Marie-Christine Labarthe

The anti-cancer agents lenalidomide and pomalidomide inhibit the proliferation and function of T regulatory cells

Lenalidomide (Revlimid®; CC-5013) and pomalidomide (CC-4047) are IMiDs® proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-α is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependant adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-β or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

Immunotherapeutic potential of whole tumour cells

Abstract. Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.

Thalidomide-derived immunomodulatory drugs as therapeutic agents.

Thalidomide, a drug originally used to treat morning sickness, was removed from the market place in the early 1960s after it was found to cause serious congenital birth defects. However, thalidomide has recently been investigated in a new light following its activity in a number of chronic diseases. Moreover, like thalidomide itself, its second-generation immunomodulatory drug (IMiD®) analogues have been shown to act as powerful anticancer agents and are clearly active in the treatment of patients with relapsed multiple myeloma. These new drugs, in particular the second-generation IMiDs, lenalidomide (CC-5013, REVLIMID™; Celgene Corp., NJ, USA) and CC-4047 (ACTIMID™; Celgene Corp.), offer improvements over thalidomide (a first-generation IMiD) in terms of efficacy and safety in human studies. The key to the therapeutic potential of IMiDs lies in the fact that the drugs have multiple mechanisms of action, which may produce both anti-inflammatory and antitumour effects. These effects are probably contextual, depending both on the cell type and the stimulus involved. Mechanisms associated with IMiD activity include TNF-α-inhibitory, T cell costimulatory and antiangiogenic activities. Studies of the mechanisms of action of these drugs are ongoing and will facilitate the continued development of this class of compound in a number of diseases.

Enhanced cross-priming of naive CD8+ T cells by dendritic cells treated by the IMiDs® immunomodulatory compounds lenalidomide and pomalidomide

The IMiDs® immunomodulatory compounds lenalidomide and pomalidomide are agents with anti‐inflammatory, immunomodulatory and anti‐cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti‐tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow‐derived DCs treated with 5 or 10 μm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD® immunomodulatory compounds increased expression of Class I (H2‐Kb), CD86, and pomalidomide also increased Class II (I‐Ab) expression in bone marrow‐derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 μm pomalidomide or lenalidomide compared with non‐treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin‐specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8+ T‐cell cross‐priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4+ T‐cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.

Regulation of the stem cell marker CD133 is independent of promoter hypermethylation in human epithelial differentiation and cancer

BackgroundEpigenetic control is essential for maintenance of tissue hierarchy and correct differentiation. In cancer, this hierarchical structure is altered and epigenetic control deregulated, but the relationship between these two phenomena is still unclear. CD133 is a marker for adult stem cells in various tissues and tumour types. Stem cell specificity is maintained by tight regulation of CD133 expression at both transcriptional and post-translational levels. In this study we investigated the role of epigenetic regulation of CD133 in epithelial differentiation and cancer.MethodsDNA methylation analysis of the CD133 promoter was done by pyrosequencing and methylation specific PCR; qRT-PCR was used to measure CD133 expression and chromatin structure was determined by ChIP. Cells were treated with DNA demethylating agents and HDAC inhibitors. All the experiments were carried out in both cell lines and primary samples.ResultsWe found that CD133 expression is repressed by DNA methylation in the majority of prostate epithelial cell lines examined, where the promoter is heavily CpG hypermethylated, whereas in primary prostate cancer and benign prostatic hyperplasia, low levels of DNA methylation, accompanied by low levels of mRNA, were found. Moreover, differential methylation of CD133 was absent from both benign or malignant CD133+/α2β1integrinhi prostate (stem) cells, when compared to CD133-/α2β1integrinhi (transit amplifying) cells or CD133-/α2β1integrinlow (basal committed) cells, selected from primary epithelial cultures. Condensed chromatin was associated with CD133 downregulation in all of the cell lines, and treatment with HDAC inhibitors resulted in CD133 re-expression in both cell lines and primary samples.ConclusionsCD133 is tightly regulated by DNA methylation only in cell lines, where promoter methylation and gene expression inversely correlate. This highlights the crucial choice of cell model systems when studying epigenetic control in cancer biology and stem cell biology. Significantly, in both benign and malignant prostate primary tissues, regulation of CD133 is independent of DNA methylation, but is under the dynamic control of chromatin condensation. This indicates that CD133 expression is not altered in prostate cancer and it is consistent with an important role for CD133 in the maintenance of the hierarchical cell differentiation patterns in cancer.

A novel murine model of allogeneic vaccination against prostate cancer.

