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MazF is an Escherichia coli-derived endoribonuclease that selectively cleaves ACA sequences of mRNA prevalent in HIV. We administered a single infusion of autologous CD4 T lymphocytes modified to express a Tat-dependent MazF transgene to 10 HIV-infected individuals (six remaining on antiretroviral therapy [ART]; four undergoing treatment interruption post-infusion) in order to provide a population of HIV-resistant immune cells. In participants who remained on ART, increases in CD4 and CD8 T cell counts of ~200 cells/mm(3) each occurred within 2 weeks of infusion and persisted for at least 6 months. Modified cells were detectable for several months in the blood and trafficked to gastrointestinal lymph tissue. HIV-1 Tat introduced ex vivo to the modified CD4(+) T cells induced MazF expression in both pre- and post-infusion samples, and MazF expression was detected in vivo post-viral-rebound during ATI. One participant experienced mild cytokine release syndrome. In sum, this study of a single infusion of MazF-modified CD4 T lymphocytes demonstrated safety of these cells, distribution to lymph tissue and maintenance of Tat-inducible MazF endoribonuclease activity, as well as sustained elevation of blood CD4 and CD8 T cell counts. Future studies to assess effects on viremia and latent proviral reservoir are warranted.

BackgroundBecause of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive.MethodsWe conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide.ResultsIn vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels.ConclusionsThe trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS.Trial registration numberNCT02366546.

As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies by a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARSCoV-2, and pseudo-virus assay, TCID assay with live SARS-CoV-2. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits. Finally, DNA vaccine protected hamsters form SARSCoV-2 infection. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic. Funding: This study was supported by Project Promoting Support for Drug Discovery grants (JP20nk0101602 and JP21nf0101623h102) from the Japan Agency for Medical Research and Development and Panasonic Co. (Japan). To fight against the worldwide COVID-19 pandemic, the development of an effective and safe The Department of Health Development and Medicine is an endowed department supported by Anges, Daicel, and FunPep. The Department of Clinical Gene Therapy is financially supported by Novartis, AnGes, Shionogi, Boeringher, Fancl, Saisei Mirai Clinics, Rohto and Funpep. Declaration of Interest: R.M. is a stockholder of FunPep and Anges. T.O. T.K. and Y.S. are employees of Anges. R.I, A.T, H.K, S.K, E.T, S.M, and H.T are employees of FunPep. R.M, H.T, and A.T. are FunPep stockholders. All other authors declare no competing interests. Ethical Approval: All experiments were approved by the Ethical Committee for Animal Experiments of the Osaka University Graduate School of Medicine.

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

The current antiretroviral therapy cannot cure the patients infected with human immunodeficiency virus type 1 (HIV-1) due to the existence of latently infected cells capable of virus production from harboring proviral DNA. MazF is an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli. The conditional expression of MazF by binding of HIV-1 Tat to the promoter region of a MazF-expression vector has previously been shown to selectively inhibit HIV-1 replication in acutely infected cells. The expression of MazF significantly suppressed tumor necrosis factor (TNF)-α-induced HIV-1 production and viral RNA expression in the HIV-1 latently infected cell line OM-10.1 transduced with the MazF-expression vector (OM-10.1/MFR). Moreover, the viability of OM-10.1/MFR cells decreased with increasing concentrations of TNF-α, whereas such decrease was not observed for HL-60 cells transduced with the MazF-expression vector (HL-60/MFR), the uninfected parental cell line of OM-10.1. TNF-α increased the expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase in OM-10.1/MFR cells, indicating that the cell death was caused by the induction of apoptosis. TNF-α-induced expression of MazF mRNA was detected in OM-10.1/MFR but not HL-60/MFR cells, suggesting that TNF-α-induced apoptosis of latently infected cells was due to the expression of MazF. Thus, the anti-HIV-1 gene therapy using the MazF-expression vector may have potential for the cure of HIV-1 infection in combination with suitable latency reversing agents through reducing the size of latently infected cells without viral reactivation.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

