Carboplatin enhances lymphocyte-endothelial interactions to promote CD8+ T cell trafficking into the ovarian tumor microenvironment.

OBJECTIVES: Standard chemotherapy agents, including carboplatin, have known immunogenic properties. We sought to determine how carboplatin may influence lymphocyte trafficking to tumor sites. METHODS: Murine models of ovarian cancer were utilized to examine lymphocyte trafficking with common clinically used agents including carboplatin, anti-PD-1 antibody, or anti-VEGFR-2 antibody. Adhesion interactions of lymphocytes with tumor vasculature were measured using intravital microscopy, lymphocyte homing with immunohistochemistry, and treatment groups followed for overall survival. RESULTS: Carboplatin chemotherapy profoundly alters the tumor microenvironment to promote lymphocyte adhesive interactions with tumor vasculature and resultant improvement in lymphocyte trafficking. The measured results seen with carboplatin in the tumor microenvironment were superior to anti-PD-1 treatment or anti-VEGFR-2 which may have contributed to increased overall survival in carboplatin treated groups. CONCLUSIONS: These novel findings suggest a role for chemotherapeutic agents to broadly influence anti-tumor immune responses beyond the induction of immunogenic tumor cell death.

CXCL12/CXCR4 blockade by oncolytic virotherapy inhibits ovarian cancer growth by decreasing immunosuppression and targeting cancer-initiating cells.

Signals mediated by the chemokine CXCL12 and its receptor CXCR4 are involved in the progression of ovarian cancer through enhancement of tumor angiogenesis and immunosuppressive networks that regulate dissemination of peritoneal metastasis and development of cancer-initiating cells (CICs). In this study, we investigated the antitumor efficacy of a CXCR4 antagonist expressed by oncolytic vaccinia virus (OVV) against an invasive variant of the murine epithelial ovarian cancer cell line ID8-T. This variant harbors a high frequency of CICs that form multilayered spheroid cells and express the hyaluronan receptor CD44, as well as stem cell factor receptor CD117 (c-kit). Using an orthotopic ID8-T tumor model, we observed that i.p. delivery of a CXCR4 antagonist-expressing OVV led to reduced metastatic spread of tumors and improved overall survival compared with oncolysis alone. Inhibition of tumor growth with the armed virus was associated with efficient killing of CICs, reduced expression of ascitic CXCL12 and vascular endothelial growth factor, and decreases in i.p. numbers of endothelial and myeloid cells, as well as plasmacytoid dendritic cells. These changes, together with reduced recruitment of T regulatory cells, were associated with higher ratios of IFN-γ(+)/IL-10(+) tumor-infiltrating T lymphocytes, as well as induction of spontaneous humoral and cellular antitumor responses. Similarly, the CXCR4 antagonist released from virally infected human CAOV2 ovarian carcinoma cells inhibited peritoneal dissemination of tumors in SCID mice, leading to improved tumor-free survival in a xenograft model. Our findings demonstrate that OVV armed with a CXCR4 antagonist represents a potent therapy for ovarian CICs with a broad antitumor repertoire.

Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases.

Oncolytic viruses hold promise for the treatment of cancer, but their interaction with the tumor microenvironment needs to be elucidated for optimal tumor cell killing. Because the CXCR4 receptor for the stromal cell-derived factor-1 (SDF-1/CXCL12) chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their stromal microenvironment, we used oncolytic virotherapy with a CXCR4 antagonist to target the CXCL12/CXCR4 signaling axis in a triple-negative 4T1 breast carcinoma in syngeneic mice. We show here that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with orthotopic tumors attains higher intratumoral concentration than its soluble counterpart and exhibits increased efficacy over that mediated by oncolysis alone. A systemic delivery of the armed virus after resection of the primary tumor was efficacious in inhibiting the development of spontaneous metastasis and increased overall tumor-free survival. Inhibition of tumor growth with the armed virus was associated with destruction of tumor vasculature, reductions in expression of CXCL12 and VEGF, and decrease in intratumoral numbers of bone marrow-derived endothelial and myeloid cells. These changes led to induction of antitumor antibody responses and resistance to tumor rechallenge. Engineering an oncolytic virus armed with a CXCR4 antagonist represents an innovative strategy that targets multiple elements within the tumor microenvironment. As such, this approach could have a significant therapeutic impact against primary and metastatic breast cancer.

