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Immunochemotherapy of human malignant melanoma with chlorambucil-carrying antibody
Europ. J. Cancer Vol. 1I, pp. 321-326. Pergamon Press 1975. Printed in Great Britain
Immunochemotherapy of Human Malignant Melanoma with Chlorambucil-carrying Antibody* T. GHOSE, S. T. NORVELL, A. G U C L U and A. S. MACDONALD Departments of Pathology and Surgery, Faculty of Medicine, Dalhousie University and Victoria General Hospital, Halifax, Nova Scotia, Canada. Abstract-- Heterologous anti-melanoma sera produced against any one patient's tumour cross-react, to varying degrees, with both the cytoplasmic and surface antigens of melanoma cells from other patients. Intravenous injections of cross-reacting antimelanoma antibodies bound to chlorambucil were followed by the regression of a considerable number of skin metastases in a patient with disseminated malignant melanoma. When immunochemotherapy had to be discontinued because of anaphylaxis, many of the metastases which had almost completely regressed, reappeared and grew progressively in spite of chemotherapy. One metastatic nodule which had not regressed and another which appeared during immunochemotherapy contained a much larger proportion of tumour cells lacking the cross-reacting surface antigen, when compared to tumour cells in metastatic nodules excised before immunochemotherapy.
Our study of 52 heterologous anti-sera produced against 18 human malignant melanomas, reveals that human malignant melanoma cells, in contrast to other reports [3], have surface antigens which they share with other human malignant melanoma cells but neither with any normal adult human tissues nor with several other types of h u m a n malignant tumours. As tumour tissue adequate for animal immunization was not available from this patient, cross-reacting anti-melanoma sera were used for immunochemotherapy.
INTRODUCTION
CHLORAMBUCIL can be non-covalently bound to anti-tumour antibodies without losing its alkylating activity or affecting the immunological reactivity of antibodies, and such chlorambucil-bound anti-tumour antibodies are more effective tumour inhibitors than chlorambucil or antibodies alone [1, 2]. We have reported the regression of metastatic nodules in a malignant melanoma patient after i.v. injections of chlorambucil-bound goat anti-melanoma globulin produced against the patient's own tumour [2]. We report here the regression of a number of metastatic nodules in the second melanoma patient treated with chlorambucilbound anti-melanoma globulin.
MATERIAL AND METHODS Anti-human melanoma sera
Surgically excised, malignant melanoma specimens were received in sterile Hank's balanced salt solution at 4°C. After removal of skin, fibro-fatty and other non-neoplastic tissues, tumours were chopped into pieces about 5 mm 3,
Accepted 5 December, 1974. *This work was supported by grants from the National Cancer Institute of Canada and Cancer Research Fund, Faculty of Medicine, Dalhousie University. 321
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washed several times with phosphate buffered saline (PBS, 0.1 M, p H 7.1) at 4°C, and then transferred to Ca and Mg free balanced salt solution containing E D T A [2]. As many dissociated tumour cells as possible were obtained from the tumour pieces by mechanical teasing. T u m o u r cells suspended in 2 ml PBS and mixed with 2 ml of Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan) were injected i.m. into both flanks and both shoulders of adult goats or New Zealand white rabbits ( 4 x 108 viable tumour cells/animal). Identical injections were repeated after 1 week using tumour ceils thawed and washed in PBS after storage in 10 % dimethyl sulfoxide at - 1 7 6 ° C . From one week after the second set of injections, the goats and rabbits were injected i.m. twice a week respectively with 100 mg and 5 mg of the tumour homogenate (prepared with a Waring blender and stored at - 7 0 ° C ) without any adjuvant. Any individual goat or rabbit received tumour materials from only one patient. Three days after the tenth injection of tumour homogenate, 5-10 ml of blood were obtained from all animals (test-bleeding). Testbleeding sera were inactivated (56°C, 0.5 hr), then absorbed with AB red cells and then repeatedly with homogenates of pooled normal h u m a n tissues and tested, thereafter, by immunofluorescence, for specific anti-melanoma activity [2]. I m m u n e sera were obtained from all animals showing titers of 1/64 or above of specific anti-melanoma activity. The resulting sera were inactivated and then successively absorbed with AB red cells and homogenates of normal h u m a n liver, lungs, kidney and spleen. Absorption with every tissue (tissue to serum ratio 1:3 by volume) was carried out at 37°C for 4 hr followed by another 8 hr absorption at 4°C. Absorbed sera were separated from the homogenates by centrifugation (20,000 x g) for 2 hr at 4°C. Absorptions were repeated (usually 2 absorptions each with human liver, spleen and lung homogenates and at least 4 absorptions with kidney homogenate) till the sera reacted only with the immunizing melanoma cells and not with the patient's own normal skin and peripheral blood lymphocytes or with other normal adult human tissues. Specific anti-melanoma sera (i.e. sera which after appropriate absorptions reacted only with malignant melanoma cells) against 18 malignant melanomas were produced in 14 goats and 38 rabbits. Immunofluorescence was performed either on 5/~m cryostat sections of melanoma and other human tissues snapfrozen at - 1 7 6 ° C or on smears of unfixed snap-frozen and thawed tumour cells by the
"sandwich method" using fluorescein isothiocyanate conjugated goat anti-rabbit, rabbit anti-goat or rabbit anti-human globulins with appropriate controls [1, 2]. For membrane immunofluorescence on viable melanoma cells, peripheral blood lymphocytes and cultured skin fibroblasts, the method of Phillips and Lewis [4] was used. Cytotoxicity of sera was assessed by the trypan blue exclusion method [2] using human AB blood group sera and a non-cytotoxic normal rabbit serum as complement sources [2]. Lymphocytes were isolated from sterile peripheral venous blood with carbonyl iron using a mechanical lymphocyte separator. Melanoma cells and normal skin fibroblasts were grown in Falcon flasks using medium 199 supplemented with human serum or 10 % foetal calf serum [2].
Cross-reaction studies All specific anti-melanoma sera were tested by immunofluorescence on smears from each of the 18 malignant melanomas. Five-tenths ml aliquots of four specific goat anti-melanoma sera were further absorbed with increasing amounts of homogenized melanoma cells or normal skin (2.5, and 5 and 10 mg/ml wet weight) from eight different patients. After absorption, increasing dilutions of sera were tested to find the titer of reactivity with cells from these 18 tumours.
