CANCER IMMUNOTHERAPY: ARE THE RESULTS DISCOURAGING? CAN THEY BE IMPROVED? Eli Kedar* and Eva Kleint T h e Lautenberg Center for General and Tumor Immunology, the Hebrew University-Hadassah Medical School, Jerusalem, Israel 'Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden
I. Introduction 11. Critical Factors in Cancer Immunotherapy A. Tumor Immunogenicity and T Cell Response B. Tumor Burden and Location C. Heterogeneity of the Tumor Cell Population 111. Current Immunotherapy Strategies A. Active Specific Immunotherapy B. Active Nonspecific Immunotherapy: Cytokines C. Adoptive Immunotherapy: Lymphokine-Activated Killer (LAK) Cells and Tumor-Infiltrating Lymphoctyes (TIL) D. Chemoimmunotherapy IV. Attempts to Improve Cancer Immunotherapy A. Gains from Experimental Models B. Selection of Patients C. Tumor Debulking D. Active Specific Immunotherapy E. Elimination of Suppressor Cells/Factors F. Active/Adoptive Immunotherapy V. Conclusions References
1. Introduction Over the past three decades, cancer immunotherapy has passed through many cycles of enthusiasm and disappointment. Whereas much progress has been made with animal models and in uitro systems, the clinical experience did not live up to the expectations. During the search for effective therapeutic measures, treatment strategies have changed several times. Initially, active immunization with autologous or allogeneic tumor tissue or extracts, and nonspecific stimulation of the immune system with crude agents (e.g., bacterial toxins, BCG, MER, Clostridium parvum) were attempted. Recently, a new approach-adoptive cellular immunotherapy-evolved as a consequence of developments in in uitro activation and propagation of lymphocytes. This ap245 ADVANCES I N CANCER RESEARCH, VOL. 59
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proach was generated with optimism and received much attention. However, the difficulty of obtaining from the patients sufficient numbers of lymphoid cells with seemingly specific reactivity with their tumor redirected the attention to nonspecific measures (reviewed in Weiss, 1980a; Kedar and Weiss, 1983; Rosenberg et al., 1989a; Borden and Sondel, 1990). The identification and large-scale production of immunostimulatory and growth-inhibitory lymphokines/cytokines, such as interleukin-2 (IL2), interferons (IFN), and tumor necrosis factor (TNF), and the development of monoclonal antibodies directed against cancer cells have led to new avenues (Oldham, 1986; Mitchell, 1988; Talmadge, 1988; Baldwin and Byers, 1989; Balkwill and Burke, 1989; Foon, 1989; Fridman, 1989; Janson et al., 1989; Kelso, 1989; Malkovska et al., 1989; Rosenberg et al., 1989a,b; Borden and Sondel, 1990; Gilewski and Golomb, 1990; DeVita et ‘al., 1991; Oldham, 1991). In particular, the IL2-induced activation and propagation of nonselective cytotoxic lymphocytes [lymphokine-activatedkiller (LAK) cells], with the capacity to damage cancer cells i n vitro, was met with enthusiasm and such cells were administered to several groups of patients (Adler et al., 1984; Rosenberg, 1984, 1986, 1988, 1990, 1991a; Herberman, 1987, 1989; Hersey and Bolhuis, 1987; Rosenberg et al., 1989a,b; Smith, 1988; Yagita and Grimm, 1988; Lotzova, 1989; Sondel and Hank, 1989; Stevenson, 1989; West, 1989; Borden and Sondel, 1990; Lotze and Finn, 1990; Masucci and Mellstedt, 1990; Parkinson, 1990; Rees and Wiltrout, 1990; Robertson and Ritz, 1990; Semenzato, 1990a; Sosman et al., 1990; Dillman et al., 1991b). While the preclinical results with cytokines, with and without LAK cells, were remarkable, the majority of cancer patients did not benefit from such treatments, and the toxic effects (e.g., with TNFa or IL-2) outweighed the slight therapeutic gains (Siege1 and Puri, 1991). The clinical results with LAK cells were rather disappointing and the interest returned to putatively tumor-specific T cells. It was assumed that such cells can be isolated from the tumor tissue [tumor-infiltrating lymphocytes (TIL)],and when amplified in number and activated by I L 2 they can be reinjected (Rosenberg et al., 1986, 1988, 1989b; Rosenberg, 1991a; Itoh et al., 1988; Whiteside et al., 1988; Kradin et al., 1989a; Maleckar et al., 1989; Radrizzani et al., 1989; F’armiani et al., 1990; Shimizu et al., 1990; Dillman et al., 1990a, 1991a,c). Other recent endeavors use combinations of cytokines (e.g., IL-2, IFN, TNF) (Brunda et al., 1987; Winkelhake et al., 1987; Agah et al., 1988; Watanabe et al., 1988; McIntosh et al., 1989; Rosenberg et al., 1989c; Fox et al., 1990), and both new and already well-known non-
specific immunomodulators (e.g., muramyl ddtripeptide, levamisole, bacterial products, and “synthetic” adjuvants), given alone and in conjunction with chemotherapy or tumor vaccines (Watanabe and Iwa, 1987; Mitchell et al., 1988a, 1990; Foon, 1989; Hoover and Hanna, 1989; Lise and Audibert, 1989; Rosenberg et al., 1989a; Sarosdy and Lamm, 1989; Bauer et al., 1990; Moertel et al., 1990; Urba et al., 1990; Kleinerman, 1991). This short survey of the therapeutic attempts shows the steady search for new strategies and returns with modifications, indicating thus the discontent. However, when the therapeutic effects are evaluated separately on the various types of malignancies, the view is somewhat less gloomy. Immunobiotherapy was indeed beneficial in certain types of malignancies. Good results were obtained in several hematological malignancies with IFNa (response rate >50%) (reviewed in Balkwill, 1989; Foon, 1989; Rosenberg et al., 1989a; Billard and Wietzerbin, 1990). The 25% response rate in metastatic melanoma and renal cell carcinoma patients (with up to 10% of the patients brought to a tumor-free state for at least several months) treated with IL-2, with or without LAK cell administration, can also be regarded as success (Rosenberg et al., 1989b,Rosenberg, 1990).Anecdotal responses to IL2-based treatments have been reported in several other cancers (Oliver, 1988; Rosenberg et al., 1989a,b; Rosenberg, 1990; Foon, 1989; West, 1989; Borden and Sondel, 1990; Lotze and Finn, 1990; Clamon et al., 1991; Dillman et al., 1991a,b). The vast majority of patient groups with the frequently occurring tumors (i.e., colon, lung, and breast carcinoma) did not benefit, however, from the current immunotherapeutic manipulations. Within the melanoma or renal cell carcinoma patient groups, the individual response is highly variable. While some patients responded to various immunotherapeutic modalities, with most or all tumor foci completely regressing, others did not respond at all. The differences may be ascribed to tumor subtypes, to variations in the antigenicity/ immunogenicity of the tumor, and to differences in the tumor-reactive T cell repertoire of the host, influenced by the HLA phenotype. After reviewing the experimental and clinical results, we conclude that the existence of an immune response against the tumor is a prerequisite for the therapeutic effects, and the biological response modifiers (BRMs) act by improving it. We discuss here both immunotherapy in the classical sense and biotherapy with BRMs, as the majority of these agents are immunopotentiators (reviewed in Oldham, 1983, 1986, 1991; Mitchell, 1988; Talmadge, 1988; Foon, 1989; Hadden, 1989; Rosenberg et al., 1989a).
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Several reviews and books on various aspects of cancer immunology and immunotherapy have been published recently (Greenberg et al., 1988; Mitchell, 1988; Rosenberg, 1988, 1990, 1991a; Rosenberg et al., 1989a; Talmadge, 1988; Foon, 1989; Fridman, 1989; Herberman, 1989; Schreiber, 1989; Borden and Sondel, 1990; Bystryn, 1990; Lotze and Finn, 1990; Oettgen, 1990, 1991a; Osband and Ross, 1990; Parmiani, 1990; DeVita et al., 1991; Greenberg, 1991; Longo, 1991; Old, 1991; Oldham, 1991; Vanky and Klein, 1991; Wadler, 1991; Melief, 1992). We have restricted our survey to recent pertinent studies on nonhematologic neoplasms, and to the relatively newly proposed strategies. The article certainly bears the marks of experimentalists without experience at the bedside.
II. Critical Factors in Cancer lmmunotherapy A large body of studies in experimental models suggests the importance of the following parameters for the outcome of immunological intervention: (1) immunogenicity of the tumor, to which the expression of the major histocompatibility complex (MHC) antigens contribute, (2) the size and location of the tumor, (3) heterogeneity of the tumor cell population, and (4) the immunocompetence status of the host and, most importantly, its ability to mount cell-mediated responses. The studies have demonstrated that: (1) the therapeutic efficacy of a regimen is determined by the potentiation of an existing tumor-specific cellular immunity; (2) combination treatment modalities are more effective than single ones; and (3) intense treatments can lead to adverse effects (reviewed in Kedar and Weiss, 1983; Greenberg et al., 1988; Mitchell, 1988; Talmadge, 1988; Bergmann, 1989; Fridman, 1989; Herberman, 1989; Rosenberg et al., 1989a; Borden and Sondel, 1990; Osband and Ross, 1990; Parmiani, 1990; Parmiani et al., 1990; Rosenberg, 1990). A. TUMOR IMMUNOGENICITY AND T CELLRESPONSE 1. Experimental Tumors The importance of tumor immunogenicity and a T cell response for the success of immunotherapy is clearly demonstrated in the experimental models (reviewed in Kedar and Weiss, 1983; Rosenberg, 1984, 1986; Robins and Baldwin, 1985; Greenberg et al., 1988; Rosenberg et al., 1989a; Schreiber, 1989; Greenberg, 1991). The evidence is as follows: (1) Adoptive transfer of tumor-reactive helper (Th) or cytotoxic (CTL) T cell populations, or CD4 T h and/or CD8 CTL clones (together with
IL2), led to rejection of tumor grafts of high or low immunogenicity (Eberlein et al., 1982; Engers et al., 1984; Palladino et al., 1984; Chou et al., 1988; Greenberg et al., 1988; Kast et al., 1989; Klarnet et al., 1989; Ellenhorn et al., 1990). (2) Marked effects were obtained in mice with established tumors of low immunogenicity with either IL-2-based cellular (LAK/TIL) immunotherapy, or with cytokines (IL2, IFNa, TNFol), with and without adjunct chemotherapy; however, the same regimens were not effective against nonimmunogenic tumors (Rosenberg et al., 1986; Mule et al., 1987; Spiess et al., 1987; Cameron et al., 1988; Kedar et al., 1988a, 1989; Papa et al., 1988; McIntosh et al., 1989; Krosnick et al., 1989a). ( 3 ) In some cases, immunotherapy of murine tumors with proven immunogenicity was more successful if initiated relatively late after tumor inoculation, when the antitumor response has already developed, than if the treatments began earlier (Rosenberg et al., 198513; Thompson et al., 1986; Maas et al., 1989; Maekawa et al., 1989; Ciolli et al., 1991). (4) However, a tumor with poor immunogenicity could be controlled only when IL-2 was introduced shortly after tumor inoculation (Slavin et al., 1989; Ackerstein et al., 1991). (5) Mice in which weakly immunogenic tumor grafts regressed after treatment with I L 2 with or without LAK cells or chemotherapy, acquired long-lasting specific immunity (Mule et al., 1986; Formelli et al., 1988; Hornung et al., 1988; Maas et al., 1989; Kedar et al., 1990, 1992). (6) I n vivo depletion of either CD4 or CD8 T cells [but not of natural killer (NK) cells] prior to cytokine (IL-1, IL-2, or IL-4) administration reduced the therapeutic effects in tumor-bearing mice (Mule et al., 1987; Peace and Cheever, 1989; Bosco et al., 1990; Ciolli et al., 1991; Kedar et al., 1992). (7) Splenocytes of mice taken 3-6 months after cyclophosphamide and I L 2 treatment, which led to complete regression of weakly immunogenic tumors, conferred specific immunity to naive recipients, with most of the protective activity residing in the CD8 T cells. The frequency of antitumor CTL precursors in such splenocyte populations was 5 to 20 times higher compared to that of control mice (Kedar et al., 1990, 1992). (8)The therapeutic effects of I L 2 , I L 4 , I L 6 , or IFN in tumor-bearing mice were markedly reduced when applied after immunosuppression with corticosteroids, cyclosporin A, or radiation (Papa et al., 1986; Rosenberg, 1988; Cameron et al., 1988; Bosco et al., 1990; Mule et al., 1990). (9) Combination treatments consisting of chemotherapy, with I L 2 , with or without IFNa, induced tumor regression in euthymic mice, but were relatively ineffective in nude mice carrying human tumor xenografts. Addition of LAK cells to these regimens increased the therapeutic efficacy in nude but not in conventional mice (Kedar et al., 1988a, 1990, 1992; Gazit et al., 1992), suggesting that the LAK cells are efficient only when T cell substitution
