tumour in study. The need of such antibody became of general importance to study the biological role of this antigen on the c e l l surface. The mode of recognition of the immune cytotoxic lymphocytes to their targets (tumour cells) is poorly understood. The restriction of cytotoxic lymphocytes to the foreign antigen to which they were raised and to the products of the genes associated with the major histocompatibility complex, H-2 on the target cells is well documented only in v i r a l l y induced tumours (Blank et a l . , 1977; Doherty et a l . , 1976). However, antiserum directed to the v i r a l antigen on these targets did not block the syngeneic T-lymphocyte mediated cytolysis of the target.cells whereas anti-H-2^ sera were effective (Zarling e_ a l . , 1978). Syngeneic blocking of CTL-mediated lys i s by certain alloantisera specific for the target tumour cells have been reported (Schrader e_ a l . , 1976; Germain et a l . , 1975), suggesting that H-2 antigens in both effector cells as well as the target cells are important in the lys i s of syngeneic c e l l s . The tumour system under study is the murine mastocytoma P815 syngeneic for DBA/2J and originally induced by methylcholanthrene. This tumour does not express strong tumour specific transplantation antigen. However, T cell-mediated cytotoxicity against P815 can be demonstrated in spleno-cytes of animals bearing subcutaneous early localized tumours (Takei e_t a l . , 1976) or can be induced in vitro (Levy _ t a l . , 1979). 122 During this course of study, we raised a tumour specific antibody to the tumour .antigen of P815 c e l l line in xenogeneic animal following the feedback inhibition method (Al-Rammahy, 1979a). This antiserum was used to monitor the enrichment and purification of the antigen associated with this tumour (submitted). The antigen was shown to constitute one major peak eluting from protein A columns and proved to be one band on analytical gradient gel electrophoresis having a molecular weight of 65-70000. We report here that the high t i t e r rabbit antibody raised to these molecules is tumour specific and can block the k i l l i n g by syngeneic cytotoxic cells but not the allogeneic cytotoxic c e l l s . Materials and Methods Preparation of Antisera Antisera to normal DBA/2J membrane antigens were prepared by immunizing a rabbit with the crude membrane extracts prepared by salt extraction with 3 M KC1 as described previously (Al-Rammahy et_ a l . , 1979a). This was done by intramuscular injections of 1:1 emulsion of antigen and complete Freund's adjuvant (CFA, Difco). Animals were immunized at 3 week intervals and bled 10-14 days later. The antiserum raised to the crude membrane extract was highly cytotoxic to a l l DBA/2J cells or c e l l lines in the presence of guinea pig complement. Antisera to the tumour antigen of the P815 c e l l line were raised by immunizing a rabbit with the purified antigen recovered from acryl-amide gels. The purification procedure is described elsewhere (submitted, Chapter III). Briefly, KC1 extracts of P815 cells were subjected to ion exchange chromatography on DEAE cellulose and 123 chromatographed by stepwise elution with increasing molarities of phosphate buffer. Active material was monitored by complement f i x -ation (CF) with tumour specific anti-P815 antiserum raised by the feedback inhibition method described previously (Al-Rammahy, 1979a). The enriched material was pooled, concentrated and re-chromatographed again using smaller increments of phosphate buffer molarities and again active peaks were monitored by CF. The DEAE, TAA enriched material was fractionated on Sephadex G-150. The TAA containing fractions were identified by CF using the feedback antibody. The P815-TAA rich fraction was radiolabelled and allowed to react with the anti-P815 antiserum and the mixture was passed over a Sepharose-protein A column. The eluted material was tested on acrylamide gradient gel electrophoresis. The gels,on completion,were autoradiographically developed at -70°C. One major unique P815 band which was missing from the normal tissue and other controls that had been treated similarly