Prostate cancer continues to be a major cause of death in men. Surgical and medical treatments of the disease have improved, but metastasic disease remains a significant clinical problem. Novel therapies such as whole cell vaccination offer the potential of treating disease by stimulating the immune system. To study the efficacy of a whole cell vaccine in prostate cancer two strains of mice were used: C57BL/6 (H-2Kb) and C3H/HeJ (H-2Kk) in combination with four different cell lines. Thus, a model was constructed of allogeneic and syngeneic vaccine, as well as a challenge tumour for each strain. Two novel cell lines were developed during this study. Firstly, the non tumourigeneic PMC-1 was derived from a normal mouse prostate and immortalized with HPV16. Secondly, the tumourigeneic PMC-1 C6ras1p1 was transformed with human ras gene which formed tumours in both SCID and C3H/HeJ mice. Protection, and the nature of the immune response to syngeneic and allogeneic vaccine, in males and females was examined in both strains. Vaccination with both syngeneic and allogeneic irradiated whole cell vaccines induced protection from syngeneic challenge in females. However, no protection was observed when allogeneic vaccine was given to male mice. This correlated with the immune response. Two types of cellular immune responses were generated in females. A NK-mediated response was observed in C57BL/6 mice, whilst C3H/HeJ mice developed a CTL response. Little or no cellular immune response was observed in males. The cytokine profile in C3H/HeJ females was a mixture of Th1 and Th2 whilst a mainly Th1 profile was observed in C57BL/6 mice. Male mice showed a diminished cytokine secretion compared to females which was further depressed after challenge. The difference in immunity was largely as expected, since tolerance to prostate antigens should not normally develop in female mice. However, this makes this model particularly relevant clinically since it directly mimics the human situation and thus may accelerate the development of whole cell vaccines for clinical use.

The Immunomodulatory Drug (IMID®) CC-4047 Enhances the Proliferation and Anti-Tumor Function of Gamma Delta T Cells

The Immunomodulatory Drug (IMID ) CC-4047 Enhances the Proliferation and Anti-Tumor Function of Gamma Delta T Cells Christine Galustian, Debbie Klaschka, Marie-Christine Labarthe, J Blake Bartlett, Angus G Dalgleish. Department of Oncology, St Georges Hospital, Tooting, London, United Kingdom; Celgene Corporation, Warren, NJ. The immunomodulatory thalidomide derivatives (IMiDs ) lenalidomide (CC-5013, REVLIMID), and CC-4047 (ACTIMID) are currently in clinical development for the treatment of a variety of inflammatory and oncological diseases with particularly successful results seen in multiple myeloma (MM) patients. These compounds are less toxic than thalidomide and have been shown to enhance tumor-specific immunity and to exert antiangiogenic effects in-vivo. The mechanisms of IMiD-induced enhancement of anti-tumor immunity are not well understood, but are thought to involve enhancement of both innate and adaptive immunity. In this study we have investigated the potential for the potent costimulatory IMiD , CC-4047, to influence gamma delta (gd) TCR+ T cell mediated anti-tumor immunity. Gamma delta (gd) T cells have been shown to play a role in innate surveillance function against tumors and are directly cytotoxic for a variety of tumor cell lines in vitro. We have now demonstrated that CC-4047 strongly costimulates both aCD3-activated fresh gd T cells and purified gd T cell lines expanded and activated with the physiological gd T cell ligand, isopentenyl pyrophosphate (IPP). Costimulation with CC-4047 favors production of IFN-g and TNF-a, but not the Th2 cytokine IL-4, and leads to early and enhanced production of pro-inflammatory cytokines in response to co-culture with a variety of tumor cells. The proliferation of gd T cells in response to IPP is strongly enhanced (by ;40%) in CC-4047 treated PBMC cultures over a period of 8–12 days. This is most likely due to a protective effect of CC-4047 on the gd T cells, since treatment results in an increase in the percentage of live gd T cells by 40% over a period of 6 or 7 days compared to gd T cells treated with IPP alone. Furthermore, we also demonstrate that CC-4047 can increase the cytotoxic activity of gd T cells against the MM cell target RPMI-8226 by up to 50%. The increase in MM cell killing by gd T cells is associated with an increase in NKG2D expression. The optimization and further investigation of the effects of IMiDs on gd T cells and other cells of the innate immune system may provide an important contribution to understanding the anti-tumor activities associated with successful treatment of cancer with this class of compound.

A novel murine model of allogeneic vaccination against renal cancer

To develop a murine model for whole‐cell allogeneic vaccination in renal cancer, as such vaccines aim to direct immune responses against patient tumour cells, due to shared antigens between the vaccine and tumour cells.