Pharmacodynamic aspects of peptide administration biological response modifiers

Advanced Drug Delivery Reviews 33 (1998) 241–252

L

Pharmacodynamic aspects of peptide administration biological response modifiers James E. Talmadge* University of Nebraska Medical Center, 600 South 42 nd Street, Omaha, Nebraska 68198 -5660, USA Received 29 September 1997; received in revised form 7 December 1997; accepted 13 February 1998

Abstract Cytokines, growth factors and other recombinant proteins have been one of the most rapidly growing areas of pharmaceuticals. Further, the development of these bio-engineered drugs is occurring at an astonishing pace with rapid preclinical and clinical development and licensing by regulatory agencies. In addition, the availability of the gene sequences and rational drug design technologies have resulted in a rapid development of engineered genes, proteins and peptidomimetics. In contrast to traditional pharmacophores, which are developed based on the identification of the maximum tolerated dose (MTD), most recombinant proteins have abnormal biodistributions, and pharmacodynamic and pharmacokinetic attributes. Within this chapter, representative cytokines including interferon-alpha (IFN-a), IFN-g and interleukin-2 are used to discuss the pharmacodynamic aspects of protein / peptide administration that are important in the development of these drugs. This includes the conceptual need for chronic immunoaugmentation for optimal therapeutic activity; the need to consider the pharmacokinetics of administration to optimize drug delivery and the nonlinear dose response relationship, which can result in a bell shaped dose response. Furthermore, these therapeutics have maximal potential in an adjuvant protocol and their development in combination with high-dose chemotherapy and stem cell rescue is discussed. The strategies for combination chemotherapy and immunotherapy, while holding great promise, require close attention to the pharmacodynamics of protein administration in order to impact on failure free and overall survival.  1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Pharmacodynamics; Peptide; Recombinant proteins; Chemotherapy; Immunotherapy; Interferon; Dose–response; Cytokine

Contents 1. Introduction ............................................................................................................................................................................ 2. Recombinant proteins .............................................................................................................................................................. 2.1. Interferon alpha (IFN-a). The need for chronic administration for therapeutic activity........................................................... 2.2. Interferon-gamma (IFN-g)-non-linear dose response relationships for immune augmentation ................................................ 2.3. Interleukin-2 (IL-2), The importance of pharmacokinetics in the activity of IL-2 .................................................................. 3. Combination Chemotherapy And Immunotherapy...................................................................................................................... 4. Conclusion ............................................................................................................................................................................. References .................................................................................................................................................................................. *Corresponding author. Tel.: 1 1 402 5595639; fax: 1 1 402 5594990; e-mail [email protected] 0169-409X / 98 / $ – see front matter  1998 Published by Elsevier Science B.V. All rights reserved. PII: S0169-409X( 98 )00032-5

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1. Introduction The use of immunostimulants to treat human disease has its origins in the experimental use of mixed bacterial toxins to treat cancer by William B. Coley early in the century [1]. These early studies have resulted in the clinical approval of the microbially derived substance BCG for the treatment of bladder cancer in the USA. While these crude drugs induce immunopharmacologic activities, they pose considerable regulatory problems due to impurity, lot to lot variability, unreliability, and side effects. Thus, immunoregulatory and hematoaugmenting cytokines have come into their own as novel therapeutic compounds for diverse indications including oncology, infectious disease and a wide variety of congenital diseases. Indeed, the rapidity of the development of these drugs is astonishing, as is the pace with which new cytokines and growth factors are identified, cloned and moved into clinical development. Immunopharmacologic analyses of structure–activity relationships, toxicology, pharmacokinetics and immunopharmacodynamics have yielded information contributing to the more effective use of these agents. Currently it appears that the ultimate use of these drugs will be as adjuvants in combination with other therapies. Nonetheless, the use of protein adjuvants is perhaps the most exciting and rapidly advancing area of oncology therapy. This review discusses the pharmacodynamics of recombinant proteins that are currently either licensed or in active clinical trials in the United States and are presented in the context of the strategies used for their development.