Recombinant adeno-associated virus (rAAV) is a promising gene delivery vehicle that has been approved as a gene therapy drug for some genetic disorders, and is being evaluated in clinical trials. To further promote clinical research under the Food and Drug Administration Investigational New Drug application, the stability of rAAV must be assessed under various conditions. However, there is scant data concerning the stability of a variety of rAAV serotypes. We hypothesized that the difference of capsid structure causes differences in stability. To investigate this hypothesis, rAAV serotypes (rAAV1, rAAV2, rAAV8, and rAAV9) were exposed to diluents and various environmental conditions, including ultraviolet (UV) irradiation, 0.1 M sodium hydroxide (NaOH), 0.06% sodium hypochlorite (NaClO), tap water, and 70% ethanol (EtOH). The changes of the infectivity of the treated samples were assessed by transduction in HeLaRC32 cells as a criterion of stability. The infectivity between recombinant and wild-type AAV (wtAAV2) was also analyzed. The activity of all rAAV serotypes was weakened by UV irradiation and NaOH and NaClO exposure. Treatment for 10 days with tap water or 70% EtOH did not appreciably inactivate rAAV1, rAAV8, and rAAV9, but did affect the activity of rAAV2. Furthermore, the infectivity of rAAV2 did not surpass wtAAV2 infectivity. The results will be important for clinical studies for gene therapy using rAAV.

Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% ± 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 ± 0.4 × 1015 viral genomes produced from ∼3.2 × 109 HEK293EB cells. We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy. Keywords: adeno-associated virus, rAAV9, quaternary ammonium anion exchangers, ultracentrifugation-free protocol

Background Adoptive transfer of TCR-redirected T cells has been reported to exhibit efficacy in some patients with melanoma and sarcoma. However, cytokine release syndrome (CRS) or its relations to tumor response has not been well documented. This study aimed to evaluate clinical responses in association with the cell kinetics and CRSs after transfer of high-affinity NY-ESO-1 TCR-gene transduced T cells in cancer patients. Methods We developed a novel-type affinity-enhanced NY-ESO-1-specific TCR and an originally-developed retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1/TCR sequence was mutated for high affinity with replacements of G50A and A51E in CDR2 region. This is a first-in-human clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It was designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3 + 3 cohort design. Cyclophosphamide (1,500mg/m2) were administered prior to the TCR-T cell transfer as pre-conditioning. Results Nine patients were treated with the TCR-T cells that expanded in peripheral blood with a dose-dependent manner, associated with rapid proliferation within 5 days after infusion. Three patients receiving 5x109 cells developed early-onset CRSs, with elevated levels of serum IL-6, IFN-γ. The CRSs on day1 or 2 were well managed with tocilizumab treatment. Three synovial sarcoma patients exhibited tumor shrinkage and partial responses, and they all had high-expression of NY-ESO-1 in the tumor samples, namely, 75% or more. Exploratory analysis revealed that multiple chemotactic cytokines including CCL2 and CCL7, and IL-3 increased in the serum from the patients with CRS. The proportions of effector-memory phenotype T cells in the infused cell-product were significantly associated with CRS development. Conclusions The affinity-enhanced NY-ESO-1/TCR-T cell transfer exhibited early-onset CRS in association with in vivo cell proliferation and sequential tumor responses in the patients with high-NY-ESO-1-expressing synovial sarcoma. Clinical trial identification NCT02366546. Legal entity responsible for the study The authors. Funding Japan Agency for Medical Research and Development. Disclosure All authors have declared no conflicts of interest.