Binding of globulins to chlorambucil Varying amounts of chlorambucil were attached to anti-melanoma globulins at a p H of 3 + 0 . 5 when up to 90% of chlorambucil can bind to goat and rabbit globulins as observed by us [1, 2] and confirmed using similar procedures by others [5].
RESULTS
Cross reactivity of the melanoma cytoplasmic antigen All specific anti-melanoma sera reacted by immunofluorescence with the cytoplasm of melanoma cells from most but not all malignant melanomas. With a few exceptions (e.g., tumour from E.R., Table 1) melanoma cells usually reacted maximally with the specific sera produced against the test tnmour and less with sera against other melanomas. When the reactivity of antl-melanoma sera with ceils from the immunizing tumour were absorbed out with just enough homogenate of the
Fig. 1. Fluorescence photomicrograph of a snap-frozen and thawed smear of malignant melanoma cells from patient D.A. stained by the "sandwich" method with a goat anti-melanoma serum (produced against M . M . ' s tumor) absorbed with AB red cells, normal human tissue homogenates and malignant melanoma cell homogenate from S.F. (5 mg tumor homogenate/0.5 ml. serum). About half of the cells show cytoplasmic staining. Dark nuclear outline of unstained cells (marked with arrows) can also be seen. x 280. Fig. 2. Fluorescence photomicrograph of a suspension of patient T.N.'s viable melanoma cells exposed first to a chlorambucil-bound cross-reacting goat-antimelanoma globulin and then to fluorescinated rabbit antigoat globulin. Discrete granular membrane staining of the viable melanoma cells shows that the anti-melanoma globulin retained its reactivity with the surface antigen of melanoma cells after binding with chlorambucil. Similar discrete granular membrane immunofluorescence of viable melanoma cells from this patient was also seen after exposure to the cross-reacting anti-melanoma globulins not bound to chlorambucil, x 800.
(to face p. 322)
Fig. 3. Photomicrograph of a section from the recurrent lesion in the patient's right heel showing cords and nests of round, o~,oid and polyhedral cells with relatively large and hyperchromatic nuclei, x 2 0 0 . Fig. 4. Photographs of the five metastatic nodules on the anterior aspect of the right thigh of T.N., which were measured every week and photographed. A : just before immunochemotherapy, B: four months after the beginning of immunochemotherapy, C: six months after the beginning of immunochemotherapy, D: eight months after the beginning of immunochemotherapy, E: is the photograph of the right inguinal region of this patient taken six months after the beginning of immunochemotherapy showing depigmentation after regression of metastatic melanoma nodules. A total of about thirty metastatic nodules regressed during immunochemotherapy.
Immunochemotherapy of Human Malignant Melanoma i m m u n i z i n g t u m o u r , they did not react with a n y o t h e r m a l i g n a n t m e l a n o m a cells. Absorption with a p p r o p r i a t e a m o u n t s o f h o m o g e n a t e o f the m a j o r i t y o f the cross-reacting t u m o u r s r e m o v e d the serum's reactivity with the t u m o u r used for absorption, b u t only r e d u c e d the reactivity o f the serum with the i m m u n i z i n g t u m o u r or with o t h e r t u m o u r s with w h i c h the serum r e a c t e d m o r e strongly t h a n the t u m o u r used for absorption ( T a b l e 1). Occasionally, cross-absorption with a p a r t i c u l a r t u m o u r h o m o g e n a t e either virtually did not affect the reactivity o f the serum with some o f the o t h e r cross-reacting t u m o u r s or revealed two populations o f t u m o u r cells; one reacting
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m e m b r a n e i m m u n o f l u o r e s c e n c e on m e l a n o m a cells (but not on the same patient's lymphocytes or fibroblasts) from 6 out o f 7 m e l a n o m a s d e m o n s t r a t e d b y each o f these 4 anti-sera (none raised against the 7 test tumours) to possess a c o m m o n cytoplasmic antigen. V i a b l e cells from two i m m u n i z i n g m e l a n o m a s were 4 a n d 9 times m o r e effective t h a n the cells from two cross-reacting m e l a n o m a s in absorbing out the m e m b r a n e i m m u n o f l u o r e s c e n c e reactivity o f two a n t i - m a l e n o m a sera with cells from their respective i m m u n i z i n g t u m o u r . Prior exposure to autologous m e m b r a n e localizing a n t i b o d y from two patients could partially (though not completely) block the reactivity of the t u m o u r
Table I. Comparison of the reactivity of a goat anti-melanoma serum with smears of melanoma cellsfrom eight different patients, before and after absorption of this serum with tumour (10 mg tumour homogenate/ml serum) from thesepatients. This serum was produced against patient M.M.' s malignant melanoma and was rendered tumour-specific (i.e., the serum reacted with M.M.'s melanoma cells but not with ( i) cryostat sections of M.M.'s skin (ii) M.M.'s skin fibroblasts in vitro (iii) M.M.'s peripheral leukocytes and (iv) cryostat sections of other normal adult human tissues), before this cross-reactivity study, by absorptions with human AB red cells (X1), and homogenates of normal human spleen (X2), liver (X2), lungs (X2) and kidney (X4)
Tr. cells from :
Reciprocals of the highest dilution of the serum which reacted by immunofluorescence with melanoma cells after absorption of the serum with tumour homogenate from:
M.M. E.R. D.A.
NONE > 1024 > 1024 1024
O.S. S.B. H.R. ILS. S.F.
1024 512 256 256 64
M.M. ---. . --.
E.R. 4 --. .
D.A. 128 32 -. .
--.
.
. . 16 4 .
O.S. 64 64 32
S.B. 64 32 --
32 --
8 --
. . .
H.R. 64 128 4
H.S. S.F. 128 256 256 128 64 32 50% cells 50%cells approx, approx. 4
m
m
128 --
32 4
4
(-- No reactivity detectable by immunofluorescence).
a n d the o t h e r not reacting with the absorbed serum (e.g., cells from D.A. in T a b l e 1 and Fig. 1). Absorptions o f these sera with c o r r e s p o n d i n g a m o u n t s o f n o r m a l skin h o m o g e n a t e and frozen t h a w e d p e r i p h e r a l blood l y m p h o c y t e s from these patients did not affect their reactivity with m e l a n o m a cells.
