James Hodge

Reduction of the central effects of tryptophan by a decarboxylase inhibitor

Administration of L‐tryptophan to animals and man following monoamine oxidase inhibition produced characteristic neurological abnormalities which were chiefly excitatory in nature. Large doses of tryptophan in gUinea pigs also produced hyperpyrexia, and death followed in over 80 per cent of the animals. Pretreatment with a potent, centrally active decarboxylase inhibitor, Ro 4‐4602, diminished or prevented the neurological abnormalities abolished the hyperpyrexia in gUinea pigs, and reduced the animal mortality rate to below 10 per cent. Serotonin levels rose in the brains of guinea pigs after tryptophan but failed to do so when the tryptophan followed treatment with Ro 4‐4602. These results favor the conclusion that the effects seen after tryptophan administration are caused by its amine metabolites and not by the amino acid itself. The administration of L‐tryptophan to man under proper conditions provides a useful means of evaluating decarboxylase inhibition within the central nervous system in vivo and is the first such method to be described.

Hormone‐induced modifications of free tyrosine in the rat thyroid gland

The concentrations of free tyrosine in liver, muscle, kidney and thyroid gland were determined in separate groups of rats maintained on a low iodine diet and treated with intraperitoneal injections of thyroxine (T 4), thyrotrophin (TSH) and TSH plus propylthiouracil (PTU). Groups of hypophysectomized rats also were given T 4. Significant changes in tissue tyrosine were generally confined to the thyroid gland. Animals treated with T 4 showed a decrease of mean thyroid tyrosine from 113·3 ± 17·9 ( S.D.) μg/g wet weight of gland to 76·2 ± 5·7 μg/g (P < 0·01). Although there was little change in content when TSH was given alone, a significant increase in tyrosine levels was observed when TSH plus PTU were administered (P < 0·01). In hypophysectomized rats thyroid tyrosine decreased and was further lowered (to 46·0 ± 3·1 μg/g; P < 0·05) by T 4 treatment. When tyrosine concentrations were expressed in relation to ribonucleic acid (RNA) or protein content of the assayed gland, all differences in tyrosine content became greater.

Treatment with the IDO inhibitor INCB024360 increased lysis of human tumor cell targets by peptide-specific CTL

We have investigated the in vitro effects of INCB024360 on dendritic cell maturation and activation of antigen-specific T cells. INCB024360 is a novel inhibitor of indoleamine-2, 3-dioxygenase (IDO), and is currently in several ongoing clinical trials. INCB024360 effectively suppresses systemic tryptophan catabolism and tumor growth. Human dendritic cells (DC) were generated from PBMCs from healthy donors. The tryptophan (Trp) and kynurenine (Kyn) concentrations in supernatants, and the Trp/Kyn ratio, were measured by HPLC. The Trp level was 75.8% in supernatants of immature DCs with little breakdown to Kyn (Kyn/Trp ratio of 0.32). In contrast, in supernatants of matured DCs, the Kyn/Trp ratio was 5.9 for IFNγ matured DCs, and 9.2 for IFNγ/LPS matured DCs. Treatment with INCB024360 resulted in almost no breakdown of tryptophan. The expression levels of several DC activation markers did not change after treatment with INCB024360. We then compared the efficacy of antigen presentation by DCs treated with and without the inhibitor. MUC1-C peptide specific CTL were derived from a prostate cancer patient. DCs pulsed with peptide and treated with INCB024360 stimulated the CTL to produce more IFNγ and other type I cytokines than untreated DCs. A MUC1-C-specific, HLA-A24+ was stimulated using its specific MUC1 peptide and DCs treated with INCB024360. The T cells were used in a CTL assay using PC3 (human prostate carcinoma, MUC1+, HLA-A24+) as a target and ASPC-1 (human pancreatic carcinoma, MUC1+, HLA-A24NEG) as a negative control. Pre-treating the DCs with INCB024360 resulted in increased tumor cell lysis. An additional T cell line, specific for brachyury peptide and HLA-A2, was derived from another prostate cancer patient, and after stimulation with INCB treated DCs the lysis of the human breast cancer cell line increased. These results thus demonstrated that INCB024360 treatment of DCs resulted in both increased cytokine production and increased tumor cell lysis by antigen-specific CD8+ T cell lines derived from cancer patients. This suggests that INCB024360 could potentially be effectively combined with other immune modulating therapies. Murine anti-tumor studies of INCB024360 alone or in combination with vaccine are ongoing.