BiTE secretion by adoptively transferred stem-like T cells improves FRα+ ovarian cancer control.

BACKGROUND: Cancer immunotherapies can produce complete therapeutic responses, however, outcomes in ovarian cancer (OC) are modest. While adoptive T-cell transfer (ACT) has been evaluated in OC, durable effects are rare. Poor therapeutic efficacy is likely multifactorial, stemming from limited antigen recognition, insufficient tumor targeting due to a suppressive tumor microenvironment (TME), and limited intratumoral accumulation/persistence of infused T cells. Importantly, host T cells infiltrate tumors, and ACT approaches that leverage endogenous tumor-infiltrating T cells for antitumor immunity could effectively magnify therapeutic responses. METHODS: Using retroviral transduction, we have generated T cells that secrete a folate receptor alpha (FRα)-directed bispecific T-cell engager (FR-B T cells), a tumor antigen commonly overexpressed in OC and other tumor types. The antitumor activity and therapeutic efficacy of FR-B T cells was assessed using FRα+ cancer cell lines, OC patient samples, and preclinical tumor models with accompanying mechanistic studies. Different cytokine stimulation of T cells (interleukin (IL)-2+IL-7 vs IL-2+IL-15) during FR-B T cell production and the resulting impact on therapeutic outcome following ACT was also assessed. RESULTS: FR-B T cells efficiently lysed FRα+ cell lines, targeted FRα+ OC patient tumor cells, and were found to engage and activate patient T cells present in the TME through secretion of T cell engagers. Additionally, FR-B T cell therapy was effective in an immunocompetent in vivo OC model, with response duration dependent on both endogenous T cells and FR-B T cell persistence. IL-2/IL-15 preconditioning prior to ACT produced less differentiated FR-B T cells and enhanced therapeutic efficacy, with mechanistic studies revealing preferential accumulation of TCF-1+CD39-CD69- stem-like CD8+ FR B T cells in the peritoneal cavity over solid tumors. CONCLUSIONS: These findings highlight the therapeutic potential of FR-B T cells in OC and suggest FR-B T cells can persist in extratumoral spaces while actively directing antitumor immunity. As the therapeutic activity of infused T cell therapies in solid tumor indications is often limited by poor intratumoral accumulation of transferred T cells, engager-secreting T cells that can effectively leverage endogenous immunity may have distinct mechanistic advantages for enhancing therapeutic responses rates.

Reprogramming the tumor microenvironment leverages CD8+ T cell responses to a shared tumor/self antigen in ovarian cancer.

Tumor antigen-driven responses to weakly immunogenic self-antigens and neoantigens directly affect treatment efficacy following immunotherapy. Using orthotopically grown SV40 T antigen+ ovarian carcinoma in antigen-naive wild-type or TgMISIIR-TAg-Low transgenic mice expressing SV40 T antigen as a self-antigen, we investigated the impact of CXCR4-antagonist-armed oncolytic virotherapy on tumor progression and antitumor immunity. Immunostaining and single-cell RNA sequencing analyses of the peritoneal tumor microenvironment of untreated tumors in syngeneic wild-type mice revealed the presence of SV40 T antigen-specific CD8+ T cells, a balanced M1/M2 transcriptomic signature of tumor-associated macrophages, and immunostimulatory cancer-associated fibroblasts. This contrasted with polarized M2 tumor-associated macrophages, immunosuppressive cancer-associated fibroblasts, and poor immune activation in TgMISIIR-TAg-Low mice. Intraperitoneal delivery of CXCR4-antagonist-armed oncolytic vaccinia virus led to nearly complete depletion of cancer-associated fibroblasts, M1 polarization of macrophages, and generation of SV40 T antigen-specific CD8+ T cells in transgenic mice. Cell depletion studies revealed that the therapeutic effect of armed oncolytic virotherapy was dependent primarily on CD8+ cells. These results demonstrate that targeting the interaction between immunosuppressive cancer-associated fibroblasts and macrophages in the tolerogenic tumor microenvironment by CXCR4-A-armed oncolytic virotherapy induces tumor/self-specific CD8+ T cell responses and consequently increases therapeutic efficacy in an immunocompetent ovarian cancer model.

