Serotherapy of Malignant Disease

Symposium on Immunotherapy in Malignant Disease

Serotherapy of Malignant Disease Peter W. Wright, M.D.,* Karl Erik Hellstrom, M.D.,** Ingegerd E. Hellstrom, M.D.,t and Irwin D. Bernstein, M.D.+

With the accumulation of evidence for! a dominant role of cellular immune mechanisms in tumor rejection,lO, 39 and the observation that humoral antibody may enhance rather than inhibit tumor growth,40, 48 the potential immunotherapeutic role of antibodies directed against tumorassociated or histocompatibility antigens expressed on tumor cells has been perhaps prematurely disrhlssed by some, and largely overlooked by others. There remains, however, a considerable body of information in animal tumor model systems that antisera obtained from appropriately immunized donors and given to recipients under defined circumstances can lead unequivocally to inhibition oftumor growth in vivo. This experience in animal model systems is beyond the scope of the present review, and has been discussed elsewhere. 25 ,39 Nevertheless, certain salient animal experiments will be mentioned here since they provide explicit evidence for an antitumor effect of tumor-immune sera in vivo. A reevaluation of the potential therapeutic role of antibodies in the treatment of malignant disease also seems warranted in light of recent findings in basic immunology. These studies have demonstrated a complex interaction between the cellular and immune mechanisms that has been previously unappreciated. Our current understanding of this interaction is graphically represented in Figure 1. In many systems, tumor cell destruction has been shown to be mediated by specifically immune

From the Division oflmmunology, Fred Hutchinson Cancer Research Center, and the University of Washington, Seattle, Washington *Assistant Professor, Medicine, University of Washington School of Medicine; Assistant Member, Fred Hutchinson Cancer Research Center; Associate Staff, University Hospital "Professor, Pathology, University of Washington School of Medicine; Member, Fred Hutchinson Cancer Research Center tProfessor, Microbiology and Immunology, University of Washington; Member, Fred Hutchinson Cancer Research Center :j:Assistant Professor, Pediatrics, University of Washington School of Medicine; Assistant Member, Fred Hutchinson Cancer Research Center; Attending Staff, Children's Orthopedic Hospital and Medical Center Supported by Contract No. N01-CB-23887, awarded by the National Cancer Institute, National Institutes of Health.

Medical Clinics of North America-Vol. 60, No. 3, May 1976

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LYMPHOCYTE EFFECTOR CELL

TUMOR TARGET CELLS

* ~~~ 4-,'ft.. Antigen-Antibody complexes

y Free Antibody

'T blocked by Antigen

~". , by Complex

Killer T Cell

Killer K Cell Armed by Antibody

Figure 1. Tumor cell distribution may be mediated by effector T-lymphocytes or Klymphocytes. T-lymphocytes may be inhibited by soluble tumor antigen or antigen-antibody complexes.

T-Iymphocytes, which react with tumor target cells through an interaction of antibody-like receptor molecules present on the lymphocyte and tumor antigens displayed on the surface ofthe tumor cells,l° Studies from this laboratory have shown that serum "blocking" factors, presumably antigen or antigen-antibody complexes detected in tumor-bearing animals or patients, can selectively inhibit the cytotoxic effect of immune lymphocytes which could otherwise result in the destruction of tumor cells in vitro. 38 . 39 Serum "unblocking" factors, detected following tumor immunization in animals 6,31 and tumor rejection in man,35 have been shown to have an opposite effect. Unblocking factors have been demonstrated to counteract the activity of blocking serum in vitro, and lead to inhibition oftumor growth in vivo. 6 Lymphocyte-dependent antibodies which combine with effector lymphocytes through the Fe portion of the immunoglobulin molecule, have been shown to "arm" normallymphocytes and potentiate the activity of immune lymphocytes. These lymphocyte-dependent antibodies will destroy tumor cells in vitro,65, 69 and may also inhibit tumor growth in vivo. 43 Humoral antibodies and serum complement have also been shown to be cytotoxic for tumor cells in vitro,9. 80 and there is indirect evidence that complement-dependent antibodies may also be cytotoxic for tumor cells in vivo. 59 Two approaches have generally been adopted for the production of tumor-immune antisera for serotherapy trials. In the first, xenogeneic antisera have been prepared by immunization of species other than that of the tumor cell donor. This approach is technically difficult, requiring extensive adsorption to remove nontumor antibodies, and is potentially dangerous because of the frequent development of reactivity to foreign serum proteins. In the second, allogeneic antisera have been produced by immunization of animals of the same species as the tumor graft donors. Unless animals of the same inbred strain are utilized, this method will also result in the production of antibodies directed at normal histocompatibility antigens, as well as tumor-associated antigens. Nevertheless, the administration of alloantisera has generally been associated with no or minimal complications.

