Experimental models in cancer immunotherapy

JOURNAL

OF SURGICAL

RESEARCH

37, 4 15-430 (1984)

CURRENT Experimental

RESEARCH

REVIEW

Models in Cancer Immunotherapy’

DONALD S. Ross, M.D.*

AND GLENN STEELE, JR., M.D.,

PH.D.-~-~

*Department of Surgery, Southern Illinois University School of Medicine, Springfield, Illinois, and TDepartment of Surgery. Brigham & Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115 Submitted for publication November 22, 1983

of cells from a chemically induced sarcoma, larger, normally tumorigenic doses of the same sarcoma would be rejected [50]. This finding suggestedthat tumor cells did indeed possessTAA capable of stimulating host immunity. Over the next three decades, Prehn and Main [98, 991, Foley [33], and Klein [63] confirmed that tumor cells possesstumor-associated antigens distinct from major histocompatibility antigens and that these TAA could act as transplantation rejection antigens in vivo in various experimental tumor models. In Prehn and Main’s experiments [98], sarcomaswere induced in inbred mice using methylcholanthrene. The tumors were then completely excised and mice challenged with autologous tumor cells which failed to grow. Syngeneic mice not previously exposed to the tumor did not reject the subsequent autologous tumor challenge. In addition, tumor-exposed (immunized) mice received skin grafts from nonimmunized mice, and the grafts were accepted. These findings suggestedthat tumor-associated antigens were separate from major histocompatibility antigens but could function in vivo to protect against tumor isograft challenge. Such tumor-associated antigens that function in vivo are referred to as transplantation rejection antigens (TRA). These findings were supported by Gorer [44] who also demonstrated that tumor-associated antigens in some systems were located in close proximity to the major histocompatibility complex. The general finding that tumor-associated antigens ’ Supported in part by NIH Grant CA 32394. ’ To whom requests for reprints should be addressed. existed in experimental tumors led to the

Effective clinical cancer immunotherapy remains an unrealized goal. Many human trials have been reported, but, despite initial enthusiasm, there has been no consistent beneficial effect on survival [24, 54, 80, 82, 104, 106, 121, 1251. The development of potential immunotherapy strategiesparalleled the development of tumor immunology. Erlich in 1900 proposed the ground work for “surveillance” [23] by postulating that tumors had cell surface differences allowing the host to recognize them as abnormal and destroy them. In 1959, Lewis Thomas suggestedthat the phenomenon of allograft rejection was a prototype that might “represent a primary mechanism for natural defense against neoplasia” [ 1221.Basedupon the ideas of Ehrlich and Thomas, Burnet proposed what has come to be known as an “immune surveillance theory” which states that there are homeostatic mechanisms in mammals that can prevent malignant transformation of cells [ 13, 141.The presenceof purported tumor-specific antigens or tumor-associated antigens (TAA) is the central dogma of the surveillance theory and tumor immunology. All proposed immunotherapeutic modalities are based on that dogma. The actual demonstration of TAA did not occur until nearly a half century following Ehrlich’s original proposal. In 1943, Gross demonstrated in a mouse model that after inoculation of animals with small numbers

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0022-4804184$1SO Copyright 0 1984 by Academic Press. Inc. All ngbts of reproduction in any form reszwed.

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speculation that human tumors possessed similar antigens that could provide the basis for immunotherapy, despite the fact that the immunogenicity of induced animal tumors and that of “spontaneous” human tumors might vary enormously. Animal models have been used extensively to evaluate host response to malignancy and to develop strategies for immunotherapy. As summarized above, the development of inbred strains of laboratory animals resulted in the initial understanding of how transplant rejection related to tumor immunology. Inbred strains are produced by at least 20 generations of brother-sister mating and result in animals that are genetically identical (i.e., syngeneic or isogenic) [32, 1341. The use of syngeneic animals allows the free transplantation of tissues (normal or malignant) between animals of the same strain. Data regarding the immune response of syngeneic animals are easier to analyze because there is minimal interstrain variation. G. D. Snell, for instance, who first developed a number of inbred strains of mice, described early on the relationship between immunogenetics and tumor transplantation [ 1161. Table 1 lists the species of animals most often utilized in tumor immunology research and the number of inbred strains currently recognized. Mice are most widely used because of the large number of well-characterized inbred strains and the easeof maintaining them in a laboratory. Rats, guinea pigs, and hamsters are also extensively used. On the negative side, inbred strains are lesshardy than random bred strains and may have TABLE 1 SPECIESOFANIMALS COMMONLYUSEDIN TUMOR IMMUNO~YRESEAR~H No. of Species