Combining Vaccines with Therapies that Render Tumor Cells more Susceptible to Immune Mediated Killing

Therapeutic cancer vaccines are an efficient and minimally toxic way to stimulate the host’s immune system in a dynamic way such that immune responses persists beyond the period of vaccine administration. The persisting immunity can be utilized by standard of care therapies given concurrently or subsequently to vaccines in the fight against cancer. Emerging pre-clinical and clinical evidence shows that the standard of care anti-cancer therapies can “modulate”. Both the Tumor and the immune system of the host such as to potentiate the immune response induced by therapeutic cancer vaccines. This book chapter focuses on these combinatorial approaches to cancer that target the residual cancer cells in the host that have not been contained by definitive procedures such as surgery. Specifically, we discuss the biological basis of anti-tumor immunity, immunomodulation with standard of care therapies such as radiation, cytotoxic chemotherapies, hormone abrogation and small molecule inhibitors, pre-clinical and clinical evidence of synergism in combinatorial strategies, clinical trial design and published and ongoing clinical studies in that regard.

Abstract 3979: In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC A previous murine study (B. Farsaci et al, Int. J. Cancer, 2010) showed that mice administered an anti-cancer vaccine prior to treatment with the pan-BCL-2 inhibitor obatoclax (GX15-070) showed a substantial reduction of metastatic mouse lung tumors due to increased apoptotic resistance of mature CD8T cells and decreased Treg function. The purpose of this study was to evaluate activity of obatotclax on human lymphocyte populations. In vitro effects of obatoclax on human T cell subsets (naive, central memory, effector-memory and terminal effector T cells) at different maturation states (CD69+ early-activated T cells and CD69- mature T cells) and regulatory T cells (Tregs) were evaluated at multiple concentrations (0.1 - 10 uM). Purified CD8 T cells or whole PBMCs from normal donors were activated for 3-4 days with anti-CD3 and anti-CD28 monoclonal antibodies (short-term culture) or maintained in an IL7/IL15-enriched medium for 7 additional days (long-term culture). After short- or long-term cultures, isolated CD8Ts and whole PBMCs were treated with obatoclax or control (DMSO) for 24 hours, and their viability and differentiation status were analyzed by flow cytometry. The results indicate that (1) long-term cultured CD8 and CD4T cells were more resistant to obatoclax induced cell death than those in the short-term culture condition, (2) obatoclax increased the percentage of total memory CD8 and CD4T cells (central memory + effector memory) in the long-term culture condition, and (3) obatoclax increased the apoptosis of regulatory T cells (Tregs) and induced a profound down-regulation of FOXP3 in a dose dependent manner. It is postulated that first, for an optimum combination effect, an immune-stimulating agent directed at T cells needs to precede obatoclax treatment long enough to allow T cells to acquire resistance to obatoclax. Then secondly, with proper treatment scheduling, memory T cells can be viably maintained, thereby establishing a sustainable source of cytotoxic T cells to kill tumors. Finally, because human regulatory T cells are sensitive to obatoclax, there is a potential for obatoclax to tip the numerical and functional balance of effector T cells and Tregs towards effector T cells in human tumor microenvironment. These conclusions support the preclinical murine in vivo study, and provide the rationale for combining obatoclax with an immunotherapeutic regimen in clinical studies. Citation Format: Peter Kim, James Hodge, Italia Grenga, Renee Donahue, Jeffrey Schlom, Benedetto Farsaci. In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2013-3979