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is needed. Combination treatment with IFNa and LFNy was successful in euthymic but not in nude mice carrying murine tumor grafts (Sayers et al., 1990). The general experience is that only rarely can immunotherapy cure nude mice when the tumor grafts are already well established. 2. Patients A key question is why mainly patients with melanoma and renal cell carcinoma respond to IL2, with or without LAK cell therapy, and why only about one-fourth of these patients respond (Oliver, 1988; Margolin et al., 1989; West, 1989; Rosenberg, 1990, 1991a; Rosenberg et al., 1989b; Borden and Sondel, 1990; Sosman et al., 1990). Similar response rates (i.e., 20-40%, albeit generally less durable) were obtained with other immunologic manipulations, such as active specific immunotherapy in melanoma (Berd et al., 1990a; Mitchell et al., 1990; Morton et al., 1990, 1991a),or treatment with IFNa, with or without chemotherapy, in melanoma and renal cell carcinoma (McLeod et al., 1987; Bergmann, 1989; Guillou et al., 1989; Kellokumpu-Lehtinen and Nordman, 1990; Mickiewicz et al., 1990; Mulder at al., 1990; Falkson et al., 1991). It is likely that the patients for which the therapy is successful have (1) immunogenic tumors, (2) a certain level of existing antitumor response, and (3)a relatively higher capacity to be influenced by immunostimulatory measures. The immunogenicity of melanomas and the role of cellular immunity in the control of these tumors is supported by the following findings: (1) Spontaneous tumor regression (in 1-2% of the patients), and recurrences occurring late (>5 years) after surgical removal of the primary tumor (Cochran et al., 1988; Rosenberg et al., 1989a); (2) the existence of a large proportion of patients with antibodies (Old, 1981) and cytotoxic cells (reviewed in Anichini, 1989; Rosenberg et al., 1989a; Parmiani et al., 1990; Boon, 1992) reactive with their tumors in vztro; (3) tumor regression sometimes occurring weeks or months after IL-2 treatment is discontinued (Rosenberg, 1988); (4) T cells with specific cytotoxicity detectable within the regressing tumor tissue (Cohen et al., 1987; Parmiani et al., 1990); (5) generation (from about 25% of the patients) of MHCrestricted cytotoxic T cell lines or clones derived from tumor-infiltrating lymphocytes (TIL), blood, or lymph nodes, which can damage ex vzvo autologous tumor cells (Itoh et al., 1988; Slingluff et al., 1988; Maleckar et al., 1989; Topalian et al., 1989; Mukherji et al., 1990; Parmiani et al., 1990); ( 6 )preferential localization of readministered autologous TIL in tumor sites (Griffith et al., 1989); (7) the more frequent lesional (subcutaneous metastases) response to IL-2-based immunotherapy in pa-
tients whose tumor cells expressed a high level of HLA-DR antigens during treatment, and infiltration of T cells and macrophages (but not NK cells) in the regressing tumor tissue (Rubin et al., 1989; Clark et al., 1990); (8) correlation between the clinical response and the frequency of melanoma-specific CTL precursors in the blood lymphocyte population following immunization with a tumor vaccine (Mitchell et al., 1990; Mitchell, 1991). Some of these parameters indicate that about 25% of the patients with melanoma (and probably renal cell carcinoma as well) carry immunogenic tumors. This corresponds to the approximately 25% response rate to immunotherapy in these patient groups. There is no evidence for a role of the nonselective cellular effectors, such as NK/LAK cells. In the majority of the clinical trials with melanoma patients treated with IL-2 o r other immunotherapeutic modalities, no correlation was found between the clinical response and the levels of cytotoxicity in NK and LAK cell assays with blood lymphocytes (Boldt et al., 1988; Eberlein et al., 1989; Ghosh et al., 1989; Favrot et al., 1990; Dillman et al., 1991b; Isacson et al., 1992). In uitro assays indicated the immunological recognition of tumor cells in a proportion of patients with other malignancies as well. Blood lymphocytes were found to be cytotoxic and/or were stimulated by autologous ex uiuo tumor cells (Vanky et al., 1976, 1982, 1983a,b, 1987; Vose et al., 1977; Vose and Bonnard, 1982; Vanky and Klein, 1982a,b, 1989; Uchida et al., 1987, 1990; Allavena et al., 1988; Uchida and Mizutani, 1989; Parmiani et al., 1990; Ioannides et al., 1991). The analysis of the cytotoxic response indicated that it represents a T cell-mediated immunity (Vanky and Klein, 1991) and it correlated with a relatively favorable prognosis in a group of patients with adeno- and squamous cell carcinoma of the lung and with mesenchymal cancers (Vanky et al., 1986, 1987; Uchida and Mizutani, 1989; Uchida et al., 1990) (see Section IV,B). 3 . MHC Antigen Expression on Tumor Cells
Major histocompatibility complex molecules on the cell surface carry and present the antigenic peptides that are recognized by cellular immunity. Appropriate expression on the tumor cells is therefore decisive for an efficient antitumor response (Tanaka et al., 1988; Van Duinen et al., 1988; Gopas et al., 1989; Elliott et al., 1989; Vanky 1986; Vanky et al., 1986, 1988, 1990; Greenberg, 1991; Vanky and Klein, 1991). Low MHC antigen expression on the tumor cells can thus be the cause of low o r no immunogenicity. In many, but not in all, experimental models deficiency in certain class I and/or class I1 antigens was associated with increased
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take of tumor grafts and metastasizing tendency. However, for the majority of human tumor types there seems to be no solid evidence for a correlation between altered MHC antigen expression, quantitative or qualitative, and increased malignancy, defined by metastasis or survival (reviewed in Tanaka et al., 1988; Anichini, 1989; Elliott et al., 1989; Gopas et al., 1989; Schreiber, 1989; Wintzer et al., 1990; Moller et al., 1991; Ruiz-Cabello et al., 1991). In melanoma and renal cell carcinoma patients, an association between the expression of high levels of MHC class I antigens on the tumor cells and favorable prognosis was seen in some studies (Van Duinen et al., 1988; Tomita et al., 1990; reviewed in Ruiter et al., 1991). In contrast, high expression of MHC class I1 antigens was associated with unfavorable prognosis in melanoma patients (reviewed in Ruiter et al., 1991).The presence of HLA-DR and D Q antigens on primary breast carcinoma was correlated with several distinct parameters of good prognosis (Brunner et al., 1991). Selective down regulation of some alleles on the malignant cells, including often the HLA-A2 allele (present in approximately 40% of the Caucasian population), has been detected by immunofluorescence and by biochemical methods in a proportion of patients with several types of cancers (Momburg et al., 1989; Natali et al., 1989; Smith et al., 1989; Ruiz-Cabello et al., 1991; Wang et al., 1991). In a group of melanoma patients, the HLA phenotype of the patients seemed to influence the response to active immunotherapy with a vaccine prepared from cultured allogeneic melanoma cells. A higher response rate was obtained in patients with the phenotypes HLA-A2 and -28, B12s (B12, B44, and B45), and C3, particularly in combination, or with HLA-DR4 in combination with A2 or C3 (Mitchell, 1990, 1991). In patients with melanoma and renal cell carcinoma treated with I L 2 and LAK cells or I L 2 and IFNa, the frequency of HLA-AS, B44, or DR4 was higher in responders than in nonresponders (Scheibenbogen et al., 1991). In patients with colon cancer, immunization with autologous tumor cells led to better clinical response if the tumor cells expressed MHC class I1 antigens (Ransom et al., 1991). T h e effect of the changes in MHC antigen expression on melanoma cells during immunotherapy was also investigated. The lesional response (subcutaneous metastases) to IL-2-based immunotherapy correlated with DR antigen expression on the tumor cells after but not before treatment (Rubin et al., 1989). In some (Atkins et al., 1988), but not in other studies (Schwartzentruber et al., 1990),a high incidence of autoimmune thyroid dysfunction was associated with the clinical response to IL2, with and without LAK cell administration, in patients with various types of neoplasms. A similar correlation between the development of
hypothyroidism and the clinical response was observed in melanoma and renal cell carcinoma patients treated with IL-2 and IFNa (Scalzo et al., 1990). This effect may be ascribed to increased MHC antigen expression on the tumor and thyroid cells, induced by the cytokines (e.g., TNFa, IFNy) produced after IL-2 administration (Siege1 and Puri, 1991). Increased MHC class I antigen expression on a murine tumor conferred increased responsiveness to IL-2 therapy (Weber et al., 1987). Several studies reported that human tumors with high levels of MHC class I antigens had more intense T lymphocyte infiltration (Kornstein et al., 1983; Van Duinen et al., 1988; Vanky et al., 1988). T h e significance of MHC class I antigen expression on the tumor cells for their sensitivity to immune effectors was shown directly in uitro. Blood lymphocytes of patients with solid tumors damaged the autologous ex vzuo tumor cells only if the latter expressed appreciable levels of MHC class I antigens and the intercellular adhesion molecule- 1 (ICAM- 1) molecules (Vanky et al., 1988, 1990). Tumor cells acquired sensitivity concurrent with induction or elevation of these molecules on the tumor cells by exposure to TNFa and IFNy. In several experiments, cytotoxic lymphocytes generated in mixed cultures with the cytokine-treated tumor cells also damaged the unmodified, low MHC class I expressor tumor cell aliquots (Vanky et al., 1988, 1989, 1990). T h e role of adhesion molecules in tumor-T cell interaction was also demonstrated by Anichini et al. (1990). Among tumor cell clones isolated from a human metastatic melanoma, susceptibility to lysis by autologous CTL clones was associated with high expression of IGAM- 1 and the very late activation antigens (VLA)-1, -2, -3, -4, and -6.
4. Conclusions It is very likely that the MHC antigenic make-up and the expression of adhesion molecules on the tumor cells are important for the response to immunotherapy (Braakman et al., 1990; Parmiani et al., 1990; Vanky and Klein, 1991; Melief, 1992). While T cells appear to be pivotal in the development and execution of antitumor response, other cell types may participate in tumor destruction. Regressing tumors are often infiltrated by macrophages, neutrophils, and eosinophils (Pretlow et al., 1983; Fidler, 1985; Musiani et al., 1989; Bosco et al., 1990), cells that do not possess antigen-specific properties. They represent the second line of defense, presumably mobilized by the lymphokines released by the antigen-specific T cells. Thus, BRM-stimulated nondiscriminative effector cells, such as macrophages (Fidler, 1985, 1988a,b; Schreiber, 1989; Mantovani, 1990; Whitworth et al., 1990; Greenberg, 1991) and NK/LAK cells (Herberman, 1987,
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1989; Rosenberg, 1988, 1990; Rosenberg et al., 1989a),may contribute to the therapeutic effect. The preclinical studies (and to some extent the clinical studies as well) suggest that antigenicity of the tumor and the capacity to mobilize a T cell response are prerequisites for the success of immunotherapy. In extensive studies, the patients (except those with advanced disease) were not found to be immunodeficient. Therefore, the antigenicity of the tumor seems to be the main property that determines whether therapeutic effects can be achieved by measures that act on the immune system. B. TUMOR BURDEN AND LOCATION Experiments in animals demonstrated that the capacity of the immune system to cope with a growing tumor is limited-up to a certain tumor size-and there is a critical time after tumor implantation when immunotherapy should be initiated. Therefore, it is advisable to reduce the tumor load-by surgery, chemotherapy, or radiotherapy-prior to administration of immunotherapy (Fefer, 1974; Fefer et al., 1976, 1982; Kedar and Weiss, 1983; Mitchell, 1988). Usually it is not possible to eliminate all the tumor cells; it may not even be an advantage because the residual cells may serve as antigen source (Hamblin, 1989). Evidence for this is seen in leukemia patients treated with high-dose chemo/ radiotherapy followed by allogeneic bone marrow transplantation, where the residual leukemic cells evoke an immune response and are destroyed subsequently by the graft versus leukemia (GVL) response (Slavin and Kedar, 1988; Butturini and Gale, 1989; Gottlieb et al., 1989; Sullivan et al., 1989; Horowitz et al., 1990; Porwit et al., 1990).There are examples in animal models in which a larger tumor responded better to the immunological manipulation than the smaller one. In mice carrying transplanted immunogenic or weakly immunogenic tumors, responses were better when I L 2 was administered 5-10 days after rather than earlier following tumor inoculation (Rosenberg et al., 198513; Thompson et al., 1986; Maas et al., 1989). Most likely, the tumor had to reach a certain size for provision of critical amounts of antigens, and time was needed to build up the antitumor immunity, which could then be amplified by the lymphokine. In mice with metastases of weakly immunogenic tumors, TNFa was more effective against 5- to 6-mm tumor foci than against smaller ones (Mu16 et al., 1988),although this finding can also be explained by the fact that TNFa action depends, in part, on newly formed capillaries in the tumor (Manda et al., 1990).On the other hand, a complete response to immunomodulators in ovarian carcinoma patients with tumors confined to the peritoneal cavity occurred only when the tumors were smaller than 0.5 cm (Berek, 1990).
