Sebastian Tuve

A New Group B Adenovirus Receptor Is Expressed at High Levels on Human Stem and Tumor Cells

ABSTRACT CD46 is used by human group B adenoviruses (Ads) as a high-affinity attachment receptor. Here we show evidence that several group B Ads utilize an additional receptor for infection of human cells, which is different from CD46. We tentatively named this receptor receptor X. Competition studies with unlabeled and labeled Ads, recombinant Ad fiber knobs, and soluble CD46 and CD46 antibodies revealed three different subgroups of group B Ads, in terms of their receptor usage. Group I (Ad16, -21, -35, and -50) nearly exclusively uses CD46. Group II (Ad3, -7p, and -14) utilizes receptor X and not CD46. Group III (Ad11p) uses both CD46 and the alternative receptor X. Interaction of group II and III Ads with receptor X occurs via the fiber knob. Receptor X is an abundantly expressed glycoprotein that interacts with group II and III Ads at relatively low affinity in a Ca2+-dependent manner. This receptor is expressed at high levels on human mesenchymal and undifferentiated embryonic stem cells, as well as on human cancer cell lines. These findings have practical implications for stem cell and gene therapy.

Combination of Tumor Site–Located CTL-Associated Antigen-4 Blockade and Systemic Regulatory T-Cell Depletion Induces Tumor-Destructive Immune Responses

Accumulating data indicate that tumor-infiltrating regulatory T cells (Treg) are present in human tumors and locally suppress antitumor immune cells. In this study, we found an increased Treg/CD8 ratio in human breast and cervical cancers. A similar intratumoral lymphocyte pattern was observed in a mouse model for cervical cancer (TC-1 cells). In this model, systemic Treg depletion was inefficient in controlling tumor growth. Furthermore, systemic CTL-associated antigen-4 (CTLA-4) blockade, an approach that can induce tumor immunity in other tumor models, did not result in TC-1 tumor regression but led to spontaneous development of autoimmune hepatitis. We hypothesized that continuous expression of an anti-CTLA-4 antibody localized to the tumor site could overcome Treg-mediated immunosuppression and locally activate tumor-reactive CD8+ cells, without induction of autoimmunity. To test this hypothesis, we created TC-1 cells that secrete a functional anti-CTLA-4 antibody (TC-1/alphaCTLA-4-gamma1 cells). When injected into immunocompetent mice, the growth of TC-1/alphaCTLA-4-gamma1 tumors was delayed compared with control TC-1 cells and accompanied by a reversion of the intratumoral Treg/CD8 ratio due to an increase in tumor-infiltrating IFNgamma-producing CD8+ cells. When local anti-CTLA-4 antibody production was combined with Treg inhibition, permanent TC-1 tumor regression and immunity was induced. Importantly, no signs of autoimmunity were detected in mice that received local CTLA-4 blockade alone or in combination with Treg depletion.

Effect of adenovirus-mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on antitumor immune responses

Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen-presenting cells. In this study, we tested whether adenovirus-mediated overexpression of secreted or membrane-associated forms of gp96 in tumor cells would stimulate an antitumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing s.c. tumors derived from syngeneic TC-1 cells, a cell line that expresses HPV16 E6 and E7 proteins. We found that secreted gp96 can induce protective and therapeutic antitumor immune responses. Our data also indicate that the antitumor effect of sgp96 expression seems to be limited by the induction of suppressive regulatory T cells (Treg). TC-1 tumor transplantation increased the number of splenic and tumor-infiltrating Tregs. Importantly, treatment of mice with low-dose cyclophosphamide decreased the number of Tregs and enhanced the immunostimulatory effect of sgp96 expression. We also tested whether an oncolytic vector (Ad.IR-E1A/TRAIL), that is able to induce tumor cell apoptosis and, potentially, release cryptic tumor epitopes in immunogenic form, could stimulate antitumor immune responses. Although tumor cells infected ex vivo with Ad.IR-E1A/TRAIL had no antitumor effect when used as a vaccine alone, the additional treatment with low-dose cyclophosphamide resulted in the elimination of pre-established tumors. This study gives a rationale for testing approaches that suppress Tregs in combination with oncolytic or immunostimulatory vectors.

Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob

ABSTRACT Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a KD (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.

Role of Cellular Heparan Sulfate Proteoglycans in Infection of Human Adenovirus Serotype 3 and 35

Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection.

Epithelial phenotype confers resistance of ovarian cancer cells to oncolytic adenoviruses

We studied the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, we discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie-adenovirus receptor or CD46. Specifically, we found that these adenovirus receptors were trapped in tight junctions and not accessible for virus binding. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-to-mesenchymal transition (EMT). We showed that specific, thus far little-explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14) that use receptor(s) other than coxsackie-adenovirus receptor and CD46 were able to trigger EMT in epithelial ovarian cancer cells and cause efficient oncolysis. Our studies on ovarian cancer cultures and xenografts also revealed several interesting cancer cell biology features. Tumors in situ as well as tumor xenografts in mice mostly contained epithelial cells and cells that were in a hybrid stage where they expressed both epithelial and mesenchymal markers (epithelial/mesenchymal cells). These epithelial/mesenchymal cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT and differentiate into mesenchymal cells. Our study provides a venue for improved virotherapy of cancer as well as new insights into cancer cell biology.

