Dinaciclib is a novel cyclin-dependent kinase inhibitor with significant clinical activity in relapsed and refractory chronic lymphocytic leukemia.

Dinaciclib (SCH727965) is a selective CDKi chosen for clinical development based upon a favorable therapeutic index in cancer xenograft models. We performed a phase I dose escalation study of dinaciclib in relapsed and refractory chronic lymphocytic leukemia (CLL) patients with intact organ function and WBC<200 × 10(9) /l. Five separate dose levels (5 mg/m(2), 7 mg/m(2), 10 mg/m(2), 14 mg/m(2) and 17 mg/m(2)) were explored dosing on a weekly schedule × 3 with 1 week off (4-week cycles) using a standard 3+3 design with expansion cohorts to optimize safety. Fifty-two patients were enrolled with relapsed and refractory CLL. Escalation through cohorts occurred with two dose-limiting toxicity (DLTs) at the 17 mg/m(2) dose (tumor lysis syndrome (TLS) and pneumonia). The phase II expansion occurred at 14 mg/m(2) with 16 patients receiving this dose with one DLT (TLS). Additional stepped up dosing to the maximum tolerated dose was examined in 19 patients at this dose. Adverse events included cytopenias, transient laboratory abnormalities and TLS. Responses occurred in 28 (54%) of patients independent of del(17)(p13.1) with a median progression-free survival of 481 days. Dinaciclib is clinically active in relapsed CLL including those patients with high risk del(17)(p13.1) disease and warrants future study.

Update on the biology of chronic lymphocytic leukemia.

An understanding of the molecular biology of B-cell chronic lymphocytic leukemia (B-CLL) has led to the appreciation that several different B-cell diseases are represented under this name. Variability in the bcl-2 family of proteins, p53 mutation, or the presence of various chromosomal abnormalities corresponds to variability of the clinical course of disease and response to therapy. Differential expression of cell surface adhesion molecules by B-CLL cells have also been shown to influence clinical outcome, as have the expression of immune regulatory molecules (eg, CD80, CD40R, CD27 and CD79b). Recent work studying immunoglobulin-heavy chain gene rearrangement postulates at least two subsets of B-CLL originating from different stages of B-cell development and following different clinical courses. The knowledge that B-CLL is the final consequence of many different molecular perturbations may allow the development of chemotherapies, immunotherapies, and gene therapies that target the specific molecular defect in a given case of B-CLL.

Immunotherapy of bladder cancer with cytokine gene-modified tumor vaccines.

This study explored the use of cytokine gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology and responds to treatment in a manner similar to that of its human counterpart. In a previous study we have shown that interleukin 2 (IL-2)-secreting, irradiated, MBT-2 cell preparations were capable of curing animals from orthotopically established tumors and engendered protective immunological memory in the cured animals. In this study we have compared the effectiveness of several cytokines and found that while IL-1 alpha, IL-1 beta, and gamma-interferon were only weakly effective in the therapeutic vaccination protocol, granulocyte-macrophage colony-stimulating factor was almost as effective as but not superior to IL-2, as reported previously for another tumor model system. Induction of cytotoxic T-lymphocyte correlated only poorly with the therapeutic benefit of the cytokine gene-modified tumor cell preparations, questioning its prognostic value for the development of improved genetically modified tumor vaccines.

Immunotherapy of prostate cancer in the Dunning rat model: use of cytokine gene modified tumor vaccines.

Adenocarcinoma of the prostate is the most common cancer in men. The majority of cancers are discovered once they have already metastasized, and there is no effective therapy for prostatic cancer at this stage. The use of cytokine-secreting tumor cell preparations as therapeutic vaccines for the treatment of advanced prostate cancer was investigated in the Dunning rat R3327-MatLyLu prostatic tumor model. IL-2 secreting, irradiated, tumor cell preparations were capable of curing animals with s.c. established tumors, and induced immunological memory that protected animals from subsequent tumor challenge. Immunotherapy was less effective when tumors were induced orthotopically, but nevertheless led to improved outcome, significantly delaying, and occasionally preventing, recurrence of tumors after resection of the cancerous prostate. Granulocyte-macrophage colony stimulating factor secreting tumor cell preparations were less effective, and interferon-gamma secreting cells had only a marginal effect. Induction of a potent immune response in tumor bearing animals against the nonimmunogenic MatLyLu tumor supports the view that active immunotherapy warrants further investigation as a potential therapeutic approach to prostate cancer.

Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs.

Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte-macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL-2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

The role of IL-2 secreted from genetically modified tumor cells in the establishment of antitumor immunity.

Although the importance of CD4+ T cells for the induction of an effective CD8+ cytolytic T cell response is well documented, the mechanism by which MHC class II-negative tumor cells recruit CD4+ T help is not well understood. We have previously shown that IL-2 or IFN-gamma gene-modified CMS-5 tumor cells do not grow in syngeneic mice; however, mice which rejected the cytokine-secreting tumor cells develop a protective immune response against a challenge with parental tumor cells. Here we show that rejection of IL-2-secreting CMS-5 cells is not mediated by T cells. However, establishment of a protective immune response against CMS-5 tumor cells requires the presence of both CD4+ and CD8+ T cell subsets during the period of immunization with IL-2-secreting CMS-5 cells as well as during the effector phase. Extensive histologic analysis has failed to detect the presence of T cells at the site of immunization with either IL-2- or IFN-gamma-secreting CMS-5 cells. The main infiltrate at the site of inoculation with IL-2-secreting CMS-5 cells consisted of NK cells that appeared to play a role in their rejection. The predominant infiltrate at the site of inoculation with IFN-gamma-secreting CMS-5 cells consisted of macrophages. These observations argue against a direct role for the intact tumor cell in presenting either T helper or CTL epitopes to the immune system, and support the view that specialized APC are responsible for the in vivo priming of a T cell response against MHC class I-restricted Ag.

Regression of bladder tumors in mice treated with interleukin 2 gene-modified tumor cells.

This study explored the use of interleukin 2 (IL-2) and interferon gamma (IFN-gamma) gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner similar to its human counterpart. Using retroviral vectors, the human IL-2 and mouse IFN-gamma genes were introduced and expressed in MBT-2 cells. The tumor-forming capacity of the cytokine gene-modified MBT-2 cells was significantly impaired, since no tumors formed in mice injected intradermally with either IL-2- or IFN-gamma-secreting cells, using cell doses far exceeding the minimal tumorigenic dose of parental MBT-2 cells. Furthermore, mice that rejected the IL-2- or IFN-gamma-secreting tumor cells became highly resistant to a subsequent challenge with parental MBT-2 cells, but not to 38C13 cells, a B cell lymphoma of the same genetic background. To approximate the conditions as closely as possible to the conditions prevailing in the cancer patient, inactivated cytokine-secreting cells were used to treat animals bearing tumors established by orthotopic implantation of MBT-2 cells into the bladder wall of the animal. Treatment of mice carrying a significant tumor burden with IL-2-secreting MBT-2 cells had a significant inhibitory effect on tumor progression with extended survival. Moreover, in 60% of the mice the tumor regressed completely and the animals remained alive and free of detectable tumor for the duration of the observation period. Treatment of tumor-bearing animals with IL-2-secreting MBT-2 cells was superior to the use of cisplatin, a chemotherapeutic agent used in the treatment of bladder cancer. The therapeutic effect of IFN-gamma-secreting cells was minimal and treatment with unmodified MBT-2 cells had no effect on tumor growth or survival, showing that the parental MBT-2 cells were nonimmunogenic in this experimental setting. Most importantly, mice that exhibited complete tumor regression after treatment with IL-2-secreting MBT-2 cells became resistant to a subsequent challenge with a highly tumorigenic dose of parental MBT-2 cells, indicating that long-term immunological memory was established in the "cured" mice.

Antimetastatic vaccination of tumor-bearing mice with two types of IFN-gamma gene-inserted tumor cells.

