INTRODUCTION: Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. Immunotherapy presents a promising alternative by specifically ...targeting cancer cells. To date, there have been no successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting proliferation of progenitor cell populations within the developing cerebellum. Using total embryonic RNA as a source of tumor rejection antigens is attractive because it can be delivered as a single vaccine, target both known and unknown fetal proteins, and can be refined to preferentially treat distinct MB subtypes. METHODS: We have created two transplantable, syngeneic animal MB models recapitulating human SHH and Group 3 variants to investigate the immunologic targeting of different MB subtypes. We generated T cells specific to the developing mouse cerebellum (P5) and tested their reactivity to target cells pulsed with total RNA from two MB subtypes and the normal brain. Immune responses were evaluated by measuring cytokine secretion following re-stimulation of activated T cells with both normal and tumor cell targets. In vivo antitumor efficacy was also tested in survival studies of intracranial tumor-bearing animals. RESULTS: We generated T cells specific to the developing cerebellum in vitro, confirming the immunogenicity of developmentally regulated antigens. Additionally, we have shown that developmental antigen-specific T cells produce high levels of Th1-type cytokines in response to tumor cells of two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells do not show cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens also conferred a significant survival benefit in a treatment model of Group 3 tumor bearing animals. CONCLUSIONS: Developmental antigens can safely target multiple MB subtypes with equal effectiveness compared to previously established total tumor strategies.
Abstract
Recent reports describing allogeneic chimeric antigen receptor (CAR) T cell therapies have generated tremendous excitement over the possibility of a universal CAR T platform. We are ...utilizing a highly engineered meganuclease designed to target the T cell receptor (TCR) alpha chain to eliminate expression of the TCR, preventing graft versus host disease following adoptive transfer. Meganuclease cleavage efficiency is highly dependent on stimulation of T cells prior to delivery of the meganuclease. To determine optimal activation conditions for TCR knockout and cell yield, T cells were activated with Dynabeads® Human T cell Activator CD3/CD28, Immunocult™ CD3/CD28 and CD3/CD28/CD2 T Cell Activators, and MACS® GMP TransAct CD3/CD28 reagent prior to nucleofection. T cells were stained prior to and following nucleofection to identify cell activation markers and cytokine receptors (CD69, IL2R, IL7R, IL15R, IL21R). Knockout efficiency was determined by staining for CD3 expression. Our results indicate that Dynabead-stimulated T cells expressed the highest levels of activation markers at 18 hours post-stimulation, and that Dynabeads® and Immunocult™ CD3/CD28/CD2 reagents both promote robust T cell proliferation and support the highest TCR knockout efficiency following our protocol. Furthermore, expression of cytokine receptors varies both during stimulation and post-nucleofection, suggesting an optimal cocktail of cytokines may promote T cell proliferation. Future studies will focus on further optimization of T cell stimulation conditions that may promote TCR knockout and expansion of T cells post-nucleofection.
INTRODUCTION: Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. Immunotherapy presents a promising alternative by specifically ...targeting cancer cells. To date, there have been no successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting proliferation of progenitor cell populations within the developing cerebellum. Using total embryonic RNA as a source of tumor rejection antigens is attractive because it can be delivered as a single vaccine, target both known and unknown fetal proteins, and can be refined to preferentially treat distinct MB subtypes. METHODS: We have created two transplantable, syngeneic animal MB models recapitulating human SHH and Group 3 variants to investigate the immunologic targeting of different MB subtypes. We generated T cells specific to the developing mouse cerebellum (P5) and tested their reactivity to target cells pulsed with total RNA from two MB subtypes and the normal brain. Immune responses were evaluated by measuring cytokine secretion following re-stimulation of activated T cells with both normal and tumor cell targets. In vivo antitumor efficacy was also tested in survival studies of intracranial tumor-bearing animals. RESULTS: We generated T cells specific to the developing cerebellum in vitro, confirming the immunogenicity of developmentally regulated antigens. Additionally, we have shown that developmental antigen-specific T cells produce high levels of Th1-type cytokines in response to tumor cells of two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells do not show cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens also conferred a significant survival benefit in a treatment model of Group 3 tumor bearing animals. CONCLUSIONS: Developmental antigens can safely target multiple MB subtypes with equal effectiveness compared to previously established total tumor strategies.
BACKGROUND: Glioblastoma multiforme (GBM) remains invariably associated with poor patient outcomes thus necessitating the development of more targeted therapeutics. To circumvent the challenges ...associated with cellular therapeutics, we have utilized clinical-grade nanoparticle (NP) vaccine formulations that are known to traffic through the liver. Since the liver is a profoundly important lymphoid organ enriched with innate immune cells that must function in the dualistic role of tolerance when constitutively engaged by gastrointestinal ligands and heighted inflammatory responses when engaged by viral elements, it represents a profound immunological niche for re-directing the immune system in the presence of specific ‘danger’ signals. Therefore, using tumor derived RNA from intracranial gliomas, imbued with the appropriate immunomodulatory RNAs packaged into an "off the shelf" NP vaccine formulation, we aim to harness the liver's immunologic potential against GBM. METHODS: We sought to assess if vaccination with autologously derived total tumor RNA encapsulated in lipophilic NPs could transfect liver antigen-presenting cells (APCs) and induce anti-tumor immunity in pre-clinical immunocompetent murine GBM models. RESULTS: We screened clinically available nanoliposomal formulations, identified a suitable NP courier for delivery of encapsulated RNA to liver APCs, and demonstrated effective anti-tumor activity against murine GBMs. These NPs were shown to preferentially transfect hepatic cells and APCs therein generating a preponderance of antigen specific T cell immunity against tumor antigens. The percentage of antigen specific T cells was greater from harvested livers compared with harvested secondary lymphoid organs. Moreover, we demonstrated the capacity to significantly enhance the immunity and anti-tumor efficacy of this platform through incorporation of immunomodulatory RNAs encoding for cytokines such as GM-CSF. CONCLUSION: RNA-NPs represent a novel therapeutic platform for inducing potent nontoxic immunity against gliomas by harnessing the immunologic potential of the liver, thus providing a more effective and specific therapy critical in improving clinical outcomes for patients with GBM.
