Phase I Clinical Trial of Systemically Administered TUSC2(FUS1)-Nanoparticles Mediating Functional Gene Transfer in Humans

Background: Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2. Methods: Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks. Results: Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in posttreatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high posttreatmen

Functional assessment and specific depletion of alloreactive human T cells using flow cytometry

Human T-cell alloreactivity plays an important role in many disease processes, including the rejection of solid organ grafts and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. To develop a better understanding of the T cells involved in alloreactivity in humans, we developed a cytokine flow cytometry (CFC) assay that enabled us to characterize the phenotypic and functional characteristic of T cells responding to allogeneic stimuli. Using this approach, we determined that most T-cell alloreactivity resided within the CD4+ T-cell subset, as assessed by activation marker expression and the production of effector cytokines (eg, tumor necrosis factor α [TNF]α) implicated in human GVHD. Following prolonged stimulation in vitro using either allogeneic stimulator cells or viral antigens, we found that coexpression of activation markers within the CD4+ T-cell subset occurred exclusively within a subpopulation of T cells that significantly increased their surface expression of CD4. We then developed a simple sorting strategy that exploited these phenotypic characteristics to specifically deplete alloreactive T cells while retaining broad specificity for other stimuli, including viral antigens and third-party alloantigens. This approach also was applied to specifically enrich or deplete human virus-specific T cells

Cell-Autonomous Upregulation of Dendritic Cell Immunocompetence Is Antigen-Dependent

Abstract Introduction: Dendritic cells (DCs) are the master regulators of the adaptive immune response. In previous experiments, the double-loading of DCs with matched sets of MHC class I and class II repertoires (i.e. mRNA and cell lysate derived from the same source) appeared to enhance T-cell responses by maximizing the availability of T-cell help. T-cell help, provided by accessory CD4+ lymphocytes, elevates DC immunocompetence in response to antigen-specific interactions between the T-cell receptor and the peptide antigen/MHC class II complex. While T-cell help is specific and aids in the priming of only cognate CD8+ effectors, the mechanism governing this specificity is not understood. Here, we have explored the hypothesis that the specificity of T-cell help may be initiated by the DC itself upon detection of an antigen-dependent signal: the loading of the DC with matched sets of class I and class II determinants. Methods: To examine this hypothesis, we doubly-loaded DCs with either matched sets of antigenic determinants derived from acute myelogenous leukemia (AML) mRNA and lysate preparations, or, mismatched sets of antigenic preparations derived from AML products, the human TF-1a erythroblastic cell line, or the murine FBMD-1 stromal cell line. Following loading and maturation, we analyzed DCs for markers of immunocompetence such as IL-12 secretion and CD83 surface expression. Using specific siRNA oligonucleotides, we also examined a possible role for the intracellular CD63 tetraspanin in this process. DCs were generated by CD14 magnetic selection of apheresis products and six days of culture in GM-CSF and IL-4. Analysis of immature DCs at this time point by flow cytometry typically showed a CD3+ content equivalent to or less than that of the isotype control. DCs were then loaded with either matched or mismatched sets of determinants (as described above) and matured. Results: Matched-loaded DCs exhibited a 4-fold increase in IL-12 secretion over unloaded DCs while mismatch-loaded DCs showed only a 0.7-fold increase (similar to controls) (p = 0.0086). Moreover, matched-loaded DCs demonstrated a 21% increase in CD83 surface expression over unloaded/singly-loaded controls while mismatch-loaded DCs showed only a 6% increase (statistically identical to controls) (p = 0.009). In a single-antigen model system, electroporation of CD63 siRNA could reduce IL-12 secretion from matched-loaded DCs by 60% in comparison to matched-loaded DCs electroporated with non-targeting siRNAs. Secretion of non-Th-1 cytokines (i.e. IL-10) was unaffected. Conclusions: It appears that DC immunocompetence may be upregulated in a cell-autonomous, antigen-dependent fashion. Such upregulation is induced by the loading of DCs with matched sets of MHC class I and class II antigenic determinants and does not occur if DCs are loaded with mismatched determinants. CD63 appears to be involved in this process. CD63 is a member of the tetraspanin family of integral membrane proteins, molecules that facilitate the interaction of membrane and intracellular signaling complexes. In DCs, CD63 is localized exclusively to lysosomal exosomes, sites at which MHC class II, MHC class I, and phagocytosed antigens also co-localize. The data are suggestive of a cross-licensing model by which T-cell help might first be solicited by DCs loaded with matched antigenic determinants. Permission granted by a receptive DC, the helper T-cell might then license DC priming of CD8+ responses. Confirmatory studies are in progress