was cut and eluted from the acrylamide by homogenizing the gel in normal saline overnight at 4°C. The eluted material was mixed with CFA 1:1 and injected into a rabbit in four sites. The band was once injected along with the acrylamide material. New gel was prepared every time the animal needed to be boosted which was done every 3 weeks. The specificity and the biological activity of the antiserum was checked after the third boost and thereafter. Preparation of Anti-H-2 d This antiserum was prepared by immunizing BIO mice repeatedly with B10D2 splenocytes. Animals were injected I.P every week with 10^ B10D2 splenocytes for 8 weeks. Subsequently, they were bled from the retro-124 orbital sinus on alternate weeks and continually immunized over a period of 2 months. The anti-H-2^ antiserum thus prepared was 100% cytotoxic to P815 cells in the presence of complement at 1/600 dilution. Cytotoxicity Assay Single c e l l suspensions were prepared from spleens of normal DBA/2 mice by pressing the tissue through a stainless steel 60-gauge mesh in RPMI 1640 (Grand Island Biological Co., Grand Island, N.Y.) medium containing 10% heat inactivated foetal calf serum (FCS), 10.mM Hepes and 5 x 10 ^  mM of 2-mercaptoethanol. Gentamycin was also added to a fi n a l concentration of 50 Ug/ml. Suspended cells were washed through 3.0 ml of FCS, resuspended in medium and counted for viable c e l l s by using trypan blue. Tumour ce l l s were washed twice in medium before resuspension in complete medium for counting. Specific cytotoxicity against either P815 or L1210 was generated in vitro by incubating 5 x 10^ splenic lymphoid cells with 5 x 10^ mitomycin-C treated tumour cells in Linbro multiwell plates containing 24 fl a t bottom wells (No. 76-033-05) at 37°C in a humidified incubator with 5% C0 2 for 5 days. The total volume of each culture was 2.5 ml. After incubation cells were harvested, counted, resuspended in complete medium at a concentration of 10^ /ml and titrated at doubling dilutions starting at an effector to target ratio of 100:1 in a standard 18 h "^Cr release 51 assay. Spontaneous Cr release under these conditions was about 20%. When allogeneic cytotoxic cells were used, spleen cells from 5 x 10^ C57B1/6 were incubated with the mitomycin C-treated 5 x 10 6 DBA/2 spleen cells as mentioned above. The effector c e l l s were harvested 125 at 4 days and the "^Cr release assay was carried out on ^ C r labelled P815 cells in a 4 h k i l l i n g assay. "^Chromium Release and Blocking Assays The specificity of the antisera used in these studies and their a b i l i t y to lyse target c e l l s in the presence of guinea pig complement and the cytotoxicity by immune cytotoxic-T cells were assessed by standard "'"''Cr release assay. This has been described in detail (Chapter II, Takei, e_ a l . , 1977). In experiments to test the effect 51, o f antisera on cytotoxicity, Cr labelled targets, either P815 or L1210 were mixed with 1/10 dilution of rabbit anti-P815, rabbit anti-normal DBA/2 or mouse anti-H-2^. The mixtures were incubated for 90 min in an ice bath to avoid shedding of the surface antigen of the targets. Labelled targets treated either with normal rabbit serum or normal mouse serum were used as controls. The antiserum treated targets as well as the untreated control cells were added to a serial dilution of the effector cells and incubated at 37°C in an atmosphere of 5% CO2 for 4 h in the allogeneic system and for 18 h when using syngeneic CTL. After incubation, the plates were spun down and 0.1 ml aliquots of the supernatant was then carefully removed and counted in an LKB 1270 Rack-gamma-2 counter. Percentage of specific l y s i s was calculated from the formula mentioned in Chapter II. ELISA Assay This test was carried out as described previously (Voller e_ a l . , 1976; Engvall and Perlmann, 1971). Briefly, 0.2 ml of tumour or normal membrane extract in pH 9.6 carbonate buffer was attached to 126 substrate microtiter plates (Cooke Engineering Co., Alexandria, Va., No. 1-220-295) for 18 h at 4°C. After washing with PBS-tween buffer, sera to be tested for their activity against the coated