Biologic Response Modifiers: The Future of Cancer Therapy?

Clinical Management of the Cancer Patient

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Biologic Response Modifiers: The Future of Cancer Therapy?

E. Gregory MacEwen, VMD*

Traditionally, surgery and radiation therapy have been the major modalities for the control of local and regional cancer. These major modalities will remain in the forefront for the treatment of most localized cancers. Metastasis represents the major reason for failure of surgery and radiation therapy. First, by the time many cancers are diagnosed, metastasis has already taken place; therefore, most deaths are due to uncontrolled growth of metastasis resistant to therapy. Second, malignant neoplasms contain multiple cell populations exhibiting a wide range of biologic heterogeneity. Cells obtained from individual tumors exhibit differences with respect to cell surface properties, antigenicity, immunogenicity, growth rate, karyotype, sensitivity to various cytotoxic drugs, and the ability to invade and metastasize. 25 • 30 The development of this heterogeneity is multifactorial. Data indicate that metastases can arise from the nonrandom spread of specialized malignant cells that pre-exist within the primary neoplasm, some metastases can be clonal in their origin, different metastases can originate from different progenitor cells, and, in general, metastatic cells can exhibit a higher rate of spontaneous mutation than nonmetastatic but tumorigenic cells. 11 · 24· 31 · 72 Observations that cancer cells are sensitive to temperature change lead to the use of cryosurgery and hyperthermia to induce tumoricidal activity. Hyperthermia can be used locally or systemically, and it is one of the *Diplomate, American College of Veterinary Internal Medicine (Oncology and Medicine); Professor of Medicine and Oncology, Department of Medical Sciences, and Associate Dean for Clinical Affairs, School of Veterinary Medicine; Affiliate Professor, Departments of Veterinary Science and Nutritional Science, College of Agriculture and Life Sciences; and Associate Member, Wisconsin Clinical Cancer Center, Department of Human Oncology, School of Medicine, University of Wisconsin, Madison, Wisconsin Supported by the Morris Animal Foundation and American Cancer Society grant number CH-473. Veterinary Clinics of North America: Small Animal Practice-Vol. 20, No. 4, July 1990

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newest areas of intense investigation in veterinary oncology. Hyperthermia seems to be most effective when combined with other modalities, particularly radiation or chemotherapy. Chemotherapy is another area of major importance in controlling cancer at the systemic level. Chemotherapy continues to develop, with new drugs and new combinations of agents being used to improve cancer treatment. A major limiting factor in the use of chemotherapy is tissue-tolerable host-toxicity. Most chemotherapeutic agents cause nonspecific cytotoxicity which can result in profound cellular changes, in addition to destroying the tumor cells. . Immunotherapy, biologic response modification (BRM), or biologic therapy consist of agents or approaches that modifY the relationship between the host and the tumor with· resultant therapeutic effects. A rationale for the use of immunotherapy is based on both clinical observations and in vitro laboratory findings. Tumor specific antigens, which result in rejection of transplanted tumors, have been identified in murine systems. Effector cells such as natural killer (NK) cells and macrophages have the capacity to kill tumor cells in vitro. Monoclonal antibodies, which recognize tumorassociated antigens on many vertebrate cells, have been generated and used in therapeutic situations. An intact and functioning immune system and lymphoid cell infiltration into primary tumors have been correlated with a good prognosis for certain malignancies in humans and animals. The current approaches to immunotherapy include both active and passive augmentation of host response (Table 1). Nonspecific stimulation of immune effector cell function can result from interferons, interleukins, muramyl peptides, and other bacterial products. Adoptive therapy with lymphoid cells or monoclonal antibodies has also received wide attention recently. Molecules such as tumor necrosis factor/cachectin and interferons have been shown to have growth regulatory activity on tumor cells. This article will review the current status of biologic therapy as agents for the treatment of cancer. Most of the research is being done in rodent models or human clinical studies. However, when appropriate, studies performed in veterinary oncology will be presented. Because the field of biotherapy or immunotherapy is so broad, it will be impossible to cover all aspects and approaches under evaluation today. Clinical studies using crude microbials, such as BCG (bacillus CalTable l. Approaches to Cancer Therapy with Biologicals AGENTS