An important parameter is the location of the tumor and the extent of its dissemination. As with chemotherapy, patients with “widespread disease’’ and those with metastases in certain sites (e.g., in the brain, liver, o r bones), do not respond as well to immunotherapy as patients with a “limited disease” o r patients with skin, lymph node, lung, or soft-tissue metastases (Lotze et al., 1986; West et al., 1987; Mitchell et al., 1988b; Bergmann, 1989). Locoregional immunotherapy can be applied and showed some effects in patients with localized disease (Markman, 1987; Bubenik, 1989), such as head and neck (Forni et al., 1988), brain (Yagita and Grimm, 1988; Yoshida et al., 1988), bladder (Pizza et al., 1984), and abdominal tumors (Ottow et al., 1987; Urba et al., 1989). C. HETEROGENEITY OF THE TUMOR CELL POPULATION Tumor cell populations are heterogeneous, comprising cells with variable growth, metastatic and immunogenic properties, and sensitivities to chemoradiotherapy and to immunological effector mechanisms (Heppner, 1984; Nicolson, 1984; Poste, 1986; Schnipper, 1986; Clark et al., 1988; Fidler, 1988a; Woodruff, 1988; Parmiani et al., 1990). Tumor samples collected from different sites of one patient, and parallel tumor cell lines derived from one tumor, often react differently with monoclonal antibodies, cytotoxic T cell clones, LAK and TIL populations (Anichini et al., 1989; Rivoltini et al., 1989; Van den Eynde et al., 1989; Notter and Schirrmacher, 1990; Parmiani et al., 1990; Topalian el al., 1990). Variants that can escape immune attacks may be present in the tumor cell populations. It is therefore likely that immunotherapy using one monoclonal antibody, one clonal effector cell population, or one cytokine, can affect only part of the cell population, and the variant cells can take over. Therefore, “cocktails” of monoclonal antibodies and/or several T cell clones directed against different antigenic epitopes on the tumor may be required for the control of the tumor. Ill. Current lmmunotherapy Strategies
A. ACTIVESPECIFIC IMMUNOTHERAPY Immunization with tumor cells or tumor extracts preventing the growth of subsequently grafted cells provided the proof for immunogenicity of some animal tumors. Active specific immunotherapy in patients was attempted by several investigators (reviewed in Foon, 1989; Rosenberg et al., 1989a; Bystryn, 1990; Livingston, 1991a), using various vaccines (with and without adju-
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vants), such as irradiated autologous tumor cells (Schulof et al., 1988; Hoover and Hanna, 1989; Wiseman et al., 1989; Berd et al., 1990a; McCune et al., 1990); allogeneic (single or pooled) ex uzuo or cultured tumor cells, or extracts, of the corresponding histologic type (Mitchell et al., 19$8a, 1990; Mitchell, 1991; Bystryn, 1990; Morton et al., 1991a); tumor oncolysates (tumor cells infected with lytic viruses) (Cassel et al., 1983); or purified tumor antigens (Hollinshead et al., 1989; Knuth et al., 1991; Livingston, 1990, 1991b; Srivastava, 1991). Encouraging results have been reported in melanoma and carcinoma (colon and lung) patients with progressive disease (Berd and Mastrangelo, 1988a; Berd et al., 1990a; Mitchell et al., 1988a, 1990; Mitchell, 1991; Morton et al., 1990, 1991a) or as adjuvant treatment after surgery (Cassel et al., 1983, 1986; Hoover and Hanna, 1989; Hollinshead et al., 1989, 1990; Bystryn et al., 1988, 1990; Morton et al., 1991a). In some of these trials, the patients were pretreated with low-dose cyclophosphamide (to deplete suppressor cells), and the tumor vaccines were given together with BCG or other adjuvants. In some cases, indomethacin or cimetidine were also used in order to act against suppressor cells (see Section 111,D). The therapeutic benefit of these auxiliary measures has not been proven, however, in randomized trials. T h e decision whether to use autologous or allogeneic tumor cells for vaccination has not yet been reached. It can be expected that only the autologous tumor carries the relevant individually distinct epitopes (Old, 1981; Notter and Schirrmacher, 1990; Parmiani et al., 1990). However, due to heterogeneity of the tumor cell population, a proportion of cells may lack the antigens present in the autologous vaccine (Bystryn, 1990). Several arguments can be mentioned for the use of pooled allogeneic tumors o r tumor cell lines: (1) Shared tumor-associated antigens may be expressed in various quantities on the tumors (Old, 1981; Crowley et al., 1990; Mitchell, 1991). (2) Some of the contributing tumors may share HLA specificities with the patient and thus the vaccine may provide the proper antigenic peptide against which the patient can respond. This may have no importance when extracts are used; the antigens then are expected to be processed and presented by the patient’s cells. (3) The alloantigens may exert “help” that can potentiate the response against the putative tumor antigens (Mitchell, 1991). (4)A pool can be produced in large quantities, thus providing a standard immunogen. It is difficult to say which of these considerations are valid, therefore a combined preparation consisting of both autologous and allogeneic source could be preferred, when possible. T h e majority of tumor types in humans are nonimmunogenic (reviewed in Sulitzeanu, 1985; Rosenberg et al., 1989a). Various manipula-
tions, however, can enhance the expression of weak antigens or impose new antigens on the cell. Such cells can then be used for elicitation of an immune response, which may act also on the resident nonaltered cells. Human and murine tumor cells have been modified in vitro by (1) chemical/enzymatic treatment (Kedar and Lupu, 1978; Kedar et al., 1979; Wunderlich et al., 1985; Berd et al., 1990b; Wiseman et al., 1989, 1990; Ramakrishna and Shinitzky, 199l), (2) xenogenization with viruses (Kobayashi, 1986; Schirrmacher, 1989; Schirrmacher et al., 1986, 1989; Bash et al., 1990; Bohle et al., 1990; Freedman et al., 1990; Lehner et al., 1990), ( 3 ) mutagenization (Boon, 1983, 1992; Boon et al., 1989), (4) transfection of MHC class I or class I1 genes (Gopas et al., 1989; Isobe et al., 1989; Ostrand-Rosenberg et al., 1990), and (5) cytokine treatment (IFNy, TNFa, IL-4), which u p regulates MHC antigen, adhesion molecule, and perhaps also tumor-associated antigen expression (Vanky et al., 1990; Wiebke et al., 1990; Hoon et al., 1991). Chemically/enzymatically altered (Skornick et al., 1986; Berd et al., 1990b; Wiseman et al., 1989, 1990) and virus-infected (Cassel et al., 1983, 1986; Schirrmacher, 1989, 1991; Freedman et al., 1990; Bohle et al., 1990) autologous tumor cells have recently been used for immunization in patients. Results cannot be evaluated yet because a relatively small number of patients have been treated, and the observation period has been short. T h e image of tumor antigens in the form of antiidiotypic antibodies has been employed as immunogen both in animals and patients. T h e preliminary clinical results are encouraging (Herlyn et al., 1987; Campbell et al., 1988, 1990; Bhattacharya-Chatterjee and Kohler, 1989; Chatterjee et al., 1990; Levy and Miller, 1990; Mittelman et al., 1990; Frodin et al., 1991; reviewed in Lee and Hellstrom, 1988; Sikorska, 1988; Foon, 1989; Rosenberg et al., 1989a; Mellstedt, 1990; Stevenson et al., 1990). These various manipulations were often successful in eliciting a T cell response in uitro or in uivo against the unmodified tumor cells also and led to regression of experimental tumors. In some of the clinical trials, the patients that responded to active specific immunotherapy with antibodies, CTL, or delayed-type hypersensitivity (DTH) reactive with the immunizing antigen, showed a better clinical response (in patients with advanced disease), and a longer disease-free survival and fewer recurrences (in the adjuvant setting) than those that responded poorly o r not at all in these immunological tests (Berd et al., 1990a; Bystryn, 1990; Frodin et al., 1991; Mitchell, 1991; A. Mittelman and V. Schirrmacher, personal communications). Obviously, the first step of a well-planned active specific irnmunotherapy requires the identification of the relevant antigens. Recently, the group of T. Boon exploited tumor cell-reactive CTL clones and
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defined (and sequenced) a family of molecules (“MAG,”) expressed on a human melanoma cell line. While the antigens were not detected on normal cells, they were expressed on other (HLA-A1 positive) melanoma lines and also on other tumor types (Van der Bruggen et al., 1991). These, and other similarly defined antigens, can be used as a raccine source in patient groups with the appropriate class I MHC molecules. B. ACTIVENONSPECIFIC IMMUNOTHERAPY: CYTOKINES
I. Introduction Over 20 cytokines (among them 12 interleukins) are known and functionally defined. The majority of these are now produced by recombinant DNA technology and are available for therapy. The cytokines are part of a complex system; each of them can be produced by more than one cell type and one cell can produce several cytokines, in a tightly regulated way (Lebendiker et al., 1987; Sariban et al., 1988; Ketzinel et al., 1990). They have pleiotropic biological effects, and different cytokines can share certain functions. Usually these soluble factors act at a short range, in low concentrations, in both autocrine and paracrine fashions. They can influence the immunological host-tumor relationship; some of them can also affect tumor growth and differentiation and can regulate inflammatory responses (reviewed in Balkwill, 1988; Balkwill and Burke, 1989; Foon, 1989; Kelso, 1989; Mizel, 1989; Paul, 1989; Rosenberg et al., 1989a; Borden and Sondel, 1990; DeVita et al., 1991; Oettgen, 1991b; Oldham 1991; Street and Mosmann, 1991; Zwierzina, 1991).
The response to cytokines may be influenced by the genetic background of the host. Furthermore, various cell types differ in susceptibility, mainly due to differences in the expression of the relevant receptors, in the signal transduction mechanism, and in the activity of specific transcription factors (Korber et al., 1988; Landolfo et al., 1988; Steiniger et al., 1988; Gribaudo et al., 1990). Cytokines can be useful in cancer treatment by (1) exerting direct effects on the tumor (cytolysis, cytostasis, vasculature damage, terminal differentiation), (2) enhancing the expression of MHC antigens, cell adhesion molecules, and other surface moieties on the tumor cells including tumor-associated antigens, (3) recruiting, expanding, and stimulating endogenous effector cells, and (4) maintaining and even enlarging adoptively transferred lymphocyte populations.
Because of the highly complex network of cytokine and cellular interactions, the mechanisms of antitumor actions obtained in several experiments are not clear. Although thousands of cancer patients have been treated with IFNs and therapeutic effects were achieved, it is unknown whether the antiproliferative, the immunomodulatory, or the antiviral effects, alone or in combination, were important. Likewise, it is unclear what the mechanisms are of the remissions seen in IL2-treated patients (Quirt and Tannock, 1990; Sasaki et al., 1991; Siege1 and Puri, 1991). Experiments in animal models point to the possibility that cytokine-mediated antitumor effects may also be caused, in part, by a decreased food intake and alterations in host substrate metabolism (Gelin et al., 199lb).
2. Antitumor Effects by Application of One Cytokine Interleukin 2, TNFa, IFNa, and IFNy have been found effective against a wide spectrum of experimental tumors, whereas in patients mainly IL-2 and IFNa were beneficial and only in certain malignancies (reviewed in Foon, 1989; Rosenberg et al., 1989a,b; Borden and Sondel, 1990; DeVita et al., 1991; Oldham, 1991). Interleukin 2-based immunotherapy is discussed later (Section 111,C). The growth inhibitory and cytotoxic effects of IFN and TNF both in vitro and in vivo for various types of experimental and human tumor cells is well established. Tumor necrosis factor can also induce hemorrhagic necrosis in the tumor through the blockade of the blood supply, and it facilitates the influx of inflammatory cells to the tumor tissue (North and Havell, 1988; Shimomura et al., 1988; Mule et al., 1988; Balkwill et al., 1990; McIntosh et al., 1990; Semenzato, 1990b). Both IFN and TNF up regulate the expression of MHC class I and class I1 antigens, the cell adhesion molecules, and perhaps even putative tumorassociated antigens, thereby increasing immunogenicity and susceptibility of the tumor cells to T cell-mediated damage (Borden, 1988; Weber and Rosenberg, 1988; Guadagni et al., 1989; Maio et al., 1989; Stotter et al., 1989; Vanky et al., 1989, 1990; Wiebke et al., 1990). Moreover, they can stimulate T cells, macrophages, and N K cells (Faltynek and Oppenheim, 1988; Havell et al., 1988; Jaffe and Herberman, 1988; Paul and Ruddle, 1988; Talmadge et al., 1988; Asher et al., 1989; Kunkel et al., 1989; Rosenblum and Donato, 1989). Interleukin 2 probably lacks direct antitumor effects, but it is an efficient stimulator of several immune functions (Mertelsmann and Welte, 1986; Smith, 1988; Rosenberg et al., 1989a; Swain, 1991). The effect of cytokines on tumor growth may differ in vivo and in vitro. For example, TNFa (Tomazic et al., 1988; Balkwill et al., 1990;
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Semenzato, 1990b) and IFN (Morikawa et al., 1989; reviewed in Wadler and Schwartz, 1990) inhibited the growth of some tumors either in vitro or in Y ~ Z I Obut , not under both conditions. IFNy stimulated the growth of a human T cell lymphoma in vitro but inhibited its growth when grafted in nude mice (Jemma et al., 1989). Adverse effects were also reported. Treatment with IFNy seemed to increase the relapse rate of patients with stage I1 cutaneous melanoma (Meyskens et al., 1990). Interleukin 1 (Giavazzi et al., 1990; Bani et al., 1991), IL-2 (Kedar et al., 1989), T N F a (Balkwill et al., 1990; Malik et al., 1990), and IFNy (Kelly et al., 1991) enhanced the local growth and experimental metastases of both murine and human tumor grafts in euthymic and athymic mice. 3 . Cytokine Combinations: Additive and Synerptic Effects
Cytokines initiate a cascade; they trigger the production of other cytokines and the expression of their receptors. For example, patients treated with high IL-2 doses have high IL-1, I L 6 , TNFa, and IFNy plasma levels (Gemlo et al., 1988; Kasid et al., 1989; Boccoli et al., 1990; Dupere et al., 1990). Treatment with combinations of cytokines differing in their mode of action, using each at subtoxic doses, may therefore improve the therapeutic index. Cytokines have been used for tumor therapy in various combinations, alone and together with chemotherapy, with and without TIL or LAK cells. Additive o r synergistic effects have been obtained with cytokine combinations in experimental systems: IL- 1 plus IL-2 (Belardelli et al., 1989; Crump et al., 1989; Ciolli et al., 1991), IL-2 plus TNFa (Winkelhake et al., 1987; Yang et al., 1989), I L 2 plus IFNa (Brunda et al., 1987; Cameron et al., 1988), IL-2 plus IFNy (Agah et al., 1988), T N F a plus IFNy (Watanabe et al., 19SS), and IL-2 plus IL-5 (Aoki et al., 1989) synergized in the activation of NK/LAK cells in vitro and/or in the induction of antitumor effects in vivo. Additive or synergistic therapeutic effects for animal tumors have also been reported for IL-1 plus IL4 (Forni et al., 1989), TNFa plus IL-6 (Mule et al., 1990),and TNFa plus IL-2 plus IFN (Agah et al., 1988; McIntosh et al., 1989). Administration of 1L-2 and IFNa to mice enhanced the proliferation of lymphoid cells (Puri et al., 1990). In our studies (Kedar et al., 1990, 1992), the cojoint treatment with IL-2 and IFNa had additive therapeutic effects in mice with pulmonary metastases or intraperitoneal growths of weakly immunogenic tumors. This cytokine combination synergized with cyclophosphamide. I L 2 plus TNFa and IL-2 plus macrophage-colony stimulating factor (M-CSF) were considerably less efficient, alone and with chemotherapy.
Additive therapeutic effects were also obtained in nude mice carrying human colorectal carcinoma and melanoma grafts with IL-2 plus IFNa, particularly in combination with chemotherapy [5-Fluorouracil (5-FU) or dacarbazine (DTIC), respectively] (Kedar et al., 1990, 1992; Gazit et al., 1992). Impressive clinical results (an approximately 40% response) were reported in patients with metastatic melanoma and renal cell carcinoma treated with high-dose IL-2 plus IFNa (Rosenberg et al., 1989b,c). T h e effects were apparently additive, and the response correlated with the cytokine doses. This was confirmed in another study, in which 50% of the patients with renal cell carcinoma responded (Hirsh et al., 1990). In other studies, however, a lower (525%) response rate was obtained with this cytokine combination (Legha et al., 1990; Stahel et al., 1990; Bukowski et aE., 1990; Sznol et al., 1990; Atkins et al., 1991; Ilson et al., 1991; Pichert et al., 1991). In patients with metastatic renal cell carcinoma, low doses of IL-2 and IFNa, administered subcutaneously, was less toxic than, and as effective as, the high-dose intravenous regimen of IL-2 (Atzpodien et al., 1990a,b). The combination of IFNa and TNFa gave a 43% response in patients with renal cell carcinoma (Otto et al., 1990), but was ronsiderally less effective in other types of solid tumors (Fuchimoto et al., 1990). Other combinations of two cytokines, employing IL-2, IFNy, and TNFa, were also of low potency in patients with various solid tumors (Rosenberg et al., 1989b; Redman et al., 1990; Sohn et al., 1990; Dexeus et al., 1991; Dillman et al., 1991a; Smith et al., 1991b; Weiner et al., 1991; Yang et al., 1991). T h e experience in experimental models suggests that sequential administration of cytokines may be more effective than concurrent treatments. The antitumor effects in tumor-bearing mice were usually stronger when TNFa or IFNa was given 1-3 days prior to IL-2, compared to either concurrent administration or sequential treatment but in the reverse order (McIntosh et al., 1988; Zimmerman et al., 1989b; Kedar et al., 1992). The optimal sequence may be different in protocols combining these cytokines and chemotherapy. In our studies using both euthymic and athymic mice carrying mouse or human tumor grafts, respectively, the most effective combination regimen, yielding a synergistic effect, was IFNa (6 days) + chemotherapy + IL-2 (6 days), with a l-day interval between IFNa and chemotherapy and 2-3 days between chemotherapy and IL-2. The second best regimen was chemotherapy -+ I L 2 --+ IFN. T h e following schedules were less efficient: IL-2 + chemotherapy + IFN, chemotherapy + IFN + I L 2 , and chemotherapy --+ IL-2 plus IFN (Kedar et al., 1992; Cazit et al., 1992).
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The animal models suggest that the synergistic effects depend largely on the presence of tumor-specific T cells that are activated by the cytokines. The synergistic effects of I L 2 plus TNFa or I L 2 plus IFNa were obtained only with immunogenic tumors; moreover, if X irradiation or i n vivo depletion of Lyt2 T cells preceeded immunotherapy, the therapeutic effects did not occur (Cameron et al., 1988; McIntosh et al., 1988). Similarly, I L l plus I L 2 treatment was inefficient in nude mice and in euthymic mice depleted of Thy 1.2+ cells (Belardelli et al., 1989). +
4 . Cytokine Combinations: Antagonistic Effects Combinations of certain cytokines can lead to adverse effects because one cytokine may counteract the function of another. Evidence for such effects stems from i n vitro experiments. Interleukin 4 (Spits et al., 1988; Ebina et al., 1990) and transforming growth factor P (TGFP) (Grimm et al., 1988) were shown to inhibit the induction of human LAK cells by IL-2; I L l O inhibited lymphokine production by activated T cells (K. W. Moore et al., 1990; Fiorentino et al., 1991); IFNy suppressed IL4-induced IgE production (ChrCtien et al., 1990); and TNFa abolished the immunosuppressive activity of TGFP (Ranges et al., 1987).