IFN-gamma genes were introduced through retroviral vectors into the high metastatic, low H-2Kb class I expressor, and poorly immunogenic 3LL-D122 clone. Two types of IFN-gamma infectants were isolated: IFN-gamma high secretors (128 to 256 IU/ml) and IFN-gamma non- or very low secretors (< 2 IU/ml). Both manifested high expression of MHC class I Ag. IFN-gamma secretors showed significant decrease in tumorigenicity and metastatic growth, whereas IFN-gamma nonsecretors retain tumorigenicity and were more metastatic than parental D122 cells. However, both groups, when inoculated in an irradiated form, induced similar high levels of CTL and protected mice to the same degree against a subsequent graft of parental cells. This indicates that enhanced expression of MHC class I and related genes caused by IFN-gamma is the major participant in CTL induction. Immunization of mice carrying an established tumor of parental D122 cells with IFN-gamma infectants is capable of almost completely preventing lung metastasis. Immunotherapy of tumor-bearing hosts is more effective when IFN-gamma secreting cells are used as immunogens, indicating that mechanisms, in addition to CTL, are stimulated by secreted IFN-gamma. Moreover, immunization with IFN-gamma high secretors of postoperative mice, carrying established micrometastases, almost completely cured these mice. Support for the participation of non-T cell effectors in the response to IFN-gamma secretors derives from the reduced tumorigenicity of these cells in CD1 nude mice. These data provide a rationale for the use of IFN-gamma gene-transferred tumor cells as a modality for cancer therapy.

Anti-metastatic vaccination of tumor-bearing mice with IL-2-gene-inserted tumor cells.

IL-2 gene was introduced through retroviral vectors into the highly malignant and poorly immunogenic 3LL-D122 clone. Both high and low D122-IL-2 secretors showed elimination of tumorigenicity in syngeneic immune-competent mice; however, in nude mice only the high IL-2 secretor showed reduced tumorigenicity as compared with parental D122 cells. Also, following intravenous inoculation, only the high IL-2 secretor showed reduced generation of metastases, whereas the low IL-2 secretors were as highly metastatic as parental cells. These results seem to indicate that low levels of IL-2 secreted by tumor cells are sufficient to activate T cells, while higher levels are needed in order to activate non-T-cell effectors. D122-IL-2 secretors induced high levels of anti-tumor CTL, while their sensitivity to the lytic activity of these CTL was similar to the sensitivity of D122 cells, thus indicating that the elevation of immunogenicity of IL-2 secretors was essentially due to the secreted IL-2. In accordance with CTL induction, pre-immunization with IL-2 secretors protected mice against subsequent challenge of parental cells. Moreover, immunization in an "immunotherapy protocol" i.e., vaccination of mice carrying an established tumor of parental D122 cells with inactivated D122-IL2 infectants, almost completely eliminated the generation of lung metastases by D122 cells, thus providing a rationale for the use of IL-2 gene transferred tumor cells as a modality for treatment of metastasis.

Retroviral vector-mediated gamma-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity.

Retroviral vectors were used to introduce the gamma-interferon (IFN-gamma) gene into CMS-5 cells, a weakly immunogenic tumor of BALB/c origin. After selection in G418-containing medium, colonies were isolated, cloned, and expanded to cell lines. IFN-gamma secretion was assessed using a bioassay and enzyme-linked immunosorbent assay, and high (25 units/ml) and low (5 units/ml) IFN-gamma producers were isolated. Tumor growth was followed after intradermal injection, and spleen cells were isolated at different time points. IFN-gamma secretion by tumor cells abrogated their tumorigenicity and induced a persistent and specific antitumor immunity. In contrast to the normally observed cellular immunosuppression in unmodified CMS-5 tumor-bearing mice, IFN-gamma-producing tumors induced a long lasting state of T-cell immunity, as judged by rejection of a CMS-5 tumor challenge and persistence of specific cytotoxic activity in the spleen cell population. High levels of tumor-specific cytotoxic activity could also be detected if IFN-gamma-secreting tumor cells, but not unmodified CMS-5 cells, were used as targets at a time point when immunosuppression was usually seen. These studies highlight the potential advantages of localized IFN-gamma secretion to induce potent antitumor immune responses.

Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.

To study the effects of localized secretion of cytokines on tumor progression, the gene for human interleukin 2 (IL-2) was introduced via retroviral vectors into CMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of IL-2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protective immune response against a challenge with a tumorigenic dose of parental CMS-5 cells. Co-injection of IL-2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation even when highly tumorigenic doses of CMS-5 cells were used. Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumor-bearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d). High levels of tumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if IL-2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets. These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.