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Purpose:Mesenchymal stem cells (MSCs) show an inherent brain tumor tropism that can be exploited for targeted delivery of therapeutic genes to invasive glioma. We assessed whether a motile MSC-based local immunomodulation is able to overcome the immunosuppressive glioblastoma microenvironment and to induce an antitumor immune response.Experimental Design:We genetically modified MSCs to coexpress high levels of IL12 and IL7 (MSCIL7/12, Apceth-301). Therapeutic efficacy was assessed in two immunocompetent orthotopic C57BL/6 glioma models using GL261 and CT2A. Immunomodulatory effects were assessed by multicolor flow cytometry to profile immune activation and exhaustion of tumor-infiltrating immune cells. Diversity of the tumor-specific immune response as analyzed using T-cell receptor sequencing.Results:Intratumoral administration of MSCIL7/12 induced significant tumor growth inhibition and remission of established intracranial tumors, as demonstrated by MR imaging. Notably, up to 50% of treated mice survived long-term. Rechallenging of survivors confirmed long-lasting tumor immunity. Local treatment with MSCIL7/12 was well tolerated and led to a significant inversion of the CD4+/CD8+ T-cell ratio with an intricate, predominantly CD8+ effector T-cell–mediated antitumor response. T-cell receptor sequencing demonstrated an increased diversity of TILs in MSCIL7/12-treated mice, indicating a broader tumor-specific immune response with subsequent oligoclonal specification during generation of long-term immunity.Conclusions:Local MSC-based immunomodulation is able to efficiently alter the immunosuppressive microenvironment in glioblastoma. The long-lasting therapeutic effects warrant a rapid clinical translation of this concept and have led to planning of a phase I/II study of apceth-301 in recurrent glioblastoma.

Suppl. Fig. 5. Spleen weight

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.

Cell membrane-anchored (ma) antiviral peptides derived from the C-terminal heptad repeat of the HIV-1 transmembrane glycoprotein gp41 (C-peptides) and expressed from retroviral vectors were shown to efficiently inhibit HIV-1 entry into target cells. Here, we analyzed the influence of the vector backbone, the scaffold modules that anchor the peptide to the membrane and the length of the C-peptide on expression level and antiviral activity. In general, antiviral activity was determined primarily by the density of the C-peptide on the cell surface. By influencing expression levels, the scaffold elements indirectly also determined antiviral activity. Additional direct effects of the scaffold on antiviral activity were minor. At comparable expression levels, the elongated C-peptide (maC46) was found to be more potent than the shorter maC36. On the basis of these findings, a dose-response assay was established that quantifies antiviral activity relative to the expression level of the antiviral gene product. Taken together, these data demonstrate the importance of analyzing the efficacy of therapeutic genes relative to the dose of the gene product.

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen.

Drug toxicity and viral resistance limit the long-term efficacy of antiviral drug treatment for human immunodeficiency virus (HIV) infection. Thus, alternative therapies need to be explored. We tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry-inhibitory peptide (maC46). Gene-modified autologous T cells were infused into ten HIV-infected patients with advanced disease and multidrug-resistant virus during anti-retroviral combination therapy. T-cell infusions were tolerated well, with no severe side effects. A significant increase of CD4 counts was observed after infusion. At the end of the 1-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes, and bone marrow throughout the 1-year follow-up, and marking levels correlated with the cell dose. No significant changes of viral load were observed during the first 4 months. Four of the seven patients who changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking, and may improve immune competence in HIV-infected patients with advanced disease and multidrug-resistant virus.

The development of highly active antiviral therapy has resulted in greater expected life spans and slower progression to AIDS for patient infected with human immunodeficiency virus (HIV) (25, 26). However, the emergence of drug-resistant viruses during treatment and extensive subsequent intraclass cross-resistance has been a limiting factor in the success of treatment (5, 6). Due to this limitation, antiretrovirals with a novel mechanism of action have been the focus of extensive drug discovery research. Enfuvirtide is one novel HIV-1 inhibitor already in clinical use; it blocks fusion of viral and cellular membranes by binding to heptad repeat region 1 (HR1) of virus gp41 (10, 15). Enfuvirtide is homologous to a segment of the HR2 region of gp41 corresponding to amino acids 127 to 162 and binds to the HR1 region of gp41 (39, 40). Enfuvirtide interferes with the formation of the six-helix bundle, composed of an inner coiled-coil trimer of the HR1 and HR2 regions aligned in an antiparallel manner, thereby blocking the final step of virus entry, the fusion of the viral membrane with the target cell membrane (28). A slightly longer, 39-amino-acid peptide, T-1249, binds to a region of HR1 that overlaps the binding site of enfuvirtide and is active against HIV-1, HIV-2, and simian immunodeficiency virus (11). In addition, several other fusion inhibitors have been studied, for example, T-649 (corresponding to amino acids 117 to 152 of gp41) and C34 (corresponding to amino acid 117 to 150 of gp41). Both block the formation of the hairpin structure in a way similar to enfuvirtide (4, 34, 40). A wide range of susceptibility to enfuvirtide has been described in virus isolates from naive patients (13, 16, 18). Early in vitro studies using enfuvirtide showed the development of resistance is associated with changes in a conserved amino acid triad (GIV) at positions 36 to 38 in the HR1 region of gp41 (34). These findings were confirmed by site-directed mutagenesis experiments and in vivo studies which expanded the core region of functional importance to amino acids 36 to 45 (16, 30, 35, 38). Single amino acid substitutions in this region are the most common and cause various degrees of susceptibility loss. Double amino acid substitutions have also been observed and these are associated with the highest levels of resistance, some combinations (G36S/V38M) exhibiting an approximately 100-fold reduction in enfuvirtide susceptibility (30, 34, 38). In addition, changes in HR2 may equally be involved in enfuvirtide resistance as well (20, 36). Most variants that are resistant to enfuvirtide maintain susceptibility to T-1249 and T-649 (12, 34). The rapid emergence of enfuvirtide-resistant viruses and the lack of oral bioavailability are major obstacles that hinder widespread application of enfuvirtide as part of an extended therapy option (5). To overcome these problems, the enfuvirtide peptide was engineered for expression on the cell membrane, leading to a high local concentration of peptide at the site of action (14). Surface expression was achieved by fusing an N-terminal signal peptide and a C-terminal scaffold consisting of a hinge and a membrane anchor to the antiviral peptide M87. This membrane-anchored peptide was expressed from a retroviral vector (pM87) and had good antiviral activity in cell lines. Recently, we developed a retroviral vector expressing a membrane-anchored antiviral peptide that was also highly effective in primary cells and had minimal potential immunogenicity and no detectable toxicity (8). This membrane-anchored peptide was expressed from an optimized retroviral vector (pM87o) and encodes a larger antiviral peptide 46 amino acids in length (M87o). This extended membrane-anchored antiviral peptide can be viewed as a combination of the previously described antiviral peptides T-20 and T-649 (or C34) and is almost similar to DP207, a soluble peptide 45 amino acids in length shown to be a potent fusion inhibitor (40). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1NL4-3 and HIV-1BaL by serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 for the two viruses analyzed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations (L33S, I48V, and N126K) to confer resistance to M87 as well as to soluble T-20 and to T-649. In addition, replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1BaL results in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1NL4-3 did enhance viral fitness compared to the parental viruses.