APPROACH

Active Augmentation Nonspecific Specific

IL-2, IFNs, MDP, bacterial agents Tumor antigen vaccine

Adoptive Therapy Cellular Antibody

T cells, LAK cells, macrophages Monoclonals

Growth Regulation Inhibitory Stimulatory Differentiation

TNFs, IFNs CSFs Retinoids

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mette-Guerin) and Corynebacterium parvum, have provided information on the potential value of immunotherapy in animal and human cancer therapy. This article will specifically cover the interleukins, particularly IL2, cytokines such as tumor necrosis factor (TNF), interferons (IFN), clinical uses of colony stimulating factors (CSF), monoclonal antibodies, muramyl peptides, and the interaction of chemotherapy drugs with biologic response modifiers. INTERLEUKINS An important group of immunoregulating molecules is the interleukins (IL). The term was introduced to indicate their production by lymphocytes and their function of signaling between these cells. The terms lymphokines and monokines are still frequently used to denote production by lymphocytes and monocytes/macrophages, respectively. A brief compilation of interleukins and their main effects on specific immune reactions is given in Table 2. Actions of some molecules will be discussed in more detail subsequently. Interleukin-1 (IL-l) is a hormone-like polypeptide that performs many roles in inflammation and immunity. 18 Originally described as a product released from activated macrophages, IL-l-like factors have been detected in the culture supernates of many types of cells. IL-l has been associated with a diversity of actions: (1) the' endogenous mediator of fever, (2) a mitogen for thymocytes, (3) the stimulant of the acute phase response, (4) cartilage resorption, and (5) muscle wasting. Although IL-l plays a major role in regulatory functions for the immune response, no clinical trials have been conducted to evaluate it as an antitumor agent. Interleukin-2 (IL-2), originally known as T cell growth factor, was discovered in the supernates of phytohemagglutinin-stimulated lymphocyte cultures. It was shown to act as aT cell growth factor enabling cloning of T cells. The property of IL-2 responsible for the interest in its application as a cancer treatment is its ability to induce marked expansion of T lymphocytes in vitro. 56 The reason this property has excited interest is that, in certain animal tumors, T lymphocytes play an important role in tumor and allograft rejection, and it has long been known that they are more efficient than serum in transferring immunologic memory for transplantation and tumor resistance. The cells responsible are presumed to be mediated by cytotoxic T lymphocytes (CTL). 56 Biochemical and molecular analyses enabled identification and cloning of the IL-2 gene. 69 Production of large quantities of IL-2, derived from recombinant DNA technology, using the human IL-2 gene functioning within E. coli, has enabled molecular, preclinical, and clinical research to proceed. Currently, IL-2 is under intense study in human patients and to a limited extent in dogs and cats. Potential for IL-2 as a cancer treatment is based on the activation ofT cell-mediated cytolysis against tumor cells, and release of cytokines that may potentially destroy neoplastic tissue. In 1980 Rosenberg and coworkers reported that lymphoid cells could be generated that were cytotoxic in vitro against a broad range of murine

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Tahie 2. Interleukins and Some of Their Regulatory Functions i~ Specific Immunity FACTOR(S)

OTHER NAMES

SOURCE

EFFECTS

Promotes multiplication and activation ofT and B cells; activates macrophages and NK cells; induces prostaglandin synthesis in many cell types; induces IL-6 production (Co)stimulates T-cell multiplication and effector functions; costimulates B-ee!! multiplication and differentiation Stimulates mast cell multiplication; stimulates proliferation and differentiation of hemopoietic stem cells Co-stimulates B-ee!! multiplication and lgE and IgG, secretion; enhances MHC class II expression on B cells and macrophages; enhances T cell growth factor; activates macro phages Co-stimulates B-cell multiplication and antibody secretion; stimulates eosinophil differentiation Co-stimulates growth and differentiation of B cells; co-promotes IL-2 production by mature T cells

L-1

Lymphocyte activating factor

Monocytes/ macrophages, and many other cell types

L-2

T cell derived growth factor

Helper T cells

L-3

Mast cell growth factor, multiple colony stimulating

Helper T cells

L-4

B cell stimulatory factor-1, B cell growth factor II, T cell growth factor II, macrophage activating factor

Helper T cells, B cells

L-5

T cell replacing factor, B cell growth factor II, eosinophil differentiation factor

Helper T cells

L-6

Hybridoma growth factor, B cell differentiation factor, interferon-~ 2 , B cell stimulatory factor-2

T cells, monocytes, fibroblasts

From Bloksma N, Schuurman HJ: Basic mechanisms of adaptive immune system function. In Autoimmunity and Toxicology. Amsterdam, Elsevier, 1989, pp 37-65; with permission.

and human tumor targets. 42 • 77 Tumor killing was not restricted by the major histocompatibility complex (MHC) and could be demonstrated against freshly isolated tumor cells as well as tumor cell lines. Resting lymphocytes became cytotoxic within 1 to 2 days following in vitro exposure to high (1000 to 1500 units per mL) concentrations of IL-2 and exhibited maximal lysis after 3 to 6 days in culture. Although these cells were termed lymphokine activated killer (LAK) cells, the effector cell has not been