In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

ABSTRACT Gene transfer vectors containing adenovirus (Ad) serotype 35 (Ad35) fibers have shown promise for cancer and stem cell gene therapy. In this study, we attempted to improve the in vitro and in vivo infection properties of these vectors by increasing their affinity to the Ad35 fiber receptor CD46. We constructed Ad vectors containing either the wild-type Ad35 fiber knob (Ad5/35) or Ad35 knob mutants with 4-fold- and 60-fold-higher affinity to CD46 (Ad5/35+ and Ad5/35++, respectively). In in vitro studies with cell lines, the higher affinities of Ad5/35+ and Ad5/35++ to CD46 did not translate into correspondingly higher transduction efficiencies, regardless of the CD46 receptor density present on cells. However, in vivo, in a mouse model with preestablished CD46high liver metastases, intravenous injection of Ad5/35++ resulted in more-efficient tumor cell transduction. We conclude that Ad5/35 vectors with increased affinity to CD46 have an advantage in competing with non-CD46-mediated sequestration of vector particles after intravenous injection.

In situ adenovirus vaccination engages T effector cells against cancer

The efficacy of cancer immunotherapy is limited because of central and peripheral immune tolerance towards tumor-antigens. We propose a novel approach based on the fact that the immune system has not evolved tolerance towards adenoviruses (Ads) and that Ads have not evolved efficient mechanisms for immune-escape. The host-response to intratumoral Ad-vector injection in mice that were immunologically tolerant to neu-positive syngeneic mammary-cancer (MMC) was investigated. Intratumoral injection with replication-deficient, transgene-devoid Ad induced immune responses at two different anatomical sites: the tumor-draining lymph nodes and the tumor microenvironment. The lymph nodes supported the generation of both neu- and Ad-specific T effector cells, while inside the tumor microenvironment only Ad-specific T cells expanded. Importantly, Ad-specific T cells were anti-tumor-reactive despite the presence of active regulatory T cell-mediated immune tolerance inside MMC tumors and anti-tumor efficacy of Ad was increased by pre-immunization against Ad despite the production of Ad-neutralizing antibodies.

Prophylactic transfer of BCR-ABL–, PR1-, and WT1-reactive donor T cells after T cell–depleted allogeneic hematopoietic cell transplantation in patients with chronic myeloid leukemia

Donor lymphocyte infusions have been effective in patients with chronic myeloid leukemia (CML) relapsing after allogeneic stem cell transplantation, but their use is associated with the risk of graft-versus-host disease. We investigated the effects of prophylactic infusion of in vitro-generated donor T cells reactive against peptides derived from CML-associated antigens. Fourteen CML patients received conditioning therapy followed by CD34(+)-selected peripheral blood stem cells from matched siblings (n = 7) or unrelated (n = 7) donors. Donor-derived mature dendritic cells generated in vitro from CD14(+) monocytes were loaded with human leukocyte Ag-restricted peptides derived from PR1, WT1, and/or B-cell receptor-ABL and used to repetitively stimulate donor CD8(+) T cells in the presence of IL-2 and IL-7. Stimulated T cells were infused 28, 56, and 112 days after transplantation. Thirteen patients are alive and 7 remain in molecular remission (median follow-up, 45 months). Interestingly, all 4 patients receiving CD8(+) T cells displaying marked cytotoxic activity in vitro and detectable peptide-reactive CD8(+) T cells during follow-up have not experienced graft-versus-host disease or relapse. Our study reveals that prophylactic infusion of allogeneic CD8(+) T cells reactive against peptides derived from CML-associated antigens is a safe and promising therapeutic strategy. This trial was registered at www.clinicaltrials.gov as #NCT00460629.

Toward a stem cell gene therapy for breast cancer

Current approaches for treatment of late-stage breast cancer rarely result in a long-term cure. In part this is due to tumor stroma that prevents access of systemically or intratumorally applied therapeutics. We propose a stem cell gene therapy approach for controlled tumor stroma degradation that uses the pathophysiologic process of recruitment of inflammatory cells into the tumor. This approach involves genetic modification of hematopoietic stem cells (HSCs) and their subsequent transplantation into tumor-bearing mice. We show that inducible, intratumoral expression of relaxin (Rlx) either by transplanting tumor cells that contained the Rlx gene or by transplantation of mouse HSCs transduced with an Rlx-expressing lentivirus vector delays tumor growth in a mouse model of breast cancer. The antitumor effect of Rlx was mediated through degradation of tumor stroma, which provided increased access of infiltrating antitumor immune cells to their target tumor cells. Furthermore, we have shown in a human/mouse chimeric model that genetically modified HSCs expressing a transgene can access the tumor site. Our findings are relevant for cancer gene therapy and immunotherapy.