inbred strains

Mouse Rat Hamster Guinea pig

230 113 37 13

unusual susceptibility to certain diseases,carcinogens, etc. Obviously, one must be cautious in transferring data obtained from such animals directly to a clinical setting. The use of animal models in experimental tumor biology has been attended with controversy. Many reasonable questions remain to be answered. What constitutes a relevant animal model? Can immunotherapy data obtained from inbred, genetically restricted strains be extrapolated to the treatment of human malignancy? Can models that utilize syngeneic tumors maintained as isografts for multiple passagesin tissue culture be in any way relevant to the clinic [ 129]? Such tumors may retain little antigenic similarity with the originally explantated primary cancer. Are most virally or chemically induced animal tumors in any way similar to the weak immunogenicity of so-called “spontaneous” human tumors [2]? Table 2 summarizes some of the more obvious characteristics of many experimental and human malignancies. Prehn has shown that immunogenicity of experimental tumors is related to the strength of the carcinogen used [loo]. To some extent, it may be a matter of semantics to try to distinguish between a carcinogen or virusinduced tumor and a “spontaneous” or etiology-unknown human tumor. Spontaneous tumors may be induced by weak carcinogens or viral agents, but simply develop after a longer latency than most model tumors. Nevertheless, human tumors are generally weakly immunogenic compared to animal tumor models, and the development of human immunotherapy protocols basedon such animal models may be of questionable validity. Although Weiss and others have suggested that the study of spontaneous tumors occurring in domestic animals may be more relevant models for the treatment of human malignant disease[ 130, 1311,the use of such spontaneous tumors would be cumbersome and the prospects of acquiring large numbers of analyzable animals unlikely. Despite the admitted compromise involved in most animal model work, the selection of inbred

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TABLE 2 COMPARISONOFEXPERIMENTALTUMORMODELSUSEDFORIMMUNOTHERAPY RESEARCHANDHUMANTUMORS Experimental tumors

Human tumors

1. Chemical or viral carcinogen induced 2. Tumors usually transplanted 3. Short latency 4. Rapid growth rate 5. Usually sarcomas and leukemias 6. Strongly immunogenic 7. Distant metastasesinfrequent

Role of carcinogen speculative Primary tumors Long latency Slow growth rate Carcinomas predominate Weakly immunogenic Distant metastasesfrequent

strains and tumors to study for immunotherapy strategies can be optimized in striving for analogy to various human tumors. Many of the ideal characteristics in animal model work for immunotherapy protocols are listed in Table 3. Current research in experimental immunotherapy has branched into many directions. Table 4 lists some areas of current study. Discussion of most of these topics is naturally beyond this review. Instead, the most promising areas of research (from the authors’ biased perspective) will be surveyed. Particularly in relation to animal models, these areas include active immunization (specific and nonspecific), adoptive transfer of immune cells, and the manipulation of immune cell populations in tumor-bearing hosts. IMMUNIZATION

the growth of tumor transplants by prior sensitization with tumor cells from the same isograft [ 1331. The protective effect gained by using a tumor vaccine to protect against subsequent challenge with the same tumor is termed specific active immunization. The proposed utility of specific active immunization as a prototype tumor therapy model has been corroborated by many investigators. TABLE 4 CURRENTAREASOFIMMUNOTHERAPY RESEARCH 1. Immunization a. specific active immunization b. nonspecific active immunization c. combined specific and nonspecific active immunization 2. Adoptive immunotherapy a. immune lymphocyte infusions b. transfer fact0rs-e.g. immune RNA

C. 0. Jensen was one of the first to suggest Passive immunotherapy that inbred mice could be protected against 3. a. antitumor heteroantisera b. monoclonal antibodies TABLE 3 S~MEGENERALCHARACYTERISTICSOFANIDEAL ANIMAL MODELFOR~MMLJNOTHERAPY RESEARCH 1. Primary tumor rather than tumor transplant. 2. Long latency for primary tumor development after carcinogen exposure. 3. Histologic features similar to human tumor counterpart. 4. Natural history similar to human tumor. 5. Primary tumor amenable to conventional therapyi.e. surgical resection, chemotherapy, or radiation therapy.