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uniquely identified using cell surface antigens. 28 • 56 The term "LAK cell" is now commonly used, but it is merely a conventional shorthand for cells that exhibit nonrestricted tumor cell killing or, more simply, cells with LAK activity. To date, IL-2/LAK has demonstrated significant antitumor activity in humans with renal cell carcinoma and malignant melanoma. Although the overall response rates are modest (15 to 30% ), prolonged complete response rates have occurred in patients with visceral disease. High doses of IL-2 induced in vivo production of other cytokines (TNF, IFN, and probably others), and that may have contributed to the antltumor activity and/or toxicity of this regimen. Current studies are underway to compare IV bolus versus continuous infusion of IL-2 in human cancer patients. 37 Another major issue under study in human cancer patients is whether LAK cells are required. Clinical trials are currently ongoing to evaluate IL-2 without LAK cells. Although the results are preliminary, it would appear that the responses of IL-2 without LAK cells are equivalent to those in trials in which LAK cells were included. 7 Despite the antitumor responses seen in some human patients, improvements are needed to obtain more meaningful clinical benefit and improved therapeutic effect. Animal models have suggested additive or synergistic effects can be obtained by combining IL-2-induced LAK activity with other agents. One of these agents is tumor necrosis factor (TNF). Zimmerman et aF8 reported that the antitumor activity of TNF could be enhanced when given prior to IL-2. A number of phase 1 trials in humans, using TNF followed by IL-2, are currently in progress. One such study has been performed in dogs with spontaneous cancers. 8 Dogs with various malignancies were administered human recombinant TNF (130 j.tg/m 2 in 100 mL of normal saline administered IV over 30 minutes) on days 1, 2, and 3 combined with human recombinant IL-2 (0.5 mg/m 2 subcutaneously daily) on days 4 through 12. In some cases the treatment included the addition of chemotherapy and surgery. Twenty dogs with various malignancies were treated; these included transitional cell carcinomas, squamous cell carcinomas, osteosarcomas, mast cell tumors, lymphosarcomas, and melanoma. No antitumor activity or response was identified in this clinical trial. 8 However, this was a very preliminary study, and to date we do not have enough information regarding the optimal dose and the method of administration of IL-2 or TNF in dogs. More studies are warranted using single agents to evaluate for toxicity, optimal dose, route, and biologic activity. The toxicity that has been associated with the administration of IL-2, alone or in combination with LAK cells, clearly indicates that the side effects of this therapy can be serious and even life-threatening. 41 However, neither the mechanism for the induction of toxicity nor the pathophysiology are well understood. Experiments in rodents have indicated that toxicity was reduced or avoided when animals were pretreated with corticosteroids or other immunosuppressant agents. 57• 61 In humans, protocols involving treatment of patients with cyclophosphamide prior to administration of high-dose IL-2 have indicated that this combination may help reduce toxicity as well as increase therapeutic efficacy. 48 These and other data have

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suggested the possibility that IL-2-induced production of cytokines may contribute to or be completely responsible for the cardiorespiratory toxicity. There are promising approaches using IL-2 to stimulate populations of lymphocytes obtained from draining lymph nodes or directly from tumorinfiltrating populations. 60 These tumor-infiltrating lymphocytes (TIL) or tumor-derived activated killer cells (TDAK) have been shown to be 50 to 100 times more potent than LAK cells in mediating the regression of established cancer in several murine tumor models. 69 TIL have been isolated and clonally expanded from a variety of human cancers. In 1988 Rosenberg et al 60 reported on a preliminary trial in metastatic melanoma patients using a regimen of cycloph~sphamide in conjunction with adopted transfer of a large number of TIL and IL-2. Of the 20 patients treated, 1 had a complete response with a duration of greater than 13 months, and 10 had a partial response with durations between 2 and 9 months. From this study, it was concluded that the addition of TIL increased the response rates over those of either IL-2 alone or IL-2 and LAK cells. These studies are very encouraging for using locally derived immune cells for activation with IL-2 in a therapeutic regimen. In veterinary oncology the future of IL-2 awaits to be determined. The few studies that have been performed have failed to show any significant antitumor activity. However, it remains to be seen whether IL-2 can induce a lymphocyte reactivity in the dog. Preliminary results have demonstrated that IL-2 does induce IL-2 receptor activity and mitogenic activity in tumorbearing dogs. 29 Furthermore, clinical trials are indicated using combinations of IL-2 with cyclophosphamide or TIL.