Cross-reactivity of melanoma cell surface antigens Membrane-immunofluorescence. All specific antim e l a n o m a sera showed discrete g r a n u l a r staining on the surface o f viable m e l a n o m a cells from the i m m u n i z i n g t u m o u r . O n e or m o r e of the 4 a n t i - m e l a n o m a sera tested revealed
cells with the heterologous m e m b r a n e localizing antibodies. N o n e o f the specific a n t i - m e l a n o m a sera r e a c t e d with either the smear or with the viable t u m o u r cell suspensions from 4 h u m a n pulm o n a r y squamous cell carcinomas, 2 h y p e r n e p h r o m a s a n d 2 breast a d e n o c a r c i n o m a s . T h o u g h all the sera prior to absorption were cytotoxic to m e l a n o m a cells and to cultures o f a n u m b e r of cell lines of h u m a n origin, no cytotoxic activity could be detected in these sera after being r e n d e r e d m e l a n o m a specific b y absorption with AB red cells a n d h u m a n o r g a n homogenates.
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T. Ghose, S. 7". Norvell, A. Guclu and A. S. Macdonald
The case history of a melanoma patient treated by immunochemotherapy is given below: Ten months prior to admission T.N., a 69 year old white male, had noticed a "wart" on his right heel, and four months later this lesion (not histologically examined) was excised. At admission, T.N. had an ulcer containing two reddish black heaped-up spherical nodules measuring 17 mm and 8 mm in their longest diameters, at the site of excision which had never healed. An incisional biopsy showed cords and nests of round and polygonal neoplastic cells with hyperchromatic nuclei and increased mitotic activity. Some of the cells contained a small amount of melanin (Fig. 3). After excision of the ulcer twelve days later, histology revealed tumour invasion of the lymphatics in the midcorium. Enlarged lymph nodes in the right groin were removed by radical dissection 25 days later. Several subinguinal nodes contained metastatic tumour. Examination 8 months later revealed histologically proven nodular recurrences of melanoma in the healed inguinal scar, an indurated mass above the inguinal ligament and edema of the right lower limb and external genitalia. Two months later about 30 metastatic nodules varying from the size of a pin head to spheres about 1 cm din appeared in or under the skin of his right inguinal region, pubic ramus, the scrotum and the right lower limb. The patient at this time, did not react to the second strength of PPD (Connaught Laboratories, Toronto). Suspension of viable melanoma cells obtained from two metastatic nodules were tested for cytoplasmic and membrane immunofluorescence against a number of goat and rabbit anti-melanoma sera produced against malignant melanoma from other patients. One goat serum and two rabbit sera showed fairly strong (i.e., titer 1/128) cytoplasmic and membrane immunofluorescence. When bound to ehlorambuell, these anti-melanoma globulins retained alkylating activity as assessed by the Epstein method [6] as well as their reactivity with tumour cells (Fig. 2). The patient was injected i.v. with a total amount of 429 mg of?-globulin bound to 41 mg of chlorambucil through nine separate injections administered on alternate days. Five subcutaneous metastases on the anterior aspect of his right thigh were measured weekly. The two largest diameters of these five nodules approximately at right angles to one another just before the beginning of treatment, were 1 - 4 x l . 2 cm, 1 - 5 x l . 2 cm, 1-2 x 1-1 cm and 1.8 x 1-2 cm. About 8 weeks after completion of the injections all of these
five nodules and about 20 other nodules which were not regularly measured became smaller and after about 4 months, four of the five metastatic nodules under regular measurement and at least twenty other nodules in the inguinal region and the scrotum almost completely regressed (Fig. 4). Depigmentation was noticed around the regressing melanoma nodules. A month later, a new crop of nine metastases appeared in or under the skin. The patient again received, through 16 i.v. injections, a total of 800 mg of a cross-reacting goat antimelanoma globulin bound to 80 mg ofchlorambucil. The patient had a mild anaphylactic reaction after the last injection and all injections of goat gamma-globulin were stopped. From 7 days after the anaphylactic reaction, the patient received three i.v. injections containing a total of 175 mg of cross-reacting rabbit gamma-globulin bound to 17.5 mg of chlorambucil. However, after the last injection, the patient developed anaphylactic reactions to rabbit globulin as well and immunochemotherapy was stopped. During this period of immunochemotherapy, though, at least five other metastatic nodules completely disappeared leaving areas of cutaneous depigmentation, and those nodules which had already regressed did not reappear, four new metastatic nodules emerged as well. One nodule on the anterior aspect of his right thigh which was being measured regularly and which did never completely regress and another recent metastatic nodule were excised. Less than 5 ~ cells from these two nodules reacted by membrane imnmnofluoresccnce with the rabbit and goat anti-melanoma globulins with which the patient was treated. At least 6 0 ~ tumour cells in metastatic nodules excised prior to immunochemotherapy and stored in liquid nitrogen, showed membrane immunofluorescence when concurrently tested with these anti-melanoma globulins. About 80-90 ~ of the stored melanoma cells and about the same proportion of cells from these excised nodules showed cytoplasmic immunofluorescence with these three anti-melanoma globulins. The patient did not have at any time membrane localizing or cytoplasmic antibody or cytotoxic lymphocytes against his own tumour cells nor could any anti-rabbit or anti-goat globulin be detected in his serum by immunodiffusion. After the stoppage of immunochemotherapy, in spite of treatment with large doses of BCG and a number of chemotherapeutic agents including DTIC, there has not been any further regression of tumour nodules. Existing