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SEROTHERAPY IN ANIMAL TUMOR MODEL SYSTEMS The effect of antiserum treatment on the growth of allogeneic tumors has been extensively investigated. 40,48 In experiments done largely in the 1950's, it was demonstrated that both active and passive immunization could result in either enhancement or inhibition oftumor growth, depending upon the conditions of the immunization. Fractionation by column chromatography of hyperimmune mouse sera produced against allogeneic tumor cell antigens demonstrated that immunoglobulin G was responsible for eIlhancement, and immunoglobulin M for inhibition, of tumor growth in vivo. 75 More recent experiments have suggested, however, that under certain circumstances IgM antibodies may also resultin enhancement and IgG antibodies in inhibition of tumor growth.1 8. 26 Similar evidence demonstrating heterogeneity ofthe tumor antibody response has also been obtained in studies done in syngeneic tumor systems. For example, it was demonstrated that immunization of mice with crude membrane preparations from syngeneic, spontaneous mammary carcinoma cells could result in either increased resistance or increased susceptibility to a subsequent challenge with tumor grafts, and that these effects could be passively transferred by serum. 2 Antisera obtained from mice hyperimmunized with syngeneic methylcholanthrene-induced tumors were also shown capable of retarding or accelerating syngeneic tumor growth. 12. 56 Fractionation of these tumor antisera indicated that the 19S fractions (presumably IgM) were primarily responsible for the induction oftumorresistance, and that the 7S fractions (presumably IgG) were primarily responsible for the induction of tumor enhancement. l l Treatment of animals with immune serum under carefully controlled conditions has been shown to predictably result in the inhibition of growth for a variety oftumors oflymphoid origin. For example, Old et al. 61 demonstrated that passive immunization with antisera prepared against syngeneic Gross virus-positive leukemia cells in inbred rats protected mice against the growth of a syngeneic Gross virus-induced leukemia. The protective effects of serum treatment were dose related, and significant inhibitory effects of the antiserum could be seen as late as 3 days after intravenous inoculation of leukemia cells. Under certain circumstances, 100 per cent of serum treated mice could be completely protected by immune serum treatment. These protected survivors were later shown to be resistant to subsequent challenge with an otherwise lethal inoculum of tumor cells. The activity of the antiserum used was determined by measurement of antibodies cytotoxic to Gross virusinduced leukemia cells, both before and after administration. All serum treated mice developed significant antibody titers except those animals with advanced leukemia in whom free antibody was no longer present, even when these mice had received antibody the day before. 61 Inhibitory effects oftumor growth by serum have also been reported for other experimental lymphoid and hematopoietic tumor models, such as EL4,21. 28 L1210,55, 67, 71 Friend leukemia virus (FLV)-induced leukemia54 ,77 and Moloney leukemia virus (MLV)-induced leukemia,63, 64 and mouse myeloma. 82

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In a recent study reported by Hardy et al., the administration of serum from normal mice was shown to significantly retard the development of leukemias induced by murine leukemia virus. The explanation for this protective effect of normal serum was not clear, but it may be due to the presence of serum complement or antibodies naturally formed against leukemia viruses. 30 Immune serum has also been shown to inhibit the growth of several nonlymphoid tumors, including ovarian carcinoma,62 melanoma,44 and polyoma virus-induced fibrosarcomas. 59 In general, effects of serum treatment have been observed only in animals with a minimal tumor burden, and when antiserum was administered before the appearance of evident tumor. Two exceptions to this rule have been reported, however, and will be considered here in detail. Treatment of young BALB/c mice bearing small but palpable primary Moloney sarcoma virus (MSV)-induced tumors, with serum from adult BALB/c or (BALB/c x DBA)F 1 mice whose autochthonous tumors had regressed, resulted in complete tumor regression in approximately 30 to 40 per cent oftreated animals. 