Robert H. Wiltrout

Phase 0 Clinical Trial of the Poly (ADP-Ribose) Polymerase Inhibitor ABT-888 in Patients With Advanced Malignancies

PURPOSE We conducted the first phase 0 clinical trial in oncology of a therapeutic agent under the Exploratory Investigational New Drug Guidance of the US Food and Drug Administration. It was a first-in-human study of the poly (ADP-ribose) polymerase (PARP) inhibitor ABT-888 in patients with advanced malignancies. PATIENTS AND METHODS ABT-888 was administered as a single oral dose of 10, 25, or 50 mg to determine the dose range and time course over which ABT-888 inhibits PARP activity in tumor samples and peripheral blood mononuclear cells, and to evaluate ABT-888 pharmacokinetics. Blood samples and tumor biopsies were obtained pre- and postdrug administration for evaluation of PARP activity and pharmacokinetics. A novel statistical approach was developed and utilized to study pharmacodynamic modulation as the primary end point for trials of limited sample size. RESULTS Thirteen patients with advanced malignancies received the study drug; nine patients underwent paired tumor biopsies. ABT-888 demonstrated good oral bioavailability and was well tolerated. Statistically significant inhibition of poly (ADP-ribose) levels was observed in tumor biopsies and peripheral blood mononuclear cells at the 25-mg and 50-mg dose levels. CONCLUSION Within 5 months of study activation, we obtained pivotal biochemical and pharmacokinetic data that have guided the design of subsequent phase I trials of ABT-888 in combination with DNA-damaging agents. In addition to accelerating the development of ABT-888, the rapid conclusion of this trial demonstrates the feasibility of conducting proof-of-principle phase 0 trials as part of an alternative paradigm for early drug development in oncology.

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.

The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma.

The 5′ flanking region of the C3(1) component of the rat prostate steroid binding protein (PSBP) has been used to successfully target the expression of the SV40 large T-antigen (Tag) to the epithelium of both the mammary and prostate glands resulting in models of mammary and prostate cancers which histologically resemble the human diseases. Atypia of the mammary ductal epithelium develops at about 8 weeks of age, progressing to mammary intraepithelial neoplasia (resembling human ductal carcinoma in situ [DCIS]) at about 12 weeks of age with the development of invasive carcinomas at about 16 weeks of age in 100% of female mice. The carcinomas share features to what has been classified in human breast cancer as infiltrating ductal carcinomas. All FVB/N female mice carrying the transgene develop mammary cancer with about a 15% incidence of lung metastases. Approximately 10% of older male mice develop anaplastic mammary carcinomas. Unlike many other transgenic models in which hormones and pregnancy are used to induce a mammary phenotype, C3(1)/Tag mice develop mammary tumors in the mammary epithelium of virgin animals without hormone supplementation or pregnancy. Although mammary tumor development appears hormone-responsive at early stages, invasive carcinomas are hormone-independent, which corresponds to the loss of estrogen receptor-α expression during tumor progression. Molecular and biologic factors related to mammary tumor progression can be studied in this model since lesions evolve over a predictable time course. Genomic alterations have been identified during tumor progression, including an amplification of the distal portion of chromosome 6 containing ki-ras and loss of heterozygosity (LOH) in other chromosomal regions. We have demonstrated that stage specific alterations in the expression of genes which are critical regulators of the cell cycle and apoptosis are functionally important in vivo. C3(1)/Tag mice appear useful for testing particular therapies since growth of the mammary tumors can be reduced using chemopreventive agents, cytokines, and an anti-angiogenesis agent.

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells.

IL‐18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL‐18 can induce IL‐13 production in both NK cells and T cells in synergy with IL‐2 but not IL‐12, suggesting IL‐18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL‐18 can act as a cofactor to induce IL‐4, IL‐10 and IL‐13 as well as IFN‐γ production in T cells in the presence of anti‐CD3 monoclonal antibodies (mAb). IL‐18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL‐18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL‐4 knockout mice. Furthermore, the administration of IL‐18 plus IL‐2 induced approximately 70‐fold and 10‐fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL‐18 plus IL‐2 was eliminated by the pretreatment of mice with anti‐CD4 or anti‐CD154, but not anti‐CD8 or anti‐NK1.1 mAb. These results suggest that IL‐18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell‐dependent, IL‐4‐independent IgE production.