Targeting a mimotope vaccine to activating Fcgamma receptors empowers dendritic cells to prime specific CD8+ T cell responses in tumor-bearing mice.

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcgamma2a) to deliver the antigenic cassette to the activating Fcgamma receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcgamma2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcgamma2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

Induction of cell death in ovarian cancer cells by doxorubicin and oncolytic vaccinia virus is associated with CREB3L1 activation.

We have demonstrated that oncolytic vaccinia virus synergizes with doxorubicin (DOX) in inducing immunogenic cell death in platinum-resistant ovarian cancer cells and increases survival in syngeneic and xenograft tumor models. However, the mechanisms underlying the virus- and doxorubicin-mediated cancer cell death remain unknown. In this study, we investigated the effect of the oncolytic virus and doxorubicin used alone or in combination on activation of the cytoplasmic transcription factor CREB3L1 (cyclic AMP [cAMP] response element-binding protein 3-like 1) in ovarian cancer cell lines and clinical specimens. We demonstrated that doxorubicin-mediated cell death in ovarian cancer cell lines was associated with nuclear translocation of CREB3L1 and that the effect was augmented by infection with oncolytic vaccinia virus or treatment with recombinant interferon (IFN)-ß used as a viral surrogate. This combination treatment was also effective in mediating nuclear translocation of CREB3L1 in cancer cells isolated from ovarian tumor biopsies at different stages of disease progression. The measurement of CREB3L1 expression in clinical specimens of ovarian cancer revealed lack of correlation with the stage of disease progression, suggesting that understanding the mechanisms of nuclear accumulation of CREB3L1 after doxorubicin treatment alone or in combination with oncolytic virotherapy may lead to the development of more effective treatment strategies against ovarian cancer.

Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells.

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Oncolytic Maraba virus armed with tumor antigen boosts vaccine priming and reveals diverse therapeutic response patterns when combined with checkpoint blockade in ovarian cancer.

BACKGROUND: Cancer immunotherapies are emerging as promising treatment strategies for ovarian cancer patients that experience disease relapse following first line therapy. As such, identifying strategies to bolster anti-tumor immunity and limit immune suppression, while recognizing diverse patterns of tumor response to immunotherapy is critical to selecting treatment combinations that lead to durable therapeutic benefit. METHODS: Using a pre-clinical mouse model, we evaluated a heterologous prime/boost vaccine in combination with checkpoint blockade to treat metastatic intraperitoneal ovarian cancer. Vaccine-elicited CD8+ T cell responses and changes in the tumor microenvironment following treatment were analyzed and compared to treatment outcome. Kinetics of intraperitoneal tumor growth were assessed using non-invasive magnetic resonance imaging (MRI). RESULTS: Vaccine priming followed by antigen-armed oncolytic Maraba virus boosting elicited robust tumor-specific CD8+ T cell responses that improved tumor control and led to unique immunological changes in the tumor, including a signature that correlated with improved clinical outcome of ovarian cancer patients. However, this treatment was not curative and T cells in the tumor microenvironment (TME) were functionally suppressed. Combination PD-1 blockade partially overcame the adaptive resistance in the tumor observed in response to prime/boost vaccination, restoring CD8+ T cell function in the TME and enhancing the therapeutic response. Non-invasive MRI of tumors during the course of combination treatment revealed heterogeneous radiologic response patterns following treatment, including pseudo-progression, which was associated with improved tumor control prior to relapse. CONCLUSIONS: Our findings point to a key hierarchical role for PD-1 signaling and adaptive immune resistance in the ovarian TME in determining the functional fate of tumor-specific CD8+ T cells, even in the context of robust therapy mediated anti-tumor immunity, as well as the ability of multiple unique patterns of therapeutic response to result in durable tumor control.