Immunochemotherapy of Human Malignant Melanoma nodules have increased in size and a few have fungated. DISCUSSION
T h e melanoma specificity of the appropriately absorbed heterologous anti-melanoma sera is supported by: 1) the absence of reaction by membrane or cytoplasmic immunofluorescence either with cryostat sections of the respective patients' own skin or with the smears and suspensions of viable autologous peripheral blood lymphocytes and cultures of autologous skin fibroblasts from these patients, 2) absorption of the reactivity of these sera with appropriate amounts of the immunizing or other cross-reacting melanoma tissues but neither with the corresponding amounts ofhomogenates of normal skin nor with skirt fibroblasts or peripheral blood lymphocytes from these patients and, 3) absence of reactivity either with smears or viable cell suspensions from several h u m a n malignant tumours other than melanoma. The tumour-specific melanoma cell-cytoplasmic antigen cross-reacts with sera from other malignant melanoma patients [3, 7-9]. T h o u g h Lewis et al. [3] are of the opinion that the melanoma cell-surface antigen which reacts with autologous serum is individual specific, other studies using membrane immunofluorescence [7, 11], cytotoxicity tests [11, 12] or lymphocytes [10, 13, 14] support our observation that there are tumour-specific cross-reacting antigens on the surface of h u m a n malignant melanoma cells. As prior exposure to the autologous membrane localizing antibody partially inhibited the reactivity of viable melanoma cells to the heterologous cellsurface localizing antibody, the antigen on the surface of malignant melanoma cells reacting with these two antibodies appears to be similar. Our observation in several mouse t u m o u r models, that chlorambucil or l a t i bound antibodies consistently cause m u c h more tumour inhibition than antibody, chlorambucil or 131I alone [1, 2, 15, 16] has now been confirmed by others [5, 17, 18]. However, as discussed by us before [2], the precise mechanism of increased tumour inhibition by antibodies attached to cytotoxic drugs or ~311 remains to be elucidated and could be explained on the basis of: (a) synergism between anti-tumour antibody and cytotoxic drugs [5, 18] or ionizing radiations [19], (b) antibody mediated preferential localization of cell damaging agents in t u m o u r tissue [16] and (c) both [2]. None of the anti-melanoma globulins used in the treatment of this patient was cytotoxic B
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to his t u m o u r cells and it is unlikely that this antibody by itself caused tumour regression. Passive transfer of serum or plasma from cured patients has failed to cause regression of disseminated melanoma [20]. The complete or partial regression of a considerable n u m b e r of metastatic melanoma nodules during immunochemotherapy (but at no time before immunochemotherapy) and the re-emergence and progressive growth of metastases after the cessation of i m m u n o c h e m o t h e r a p y but in spite of continuing chemotherapy and BCG administration, supports the contention that the regression of these metastases was not spontaneous but causally associated with immunochemotherapy. Depigmentation has been observed after the regression of melanoma metastases in skin [20, 21]. No anti-melanoma cytoplasmic antibody (reported to be associated with the depigmentation around resolving "halo" nevus [22]) or antibodies against melanin containing cells in normal h u m a n skin could be detected in the serum of this patient before or after the appearance of these areas of depigmentation. The results of our cross-reaction study are in keeping with the concept of antigenic diversity of tumour cells [23, 24] and suggest that the tumour-specific melanoma antigens have mosaics of determinants and that different combinations of these determinants are expressed in different tumours. The considerable decrease in the proportion of surface antigen containing melanoma cells in the metastasis which did not regress and the other which appeared during i m m u n o c h e m o t h e r a p y compared to metastases excised before immunochemotherapy, suggest selective suppression of the surface antigen containing cells and survival and proliferation of the antigen negative cells or antibody induced desensitization resulting either from the loss of antigen i.e., "antigerfic modulation" [25] or from the redistribution of antigen antibody complex on the cell membrane [26]. Such selective proliferation of antigen negative and/or drug-resistant t u m o u r cells might be an impediment to the effective eradication of tumour cells by immunochemotherapy. Development of anaphylaxis against foreign globulins constitutes another limitation o f i m m u n o c h e m o t h e r a p y . At present, attempts are being made to remove the antigenic Fc fragment of the immunoglobulin molecules prior to binding with chlorambucil. Acknowledgement--We are grateful to Ms. Burroughs Wellcome and Co. for the free gift of chlorambucil (leukeran) and to Mrs. M. M a m m e n , P. Seaman and R. Sekaran for technical help.