2. Recombinant proteins Cytokines and growth factors are relatively low molecular weight proteins that are secreted in minute quantities and act in either an autocrine fashion on the cell from which they are secreted or in a paracrine manner on adjacent cells. The isolation of cDNA clones for cytokines and growth factors has permitted their production in large and reproducible quantities, which in turn has also accelerated the preclinical and clinical study of their function and therapeutic attributes. The ability to cut and rejoin

DNA at any desired site or to introduce point mutations at directed sites has resulted in the development and clinical use of mutant as well as chimeric therapeutic proteins. Thus, we can now utilize proteins that are either exact or mutated forms of the naturally occurring ones, or design proteins that are composed of various polypeptide structures derived from different sequences, for example the humanized monoclonal antibodies. The use of various point mutations has resulted in drugs with decreased toxicity, better production capabilities or higher expression levels such as the mutant IL-2, IL-3 or CSF-g molecules [2–4] where for example, serines are substituted for cysteines to reduce the development of aberrant tertiary structures. Furthermore, chimeric cytokines have been developed with properties associated with multiple parent structures such as PIXY, which is a single biologically active drug comprised of IL-3 and CSF [5,6]. Therapeutic proteins have emerged as an important class of drugs for the treatment of cancer, myelodysplasia and infectious diseases [7]. However, their development has been slowed by poorly predictive models and our superficial knowledge of the pharmacology and mechanism of action. To facilitate the development of these immunoregulatory proteins additional information is needed on their pharmacodynamics [8,9]. One approach to the development of these proteins is to identify a clinical hypothesis based on the preclinical identification of a therapeutic surrogate(s). Notably this strategy was recently formally accepted by the FDA [10]. A surrogate for clinical efficacy may be a phenotypic, biochemical, enzymatic, functional (immunologic, molecular or hematologic) or quality of life measurement which is believed to be associated with therapeutic activity. Phase I clinical trials can then be designed to identify the optimal and maximum tolerated dose or treatment schedule that maximizes the augmentation of the surrogate end point(s). Subsequent phase II / III trials can then be established to determine if the changes in the surrogate levels correlate with therapeutic activity. Table 1 lists the immunologically and hematologically active proteins that are approved for general use in the United States. The proteins that are currently in clinical trials are listed in Table 2 along with the indications under investigation. In contrast to strategies based on the identification

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Table 1 Approved biotechnology drugs Product type

Abbreviated indication

Erythropoetin

Interferon beta Interleukin-2

Anemia associated with chronic renal failure Anemia in HIV-infected patients Anemia associated with cancer chemotherapy Febrile neutropenia associated with chemotherapy Treatment of marrow transplants Treatment of severe chronic neutropenia (chemotherapy) Peripheral blood progenitor cell transplants Neutropenia associated with transplants Acceleration of myeloid recovery following autologous bone marrow transplantation Reduce immunosuppression in AML Peripheral blood progenitor cell transplants Allogenic bone marrow transplantation from HLA-matched related donors Treatment of hairy cell leukemia Treatment of AIDS-related Kaposi’s sarcoma Treatment of Philadelphia chromosome-positive CML Treatment of hairy cell leukemia Treatment of AIDS-related Kaposi’s sarcoma Treatment of patients with non-A, non-B / C hepatitis Treatment of patients with chronic hepatitis B Systemic recurrence of malignant melanoma Chronic granulomatous disease Rheumatoid arthritis Multiple sclerosis Renal cell carcinoma

of surrogates for efficacy, many strategies developed for these recombinant proteins have been predicted on practices developed for conventional low molecular weight drugs and may not be advantageous for the development of proteins. The unique pharmacologic attributes of proteins require their selective or targeted delivery to the desired site (i.e. the bone marrow, spleen or tumor [11,12]) and consideration of their pharmacokinetics. Optimally administering proteins as drugs and assuring their targeting are the primary challenges for their development. One further difficulty in the development of a recombinant protein is that in many instances there is little relationship between the dose administered and biologic effect. Indeed, in some instances there is a nonlinear dose relationship that has been described as bell shaped [13]. This dose response relationship, or lack thereof, may be due to the nonlinear way in which these drugs are dispersed in the body; a poor ability to enter into a saturable receptor mediated