INTERFERONS Interferons are molecules which can modulate the host response to a neoplasm and inhibit tumor cell growth. They can regulate gene expression, modulate expression of proteins on the cell surface, and induce synthesis of new enzymes. On a cellular basis, these effects translate into an alteration of the state of cellular differentiation, the rate of proliferation, and functional activity of several cell types. A large-scale assessment of interferons as treatment for spontaneous malignancies began in 1979 with the American Cancer Society program. Interferons have substantial single agent activity in hematologic malignancies 6 (Table 3). By far, the most significant response has been seen in human patients with hairy cell leukemia. More than 85% of patients have objective evidence of partial or complete hematologic response. In nodular lymphomas, a high-frequency objective response (up to 50%) can occur in patients refractory to other modalities of treatment. Interferons can significantly augment the impact of chemotherapy. Currently, studies are ongoing in humans to evaluate combined interferon alpha-2 with chemotherapy. In chronic myelogenous leukemia, interferon alpha-2 has both clinical and biologic impact on persistence of the disease process. Objective response rates of greater than 50% occur with a decrease in peripheral blood counts and an improvement in bone marrow infiltration.

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Table 3. Human Malignancies in Which Interferon Alpha Has Demonstrated Antitumor Effectiveness Chronic leukemias Myeloid Hairy cell Lymphomas Nodular T cell Multiple myeloma

Renal carcinoma Malignant melanoma Carcinoids Gliomas Ovarian carcinomas Basal cell carcinomas Bladder carcinomas Kaposi's sareoma

In solid tumors the antitumor activity of interferon has been established in metastatic renal carcinoma with partial and complete response rates to 30%. Overall, in a collected series of 228 patients, 22% have had objective regression of metastatic renal carcinoma. 58 In other solid tumors, interferons have also shown effectiveness. For example, in endocrine tumors of the gastrointestinal tract, interferons have proved useful in controlling both objective and subjective symptoms. 53 In Zollinger-Ellison syndrome and nonfunctioning pancreatic malignancies, approximately a 20% response rate has been observed. In carcinoid tumors, approximately half of the patients have improvement in biochemical parameters, and 10 to 15% have a reduction in measurable disease. 54 Finally, in human immunodeficiency virus infection-induced Kaposi's sarcoma, responses have been seen with interferon. 38 Few clinical trials have been performed using available human recombinant interferons in veterinary medicine because of the species specificity of the interferons. However, there has been a report of the use of human interferon alpha in catsY Twenty-one feline leukemia virus (FeLV)-susceptible cats were inoculated with the Rickard strain of FeLV. Cats given oral human interferon alpha survived significantly longer than untreated FeLVinfected cats. Moreover, only 4 of 13 (30.8%) human interferon alphatreated cats developed clinical disease during the course of the study, whereas 100% of the untreated controls developed fatal FeLV-related disease. Thus, in an experimental retroviral infection, heterologous species human interferon alpha provided significant clinical benefits. To date, this is the only encouraging clinical study showing potential activity for the human recombinant interferons in veterinary medicine. CYTOKINES Cells of the monocyte/macrophage lineage play a central role in cytokine (monokine) production, which can modulate many aspects of the inflammatory and antitumor response. IL-l alpha and beta and a second molecule widely known as cachectin or TNF alpha are among the best studied of all cytokines. TNF alpha and the related molecule lymphotoxin TNF beta directly inhibit proliferation of many malignant cell lines. TNF was first identified as an activity in serum of bacillus Calmette-Guerininfected mice challenged with endotoxin. 9 The serum of these mice con-

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tained the factor which upon direct inoculation (or intravenous injection) into certain tumors resulted in necrosis of these tumors. Further studies demonstrated that TNF production by endotoxin resulted from stimulation of monocytes, which is responsible for the production of this antitumor factor. The mechanism by which TNF mediates tumor regression in vivo remains unclear. Although it is tempting to ascribe antitumor toxicity to direct antiproliferative effects, the situation is more complex. Antitumor effects in vivo have been seen for cells resistant to the antiproliferative effects in vitro. These findings suggest that part of the effect of TNF is through the host response. Both monocyte cytotoxicity and peroxide generation can be augmented by TNF. TNF probably accounts for the tumor cell cytotoxicity which has been associated with monocytes and macrophages. TNF has also been shown to induce a hemorrhagic necrosis which might reflect generalized vascular collapse and could be related to infarcting tumors by ischemia. TNF down regulates endothelial cell expression of thrombomodulin and causes the elaboration of a procoagulant activity as well as the release of IL-l from endothelial cells in vitro. 49• 71 TNF also inhibits the growth of vascular endothelium and causes morphologic changes. In clinical trials the major limitation of TNF has been the severe toxicity. The limiting toxicity has been hypotension; other side effects have included fever, nausea, and vomiting. Tumor regressions have been observed in phase 1 trials in colorectal carcinoma and soft-tissue sarcoma. The few studies that have been performed in veterinary medicine have failed to demonstrate any antitumor activity. It seems likely that a therapeutic role in malignancy will be identified eventually.