4. Manipulation of immune cell subsets a. infusion of immune effector cells b. depletion of suppressorT cells 5. Immunomodulators a. thymosin b. interferon c. prostaglandin synthetase inhibitors d. lymphokines 6. Depletion of immunosuppressive substances a. plasmapheresis b. immunoadsorption 7. lmmunopotentiators

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In general, tumor vaccines made from induced animal tumors (such as methylcholanthrene sarcomas in rodents) will not protect against challenge from another chemically induced tumor, even if the second tumor were produced by the same carcinogen in the same animal [ 1, 5, 39, 881. Hence, chemically induced tumors have been purported to contain TAA that are predominantly individual or tumor specific [l, 88, 51. In contrast, virus-induced tumors bear TAA that have been found to protect against subsequent challenge from all tumors that were induced by the same virus even if the tumor came from a different strain of animal or occasionally even from a different species [5 1, 112,401. The cross-reactivity seenamong these viral tumors was due in part to the presence of TAA that were actually products of a common viral genome. Sjogren and Steele subsequently reported that chemically induced tumors did in fact possessnot only tumor-specific or individually distinct membrane antigens but also contained cross-reactive antigens that were in all probability differentiation and/or “tissue-type” specific antigens, and that these cross-reactive antigens were immunogenic [ 117, 1181.Thus, the cross-reacting antigens among a group of rat colon cancers could be used as a vaccine to protect against subsequent challenge using any other rat colon cancer. Most models that have been used to test for immunogenicity of various animal tumors consist of sensitization with the immunogen and subsequent isografi tumor challenge [72, 1021. Naturally, such models using prior immunization bear little relevance to the treatment of human cancers in which immunotherapy is generally regarded as an adjuvant to surgical or chemotherapeutic approaches. One exception will be the use of tumor vaccines in high-risk populations of patients (such as those in Africa who have an elevated incidence of hepatoma associated with HAA-positive hepatitis). In general, however, the use of prior immunization is not feasible becauseof our inability to predict populations at risk and the unavailability of

known human TAAs from most solid and nonsolid cancers. If adjuvant immunotherapy is to succeed at all, it will probably be in the setting of minimal residual disease since experimental immunotherapy has seldom been effective against large tumor burden [42, 781. For instance, Mathe has reported extensively on the use of specific active immunization in the treatment of mice with a chemically induced leukemia [75-771. Mice with L1210 leukemia treated first by chemotherapy to reduce the tumor burden and then immunized with irradiated leukemia cells had a significantly prolonged survival compared to nonimmunized controls [77]. Unfortunately, extrapolation to various immunotherapy maintenance protocols in humans with leukemia has not been a straightforward success. Steele and associateshave reported the use of specific active immunization in a minimal residual diseasemodel of human colon cancer [52, 119, 1131. Primary colon carcinomas induced by 1,2-dimethylhydrazine (DMH) in Wistar/Furth rats are “curatively” resected. Resected rats were immunized with a tumor vaccine prepared from one of an array of syngeneic tumor isografts differing in histology and differing in the presence of crossreacting or “tissue-type” specific antigens. Isografts used were DMH-W 163-an adenocarcinoma bearing antigens cross-reactive with other rat colon carcinomas, DMHW 15-a sarcoma with no cross-reacting antigens, DMH-W49-a carcino-sarcoma with an intermediate array of colon cancer crossreacting antigens, and SPK-a spontaneous (non-DMH induced) Wistar/Furth renal cell carcinoma. The results of immunization protocols are summarized in Fig. 1. Seventy-five percent of rats with no adjuvant immunotherapy recurred within 24 weeks. DMHW163 immunized rats had a 33% recurrence rate compared to 75% for DMH-W 15 treated rats (P < 0.05). DMH-W49 treated rats showed intermediate protection with approximately a 50% recurrence rate. Rats treated with SPK after curative resection of primaries were not protected compared to control or

ROSS AND STEELE: CANCER IMMUNOTHERAPY

FIG. I. Tumor recurrence after “curative” resection of primary bowel adenocarcinomas in nonimmunized and immunized W/Fu rats. The numbers in the figure refer to animals followed for the full 24-week period, and the curves therefore represent actual survival.

DMH-W15 immunized rats and had a 63% recurrence rate. Effective immunization in this model, therefore, depends upon the presence of cross-reacting or “tissue-type” specific antigens on the tumor cells used in the vaccine [52, 119, 1071.There are, of course, potential problems in this model. The use of a syngeneic tumor vaccine clearly has no direct relevance to humans unless autologous tumor is to be used for an immunization protocol. If, however, allogeneic tumor vaccines are utilized in human trials, the effect of MHC antigen differences would have to be assessed.Likewise, in this model, distant metastasesare rare. Beneficial effect of immunization after primary tumor resection in this model is reflected in a decrease in local recurrence. Whether or not immunotherapy would protect against visceral metastases is unknown. An interesting animal system described by Goldrosen and Lewis may help to answer some of the questions concerning widespread or distant metastases in immunization protocols [41]. They have described the orthotopic transplantation of a syngeneic murine colon carcinoma into the submucosa of the cecum, resulting in metastasesto the regional mesenteric lymph nodes and to the liver. Their model presumably could be used to test the effect of immunization utilizing tissue-type specific vaccines as immunogens