24 More recent in vitro studies have indicated that sera from regressor mice contain complement-dependent cytotoxic antibody, unblocking antibody, and lymphocyte-dependent antibody. Any or all of these antibodies may play a role in inhibition oftumor growth. In addition, since the growth of tumors induced by Moloney sarcoma virus may involve continuous transformation and recruitment of neoplastic cells by infectious virus released from a tumor, the therapeutic effect of immune serum could be due to its virus-neutralizing activity. 52 An antitumor effect of serum treatment has also been shown in rats bearing primary or transplanted sarcomas induced by polyoma virus. Sjogren and co-workers have demonstrated a correlation between antitumor immune parameters in vitro, and tumor growth in vivo. 7 , 70 W/Fu rats bearing primary or transplantable syngeneic polyoma virus-induced tumors develop specific lymphocyte-mediated cytotoxicity to polyoma virus-induced tumor target cells in vitro. Serum from tumor-bearing animals was shown to selectively block the cytotoxic effect of immune lymphocytes. Significant blocking activity was detected before the appearance of palpable tumor. Blocking activity was shown to persist and increase with progressive tumor growth, and disappear rapidly following tumor excision. 7 Inoculation of serum with blocking activity in vitro was also demonstrated to facilitate polyoma virus-induced tumor growth in vivo. 4 The immunization of BCG-primed W/Fu rats or rabbits with polyoma virus-induced W/Fu tumor cells resulted in the formation of unblocking serum factors which could counteract the effect of specific blocking sera in vitro; these factors were found to be immunoglobulins specifically reactive with polyoma virus tumor-associated antigens. The administration of an unblocking antipolyoma serum was shown to result in the regression of a syngeneic polyoma virus-induced tumor cell transplant in 4 out of 5 inoculated animals. The effect of treatment with unblocking serum was specific in that all control rats (4 out of 4) receiving inoculations of MCA-sarcoma serum developed progressively growing tumors. 5

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Unblocking antipolyoma serum was also reported to result in inhibition of growth of primary polyoma virus-induced tumors in W/Fu rats. 6 In these experiments, W/Fu rats were inoculated with polyoma virus within 24 hours of birth. At approximately 30 to 40 days of age, when primary kidney sarcomas became grossly palpable, the animals were explored and their tumors and kidneys inspected, measured, and photographed. Rats were subsequently treated with either unblocking rat serum or normal CW/Fu) rat serum. The effects of serum inoculation were monitored using in vitro tests of serum blocking and unblocking activity. Serum blocking activity decreased or disappeared, and serum unblocking activity was detected in some experimental rats treated with unblocking serum. The survival time of 9 of 11 rats treated with unblocking serum was significantly prolonged when compared to controlrats. In additional experiments, inoculation of unblocking sera was also shown to potentiate the effect of BCG therapy, and significantly delay the appearance of pulmonary metastases in W/Fu rats inoculated with syngeneic, transplantable, polyoma virus-induced tumors.8 In each of these experiments, the unblocking antipolyoma serum was pretested for anti-tumor activity in vitro, and these same in vitro assays were used to monitor the effects of immune serum treatment in vivo. Sufficient unblocking antipolyoma tumor serum was administered to result in the decrease or disappearance of serum blocking activity and the appearance of unblocking activity in the serum recipient. Since rat tumors induced by polyoma virus fail to release infectious virus, and because antisera produced against polyoma viral antigens are not cytotoxic to tumors induced by polyoma virus,29 the effects of the unblocking polyoma sera used in these experiments are presumably due to antibodies directed at polyoma virus tumor-associated antigens rather than polyoma virion antigens.