Recruitment of Intratumoral CD103+ Dendritic Cells by a CXCR4 Antagonist-Armed Virotherapy Enhances Antitumor Immunity.

Intratumoral dendritic cells play an important role in stimulating cytotoxic T cells and driving antitumor immunity. Using a metastatic ovarian tumor model in syngeneic mice, we explored whether therapy with a CXCR4 antagonist-armed oncolytic vaccinia virus activates endogenous CD103+ dendritic cell responses associated with the induction of adaptive immunity against viral and tumor antigens. The overall goal of this study was to determine whether expansion of CD103+ dendritic cells by the virally delivered CXCR4 antagonist augments overall survival and in situ boosting with a tumor antigen peptide-based vaccine. We found that locoregional delivery of the CXCR4-A-armed virus reduced the tumor load and the immunosuppressive network in the tumor microenvironment, leading to infiltration of CD103+ dendritic cells that were capable of phagocytic clearance of cellular material from virally infected cancer cells. Further expansion of tumor-resident CD103+ DCs by injecting the FMS-related tyrosine kinase 3 ligand, the formative cytokine for CD103+ DCs, provided a platform for a booster immunization with the Wilms tumor antigen 1 peptide-based vaccine delivered intraperitoneally with polyriboinosinic:polyribocytidylic acid as an adjuvant. The vaccine-induced antitumor responses inhibited tumor growth and increased overall survival, indicating that expansion of intratumoral CD103+ dendritic cells by CXCR4-A-armed oncovirotherapy treatment can potentiate in situ cancer vaccine boosting.

Modulation of the Tumor Microenvironment by CXCR4 Antagonist-Armed Viral Oncotherapy Enhances the Antitumor Efficacy of Dendritic Cell Vaccines against Neuroblastoma in Syngeneic Mice.

The induction of antitumor immune responses in tumor-bearing hosts depends on efficient uptake and processing of native or modified tumors/self-antigens by dendritic cells (DCs) to activate immune effector cells, as well as the extent of the immunosuppressive network in the tumor microenvironment (TME). Because the C-X-C motif chemokine receptor 4 (CXCR4) for the C-X-C motif chemokine 12 (CXCL12) is involved in signaling interactions between tumor cells and their TME, we used oncolytic virotherapy with a CXCR4 antagonist to investigate whether targeting of the CXCL12/CXCR4 signaling axis in murine neuroblastoma cells (NXS2)-bearing syngeneic mice affects the efficacy of bone marrow (BM)-derived DCs loaded with autologous tumor cells treated with doxorubicin for induction of immunogenic cell death. Here, we show that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with neuroblastoma tumors augmented efficacy of the DC vaccines compared to treatments mediated by a soluble CXCR4 antagonist or oncolysis alone. This study is the first demonstration that modulating the tumor microenvironment by an armed oncolytic virus could have a significant impact on the efficacy of DC vaccines, leading to the generation of effective protection against neuroblastoma challenge.

Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library.

Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.

DNA vaccine expressing the mimotope of GD2 ganglioside induces protective GD2 cross-reactive antibody responses.

The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.

Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy.

Ovarian cancer remains the most lethal gynecologic malignancy owing to late detection, intrinsic and acquired chemoresistance, and remarkable heterogeneity. Here, we explored approaches to inhibit metastatic growth of murine and human ovarian tumor variants resistant to paclitaxel and carboplatin by oncolytic vaccinia virus expressing a CXCR4 antagonist to target the CXCL12 chemokine/CXCR4 receptor signaling axis alone or in combination with doxorubicin. The resistant variants exhibited augmented expression of the hyaluronan receptor CD44 and CXCR4 along with elevated Akt and ERK1/2 activation and displayed an increased susceptibility to viral infection compared with the parental counterparts. The infected cultures were more sensitive to doxorubicin-mediated killing both in vitro and in tumor-challenged mice. Mechanistically, the combination treatment increased apoptosis and phagocytosis of tumor material by dendritic cells associated with induction of antitumor immunity. Targeting syngeneic tumors with this regimen increased intratumoral infiltration of antitumor CD8+ T cells. This was further enhanced by reducing the immunosuppressive network by the virally-delivered CXCR4 antagonist, which augmented antitumor immune responses and led to tumor-free survival. Our results define novel strategies for treatment of drug-resistant ovarian cancer that increase immunogenic cell death and reverse the immunosuppressive tumor microenvironment, culminating in antitumor immune responses that control metastatic tumor growth.