5 . Toxicity The currently used cytokines can cause moderate or severe toxicity. This can be attributed to the extremely large doses administered, which are several orders of magnitude higher than the physiologic levels. Most serious is the vascular leak syndrome, induced mainly by I L 2 and TNFa (S. A. Rosenberg et al., 1987a, 1989a,b; Margolin et al., 1989; Semenzato, 1990b; Dillman et al., 1991b; Siege1 and Puri, 1991). High doses of I L 2 or IFN also caused transient immunosuppression in both animals and patients (Talmadge et al., 1987; Kedar et al., 1988b; Wiebke et al., 1988; Kradin et al., 1989b; Hank et al., 1990b). Several attempts were made to search for less toxic cytokines. Impressive antitumor effects, similar to those seen with high-dose IL-2, but with negligible toxicity, were obtained in mice treated with I L 6 (Mule et al., 1990). A nontoxic derivative of I L 1 maintained the immunomodulatory activity (Forni et al., 1989). Chemically altered recombinant human TNFa had a broader cytotoxic spectrum for human and mouse tumor cells in vitro and stronger antitumor effects in mice, with fewer side effects, than the unmodified cytokine (Noguchi et al., 1991). It is also possible to administer agents that counteract toxicity. Indomethacin (inhibits prostaglandin synthesis) and bismuth subnitrite (blocks oxygen-free radical production) have been shown to reduce TNFa toxicity in rodents (Haranaka, 1988; Talmadge, 1988; Krosnick et
al., 1989b). Corticosteroids decreased I L 2 toxicity in patients (Vetto et al., 1987; Mier et al., 1990). Their effect on the tumor response has not been determined; in mice, however, the therapeutic efficacy was also reduced (Papa et al., 1986). Certain cytokines can counteract the toxicity of other cytokines. Interleukin 1 administered together with I L 2 reduced the vascular leakage, without impairing the antitumor activity in mice (Puri et al., 1989). In culture, pretreatment of human endothelial cells with I L 1, TNFa, or IFNy increased their resistance to LAK cell-mediated lysis (Mier et al., 1989). 6. Dose Response
The majority of the cytokines show a direct antitumor dose-response relationship in vivo. In studies with mice carrying weakly immunogenic tumors and also in patients, the beneficial response to systemic I L 2 treatment was usually dose dependent and required high doses (Rosenberg, 1986; Bradley et al., 1987; West, 1989). This was also the case with TNFa in experimental models (McIntosh et al., 1988). For systemic treatment of mice carrying immunogenic tumors (Talmadge et al., 1987; Talmadge, 1988)and for local treatment of murine and human tumors (Vaage, 1987; Forni et al., 1988; Musiani et al., 1989),even very low I L 2 doses (25- 1000 U/day) were sufficient to cause partial or complete tumor regression. Interferon? was shown to have a bell-shaped therapeutic and immunomodulating dose-response curve, both in the experimental and clinical studies (Jaffe and Herberman, 1988; Maluish et al., 1988; Talmadge, 1988; Frick and Aulitzky, 1990; Wadler and Schwartz, 1990). Its optimal therapeutic and immunomodulatory doses were usually lower than the maximum tolerated dose.
7. Mode of Cytokine Administration Because of the nonspecific biodistribution and the short plasma halflives (several minutes in mice and up to approximately 4 hr in humans), the in vivo effects of systemically administered cytokines depend on the mode and route of administration. It was suggested that the antitumor effects of IL-2 correlate more with the duration of exposure to a certain level than with the peak plasma level, whereas toxicity was mainly correlated with the latter (Cheever et al., 1985; Ettinghausen and Rosenberg, 1986; Zimmerman et al., 1989a). Bolus intravenous (iv) administration leads rapidly both to high plasma levels and clearance. In melanoma patients treated with high-dose I L 2 by either bolus or continuous iv infusion, the former mode appeared slightly more toxic and less immunostimulatory, but more efficient therapeutically, although
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the total I L 2 dose administered per day was usually higher with the bolus administration (Rosenberg et al., 1987a; West et al., 1987; Sosman et al., 1988; Thompson et al., 1989; Clark et al., 1990; Dutcher et al., 199 1). Comparison of the therapeutic efficacy and toxicity between bolus and continuous infusion of IL-2 has been carried out in only a few randomized clinical trials. T h e two ways of administration were equally effective in renal cell carcinoma, whereas the bolus treatment was more beneficial in melanoma patients (reviewed in Parkinson, 1990). In mice carrying intraperitoneal (ip) tumor grafts, three daily ip injections of IL-2 for 7 days was significantly more efficient therapeutically than continuous administration with the Alzet microosmotic pump (E. Kedar, unpublished observations). Bolus iv administration of IL-2 was also more efficient than continuous infusion in other experimental models (Zimmerman et al., 1990; S. Slavin, personal communication). Additional studies are also warranted to evaluate single or a few highdose courses versus multiple, low-dose treatments extended in time. A possible drawback of the latter schedule is that the therapeutic efficacy may gradually decline due to increasing serum levels of soluble cytokine receptors (Lotze et al., 1987) and/or the appearance of antibodies against the cytokines (Oberg et al., 1989; Kirchner et al., 1991). It may be possible to reduce levels of such antibodies by alternate cycles of recombinant and natural cytokines (Wussow et al., 1990). In one study, local continuous infusion of natural IL-2, but not recombinant I L 2 , led to complete regressions of advanced bladder carcinomas in several patients (Huland et al., 1990). It is still unknown whether or not the soluble cytokine receptors (Fernandez-Botran, 1991) or anti-cytokine antibodies jeopardize the therapeutic efficacy in patients. Due to the short plasma half-life, continuous iv infusions or frequent repeated bolus iv administrations of high (and toxic) doses of cytokines (in particular, IL-2) have been commonly used in patients. Therefore, in order to reduce toxicity and also to simplify the treatment, alternative routes were tested, such as ip or subcutaneous (sc) administrations, already shown to be effective in the prolonged maintenance of high cytokine levels (Gustavson et al., 1989; Urba et al., 1989; Cebon et al., 1990). High ip cytokine levels may be advantageous for patients with tumors confined to the peritoneal cavity, such as in ovarian and colon carcinomas (Berek, 1990; Steis et al., 1990). However, a common side effect of the ip infusion of several cytokines is the formation of local fibrosis (reviewed in Kovacs, 1991). The sc route, on the other hand, may facilitate production of neutralizing anti-cytokine antibodies (Kolitz et al., 1988; Oberg et al., 1989; Atzpodien et al., 1990a; Whitehead et al., 1990). T h e use of infusion pumps, similar to those introduced to pa-
tients for the local continuous instillation of chemotherapy, may be considered also for cytokines. T h e impact of the route of cytokine administration was studied in tumor-bearing mice. Comparing the ip and iv routes for IL-2, the former was more effective for ip tumors, whereas for pulmonary metastases the latter was superior (Kedar et al., 1988a; R. Zimmerman, personal communication). In mice with spontaneous pulmonary metastases, TNFa was effective when administered iv but not ip (Talmadge, 1988). These observations can be explained by the fact that the route of administration markedly influences the tissue distribution, and by the different mechanisms of action of the various cytokines. For tumors confined to accessible compartments, a locoregional administration (intratumoral, periturnoral, perilymphatic) may be effective. Periturnoral injections of IL-1, IL-2, and I L 4 , individually or in various combinations, caused marked responses in mice (Vaage, 1988; Bubenik, 1989; Forni et al., 1989; Bosco et al., 1990). In patients with head and neck tumors, local administration of IL-2 caused transient tumor regression (Musiani et al., 1989) and local activation of specific and nonspecific killer cells (Rivoltini et al., 1990). Beneficial effects were also reported with I L 2 with or without LAK cells administered ip (in colon and ovarian carcinoma; Urba et al., 1989; Stewart et al., 1990; Steis et al., 1990), intrapleurally (in mesothelioma; Yasumoto et al., 1987; Eggermont et al., 1991), intravesically (in bladder carcinoma; Pizza et al., 1984; Huland and Huland, 1989; Huland et al., 1990), and intracerebrally (in brain tumors; Yagita and Grimm, 1988; Yoshida et al., 1988). Local administration of TNFa, IFNP, or IFNy was also found effective as a palliative treatment in patients with different types of solid neoplasms (Bezwoda and Dansey, 1990; Schmid et al., 1990; Wildfang et al., 1990; Boutin et al., 1991; Pujade-Lauraine et al., 1991; Rath et al., 1991). For disseminated diseases, the local treatment may be supported by systemic treatment. 8. New Methods f o r Delivery of Cytokines and Other Biologzcal Response Modifiers
The problems raised by the short plasma half-lives, requiring continuous infusion or frequent bolus administration of high doses of cytokines, as well as the nonspecific biodistribution and systemic toxicity, may be alleviated by resorting to controlled-release vehicles, using techniques already available for the delivery of conventional drugs. These include ( 1) incorporation into microvesicles (phospholipid liposomes) that are introduced systemically, (2) entrapment within polymeric materials (minipellets) or pumps, which are placed in direct contact with the
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tumor, and (3) modification by chemical means (reviewed in Langer, 1990). As opposed to other slow-release vehicles, the distribution of liposomes in the various tissues can be controlled by altering their lipid composition and size and by using various administration routes (Gregoriadis, 1990). “Classical” liposomes (negatively charged and larger than 0.1 pm) are rapidly taken up by phagocytes (mainly in liver, spleen, and lungs) and thus can be effective for tumors in these organs through local activation of macrophages (Schroit et al., 1986; Brodt et al., 1989; Phillips, 1989; Whitworth et al., 1990; Nii et al., 1991). In contrast with the classical liposomes, the recently introduced “stealth” liposomes [containing specific glycolipids and phospholipids of high phase-transition temperature ( T J , or polyethylene glycol (PEG)-derivatized phospholipids and lipids of either high or low T,, with or without cholesterol, and smaller than 0.1 pm] can evade the reticuloendothelial system, thereby achieving prolonged circulation time and enhanced accumulation in tumors localized in various body compartments (Gabizon, 1989, 1991; Gabizon and Papahadjopoulos, 1988; Ranade, 1989; Gabizon et al., 1990; Gregoriadis, 1990; Papahadjopoulos and Gabizon, 1990; Mayhew and Lasic, 1991; Papahadjopoulos et al., 1991). The entrapped cytokines may even be taken up through endocytosis by cells that lack surface cytokine receptors (Fidler et al., 1985).The poor extravasation capacity of certain types of liposomes (Matzku et al., 1990) can be overcome by administering them simultaneously with cytokines that increase vascular permeability, such as TNFa (Suzuki et al., 1990) and I L 2 (Schultz et al., 1991).Other agents with vasoactive activity [e.g., prostaglandin E, (PGE,), histamine, leukotrienes] (Hennigan et al., 1991), or hyperthermia, may also be useful. Even if large “stealth” liposomes fail to extravasate, they can still function as circulating microreservoirs, slowly releasing the entrapped BRM. The availability of small stealth liposomes with prolonged circulation time and good extravasation capacity opens the possibility of targeting liposomes to tumors in various locations by using them equipped with monoclonal antibodies [or the F(ab’) fragments] directed against tumor antigens (immunoliposomes)(Hashimoto et al., 1983; Freeman and Mayhew, 1986; Papahadjopoulos and Gabizon, 1987; Bankert et al., 1989; Singh et al., 1989; Akaishi et al., 1990; Kumai et al., 1990; Yemul et al., 1990). The immunoliposomes can be administered systemically or locally (such as in ovarian carcinoma; Nassander, 1991). In addition to the “classical”putative tumor-associated antigens, other molecules expressed on the tumor cells can be exploited for targeting. Theoretically, for tumor cells “overexpressing”growth factor receptors
[e.g., for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), estrogen, transferrin, bombesin] (reviewed in Sat0 and Sato, 1989; Herlyn et al., 1990; Cantley et al., 1991; Hunter, 1991; Lippman, 1991), targeting may be enhanced by coupling to the liposomes the respective growth factor or growth factor analog (e.g., tamoxifen in breast carcinoma), or antibodies directed to the receptor. Antibodies to cell surface adhesion molecules may also be used for targeting. Increased binding to tumor cells in vitro was demonstrated with nerve growth factor (M. B. Rosenberg et al., 1987) and anti-laminin receptor antibody (Rahman et al., 1989)-conjugated liposomes. The same possibility applies to tumor cells displaying on their surface high levels of oncogene products (e.g., HER-2lneu) (Hellstrom and Hellstrom, 1989; Yarden, 1990; Tecce et al., 1991; Xu et al., 1991). Thus, compared with free, soluble BRMs, the treatment with BRMcontaining targeted liposomes for site-specific delivery is likely to be more efficient, more cost effective, and less toxic. Other advantages of liposome-encapsulated cytokines are their relatively long shelf life (at 4"C), allowing preparation of large batches sufficient for several months (E. Kedar and Y. Barenholz, unpublished observations), and protection from antibodies in uivo (Debs et al., 1989). Liposome-encapsulated cytokines and other BRMs have already been utilized in experimental systems and in patients (Fidler, 1988b; Brenner, 1989; Whitworth et al., 1990; Kleinerman, 1991). The antiproliferative activity of IFNa on human tumor cell lines in uitro was markedly enhanced when added in liposomes (Killion et al., 1989; Shin et d., 1990). In animal models, better antitumor responses were obtained with encapsulated TNFa (Debs et al., 1990) and IL-2 (Anderson et al., 1990; Loeffler et al., 1991a; E. Kedar and Y. Barenholz, unpublished results) than with the soluble ones. Similarly, encapsulated muramyl dipeptide (MDP; Phillips et al., 1987; Fidler, 1988b) and muramyl tripeptidephosphatidylethanolamine (MTP-PE, MacEwen et al., 1989; Kleinerman, 1991), exhibited stronger therapeutic effects and the effects were obtained with much lower doses, as compared with the unmodified agents. In an attempt to further enhance therapeutic efficacy, combinations of cytokines and encapsulated BRMs have been tested recently. Treatment with liposomal MTP-PE and granulocyte/macrophage-CSF (GM-CSF) increased the survival of nude mice carrying human ovarian cancer xenografts, compared to mice given either agent singly (Malik et al., 1991). A synergistic antitumor effect was also found in mice treated with liposomal MTP-PE and IL-2 (Dinney et al., 1991). In clinical trials, encapsulated MDP was beneficial in patients with prostate cancer (Vosika et al., 1990), and phase 1/11 trials with encapsu-
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lated MTP-PE began recently in patients with advanced cancer, alone (Kleinerman et al., 1989; Creaven et al., 1990; Urba et al., 1990; Frohmiiller et a!., 1991; Kleinerman, 1991) and in combination with IFNy (Lautersztain et al., 1991). Encouraging results were recently obtained in children with osteosarcoma receiving encapsulated MTP-PE as postsurgery adjuvant treatment (I. Fidler, personal communication; Mihich, 1991). Another novel approach for regional delivery of cytokines to tumor sites is the use of solid-phase cytokines. Interleukin 2 coupled to polystyrene beads was more efficient than soluble I L 2 in vivo in the induction of cytotoxic activity and local tumor growth inhibition in rats (Crum and Kaplan, 1991). Prolongation of the IL-2 (Katre et al., 1987) and TNFa (Noguchi et al., 1991) half-life in uiuo has been achieved by coupling them to a carrier molecule. In mice, polyethylene glycol (PEG)-conjugated IL-2 had a 20fold longer plasma half-life, compared to soluble IL-2. Moreover, the antitumor effect of PEG-IL2 was superior, at equitoxic doses, and it could be obtained with fewer and less frequent administrations (Katre et al., 1987; Zimmerman et al., 1989a;J. C. Yang et al., 1990b; E. Kedar, unpublished results). Recently, w e have found that the LAK cell activity of peritoneal cells and splenocytes of mice inoculated ip or iv with PEGIL-2, or liposomal I L 2 (each given in two doses, 3-4 days apart) was 2030 times greater than that of cells derived from mice given the same total amount (1-2 x lo5 Cetus units) of the unmodified cytokine (introduced once daily over 5 days) (E. Kedar and Y. Barenholz, unpublished observations). Polyethylene glycol-IL-3 and polyethylene glycol-G-CSF were recently found to increase the blood leukocyte count in animals more efficiently compared to the unmodified cytokines (Bree et at., 1991; Tanaka et al., 1991). C. ADOPTIVE IMMUNOTHERAPY: LYMPHOKINE-ACTIVATED KILLER(LAK) CELLS AND TUMOR-INFILTRATING LYMPHOCYTES (TIL) The combination treatment with I L 2 and LAK cells (Rosenberg et al., 1985a, 1989b), which led to complete regression of advanced tumors in some patients with melanoma and renal cell carcinoma, raised criticism because of the severe toxicity and the low rate of success (Moertel, 1987; Quirt and Tannock, 1990). T h e treatment with activated autologous lymphocytes (LAK or TIL) entails complicated logistics and its practice is limited. Therefore it is
important to summarize the available experience in order to ascertain whether such efforts are justified.