HUMAN IMMUNOTHERAPY A variety of clinical observations has suggested the importance of immunologic host factors in tumor growth. These include, for certain malignancies, well-documented cases of spontaneous regression or of regression following minimal or inadequate therapy, 22 and examples of prolonged remission of several years' duration before ultimate relapse. 73 • 78 More recently, evidence of a host immune response to tumorassociated antigens has come from in vitro tests done in the laboratory. Circulating antibodies which react with autologous and allogeneic tumor cells have been demonstrated by a variety of techniques. Specific cellular immunity has been shown by delayed cutaneous hypersensitivity in vivo, and by a variety of techniques in vitro, most notably the microcytotoxicity assay and the macrophage migration inhibition test. This evidence has been extensively reviewed. 39 The first published studies of attempted serotherapy in the treatment of malignancy were reported in the late nineteenth century. In general, these attempts involved the production of xenogeneic antisera in animals immunized with human tumor cells. Both objective as well as subjective

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benefits of antiserum administration were claimed in these early articles. For example, Lindstr6mm produced antisera in rabbits by immunization with cells from patients with acute myelogenous leukemia. Four of 11 patients receiving "myelotoxic sera" with splenic irradiation and arsenic achieved clinical remission of several months' duration. 53 Hueper and Russell inoculated rabbits with buffy coat cells from a patient with chronic myelogenous leukemia. The antiserum produced was shown to be cytotoxic for lymphocytes in vitro. When administered to the leukemic cell donor, the serum resulted in a significant decrease in the peripheral leukocyte count, and some decrease in spleen size. Significant complications of serum therapy, including erythematous skin eruptions, and unconsciousness of unknown cause, were described.47 The results of these early attempts at serotherapy have been recently reviewed in detail. 3.19.57 Altogether, it seems fair to conclude that in none of these reports were effects of unequivocal benefit observed that could be ascribed to treatment with serum alone. In more modern times, Murray has reported therapy of more than 60 patients with metastatic breast carcinoma with antiserum produced in horses immunized with human breast cancer tissue. Relief of bone pain, recalcification of osteolytic metastatic lesions, regression of intracranial and pulmonary metastases, and resolution of pleural effusions were reported with minimal complications of therapy.58 DeCarvalho produced antisera in horses hyperimmunized with a tumor or leukemic tissue "cocktail." Treatment with these sera was reported to result in objective remission in 11 of 15 patients with leukemia and significant benefits, including objective tumor regression, in 15 of 16 patients with a variety of solid tumors. 20 Sumner and Foraker reported that the transfusion of250 ml of whole blood from a patient previously reported to have undergone a spontaneous regression of malignant melanoma resulted in complete regression of all evident tumor in a second patient with malignant melanoma. 74 Additional sporadic reports suggesting a possible benefit of serotherapy have also been reported, but remain unsubstantiated. The fact that these reports have not been confirmed in the 12 to 16 years since they were published suggests that the approach used in these studies was not as uncomplicated, and the results of therapy were not as dramatic, as originally described. The remainder of this review will be concerned with more recent and more systematic trials of serum therapy. In most ofthese studies, the effects of serotherapy were monitored by simultaneous in vitro tests, and/or compared to an untreated control population.

Leukemia and Lymphoma Patients with leukemia and/or lymphoma have been treated with antisera obtained from human donors immunized either against histocompatibility antigens or putative leukemia-associated antigens. Laszlo et al. 51 treated patients with chronic lymphocytic leukemia by infusion of plasma obtained from volunteer donors immunized against normallymphocytes. 51 Hyperimmune donor plasma was shown to contain cytotoxic antibody by an in vitro assay. Multiple injections of 5 different cytotoxic plasma preparations were administered in volumes rang-

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ing from 20 to 300 ml to 3 patients with chronic lymphocytic leukemia. A prompt fall in the number ofleukemic lymphocytes in the peripheral blood was generally observed within 24 hours following infusion. After most serum injections, the effect observed was transient, with return of peripheral blood counts to preinfusion levels within a few days. In one patient, however, the effect of plasma infusion on the peripheralleukemic cell count was of greater duration, reportedly lasting for several weeks. The fall in the white cell count was associated with a decrease in lymph node size. There was no significant change in the red cell or platelet count in treated patients. A change in bone marrow status was not reported. The turnover rate of labelled alloantibody (IgG) was significantly more rapid than that of normal IgG. Plasma infusion had no effect when given to a single patient in blastic crisis of acute myelogenous leukemia. Similar findings were later reported by Herberman et al. 42 Two of 3 patients with chronic lymphocytic leukemia receiving intravenous infusion of cytotoxic alloantisera demonstrated a rapid decrease in peripheral leukocyte count (less than 50 per cent of preinfusion levels), and a decrease in lymph node diameter (50 to 90 per cent). Effects of serum treatment on the leukocyte count were transient, however, with a return to preinfusion values reported within 12 to 24 hours. A comparable response to plasma infusion was observed in only 1 of 4 patients with lymphoma. Administration of the alloantisera, in volumes up to 50 ml, were associated with significant complications in a majority of patients. Chills, fever, and nausea and vomiting were commonly reported. Two patients with leukocyte counts of over 70,000 per cu mm, developed marked pulmonary distress. These studies demonstrated that although alloantisera can transiently reduce leukocyte counts and peripheral node size in some patients, their administration is also associated with potentially serious complications. Skurkovich et al. 68 treated 10 patients with acute leukemia in remission with repeated infusion of autologous plasma and autologous leukocytes. A significant increase in remission duration was seen in the immunotherapy group (491± days) when compared to an historical control group (171± days).68 Contrary to their findings, however, Albo et al. 1 reported results obtained by the Children's Cooperative Group A, who showed no significant effect of parental plasma in patients with acute lymphocytic leukemia in remission. 1 In general, the effects of serotherapy in leukemia, although definite, have been oflimited duration, and judged to be of little or no clinical significance.