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after implantation of the cecal tumor but before metastaseshad occurred. Because some tumor immunogens are weaker than others in their ability to stimulate host immune response,a number of methods have been used to enhance immunogenicity. These have included mixing tumor cells with immunoadjuvants such as BCG or Corynebacterin parvum [34, 1291. Attempts have also been made to enhance immunogenicity by altering the cell surfaces of tumors. Neuraminidase (derived from vibrio cholerae as well as other sources) has been shown to increase the host immune response to a variety of experimental tumors [26, 1lo]. Neuraminidase is believed to act by exposing otherwise hidden cell-surface antigens or altering those that are present [ 171. Another interesting approach to specific active immunization is the use of tumor cell membrane extracts to present to the host the specific tumor antigen desired as an immunogen, thus reducing contamination by unwanted antigens. Cell extracts have been prepared using a variety of agents including 3MKCL, various detergents such as Nonidetphosphate 40 (NP-40), and 1 butanol [71, 73, 74, 85, 971. Numerous methods for the purification of such extracts have been reported [7 1, 731. Kahan and associates have reported the use of 3MKCL tumor cell membrane extracts assayed successfully in vitro and in vivo in immunoprotection assays[6 1, 74, 93, 941. These investigators have found that purification of the cell membrane extracts with preparative isoelectric focusing can produce fractions that enhance tumor growth in vivo and other fractions that are immunoprotective [ 1351. Using C3H/HeJ mice and a syngeneic methylcholanthrene-induced sarcoma, Kahan et al. immunized animals with the immunoprotective Fraction 15 of the sarcoma extract and subsequently challenged them with a known tumorigenic dose of the same tumor. Eighty percent of the mice that received three weekly inoculations of Fraction 15 were alive after 40 days compared to 15% of those that received no treatment or only 1 or 2 weekly inoculations of the extract

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[ 1351. In addition, mice that underwent excision of small tumors and subsequent immunization with the extract had significantly fewer recurrences than those who received no treatment. Jessup and Kahan have proposed a mechanism for the apparent tumor enhancement of certain tumor antigens [60]. In their methylcholanthrene-induced mouse sarcoma model, mice were immunized with Fraction 1 of purified 3MKCL tumor extract. It was found that splenic nonlymphocyte suppressor cells (i.e., macrophages) were activated and that subsequent attempts to immunize these mice with the immunoprotective Fraction 15 was not possible. LaGrue, Kahan, and associateshave demonstrated the use of cell membrane extracts prepared by 1-butanol in immunoprotection assays1741.I-Butanol extracts were prepared from two syngeneic but antigenically distinct methylcholanthrene-induced sarcomas. Two groups of mice were than immunized with various doses of one or the other of the tumor extracts. Ten days later, the mice were challenged with the corresponding tumor (a known tumorigenic dose) and subsequent tumor growth was measured. Both extracts, used as immunogens, resulted in significantly decreasedrates of growth of the corresponding tumor. There was no cross-protection, however, when immunized mice were challenged with the antigenically distinct tumors. Another significant finding was that I-butanol releases only the tumor-associated antigens but not major histocompatibility antigens. This finding is important since in any clinical setting (unless autologous tumor would be used), allogeneic tumor would be necessary as an immunogen. Allogeneic immunization has met with mixed successin experimental models and as reviewed by Hosokawa and Kobayashi [56] has been shown to be unpredictable presumably because of the presence of a variable MHC incompatibility. The use of tumor extracts to present to the host precisely those antigens that will stimulate the desired immune response has considerable merit, particularly if extracts can be prepared not containing irrelevant MHC components. Questions to be answered

in all of the models described above are (1) will extracts from histologically similar tumors in different animals of the same strain result in immunoprotection or will the use of autologous tumor extracts be necessary? (2) Will tumor-associated antigens from allogeneic tumors be effective in immunoprotection experiments? (3) Will extracts from “spontaneous” tumors, which are generally weakly immunogenic, stimulate a significant immune response? (4) Can host immune response be predicted by in vitro tests after exposure to the desired immunogen assuring that the desired immune response has been achieved? These questions will need to be addressed before moving into any clinical trials. NONSPECIFIC

ACTIVE

IMMUNIZATION

Nonspecific active immunization is defined as the stimulation of host immunity by an agent that presumably bears no immunologic identity to a specific antigenic target. Bacille Calmette Guerin (BCG) and Corynebucterum purvum (C. Purvum) are the most common agents used to stimulate host immunity nonspecifically. Pearl in 1929 noted that patients suffering from tuberculosis appeared to have a lower incidence of malignancy [92]. BCG was originally developed for the immunoprophylaxis of tuberculosis. Old, Benacerraf, and co-workers reported in 1959 that mice with prior BCG infections were resistant to syngeneic tumor transplants [87]. Many reports have described the use of BCG alone or as an adjuvant to chemotherapy or to surgical treatment in various experimental tumor models [22, 116, 78, 137, 136, 70, 1261. BCG may also be given mixed with tumor cells vaccines. BCG is believed to potentiate the host immune response by a variety of mechanisms as reviewed by Weiss [ 1291and Florentin [34]. It may act (1) by modifying weak antigens to increase their immunogenicity, (2) by creating an inflammatory environment which increases the effectiveness of contact between antigen and antigen-processing cells, or (3) by altering the behavior of certain immune cell populations, especially