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T. GHOSE and S. NIGAM, Antibody as carrier of chlorambucil. Cancer 29, 1398 (1972). T. GHOSE, S. T. NORVELL, A. GUCLU, D. CAMERON, A. BODURTHAand A. S. MACDONALD, Immunochemotherapy of cancer with chlorambucil-carrying antibody. Brit. med. J. 3, 495 (1972). M . G . LEwis, R. L. IKONOPISOV,R. C. NAIRN,T. M. PHILLIPS,G. HAMILTON FAIRLEY, D. C. BODENHANand P. ALEXANDER,Tumour-specific antibodies in human malignant melanoma and their relationship to the extent of the disease. Brit. reed. J. 3, 547 (1969). W . M . PHILLIPS and M. G. LEWIS, A system of immunofiuorescence in the study of tumour cells. Rev. europ, dtudes din. et biol. 15, 1016 (1970). D . A . L . DAVIES and G. J. O'NEIL, In vivo and in vitro effects of tumourspecific antibodies with chlorambucil. Brit. J. Cancer 28 (suppl.), 285 (1973). J. EPSTEIN,R. W. ROSENTHALand R . J . Ess, Use of 7-(4-nitrobenzyl) pyridine as analytical reagent for ethylenimines and alkylating agents. Anal. Chem. 27, 1435 (1955). D . L . MORTON, R. A. MALMOREN, E. C. HOLMESand A. S. KETCHAM,Demonstration of antibodies against human malignant melanoma by immunofluorescence. Surgery 64, 233 (1968). H . F . OETTGEN, T. AOKI, L. J. OLD, E. A. BOYSE, E. DEHARVEN and G. M. MILLS, Suspension cultures of a pigment-producing cell line derived from a human malignant melanoma. J. natl. Cancer Inst. 41, 827 (1968). N . M . MUNA, S. MARCUS and C. SMART, Detection by immunofluorescence of antibodies specific for human malignant melanoma cells. Cancer 29, 88 (1969). G. FOSSATI, M. I. COLNAOHI and G. DELLA PORTA, Cellular and humoral immunity against human malignant melanoma. Int. J. Cancer 8, 344 (1971). R . S . METZOAR, P. M. BERGOC, M. A. MORENO and H. F. SEIOLER, Melanomaspecific antibodies produced in monkeys with human melanoma cell lines. J. natl. Cancer Inst. 50, 1065 (1973). D . P . GOODWIN, M. O. HORNUNG, S. P. L. LEONO and E. T. KREMENTS, Immune responses induced by human malignant melanoma in the rabbit. Surgery 72, 737 (1972). J. E. DEVRIES, P. RUMKE and J. L. BERNHEIM, Cytotoxic lymphocytes in melanoma patients. Int. J. Cancer 9, 567 (1972). K. E. HELLSTROM and I. HELLSTROM, Immunity to neuroblastomas and melanomas. Ann. Rev. Med. 23, 19 (1972). T. GI-IOSE,M. CERINI, M. CARTER and R. C. NAIRN, Immunoradiative agent against cancer. Brit. Med. J. l, 90 (1967). T. GHOSE and A. GUCLU, Cure of a mouse lymphoma with radio-iodinated antibody. Europ. J. Cancer. In press (1974). I. FLECHNER, The cure and concomitant immunization of mice bearing Ehrlich ascites tumours by treatment with an antibody-alkylating agent complex. Europ. d. Cancer 9, 741 (1973). R . D . RUBENS and R. DULBECCO,Augmentation of cytotoxic drug action by antibodies directed at cell surface. Nature (Lond.) 248, 81 (1974). T. GHOSE and M. CERINI, Radiosensitization of Ehrlich ascites tumour cells by a specific antibody. Nature (Lond.) 222, 993 (1969). T . C . EVERSON and W. H. COLE, Spontaneous Regression of Cancer, p. 197. Saunders, Philadelphia (1966). W . C . SUMNER and A. G. FORAKER,Spontaneous regression of human melanoma. Cancer 13, 79 (1960). P . W . M . COPEMAN,M. G. LEWIS, T. M. PHILLIPS and P. G. ELLIOTT, Immunological associations of the halo naevus with cutaneous malignant melanoma. Brit. J. Derrn. 88, 127 (1973). J. DAVIDSOHN,L. Y. NI and R. STEJSKAL,Tissue iso-antigens A, B, and H in carcinoma of the stomach. Arch. Path. 92, 456 (1971). E . V . SUGARBAKER and A. M. COHEN, Altered antigenicity in spontaneous pulmonary metastases from an antigenic murine sarcoma. Surgery 72, 155 (1972). E . A . BOYSE, L. J. OLD and S. LU~LL, Antigenic properties of experimental leukemias. II. Immunological studies in vivo with C57BL/6 radiation induced leukemias, or. nat. Cancer Inst. 31, 987 (1963). M . C . RAFV and S. DEPETRIS, Ligand induced redistribution of membrane macromolecules: Some possible implications for cancer. J. clin. Path. 27 (suppl. 7), 31 (1974).
Immunochemotherapy of Human Malignant Melanoma with Chlorambucil-carrying Antibody* T. GHOSE, S. T. NORVELL, A. G U C L U and A. S. MACDONALD Departments of Pathology and Surgery, Faculty of Medicine, Dalhousie University and Victoria General Hospital, Halifax, Nova Scotia, Canada. Abstract-- Heterologous anti-melanoma sera produced against any one patient's tumour cross-react, to varying degrees, with both the cytoplasmic and surface antigens of melanoma cells from other patients. Intravenous injections of cross-reacting antimelanoma antibodies bound to chlorambucil were followed by the regression of a considerable number of skin metastases in a patient with disseminated malignant melanoma. When immunochemotherapy had to be discontinued because of anaphylaxis, many of the metastases which had almost completely regressed, reappeared and grew progressively in spite of chemotherapy. One metastatic nodule which had not regressed and another which appeared during immunochemotherapy contained a much larger proportion of tumour cells lacking the cross-reacting surface antigen, when compared to tumour cells in metastatic nodules excised before immunochemotherapy.
Our study of 52 heterologous anti-sera produced against 18 human malignant melanomas, reveals that human malignant melanoma cells, in contrast to other reports [3], have surface antigens which they share with other human malignant melanoma cells but neither with any normal adult human tissues nor with several other types of h u m a n malignant tumours. As tumour tissue adequate for animal immunization was not available from this patient, cross-reacting anti-melanoma sera were used for immunochemotherapy.
INTRODUCTION
CHLORAMBUCIL can be non-covalently bound to anti-tumour antibodies without losing its alkylating activity or affecting the immunological reactivity of antibodies, and such chlorambucil-bound anti-tumour antibodies are more effective tumour inhibitors than chlorambucil or antibodies alone [1, 2]. We have reported the regression of metastatic nodules in a malignant melanoma patient after i.v. injections of chlorambucil-bound goat anti-melanoma globulin produced against the patient's own tumour [2]. We report here the regression of a number of metastatic nodules in the second melanoma patient treated with chlorambucilbound anti-melanoma globulin.