Burkitt's Lymphoma Burkitt's tumor, an undifferentiated malignant lymphoma with distinct clinical and pathological features was first described among children in East Africa in 1958.1 3 Well-documented cases of spontaneous remission, 16 long-term remissions following chemotherapy, 14, 17 as well as the results of direct immunologic assays of Burkitt's lymphoma cells tested in vitr0 50 have suggested that immunologic host factors play a significant role in the pathogenesis of this disease. The evidence of humoral and cellular immunity to Burkitt's lymphoma-associated antigens has been the subject of several recent review articles. 41,49

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N gu reported general clinical improvement and objective tumor re- . gression in a 4 year old boy treated with plasma and blood from a 10 year old girl in complete clinical remission 4 years after treatment of Burkitt's lymphoma with systemic chemotherapy. GO A similar experience was noted by Burkitt et al. 15 These anecdotal studies prompted a controlled trial to evaluate the possible clinical effectiveness of serotherapy.23 In this trial, 8 patients with previously untreated Burkitt's lymphoma were given plasma from patients with Burkitt's lymphoma in remission or from normal volunteers. Remission plasma was shown to have antibody activity against Epstein-Barr virus and against Burkitt's lymphoma cells in vitro. Control plasma lacked this activity. Five patients received remission Burkitt plasma; 3 received plasma from normal volunteers. Only 1 recipient had localized (stage I) disease. Plasma was given in a single intravenous dose of 5 ml per kg of body weight. The total plasma administered ranged from 55 to 160 ml. The patients were observed for a 7 day period. In the group of patients who received Burkitt's remission plasma, tumors enlarged in 2, and remained unchanged in 2. The response in 1 patient could not be evaluated because of chemotherapy given 24 hours after plasma infusion. In the group of3 patients who received control plasma, tumors enlarged in 2, and remained unchanged in 1. Anti-EB virus antibody was detected in the serum of each Burkitt remission plasma recipient. No correlation between the patient's clinical course and antibody tit er was observed. The ultimate fate and survival of the patients was not reported. Because of the fact that so few patients were included in this study, because only 1 patient had localized disease, and because only a small amount of plasma was given, any conclusions concerning serotherapy in Burkitt's lymphoma would be premature. Clearly, studies with larger numbers of patients with more limited disease, given larger amounts of plasma over a longer period of time will be needed to determine the potential efficacy of serotherapy for this disease.

Clear-Cell Carcinoma of the Kidney Evidence for the role of immunologic host factors in clear-cell carcinoma (hypernephroma) of the kidney has been recently reviewed. 45 Regression of pulmonary metastases following excision of a primary renal carcinoma has been well-documented. 22,27 Similarly, regression of established pulmonary metastases following treatment with hormones has also been reported, although the incidence of an objective response with this treatment modality is probably less than 10 per cent. GG Metastatic clear-cell carcinoma has generally been reported to be refractory to treatment with standard chemotherapeutic agents.79 Experience with immunotherapy of hypernephroma has been limited. Studies involving 4 patients in one family, all with clear-cell carcinoma,32, 4G have been sufficiently dramatic and well-documented, however, to warrant further elaboration. One patient, H.H., who developed pulmonary metastases 16 months after nephrectomy and subsequently underwent thoracotomy and left upper lobe lobectomy demonstrating evidence of multiple pulmonary metastases and extensive regional lymph node involvement, was treated with remission plasma from an uncle who

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had himself undergone nephrectomy for clear-cell carcinoma 2 years before, and who had no evidence of recurrence. Approximately 500 ml of plasma were administered intravenously each week for 12 weeks and then biweekly thereafter. This patient remained clinically free of tumor for a period of 20 months following the onset of therapy, at which time he developed a cerebral metastasis with which he ultimately died, 6 months later. At autopsy, there was no evidence of tumor outside the central nervous system, except for a 15 mm metastatic lesion in the diaphragm. Another patient, B.S., was similarly treated with the inoculation of plasma from a fourth family member clinically free of tumor following surgical removal of a clear-cell carcinoma of the kidney. Plasma therapy was begun when the patient had radiologic ally detected pulmonary metastases which were considered-too extensive for surgical resection. Complete regression of 1 of 3 pulmonary nodules was detected during a 6 month period. The other 2 nodules remained stable. The patient received plasma as her only therapy at this time. Approximately 9 months following the onset of plasma treatment, this patient too developed central nervous system metastases. She was given x-irradiation and then transfer factor therapy. She clinically improved over a course of 10 months, but ultimately died 2 years later. The two remaining pulmonary metastases were unchanged throughout the period of her therapy. The effects of serum therapy in these patients was monitored by serial tests of cell-mediated immunity and serum blocking activity to tumor cell antigens in vitro. Serum obtained from the recipients before the onset of immunotherapy contained significant serum blocking activity. The donor remission plasma was demonstrated to have specific unblocking activity. Sera obtained from the immunotherapy recipients at a time when they were judged to have no or stable tumor showed unblocking activity. Sera taken when these patients had clinically detectable metastases to the central nervous system again had blocking activity. The experience with these 2 patients is in many ways parallel to the experience described previously for immunotherapy of rats bearing polyoma-induced tumors. Any conclusions drawn from this latter experience must be tempered with the realization that only 2 patients from one family were investigated. The clinical benefits of serotherapy, if any, were obviously of limited value in that both patients ultimately died with tumor. A larger number of patients, including patients given control therapy, must be evaluated before any conclusions can be drawn with regard to the efficacy of serotherapy. In addition, a larger number of in vitro tests done at more frequent intervals would be necessary to establish the relationship between serum administration and the patient's response to therapy.