Cancer vaccine with mimotopes of tumor-associated carbohydrate antigens.

The GD2 ganglioside, displayed by five carbohydrate Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glcbeta residues attached to a ceramide chain that anchors the ganglioside in the cell membrane, is expressed on neuroectodermally derived tumors. GD2 has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have generated 47-LDA mimotope of GD2 by screening a phage display peptide library with anti-GD2 mAb 14G2a and reported that vaccination with the 47-LDA mimotope elicited GD2 cross-reactive IgG antibody responses as well as MHC class I-restricted CD8(+) T cells to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated antigen cross-reacting with 47-LDA. Immunoblotting studies using 14G2a mAb demonstrated that this antibody cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional (3D) collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by 105 kDa activated leukocyte cell adhesion molecules (ALCAM/CD166). The CD166 glycoprotein was shown to be recognized by 14G2a antibody, and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166 and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate antigens.

The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The majority of children suffers from high risk neuroblastoma and has disseminated disease at the time of diagnosis. Despite recent advances in chemotherapy, the prognoses for children with high risk NB remain poor. Therefore, new treatment modalities are urgently needed. GD2 ganglioside is an antigen that is highly expressed on NB cells with only limited distribution on healthy tissues. Consequently, it appears to be an ideal target for both active and passive immunotherapy. The immunological effector mechanisms mediated by anti-GD2 monoclonal antibodies (mAbs) have been already well characterized. However, a growing number of reports suggest that GD2-specific antibodies may exhibit anti-proliferative effects without the immune system involvement. Here, we have shown that anti-GD2 14G2a mAb is capable of decreasing survival of IMR-32 human neuroblastoma cells in a dose-dependent manner. Death induced by this antibody exhibited several characteristics typical for apoptosis such as increased number of Annexin V- and propidium iodide-positive cells, cleavage of caspase 3 and prominent rise in caspase activity. The use of a pan caspase inhibitor Z-VAD-fmk suggested that the killing potential of this mAb is partially caspase-dependent. 14G2a mAb was rapidly endocytosed upon antigen binding. Employment of chloroquine, an inhibitor of lysosomal degradation, did not rescue IMR-32 cells from antibody-induced cell death suggesting lack of ceramide involvement in the observed effect. Most importantly, our studies showed that at particular drug concentrations 14G2a mAb exerts a synergistic effect with doxorubicin and topotecan, as well as an additive effect with carboplatin in killing IMR-32 cells in vitro. Our results provide guidance regarding how to best combine GD2-specific 14G2a antibody with existing cancer therapeutic agents to improve available treatment modalities for neuroblastoma.

Therapeutic effect of a T helper cell supported CTL response induced by a survivin peptide vaccine against murine cerebral glioma.

Survivin is a tumor-associated antigen (TAA) that has significant potential for use as a cancer vaccine target. To identify survivin epitopes that might serve as targets for CTL-mediated, anti-tumor responses, we evaluated a series of survivin peptides with predicted binding to mouse H2-K(b) and human HLA-A*0201 antigens in peptide-loaded dendritic cell (DC) vaccines. H2-K(b)-positive, C57BL/6 mice were vaccinated using syngeneic, peptide-loaded DC2.4 cells. Splenocytes from vaccinated mice were screened by flow cytometry for binding of dimeric H2-K(b):Ig to peptide-specific CD8+ T cells. Two survivin peptides (SVN(57-64) and SVN(82-89)) generated specific CD8+ T cells. We chose to focus on the SVN(57-64) peptide because that region of the molecule is 100% homologous to human survivin. A larger peptide (SVN(53-67)), containing multiple class I epitopes, and a potential class II ligand, was able to elicit both CD8+ CTL and CD4+ T cell help. We tested the SVN(53-67) 15-mer peptide in a therapeutic model using a peptide-loaded DC vaccine in C57BL/6 mice with survivin-expressing GL261 cerebral gliomas. This vaccine produced significant CTL responses and helper T cell-associated cytokine production, resulting in a significant prolongation of survival. The SVN(53-67) vaccine was significantly more effective than the SVN(57-64) core epitope as a cancer vaccine, emphasizing the potential benefit of incorporating multiple class I epitopes and associated cytokine support within a single peptide.