antigens were added to the wells as 0.2 ml aliquots. Following incubation for 2 h at room temperature and subsequent washing, the developing alkaline phosphatase-linked goat anti-rabbit Ig (GARIg) at a dilution of 1:400 was added in 0.2 ml aliquot. After further 2 h incubation and fi n a l washing with buffer, 0.2 ml of the enzyme substrate solution (Sigma-104-105, p-nitrophenyl phosphate disodium) was added to each well and the enzyme substrate reaction was allowed to continue for 30 min at room temperature. The reaction was terminated by the addition of .05 ml of 3 M NaOH to each well. The contents of each well were transferred to tubes containing 0.75 ml of d i s t i l l e d water and read for absorbance at 400 nm in a Beckman DBG spectrophotometer. A l l tests were done in tr i p l i c a t e . Results Anti-P815 Specificity The specificity of the anti-P815 serum raised in rabbits to tumour specific material taken from acrylamide gels were tested i n i t i a l l y by testing i t s a b i l i t y to k i l l either 5 l C r labelled P815 cells or DBA/2 Con A blasts in the presence of guinea pig complement. The results are shown in Fig. 21 in which i t can be seen that the anti-P815 serum ki l l e d only the P815 cells whereas the anti-DBA/2 serum effectively k i l l e d both target c e l l s . These results also indicate that these 127 Figure 21. Cytotoxicity of anti-P815-4 in comparison to anti-DBA/2 serum. Target c e l l s were either DBA/2 con A blasts or P815 ce l l s both of which had been labeled with 5 1 C r . o — — O > anti-DBA/2; # • , anti-P815-4 antisera were tested in the presence of commercial guinea pig complement (1:10). 128 i antisera are also directed toward surface antigens. The specificity of antisera was assessed by the ELISA assay. As shown in Fig. 22 and 23, this antiserum was reactive only with the tumour extracts while anti-normal DBA/2 reacted with both normal and tumour extracts. It was concluded that the material eluted from the gel did indeed contain a tumour associated components not found in normal DBA/2 ce l l s . Blocking of the Syngeneic K i l l i n g In order to test the correlation between the H-2 compatibility of the CTL and the target cells during the process of lympholysis, and the role of the tumour antigen in this process, f i r s t we examined whether the specific anti-P815 antiserum would inhibit l y s i s of tumour ce l l s by H-2 compatible CTL and secondly, whether both tumour specific and H-2 antigens on the tumour cells serve as determinants for syngeneic CTL. The "'"'"Chromium labeled targets (P815) cells were treated with either rabbit anti-P815, rabbit anti-DBA/2 or B10BR anti-B10D2 (anti-H-2^) antisera before mixing with the syngeneic CTL cells which were raised in vitro. The results are shown in Fig. 24. The data show inhibition of the ly s i s using a l l the antisera that were tested. This demonstrates that the H-2 as well as the tumour antigenic deter-minants sera are necessary for CTL to become ful l y cytotoxic. These sera were titrated over dilutions between 1:3 and 1:100, the effect of the serum dilution are summarized in Fig. 25, showing relative k i l l i n g of the different preparations at effector to target ratio of 25:1. The anti-P815 serum showed inhibitory activity even at dilutions of 1:100 at which dilution anti-DBA/2 showed relatively weak blocking. 129 F i g u r e 2 2 . T i t r a t i o n o f P815 e n r i c h e d a n t i g e n , # . # and DBA/2 a n t i g e n , O O w i t h t h e a b s o r b e d ( 1 : 3 0 0 d i l u t i o n ) a n t i - P 8 1 5 - 4 and a n t i - D B A / 2 serum i n an E L I S A a s s a y . A n t i - n o r m a l DBA/2 w i t h P 8 1 5 , • A and NRS A A w i t h P815 a n t i g e n . 130 Figure 23. Titration of anti-P815-4 serum with the enriched P815 antigen in an ELISA assay. The optimum concentration of the antigen was shown to be 375 ng/well (Fig. 2). Anti- P815-4, £ £ NRS, 131 125/1 25/1 50/1 100/1 E/T RATIO Figure 24. Blocking activity of either rabbit anti-P815 or anti-DBA/2 serum on ^ C r labeled target cells incubated with CTL raised to P815 ce l l s . Target cells were preincubated for 90 min with 1:10 dilutions of either antisera or NRS, following which they were washed prior to addition to the -^Cr release assay, o -O > NRS treated target c e l l s ; • • , anti-DBA/2 treated c e l l s ; A A , anti-P815-treated target c e l l s . 