Malignant Melanoma Evidence for a host response to tumor-associated antigens in malignant melanoma has been mentioned above, and a summary of various studies showing humoral and cellular immunity to melanoma has recently been published. 38 A variety of approaches to the immunotherapy of melanoma have been adopted and are discussed elsewhere in this volume.

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The earlier report of Sumner and Foraker demonstrating complete regression of metastatic melanoma in a patient receiving 250 ml of whole blood from a patient who had previously undergone a spontaneous regression of melanoma has been mentioned above. 74 A similar experience has been reported by Teimourian and McKuhn who described partial regression of pulmonary metastases in a patient transfused with blood (amount unspecified) from a donor with a melanoma of the back and bilateral axillary metastases in remission 10 years following surgery.76 These sporadic case reports and the more recent evidence for reactivity to melanoma-associated antigens in vitro have prompted us to undertake a more systematic evaluation of serotherapy in patients with malignant melanoma. The design of these studies was based on the observations that lymphocytes from melanoma patients usually show significant cytotoxicity against melanoma target cells in vitro, although the cytotoxic activity of lymphocytes in patients with advanced disease may be quantitatively depressed compared to patients with more localized tumor, or those with no evident disease. 33 Measurement of serum blocking activity has been shown to parallel disease activity.33. 37 Significant serum blocking activity has been detected in a majority of patients with clinically evident disease. By contrast, serum blocking activity has infrequently been present in patients considered to be clinically in remission. The measurement of serum blocking activity has also been shown to be of potential prognostic significance. In one study, in which serum samples were obtained within 3 months following surgery at a time when all patients were judged to be free of evident tumor, 93 per cent of the patients whose serum showed significant blocking activity developed a recurrence within one year. By comparison, 87 per cent ofthe patients whose serum lacked blocking activity following surgery remained disease-free at 1 year. 34 This study and others demonstrating a correlation between the patient's clinical course and the measurement of serum blocking activity have suggested that serum blocking factors which specifically inhibit cellular immune responses in vitro may exert a similar effect in vivo, serving to prevent immunologic destruction of tumor cells. This postulate was strengthened by the demonstration of "unblocking" activity in the serum of a patient who had undergone a spontaneous regression of melanoma. Sera from this patient could be shown to specifically counteract the blocking effect of serum taken from other patients with evident melanoma. 3s The demonstration of unblocking antibody in the serum of a patient who had undergone spontaneous regression of melanoma raised the possibility that transfer of such serum to patients with evident tumor might have some potential therapeutic benefit. The availability of unblocking plasma from melanoma patients was severely limited, however. A serotherapy trial with unblocking plasma could not be seriously considered, therefore, until the chance observation that peripheral blood lymphocytes from many normal, healthy Blacks (North American Negro) were specifically cytotoxic for melanoma target cells in vitro, and that serum from these same Black donors usually had unblocking activity to melanoma-associated antigens. 36 The specificity of the unblocking antibody obtained from Black plasma appeared to be identical

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to that observed in melanoma patients who had undergone spontaneous regression of their tumor. With this background, we undertook a limited feasibility trial to evaluate the potential therapeutic efficacy of the administration of unblocking (Black) plasma in patients with disseminated melanoma. The effects of plasma infusion were systematically monitored using in vitro tests oflymphocyte-mediated cytotoxicity and serum blocking activity to melanoma-associated antigens, in addition to established clinical and laboratory parameters. Seven patients were included in this initial feasibility trial. All patients had evidence of disseminated melanoma, and were judged to be resistant to conventional therapeutic modalities. The patients were treated with BCG administration at scarified skin site~ (Tice strain BCG, approximately 108 colony-forming units per scarification), and