Molecular Therapy Oncolytics最新文献

To address CD19 loss from lymphoma after anti-CD19 chimeric antigen receptor (CAR) T cell therapy, we designed a bicistronic construct encoding an anti-CD19 CAR and an anti-CD20 CAR. We detected deletions from the expected bicistronic construct sequence in a minority of transcripts by mRNA sequencing. Loss of bicistronic construct transgene DNA was also detected. Deletions of sequence were present at much higher frequencies in transduced T cell mRNA versus gamma-retroviral vector RNA. We concluded that these deletions were caused by intramolecular template switching of the reverse transcriptase enzyme during reverse transcription of gamma-retroviral vector RNA into transgene DNA of transduced T cells. Intramolecular template switching was driven by repeated regions of highly similar nucleic acid sequence within CAR sequences. We optimized the sequence of the bicistronic CAR construct to reduce repeated regions of highly similar sequences. This optimization nearly eliminated sequence deletions. This work shows that repeated regions of highly similar nucleic acid sequence must be avoided in complex CAR constructs. We further optimized the bicistronic construct by lengthening the linker of the anti-CD20 single-chain variable fragment. This modification increased CD20-specific interleukin-2 release and reduced CD20-specific activation-induced cell death. We selected an optimized anti-CD19/CD20 bicistronic construct for clinical development.

New treatment strategies are urgently needed for glioblastoma (GBM)-a tumor resistant to standard-of-care treatment with a high risk of recurrence and extremely poor prognosis. Based on their intrinsic tumor tropism, adoptively applied mesenchymal stem cells (MSCs) can be harnessed to deliver the theranostic sodium/iodide symporter (NIS) deep into the tumor microenvironment. Interleukin-6 (IL-6) is a multifunctional, highly expressed cytokine in the GBM microenvironment including recruited MSCs. MSCs engineered to drive NIS expression in response to IL-6 promoter activation offer the possibility of a new tumor-targeted gene therapy approach of GBM. Therefore, MSCs were stably transfected with an NIS-expressing plasmid controlled by the human IL-6 promoter (IL-6-NIS-MSCs) and systemically applied in mice carrying orthotopic GBM. Enhanced radiotracer uptake by ¹⁸F-Tetrafluoroborate-PET/magnetic resonance imaging (MRI) was detected in tumors after IL-6-NIS-MSC application as compared with mice that received wild-type MSCs. Ex vivo analysis of tumors and non-target organs showed tumor-specific NIS protein expression. Subsequent ¹³¹I therapy after IL-6-NIS-MSC application resulted in significantly delayed tumor growth assessed by MRI and improved median survival up to 60% of GBM-bearing mice as compared with controls. In conclusion, the application of MSC-mediated NIS gene therapy focusing on IL-6 biology-induced NIS transgene expression represents a promising approach for GBM treatment.

Colorectal cancer (CRC) has the third highest incidence and the second highest mortality in the world, which seriously affects human health, while current treatments methods for CRC, including systemic therapy, preoperative radiotherapy, and surgical local excision, still have poor survival rates for patients with metastatic disease, making it critical to develop new strategies for treating CRC. In this article, we found that the gut microbiota can modulate the signaling pathways of cancer cells through direct contact with tumor cells, generate inflammatory responses and oxidative stress through interactions between the innate and adaptive immune systems, and produce diverse metabolic combinations to trigger specific immune responses and promote the initiation of systemic type I interferon (IFN-I) and anti-viral immunity. In addition, oncolytic virus-mediated immunotherapy for regulating oncolytic virus can directly lyse tumor cells, induce the immune activity of the body, interact with interferon, inhibit the anti-viral effect of IFN-I, and enhance the anti-tumor effect of IFN-II. Interferon plays an important role in the anti-tumor process. We put forward that exploring the effects of intestinal flora and oncolytic virus on interferon to treat CRC is a promising therapeutic option.