132 01 I I L I I 1/3 1/10 1/30 1/100 SERUM DILUTION Figure 25. Titration of various antisera with Cr labeled P815 targets prior to their addition to assay for CTL. Effector:target ratios in each case were 50:1, and serum dilutions were for 1:3 to 1:100. O O , NRS-treated target c e l l s ; % 9 , anti-DBA/2-treated target c e l l s ; A A , anti-P815-treated target c e l l s . 133 1 T I 1 1 12.5/1 25/1 50/1 100/1 E/T RATIO Figure 26. Blocking activity of either rabbit anti-P815 or anti-H-2 antiserum on Cr labeled target cells incubated with CTL raised to P815 c e l l s . Target cells were pre-incubated for 90 min with 1:10 dilutions of either antisera or NMS (from B10 mice) following which they were washed prior to addition go the -^Cr release assay, Q Q » NMS; Q Q , anti-H-2 serum; _ , anti-P815 serum. 134 The effect of antiserum specific to H-2U antigens was also tested for i t s a b i l i t y to block syngeneic k i l l i n g . The results are shown in Fig. 26 in which i t can be seen that both anti-P815 and anti-H-2^ serum block k i l l i n g , although again the anti-P815 serum appeared to be more effective. In order to test the specificity of blocking of the anti-P815 serum, i t s a b i l i t y to block k i l l i n g of syngeneic CTL raised against L1210 tumour were tested (Fig. 27). In this case, the anti-DBA/2 and anti-H-2^ sera exhibited some blocking whereas the anti-P815 serum did not, indicating that this antiserum was recognizing and binding to a site present on the P815 c e l l surface but not on L1210. Blocking of Allogeneic K i l l i n g To investigate whether tumour antigen and/or H-2 complex on the target cells are involved in the l y s i s of allogeneic CTL, the above experiments were repeated but instead the CTL were generated by mixing mitomycin C-treated DBA/2 c e l l with allogeneic spleen cells (C57B1/6, H-2b). Figure 28 demonstrates that anti-P815 antiserum did not have any activity in inhibiting the k i l l i n g of P815 targets in the presence of allogeneic CTL while anti-DBA/2 and H-2^ strongly inhibited the l y s i s by those CTL in a l l ratios of effector:target used. This indicates that H-2 antigens play a major role in the allogeneic CTL l y s i s of the H-2 incompatible targets by allogeneic CTL and that the P815 TAA does not constitute a form of modified H-2 antigen. 135 80h 20h 1 1 r-12.5/1 25/1 50/1 100/1 E/T R A T I O Figure 27. Blocking activij:^ of rabbit anti-P815, anti-H-2" or rabbit anti-DBA/2 serum on Cr labeled target cells (L1210) incubated with CTL raised to L1210. Target cells were preincubated for 90 min with 1:10 dilutions of serum, following which they were washed prior to addition to the -^Cr release assay, Q Q , NMS; <• • , anti-H-2^; _, • > anti-DBA/2; A A » anti-P815 serum. 136 80 Figure 28. B l o c k i n g a c t i v i t y of r a b b i t anti-P815, a n t i - H - 2 Q or r a b b i t a n t i -DBA/2 serum on Cr l a b e l e d target c e l l s (P815) incubated w i t h C57B1/6 CTL r a i s e d to DBA/2 spleen c e l l s . Target c e l l s were preincubated f o r 90 min w i t h 1:10 d i l u t i o n s of serum f o l l o w i n g which they were washed p r i o r to a d d i t i o n to the 4 h ^ Cr r e l e a s e assay. O — O » NMS; A A , anti-P815; ^ ^ , a n t i - H - 2 d ; A • , anti-DBA/2 serum. 137 Discussion We have shown previously that P815 mastocytoma c e l l s , a chemically induced tumour of the DBA/2 mouse carry on their surface tumour specific antigens by raising a xenoantibody specific to this antigen (Chapter II). Using that antibody to monitor the enrichment of the tumour specific antigen after biochemical fractionation and i t was possible to purify the tumour associated antigen. Further evidence for the existence of the tumour antigen i s shown in this chapter using an antibody raised to the purified material. This antiserum is P815 tumour specific, since i t reacted strongly with the P815 extract in an ELISA assay but