Oral vaccination with modified vaccinia virus Ankara attached covalently to TMPEG-modified cationic liposomes overcomes pre-existing poxvirus immunity from recombinant vaccinia immunization.

Development of a safe and effective vaccine for induction of mucosal immunity to the human immunodeficiency virus (HIV) envelope glycoprotein (Env, gp160) represents the best hope for containing the spread of an HIV epidemic worldwide. The highly attenuated modified vaccinia virus Ankara (MVA) is a laboratory virus well suited as a safe vaccine vector. However, the presence of pre-existing immunity to Vaccinia virus in the adult population represents a hindrance that limits the application of the MVA vector for inducing immunity to HIV antigens. Here, cationic liposomes were covalently attached to the surface of recombinant MVA expressing the HIV-1 strain IIIB Env glycoprotein and beta-galactosidase (MVA(IIIB/beta-gal)) using tresylmonomethoxypolyethylene glycol (TMPEG) grafted into a lipid membrane without compromising viral infectivity in vitro and in vivo. The orally administered MVA(IIIB/beta-gal)-TMPEG/liposome complexes were capable of delivering the transgenes to mucosal tissues in mice with pre-existing poxvirus immunity based on beta-galactosidase gene expression in intestinal tissues measured 18 h after infection. Importantly, the MVA(IIIB/beta-gal)-TMPEG/liposome complexes enhanced Env-specific cellular and humoral immune responses in the mucosal and systemic tissues after repeated oral immunization of BALB/c mice. This approach may prove useful for induction of protective immunity against infectious diseases and cancer in populations with pre-existing immunity to vaccinia from smallpox vaccination.

Induction of protective immune responses against NXS2 neuroblastoma challenge in mice by immunotherapy with GD2 mimotope vaccine and IL-15 and IL-21 gene delivery.

The GD2 ganglioside expressed on neuroectodermal tumor cells is weakly immunogenic in tumor-bearing patients and induces predominantly IgM antibody responses in the immunized host. Using a syngeneic mouse challenge model with GD2-expressing NXS2 neuroblastoma, we investigated novel strategies for augmenting the effector function of GD2-specific antibody responses induced by a mimotope vaccine. We demonstrated that immunization of A/J mice with DNA vaccine expressing the 47-LDA mimotope of GD2 in combination with IL-15 and IL-21 genes enhanced the induction of GD2 cross-reactive IgG2 antibody responses that exhibited cytolytic activity against NXS2 cells. The combined immunization regimen delivered 1 day after tumor challenge inhibited subcutaneous (s.c.) growth of NXS2 neuroblastoma in A/J mice. The vaccine efficacy was reduced after depletion of NK cells as well as CD4(+) and CD8(+) T lymphocytes suggesting involvement of innate and adaptive immune responses in mediating the antitumor activity in vivo. CD8(+) T cells isolated from the immunized and cured mice were cytotoxic against syngeneic neuroblastoma cells but not against allogeneic EL4 lymphoma, and exhibited antitumor activity after adoptive transfer in NXS2-challenged mice. We also demonstrated that coimmunization of NXS2-challenged mice with the IL-15 and IL-21 gene combination resulted in enhanced CD8(+) T cell function that was partially independent of CD4(+) T cell help in inhibiting tumor growth. This study is the first demonstration that the mimotope vaccine of a weakly immunogenic carbohydrate antigen in combination with plasmid-derived IL-15 and IL-21 cytokines induces both innate and adaptive arms of the immune system leading to the generation of effective protection against neuroblastoma challenge.

Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery.

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env(121-129) epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.