I . Lymphokine-Activated Killer Cells: Experimental Models Lymphokine-activated killer cells (or TIL) may inflict tumor damage directly and/or indirectly by releasing immunopotentiating (e.g., I L 2 , I L 4 , IL-6, IFNy) and antiproliferativeicytolytic (e.g., TNFa, TNFP, TGFP, perforin, serine esterases) molecules (Henkart and Yue, 1988; Berke, 1989; Hersh et al., 1989; Larisch-Bloch et al., 1989, 1990; Mazzocchi et al., 1990; Barth et al., 1991). In mice with a relatively small tumor burden, LAK cells and IL-2 were shown to act synergistically; treatment with LAK cells without IL-2 had no or minimal therapeutic effects (reviewed in Rosenberg, 1986, 1988, 1991a; Rosenberg et al., 1989a). In an experimental model of postsurgical adjuvant immunotherapy, treatment with LAK cells improved the results in mice receiving low, but not high, doses of IL-2 (Rodolfo et al., 1990). In mice with advanced sarcoma or carcinoma, both of low immunogenicity, cojoint administration of LAK cells did not improve the cure rate achieved by a combination of cyclophosphamide and intermediate doses of IL-2; moreover, treatment with IL-2 and LAK cells without cyclophosphamide had no effect at all (Kedar et al., 1988a, 1989). Thus in animals with bulky disease, the therapeutic gain from LAK cell administration seems to be minimal, particularly when given in combination with high-dose I L 2 or with I L 2 and cytotoxic chemotherapy.
2. Lymphkine-Activated Killer Cells: Clinical Trials In many of the clinical trials, mostly nonrandomized, the objective response rate [partial and complete remissions (PR and CR, respectively)] was about equal for IL-2 given with and without LAK cells (Boldt et al., 1988; Bergmann, 1989; West, 1989; Margolin et al., 1989; Parkinson, 1990; IMlman et al., 1991b; McCabe et al., 1991; Oliver, 1991). In a few studies, LAK cell infusions to IL-2-treated patients slightly increased the incidence of complete remission and the duration of response in patients with melanoma and renal cell carcinoma (Nkgrier et al., 1989; Rosenberg, 1990, 1991a; Rosenberg et al., 1989a,b; Parkinson, 1990; Dillman et al., 1991a; Escudier et al., 1991). In patients with melanoma and renal cell carcinoma, the success with IL-2 treatment, with and without LAK cells, differs in various trials. T h e relatively high response rate (CR plus PR, 20-35%) reported in several studies (Rosenberg et al., 198913; Rosenberg, 1990; Parkinson et al.,
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1990b; Thompson et al., 1991a) was not confirmed (0-20% response, or no CR) in other studies (Dutcher et al., 1989, 1991; Sondel and Hank, 1989; Abrams et al., 1990; Bar et al., 1990; Clark et al., 1990; Gaynor et al., 1990; Hercend et al., 1990; Parkinson et al., 1990a; Dillman et al., 1991b; Kim and Louie, 1991; McCabe et al., 1991). Since the patient groups were heterogeneous and they were treated with different I L 2 doses, administered at various schedules, sometimes even in combination with other agents (e.g., cyclophosphamide, indomethacin), comparisons and judgments for the optimal treatment conditions are not possible. The higher response rates reported in several studies may be attributed to more aggressive treatments (i.e., higher amounts of I L 2 andlor LAK cells; bolus vs continuous I L 2 infusion) and to differences in the criteria for selection of the patients. The review of these reports leads to the conclusion that the therapeutic value of LAK cells as an adjunct to I L 2 treatment is questionable. In fact the cells may even aggravate the toxicity (Ettinghausen et al., 1988; Albertini et al., 1990; Siegel and Puri, 1991), by damaging normal tissues. Mouse and human LAK cells were shown to be moderately cytotoxic in uitro for normal cells, such as lymphocytes, endothelial cells, and monocytes (Kedar et al., 1982, 1983; Damle et at., 1987; Djeu and Blanchard, 1988; Borden and Sondel, 1990; Zambello et al., 1990; Siegel and Puri, 1991). In spite of the discouraging clinical results with LAK cell therapy, the use of nonspecifically activated cells has not been abandoned. Recently, patients with metastatic renal cell carcinoma were treated with autologous blood lymphocytes, activated in uitro by crude supernatants of OKT3 antibody (a T cell mitogen)-stimulated autologous lymphocytes. A response rate of 2 1%, a clear survival benefit, and minimal toxicity were reported (Osband et al., 1990, 1991).In another approach, patients with metastatic carcinoma and melanoma were treated with autologous blood-derived monocytes briefly activated in uitro with IFNy (Keller, 1989). In several patients with malignant ascites, ip infusions of the cells led to disappearance of the local tumor (Andreesen et al., 1990). 3 . Tumor-InfiltratingLymphocytes: Experimental Systems and Clinical Trials
The limited therapeutic capacity of blood-derived LAK cells motivated the search for more potent effector cells. It can be assumed that T cell populations collected from the tumor tissue (TIL) would be enriched in lymphocytes with specificity for the tumor (Vose and Moore, 1985; Rosenberg et al., 1986, 1989a,b; Rosenberg, 1991a; Itoh et al., 1988; Whiteside et al., 1988; Maleckar et al., 1989; Topalian et al., 1989;
Parmiani et al., 1990). In mice with tumors of low immunogenicity treated with low-dose I L 2 and TIL or LAK cells, the former cells were 50- 100 times more potent in eradicating micrometastatic disease (Rosenberg et al., 1986). Such an effect was not seen, however, in mice with nonimmunogenic tumors (Spiess et al., 1987). In a model for the clinical situation, treatment of mice carrying spontaneous metastases (after resection of the primary tumor) with IL-2 and tumor-specific T cells, generated from splenocytes of immune mice in mixed lymphocyte-tumor cell cultures, was considerably more effective than with LAK cells (Rodolfo et al., 1990). In the limited number of clinical trials, the results with TIL were disappointing. In melanoma and renal carcinoma patients, as well as in other malignancies, the response rate to I L 2 and TIL (10-30%) was not better than that reported for I L 2 and LAK cells (Kradin et al., 1989a; Dillman et al., 1990a, 1991a,c; Hanson et al., 1991; Markowitz et al., 1991; Thompson et al., 1991b). A higher response rate (55%)was obtained in 20 melanoma patients by Rosenberg et al. (1988), but the responses were mainly partial and of short duration. In addition to the questionable therapeutic benefit of TIL, the possibility for their use is very limited. Tumor-infiltrating lymphocytes in sufficient numbers can be collected only rarely, and extensive enlargement of the cell population in uitro must be interposed before their use. This step is not always successful. Prolonged cultivation of the lymphocytes with IL-2 (usually 1-2 months is required) can abrogate selectivity for the tumor. The “contaminating” nonselective killer cells (NK) expand also (Vanky et al., 1982; Whiteside et al., 1988; Topalian et al., 1989; Lotzova et al., 1990), and even the tumor-selective T cells can acquire broader reactivity when exposed to IL2. Moreover, only a small fraction of the readministered human TIL reaches the tumor (Griffith et al., 1989). In control mice (i.e., without tumors), iv-administered T I L localized preferentially in the liver and lungs (Wong et al., 1991). In our studies, iv-injected cultured TIL and spleen-derived LAK cells accumulated mainly in the liver and lungs of both control and tumor-bearing mice (with pulmonary metastases), while the corresponding freshly explanted lymphoid cells localized mainly in the spleen (E. Kedar, unpublished results; Gazit et al., 1992). Although broadening of the cytotoxic potential for allogeneic tumor cells (which is often seen in T cell cultures) is not relevant for the therapeutic effects, damage inflicted on normal cells and poor tumor localization impose serious limitations for the use of lymphocytes activated and expanded in culture. In recent studies, a small fraction of readministered TIL was traced in the tumor tissue of mice and patients for several weeks or months.
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Human TIL transfected in uitro with the neomycin-resistance gene and returned to the patients intravenously could be detected by the highly sensitive polymerase chain reaction (PCR) technique. In three of five melanoma patients, the cells were traced for 6 to >60 days in cutaneous tumor biopsies (Rosenberg et al., 1990). In mice with pulmonary metastases, readministered TIL were found in the lungs 4 months later (Alexander and Rosenberg, 1991). In an attempt to use TIL on a rational basis, their in vitro performance was tested in a group of melanoma patients treated with TIL and IL-2. T h e cells of those who showed a clinical response had a stronger autotumor cytotoxicity in vitro than did TIL of the nonresponding patients (median cytotoxicity 18 vs. 4.5%,respectively) (Aebersold et al., 1991). In similar murine studies, however, the therapeutic effect of TIL correlated better with secretion of IFNy and TNFol on exposure to the relevant tumor cells than with the autotumor cytotoxic activity in vitro (Barth et al., 1991). It seems therefore that the recognition of tumor cells in nitro by the TIL population has a predictive value. T h e employment of tumor-reactive cells can be further refined. Cytotoxic or helper T cell clones specific for the tumor cells could represent the ideal tools for immunotherapy (Greenberg et al., 1988; Greenberg, 1991; Melief, 1992). However, due to the difficulty of establishing such clones from patients and the limitations in their expansion, this strategy may be possible only in highly specialized centers and for only a small number of patients.
4. Genetically Enganeered Tumor-In.h a t i n g Lymphocytes Readministered T cells may be exploited as vehicles for genes encoding cytokines/cytotoxins that can be inserted into the cells by the retroviral-mediated gene transfer technique (Kohn et al., 1987; Blease, 1991). Even if only a small proportion of the cells localize in the tumor, the engineered cells may release large amounts of the products of the transfected genes for extended periods (Kasid et al., 1990; Rosenberg et al., 1990; Rosenberg, 1991a; Morecki et al., 1991; Culver et al., 1991a; reviewed in Kinnon and Levinsky, 1990; Russell, 1990; Friedmann, 1991). It can be expected that such lymphocytes may release quantities of the cytokine in the vicinity of the tumor that highly exceed the local concentration achievable by systemic administration, without causing severe systemic toxicity. However, because the majority of the cells do not reach tumor sites (Griffith et al., 1989), the cytokines released constitutively at other sites may inflict damage on normal cells as well. Theoretically, toxicity could be minimized if the transduced gene could be regulated
and expressed only if the lymphocytes are activated through confrontation with the tumor cells. T h e provision of cytokines inducing or potentiating endogenous effector cells at the tumor site, and of cytokines with a direct effect on tumor growth, may even help generate a response against poorly immunogenic and “nonimmunogenic” tumors. Several animal models suggest that “local help” can induce efficient antitumor responses (Fearon et al., 1990; reviewed in Kinnon and Levinsky, 1990; Russell, 1990; Blankenstein et al., 1991a). Cells from an antitumor CTL clone transfected with the IFNy gene had a stronger antitumor effect in mice than the unmodified lymphocytes (Miyatake et al., 1990). Tumor rejection was reported in euthymic and athymic nude mice when lymphocytes or fibroblasts transfected with the I L 2 (Bubenik et al., 1988, 1990) or the IFNy gene (Ogura et al., 1990) were deposited in its vicinity. This new approach must be exercised with caution, however, because it can lead to adverse effects. Tumors may become more aggressive under the influence of certain cytokines, as it has recently been shown in I L 2 (Kedar et al., I989), IL-1, or TNFa (Bani et al., 1991) treated euthymic and athymic mice, and also with tumor cells transfected with the TNFa gene (Malik et al., 1990). The growth-promoting activity of TNFa in tumor-bearing mice has also been shown in other studies. Antibodies against TNFa inhibited local tumor growth (Gelin et al., 1991a) and metastasis (D. Mannel, personal communication). 5. Adoptive Immunotherapy with Monoclonal Antibodies
The clinical experience with antitumor monoclonal antibodies (MAbs) in patients with solid tumors has been, generally, disappointing (reviewed in Catane and Longo, 1988; Foon, 1989; Rosenberg et al., 1989a; Chapman et al., 1991; Mach et al., 1991). However, MAbs can be useful in combination with LAK cells, T cells, and cytokines in inducing antibody-dependent cellular cytotoxicity (ADCC). Treatment with IL-2 or other cytokines, with and even without exogenous LAK cell administration, was more efficient in tumor-bearing euthymic and athymic mice when MAbs were also administered (Eisenthal et al., 1987, 1988; Kawase et al., 1988; Eisenthal and Rosenberg, 1989; Gill et al., 1989; Junghans, 1990; Schultz et al., 1990; Pendurthi et al., 1991; Van Dijk et al., 1991; Gazit et al., 1992). In patients, systemic treatment with tumorreactive antibodies and IL-2 was not effective (Rosenberg et al., 1989b; Bajorin et al., 1990; Ziegler et al., 1991), whereas local administration of antibody-coated LAK cells (but not untreated LAK cells) caused complete tumor regression in several patients with glioma (Nitta et al., 1990).
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This strategy depends on the availability of tumor-reactive antibodies with the appropriate isotype (Vuist et al., 1990).High levels of circulatory soluble tumor antigens can interfere with this treatment. It is possible that once satisfactory MAbs are available, the administration of effector cells may be superfluous, unless the patient’s cellular functions are deficient.
