Safety of Repeated Administration of Xenogeneic Human Apoptotic State (Allocetra-OTS) in Sprague Dawley Rats.

Apoptotic cells possess immunomodulatory effects that can be utilized to treat imbalanced immune conditions. Information on the preclinical safety of such treatment is sparse. In this study, the safety of apoptotic cells (Allocetra-OTS) was assessed in a GLP toxicological study on Sprague Dawley rats. Three doses of Allocetra-OTS or vehicle were administered intravenously (IV) for 3 consecutive days. Animals in the main study were sacrificed on day 4, while animals from the recovery groups were kept for 14 or 28 days. Allocetra-OTS was well tolerated, and no adverse effects were observed in terms of body weight, clinical signs, food consumption, or ophthalmologic observation. Thus, the No Observed Adverse Effect Level (NOAEL) dose was determined as the highest dose administered. An observed elevation in immune cells was suspected to be due to Allocetra-OTS, similarly to other clinical chemistry parameters; however, it was resolved in the recovery phases. Splenomegaly and dose-related extramedullary hematopoiesis (EMH) in the red pulp were observed, with no adverse events, and were considered to be a normal and expected reaction following the IV administration of cell-based therapies. In conclusion, under the conditions of this study, Allocetra-OTS was concluded to be safe, further supporting its potential candidacy for clinical studies.

Novel immunotherapy for malignant melanoma with a monoclonal antibody that blocks CEACAM1 homophilic interactions.

CEACAM1 (biliary glycoprotein-1, CD66a) was reported as a strong clinical predictor of poor prognosis in melanoma. We have previously identified CEACAM1 as a tumor escape mechanism from cytotoxic lymphocytes. Here, we present substantial evidence in vitro and in vivo that blocking of CEACAM1 function with a novel monoclonal antibody (MRG1) is a promising strategy for cancer immunotherapy. MRG1, a murine IgG1 monoclonal antibody, was raised against human CEACAM1. It recognizes the CEACAM1-specific N-domain with high affinity (K(D) ~ 2 nmol/L). Furthermore, MRG1 is a potent inhibitor of CEACAM1 homophilic binding and does not induce any agonistic effect. We show using cytotoxicity assays that MRG1 renders multiple melanoma cell lines more vulnerable to T cells in a dose-dependent manner, only following antigen-restricted recognition. Accordingly, MRG1 significantly enhances the antitumor effect of adoptively transferred, melanoma-reactive human lymphocytes using human melanoma xenograft models in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice. A significant antibody-dependent cell cytotoxicity response was excluded. It is shown that MRG1 reaches the tumor and is cleared within a week. Importantly, approximately 90% of melanoma specimens are CEACAM1(+), implying that the majority of patients with melanoma could be amenable to MRG1-based therapy. Normal human tissue microarray displays limited binding to luminal epithelial cells on some secretory ducts, which was weaker than the broad normal cell binding of other anticancer antibodies in clinical use. Importantly, MRG1 does not directly affect CEACAM1(+) cells. CEACAM1 blockade is different from other immunomodulatory approaches, as MRG1 targets inhibitory interactions between tumor cells and late effector lymphocytes, which is thus a more specific and compartmentalized immune stimulation with potentially superior safety profile.

Regulation of cancer aggressive features in melanoma cells by microRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.

Establishment and large-scale expansion of minimally cultured "young" tumor infiltrating lymphocytes for adoptive transfer therapy.

Treatment of metastatic melanoma patients with adoptively transferred tumor infiltrating lymphocytes (TIL) has developed into an effective therapy. Various studies reported objective responses of 50% and more. The use of unselected, minimally cultured, bulk TIL (Young-TIL) has simplified the TIL production process and may therefore, allow the accessibility of this approach to cancer centers worldwide. This article describes the precise process leading to the large-scale production of Young-TIL for therapy. We have enrolled 55 melanoma patients and optimized their Young-TIL generation process. Young-TIL cultures were successfully established for 51 of 55 (93%) patients in 16.7 ± 5.5 days. In a large-scale expansion procedure Young-TIL of 32 patients were further expanded to treatment levels, resulting in a final number of 4.5 x 10¹° ± 2.0 x 10¹° TIL. Fifteen of 31 (48%) patients, who were evaluated, achieved a clinical response, including 4 complete and 11 partial responses. We confirmed the significant correlation between short culture duration, high number of infused cells, and tumor regression. A high percentage of CD8 T cells in the infusion product was beneficial to achieve an objective response. All responding patients were treated with Young-TIL cultures established in < 20 days. In summary, we describe here an efficient and reliable method to generate Young-TIL for adoptive transfer therapy, which may easily be adopted by other cancer centers and can lead to objective responses in 50% of refractory melanoma patients. In the future this approach may be used also in other types of malignancies.

Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients.

PURPOSE: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) has shown promising results in metastatic melanoma patients. Although objective response rates of over 50% have been reported, disadvantages of this approach are the labor-intensive TIL production and a very high drop-out rate of enrolled patients, limiting its widespread applicability. Previous studies showed a clear correlation between short TIL culture periods and clinical response. Therefore, we used a new TIL production technique using unselected, minimally cultured, bulk TIL (Young-TIL). The use of Young-TIL is not restricted to human leukocyte antigen (HLA)-A2 patients. The purpose of this study is to explore the efficacy and toxicity of adoptively transferred Young-TIL following lympho-depleting chemotherapy in metastatic melanoma patients, refractory to interleukin-2 and chemotherapy. EXPERIMENTAL DESIGN: Young-TIL cultures for 90% of the patients were successfully generated, enabling the treatment of most enrolled patients. We report here the results of 20 evaluated patients. RESULTS: Fifty percent of the patients achieved an objective clinical response according to the Response Evaluation Criteria in Solid Tumors, including two ongoing complete remissions (20+, 4+ months) and eight partial responses (progression-free survival: 18+, 13+, 10+, 9, 6+, 4, 3+, and 3 months). All responders are currently alive. Four additional patients showed disease stabilization. Side effects were transient and manageable. CONCLUSION: We showed that lympho-depleting chemotherapy followed by transfer of short-term cultured TIL can mediate tumor regression in 50% of metastatic melanoma with manageable toxicity. The convincing clinical results combined with the simplification of the process may thus have a major effect on cell therapy of cancer.

Focus on adoptive T cell transfer trials in melanoma.

Adoptive Cell Transfer (ACT) of Tumor-Infiltrating Lymphocytes (TIL) in combination with lymphodepletion has proven to be an effective treatment for metastatic melanoma patients, with an objective response rate in 50%-70% of the patients. It is based on the ex vivo expansion and activation of tumor-specific T lymphocytes extracted from the tumor and their administration back to the patient. Various TIL-ACT trials, which differ in their TIL generation procedures and patient preconditioning, have been reported. In the latest clinical studies, genetically engineered peripheral T cells were utilized instead of TIL. Further improvement of adoptive T cell transfer depends on new investigations which seek higher TIL quality, increased durable response rates, and aim to treat more patients. Simplifying this therapy may encourage cancer centers worldwide to adopt this promising technology. This paper focuses on the latest progress regarding adoptive T cell transfer, comparing the currently available protocols and discussing their advantages, disadvantages, and implication in the future.

Adrenal 20alpha-hydroxysteroid dehydrogenase in the mouse catabolizes progesterone and 11-deoxycorticosterone and is restricted to the X-zone.

The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) is a progesterone-catabolizing enzyme that is highly expressed in mouse ovaries and adrenals. Although the functional significance of ovarian 20alpha-HSD for the induction of parturition has been defined, regulation and distribution of 20alpha-HSD in the adrenal gland has not been determined. We demonstrate that the expression of adrenal 20alpha-HSD is restricted to the X-zone, a transient zone between the adrenal cortex and the medulla of yet unknown function. Adrenal 20alpha-HSD activity in male mice peaks at 3 wk of age and disappears thereafter, whereas 20alpha-HSD enzyme activity is maintained in adrenals from nulliparous female animals. Testosterone treatment of female mice induces rapid involution of the X-zone that is associated with the disappearance of the 20alpha-HSD-positive cells. Conversely, reappearance of 20alpha-HSD expression and activity in male animals is evident after gonadectomy. Moreover, pregnancy, but not pseudopregnancy, is accompanied by X-zone regression and loss of 20alpha-HSD activity. Pregnancy-induced X-zone regression and -abolished 20alpha-HSD expression is partially restored in animals that were kept from nursing their pups. We found that in addition to its progesterone-reducing activity, 20alpha-HSD also functions as an 11-deoxycorticosterone-catabolizing enzyme. The unaltered growth kinetics of the X-zone in 20alpha-HSD knockout animals suggests that 20alpha-HSD is not required for the regulation of X-zone growth. However, 20alpha-HSD expression and enzymatic activity in all experimental paradigms is closely correlated with the presence of the X-zone. These findings provide the basis for 20alpha-HSD as a reliable marker of the murine X-zone.