cDNA microarray assays to evaluate immune responses following intracranial injection of baculoviral vectors in non-human primates.

Recombinant insect baculoviral vectors efficiently transduce several types of cells in the brain and can possibly be used in gene therapy for brain disorders. However, together with contaminating insect cell proteins, they trigger immune responses that might damage host brain cells. To substantially reduce unwanted immune responses due to the insect cell impurities, we purified and concentrated baculoviral vectors by combining an ion-exchange membrane chromatography method with high-speed centrifugation and demonstrated reduced immune responses of the vector preparations in the mouse brain. To verify the suitability of using these viral vectors for gene therapy strategies in the brain, we evaluated immune reactions and vector toxicity upon acute administration of baculoviral vectors into the brains of cynomolgus macaques. We demonstrated that the virus inoculation caused no abnormalities to non-human primates but induced host anti-viral responses in the brain. With global cellular gene expression profiling, using cDNA microarray technology, we detected that the affected genes were mainly associated with innate immunity, involving the genes of RIG-1-like receptor signaling pathway as the major interferon production pathway. These findings in non-human primates, which share close physiological and genomic similarities with man, may better mimic the responses to baculoviral transduction in the human brain and should be useful in guiding rational therapeutic applications of baculoviral vectors to treat brain disorders.

Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy.

Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer.

Manufacturing of AcMNPV baculovirus vectors to enable gene therapy trials.

Over the past two decades, baculoviruses have become workhorse research tools for transient transgene expression. Although they have not yet been used directly as a gene therapy vector in the clinical setting, numerous preclinical studies have suggested the highly promising potential of baculovirus as a delivery vector for a variety of therapeutic applications including vaccination, tissue engineering, and cancer treatment. As such, there is growing interest in using baculoviruses as human gene therapy vectors, which has led to advances in baculovirus bioprocessing methods. This review provides an overview of the current approaches for scaled-up amplification, concentration, purification, and formulation of AcMNPV baculoviruses, and highlights the key regulatory requirements that must be met before gene therapy clinical trials can be initiated.