Characterization of optimal T Cell/Dendritic Cell (DC) Co-Culture Conditions for Ex Vivo Expansion of Antigen-Specific Human T Cells

Abstract Adoptive T cell immunotherapy has demonstrated clinical efficacy in controlling reactivation of human herpesviruses (e.g., cytomegalovirus, CMV and Epstein-Barr Virus, EBV) that are known etiologic agents of life-threatening illness in immunocompromised populations. Early approaches utilized in the expansion of large numbers of human virus-specific CD4+ and CD8+ T cell often involved laborious culture techniques impractical for widespread clinical use in populations at risk, including stem cell transplant (SCT) recipients. Advances in our understanding of the biology of myeloid dendritic cells (DC) can now facilitate more practical approaches for ex vivo antigen-specific T cell expansion. These advances have included novel approaches to isolate human peripheral blood monocytes (e.g., CD14 selection) and the optimization of cytokine cocktails inducing DC maturation. To define the optimal culture conditions for human antigen-specific T cell expansion, we conducted an iterative series of pairwise CMV-specific T cell expansions, in which important determinants of the expansion process could be directly compared. T cells, derived from apheresis products obtained from CMV-seropositive healthy donors, were expanded under GLP-compliant conditions, following incubation with autologous myeloid DC pulsed with overlapping pentadecapeptide pools spanning the CMV pp65 protein. In each case, we assessed the expanded CMV-specific T cell populations by immunophenotyping of the generated DC, and by phenotypic analysis, cytokine flow cytometry (CFC), ELISPOT analysis and HLA-peptide tetramer staining of the expanded T cells. Specifically, we compared:DC generated from monocytes isolated via plastic adherence to those isolated via positive selection of CD14+ T cells using magnetic beads;DC matured using tumor necrosis factor-α (TNFα) alone or in combination with other cytokines (ITIP, containing TNFα, IL-1β, IL-6 and PGE2);the quality of expansions derived from a starting lymphocyte population selected by non-adherence to plastic vs. the CD14-negative fraction in the monocyte selection process.From this series of experiments, we can conclude that while CD14 selection of monocytes results in a more phenotypically homogeneous population of DC, that this does not improve the quantity or quality of expanded virus-specific T cells. When we compared TNFα to ITIP maturation of DC, we found that while ITIP induced significantly higher CD83 expression on DC, TNFα-matured DC favored the maintenance of CMV-specific CD4+ T cells in culture resulting in ultimately greater numbers and functional proportions of tetramer-stained CD8+ T cells. Finally, we consistently found that the use of non-adherent PBMC resulted in dramatically better quantitative expansions of CMV-specific CD4+ and CD8+ T cells, vs. a lymphocyte population obtained following depletion of monocytes using CD14 positive selection. These results suggest a set of optimal T cell/DC co-culture conditions that lead to the generation of the largest numbers of expanded, functional CD4+ and CD8+ T cells, and are likely to have relevance for human trials wherein adoptive immunotherapy and/or DC immunization is planned

Regulatory and Naïve T Cells in Unmanipulated Donor Grafts Are Not Associated with Acute Graft Vs Host Disease in Matched Sibling Transplants for AML