with one unit of unblocking (Black) plasma. BCG and plasma were given on alternate weeks. Four patients with advanced disease expired within a few weeks of treatment with no discernible effects of the treatment on either clinical or laboratory parameters. The 3 remaining patients with less advanced disease showed a decrease or disappearance of serum blocking activity, an increase in specific cell-mediated immunity, and the appearance of unblocking activity in their serum. The results of this preliminary trial indicated that the administration of BCG and unblocking (Black) plasma on alternate weeks to patients with a relatively limited tumor burden could be associated with changes in the patient's immunologic reactivity to melanoma-associated antigens in vitro. No significant adverse effects of therapy were encountered. The number of patients involved in this feasibility trial was too small to determine the clinical effectiveness of serotherapy. On the basis of these findings, we have recently undertaken a prospective, randomized, controlled clinical trial of unblocking (Black) plasma in patients with malignant melanoma. Patients include individuals with regional lymph node involvement (stage ll) or disseminated (stage Ill) disease. Patients are randomly allocated to treatment with either (1) BCG and unblocking (Black) plasma; (2) BCG and normal (Caucasian) plasma; or (3) "conventional" therapy. Conventional therapy may include no treatment for patients with stage II disease, or treatment with surgery, radiotherapy, or chemotherapy (but no form of immunotherapy) in patients with stage III disease. The plasma is coded in a "blind" fashion, its source unknown to anyone associated with the clinical trial. This particular design was selected not only because it would presumably allow for an evaluation of the effect of treatment with unblocking (Black) versus normal plasma, but also because it would provide a comparison between groups receiving BCG and no form of immunotherapy. At the time of the preparation of this manuscript, insufficient numbers of patients had been randomized, and insufficient time had elapsed to allow any conclusions concerning the efficacy of these forms of immunotherapy. Immunologic studies performed on patients in this trial, however, have again demonstrated a correlation between the immunologic parameters observed and the patient's clinical course, thus strengthening the impression that these parameters reflect mechanisms of biological importance in tumor growth in vivo.

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CONSIDERATIONS FOR FUTURE SEROTHERAPY TRIALS As noted in the preceding section, although inhibition of tumor growth in vivo by treatment with antiserum has been clearly documented in animal model tumor systems, beneficial effects of serum treatment in man have not been unequivocally established. This conclusion has certain practical implications for future serotherapy trials: 1. Serotherapy in man must be considered experimental. If serum therapy is to be given at all, such treatment must be given under defined, well-controlled circumstances, and administered by individuals experienced in clinical research. As noted in the preceding sections, there have been many sporadic, uncontrolled attempts at serotherapy in the past century. These anecdotal, often emotional reports have provided little information of scientific value. The potential therapeutic efficacy of serotherapy is unknown, and will remain so until competent, controlled clinical trials are conducted. 2. Future serotherapy trials in man should be based on the best available animal model tumor systems. For example, Hershey has recently described methods for producing xenogeneic antisera which were specific after appropriate adsorption for antigens expressed on leukemic, but not normal, rat cells. Administration of this antiserum had a clear antileukemic effect in vivo. 43 The procedures described by Hershey could well be adapted for the production of antisera to human leukemic cells, and would seem to provide intelligent guidelines were such an approach adopted in the treatment of human leukemia. The experience with animal model systems has already provided clear evidence that serotherapy, if there is to be any realistic hope of a clinical effect, must be administered to patients with antigenic tumors with a minimal tumor burden at the time ofimmunotherapy. Presumably to achieve these conditions, serotherapy must be given in conjunction with other treatment modalities, such as surgery, radiotherapy, or chemotherapy. Preliminary attempts have been made in animal model systems to better define the interaction ofimmunologic mechanisms with the effects of chemotherapy and radiotherapy, 25 but additional studies are clearly needed to provide a better understanding of these complex interactions. 