COLONY-STIMULATING FACTORS

The CSF are glycoproteins that regulate the proliferation and differentiation of progenitor cells. The existence of such factors was suspected when it was found that the formation of colonies of granulocytes or macrophages from bone marrow cultured in soft agar required the presence of an underlayer of accessory cells, serum, urine, or condition medium. 20 The CSF have been difficult to classify in terms of their cellular origin (Table 4). They are not strictly lymphokines, because their cellular sources may be quite diverse. The major classifications for the CSF are as follows: IL-3 or multi-CSF, granulocyte-macrophage CSF (GM-CSF), granulocyte Table 4. Characteristics of Colony Stimulating Factors NAME

G-CSF GM-CSF IL-3 (multi-CSF) M-CSF

PROTEIN SIZE (KD)

18-20 14-35 14-28 45 (monomer)

CELLULAR SOURCES

Monocytes, fibroblasts T cells, endothelial cells, fibroblasts T cells Monocytes, fibroblasts, endothelial cells

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CSF (G-CSF), macrophage CSF (M-CSF), and erythropoietin (EPO). Other growth factors which have been identified are IL-4 or B cell growth factor1, IL-5 eosinophil differentiation factor, and IL-6 B cell differentiating factor (see Table 2). Since the advent of genetic engineering and the ability to clone these genes from humans, recombinant CSF are now available for study. The availability of recombinant GM-CSF has led to several in vivo studies of its effects in nonhuman primates and dogs. 16• 17 · 39• 52 Th~ initial investigations by Donahue et aP 6 used a continuous IV infusion over varying periods of time. Typically, the rise in white blood cells (WBCs) occurred by day 3 and reached a plateau on days 7 to 9. The leukocytosis was composed of eosinophils, neutrophils, lymphocytes, and monocytes. Withdrawal of the drug led to a prompt decline of leukocytosis within several days. In our laboratory we have also looked at the effect of recombinant GM-CSF in normal dogs. We found that the leukocytosis peaked around day 7 and then declined over the next 2 weeks, where it was close to its pretreatment level at day 14. Other studies using recombinant GM-CSF in normal canines have been reported with similar findings noted. 47 Also, the peak response in neutrophil counts occurred around days 5 to 8 and subsequently declined. Between 12 and 21 days of treatment, all dogs treated in this study developed antibodies to recombinant GM-CSF, and further response to the growth factor ceased. Additional studies in our laboratory using a canine recombinant GCSF have demonstrated that the effect can be sustained for longer periods of time. 39 We have shown that canine recombinant G-CSF in dogs will maintain a significant leukocytosis (approximately 80,000 to 100,000 WBCs) for as long as 8 weeks of treatment. When the drug is stopped, there is a prompt decline in leukocytosis within a few days. In this study it did not appear that dogs developed any antibodies to the G-CSF and thus continued response to the agent was seen. There are two broad areas of development for the growth factors: (1) their use as single agents, and (2) their use with each other. As single agents, they could be used as an adjunct to the treatment of neutropenic sepsis, and the use of these recombinant growth factors could be very applicable in myelosuppressive chemotherapeutic protocols. In addition, the growth factors have been shown to be useful when combined with total body radiation for bone marrow transplantation. Additional uses for these growth factors may be in bone marrow dysplasias such as aplastic anemia and in patients with myelodysplasia. The other use for these growth factors may be in combination with each other. Recently, IL-6, which is also called B cell stimulating factor, has been reported to act as a priming agent on early bone marrow progenitors. 75 Its mode of action appears similar to that of IL-l in that it sensitizes or primes the earlier progenitors and makes them more capable of proliferating in response to later-acting, lineagespecific CSFs. It would seem appropriate then to combine IL-6 with either G-CSF or GM-CSF. There is considerable excitement and promise in the clinical application of hematopoietic growth factors for a variety of disease states. Other avenues