D. CHEMOIMMUNOTHERAPY 1. Introduction Efficient chemotherapy must eradicate all the tumor cells. This cannot be achieved even in cases where the tumor cells are sensitive to the drug. There is a limitation to the tolerated dose and, frequently, variant cells that are resistant to the drugs arise in the population. Immunotherapy triggers the host defense mechanisms. These modalities rarely have overlapping toxicities, therefore a combination of the two can increase the therapeutic index (Mitchell, 1988; LoRusso et al., 1990). Moreover, drug-resistant tumor cells are still sensitive to an immunological attack (Gambacorti-Passerini et al., 1988). There are several differences between patient response to chemotherapy and to immunotherapy: (1) In general, only those patients achieving a “complete response” to chemotherapy experience a significant increase in survival, whereas even partial responses to immunotherapy can result in prolonged survival; (2) while the effect of chemotherapy is over with the cessation of treatment, clinical responses can occur weeks or months after immunotherapy (Hamblin, 1989); (3) response to chemotherapy, in most cases, is dose dependent, whereas with immunotherapy doseresponse relationships are less marked (Talmadge, 1988); and (4) chemotherapy is more effective than immunotherapy for bulky disease, whereas immunotherapy is probably more effective for minimal residual disease (Wadler, 1991). The goal is thus to reduce the tumor load by chemotherapy, and perhaps also to diminish suppressor cell activity, whereby the chance for the endogenously generated or adoptively transferred effector cells of coping with the residual disease is increased (Fefer, 1974; Fefer et al., 1976, 1982; Kedar and Weiss, 1983; Mitchell, 1988; Greenberg, 1991; Longo, 1991). Since many of the chemotherapeutic agents are immunosuppressive, the protocols of chemoimmunotherapy need to be carefully designed. With a few exceptions (see below), the cytoreductive therapy must be given prior to the immunological measures, at doses close to the maximal tolerance, with intervals allowing the recovery of immunohematopoietic functions but avoiding tumor regrowth (Mitchell,
1988). However, in many clinical trials, chemotherapy and immunostimulating agents have been applied concurrently, and when sequentially, without attention to the optimal intervals and/or sequences. Therefore immunohematopoietic recovery must be followed in order to design the treatment schedule optimally.
2. Experimental Systems: Chemoimmunotherapy with IL-2 Additive or synergistic effects of chemotherapy (mostly cyclophosphamide) followed by IL-2, with or without LAK cells or CTLs, in tumor-bearing mice were reported by several groups (Kedar et al., 1984a, 1988a, 1989, 1990; Rosenberg et al., 1986; Silagi and Schaefer, 1986; Greenberg et al., 1988; Wiltrout and Salup, 1988; Eggermont and Sugarbaker, 1988; Papa et al., 1988; Formelli et al., 1988; LoRusso et al., 1990; Greenberg, 1991). T h e importance of the sequence and timing in chemoimmunotherapy protocols was studied by us in mice with advanced, weakly immunogenic carcinomas and sarcomas. T h e treatment consisted of cyclophosphamide (100-150 mg/kg) and I L 2 (5-10 x lo4 Cetus U/day, for 5-6 days). Synergistic effects were observed only when IL-2 was given after cyclophosphamide, optimally 2-4 days later, whereas the reverse sequence was ineffective (Kedar et al., 1988a, 1989, 1990). Similar findings were reported by other investigators (Hosokawa et al,, 1988). It is likely that with the former schedule, chemotherapy reduced the tumor load (and perhaps also eliminated suppressor cells), thereby allowing the IL-2-stimulated effector cells to act more efficiently on the smaller number of targets, whereas in the reverse sequence the IL-2induced lymphocyte activation and proliferation was counteracted by the chemotherapy. In other reports, however, a potentiation of antitumor response was shown when I L 2 was given before but not after chemotherapy (Rinehart et al., 1990; Wolmark et al., 1990). The contradictory findings may be due to differences in several parameters, such as the dosages, the treatment schedules, the drugs, and the tumor models, and show the difficulties in the design of treatment protocols in the clinic. T h e immunosuppressive effect of the given chemotherapy regimen should thus be considered when planning the sequences and intervals. Thus, cytokines whose lymphoproliferative effect is exploited should not be given immediately prior to immunosuppressive chemotherapy. When the chemotherapy drugs are weakly immunosuppressive (e.g., doxorubicin, dacarbazine) (Mitchell, 1988), or when cytokines with other effects are used (e.g., I L 1 ) (Nakamura et al., 1991), the sequence and timing may be less important.
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3 . Experimental System: Chemoimmunotherapy with Interferon and Tumor Necrosis Factor In several animal models (using, however, mostly tumors with proven immunogenicity), sequential treatment with chemotherapeutic agents and TNFa o r IFN gave additive or synergistic effects. These cytokines do not induce lymphoproliferation, and in mice they were found almost always to be more effective when applied shortly before chemotherapy (Borden et al., 1988; Krosnick et al., 1989a; Kedar et al., 1992). In our experiments with mouse and human tumor grafts in euthymic and athymic mice, respectively, the best effects were obtained with IFNa given before and I L 2 given after chemotherapy (Gazit et al., 1992; Kedar et al., 1992). It can be assumed therefore that IFN and TNF may act better in patients if applied prior to chemotherapy rather than concomitantly (which is, however, the current procedure). These cytokines can also protect the patient from myelosuppression because they inhibit the proliferation of hematopoietic cells (Mitchell, 1988; Talmadge, 1988; Richman et al., 1990; Wadler and Schwartz, 1990). Pretreatment with low-dose IFN or TNF could thus allow the delivery of cytotoxic agents more frequently and/or in higher doses. While the inhibitory effect on cell proliferation can decrease sensitivity to drugs acting on cycling cells, tumor cells exposed to IFN (Elias and Sandoval, 1989; Yoneda et al., 1989; Wadler and Schwartz, 1990; Scala et al., 1991) o r to leukoregulin (Baker and Evans, 1990) had increased sensitivity to chemotherapy. This effect seems to depend on (1) increased drug uptake due to down regulation of the multidrug resistance-associated p 170 glycoprotein, and (2) modifications in intracellular drug metabolism. 4 . Clinical Trials with Various Cytokines
In several clinical trials, chemoimmunotherapy was not more effective than immunotherapy alone. The response rate (approximately 25%) in patients with metastatic melanoma treated with low- or high-dose I L 2 in combination with either DTIC (Shiloni et al., 1989; Dillman et al., 1990b; Flaherty et al., 1990a; Stoter et al., 1991; Isacson et al., 1992), doxorubicin (F'aciucci et al., 1990), or low-dose cyclophosphamide (used mainly as an immunomodulator) (Mitchell et al., 1988b; Lindemann et al., 1989; Rosenberg et al., 1989b)was similar to that achieved with high-dose l L 2 alone. Other combinations were more effective, however. In melanoma patients, approximately 40% responded to I L 2 combined with cisplatin (Atkins et al., 1990) or cisplatin and DTIC (Flaherty et al., 1990b; Blair et al., 1991), and to IFNa combined with DTIC (Breier et al., 1990; Mulder
et al., 1990; Falkson et al., 1991). Higher response rates (60433%) were recently reported when I F N a was combined with DTIC and 5-FU (Mulder et al., 1991) and for IL-2 plus I F N a together with combination chemotherapy (Hamblin et al., 1991; Richards et al., 1991). T h e results are also encouraging in patients with colon cancer. A higher response rate and longer disease-free survival, compared to chemotherapy or biotherapy alone, were achieved with two combinations: (1) 5-FU plus I F N a , which gave a response rate of 20-40% (and up to 76%), including a few complete responses in metastatic disease (Wadler et al., 1989, 1991; Kemeny et al., 1990; Pazdur et al., 1990; Wadler and Schwartz, 1990; Inoshita et al., 1991; Wadler, 1991), and (2) 5-FU plus levamisole as postsurgery adjuvant treatment in Duke’s C colon cancer (Moertel et al., 1990). T h e combination of I F N a and 5-FU or other drugs was also effective for urothelial tumors (Logothetis et al., 1991; Ruther et al., 1991), stage IV head and neck cancer (Vokes et al., 1991), and follicular lymphoma (Solal-Celigny et d., 1991). A response rate of 30-40% was obtained in patients with renal cell carcinoma treated with IFNa and vinblastine (Dal Ri et al., 1990; Nordman and Kellokumpu-Lehtinen, 1990; Wadler and Schwartz, 1990). These responses appeared to be at least additive, as compared with single-modality treatments. In large groups of patients with various tumors, alternating chemotherapy with LAK cells and IL-2 gave better results than without chemotherapy (Dillman p t al., 1991a).
5. Immunomodulatory Efects of Chemotherapy Drugs used for chemotherapy can inhibit but also stimulate immune functions. Their effect is strongly influenced by the dose and the treatment schedules. Enhanced I L 2 production and NK/LAK cell activity, and amplification of T cell responses, were observed in mice treated with low doses of cyclophosphamide, doxorubicin, or cisplatin (Ehrke et al., 1982, 1986, 1988; Kedar et al., 1984b, 1986; Mitchell, 1988; Lafreniere et al., 1989). In our recent experiments (Kedar and Gazit, 1992), high-dose (100 o r 200 mg/kg) cyclophosphamide suppressed the in uitro proliferative and cytotoxic T cell responses to allogeneic cells and reduced the number of LAK cell precursors in mouse splenocytes, tested 14 days after its administration. On the other hand, these responses were markedly enhanced (2-20 times the normal levels) when tested 67 and 9-10 days, respectively, later. A transient decrease of LAK cell activity followed by an increase in vitro and in viuo was seen in athymic nude mice treated with 100 mg/kg of 5-FU or 100 mg/kg of dacarbazine. T h e postchemotherapy “overshot” effect, lasting for 2-4 days in mice, might be the optimal time window for administration of immunother-
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apy. Indeed, the therapeutic effect was found to be maximal when I L 2 was administered daily to tumor-bearing euthymic mice on days 3-8 after treatment with 100 mg/kg cyclophosphamide (Kedar et al., 1989, 1990). Similar chemotherapy-induced fluctuations in immune functions were also reported by other groups in animals and humans (Hosokawa et al., 1988; Cramer et al., 1989; Kim et al., 1989; Sarneva et al., 1989; Allavena et al., 1990; Karimine et al., 1990; Katsanis et al., 1990; Avner et al., 1990; Sensi et al., 1990). When lymphocytes of cancer patients were collected 5-7 days after treatment with mitomycin C and cultured with I L 2 they had a stronger cytotoxic activity than pretreatment lymphocytes cultured the same way (Nanbara et al., 1989).Ex vivo blood lymphocytes of patients 1 month after chemotherapy (without IL2) had LAK cell-like activity (Kiyohara et al., 1988) and an increased capacity to be triggered for cytotoxicity in culture, to respond to mitogens, and to produce I L 2 (Onodera et al., 1990). Changes in the composition of the cell population, including the elimination of suppressor cells (see the next section), may explain the elevation of certain immune functions (Berd et al., 1984; North, 1984; Mokyr and Dray, 1987; Awwad and North, 1989; Hoover et al., 1990). 6. Effects of Chemotherapy and Other Drugs on Suppressor Cells
Low-dose chemotherapy can eliminate suppressor cells or counteract their activation (Fefer et al., 1982; Kedar et al., 1984b, 1986; Livingston et al., 1987; Mokyr and Dray, 1987; Berd and Mastrangelo, 1988b; North et al., 1989; Hoon et al., 1990; reviewed in Naor et al., 1989). The existence of chemosensitive tumor-specific suppressor T cells and nonspecific suppressor cells has been documented in animals with advanced tumors. Depletion of such cells by low-dose cytoreductive therapy (primarily by cyclophosphamide), prior to adoptive cellular immunotherapy, enhanced the antitumor response in mice (Fefer et al., 1976, 1982; North, 1984; Awwad and North, 1988a,b), and in some studies (Berd et al., 1990a,b; Hoon et al., 1990), but not others (Morton et al., 1991a) appeared to improve the clinical response in patients treated with a melanoma cell vaccine. It is still not clear whether suppressor cells have any impact on immunotherapy in patients. Patients with solid tumors usually do not show impaired immune functions and they do not have circulating active suppressor cells (reviewed in Naor et al., 1989). Suppressor cells may be present, however, in the tumor or in the draining~lymphnodes (Parmiani et al., 1990).
