Aberrant angiogenesis in ovarian cancer (OC), driven by excessive vascular endothelial growth factor (VEGF) and other proangiogenic mediators, gives rise to structurally and functionally abnormal tumor vasculature that hinders effective T-cell infiltration. To overcome these barriers, we investigated how modulation of the perivascular niche influences antitumor T-cell trafficking and function in OC. T cells expressing a rearranged TCR transgene specific for SV40 T antigen (TAG) were adoptively transferred into TAG+ MOVCAR 5009 ovarian tumor-bearing SCID mice or syngeneic TgMISIIR-TAg-Low transgenic mice, which express TAG as a self-antigen in the fallopian tube epithelium. Transfers were performed either alone or following treatment with an oncolytic vaccinia virus expressing a CXCR4 antagonist (OV-CXCR4-A) or a control virus (OV-Fc). Compared with OV-Fc, OV-CXCR4-A treatment remodeled the tumor vasculature, inhibited recruitment of VEGF-producing myeloid-derived suppressor cells, and disrupted the proangiogenic microenvironment. These changes enhanced infiltration of adoptively transferred TCRTAG T cells within the perivascular niche, correlating with improved antitumor activity and survival. Collectively, our findings demonstrate that CXCR4 blockade-mediated reprogramming of the perivascular tumor microenvironment promotes effective T-cell trafficking and function, providing a mechanistic rationale for combining oncolytic virotherapy with adoptive cell transfer in OC.

The treatment-induced activation level within the perivascular tumor microenvironment (TME) that supports T-cell trafficking and optimal T-cell differentiation is unknown. We investigated the mechanisms by which inflammatory responses generated by tumor-specific T cells delivered to ovarian tumor-bearing mice alone or after oncolytic vaccinia virus-driven immunogenic cancer cell death affect antitumor efficacy. Analyses of the perivascular TME by spatially resolved omics technologies revealed reduced immunosuppression and increased tumoricidal T-cell trafficking and function after moderate inflammatory responses driven by a CXCR4 antagonist-armed oncolytic virus. Neither weak nor high inflammation created a permissive TME for T-cell trafficking. Notably, treatment-mediated differences in T-cell effector programs acquired within the perivascular TME contrasted with comparable antigenic priming in the tumor-draining lymph nodes regardless of the activation mode of antigen-presenting cells. These findings provide new insights into combinatorial treatment strategies that enable tumor-specific T cells to overcome multiple barriers for enhanced trafficking and control of tumor growth. .

Tumor antigen-driven responses to weakly immunogenic selfantigens and neoantigens directly affect treatment efficacy following immunotherapy. Using orthotopically grown SV40 T antigen+ ovarian carcinoma in antigen-naive wildtype 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 wildtype 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 CXCR4antagonist-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 responses and consequently increases therapeutic efficacy in an immunocompetent ovarian cancer model.

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.

EEG recordings confirm hemispheric lateralization of brain activity during cognitive tasks. The aim of the present study was to investigate spontaneous EEG lateralization under two conditions, waking and REM sleep. Bilateral monopolar EEG was recorded in eight participants using a 12-electrode montage, before the night (5 min eyes closed) and during REM sleep. Spectral analysis (0.75-19.75 Hz) revealed left prefrontal lateralization on total spectrum amplitude power and right occipital lateralization in Delta activity during waking. In contrast, during REM sleep, right frontal lateralization in Theta and Beta activities and right lateralization in occipital Delta activity was observed. These results suggest that spontaneous EEG activities generated during waking and REM sleep are supported in part by a common thalamo-cortical neural network (right occipital Delta dominance) while additional, possibly neuro-cognitive factors modulate waking left prefrontal dominance and REM sleep right frontal dominance.

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)-β 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.

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.

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 CD10(3)+ 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.

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.