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of research or clinical application may be the use of these growth factors combined with biologic response modifiers. MONOCLONAL ANTIBODIES Monoclonal antibodies are promising for cancer therapy because of their potential selectivity and specificity. There are a variety of mechanisms by which antibodies may be useful when administered systemically as cancer therapy. Monoclonal antibodies may be directly cytotoxic by the interaction of the Fe portiqn of the immunoglobulin molecule with complement or cytotoxic effector cells. They may also have direct antiproliferative effects by competing at the level of growth factor receptors and/or by altering the regulation of cell proliferation. Antibodies may also be useful as carriers of cytotoxic agents by creating immunoconjugates in which antibodies are linked to radioisotopes, natural toxins, chemotherapeutic agents, or cytotoxic cells. 27 • 55 Most monoclonal antibodies are produced in murine systems and are antibodies of murine origin. Clinical results with murine monoclonal antibodies alone have generally been disappointing. Even when murine monoclonal antibodies have been effective, their efficacy may have been limited by the development of human antimouse antibodies _57 Because of the technical difficulties involved with producing speciesspecific antibodies, most of the work to date is still based on murine monoclonal antibodies. Owing to the heterogeneity of cancers, even if a tumor-specific antigen were found, it is reasonable to assume that not all the tumor cells would be reactive to a particular monoclonal antibody. Therefore, to ensure that all tumor cells can be recognized and subsequently destroyed, a "cocktail" of monoclonal antibodies should be formulated to better ensure the identification of all cancer cells. Clinical trials using cocktails of murine monoclonal antibodies are already in progress, and those with human-derived monoclonal antibodies will commence in the near future. 55 Because of some of the inherent limitations in generating speciesspecific antibodies (that is, human hybridomas), some investigators have approached the problem from another direction by constructing chimeric monoclonal antibodies. 27 These have consisted of attaching a mouse variable portion or a human variable portion of the immunoglobulin molecule to a different human heavy chain. It is hoped that such a predominantly human construct will be less immunogenic, as compared to the mouse immunoglobulin, because humans have been shown to mount an immune response against immunologically foreign murine antibodies. 67 The human Fe portion may interact more efficiently with human complement and/or effector cells. The mechanisms involved in antibody-dependent tumor cell destruction are not well characterized. Lysis of tumor cells may result from the interaction of monoclonal antibodies with one or more of the effector cells including lymphocytes, macrophages, natural killer cells, or killer cells. The success of monoclonal antibody therapy will depend on certain selection criteria used when these antibodies are developed. It seems reasonable to consider the following in selecting antibodies for in vivo

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application 55 : (1) antibody binds to an antigen on the cell surface; (2) antigen is highly expressed on cell surface and found on most, preferably all, cells in the tumor; (3) antibody binds with high affinity; (4) antibody is expressed at very low levels on a limited number of normal tissues and/or is found on occasional cells in normal tissues; (5) antigen antibody complexes are internalized; (6) biodistribution studies reveal localization in tumor far in excess of uptake by the reticuloendothelial system; and (7) antibody mediates antibody-dependent cellular cytotoxicity. In addition, antibodies must reach the tumor bed to be affected. Smaller antibodies (IgG rather than IgM) or antibody fragments may be more likely to diffuse from the vascular compartme'nt into the tumor bed. Although entry into the tumor mass is clearly critical, retention within the tumor may be equally or more important. Thus, fragments may diffuse more quickly into the tumor nodule, but whole antibody may be retained for a longer time within that same tumor nodule. In veterinary oncology, few monoclonal antibodies have been developed against canine tumor cells. One antibody which has been shown to be an anticanine lymphoma cell antibody is currently under study in a clinical trial in dogs with lymphoma. 59• 70 Although the results are preliminary, it appears that those dogs receiving the canine lymphoma monoclonal antibody are maintaining a longer remission than those receiving chemotherapy alone (Jeglum AK: Personal communication, 1989). Although these results appear positive, the current trend is that monoclonal antibodies will be most effective when they are used as immunoconjugates. Developing immunoconjugates such as chemotherapy agents or toxins is a very active area of clinical and experimental research in both human and veterinary medicine. Ongoing trials in human medicine will determine the overall importance of this therapy for cancer. MURAMYL PEPTIDES

Muramyl peptides contained within Freund's adjuvant (killed mycobacteria in a water-in-oil emulsion with paraffin oil and an emulsifier) are considered to be one of the most efficient adjuvants for increasing antibody production and establishing cellular immunity to an antigen. This biologic activity of the whole mycobacterium cell is mainly due to its cell wall and, in particular, its peptidoglycan. In 1974 Ellouz et al2 1 showed that natural muramyl peptides and the synthetic muramyl dipeptide (MDP) could replace whole mycobacterium for adjuvant activity. The precise mechanism of action of muramyl peptides is not yet understood. The direct effect on different cells has been demonstrated in experimental models. 40 • 51 • 73 In particular, MDP-treated macrophages have killing capacity for tumor cells and various microbial agents. Macrophages undoubtedly are involved in the various biologic effects of MDP. Their stimulation has been assessed by a number of parameters, including metabolic, biochemical, and functional changes (Table 5). The increased capacity of macrophages to produce oxygen metabolites, to secrete mediators, and to kill microbial agents in tumor cells is of importance in