The role of suppressor cells in adoptive immunotherapy was assessed in mice treated with IL-2 and TIL as an adjunct to cyclophosphamide, whole-body low-dose irradiation, o r local tumor irradiation (Cameron et al., 1990). T h e immunotherapeutic effect was largely correlated with the reduction of the tumor load rather than with the elimination of suppressor cells. Prior treatment with cyclophosphamide or low-dose irradiation was essential for the effect of specifically sensitized T cells but not of LAK cells (Cameron et al., 1990). In mice carrying weakly immunogenic tumors treated with several chemotherapeutic agents at various doses in combination with IFNa and/or I L 2 , or with tumor-reactive T cells, additive/synergistic effects were obtained only when the chemotherapy itself had an antitumor effect (Formelli et al., 1988; Papa et al., 1988; E. Kedar, unpublished observations). Other drugs that were found to counteract suppressor cell functions in experimental models, in particular the type-2 histamine (H2) receptor antagonist, cimetidine (which inhibits the activation of H2 receptor-bearing suppressor cells), and the cyclooxygenase inhibitor, indomethacin (which blocks the production of the immunosuppressive mediator PGE,), were also employed in therapy. In mice, cimetidine was efficient in several tumor systems (Gifford et al., 1981; Osband et al., 1981), and it enhanced the therapeutic efficacy of I L 2 (Nakajima and Chu, 1991). Also, in patients with various types of solid tumors, cimetidine combined with histamine was beneficial (Burtin et al., 1988). In patients with metastatic melanoma, cimetidine improved the antitumor effect of IFNa in some (Flodgren et al., 1983) but not in other (Creagan et al., 1985) trials. In melanoma patients immunized with a melanoma cell vaccine, pretreatment with cimetidine augmented cellular and humoral responses to melanoma cells and appeared to improve the clinical response (Morton et al., 1991b). Indomethacin had therapeutic effects in several experimental tumor systems (Lynch et al., 1978; Fulton, 1988; Gelin et al., 1991b). It also enhanced the antitumor effects of I L 2 and IFNa, administered with and without chemotherapy, in euthymic and athymic mice (Kedar et al., 1984a; Lala and Parhar, 1988; Kim and Warnaka, 1989; Lala et al., 1990). However, patients treated with I L 2 (Sosman et al., 1988) or IFNa (Miller et al., 1989) did not benefit from indomethacin. It is possible that continuous, extended treatment with indomethacin is required (Lala et al., 1990), which was not the case in these clinical studies. Indeed, in a recent study on 25 patients with advanced melanoma receiving indomethacin (together with ranitidine) for several days, 2 patients showed an objective response (1 CR, 1 PR) even before I L 2 was administered (Mertens et al., 1991). The same treatment regimen with I L 2 was not more
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effective, however, than treatment with 1 L 2 alone in patients with renal cell carcinoma (Bramwell et al., 1991). Indomethacin was also found to potentiate IL-2 production (Kedar et al., 1986), LAK cell induction, and ADCC activity in murine splenocytes (Eisenthal, 1990). Interestingly, indomethacin and other cyclooxygenase inhibitors (e.g., aspirin) diminished the toxicity of high doses of I L l , I L 2 , IFNy, or TNFa in tumor-bearing mice and rats without reducing the therapeutic efficacy (Haranaka, 1988; Talmadge, 1988; E. Kedar, unpublished observations). 7. Protection against MyelofImmunosu#n-ession with Cytokines
Certain cytokines can protect patients from the myelo/immunosuppression caused by high-dose chemotherapy, or radiotherapy. By accelerating the recovery of immunohematopoietic functions, they can shorten the intervals of cytoreductive therapy o r immunotherapy administrations, and may even allow a dose increment of chemotherapy, thereby enhancing antitumor effects. Administration of colony-stimulating factors (CSFs)-IL-3, GM-CSF, G-CSF, and M-CSF-as well as IL-1, IL-6, and IFN, individually or combined, to normal and tumor-bearing animals shortly before and/or after chemol radiotherapy was found to facilitate immunohematopoietic reconstitution and to enhance the therapeutic efficacy (Neta, 1988; Neta and Oppenheim, 1988; Kedar et al., 1988c; Slavin and Kedar, 1988; Futami et al., 1990; M. A. S. Moore et al., 1990). In cancer patients treated with cytoreductive therapy and CSFs, the number and severity of infections and hospitalization time were decreased, the patients required fewer blood or platelet transfusions, and chemotherapy could be administered more frequently or at higher doses, compared to patients not receiving CSFs (reviewed in Griffin, 1989; Groopman et al., 1989; Laver and Moore, 1989; Metcalf, 1989a,b; Bronchud, 1990; Demetri and Griffin, 1990; Ganser et al., 1990; Gianni et al., 1990; Golde, 1990; Kelso and Metcalf, 1990; Monroy et al., 1990; Richman et aL, 1990; Antman, 1991; Moore, 1991). In addition to the myeloprotective effects, CSFs and other cytokines (e.g., TNFa, IFNy) can amplify antitumor effector mechanisms by augmenting cytotoxic and phagocytic activities of monocytesfmacrophages and granulocytes. T h e beneficial effect of levamisole in combination with 5-FU in patients with colon cancer has been attributed, in part, to improved immunohematopoietic recovery, probably through inducing production of CSFs and other cytokines (Grem and Allegra, 1989). It is thus likely that optimal treatment with certain cytokines will
provide both myeloprotection and immunostimulation, allowing the use of more intensive cytoreductive therapy, which may lead to a higher response rate. It should be noted, however, that CSFs must be employed with caution, since they may stimulate residual tumor growth, not only leukemias (Laver and Moore, 1989; Aglietta et al., 1990; Glaspy and Golde, 1990; Moore, 1991) but, as shown thus far in vztro, some types of solid tumors as well (Foulke et d.,1990; Joraschkewitz et al., 1990; Marshall and von Hoff, 1990; Pedrazzoli et al., 1990; Vellenga et al., 1991). 8. Conclusions
In addition to tumor debulking, the elimination of suppressor cells, and the transient increase in certain immune functions, the chemotherapeutic agents may also potentiate immunotherapy by: (1) increasing the sensitivity of‘ tumor cells to immunological attack, by arresting them in the G,/G, phase and/or by inducing biochemical alterations of the cell membrane, (2) imposing antigenic changes on the cells by acting as a hapten, (3) facilitating infiltration of effector cells to tumor sites, (4) potentiating stimulation of effector cells as a consequence of massive release of tumor antigens, (5) making “space” for adoptively transferred effector cells, and (6) protecting against toxicity induced by some cytokines (e.g., I L 2 ) (Giampietri et al., 1981; Mokyr and Dray, 1987; Mitchell, 1988; Hosokawa et al., 1988, 1990; Lee et al., 1988; Papa et al., 1988; Wiltrout and Salup, 1988; Kovach, 1991). Although not yet proven in clinical studies, chemotherapy may also reduce production of neutralizing antibodies to exogenously administered recombinant cytokines, and suppress the human anti-mouse antibody (HAMA) response in patients treated with mouse monoclonal antitumor antibodies. In order to best exploit these various actions of chemotherapy, the drugs must be carefully selected and their schedule and dosage should be adjusted to the individual, with evaluation of the immunosuppressive and immunostimulatory effects. During treatment with either cytokines or effector cells, repeated low-dose chemotherapy courses may also be required to maintain the reduced tumor burden. Better clinical results with chemoimmunotherapy may be obtained with new combination chemotherapy protocols based on the above considerations. It should be noted that no systematic clinical studies have yet been carried out to determine the optimal schedule, sequence, time interval, and duration of treatment for combined chemoimmunotherapy regimens, which may explain, in part, the meager success in many trials.
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IV. Attempts to Improve Cancer lrnrnunotherapy
Based on the new developments in biotechnology and on the results in experimental systems and in clinical trials, we summarize the considerations that can lead to improvement of cancer immunotherapy. These are outlined below and rely on the data reviewed in the previous sections. Considerations in Cancer Immunotherapy A. Basis for patient selection 1 . Tumor load/location
2. Karnofsky performance 3. MHC antigen expression on the tumor cells 4. Cell-mediated recognition of the tumor cells in vitro
B. Tumor debulking 1. Surgery 2. Radiotherapy/chemotherapy a. Supported by administration of colony stimulating factors (CSF) b. Periodic monitoring of general immune competence C. Active immunotherapy 1. Tumor vaccines a. Supported by adjuvants b. Inhibition of suppressor cells 2. Cytokines and other BRMs a. Combinations b. Targeted delivery D. Passive/adoptive immunotherapy 1 . Tumor-reactive monoclonal antibodies (MAbs) 2. Tumor-reactive T-cells
A. GAINSFROM EXPERIMENTAL MODELS
The rationale of immunotherapy was based on results obtained in experimental systems. It can be argued that the majority of animal models do not represent clinical situations (Hewitt, 1978, 1982; Weiss, 1980b; Herberman, 1983a,b). In many of the preclinical studies, long-passaged immunogenic tumors were selected and usually grafted at sites other than the tissue of origin. Moreover, immunotherapy was frequently initiated concomitantly or soon after tumor grafting. Immunotherapeutic manipulations were usually not effective in animals with established tumors, particularly if the latter were poorly or nonimmunogenic. This criticism is, however, only partly valid, because the relevant model system
is hardly available (Scott, 1991). In addition, a considerable body of information has been collected in the animal experiments. Among these are the importance of tumor immunogenicity and the T cell response for the outcome of immunotherapy, the potential of BRMs, and the analysis of critical factors in combined immunotherapy regimens. T h e natural history of the majority of human tumors, and the fact that the common cancers do not show higher incidence in immunodeficient individuals, indicates that these tumors are rarely recognized by the immune system. T h e tumor cells may lack immunogenic determinants; and if they carry such, the host may have developed tolerance. Therefore, animal models using low or nonimmunogenic metastatic tumors must be concentrated upon (McIntosh et al., 1990; Rodolfo et al., 1990; Sakai et al., 1990), and attempts should be made to induce the immunological recognition of tumor cells. Human tumors can be studied in athymic nude mice (Giovanella and Fogh, 1985; Giavazzi et al., 1986; Ortaldo et al., 1986; Fidler, 1990; Kedar et al., 1990; Gazit et al., 1992) and in severe combined immunedeficient (SCID) mice (Mueller and Reisfeld, 1990; Waller et al., 1990; Mule et al., 1991; Schmidt-Wolf et at., 1991), in which the function of autologous effector cell populations can also be tested (Crowley et al., 1992). Although these xenogeneic systems may not permit optimal interaction between the host and the grafted cells due to differences in species-specific adhesion molecules, homing receptors, and extracellular matrix proteins (Albeda and Buck, 1990; Michl et al., 1991; Van Seventer et al., 1991), these models can have advantages over the test tube experiments. Many experimentalists (and clinicians) use the reduction in tumor size or in number of metastases as parameters for assessing the efficacy of immunotherapy. Although these are indicative of antitumor effects, critical end-point evaluation of treatment benefit should be based on prolongation of survival and lack of disease progression (Osband and Ross, 1990). Despite all the efforts involved in creating “relevant” animal models, even these may not predict the value of therapeutic manipulations in patients, because (1) the response to, and the toxicity of, various BRMs (e.g., TNFa) in animals and humans may be different (Rosenberg, 1991a), and (2) the patient groups may be immunobiologically heterogeneous. Extrapolation from the preclinical models to the clinical setup can be facilitated by better characterization and grouping of the patients, and by identification of prognostic factors predictive of response to immunotherapy (reviewed in Osband and Ross, 1990; Parkinson, 1990).
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B. SELECTION OF PATIENTS Apart from general parameters, such as the physical condition of the patient and the type of tumor and disease stage, specific, reliable criteria for selection of candidates for immunotherapy do not exist. Discovery of meaningful predictive parameters is therefore an important goal. Patients in general good physical condition (e.g., with a Karnofsky performance status of 270%) are expected to withstand the toxic effects accompanying most of the combined immunotherapeutic regimens. Patients with severe leukopenia, severe cardiovascular, respiratory, renal, or liver disorders, neurological disorders, or brain metastases, cannot receive certain types of treatment (e.g., high-dose IL2 or IFN). With age, both the capacity for immune response and the tolerance for chemotherapy decline. When priority must be exercised, patients presenting with a small tumor burden, o r those whose large tumor load can be decreased by conventional measures, are to be selected. Accordingly, patients with metastatic renal cell carcinoma likely to respond to I L 2 therapy are those in risk group 1, whose primary tumor had been removed by nephrectomy, and who have only pulmonary metastases and not a bulky disease (reviewed in Parkinson, 1990).A considerably higher response rate to various forms of immunotherapy and longer remissions have been obtained in melanoma patients with subcutaneous nodules than in patients with visceral metastases. Since partial responses and long-lasting disease stabilization have also been achieved with immunotherapy in some patients with advanced bulky disease, protocols for these patients should be designed according to their special needs and evaluated separately. Even for the assumed immunogenic tumors, melanoma and renal cell carcinoma, no specific parameters are available that can predict the response to immunotherapy. Demonstration of a specific antitumor T cell response may be valuable. This can be tested in (1) cytotoxicity assays [autologous lymphocyte cytotoxicity (ALC)],using ex vivo tumor cells and blood- or lymph node-derived lymphocytes, (2) proliferation assays [autologous tumor stimulation (ATS)](see Section II,A), and (3) a skin test (i.e., DTH response) with autologous tumor cells or extracts. In view of their importance for recognition by T cells, the level of MHC class 1/11 antigen expression on the tumor cells can also be of predictive value. These assays cannot be performed regularly, however, due to the requirement for tumor cells in sufficient quantity and good quality. Furthermore, because of the heterogeneity of the tumor cell population, the results may not cover reactivities to all tumor cells. As mentioned before (Section II,A), a relatively favorable prognosis
was associated with the autotumor cytotoxicity (ALC), tested at the time of surgery, in patients with squamous cell- and adenocarcinoma of the lung o r with malignant mesenchymal tumors without apparent metastases (Vanky et al., 1983a,b, 1986, 1987; Uchida and Mizutani, 1989; Uchida et al., 1990, 1991; Ortaldo and Wiltrout, 1990). There was no correlation between the autotumor cytotoxicity and other in vitro immune functions of blood lymphocytes, including N K activity, proliferation to autologous fresh tumor cells, to mitogens, and to allogeneic leukocytes, and production of cytokines (Uchida et al., 1990). In order to establish whether the cytotoxicity of blood lymphocytes against autologous tumor cells could be used for patient selection, it would be important to correlate the results of this test with the immunotherapeutic effects. Coexpression of high levels of MHC class I antigens and the adhesion molecules ICAM-1 and certain VLA antigens on the tumor cells (Vanky and Klein, 1989, 1991; Anichini et al., 1990; Parmiani et al., 1990; RuizCabello et al., 1991) may also be indicative for the possible response to immunotherapy. Patients with particular HLA haplotypes may be more likely to respond, as recently demonstrated for melanoma patients treated with a tumor vaccine (Mitchell, 1990, 1991) or with IL-2 based immunotherapy (Scheibenbogen et al., 1991) (see Section 11,A). It would be of interest, therefore, to determine whether such a correlation exists in patients treated with other immunotherapy modalities. Usually cancer patients do not have impaired N K and LAK activities, and these functions do not correlate with the clinical response in IL-2treated patients (Boldt et al., 1988; Eberlein et al., 1989; Ghosh et al., 1989; Favrot et al., 1990; Dillman et al., 1991b). We tested a group of patients with metastatic melanoma who received chemotherapy (dacarbazine) and IL-2 in alternating cycles for the composition of the lymphocyte populations in the blood, mitogenic responsiveness, IL- 1, IL-2, TNFa, and soluble I L 2 receptor levels in the serum, and N K and LAK cell activity, before and during treatment (Isacson et al., 1992). None of these had any prognostic value. The only parameter that correlated with the clinical response was the greater increase (relative to pretreatment level) in the proportion of lymphocyte populations expressing the CY chain of the IL-2 receptor (CD25) in the responders, 2 days after the IL-2 infusion. T h e number of patients (CR + PR = 4/18) was small, however. A similar observation was made in patients with renal cell carcinoma who responded to IL-2 and indomethacin (Banerjee et al., 1991). In another group of patients with renal carcinoma, the clinical response to IL-2-based immunotherapy was correlated with the I L 1 and
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TNFa serum levels and the capillary leak syndrome (Blay et ul., 1991). It should be noted that in all these studies, the differences in the nonspecific immune parameters between responders and nonresponders were seen only posttreatment, usually shortly after the first one to three cycles of IL-2, but not after additional cycles. Differences in these and other nonspecific parameters before treatment were not observed. It is thus highly desirable to identify specific immunological parameters that can be used for patient selection. C. TUMOR DEBULKING In patients with a large tumor burden, immunotherapy must be applied after surgery and/or chemo/radiotherapy. There is no guideline, however, for the optimal schedules. It is not known whether (1) immunotherapy should be given after the completion of cytoreductive therapy, or (2) chemo/radiotherapy and immunotherapy should be applied in several alternating cycles. Obviously, if the cytoreductive treatments are immunosuppressive, immunohematopoietic recovery is essential. In addition to blood counts, composition of the lymphocyte populations, proliferative responses to allogeneic cells and to mitogens, skin tests with common test antigens [e.g., Candida, purified protein derivative (PPD), dinitrochlorobenzene (DNCB)], antibody response to recall antigens (e.g., Tetanus toxoid), and phagocytic cell functions can be performed in order to decide the timing of immunotherapy. Testing for the presence of suppressive factors (e.g., TGFP and others) (Itoh et al., 1985; Ebert et al., 1990; Hirte and Clark, 1990; Reynolds et al., 1990) in the serum can also be informative. For efficient tumor reduction, high-dose (lethal) chemotherapy may be administered followed by autologous bone marrow or derived blood stem-cell transplantation (Dicke et al., 1989; Antman, 1991; Henon et al., 1991; Kessinger and Armitage, 1991; Spitzer et al., 1991). However, since this procedure is burdened with risks (with a mortality rate of approximately lo%), it can be justified only in patients with a poor prognosis whose tumors have a good response to chemotherapy. A supportive treatment with CSFs and other hematopoietic-stimulating cytokines (e.g., I L l , IL4,ILS)could alleviate myelotoxicity and potentiate the therapeutic effects (Demetri and Griffin, 1990; Ganser et al., 1990; Gianni et al., 1990; Glaspy and Golde, 1990; Golde, 1990; Gutterman et al., 1990; Monroy et al., 1990; Antman, 1991; Demetri et al., 1991; Grem et al., 1991; Luikart et al., 1991; Moore, 1991; Nemunaitis et al., 1991; Postmus et al., 1991; Shank and Balducci, 1991). A promising additive to the arsenal of hematopoietic factors is the
recently discovered stem cell factor (also known as c-kit ligand, or mast cell growth factor), which appears to act on primitive hematopoietic cells, particularly in combination with other cytokines (de Vries et al., 1991; Scadden et al., 1991). D. ACTIVESPECIFIC IMMUNOTHERAPY
Immunization with various tumor vaccines has been performed in patients with several types of cancer (Section 111,A). Active immunization is expected to stimulate the tumor-reactive T cell population as well as the nonspecific effector cells. These populations can be enlarged and further activated by administration of cytokines. In patients treated with high-dose chemotherapy, an interval of several weeks to months may be needed before active immunization (Ahlert et al., 1990). In contrast, lowdose chemotherapy given prior to active immunization may improve vaccine efficacy, as discussed earlier. Several measures are known that can elevate or impose immunogenicity on the tumor cells. We mentioned earlier manipulations that could augment immunogenicity (see Section 111,A). Further possibilities that can elicit or amplify the response have been tested in experimental systems, also with tumor antigens; some of these have not yet been applied in patients: 1. Antigens encapsulated in liposomes (Gregoriadis, 1990; Harding et al., 1991): In patients with stage 111 melanoma, immunization with autologous tumor material incorporated in liposomes led to complete or partial tumor regressions in 5 of 13 patients (Phillips et al., 1990). 2. Administration of the immunogen together with cytokines, such as IL-1 (McCune and Marquis, 1990), IL2 (Naito et al., 1988; Freedman et al., 1990; Thiele et al., 1990), liposomal IL-2 (Sencer et al., 1991), IL6 (Naito et al., 1991), IFNy (Giovarelli et al., 1986), or IL-2 plus IFNol (Arroyo et al., 1990). 3 . Administration of the immunogen together with improved adjuvants [e.g., detoxified endotoxin (DETOX)] (Mitchell et al., 1990; Mitchell, 1991) or with BCG (Hoover and Hanna, 1989; Berd et al., 1990a; Russel et al., 1990; Morton et al., 1991a): Other BRMs that may potentiate the effect of specific immunization are muramyl di/tripeptide (Kleinerman et al., 1989), bestatin (Inoue et al., 1990; Sawada et al., 1990), bryostatin (Schuchter et al., 1991), levamisole (Moertel et al., 1990), OK-432 (Watanabe and Iwa, 1987; Nakagami et al., 1990), PSK (Koike et al., 1990; Matsushima et al., 1990; Mitomi and Noto, 1990; Mitomi et al., 1990; Torisu et al., 1990), and AS101 (Sredni et al., 1987, 1990, 1991; Tichler et al., 1990; Kalechman et al., 1991).