Albumin is an attractive candidate carrier for the development of novel therapeutic drugs. Gemcitabine has been FDA approved for the treatment of solid tumors; however, new drugs that optimize gemcitabine delivery are not available for clinical use. The aim of this study was to test the efficacy of a novel albumin-encapsulated gemcitabine prodrug, JNTX-101, and investigate whether Cav-1 expression predicts the therapeutic efficacy of JNTX-101. We first determined the treatment efficacy of JNTX-101 in a panel of pancreatic/lung cancer cell lines and found that increases in Cav-1 expression resulted in higher uptake of albumin, while Cav-1 depletion attenuated the sensitivity of cells to JNTX-101. In addition, decreased Cav-1 expression markedly reduced JNTX-101-induced apoptotic cell death in a panel of cells, particularly in low-serum conditions. Furthermore, we tested the therapeutic efficacy of JNTX-101 in xenograft models and the role of Cav-1 in JNTX-101 sensitivity using a Tet-on-inducible tumor model in vivo. Our data suggest that JNTX-101 effectively inhibits cell viability and tumor growth, and that Cav-1 expression dictates optimal sensitivity to JNTX-101. These data indicate that Cav-1 correlates with JNTX-101 sensitivity, especially under nutrient-deprived conditions, and supports a role for Cav-1 as a predictive biomarker for albumin-encapsulated therapeutics such as JNTX-101.

T cell lymphoma (TCL) is a highly heterogeneous group of diseases with a poor prognosis and low 5-year overall survival rate. The current therapeutic regimens have relatively low efficacy rates. Clinical studies of single-target chimeric antigen receptor T cell (CAR-T cell) therapy in T lymphocytes require large and multiple infusions, increasing the risks and cost of treatment; therefore, optimizing targeted therapy is a way to improve overall prognosis. Despite significant advances in bispecific CAR-T cell therapy to avoid antigen escape in treatment of B cell lymphoma, applying this strategy to TCL requires further investigation. Here, we constructed an alpaca nanobody (Nb) phage library and generated high-affinity and -specificity Nbs targeting CD30 and CD5, respectively. Based on multiple rounds of screening, bispecific NbCD30-CD5-CAR T cells were constructed, and their superior anti-tumor effect against TCL was validated in vitro and in vivo. Our findings demonstrated that Nb-derived bispecific CAR-T cells significantly improved anti-tumor efficacy in TCL treatment compared with single-target CAR-T cells and bispecific single chain variable fragment (scFv)-derived CAR-T cells. Because Nbs are smaller and less immunogenic, the synergistic effect of Nb-based bispecific CAR-T cells may improve their safety and efficacy in future clinical applications.

Unraveling the complexities of the tumor microenvironment (TME) and its correlation with responsiveness to immunotherapy has become a main focus in overcoming resistance to such treatments. Targeting tumor-intrinsic retinoic acid-inducible gene-I (RIG-I), a sensor for viral RNA, was shown to transform the TME from an immunogenically "cold" state to an inflamed, "hot" lesion, which we demonstrated previously to be a crucial mediator of the efficacy of immune checkpoint inhibition with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In this study, we focus on the chimeric oncolytic virus vesicular stomatitis virus (VSV)-Newcastle disease virus (NDV), comprised of genetic components of VSV and NDV, and we investigate its utility to support tumor-intrinsic RIG-I-dependent therapy with anti-CTLA-4. Overall, we demonstrate that treatment with VSV-NDV efficiently delays tumor growth and significantly prolongs survival in a murine model of malignant melanoma, which was further enhanced in combination with anti-CTLA-4. Although the direct oncolytic and pro-inflammatory effects of VSV-NDV therapy were independent of RIG-I activation, the synergism with anti-CTLA-4 therapy and associated activation of tumor-specific T cells was critically dependent on active RIG-I signaling in tumor cells. This work highlights the therapeutic value of utilizing an immune-stimulatory oncolytic virus to sensitize tumors to immune checkpoint inhibition.

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR T cell activity to prevent incoming graft rejection. Transient expression of an anti-CD117 CAR by mRNA conferred T cells the ability to eliminate CD117+ targets in vitro and in vivo. As an alternative approach, we used a Sleeping Beauty transposon vector for the generation of CAR T cells incorporating an inducible Caspase 9 safety switch. Stable CAR expression was associated with high proportion of T memory stem cells, low levels of exhaustion markers, and potent cellular cytotoxicity. Anti-CD117 CAR T cells mediated depletion of leukemic cells and healthy hematopoietic stem cells in NSG mice reconstituted with human leukemia or CD34+ cord blood cells, respectively, and could be terminated in vivo. The use of a non-viral technology to control CAR T cell pharmacokinetic properties is attractive for a first-in-human study in patients with acute myeloid leukemia prior to hematopoietic stem cell transplantation.