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Table 5. Effect of Muramyl Dipeptide (MDP) on Macrophage Function and Metabolism Glucose oxidation and glucosamine uptake Adherence and spreading Chemotaxis, inhibition of migration Activation of killing capacity Phagocytosis Production of peroxides Secretion of monokines (CSF, IL-l, TNF) Production of collagenases

increasing the resistance of the host to tumors after the administration of muramyl peptides. Activation of macrophages, however, generally requires the presence of other compounds, such as minute concentrations of endotoxins, and the use of lipophilic vesicles (liposomes) for in vivo effects. 26 When free MOP is administered parenterally, it is excreted very rapidly in the urine. To improve its pharmacokinetics, MOP has been encapsulated within liposomes that are delivered to macrophages, preventing its rapid elimination and allowing a 500 to 1000 fold reduction of the amount of drug needed for macrophage activation. To enhance the efficacy of delivery and encapsulation, a lipophilic muramyl peptide (muramyl tripeptide, MTP) has been used in experiments and clinical trials. 26 · 46 Muramyl peptides can interact with cell membranes directly owing to the presence of cell surface receptors on macrophages or enter the cell membrane via endocytosis of attached liposornes. MTP acts primarily at the intracellular level. MTP can induce rapid changes in the expression of various genes' coding, for example, IL-l and major histocompatibility complex class II antigens. 1• 26 · 51 It has been suggested repeatedly that IL-l released by MTP-stimulated macrophages is responsible for its adjuvant activity. 1 B cells, however, have also been considered as an essential target for this effect. For example, MTP has been shown to synergize with various B cell mitogens and polyclonal activators, leading to increased proliferation and secretion of polyclonal antibodies. 19 Liposome-encapsulated MOP and MTP have been shown to have antitumor activity in rodent tumor models. 26 In studies utilizing the B-16 melanoma cell line in mice, it was found that administration of liposome MOP resulted in regression of spontaneous lung metastases when compared to mice treated with MTP liposomes, saline, and free muramyl peptides. 26 · 63 Similar studies of other murine metastatic tumors involving systemic administration of liposomes containing different immunomodulators have shown similar antitumor activity. Liposome-encapsulated MTP has been shown to activate peripheral blood monocytes when injected into humans. 35 Recent, yet unpublished, studies in our laboratory show that when liposome-encapsulated MTP is injected into normal dogs, it will induce adherent mononuclear cells to become cytostatic to canine osteosarcoma cells. The phospholipid composition of liposomes will influence the success of their being phagocytosed or endocytosed by the macrophage.

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Liposomes containing negatively charged phospholipids have enhanced uptake by blood monocyte tissue macrophages. 26 In addition, the presence of phosphatidylserine in the liposome has been shown to result in enhanced binding and endocytosis to all cells of the re ticuloendothelial system . Negatively charge d liposomes containing phosphatidylserine are endocytosed up to 10-fold faster than are positively charged liposomes of the same size and configuration. 26 We have been conducting studies to evaluate the effect of liposomeencapsulated MTP as a treatment for micrometastatic disease in dogs with osteosarcoma. Canine osteosarcoma is a rapidly metastasizing tumor, and probably all afflicted dogs have micrometastases at the time of diagnosis. The most commonly reported metastatic site in the dog is the lung. Despite various therape utic regimens, mostly involving amputation, survival times remain short. The median survival times usually range from 3 to 4 months, and only about 10% of the dogs survive 1 year or longer. We have recently completed a randomized double-blind study to evaluate a liposome-encapsulated MTP combined with amputation compared to amputation alone . 46 All dogs underwent a thorough evaluation for evidence of metastasis prior to surgery. The liposome-MTP combination was administered at a dose of 2 mg/m 2 twice weekly for an 8-week period. The liposomes used in this study consisted of phosphatidylserine and phosphatidylcholine at a 3:7 molar ratio. After the completion of therapy, dogs had thoracic radiographs at 2-month intervals to monitor for the development of metastases.

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Days Post Surgery Figure l. Metastasis-free intervals of dogs with osteosarcoma receiving liposome MTP vs. those receiving surgery alone , P < 0.02. (From MacE wen E G, Kurzman ID, Rosenthal HC, et al: Therapy for osteosarcoma in dogs with intravenous injection ofliposome-enc::a psulated muramyl tripeptide . J Nat! Cancer Inst 81:935, 1989. )

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Twenty-seven dogs with osteosarcoma were admitted into this study. Of these, 14 received liposome-encapsulated MTP and 13 received surgery alone. All dogs tolerated the treatment very well. The median time from surgery to the development of metastases was significantly longer in the liposome-encapsulated MTP group. For those dogs getting liposome MTP, median time to metastases was 168 days, with a range of 54 to 974 + days, versus 58 days, range of 31 to 227 days, for the surgery-only group (P = .02) (Fig. 1). The median survival time for the dogs receiving liposome MTP was 222 days, versus 77 days for those receiving just amputation alone (P = 0.002) (Fig. 2). In the liposome MTP group, 4 dogs were alive longer than 1 year and 2 were free of metastases for longer than 2 years. In the amputation-alone group, all 13 dogs died of metastatic disease. This study is an extepsion of the studies done in murine systems, demonstrating that liposome-encapsulated MTP has significant activity in delaying or preventing metastases. Current studies are underway in our clinic combining amputation plus cisplatin chemotherapy with liposomeMTP therapy. Although those studies are preliminary, it appears that the addition of liposome-encapsi.Ilated MTP to cisplatin achieves better results than cisplatin alone. The optimal conditions for systemic therapy with liposome-encapsulated immunomodulators and the efficacy of this modality alone and in combination with other treatment modalities are now being defined. As