Adoptive cellular therapy after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has previously been limited to melanoma due to a lack of reliable methods for ...expanding tumor-specific lymphocytes from the majority of other cancers. We demonstrate that tumor RNA-pulsed dendritic cells can be used to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative adoptive cellular therapy in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative conditioning and hematopoietic stem cell transfer, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed dendritic cell (DC) vaccines. By employing this platform we activate a polyclonal population of tumor reactive T cells and have further identified tumor-specific repertoires of tumor-reactive CD8+ T lymphocytes. Furthermore, we have observed a significant increase of clonotypic frequency of tumor-specific CD8+ cells by following TCR β chain expression in mice that are cured of established tumor by adoptive cellular therapy, indicating the ability to potentially predict treatment outcomes in a preclinical setting. Clinical studies exploring the feasibility and potential survival benefit of this platform against both pediatric and adult CNS malignancies are currently underway in our institution. Our adoptive cellular therapy platform has demonstrated superior immunologic treatment of malignant gliomas and holds potential against other solid tumors.
Adoptive cellular therapy after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has previously been limited to melanoma due to a lack of reliable methods for ...expanding tumor-specific lymphocytes from the majority of other cancers. We demonstrate that tumor RNA-pulsed dendritic cells can be used to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative adoptive cellular therapy in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative conditioning and hematopoietic stem cell transfer, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed dendritic cell (DC) vaccines. By employing this platform we activate a polyclonal population of tumor reactive T cells and have further identified tumor-specific repertoires of tumor-reactive CD8+ T lymphocytes. Furthermore, we have observed a significant increase of clonotypic frequency of tumor-specific CD8+ cells by following TCR beta chain expression in mice that are cured of established tumor by adoptive cellular therapy, indicating the ability to potentially predict treatment outcomes in a preclinical setting. Clinical studies exploring the feasibility and potential survival benefit of this platform against both pediatric and adult CNS malignancies are currently underway in our institution. Our adoptive cellular therapy platform has demonstrated superior immunologic treatment of malignant gliomas and holds potential against other solid tumors.
Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. The heterogeneity of MB molecular subtypes as well as the failure of standard ...therapies to treat metastatic or recurrent disease necessitates more potent targeted approaches that minimize collateral toxicity. Immunotherapy presents a promising strategy by specifically targeting cancer cells and to date, there have been few successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting the proliferation and differentiation of progenitor cell populations within the developing cerebellum. To test the developing cerebellum as a source of tumor rejection antigens, we adapted two animal models of MB recapitulating human Sonic Hedgehog (SHH) and Group 3 tumors for immunotherapeutic evaluation. Immunologic characterization of these murine models revealed subtype-specific differences in the tumor microenvironment and a differential response to immune checkpoint blockade. We used total embryonic RNA from the developing mouse cerebellum (P5) to generate antigen-specific T cells and confirmed the immunogenicity of targeting developmentally regulated antigens in vitro. Developmental antigen-specific T cells produced high levels of Th1-type cytokines in response to two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells did not show any cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens conferred a significant survival benefit and long term cures in intracranial treatment models of SHH and Group 3 tumor bearing animals. We additionally tested whether the enrichment of select developmental antigens through the exclusion of normal brain transcripts would potentiate antitumor responses in both animal models. Finally, we evaluated the relevance of targeting fetal antigens across human MB subtypes. Our studies demonstrate that developmental antigens can safely target multiple MB subtypes and can be further refined to preferentially target individual subgroups. Further studies targeting immunogenic developmental antigens and leveraging this strategy with specific immune modulatory interventions represent a novel approach at utilizing patient molecular classification information to mediate safe and effective immunotherapy.
Plastoglobulins (PGL) are the predominant proteins of lipid globules in the plastids of flowering plants. Genes encoding proteins similar to plant PGL are also present in algae and cyanobacteria but ...in no other organisms, suggesting an important role for these proteins in oxygenic photosynthesis. To gain an understanding of the core and fundamental function of PGL, the two genes that encode PGL-like polypeptides in the cyanobacterium Synechocystis sp. PCC 6803 (pgl1 and pgl2) were inactivated individually and in combination. The resulting mutants were able to grow under photoautotrophic conditions, dividing at rates that were comparable to that of the wild-type (WT) under low-light (LL) conditions (10 microeinsteins x m(-2) x s(-1)) but lower than that of the WT under moderately high-irradiance (HL) conditions (150 microeinsteins x m(-2) x s(-1)). Under HL, each Deltapgl mutant had less chlorophyll, a lower photosystem I (PSI)/PSII ratio, more carotenoid per unit of chlorophyll, and very much more myxoxanthophyll (a carotenoid symptomatic of high light stress) per unit of chlorophyll than the WT. Large, heterogeneous inclusion bodies were observed in cells of mutants inactivated in pgl2 or both pgl2 and pgl1 under both LL and HL conditions. The mutant inactivated in both pgl genes was especially sensitive to the light environment, with alterations in pigmentation, heterogeneous inclusion bodies, and a lower PSI/PSII ratio than the WT even for cultures grown under LL conditions. The WT cultures grown under HL contained 2- to 3-fold more PGL1 and PGL2 per cell than cultures grown under LL conditions. These and other observations led us to conclude that the PGL-like polypeptides of Synechocystis play similar but not identical roles in some process relevant to the repair of photooxidative damage.
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