MATERIAL AND METHODS Anti-human melanoma sera
Surgically excised, malignant melanoma specimens were received in sterile Hank's balanced salt solution at 4°C. After removal of skin, fibro-fatty and other non-neoplastic tissues, tumours were chopped into pieces about 5 mm 3,
Accepted 5 December, 1974. *This work was supported by grants from the National Cancer Institute of Canada and Cancer Research Fund, Faculty of Medicine, Dalhousie University. 321
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T. Ghose, S. T. Norvell, A. Guclu and A. S. Macdonald
washed several times with phosphate buffered saline (PBS, 0.1 M, p H 7.1) at 4°C, and then transferred to Ca and Mg free balanced salt solution containing E D T A [2]. As many dissociated tumour cells as possible were obtained from the tumour pieces by mechanical teasing. T u m o u r cells suspended in 2 ml PBS and mixed with 2 ml of Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan) were injected i.m. into both flanks and both shoulders of adult goats or New Zealand white rabbits ( 4 x 108 viable tumour cells/animal). Identical injections were repeated after 1 week using tumour ceils thawed and washed in PBS after storage in 10 % dimethyl sulfoxide at - 1 7 6 ° C . From one week after the second set of injections, the goats and rabbits were injected i.m. twice a week respectively with 100 mg and 5 mg of the tumour homogenate (prepared with a Waring blender and stored at - 7 0 ° C ) without any adjuvant. Any individual goat or rabbit received tumour materials from only one patient. Three days after the tenth injection of tumour homogenate, 5-10 ml of blood were obtained from all animals (test-bleeding). Testbleeding sera were inactivated (56°C, 0.5 hr), then absorbed with AB red cells and then repeatedly with homogenates of pooled normal h u m a n tissues and tested, thereafter, by immunofluorescence, for specific anti-melanoma activity [2]. I m m u n e sera were obtained from all animals showing titers of 1/64 or above of specific anti-melanoma activity. The resulting sera were inactivated and then successively absorbed with AB red cells and homogenates of normal h u m a n liver, lungs, kidney and spleen. Absorption with every tissue (tissue to serum ratio 1:3 by volume) was carried out at 37°C for 4 hr followed by another 8 hr absorption at 4°C. Absorbed sera were separated from the homogenates by centrifugation (20,000 x g) for 2 hr at 4°C. Absorptions were repeated (usually 2 absorptions each with human liver, spleen and lung homogenates and at least 4 absorptions with kidney homogenate) till the sera reacted only with the immunizing melanoma cells and not with the patient's own normal skin and peripheral blood lymphocytes or with other normal adult human tissues. Specific anti-melanoma sera (i.e. sera which after appropriate absorptions reacted only with malignant melanoma cells) against 18 malignant melanomas were produced in 14 goats and 38 rabbits. Immunofluorescence was performed either on 5/~m cryostat sections of melanoma and other human tissues snapfrozen at - 1 7 6 ° C or on smears of unfixed snap-frozen and thawed tumour cells by the
"sandwich method" using fluorescein isothiocyanate conjugated goat anti-rabbit, rabbit anti-goat or rabbit anti-human globulins with appropriate controls [1, 2]. For membrane immunofluorescence on viable melanoma cells, peripheral blood lymphocytes and cultured skin fibroblasts, the method of Phillips and Lewis [4] was used. Cytotoxicity of sera was assessed by the trypan blue exclusion method [2] using human AB blood group sera and a non-cytotoxic normal rabbit serum as complement sources [2]. Lymphocytes were isolated from sterile peripheral venous blood with carbonyl iron using a mechanical lymphocyte separator. Melanoma cells and normal skin fibroblasts were grown in Falcon flasks using medium 199 supplemented with human serum or 10 % foetal calf serum [2].
Cross-reaction studies All specific anti-melanoma sera were tested by immunofluorescence on smears from each of the 18 malignant melanomas. Five-tenths ml aliquots of four specific goat anti-melanoma sera were further absorbed with increasing amounts of homogenized melanoma cells or normal skin (2.5, and 5 and 10 mg/ml wet weight) from eight different patients. After absorption, increasing dilutions of sera were tested to find the titer of reactivity with cells from these 18 tumours.
Binding of globulins to chlorambucil Varying amounts of chlorambucil were attached to anti-melanoma globulins at a p H of 3 + 0 . 5 when up to 90% of chlorambucil can bind to goat and rabbit globulins as observed by us [1, 2] and confirmed using similar procedures by others [5].
RESULTS
Cross reactivity of the melanoma cytoplasmic antigen All specific anti-melanoma sera reacted by immunofluorescence with the cytoplasm of melanoma cells from most but not all malignant melanomas. With a few exceptions (e.g., tumour from E.R., Table 1) melanoma cells usually reacted maximally with the specific sera produced against the test tnmour and less with sera against other melanomas. When the reactivity of antl-melanoma sera with ceils from the immunizing tumour were absorbed out with just enough homogenate of the
Fig. 1. Fluorescence photomicrograph of a snap-frozen and thawed smear of malignant melanoma cells from patient D.A. stained by the "sandwich" method with a goat anti-melanoma serum (produced against M . M . ' s tumor) absorbed with AB red cells, normal human tissue homogenates and malignant melanoma cell homogenate from S.F. (5 mg tumor homogenate/0.5 ml. serum). About half of the cells show cytoplasmic staining. Dark nuclear outline of unstained cells (marked with arrows) can also be seen. x 280. Fig. 2. Fluorescence photomicrograph of a suspension of patient T.N.'s viable melanoma cells exposed first to a chlorambucil-bound cross-reacting goat-antimelanoma globulin and then to fluorescinated rabbit antigoat globulin. Discrete granular membrane staining of the viable melanoma cells shows that the anti-melanoma globulin retained its reactivity with the surface antigen of melanoma cells after binding with chlorambucil. Similar discrete granular membrane immunofluorescence of viable melanoma cells from this patient was also seen after exposure to the cross-reacting anti-melanoma globulins not bound to chlorambucil, x 800.
(to face p. 322)
Fig. 3. Photomicrograph of a section from the recurrent lesion in the patient's right heel showing cords and nests of round, o~,oid and polyhedral cells with relatively large and hyperchromatic nuclei, x 2 0 0 . Fig. 4. Photographs of the five metastatic nodules on the anterior aspect of the right thigh of T.N., which were measured every week and photographed. A : just before immunochemotherapy, B: four months after the beginning of immunochemotherapy, C: six months after the beginning of immunochemotherapy, D: eight months after the beginning of immunochemotherapy, E: is the photograph of the right inguinal region of this patient taken six months after the beginning of immunochemotherapy showing depigmentation after regression of metastatic melanoma nodules. A total of about thirty metastatic nodules regressed during immunochemotherapy.