Bispecific T cell engagers (BiTEs) represent a promising immunotherapy, but their efficacy against immunologically cold tumors such as pancreatic ductal adenocarcinoma remains unclear. Oncolytic viruses (OVs) can transform the immunosuppressive tumor microenvironment into the active state and also serve as transgene vectors to selectively express the desired genes in tumor cells. This study aimed to investigate whether the therapeutic benefits of tumor-targeting Claudin18.2 BiTE can be augmented by combining cancer selectively and immune-potentiating effects of OVs. Claudin18.2/CD3 BiTE was inserted into herpes simplex virus type 1 (HSV-1) to construct an OV-BiTE. Its expression and function were assessed using reporter cells and peripheral blood mononuclear cell (PBMC) co-culture assays. Intratumoral application of OV-BiTE restrained tumor growth and prolonged mouse survival compared with the unarmed OV in xenograft models and syngeneic mice bearing CLDN18.2-expressing KPC or Pan02 pancreatic cancer cells. Flow cytometry of tumor-infiltrating immune cells suggested both OV-BiTE and the unarmed OV remodeled the tumor microenvironment by increasing CD4+ T cell infiltration and decreasing regulatory T cells. OV-BiTE further reprogrammed macrophages to a more pro-inflammatory antitumor state, and OV-BiTE-induced macrophages exhibited greater cytotoxicity on the co-cultured tumor cell. This dual cytotoxic and immunomodulatory approach warrants further development for pancreatic cancer before clinical investigation.

Prostate cancer (PCa) incidence is increasing and endangers men's lives. Early detection of PCa could improve overall survival (OS) by preventing metastasis. The prostate-specific antigen (PSA) test is a popular screening method. Several advisory groups, however, warn against using the PSA test due to its high false positive rate, unsupported outcome, and limited benefit. The number of disease-related biopsies performed annually far outweighs the number of diagnoses. Thus, there is an urgent need to develop accurate diagnostic biomarkers to detect PCa and distinguish between aggressive and indolent cancers. Recently, non-coding RNA (ncRNA), circulating tumor DNA (ctDNA)/ctRNA, exosomes, and metabolomic biomarkers in the liquid biopsies (LBs) of patients with PCa showed significant differences and clinical benefits in diagnosis, prognosis, and monitoring response to therapy. The analysis of urinary exosomal ncRNA presented a substantial correlation among Exos-miR-375 downregulation, clinical T stage, and bone metastases of PCa. Furthermore, the expression of miR-532-5p in urine samples was a vital predictive biomarker of PCa progression. Thus, this review focuses on promising molecular and metabolomic biomarkers in LBs from patients with PCa. We thoroughly addressed the most recent clinical findings of LB biomarker use in diagnosing and monitoring PCa in early and advanced stages.

Integration site (IS) analysis is essential in ensuring safety and efficacy of gene therapies when integrating vectors are used. Although clinical trials of gene therapy are rapidly increasing, current methods have limited use in clinical settings because of their lengthy protocols. Here, we describe a novel genome-wide IS analysis method, "detection of the integration sites in a time-efficient manner, quantifying clonal size using tagmentation sequencing" (DIStinct-seq). In DIStinct-seq, a bead-linked Tn5 transposome is used, allowing the sequencing library to be prepared within a single day. We validated the quantification performance of DIStinct-seq for measuring clonal size with clones of known IS. Using ex vivo chimeric antigen receptor (CAR)-T cells, we revealed the characteristics of lentiviral IS. We then applied it to CAR-T cells collected at various times from tumor-engrafted mice, detecting 1,034-6,233 IS. Notably, we observed that the highly expanded clones had a higher integration frequency in the transcription units and vice versa in genomic safe harbors (GSH). Also, in GSH, persistent clones had more frequent IS. Together with these findings, the new IS analysis method will help to improve the safety and efficacy of gene therapies.