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EMPTY MTP-PE

60 50 40 30

E

20

0

10

::s

----o-e

0

0

100 200 300 400 500 600 700 800 900 1000

Days Post Surgery Figure 2. Survival of dogs with osteosarcoma receiving liposome MTP vs. those receiving empty lysosomes, P < .002. (From MacEwen EG, Kurzman ID, Rosenthal RC , eta!: Therapy for osteosarcoma in dogs with intravenous injection of liposome-encapsulated muramyl tripeptide. J Nat! Cancer Inst 81:935, 1989.)

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BIOLOGIC RESPONSE MODIFIERS: THE FUTURE OF CANCER THERAPY?

with many other antitumor therapies, optimal application of macrophage treatment for metastases will require combination with other antitumor agents. Indeed, the most likely role for tumoricidal macrophages is in the destruction of micrometastases or new tumor cells that remain after treatment with conventional adjuvant therapies such as chemotherapy. Other bacterial products that have shown promise as anticancer agents in veterinary medicine are the use of Corynebacterium parvum in dogs with malignant melanoma, 45 and the use of Staphylococcus protein A for the treatment of cats with feline lymphoma and leu~emia. 14• 22 • 34• 76 The current trend in BRM therapy has been away from the crude bacterial products to more defined chemical immunomodulators or the use of lymphokines, cytokines, and CSF. However, the anecdotal observations of the importance of bacterial products as antitumor agents cannot be ignored. Even today, studies are in effect to continue to look at bacterial agents for their antitumor activity. 3 Additionally, other approaches which have merit for study as immunotherapeutic agents include tumor vaccines, 32• 33 plasma components, 43 • 44 and approaches directed at inducing cellular differentiation. 5, 66

INTERACTIONS OF CYTOTOXIC DRUGS WITH BIOLOGIC RESPONSE MODIFIERS Chemotherapeutic agents can play a very important role as immunomodulators and can influence the activity of other BRMs. A few of the chemotherapeutic agents which have been studied for their role as immunomodulators have been cyclophosphamide, 6-mercaptopurine (6-MP), and doxorubicin (Table 6). Cyclophosphamide has been shown to affect both the cellular and the humoral immune systems. Low doses of cyclophosphamide will augment antibody production in mice, and higher doses will suppress antibody production. 4 • 10 It appears that cyclophosphamide augments cellular immunity by a selective toxicity for suppressor T-cells. 23 • 5°Cyclophosphamide has been used to augment the use of IL-2/LAK cell immunotherapy. 60 • 68 6-MP is a potent immunosuppressive agent which inhibits both antibody synthesis and delayed cellular hypersensitivity reactions as well as retards the rejection of allografts. 64 • 65 However, later studies demonstrated that 6-MP could be used to augment antibody formation. 15 This property may make 6-MP a useful agent to combine with immune modulators, particularly those dependent on B-cell stimulation. Table 6. The Role of Chemotherapy Agents in Immunomodulation DELAYED DRUG

Cyclosphosphamide 6-MP Doxorubicin

MACROPHAGE/

T-CELL

HYPERSENSITIVITY

B-CELL

MONOCYTE

ACTIVITY

REACTION

ACTIVITY

ACTIVITY

~ No effect

No effect

t t

t

t

~

~

t t

No effect

1070

E.

GREGORY MACEWEN

Doxorubicin has been shown to effectively augment cytotoxicity of peritoneal cells in various mouse strains. 62 Other studies have suggested that doxorubicin may enhance monocyte-macrophage cell-mediated immunity and, in addition, increase T-helper cells and increase IL-2 production. 2. 36, 74 The role of chemotherapy as an immune modulator or as a potentiator for other BRMs has recently gained interest. The potential of using these drugs in these ways awaits further exploration.

SUMMARY The major shift today has been away from nonspecific compounds acting on immune mechanisms to using biologics which have specific, defined roles in acting on the immune response. The field of biologic response modification is progressing very rapidly. New peptides are being identified, as are receptors for these peptides, autocrines, lymphokines, cytokines, growth fractors, differentiation factors, hormones, and so on-all of which will control body function, cell populations, and cell to cell interactions. This rapidly advancing area of research in cancer biology and cancer therapy may hold the key to the future of successful therapy.

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Address reprint requests to E. Gregory MacEwen, VMD Department of Medical Sciences School of Veterinary Medicine University of Wisconsin Madison, WI 53706