Abstract Background: Accumulating evidence indicates that breast cancer progression, resistance to therapies and development of metastasis are driven by cancer cells with stem cell-like characteristics. The increased numbers of cancer stem-like cells (CSCs) have been reported in triple negative breast cancers (TNBCs). Therefore, novel methods for targeting CSC population are needed. Aurora-A kinase (AURKA) has been shown to play a key role in acquisition of stem cell-like properties by breast cancer cells. Furthermore, the CSCs have been demonstrated to have increased sensitivity to AURKA specific inhibitor, MLN8237 (Alisertib). Our group has previously reported that oncolytic vaccinia virus (OVV) preferentially infects CSCs. Therefore we hypothesize that the combined treatment of OVV and AURKA inhibitor will result in overall enhanced anti-tumor activity due to improved elimination of CSCs. Results: We confirmed that AURKA overexpression in MCF-7 breast cancer cell line (MCF-7 AURKA) resulted in increased number of CSCs as shown by upregulation of CD44 antigen and decreases in expression of CD24 antigen. The MCF-7 and MCF-7 AURKA cells were subsequently infected with 0.1 pfu/cell of OVV expressing EGFP (OVV-EGFP). After 72h of incubation, viral replication was analyzed by fluorescent microscopy and standard plaque-forming assay. The analysis revealed over 2-fold increases in viral replication in MCF-7 AURKA cells as compared to the parental counterparts. The enhanced viral replication resulted in decreased viability of MCF-7 AURKA cells when compared to OVV-treated MCF-7 (64% vs 81%, p<0.031). The efficacy of combined treatment of OVV-EGFP and alisertib was next analyzed in MCF-7AURKA cells. The MTT assay analysis revealed 73% decreases in viability of cells treated with combination of oncolytic viortherapy and alisertib, whereas the effect of single treatment resulted in 34% and 52% reduction of viability for OVV-EGFP and alisertib, respectively (p <0.01 and p< 0.024). We next tested the efficiency of OVV-EGFP and Alisertib combination treatment against TNBC cell line, BT549. The cells treated with combination of OVV-EGFP and alisertib showed 63% decreases in cell viability compared to 25% and 46% reductions mediated by OVV-EGFP and alisertib, respectively (p<0.003 and p< 0.03). Conclusions: Combination therapy of alisertib and OVV has a significantly higher anti-cancer cell activity in vitro than either agent used alone, and it is particularly applicable for treatment of cells with CSC phenotype. In vivo studies are currently in progress. The combined treatment of alisertib and OVV represents novel therapeutic approach in treating breast cancer with increased numbers of CSCs, resistance to standard therapies and worse clinical outcomes. The results present a novel treatment approach with high translational potential. Citation Format: Malgorzata Gil, Marcin Komorowski, Danuta Kozbor, Mateusz Opyrchal. Concurrent use of alisertib and vaccinia virus demonstrate increased in vitro efficacy against breast cancer models with elevated numbers of cancer stem-like cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B35.

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.

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.

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.

The technology for production of murine monoclonal antibodies has ad-vanced enormously since its introduction by Köhler and Milstein (1975). However, the production of human monoclonal antibodies by fusion technologies has been hampered, mainly by the current scarcity of suitable human cell lines as fusion partners. Hypoxanthine-aminopterin-thymidine (HAT)-sensitive murine plasmacytomas have been fused with human lymphocytes to yield mouse—human hybrids that secrete human antibody against the Forssman antigen (Nowinski et al., 1980), keyhole limpet hemocyanin (KLH)(Lane et al., 1982), tetanus toxoid (Kozbor et al., 1982b; Butler et al., 1983), human tumor-associated antigen (Schlom et al., 1980, Sikora and Wright, 1981; Sikora and Phillips, 1981), and multiple endocrine organs (Satoh et al., 1983). These interspecies hybridomas preferentially segregate human chromosomes, making it difficult to derive stable lines secreting human antibody. However, the loss of human chromosomes from mouse—human hybridomas is not random. It is known, for example, that human chromosomes 14 (heavy chain) and 22 (A light chain) are preferentially retained, whereas chromosome 2 (K chain) is preferentially lost (Croce et al., 1979; Erikson et al., 1981).

Background: Therapies targeted towards the tumour vasculature can be exploited for the purpose of improving the systemic delivery of oncolytic viruses to tumours. Photodynamic therapy (PDT) is a clinically approved treatment for cancer that is known to induce potent effects on tumour vasculature. In this study, we examined the activity of PDT in combination with oncolytic vaccinia virus (OVV) against primary and metastatic tumours in mice. Methods: The effect of 2-[1-hexyloxyethyl-]-2-devinyl pyropheophorbide-a (HPPH)-sensitised-PDT on the efficacy of oncolytic virotherapy was investigated against subcutaneously implanted syngeneic murine NXS2 neuroblastoma and human FaDu head and neck squamous cell carcinoma xenografts in nude mice. Treatment efficacy was evaluated by monitoring tumour growth and survival. The effects of combination treatment on vascular function were examined using magnetic resonance imaging (MRI) and immunohistochemistry, whereas viral replication in tumour cells was analysed by a standard plaque assay. Normal tissue phototoxicity following PDT-OV treatment was studied using the mouse foot response assay. Results: Combination of PDT with OVV resulted in inhibition of primary and metastatic tumour growth compared with either monotherapy. PDT-induced vascular disruption resulted in higher intratumoural viral titres compared with the untreated tumours. Five days after delivery of OVV, there was a loss of blood flow to the interior of tumour that was associated with infiltration of neutrophils. Administration of OVV did not result in any additional photodynamic damage to normal mouse foot tissue. Conclusion: These results provide evidence into the usefulness of PDT as a means of enhancing intratumoural replication and therapeutic efficacy of OV.

The GD2 ganglioside, displayed by five carbohydrate Neu5Acα2-8Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ 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.

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-Fcγ2a) to deliver the antigenic cassette to the activating Fcγ 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-Fcγ2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcγ2a 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.

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.

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.