Preclinical activity of melflufen (J1) in ovarian cancer

Ovarian cancer carries a significant mortality. Since symptoms tend to be minimal, the disease is often diagnosed when peritoneal metastases are already present. The standard of care in advanced ovarian cancer consists of platinum-based chemotherapy combined with cytoreductive surgery. Unfortunately, even after optimal cytoreduction and adjuvant chemotherapy, most patients with stage III disease will develop a recurrence. Intraperitoneal administration of chemotherapy is an alternative treatment for patients with localized disease. The pharmacological and physiochemical properties of melflufen, a peptidase potentiated alkylator, raised the hypothesis that this drug could be useful in ovarian cancer and particularily against peritoneal carcinomatosis. In this study the preclinical effects of melflufen were investigated in different ovarian cancer models. Melflufen was active against ovarian cancer cell lines, primary cultures of patient-derived ovarian cancer cells, and inhibited the growth of subcutaneous A2780 ovarian cancer xenografts alone and when combined with gemcitabine or liposomal doxorubicin when administered intravenously. In addition, an intra- and subperitoneal xenograft model showed activity of intraperitoneal administered melflufen for peritoneal carcinomatosis, with minimal side effects and modest systemic exposure. In conclusion, results from this study support further investigations of melflufen for the treatment of peritoneal carcinomatosis from ovarian cancer, both for intravenous and intraperitoneal administration

Increased Memory Conversion of Naïve CD8 T Cells Activated during Late Phases of Acute Virus Infection Due to Decreased Cumulative Antigen Exposure

Background: Memory CD8 T cells form an essential part of protective immunity against viral infections. Antigenic load, costimulation, CD4-help, cytokines and chemokines fluctuate during the course of an antiviral immune response thus affecting CD8 T cell activation and memory conversion. Methodology/Principal Findings: In the present study, naïve TCR transgenic LCMV-specific P14 CD8 T cells engaged at a late stage during the acute antiviral LCMV response showed reduced expansion kinetics but greater memory conversion in the spleen. Such late activated cells displayed a memory precursor effector phenotype already at the peak of the systemic antiviral response, suggesting that the environment determined their fate during antigen encounter. In the spleen, the majority of late transferred cells exhibited a central memory phenotype compared to the effector memory displayed by the early transferred cells. Increasing the inflammatory response by exogenous administration of IFNc, PolyI:C or CpG did not affect memory conversion in the late transferred group, suggesting that the diverging antigen load early versus later during acute infection had determined their fate. In agreement, reduction in the LCMV antigenic load after ribavirin treatment enhanced the contribution of early transferred cells to the long lasting memory pool. Conclusions/Significance: Our results show that naïve CD8 cells, exposed to reduced duration or concentration of antigen during viral infection convert into memory more efficiently, an observation that could have significant implications fo

BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to <i>In Vitro</i> Differentiation into Functional Foxp3⁺ T Regulatory Cells

<div><p>The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, <i>in vitro</i> differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4⁺ T cells of the BDC12-4.1 clone to convert into Foxp3⁺ iTreg cells. We found that <i>in vitro</i> polarization toward Foxp3⁺ iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3⁺ BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, <i>in vitro</i> polarization of insulin-specific BDC12-4.1 TCR transgenic CD4⁺ T cells toward Foxp3⁺ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an <i>in vitro</i> acquired Foxp3⁺ cell phenotype and its associated <i>in vivo</i> regulatory potential.</p></div

BDC12-4.1 T cells expand and display memory and Treg cell phenotypes in the periphery.

<p>(A and B) Age-associated increases in total CD4⁺ T cell numbers in the spleen and PLN of BDC12-4.1.RAG^KO mice. Splenocytes and PLN lymphocytes from BDC12-4.1.RAG^KO mice were analyzed at different ages by flow cytometry for the frequency of CD4⁺Vβ2⁺ T cells. The percentage of CD4⁺Vβ2⁺ cells was multiplied with the total number of cells isolated from the spleen or PLN to calculate the total number of CD4⁺ T cells. Trypan blue was used for the exclusion of dead cells prior to counting. (C and D) Representative FACS plots and data for activated T cells, CD44^hiCD62L^low, and Treg cells, CD25⁺Foxp3⁺, from the spleens of donor mice that were used to generate Foxp3⁺ Treg cells in vitro are shown.</p

<i>In vitro</i> polarization induces Foxp3 expression in memory BDC12-4.1 T cells but does not endow <i>in vivo</i> regulatory functions.

<p>CD4⁺CD25⁺ cells from splenocytes of BDC12-4.1 mice polarized into Treg cells (A) <i>in vitro</i> in the presence of a-CD3 (left panel) or InsB:9-23 peptide (right panel). Expanded T cells were gated on CD4⁺CD25⁺ cells (not shown) and expression of CD127 and Foxp3 were determined. (B). 1×10⁶ Tregs polarized with anti-CD3 (left panel) or InsB:9-23 peptide (right panel) were adoptively transferred into prediabetic 8-wk old NOD mice and diabetes development was monitored. Unmanipulated NOD mice monitored on a regular basis in our animal colony served as negative controls with cumulative diabetes incidence of ∼90% by 27 wks of age. Representative results from one of three independent experiments are shown.</p

T cells with InsB:9-23 specific responses increase in frequencies with age.

<p>Total splenocytes from BDC12-4.1.RAG^KO mice were obtained at different ages. 250,000 splenocytes per well were incubated with or without native InsB:9-23 peptide in triplicates. Following 3-day incubation, IFN-γ, IL-2, and IL-4 production were determined by ELISpot assay. Representative images of cytokine production from BDC12-4.1.RAG^KO T-cells with (bottom row) or without antigen (top row) stimulation is shown in A. Background (media) subtracted IFN-γ spot numbers produced in response to antigen stimulation, from BDC12-4.1.RAG^KO mice of different age groups are shown on the Y-axis in B. Representative means ± SEM data from one of two independent experiments with n = 3 per each group with similar results are shown.</p

Minimal Effect of CD103 Expression on the Control of a Chronic Antiviral Immune Response

Impaired antiviral CD8 and CD4 T-cell responses are often associated with chronic viral infections. Cell-intrinsic as well as cell-extrinsic mechanisms are thought to dampen such responses, for example programmed death 1 receptor (PD-1) expression on T cells, and interleukin (IL)-10 production primarily by dendritic cells (DCs), have been shown to support viral persistence by suppressing immune responses. Here we demonstrate that CD103, an alpha E integrin necessary for T-cell homing and retention in the gut and other epithelia expressed by the majority of naïve CD8+, and CD4+CD25+ T cells and some DC subsets, is unnecessary for controlling T-cell responses during chronic lymphocytic choriomeningitis virus clone 13 (LCMV cl13) infection. T-cell analysis following viral infection showed that the primary as well as the memory CD8+ and CD4+ T-cell responses among CD103-sufficient and CD103-deficient mice were identical. In addition, no rescue of cytokine production by virus-specific T cells or alterations in viral titers in the absence of intrinsic CD103 expression was observed. Interestingly, CD103 levels on the effector CD8+ T cells became reduced soon after virus infection, with a small proportion of cells co-expressing PD-1 and CD103. In contrast, although no substantial differences in the frequency and number of the CD4+CD25+ cell population were seen, CD103 expression increased significantly over time in this population, correlating with viral persistence. Thus, a lack of CD103 expression does not affect functional impairment of effector T-cell responses during chronic viral infection