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macrophages [ 128, 1291. C. parvum is believed to affect host immune response in a similar manner [ 1291. One drawback in the use of BCG and other biologic immunomodulating substances is that they vary in their ability to stimulate host responsesdepending upon their source, their method of preservation, and the dose and route of administration [34, 1361. In addition, because the effects upon the immune system are so varied, one cannot predict which compartment of the complicated host immune response will be stimulated. Wepsic and colleagues using inbred guinea pigs bearing syngeneic hepatoma isografts found that injection of BCG on the day after tumor inoculation or 7 days later resulted in enhancement of tumor growth compared to controls [ 1321. Drukar, Wepsic, and colleagues [ 191 reported a severely depressed mixed lymphocyte response and a decreased response to the mitogen Con A in animals immunized with BCG intraperitoneally. Removal of plastic adherent cells (i.e., macrophages) from the lymphocyte preparation resulted in complete restoration of the response to Con A and a partial return of MLR reactivity. An alternative mechanism for tumor enhancement by BCG was advanced by Bansal and Sjogren who suggested that the increased growth of polyoma virusinduced tumors in rats might result from the production of “blocking factors” shielding tumor cells from immune defense mechanisms [3]. BCG might potentiate humoral blocking of cell-mediated responses or increase suppressor activity of T-cell subsets. Thus, the use of immunopotentiating substances has potential in the development of immunotherapy strategies,but understanding that such agents do not act upon a single immune compartment and indeed may have adverseeffects,implies that more work should be done on experimental models before rational clinical trials can be designed. ADOPTIVE

IMMUNOTHERAPY

Adoptive immunotherapy is defined as the transfer of immunity from one animal to another by immune cells and/or serum fac-

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tors. In most experimental cancer immunotherapy models, lymphocytes have been transferred becausethe cellular responseshave been thought to be central to host antitumor attack. As stated by Fefer and Kedar [28], the use of lymphocytes in adoptive immunotherapy is predicated on the assumption that tumor antigens exist and that they are capable of serving as a target for an immune response.Likewise, there must be a presumed defect in the host antitumor response that may be correctable by the infusion of specifically targeted immunocytes. Classically, lymphocytes from a syngeneic animal that has been immunized with a particular tumor are infused into another animal of the same strain before or shortly after challenge with that sametumor ([28,62]. Successfuladoptive transfer results in the prevention or delay of tumor development. The sensitized lymphocytes may also be used in cell-mediated cytotoxicity assaysagainst the same or different tumor targets to demonstrate specificity or immunologic cross-reactivity. Antigen-exposed lymphocytes may also be secondarily sensitized in vitro by coculture with irradiated tumor cells in order to increase activity in vivo and in vitro [ 15, 1051. The use of adoptive immunotherapy in pretreatment models is, of course, not relevant to the treatment of human cancers since most patients have well-established or disseminated tumors at the time they need therapy. Adoptive immunotherapy in animal models with advanced cancer has been uniformly unsuccessful [ 11, 621. Borberg and colleagues [ 111 demonstrated that lymphocytes from mice immunized with a chemically induced sarcoma were able to inhibit the rate of growth of the same tumor in a syngeneic mouse. However, the infusion of 1 X lo9 cells was necessary to achieve this effect. Most transient therapy successesin animal experiments have been demonstrated in syngeneic systems. Until recently, allogeneic lymphocytes have presumed to be necessary in adoptive immunotherapy of human cancer. Despite some reports of successful adoptive transfer using allogeneic lymphocytes, in general allogeneic lymphocytes do not survive