transport process; chemical instability; sequence of administration with other agents or an incorrect time of administration; and or due to an inappropriate location and response of the target cells. Further, a ‘bell shaped’ dose response curve could be associated with the tachyphylaxis of receptor expression or a signal transduction mechanism whereby the cells become refractory to subsequent receptor mediated augmentation. Because the regulation of biological control can and likely will lead to numerous physiologically untoward events, it is important that both the physical–chemical structure and the mechanism of administration of recombinant proteins be tailored so as to ensure the desired physiological activity. Several paradigms distinguish the therapeutic activity of proteins from classical low molecular weight drugs. These differences are predominantly associated with the pharmacodynamic attributes of the proteins. Thus, it is critical to understand the pharmacology of these drugs so as to optimize their

CSF-G

CSF-GM

Interferon alpha 2a

Interferon alpha 2b

Interferon gamma 1

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Table 2 Biotechnology drugs in development – approved drugs Product Type Colony stimulating factors CSF-GM CSF-GM Sargramostim (CSF-GM) CSF-M CSF-M Filgrastim (R-CSF-G) Sargramostim (CSF-GM) PIXY Stem Cell Factor (SCF) TPO Flt3 ligand Erythropoietins epoetin beta epoetin alpha

Abbreviated indication

U.S. Status

adjuvant to chemotherapy low blood cell counts allogeneic bone marrow transplantations, chemotherapy adjuvant adjuvant to AIDS therapy cancer, fungal disease cancer, hematologic neoplasms, bone marrow transplantations AIDS, leukemia aplastic anemia neutropenia to secondary chemotherapy Neutropenic neutropenia / thrombocytopenia thrombocytopenia

Phase I / II Submitted Phase III Phase I Phase I Submitted Phase III Phase I / II Phase II Phase I

neutropenia / thrombocytopenia

Phase I

anemia secondary to kidney disease autologous transfusion anemia of cancer and chemotherapy anemia of surgical blood loss, autologous transfusion

Submitted Phase II / III Submitted Phase

small-cell lung cancer, atop dermatitis trauma-related infections, renal cell carcinoma asthma and allergies ARC, AIDS cancer rheumatoid arthritis venereal warts cancer, infectious disease cancer, infectious disease superficial bladder cancer; basal cell carcinoma; chronic hepatitis B, delta hepatitis acute hepatitis B, delta hepatitis, acute chronic myelogenous leukemia HIV (with Retrovir) unresponsive malignant diseases colorectal cancer (with 5-fluorouracil) chronic, acute hepatitis b; non-A, non-B hepatitis; chronic myelogenous leukemia; HIV positive, ARC, AIDS (with Retrovir)

Phase III Phase II Phase I / II Phase I / II Phase II / III Phase II Phase II / III Phase II Submitted Phase III Phase I Phase I Phase II

AIDS (with Retrovir) cancer Kaposi’s sarcoma (with Retrovir) bone marrow suppression (chemo-, radiotherapy) bone marrow suppression, melanoma, immunotherapy wound healing cancer immunotherapy cancer immunotherapy (with Roferon-A) bone marrow failure, platelet deficiencies, autologous marrow transplant, chemotherapy adjuvant peripheral stem cell transplant immunodeficient disease, cancer therapy, vaccine adjuvant, immunization cancer immunomodulator platelet deficiencies thrombocytopenia thrombocytopenia neoplasia

Phase Phase Phase Phase

Phase Phase Phase Phase Phase Phase Phase Phase

cancer cancer

Phase II Phase II

AML, CML, inflammatory bowel disease, rheumatoid arthritis, sepsis, septic shock

Phase II

III Interferons interferon gamma-1b interferon alpha-n interferon beta interferon gamma

3

interferon consensus interferon gamma interferon alpha 2b

interferon beta interferon alpha-2a Interleukins PEG IL-2 aldesleukin (IL-2) human IL-1 alpha human IL-1 beta human IL-2 human IL-2 human IL-3

human IL-4 human IL-4 human IL-6 human IL-9 human IL-11 Human IL-12 Tumor Necrosis Factors TNF TNF-b Others Anakinra (IL-1 receptor antagonist)

I II / III I I / II

Phase III In clinical trials II / III II I / II II II I / II II I

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therapeutic activity, or as more generally is the case, to identify their therapeutic activity. These paradigms include: The short half life of proteins and the requirement for subcutaneous or continuous infusion delivery for maximal activity, The apparent bell shaped dose response curve, The need for chronic administration which is associated with the perceived mechanism of action by these agents, The optimal activity of these agents is as an adjuvant therapeutic administered in conjunction with chemotherapy and or radiotherapy and The maximum adjuvant immunotherapy is found in patients with minimal residual disease. This chapter discusses these paradigms using individual cytokines as examples with those agents that have been approved and / or are currently within clinical trials.