Abstract Acute graft-versus-host disease (aGVHD) occurs in approximately 20–50% of matched-related stem cell transplants (HCT). The presence of increased frequencies of CD4+CD25+ regulatory T cells (Treg) in murine donor grafts has been shown to ameliorate aGVHD. In similar models, several groups have demonstrated that the transfer of equivalent numbers of naïve T cells (vs. various memory subsets) confers a relatively greater risk for aGVHD. To test the hypothesis that natural variations in donor graft naïve and Treg content would be associated with the incidence of aGVHD in graft recipients, we conducted a detailed analysis of donor graft T cells within unmanipulated grafts of 70 SCT recipients transplanted for AML/MDS following the administration of a uniform fludarabine/busulfan conditioning regimen. Median follow up time was 38 months. 19% (n=13) of recipients experienced grades II–IV aGVHD (n=3 grades III–IV). Cryopreserved donor grafts samples were analyzed using 9-color flow cytometry (with a panel including a viability dye and MAbs recognizing CD4, CD8, CD25, CD127, CD45RA, CD27, Foxp3, and Ki-67). Tregs were defined as being CD4+CD25+CD127loFoxp3+. CD45RA and CD27 were used to measure naïve (CD45RA+CD27+) and memory conventional CD4+ and CD8+ T cells. In addition, the proliferating fraction of all cells was defined by Ki-67 nuclear-antigen expression. We conducted analyses based on donor graft T cells assessed both continuously and by quartiles, with similar results. No significant associations were found between aGVHD incidence and total Tregs (HR 1.01, 95%CI 0.9–1.2, p 0.8), nor with proliferating Tregs (HR 1.2, 95%CI 0.2–5.9, p 0.8). Since murine models typically fix the dose of Tregs vs. conventional T cells (Tcon), we also analyzed GVHD risk by donor graft Treg:Tcon ratio, finding no association (HR 1.03, 95%CI 0.8–2.1, p 0.3). Although patients in the highest Treg:Tcon quartile received Treg doses known in vitro to induce suppression (>1:21.5), there was no reduction in aGVHD (HR 1.5 Quartile 4 vs others, 95%CI 0.5–4.9, p 0.5). Additionally, no significant association with aGVHD was found with total naïve T cells (HR 1, 95%CI 0.9–1.0, p 0.9), naïve CD4+ (HR 1, 95%CI 0.9–1.03, p 0.6), or naïve CD8+ cells (HR 0.98, 95%CI 0.9–1.03, p 0.6). Because naïve:memory cell ratios were also fixed in murine models demonstrating the importance of naïve T cells in GVHD, we also assessed naïve:memory T cell ratios within donor grafts with respect to recipient aGVHD. No reduction in aGVHD was observed considering naïve:memory ratios analyzed continuously (HR 1.1, 95%CI 0.3–4.2, p 0.8) or in the quartile receiving the lowest naïve:memory ratio (HR 0.5 Quartile 1 vs others, 95%CI 0.1–2.2, p 0.3). Our results demonstrate that donor graft Treg and naïve T cell content, assessed in the context of unmanipulated PBSC transplantation in matched siblings, does not predict recipient aGVHD incidence. These data suggest that the therapeutic efficacy of Treg enrichment and/ or naïve T cell depletion may require grafts to be manipulated to supraphysiologic levels for maximal therapeutic benefit

Double loading of dendritic cell MHC class I and MHC class II with an AML antigen repertoire enhances correlates of T-cell immunity in vitro via amplification of T-cell help

Therapeutic vaccination with dendritic cells presenting tumor-specific antigens is now recognized as an important investigational therapy for the treatment of neoplastic disease. Dendritic cell cross-presentation is credited with the ability of tumor lysate-loaded dendritic cells to prime both CD4 and CD8-specific T-lymphocyte responses, enabling the generation of cancer specific CTL activity without the loading of the classical MHC class I compartment. Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have doubly-loaded human dendritic cells with both AML-specific tumor lysate and AML-specific tumor mRNA. Our results show that these doubly-loaded dendritic cells can mediate superior primary, recall, and effector lytic responses in vitro in comparison to those of dendritic cells loaded with either tumor lysate or tumor mRNA alone. Enhanced recall responses appeared to be influenced by CD40/CD40L signaling, underscoring the importance of T-cell help in the generation and perpetuation of the adaptive immune response