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long enough in the host to affect tumor. Allolymphocytes are rapidly trapped in the host spleen or liver and destroyed [79]. In addition, the infusion of large numbers of allogeneic lymphocytes may result in graft versus host disease,particularly if hosts have been pretreated to prevent early destruction of the transplanted immunocytes. Recognition that lymphocytes require a growth factor (IL-2) which can sustain clonal expansion has resulted in a significant advance in adoptive transfer immunotherapy schemes. Interleukin-2 is elaborated by amplifier T lymphocytes and can be harvested in large quantities and applied to lymphocyte cultures allowing for their long-term maintenance and clonal expansion [35, 36, 1091. Such lymphocyte cultures retain their antigenie specificity without the continued presence of antigen [35]. Eberlein and Rosenberg have reported the use of an adoptive transfer immunotherapy regimen in a mouse tumor model [20]. C57BL/6 mice bearing the tumor FVL-3, a Friend virus-induced lymphoma, were treated by adoptive transfer of antigencommitted lymphocytes. Lymphocytes from immune donors were sensitized secondarily in vitro by incubation with an irradiated FVL-3 tumor cell population. After 5 days, lymphocytes were infused into tumor-bearing mice at different doses. Those lymphocytes not infused after 5 days were further expanded in vitro with IL-2. Eleven of 14 tumorbearing mice were “cured” compared to O/ 16 nontreated controls. Mice treated with 14-day IL-2 cultured lymphocytes had a cure rate of 93% compared to 0% in the control group. In both the IL-2 and non-IL-2 cultured lymphocyte groups, 5 X lo7 cells gave the optimum response. Eberlein and Rosenberg found that IL-2 expanded lymphocyte clones in the FVL3 mouse model were more effective at prolonging survival when used in combination with Cytoxan [21]. This may be due to the fact that Cytoxan, in low doses,selectively depletes T-suppressor cells, enhancing the antitumor effect of the transferred antigen-committed lymphocyte effecters.Other investigators have confirmed the effectivenessof IL-2 expanded

lymphocyte clones in other experimental tumor systems [15, 1051. MANIPULATION OF LYMPHOCYTE SUBSETS

The tumor-bearing host has been shown to have a number of immunologic derangements that may result in the inability to mount a successful antitumor response. Increased suppressor activity may be reflected by diminished lymphocyte response to mitogens and alloantigens [27, 1031. A more generalized immune depression has been easily reflected using numerous cell-mediated cytotoxicity assay techniques [66, 691. Sup pressor T cells have been implicated in immune suppression monitored by various in vitro assays [7, 31, 67, 69, 831. Monoclonal antibodies that can distinguish T helpers from T suppressors have been utilized in humans and animal models to analyze specific compartment changes in host immune response to cancers. A possibility, therefore, now exists that correction of a precise imbalance between T-helper and T-suppressor subsets might shift the balance in favor of the host and against the cancer. Femandez-Cruz, Feldman, and associates have shown that the adoptive transfer of T helpers that have been presensitized against a rat sarcoma will cure an already growing sarcoma [37]. Inbred Brown Norwegian rats were immunized with irradiated Moloney virus-induced sarcoma cells and subsequently challenged with a known tumorigenic dose of viable cells. Splenic lymphocytes from these immune rats were then further sensitized in vitro by coculturing with mitomycintreated sarcoma cells for 7 days. Normal, nonimmunized rats were then inoculated with a known tumorigenic dose of the sarcoma, and tumors were allowed to reach a diameter of 1.5 cm2. The immune splenic lymphocytes were separated by monoclonal antibodies into T-helper cells and non-T helper populations, or left unfractionated. B lymphocytes (i.e., T-cell depleted) were infused into tumorbearing rats and had no effect on tumor growth compared to nontreated controls. Unfractionated cells resulted in a slower tu-

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mor growth but no cures. T-helper cells resulted in rejection of the growing tumor and no recurrence after 18 months. The fraction containing non-T helper cells enhanced tumor growth (apparently based on the presence of suppressor T cells) when infused. In an earlier report, Fernandez-Cruz and associatesdemonstrated that the use of adoptively transferred sensitized T-helper cells caused the regression of chemically induced sarcomas [29] as well as virus-induced tumors [30]. Greene, Benacerraf, and associates have shown that T-suppressor activity in the mouse can be abrogated in vivu by the infusion of monoclonal antibodies. In their mouse model, the I-J locus of the major histocompatibility complex has been shown to encode antigens that are located on the surface of suppressor T cells. These gene products are of functional significance [46]. Greene and co-workers have injected anti-I-J alloantisera into tumorbearing mice and demonstrated the abrogation of suppressor activity in vitro and the inhibition of tumor growth in vivo [45]. Debrin and co-workers have recently utilized newly available anti-I-J monoclonal antibodies to successfully treat mice bearing a methylcholanthrene-induced fibrosarcoma [ 181.A/ J mice were inoculated with a tumorigenic dose of the fibrosarcoma cells and treated with either anti-I-J monoclonal antibody or placebo. Anti-I-J treated mice had a significantly decreased rate of tumor growth compared to control mice. It is important to note that tumor growth rates were altered in these experiments, but no mice were cured by the treatment. It is not clear from these studies whether the abrogation of T-suppressor activity is permanent or temporary, although the fact that no complete tumor regressionswere noted suggeststhe latter. Perhaps, the use of anti-I-J antibodies may be more effective if combined with primary surgical resection or effective chemotherapy. OTHER