Immunochemotherapy of Human Malignant Melanoma i m m u n i z i n g t u m o u r , they did not react with a n y o t h e r m a l i g n a n t m e l a n o m a cells. Absorption with a p p r o p r i a t e a m o u n t s o f h o m o g e n a t e o f the m a j o r i t y o f the cross-reacting t u m o u r s r e m o v e d the serum's reactivity with the t u m o u r used for absorption, b u t only r e d u c e d the reactivity o f the serum with the i m m u n i z i n g t u m o u r or with o t h e r t u m o u r s with w h i c h the serum r e a c t e d m o r e strongly t h a n the t u m o u r used for absorption ( T a b l e 1). Occasionally, cross-absorption with a p a r t i c u l a r t u m o u r h o m o g e n a t e either virtually did not affect the reactivity o f the serum with some o f the o t h e r cross-reacting t u m o u r s or revealed two populations o f t u m o u r cells; one reacting
323
m e m b r a n e i m m u n o f l u o r e s c e n c e on m e l a n o m a cells (but not on the same patient's lymphocytes or fibroblasts) from 6 out o f 7 m e l a n o m a s d e m o n s t r a t e d b y each o f these 4 anti-sera (none raised against the 7 test tumours) to possess a c o m m o n cytoplasmic antigen. V i a b l e cells from two i m m u n i z i n g m e l a n o m a s were 4 a n d 9 times m o r e effective t h a n the cells from two cross-reacting m e l a n o m a s in absorbing out the m e m b r a n e i m m u n o f l u o r e s c e n c e reactivity o f two a n t i - m a l e n o m a sera with cells from their respective i m m u n i z i n g t u m o u r . Prior exposure to autologous m e m b r a n e localizing a n t i b o d y from two patients could partially (though not completely) block the reactivity of the t u m o u r
Table I. Comparison of the reactivity of a goat anti-melanoma serum with smears of melanoma cellsfrom eight different patients, before and after absorption of this serum with tumour (10 mg tumour homogenate/ml serum) from thesepatients. This serum was produced against patient M.M.' s malignant melanoma and was rendered tumour-specific (i.e., the serum reacted with M.M.'s melanoma cells but not with ( i) cryostat sections of M.M.'s skin (ii) M.M.'s skin fibroblasts in vitro (iii) M.M.'s peripheral leukocytes and (iv) cryostat sections of other normal adult human tissues), before this cross-reactivity study, by absorptions with human AB red cells (X1), and homogenates of normal human spleen (X2), liver (X2), lungs (X2) and kidney (X4)
Tr. cells from :
Reciprocals of the highest dilution of the serum which reacted by immunofluorescence with melanoma cells after absorption of the serum with tumour homogenate from:
M.M. E.R. D.A.
NONE > 1024 > 1024 1024
O.S. S.B. H.R. ILS. S.F.
1024 512 256 256 64
M.M. ---. . --.
E.R. 4 --. .
D.A. 128 32 -. .
--.
.
. . 16 4 .
O.S. 64 64 32
S.B. 64 32 --
32 --
8 --
. . .
H.R. 64 128 4
H.S. S.F. 128 256 256 128 64 32 50% cells 50%cells approx, approx. 4
m
m
128 --
32 4
4
(-- No reactivity detectable by immunofluorescence).
a n d the o t h e r not reacting with the absorbed serum (e.g., cells from D.A. in T a b l e 1 and Fig. 1). Absorptions o f these sera with c o r r e s p o n d i n g a m o u n t s o f n o r m a l skin h o m o g e n a t e and frozen t h a w e d p e r i p h e r a l blood l y m p h o c y t e s from these patients did not affect their reactivity with m e l a n o m a cells.
Cross-reactivity of melanoma cell surface antigens Membrane-immunofluorescence. All specific antim e l a n o m a sera showed discrete g r a n u l a r staining on the surface o f viable m e l a n o m a cells from the i m m u n i z i n g t u m o u r . O n e or m o r e of the 4 a n t i - m e l a n o m a sera tested revealed
cells with the heterologous m e m b r a n e localizing antibodies. N o n e o f the specific a n t i - m e l a n o m a sera r e a c t e d with either the smear or with the viable t u m o u r cell suspensions from 4 h u m a n pulm o n a r y squamous cell carcinomas, 2 h y p e r n e p h r o m a s a n d 2 breast a d e n o c a r c i n o m a s . T h o u g h all the sera prior to absorption were cytotoxic to m e l a n o m a cells and to cultures o f a n u m b e r of cell lines of h u m a n origin, no cytotoxic activity could be detected in these sera after being r e n d e r e d m e l a n o m a specific b y absorption with AB red cells a n d h u m a n o r g a n homogenates.
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T. Ghose, S. 7". Norvell, A. Guclu and A. S. Macdonald
The case history of a melanoma patient treated by immunochemotherapy is given below: Ten months prior to admission T.N., a 69 year old white male, had noticed a "wart" on his right heel, and four months later this lesion (not histologically examined) was excised. At admission, T.N. had an ulcer containing two reddish black heaped-up spherical nodules measuring 17 mm and 8 mm in their longest diameters, at the site of excision which had never healed. An incisional biopsy showed cords and nests of round and polygonal neoplastic cells with hyperchromatic nuclei and increased mitotic activity. Some of the cells contained a small amount of melanin (Fig. 3). After excision of the ulcer twelve days later, histology revealed tumour invasion of the lymphatics in the midcorium. Enlarged lymph nodes in the right groin were removed by radical dissection 25 days later. Several subinguinal nodes contained metastatic tumour. Examination 8 months later revealed histologically proven nodular recurrences of melanoma in the healed inguinal scar, an indurated mass above the inguinal ligament and edema of the right lower limb and external genitalia. Two months later about 30 metastatic nodules varying from the size of a pin head to spheres about 1 cm din appeared in or under the skin of his right inguinal region, pubic ramus, the scrotum and the right lower limb. The patient at this time, did not react to the second strength of PPD (Connaught Laboratories, Toronto). Suspension of viable melanoma cells obtained from two metastatic nodules were tested for cytoplasmic and membrane immunofluorescence against a number of goat and rabbit anti-melanoma sera produced against malignant melanoma from other patients. One goat serum and two rabbit sera showed fairly strong (i.e., titer 1/128) cytoplasmic and membrane immunofluorescence. When bound to ehlorambuell, these anti-melanoma globulins retained alkylating activity as assessed by the Epstein method [6] as well as their reactivity with tumour cells (Fig. 2). The patient was injected i.v. with a total amount of 429 mg of?-globulin bound to 41 mg of chlorambucil through nine separate injections administered on alternate days. Five subcutaneous metastases on the anterior aspect of his right thigh were measured weekly. The two largest diameters of these five nodules approximately at right angles to one another just before the beginning of treatment, were 1 - 4 x l . 2 cm, 1 - 5 x l . 2 cm, 1-2 x 1-1 cm and 1.8 x 1-2 cm. About 8 weeks after completion of the injections all of these