2.1. Interferon alpha ( IFN-a). The need for chronic administration for therapeutic activity The initial, nonrandomized, clinical studies with IFN-a suggested that it had therapeutic activity for malignant melanoma, osteosarcoma, and various lymphomas [14]. However, subsequent randomized trials demonstrated significant therapeutic activity only against less common tumor histiotypes including hairy cell leukemia, multiple myeloma leukemia and chronic myelogenous leukemia (CML) [14–16]. Subsequently, the list of responding indications was expanded to include renal cell carcinoma [17,18], AIDS and Kaposi’s sarcoma [19], genital warts [20], hepatitis [21], and most recently, malignant melanoma [22–24]. It also appears to have activity for low grade non-Hodgkin’s lymphoma [25], bladder papillomatosis [26], cutaneous T-cell lymphoma [27] and adult T cell leukemia / lymphoma [28]. Thus, it has taken almost three decades to translate the concept of IFN-a as an anti-viral to its routine utility in clinical oncology and infectious diseases and as an antiviral. Despite extensive clinical studies, the development of IFN-a is still in its early stages, and such basic parameters as optimal dose and therapeutic schedule remain to be determined [15,16]. The mechanism of activity is also controversial since IFN-a has been shown to have dose-

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dependent antitumor activities in vitro, yet be active at low doses in hairy cell leukemia [15,16]. Immunomodulation as the mechanism of therapeutic activity with IFN-a is perhaps best supported by its action against hairy cell leukemia. Treatment with IFN-a in this disease is associated with a 90–95% response rate; however, this is not fully achieved until the patients have been on the protocol for a year, and it appears that low doses of IFN-a are as active as higher doses [29]. In multiple myeloma the mechanism(s) by which IFN-alpha 2b prolongs remission is also unknown. In clinical studies of IFN-a, 29,59oligoadenylate synthetase (2,5-A synthetase) has been used as an objective indicator of in vivo activity. This enzyme has been assayed in cytosol preparations of peripheral blood mononuclear cells (MNCs) in one trial of 111 patients [30] who received IFN- alpha 2b and 54 patients who did not. In this study the level of 2,5-A synthetase activity was compared with the response to intensive therapy and with duration of maintenance therapy. Seventythree per cent of patients had measurable amounts of 2,5-A synthetase during the first 6 months of maintenance therapy. However, there was no difference between the magnitude of enzyme induction amongst patients who were in complete remission, partial response or who had no change in disease status following intensive therapy. Thus, the studies to date suggest that immune modulation as measured by the levels of 2,5-A synthetase in patients with multiple myeloma is not indicative of a clinical response to IFN-alpha 2b. Initial dose finding studies by Quesada et al. determined that a dose of 12 3 10 6 U / M 2 of r-IFN-a was not tolerable for patients with hairy cell leukemia [16]. Subsequently they found that a dose of 2 3 10 6 U / M 2 was both well tolerated and effective when administered three times per week. Smalley et al., [31] demonstrated that highly purified natural IFN-a at a dose of 2 3 10 6 U / M 2 when administered for 28 days was well tolerated in most patients with hairy cell leukemia but suggested that this dose might be myelosuppressive in some patients as well as neurotoxic or cardiotoxic. In their studies, a lower dose of 2 3 10 5 U / M 2 was also administered for 28 days and it was found that this dose was found to be better tolerated and also induced improvements in peripheral neutrophil and