Monoclonal

AREAS

OF

INVESTIGATION

Antibodies

The development of monoclonal antibody technology by Kohler and Milstein [64] has

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raised the promise of many potential clinical applications for tumor immunodiagnosis and immunotherapy. Monoclonal antibodies are produced by the fusion of immunized splenocytes and myeloma cells. The resultant hybrid cells produce antibodies coded by the sensitized lymphocytes. The resultant antibody is specific for a single antigenic epitope. A number of experimental models have been used in which antibodies with a high degree of specificity for a particular tumor have been coupled with chemotherapeutic agents or radioisotopes [lo, 37, 811. Monoclonal antibody serotherapy has been used to treat numerous experimental tumors, mainly leukemias [89]. Monoclonal antibodies have been used, coupled to radioisotopes, to localize tumors in experimental models [53]. Problems with the use of monoclonal antibodies include the continuing inability to define tumor-specific antigens, i.e., antigens that do not appear on normal tissues. In addition, large amounts of antibody are required to ensure a state of antibody excess so that antibody reaches the tumor target before it complexes with circulating antigen and is cleared by the host. An additional interesting problem has been defined by Old and associates.They have found that treatment of mouse leukemia with monoclonal serotherapy resulted in the loss of the target antigen from the tumor cell surface [89]. This alteration in the malignant cell surface after antibody treatment has been termed “immunomodulation,” has been shown to last as long as the antibody is present, and represents still another possible mechanism of tumor escapefrom host immune response Removal of Circulating

Suppressor Factors

A variety of mechanisms have been implicated in the immune suppression of tumorbearing animals. In addition to the possible presence of suppressor T cells, suppressor macrophages, and circulating immune complexes, a variety of low-molecular-weight suppressor factors have been described in tumor bearers [3, 7, 83, 86, 103, 108, 1111. A number of investigators have shown that the serum from a tumor-bearing animal will

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suppress the lymphocyte responses of nontumor-bearing animals when monitored in various in vitro assays.The presence of circulating immune complexes have been associated with this immunosuppression or “blocking” of in vitro cell-mediated antitumor activity. A number of therapies have been developed with the aim of removing circulating immune complexes from the plasma of tumor-bearing animals or humans. Stuphy/ococcus aureus, Cowan I strain, has been found to contain a cell wall protein called Protein A which binds with the Fc portion of certain immunoglobulins, whether free or in complex. Using this strategy, Steele and associates demonstrated that adsorbing tumor-bearing serum with staphylococcal Protein A abrogated “blocking” activity in vitro [ 1201.Ray and co-workers as well as Termin et al. have demonstrated that adsorbing plasma from various experimental animals bearing chemically or virally induced carcinomas results in a significant regression of the primary tumor [ 101, 551. However, these investigators have noted that adsorption was more effective against subcutaneous tumors than visceral metastases.Clinical application of various Protein A adsorption schemes have produced interesting but generally uninterpretable results.

eluding the enhancement of T-cell responses to alloantigens, the enhancement of delayedtype hypersensitivity reactions, and the potentiation of cytotoxicity mediated by natural killer (NK) cells [48]. Interferon has also been shown to either enhance or suppress immunoglobulin production by B cells in vitro and in vivo depending upon the timing of interferon treatment after antigen exposure [ 121. Likewise, when added to cocultures of lymphocytes and autologous tumor cells, interferon results in a depressedgeneration of cytotoxic T cells. In short, interferon has a myriad of effects on the different compartments of host immune response.Inconsistent results seen in human trials to date probably relate to the as yet incomplete characterization of interferon’s multiple effectsin relevant animal models.