five nodules and about 20 other nodules which were not regularly measured became smaller and after about 4 months, four of the five metastatic nodules under regular measurement and at least twenty other nodules in the inguinal region and the scrotum almost completely regressed (Fig. 4). Depigmentation was noticed around the regressing melanoma nodules. A month later, a new crop of nine metastases appeared in or under the skin. The patient again received, through 16 i.v. injections, a total of 800 mg of a cross-reacting goat antimelanoma globulin bound to 80 mg ofchlorambucil. The patient had a mild anaphylactic reaction after the last injection and all injections of goat gamma-globulin were stopped. From 7 days after the anaphylactic reaction, the patient received three i.v. injections containing a total of 175 mg of cross-reacting rabbit gamma-globulin bound to 17.5 mg of chlorambucil. However, after the last injection, the patient developed anaphylactic reactions to rabbit globulin as well and immunochemotherapy was stopped. During this period of immunochemotherapy, though, at least five other metastatic nodules completely disappeared leaving areas of cutaneous depigmentation, and those nodules which had already regressed did not reappear, four new metastatic nodules emerged as well. One nodule on the anterior aspect of his right thigh which was being measured regularly and which did never completely regress and another recent metastatic nodule were excised. Less than 5 ~ cells from these two nodules reacted by membrane imnmnofluoresccnce with the rabbit and goat anti-melanoma globulins with which the patient was treated. At least 6 0 ~ tumour cells in metastatic nodules excised prior to immunochemotherapy and stored in liquid nitrogen, showed membrane immunofluorescence when concurrently tested with these anti-melanoma globulins. About 80-90 ~ of the stored melanoma cells and about the same proportion of cells from these excised nodules showed cytoplasmic immunofluorescence with these three anti-melanoma globulins. The patient did not have at any time membrane localizing or cytoplasmic antibody or cytotoxic lymphocytes against his own tumour cells nor could any anti-rabbit or anti-goat globulin be detected in his serum by immunodiffusion. After the stoppage of immunochemotherapy, in spite of treatment with large doses of BCG and a number of chemotherapeutic agents including DTIC, there has not been any further regression of tumour nodules. Existing
Immunochemotherapy of Human Malignant Melanoma nodules have increased in size and a few have fungated. DISCUSSION
T h e melanoma specificity of the appropriately absorbed heterologous anti-melanoma sera is supported by: 1) the absence of reaction by membrane or cytoplasmic immunofluorescence either with cryostat sections of the respective patients' own skin or with the smears and suspensions of viable autologous peripheral blood lymphocytes and cultures of autologous skin fibroblasts from these patients, 2) absorption of the reactivity of these sera with appropriate amounts of the immunizing or other cross-reacting melanoma tissues but neither with the corresponding amounts ofhomogenates of normal skin nor with skirt fibroblasts or peripheral blood lymphocytes from these patients and, 3) absence of reactivity either with smears or viable cell suspensions from several h u m a n malignant tumours other than melanoma. The tumour-specific melanoma cell-cytoplasmic antigen cross-reacts with sera from other malignant melanoma patients [3, 7-9]. T h o u g h Lewis et al. [3] are of the opinion that the melanoma cell-surface antigen which reacts with autologous serum is individual specific, other studies using membrane immunofluorescence [7, 11], cytotoxicity tests [11, 12] or lymphocytes [10, 13, 14] support our observation that there are tumour-specific cross-reacting antigens on the surface of h u m a n malignant melanoma cells. As prior exposure to the autologous membrane localizing antibody partially inhibited the reactivity of viable melanoma cells to the heterologous cellsurface localizing antibody, the antigen on the surface of malignant melanoma cells reacting with these two antibodies appears to be similar. Our observation in several mouse t u m o u r models, that chlorambucil or l a t i bound antibodies consistently cause m u c h more tumour inhibition than antibody, chlorambucil or 131I alone [1, 2, 15, 16] has now been confirmed by others [5, 17, 18]. However, as discussed by us before [2], the precise mechanism of increased tumour inhibition by antibodies attached to cytotoxic drugs or ~311 remains to be elucidated and could be explained on the basis of: (a) synergism between anti-tumour antibody and cytotoxic drugs [5, 18] or ionizing radiations [19], (b) antibody mediated preferential localization of cell damaging agents in t u m o u r tissue [16] and (c) both [2]. None of the anti-melanoma globulins used in the treatment of this patient was cytotoxic B
325
to his t u m o u r cells and it is unlikely that this antibody by itself caused tumour regression. Passive transfer of serum or plasma from cured patients has failed to cause regression of disseminated melanoma [20]. The complete or partial regression of a considerable n u m b e r of metastatic melanoma nodules during immunochemotherapy (but at no time before immunochemotherapy) and the re-emergence and progressive growth of metastases after the cessation of i m m u n o c h e m o t h e r a p y but in spite of continuing chemotherapy and BCG administration, supports the contention that the regression of these metastases was not spontaneous but causally associated with immunochemotherapy. Depigmentation has been observed after the regression of melanoma metastases in skin [20, 21]. No anti-melanoma cytoplasmic antibody (reported to be associated with the depigmentation around resolving "halo" nevus [22]) or antibodies against melanin containing cells in normal h u m a n skin could be detected in the serum of this patient before or after the appearance of these areas of depigmentation. The results of our cross-reaction study are in keeping with the concept of antigenic diversity of tumour cells [23, 24] and suggest that the tumour-specific melanoma antigens have mosaics of determinants and that different combinations of these determinants are expressed in different tumours. The considerable decrease in the proportion of surface antigen containing melanoma cells in the metastasis which did not regress and the other which appeared during i m m u n o c h e m o t h e r a p y compared to metastases excised before immunochemotherapy, suggest selective suppression of the surface antigen containing cells and survival and proliferation of the antigen negative cells or antibody induced desensitization resulting either from the loss of antigen i.e., "antigerfic modulation" [25] or from the redistribution of antigen antibody complex on the cell membrane [26]. Such selective proliferation of antigen negative and/or drug-resistant t u m o u r cells might be an impediment to the effective eradication of tumour cells by immunochemotherapy. Development of anaphylaxis against foreign globulins constitutes another limitation o f i m m u n o c h e m o t h e r a p y . At present, attempts are being made to remove the antigenic Fc fragment of the immunoglobulin molecules prior to binding with chlorambucil. Acknowledgement--We are grateful to Ms. Burroughs Wellcome and Co. for the free gift of chlorambucil (leukeran) and to Mrs. M. M a m m e n , P. Seaman and R. Sekaran for technical help.
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