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platelet counts as rapidly as the standard dose. In this trial substantial clinical improvement, primarily in terms of increased platelet and / or neutrophil counts, was observed within the first 4 to 8 weeks of treatment in patients receiving the low dose of IFNa. This resulted in an improved quality of life and a decrease in cardiac and neurologic toxicity, flu-like syndromes, myelosuppression, the need for platelet transfusions, and a reduced incidence of bacterial infections. They suggested that once such an improvement was obtained at 2 3 10 5 U / M 2 and patients become tolerant to the acute toxicity associated with IFN-a, the dose could then be increased to the standard 2 3 10 6 U / M 2 to obtain the greater anti-leukemia effect. It appears that significant improvements in thrombocytopenia and neutropenia can be rapidly induced rapidly in the majority of patients when low and minimally toxic doses of IFN-a are used. However, there is also a therapeutic dose response effect, whereby higher doses of IFN-a will induce a quantitatively greater antileukemic response than that observed with low doses of IFNa.

2.2. Interferon-gamma ( IFN-g)-non-linear dose response relationships for immune augmentation Preclinical studies have suggested that recombinant IFN-g (r-IFN-g) has significant therapeutic activity in animal models of experimental and spontaneous metastasis which occurs with a reproducible bell-shaped dose response curve [13]. Studies of the immune response in normal animals have revealed the same bell-shaped dose response curve for the augmentation of macrophage tumoricidal activity [13,32]. Further, optimal therapeutic activity is also observed at the same dose and protocol with significantly less therapeutic activity observed at lower and higher doses. Thus, a significant correlation between macrophage augmentation and therapeutic efficacy has been reported [32] in these preclinical models, suggesting that immunological augmentation provides an indirect mechanism for the therapeutic effect of r-IFN-g. In addition this observation supports the hypothesis that treatment with the MTD of r-IFN-g may not be optimal in an adjuvant setting. The preclinical hypothesis of a bell-shaped dose response curve for r-IFN-g has been confirmed in

numerous clinical studies of the immunoregulatory effects of r-IFN-g which defined an optimal immunomodulatory dose (OID) [33,34]. In general, the OID for r-IFN-g has been found to be between 0.1 and 0.3 mg / M 2 following intravenous (i.v.) or intramuscular injection. In contrast, the MTD for r-IFN-g may range from 3 to 10 mg / M 2 depending upon the source of the r-IFN-g and / or the clinical center. The identification of an OID for r-IFN-g in patients with minimal tumor burden has resulted in the development of clinical trials to test the hypothesis that the immunological enhancement induced by r-IFN-g will result in prolongation of the disease-free period and overall survival of patients in an adjuvant setting [34]. Jett et. al. [35] used the OID for IFN-g (0.2 mg / M 2 ) subcutaneously (s.c.) daily for 6 months as an adjuvant therapy for patients with clinical complete response or an initial response to combination chemoradiotherapy for small cell lung cancer. In this trial 100 patients were randomized to IFN-g or observation with the finding that IFN-g had no effect on disease free or overall survival. However, r-IFN-g was active in this study as macrophage activation was observed [36]. In contrast, it should be noted that r-IFN-g was found, on an empirical basis, to have therapeutic activity in chronic granulomatous disease (CGD) [37] and it was for this indication that the FDA approved r-IFN-g. The studies in CGD suggested that the mechanism of therapeutic activity for r-IFNg is associated with enhanced phagocytic oxidase activity and increased superoxide production by neutrophils. However, more recent data suggests that the majority of CGD patients obtain clinical benefit by prolonging r-IFN-g therapy and the mechanism of action may not be due to enhanced neutrophils oxidase activity but rather to the correction of a respiratory burst deficiency in a subset of monocytes [38]. In addition to its licensing for CGD, IFN-g has also been approved for the treatment of rheumatoid arthritis in Germany.