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4. Oncogene products: Some of the oncoproteins, such as Ad E l and HER-2/neu, were shown to elicit T cell responses in vitro and in vivo (Bernards et al., 1987; Hellstrom and Hellstrom, 1989; Kast et al., 1989; Fendly et al., 1990; Melief and Kast, 1990a,b; Talarico et al., 1990; Jung and Schluesener, 1991). Many tumors carry mutated p53 and p21 (ras) protooncogenes (Bos, 1989; Bishop, 1991), which may also evoke an antitumor response. In a recent study, T cells specific for the p21 ras protein were detected in mice immunized with synthetic peptides corresponding to the mutated regions of activated p21 ras proteins (Peace et al., 1991). 5 . Genetically engineered tumor cells: Immunogenicity of the tumor cells may be potentiated by introduction of cytokine-encoding genes with the help of retroviral vectors (reviewed in Kinnon and Levinsky, 1990; Russell, 1990; Blankenstein et al., 1991; Rosenberg, 1991a). The tumor cells can then release cytokines that can act on an autocrine or paracrine basis. In this way the tumor cells can induce “local help” for amplification of an immune response (Fearon et al., 1990). Mice grafted with weakly immunogenic tumor cells transfected with the genes encoding IL-1 (Blease, 1990; Douvdevani et al., 1992), IL-2 (Gansbacher et al., 1990b; Fearon et al., 1990; Ley etal., 1991), IL-4 (Tepper et al., 1989; Li et al., 1990), IFNy (Watanabe et al., 1989; Gansbacher et al., 1990a), TNFa (Asher et al., 1991; Blankenstein et al., 1991b; Vanhaesebroeck et al., 1991), o r G-CSF (Colombo et al., 1991) rejected the tumor and developed immunity against a subsequent challenge with the nontransduced tumor cells. T h e regression of such tumor cells correlated with the amounts of cytokine they produced. In a recent study, even an established wild-type tumor was rejected following administration of the transduced tumor cells (Golumbek et al., 1991). When used for vaccination, care must be taken, because such tumor cells may acquire a higher growth or metastatic potential (Malik et al., 1990; Blankenstein et al., 1991a; see also Section 111,B). 6. Tumor cells transfected with exogenous DNAs specifying foreign antigens (e.g., viral antigens, MHC antigens, tumor antigens) (reviewed in Nowak et al., 1991). Such tumor cells demonstrated increased immunogenicity in animals. 7. Recombinant viruses (e.g., vaccinia virus constructs) that express human tumor antigens (Estin et al., 1988; Kahn et al., 1991).
Thus, new approaches for cancer vaccines have been developed, but the majority of these have not yet been tested in patients. Active immunotherapy can be followed by adoptive measures. The population of tumor-reactive T cells generated by active immunization may be first en-
larged in vitro and readministered to the patient. In a presently conducted clinical trial at the National Institutes of Health (Bethesda, MD), viable, autologous melanoma cells transduced with the TNFa gene are injected subcutaneously and intradermally, in an attempt to enlarge the number of tumor specific T cells in the regional lymph nodes. The latter are removed 2-3 weeks later, and the lymphocytes expanded in culture with IL2 for readministration (R Rosenberg, personal communication).
E. ELIMINATION OF SUPPRESSOR CELLS/FACTORS Although tumor reduction may decrease the levels of suppressor cells and/or soluble factors (see Section III,D), additional measures could be applied prior to or during immunotherapy. Inhibitors of the cyclooxygenase pathway (indomethacin, ibuprofen, aspirin) (Kedar et al., 1984a,b, 1986; Israel et al., 1990; Khoo et al., 1990; Lala et al., 1990), the histamine H2 receptor blocker, cimetidine (Burtin et al., 1988), low-dose cyclophosphamide or doxorubicin (Berd and Mastrangelo, 1988b; Mitchell, 1988; North et al., 1989), and monoclonal antibodies directed to T suppressor cells (North et al., 1989) had beneficial therapeutic effects in rodents. However, their therapeutic value in patients is still not substantiated (see Section 111,D). It is possible that administration of monoclonal antibodies reactive with immunosuppressive cytokines (e.g., TGFP) (Sporn and Roberts, 1990; Wahl et al., 1990; Tada et al., 1991), which are secreted in large quantities by certain cancer cells, or removal of such factors by plasmapheresis may be beneficial.
F. ACTIVE/ADOPTIVE IMMUNOTHERAPY I . Cpokines After the reduction of tumor burden (and putative suppressor cells), and following active specific immunization (if possible), further potentiation of effector mechanisms may be achieved with cytokines, singly o r combined (see Section 111,B).Based largely on the results in experimental systems, the most effective combinations tested thus far are IL-2 and IFNa (Cameron et al., 1988; Rosenberg et al., 1989b,c; Kedar et al., 1990, 1992) and IL-2, IFNa and TNFa (McIntosh et al., 1989). I n recent clinical trials, tumor regressions were also achieved with IL-1 in melanoma (Smith et al., 1991a; Starnes et al., 1991), and with I L 4 in lymphomas (Davis et al., 1991). Other cytokines that may prove effective in patients are IFNP, IL-6, and IL7. Another novel approach to potentiate cytokine activity is the use of
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hybrid or chimeric molecules, composed of parts of several cytokines, such as the consensus IFNa and the GM-CSF/ILS (PIXY 321) molecules (Curtis et al., 1991; Johnston, 1991). Combination of IL-2 (or other cytokines) and the monoclonal antiCD3 reagent can activate both T and LAK cells. This approach has been successful in animal models, and clinical trials have begun recently (Anderson et al., 1988, 1989; Yun et al., 1989; Ellenhorn et al., 1990; Gallinger et al., 1990; Lafreniere et al., 1990; Schoof et al., 1990; Stohl et al., 1990; S. C. Yang et al., 1990; Gambacorti-Passeriniet al., 1991; Loeffler et al., 1991b; Ochoa et al., 1991; Weil-Hillman et al., 1991). A novel approach for damaging tumor cells that express high levels of cytokine receptors is the use of relevant cytokine molecules conjugated to cytotoxic agents (Strom et al., 1990; Pastan and FitzGerald, 1992). A recombinant fusion protein, made by replacement of the diphtheria toxin gene receptor-binding domain with the gene for human IL2, was found effective in patients with hematologic malignancies that express the IL2 receptor (LeMaistre et al., 1991a,b). The cytokines IL4,IL-6, and TGFa fused with the Pseudomonas exotoxin exhibited impressive antitumor effects in mice (Pai et al., 1991; Puri et al., 1991; Siegall et al., 1991). Patients with localized tumors (e.g., bladder carcinoma, mesothelioma, abdominal carcinomatosis, head/neck, and brain tumors) could benefit from locoregional administration, concurrent with systemic treatment with cytokines. The combined 1ocaUsystemic regimen has not yet been practiced in clinical trials. The therapeutic effects may be improved and toxicity may be reduced by delivery of the cytokines in liposomes or by their chemical modification (see Section 111,B). 2 . Cells The clinical experience of the past 8 years with several thousands of patients does not support the use of LAK cells (see Section 111,C). A possibility yet to be explored is the targeted cellular cytotoxicity. Lymphokine-activated killer cells (or T cells), with or without IL2, could be administered together with monoclonal antitumor antibodies active in ADCC, or with bispecific, heteroconjugate, or hybrid antibodies directed against molecules on the plasma membrane of effector cells (such as CD2, CD3, CD16, CD28, CD59) and the tumor cells. Such antibodies synergized in antitumor effects with adoptively transferred cells in animal models, and enhanced the cell-mediated lysis of human tumor cells in vitro (Titus et al., 1987; Eisenthal et al., 1988; Donohue et al., 1990; Fanger et al., 1990, 1991; Hank et al., 1990a; Kerr et al., 1990; Segal et al., 1990; Goldenberg, 1991; Nitta et al., 1991; Reid et al., 1991a,b). Encour-
aging results in glioma patients were reported with bispecific (antiCD3/anti-glioma) antibody-coated LAK cells (Nitta et al., 1990). Such cell-antibody combinations may be much more efficient when administered together with IFN or TNF, because these can enhance the expression of tumor antigens and adhesion molecules on the tumor cells, thereby potentiating the interaction between the effector and tumor cells (Murray et al., 1990).The use of “humanized”or human MAb instead of murine MAb should improve these possibilities. The results with TIL do not appear to be superior to those with LAK cells in clinical trials (see Section 111,C). Moreover, TIL-based immunotherapy can be carried out only in a limited number of patients. Tumor cell-selectivesyngeneic T cell clones gave excellent results in animal models, and they could exert the most efficient antitumor effects. However, even if such human T cells were selected in culture, they could rarely proliferate to reach sufficient numbers for readministration. The aims are usually to develop CD8 cytotoxic cells, but it is possible that cytokine-producing CD4 clones may give better therapeutic effects (Greenberg, 1991). Attention should be directed to recent methodological improvements that may render this approach feasible. These include: (1) selection of T cell populations from the tumor tissue by using anti-T cell antibodies bound to beads (Morecki et al., 1990;J. C. Yang et al., 1990a),(2) repeated in uitro stimulation of the T cells with irradiated tumor cells and lowdose IL2 (McKinnon et al., 1990; Skornick et al., 1990), or by monoclonal anti-CD3 antibodies (Nijhuis et al., 1990; Yoshizawa et al., 1991), and (3) use of several cytokines (e.g., IL-2 and IL-4, IL-2 and TNFa, or IL2, and TNFa and IFNa or IFNy), with and without anti-CD3, instead of IL-2 alone in the T cell cultures (Finke et al., 1991;Jadus et al., 1991; Shimizu et al., 1991). Expansion of adoptively transferred tumor-specific T cells was obtained in mice by repeated administrations of the tumor antigen together with low-dose IL-2 (Chen et al., 1990). It is possible that such a manipulation can be effective in patients. Propagation of mouse T cells for an extended period of time, with or without I L 2 , and without the need for repeated exposure to the antigen, was recently achieved by introduction of the protein kinase C y gene, using a retroviral vector (Finn et al., 1991). Such transduced CTL clones maintained their specific cytotoxic activity in uitro and antitumor effect in uiuo without being tumorigenic (Chen et al., 1991). The therapeutic effects of T cells transduced with cytokine-encoding genes are presently being tested, based on the assumption that they can specifically accumulate and release large amounts of cytokines at tumor sites (Rosenberg et al., 1990; Culver et al., 1991a) (see Section 111,C).
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Melanoma patients are now being treated with autologous TIL transduced with the TNFa gene (Rosenberg, 1991a,b). The value of this approach has not yet been substantiated, however, in animal models. In mice, IL1 gene-carrying CD8 CTL had enhanced tumor cytotoxicity in vitro, but the therapeutic effects were not superior, compared with the unmanipulated T cell population. In other experiments, TNFa genecarrying T cells lacked antitumor activity in uiuo (Culver et al., 1991b; Fox et al., 1991). T h e failure may be ascribed to a low level of cytokine secretion o r poor localization of the cells in tumor sites. Thus, this approach needs to be tested further in animal models for efficacy and toxicity before it can be applied to patients. V. Conclusions
T h e demonstration of antigenicity of experimental tumors and the beneficial effects of immunotherapy in animal models generated great expectations for cancer treatment. Various immunotherapy protocols have been reported to be moderately effective in patients with certain types of cancers. T h e value of many of these trials cannot be judged, however, because though they could stand the scrutinies of evaluation, they were not repeatable by other groups. This may be attributed to differences in patient selection and in the details of treatment protocols. T h e question can be posed whether at the present state of knowledge cancer immunotherapy can be improved. Some recommendations can be proposed. T h e experimentalists may focus on models relevant to the clinical situation, such as metastatic tumors of low or no immunogenicity, and exploit the possibility of studying the interaction of human tumor and effector cells in immunologically deficient mice. The message for the clinicians is that immunotherapy, generally, is not effective in patients with high tumor load. Therefore, it can only be an adjunct to traditional cytoreductive treatments. Tests for new treatment regimens in phase 1/11 trials ought to be performed in patients that have low tumor burden, but poor prognosis, rather than in end-stage patients with bulky disease. Imposing experimental immunotherapy regimens on such patients is hard to justify, however. New regimens should be tested first in well-designed experimental models. Obviously, priority should be given to protocols without major toxicity and with the possibility for application to large patient groups. New modalities may first be tested in melanoma and renal cell carcinoma patients, because these have been already shown to be capable of response. Since only a low proportion (