not with the normal c e l l extracts of syngeneic mice. Chromium release assay results indicate that the tumour antigen, at least in part, is located at the c e l l surface. The role of histocompatibility antigens for the determination of the specificity of CTL has been studied by many investigators. Specific lysis by immune CTL was shown in v i r a l l y transformed (Blank et_ a l . , 1977 ; Doherty et _ , 1976), chemically transformed (Germain et a l . , 1975), as well as in hapten modified c e l l s (Shearer, 1974). In a l l cases, compatible H-2 antigen on both cytotoxic cells and the targets was needed. It was of interest to determine whether the same situation exists in our chemically induced tumour and investigate the involvement of the tumour antigen to which the CTL were immuned, since there is no direct evidence in the literature showing that the tumour antigens may or may not serve as recognition structures for CTL c e l l s . Inhibition of ly s i s by specific anti-H-2 sera is another piece of evidence suggesting that H-2 antigens are directly involved in the CTL recognition of syngeneic tumour cells (Schrader et a l . , 1976; Germain et a l . , 1975). 138 The results obtained from the blocking experiments with both anti-DBA/2 and B10BR anti-B10D2 (anti-H-2 d) antiserum treated targets (Fig. 24,26) further show the validity of the H-2 antigens as target determinants for the immune CTL in order to become ful l y cytotoxic. The direct involvement of the tumour antigen in the recognition process is implicated, since strong inhibition of cytolysis was achieved by treating the target cells with specific anti-P8l5 antiserum. These data indicate that both the H-2 and the tumour antigens are important for the cellular recognition by the CTL in the syngeneic k i l l i n g . There are number of reports suggesting that TAA is linked to histo-compatibility antigens and this altered H-2 complex may e l i c i t CTL formation Schrader et a l . , 1976; Callahan et a l . , 1979). In our studies, i f both anti-P815 and anti-H-2 antisera recognize the same molecular complex on the c e l l surface of the target cells one should think that the reactivity of this antibody must be absorbed by normal lymphoid cells of the same haplo-type. This was not the case, since absorption with DBA/2 spleen cells did not abrogate the activity of the anti-P815 serum. One could argue that this antiserum was raised to the tumour molecules after the last being dissociated from H-2 complex during extraction. However, anti-P815 did not inhibit cytolysis by allogeneic CTL of the P815 target when the last were treated with anti-P815, while treatment of those targets with anti-DBA/2 or anti-H-2 d strongly inhibited the k i l l i n g by the allogeneic CTL (Fig. 28). Despite the evidence showing the physical association between v i r a l and H-2 antigens, treatment with high t i t e r anti-viral antigen antibody did not affect the cytolysis in syngeneic CTL mediated cytolysis (Zarling et a l . , 1979; Gomard et a l . , 1978). 139 From our studies, i t is more l i k e l y that both H-2 and tumour antigens are of separate entities on the surface of the target c e l l s . The possible mechanisms by which CTL recognize the target c e l l are: (1) dual recog-nition where both H-2 and tumour antigen have to exist as distinct components on the target cells or (2) modified self, in which the tumour antigen constitutes a part of the major alloantigen complex, in which case, the anti-P815 antiserum should block CTL directed to H-2d alloantigens. Henning e_t a l . (1976) reported that anti-viral antibody inhibited the ly s i s of tumour cells by anti-H-2 serum. Our data support the f i r s t possibility to be more l i k e l y . This i s in agreement with the finding that CTL to non-H-2 antigens must share the H-2 haplotype of the target c e l l s , although, H-2 and non-H-2 are presumably not on the same structure (Bevan et a l . , 1975). The blocking activity of the anti-P815 is specific since L1210 treated cells with this antibody do not resist k i l l i n g by L1210 stimulated CTL. 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