Immunotherapy of Cancer by IL-12-based Cytokine Combinations

Cancer is a multi-faceted disease comprising complex interactions between neoplastic and normal cells. Over the past decade, there has been considerable progress in defining the molecular, cellular and environmental contributions to the pathophysiology of tumor development. Despite these advances, the conventional treatment of patients still generally involves surgery, radiotherapy and/or chemotherapy, and the clinical outcome for many of these efforts remains unsatisfactory. Recent studies have highlighted the feasibility of using immunotherapeutic approaches that seek to enhance host immune responses to developing tumors. These strategies include immunomodulatory cytokines, with TNF-α, type I or type II IFNs, IL-2, IL-12, IL-15 and IL-18 being among the most potent inducers of anti-tumor activity in a variety of preclinical studies. More recently, some exciting new cytokines have been characterized, such as IL-21, IL-23, IL-27 and their immunomodulatory and antitumor effects in vitro and in vivo suggest that they may have considerable promise for future immunotherapy protocols. The promise of cytokine therapy does indeed derive from the identification of these novel cytokines but even more fundamentally, the field is greatly benefiting from the ever-expanding amount of preclinical data that convincingly demonstrate synergistic and/or novel biologic effects, which may be achieved through the use of several combinations of cytokines with complementary immune-stimulating capabilities. One cytokine in particular, IL-12, holds considerable promise by virtue of the fact that it plays a central role in regulating both innate and adaptive immune responses, can by itself induce potent anticancer effects, and synergizes with several other cytokines for increased immunoregulatory and antitumor activities. This review discusses the antitumor activity of IL-12, with a special emphasis on its ability to synergize with other cytokines for enhancement of immune effector cell populations and regulation of host–tumor cell interactions and the overall tumor microenvironment.

IFN-γ and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

IFN-γ mediates CD4 + T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy

Protective cell-mediated immune responses in cancer are critically dependent on T-helper type 1 (TH1) cytokines such as interferon-γ (IFN-γ). We have previously shown that the combination of CD40 stimulation and interleukin-2 (IL-2) leads to synergistic antitumor responses in several models of advanced metastatic disease. We now report that after this treatment and other immunotherapy regimens, the CD4+ T-cell population, in contrast to CD8+ T cells, did not significantly increase but rather exhibited a substantial level of apoptosis that was dependent on IFN-γ. Mice immunized with tumor cells and treated with an immunotherapy regimen that was initially protective were later unable to mount effective memory responses compared with immunized mice not receiving immunotherapy. Immunotherapy given to tumor-bearing Ifngr−/− mice resulted in restoration of secondary responses. Thus, although immunotherapeutic regimens inducing strong IFN-γ responses can lead to successful early antitumor efficacy, they may also impair the development of durable antitumor responses.

Co‐expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood

Previously, we found that co‐expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3+ cells present in CD25high, CD25low and even CD25– subsets of CD4+ cells expressed high levels of TNFR2. Consequently, TNFR2‐expressing CD4+CD25+ Treg included all of the FOXP3+ cells present in the CD4+CD25high subset as well as a substantial proportion of the FOXP3+ cells present in the CD4+CD25low subset. Flow cytometric analysis of PB identified five‐fold more Treg, determined by FOXP3 expression, in the CD4+CD25+TNFR2+ subset than in the CD4+CD25high subset. In addition, similar levels of FOXP3+ cells were identified in both the CD4+CD25+TNFR2+ and CD4+CD25+CD127low/− subsets. Furthermore, the CD4+CD25+TNFR2+ subset expressed high levels of CTLA‐4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4+CD25+TNFR2+ cells were anergic and markedly inhibited the proliferation and cytokine production of co‐cultured T‐responder cells. In contrast, CD4+CD25+TNFR2– and CD4+CD25–TNFR2+ T cells did not show inhibitory activity. As some non‐Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.

Tumor-Specific CTL Kill Murine Renal Cancer Cells Using Both Perforin and Fas Ligand-Mediated Lysis In Vitro, But Cause Tumor Regression In Vivo in the Absence of Perforin

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8+ T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2Kd-restricted lysis and production of IFN-γ, TNF-α, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-γ and TNF-α increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp−/−) mice. Although these pfp−/− CTL showed reduced cytotoxic activity against Renca, their IFN-γ production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp−/− CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.

Dissociation of NKT Stimulation, Cytokine Induction, and NK Activation In Vivo by the Use of Distinct TCR-Binding Ceramides

NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is α-galactosylceramide (αGalCer). However, αGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, β-galactosylceramide (βGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with βGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with αGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, βGalCer (C12), in contrast to αGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to αGalCer, βGalCer (C12) is a poor inducer of IFN-γ, TNF-α, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, αGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while βGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show βGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.