2.3. Interleukin-2 ( IL-2), The importance of pharmacokinetics in the activity of IL-2 Over the past decade recombinant IL-2 (r-IL-2) has been used extensively in animal models and clinical trials. It was initially developed based on a chemotherapeutic paradigm and administered to

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humans at the MTD. However, three major deficiencies were identified: low response rate, severe dose-dependant tonicity and high cost of therapeutic delivery. Despite the initial promising results in patients with advanced cancer, no significant improvements or dose related efficacy have been identified. Thus, high dose r-IL-2 alone has demonstrated some activity in metastatic renal cell cancer and melanoma with objective response rates observed in 15–20% of patients. IL-2 is a T-cell proliferative cytokine, as well as a potent natural killer (NK) and lymphokine activated killer (LAK) augmenting agent, and can activate T cells. In contrast to the initial trials, the current trials are focused on the design of well-tolerated schedules with low doses of r-IL-2. In some studies cells are cultured with IL-2 in vitro for 72 h or longer and infused back into the patient. As such, IL-2 is important to all facets of T cell and NK cell augmentation and proliferation. R-IL-2 has been approved for use as a single agent in the treatment of renal cell carcinoma. In addition, it is also administered in conjunction with LAK or T-cell infiltrating lymphocytes (TILs) in adoptive cellular therapy protocols. TIL cells are T-cells obtained from tumors which are expanded in vitro with lower levels of IL-2 then used with LAK cells and in the presence of tumor antigen. The overall goal is to expand a population of tumor specific cytotoxic T cells. However, it has been questioned whether the adoptive transfer of LAK cells is necessary or adds to the clinical efficacy of r-IL-2. Indeed, there has been little indication of an improved therapeutic effect of r-IL-2 plus LAK cells versus IL-2 alone [39,40]. When the clinical trials with IL-2 are rigorously examined neither strategy has impressive (as opposed to significant) therapeutic activity [39,40]. The overall response rate with r-IL-2 is 7–14% and is associated with considerable toxicity [41]; however, it should be remarked that these responses are durable. In one of the first clinical studies [42], partial responses were observed in 4 out of 31 patients. Interestingly, these partial responders did not correspond to the patients with increased LAK or NK cell activity. The anti-tumor effect by of both TIL and LAK cells could be due to either a direct effect or secondary to the generation of other cytokine mediators. The latter mechanism is suggested by the observation that r-IL-2-stimulated

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lymphocytes produce IFN-g and tumor necrosis factor (TNF) as well as other cytokines and that the therapeutic activity of r-IL-2 may be synergistic with these cytokines [42]. Many of the i.v. r-IL-2 infusion clinical trials with or without LAK cells in metastatic renal cell carcinoma have used an MTD of r-IL-2. A recent study by the laboratory of Fefer et al., [43] compared maintenance r-IL-2 therapy at the MTD of 6 3 10 6 U / M 2 / day to 2 3 10 6 U / M 2 / day. They found that it was possible to maintain the patients for a median of 4 days at 6 3 10 6 U / M 2 / day but in the presence of severe hypotension and capillary leak syndrome. In the lower dose protocol none of the patients experienced severe hypertension or capillary leak syndrome and the median duration of maintenance r-IL-2 therapy was 9 days. In the lower dose protocol there was a total response rate of 41%. which contrasted to the higher dose protocol (with a shorter duration of administration) which had a 22% response rate. These investigators suggest that there may be an improved therapeutic activity associated with a longer maintenance protocol at lower doses. Lauria et al. [44] undertook studies of low dose, s.c. administration of r-IL-2 in 11 non-Hodgkin’s lymphoma patients. In this study two patients with residual disease after autologous bone marrow transplantation obtained complete responses following 7 and 10 months of therapy. Together these studies suggest the need not only for infusion or s.c. administration of r-IL-2 for therapeutic efficacy in cancer and AIDS but also the need for chronic administration. The latter observation echoes the studies with IFN-a in hairy cell leukemia. A similar need for pharmacodynamic considerations has been found with granulocyte colony-stimulating factor (g-CSF). Sugiura et al. [45] compared s.c. to intravenous (iv) delivery of g-CSF and found an increase in neutrophil counts in the blood after s.c. compared to i.v. administration. This occurred despite a lower area under the curve plasma concentration following s.c. compared to i.v. administration. Thus, a rational dosage regimen of g-CSF suggests that a slow constant infusion maybe more useful that a rapid infusion to maintain receptor occupancy. A recent study that examined the transcriptional regulation of cytokine mRNA levels in the PBL peripheral blood leukocytes (PBL) of cancer patients