3. Future serotherapy trials should include in vitro monitoring. The specific immunologic activity of the serum should be determined prior to, and at various times after, its administration. For example, in the experiments of Bansal and Sjogren described above, the "unblocking" antipolyoma serum was pretested for its unblocking activity prior to its administration to tumor-bearing rats. Following injection, serum recipients were carefully monitored to determine the appearance of unblocking anti-polyoma activity. Sufficient amounts of unblocking anti-polyoma serum were administered to result in the disappearance of blocking activity, and the appearance of unblocking activity in the serum recipients. 6 4. Attempts should be made in the future to better characterize and hopefully concentrate antibodies responsible for anti-tumor effects in vivo. As noted above, antisera obtained from tumor-immunemice have

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been shown to contain factors capable of either retarding or accelerating tumor growth in vivo. Fractionation ofthese sera was required to isolate those antibodies responsible for tumor growth inhibition. Fractionation of tumor sera, isolation and concentration ofthose antibodies responsible for inhibition of tumor growth in vivo, and removal of serum factors which could potentially result in enhancement of tumor gTowth may substantially increase the efficacy of such serum preparations. For example, fractionation of sera from rats bearing polyoma virus-induced sarcomas or carcinogen-induced carcinomas of the colon have shown that serum blocking activity is associated with IgG. Removal of IgG from serum by immunoadsorbents prepared with antibody to rat IgG81 or with streptococcal protein A72 have shown that the adsorbed serum has no blocking activity but retains specific unblocking activity. Utilization of selected immunoadsorbents on a larger scale may provide a suitable means of preparation of antisera with unblocking activity for future serotherapy trials. ACKNOWLEDGMENTS

The authors gratefully acknowledge the support and the varied contributions made to our serotherapy studies by Drs. William B. Hutchinson, Fred Hutchinson Cancer Research Center; Roger Moe and Robert F. Jones, University of Washington; Dennis Donohue, Puget Sound Blood Center; Glenn Warner and H. Clark Hoffman, Swedish Hospital; and Edmund R. Clarke, Jr., Group Health Cooperative of Puget Sound, Seattle, Washington.

REFERENCES 1. Albo, V., Hartmann, J., and Krivit, W.: Fresh plasma as an adjuvant to the chemotherapy of previously untreated children with acute lymphoblastic leukemia. Proc. Amer. Assoc. Cancer Res., 9:2, 1968. 2. Attia, M. A. M., and Weiss, D. W.: Immunology of spontaneous mammary carcinomas in mice. IV. Acquired tumor resistance and enhancement in strain A mice infected with mammary tumor virus. Cancer Res., 26: 1787-1798, 1966. 3. Baker, M. A., and Taub, R. N.: Immunotherapyofhumanleukemia. Mount Sinai Med. J., 39:548-565, 1972. 4. Bansal, S. C., Hargreaves, R., and Sjogren, H. 0.: Facilitation of polyoma tumor growth in rats by blocking sera and tumor eluate. Int. J. Cancer, 9:97-108, 1972. 5. Bansal, S. C., and Sjogren, H. 0.: "Unblocking" serum activity in vitro in the polyoma system may correlate with antitumor effects of antiserum in vivo. Nature New BioI., 233:76-78, 1971. 6. Bansal, S. C., and Sjogren, H. 0.: Counteraction of the blocking of cell-mediated tumor immunity by inoculation of unblocking sera and splenectomy: Immunotherapeutic effects on primary polyoma tumors in rats. Int. J. Cancer, 9:490-509, 1972. 7. Bansal, S. C., and Sjogren, H. 0.: Correlation between changes in antitumor immune parameters and tumor grbwth in vivo in rats. Federation Proc., 32: 165-172, 1973. 8. Bansal, S. C., and Sjogren, H. 0.: Regression of polyoma tumor metastasis by combined unblocking and BCG treatment: Correlation with induced alterations in tumor immunity status. Int. J. Cancer, 12:179-193, 1973. 9. Bloom, E.: Quantitative detection of cytotoxic antibodies against tumor-specific antigens of murine carcinomas induced by 3-methyl-cholanthrene. J. Nat. Cancer Inst., 45 :433-453, 1970. 10. Brunner, K. T., Mauel, J., Cerottini, J.-C., et al.: Quantitative assays of the lytic action of immune lymphoid cells on 51Cr-Iabelled allogeneic target cells in vitro: Inhibition by isoantibody and by drugs. Immunology, 14:181-196, 1968. 11. Bubenik, J., Ivanyi, J., and Koldovsky, P. P.: Participation of 7S and 19S antibodies in enhancement and resistance to methylcholanthrene-induced tumors. 'Folia Bio1., 11 :426-433, 1965.

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