Prostaglandins Prostaglandins are ubiquitous compounds found in every tissue of the body. They have been shown to be mediators of the inflammatory response. Prostaglandin E, and Ez cause vasodilation and increased vascular permeability when injected intradermally. They potentiate the action of histamine and bradykinin [6, 161.Prostaglandins also modulate most immune responses.T-lymphocyte responses to mitogens are decreased in the Interferon presence of prostaglandin Ez [43, 1141.LymInterferon is the generic term for a family phocyte responses to alloantigens and cellof glycoproteins synthesized by mammalian mediated antitumor cytotoxicity of activated cells in responseto viral infections. Interferon lymphocytes are depressedby prostaglandins is believed to act by blocking the transcription [6, 651. Synthesis of antibody by B cells and of viral RNA, thus inhibiting the translation natural killer activity are suppressedby prosof viral proteins. Its antiviral properties were taglandins [6]. A number of studies have first described by Isaac and Lindemann in indicated that macrophages are responsible 1954 [58, 591. The interferon system is non- for the synthesis of some of these prostaglanspecific in that it is activated by a variety of dins and can be stimulated by a variety of viruses and results in cellular resistance to a agents including immune complexes, BCG, variety of viruses. In 1962, Paucker and C. parvum, endotoxin, etc. to produce various associates made the initial observation that prostaglandins or prostaglandin mediators interferon inhibits cell growth and replication [49, 57, 68, 901. Prostaglandin synthetase 1911.Gresser subsequently demonstrated that inhibitors such as indomethacin, aspirin, or the growth of viral- and nom&l-induced piroxicam have been shown to abrogate the tumors in mice was inhibited by interferon effects of prostaglandin in vitro [6, 231. Pollard has written extensively on the use [47, 481. Interferon has been found to have a number of immunomodulating effects in- of prostaglandin synthetase inhibitors to treat

ROSS AND STEELE: CANCER IMMUNOTHERAPY

rats with carcinogen-induced bowel cancers [95, 961. In one such study [94], primary bowel tumors were induced in SpragueDawley rats by gastric lavage. Rats were given indomethacin ad libitum (20 mg/ml) in drinking water starting at 3, 12, or 35 days after the last of five weekly carcinogen doses. At 20 weeks after the start of the experiment, rats were sacrificed and the gastrointestinal tract examined for tumors. Regardless of the interval between the end of DMH treatment and the initiation of indomethacin, there were significantly fewer tumors in the indomethacin group when compared to nontreated controls. Narisawa and associates[84] have shown that CD/Fischer rats treated with colon carcinoma N-methylnitrosourea were protected against subsequent primary tumor development by later indomethacin treatment. The study of these effects of prostaglandin synthetase inhibition on tumor development and the treatment of already established tumors is promising. However, it has not been established that any beneficial effect noted in the various animal models is related in any way to host immune response or, rather, due to a direct antitumor activity. CONCLUSION

In the 83 years since Ehrlich postulated that normal and malignant cells were different, advances in understanding how these differences might effect immune response to malignancy have been made. The science of immunology is, however, still young. Most discoveries have underscored the enormous complexity of the immune system. Advances in knowledge about lymphokines, interferon, and prostaglandins have opened the door to various feedback mechanisms. Monoclonal antibodies have allowed the identification of numerous new subpopulations of immune cells which might have functionally distinct roles in immune response. Recent investigations into the molecular genetic aspects of immune response have led to further delineation of the ultimate control mechanisms involved. The use of immunotherapy in the treatment of malignancies is attractive because it

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makes use of the host’s natural defenses.This approach obviously has been effective against many infectious diseases.The successof such a prototype in clinical immunotherapy depends upon the presence of tumor-associated antigens on human tumor cells that are capable of acting as transplantation rejection antigens, as they do in experimental models. The disappointment of most human immunotherapy trials to date is due undoubtedly to the lack early on of relevant animal models. Most new models mentioned in this review do bear a greater resemblance to human malignancies than previously available experimental systems. Improvements in these newer models include the use of primary tumors rather than transplanted tumors, histologic features, and natural history which bear some resemblance to the human counterpart being investigated, a more analogous latency and tumor development, and the accessibility of tumors for multimodal treatment schemes which undoubtedly will be applied in the human tumor counterpart. Still, none of these models will be perfect, and it will be necessary to keep in mind potential flaws in any attempt to make a transition from immunotherapy in the laboratory to immunotherapy in the clinic. REFERENCES 1. Baldwin, R. W. Immunity to methylcholanthrene induced tumors in inbred rats following atrophy and regression of the implanted tumors. Brit. J. Cancer 9: 652, 1955. 2. Baldwin, R. W. Relevant animal models for tumor immunotherapy. Cancer Immunol. Immunother. 1: 197, 1976. 3. Bansal, S. C., Hargreaves, R., and Sjogren, H. 0. Facilitation of tumor growth in rats by blocking sera and tumor eluates. Int. J. Cancer 9: 97, 1972. 4. Bartlett, G. L. Effect of host immunity on the antigenic strength of primary tumors. J. Natl. Cancer Inst. 49: 493, 1972. 5. Basambrio, M. A. Searchfor common antigenicities among twenty-five sarcomas induced by methylcholanthrene. Cancer Res. 30: 2458, 1970. 6. Bennett, A. Effect of prostaglandin synthesis inhibitors on tumor growth in viva In Prostagkmdins and Cancer: First International Conference, New York: Alan R. Liss, 1982. P. 759. 7. Berendt, M. J., and North, R. J. T-cell mediated suppression of anti-tumor immunity. An explana-

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