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receiving r-IL-2 suggested that: (1) Doses of r-IL-2 as low as 3 3 10 4 U / day could augment T cell function, and (2) higher doses of r-IL-2 $ 1 3 10 5 U / day increase not only T cell but also macrophage function [46]. The latter was measured as TNF levels and the upregulation of TNF at the higher dose of r-IL-2 combined with the T cell production of IFN-g which occurs at the lower dose of r-IL-2 may be responsible for the toxicity of r-IL-2 [47]. Recently, renal cell cancer patients were randomized to receive a high-dose regimen (FDA approved dose), or one using one-tenth of the dose (72 000 IU / kg / 8 h) administered by the same schedule (days 1–5 and 15–19, which was repeated every 4–6 weeks). An interim report of this ongoing trial [48] reported similar response rates in the two groups – 7% complete responses (CR) and 8% partial responses (PR) in the low-dose group, vs 3% CR and 17% PR in the high-dose group. However, the toxicity of the low-dose regimen was substantially less than that of the high-dose regimen. Recently, chronic r-IL-2 administration at low doses ( | 200,000 IU / M 2 / day) has been found to increase CD4 1 cell number and the CD4:CD8 ratio in AIDS patients [48,49]. The goal of one of these studies [49] was to give asymptomatic HIV 1 individuals r-IL-2 without promoting viral replication, using an approach patterned after that described by Ritz and coworkers [50], who reported that low doses of r-IL-2 could be given to cancer patients for periods up to 3 months with minimal toxicity. The results indicate that extremely low r-IL-2 doses are nontoxic and effective in stimulating immune reactivity.

3. Combination Chemotherapy And Immunotherapy Because the cytokines and growth factors have unique mechanisms of action, they are ideal candidates for combination with therapy with chemotherapeutic agents. However, increased knowledge and consideration of the potential interactions between these two classes of drugs is necessary for clinical use. The use of high dose chemotherapy (HDT) and stem cell rescue provides the ultimate in cytoreductive therapy and post-transplant immuno-

therapy. As shown later in this chapter, stem cell transplantation provides one of the few statistically supported demonstrations of therapeutic efficacy by T-cell augmentation (comparison of allogeneic to autologous transplantation). Thus, strategies to upregulate T-cell function post-autologous stem cell transplantation are one focus for cytokine therapy post-transplantation. This is important as the return of immunologic function in transplanted patients is slow and is accompanied by depressed numbers of CD-4 1 T cells, a low CD-4 / CD-8 T cell ratio, and depressed cellular responses [51]. The role of T-cells in controlling (or ablating) neoplastic disease has been demonstrated in allotransplanted patients and described as a graft versus tumor (GVT) reaction. A significantly higher risk of relapse is associated with the use of in vitro T-cell depleted bone marrow (BM) cells or the clinical use of cyclosporine A (CSA) to prevent graft versus host disease (GVHD) [51–54]. It has been postulated that T-lymphocyte depletion of the graft increases leukemia relapse by removing the cells responsible for the GVT effect [52–55]. Similar relapse rates are observed in recipients of non-T-cell depleted transplants receiving cyclosporine A (CSA), suggesting that it inhibits the same GVT cells that are removed by T-cell depletion. Clearly, GVHD can also have unfavorable effects on transplant-related mortality. In first remission, the decreased relapse rates with acute and / or chronic GVHD are offset by the increased risk of death from other causes. Consequently, patients with GVHD have a lower risk of treatment failure, but an increased risk of morbidity due to GVHD. Thus, one approach to improving survival of cancer patients has been to use immunotherapy following HDT and stem cell transplantation to induce an autologous GVT response. Based on this strategy, studies using r-IL-2 alone following bone marrow transplantation (BMT) to induce a postinfusion lymphocytosis have shown an increase in NK cell phenotype and function [56–59]. In one such study [58] with 18 evaluable patients, three responses were observed. In another study, r-IL-2 was infused following both autologonus and allogeneic transplantation for a median of 85 days at a dose of 2 3 10 5 units / M 2 / day [59]. Toxicity was minimal and the treatment could be undertaken in the outpati-

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ent setting via a Hickman catheter. In this study no patient developed any signs of GVHD, hypotension, or pulmonary capillary leak syndrome. The treatment did not affect the absolute neutrophil count or hemoglobin level, although eosinophilia was observed. Despite the administration of this low dose of r-IL-2, significant immunological changes were noted with a 5–40-fold increase in NK cell numbers. In a similar study, it was shown that following continuous infusion of r-IL-2 in patients receiving autologous bone marrow transplant (AuBMT), the CD-3 1 and CD-16 1 cells secrete increased levels of IFN-g and TNF, following in vitro culture, and that there was a significant increase in serum levels of IFN-g but not TNF following the administration of r-IL-2 [59]. Massumoto et. al. [60] treated NHL and AML patients with IL-2 at 9 3 10 6 IU of r-IL-2 for 5 days beginning a median of 35 days following AuBMT, the patients were apharesised and the ex vivo expanded LAK cells infused. The patients also received 10 days of maintenance r-IL-2 following LAK cell infusion. In this study they observed skin GVHD which they confirmed by skin biopsy in 85% of patients. However, the association of this autologous GVHD with GVL remains to be addressed in a phase III trial. Similar post transplantation strategies with r-IFNa have been undertaken with the observation of a reduced risk of relapse and an increase in myelosuppression [61,62]. The Seattle Group [62] reported an early study of the prophylactic use of leukocyte interferon following allogeneic (Ao) BMT, and that adjuvant treatment with IFN-a had no effect on the probability or severity of cytomegalovirus (CMV) infections or GVHD in acute lymphocytic leukemia (ALL) patients who were in remission at the time of transplantation. However, in this large study, there was a significant reduction in the probability of relapse in the r-IFN-a recipients (P 5 0.004) as compared to transplant patients who did not receive r-IFN-a, although survival rates did not differ between the r-IFN-a recipients and control patients. It was suggested that the administration of r-IFN-a following transplantation reduced the risks of relapse but did not effect CMV infection, perhaps because r-IFN-a was not initiated until a median of 18 days following transplantation and was not administered chronically. Recently, Ratanatharathorn et al. [63]

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extended the approach of the induction of a GVT reaction to a combination study that utilized both CSA induced GVHD and IFN-a augmentation of this effect in autologous transplant patients. Twentytwo patients were enrolled of which 17 were considered evaluable. Thirteen of the patients who received r-Hu-IFN-a2a developed GVHD regardless of whether they received CSA, whereas only two of the four patients who received CSA alone developed detectable GVHD. Patients receiving 1 3 10 6 units / day of r-Hu-IFN-a2a concomitant with CSA showed a trend towards increased severity of clinical GVHD as compared to patients receiving CSA alone (P 5 0.06). They concluded that IFN-a administration can be safely started on day 0 of AuBMT and can both induce AuGVHD as a single agent with the potential to improve therapy. In similar studies Kennedy et al. [64] treated women with advanced breast cancer with combined therapy of CSA for 28 days and 0.025 mg / M 2 of s.c. r-IFN-g every other day on days 7–28 after HDT and AuBMT. They observed that autologous GVHD developed in 56% of the patients, an incidence comparable to that previously observed with CSA alone. The severity of GVHD was greater with CSA plus r-IFN-g than with CSA alone, as 16 patients required corticosteroid therapy for dermatologic GVHD. Note that strategies to induce an autologous GVT, while conceptually interesting, have not matured to allow a discussion of efficiency.

4. Conclusion In the last 20 years, nonspecific immunostimulation has progressed from the trials with crude microbial mixtures and extracts to more sophisticated immunopharmacologically active compounds (only a few of which are discussed here) having diverse actions on the immune system. A body of pharmacodynamic knowledge has evolved which shows substantial divergence from conventional pharmacology, particularly in terms of the relationship of dosing schedules to immunopharmacodynamics. This knowledge is important in evaluating agents and predicting appropriate use. While much remains to be learned and new compounds to be cloned, the future of immunotherapy seems bright. A number of the

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cytokines have been approved, as well as numerous supplemental indications [65], in the US, Europe and Asia. However, it is apparent that the combinations of growth factors, cytokines and BRMs will have optimal activity when used as adjuvants with more traditional therapeutic modalities.

[14]

[15]

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