2530 Background: Adoptive transfer of TCR-redirected T cells has been reported to exhibit efficacy in some of melanoma and sarcoma patients. However, there have not been well known about cytokine release syndrome (CRS) or its relations to tumor responses. This study evaluates clinical responses in association with the cell kinetics and CRSs after transfer of high-affinity NY-ESO-1 TCR-gene transduced T cells in NY-ESO-1-expressiong cancer patients (NCT02366546). Methods: We developed a novel-type affinity-enhanced NY-ESO-1-specific TCR and an originally-developed retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1/TCR sequence is mutated for high affinity with replacements of G50A and A51E in CDR2 region. This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It was designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide (1,500mg/m2) were administered prior to the TCR-T cell transfer as pre-conditioning. Results: 9 patients were treated with the NY-ESO-1/TCR-T cell transfer. The TCR-T cells expanded in peripheral blood with a dose-dependent manner, associated with rapid proliferation within 5 days after the cell transfer. 3 patients receiving 5x109 cells developed early-onset CRSs, with elevations of serum IL-6, IFN-γ. The CRSs developed on day1 or 2 after the cell transfer. They were well managed with tocilizumab treatment. 3 synovial sarcoma patients exhibited tumor shrinkages of partial responses, and they all had high-expression of NY-ESO-1 in the tumor samples, namely, 75% or more. Exploratory analysis revealed that multiple chemotactic cytokines including CCL2 and CCL7, and IL-3 increased in the serum from the patients with CRS. The proportions of effector-memory phenotype T cells in the infused cell-product were significantly associated with CRS development. Conclusions: The affinity-enhanced NY-ESO-1/TCR-T cell transfer exhibited early-onset CRS in association with in vivo cell proliferation and sequential tumor responses in the patients with high-NY-ESO-1-expressing synovial sarcoma. Clinical trial information: NCT02366546.

Transcriptional activation of gene expression directed by the long terminal repeat (LTR) of HIV-1 requires both the transactivation response element (TAR) and Tat protein. HIV-1 mutants lacking a functional tat gene are not able to proliferate. Here we take a genetic approach to suppress HIV-1 replication based on Tat-dependent production of MazF, an ACA-specific endoribonuclease (mRNA interferase) from Escherichia coli. When induced, MazF is known to cause Bak- and NBK-dependent apoptotic cell death in mammalian cells. We first constructed a retroviral vector, in which the mazF (ACA-less) gene was inserted under the control of the HIV-1 LTR, which was then transduced into CD4+ T-lymphoid CEM-SS cells in such a way that, upon HIV-1 infection, the mazF gene is induced to destroy the infecting HIV-1 mRNA, preventing HIV-1 replication. Indeed, when the transduced cells were infected with HIV-1 IIIB, the viral replication was effectively inhibited, as HIV-1 IIIB p24 could not be detected in the culture medium. Consistently, not only cell growth but also the CD4 level was not affected by the infection. These results suggest that the HIV-1-LTR-regulated mazF gene was effectively induced upon HIV-1 IIIB infection, which is sufficient enough to destroy the viral mRNA from the infected HIV-1 IIIB to completely block viral proliferation in the cells, but not to affect normal cell growth. These results indicate that the T cells transduced with the HIV-1-LTR-regulated mazF gene acquire HIV-1 resistance, providing an intriguing potential for the use of the HIV-1-LTR-regulated mazF gene in anti-HIV gene therapy.

Adoptive T-cell transfer of in vitro cultured T cells derived from cancer patients with naturally developed immune responses has met with some success as an immunotherapeutic approach, although only a limited number of patients showed spontaneous immune responses. To find alternative ways, such as cancer-specific T-cell receptor (TCR) gene transfer, in preparation for sufficient numbers of antigen-specific T cells is an important issue in the field of adoptive T-cell therapy. Given the inherent disadvantage of alphabeta TCR transfer to other alphabeta T cells, namely the possible formation of mixed TCR heterodimers with endogenous alpha or beta TCR, we employed gammadelta T cells as a target for retroviral transfer of cancer-specific TCR and examined whether gammadelta T cells were useful as an alternative population for TCR transfer. Although retroviral transduction to gammadelta T cells with TCR alphabeta genes alone, isolated from a MAGE-A4(143-151)-specific alphabeta CD8(+) cytotoxic T lymphocyte (CTL) clone, did not provide sufficient affinity to recognize major histocompatibility (MHC)-peptide complexes due to the lack of CD8 co-receptor, gammadelta T cells co-transduced with TCR alphabeta and CD8 alphabeta genes acquired cytotoxicity against tumor cells and produced cytokines in both alphabeta- and gammadelta-TCR-dependent manners. Furthermore, alphabeta TCR and CD8-transduced gammadelta T cells, stimulated either through alphabeta TCR or gammadelta TCR, rapidly responded to target cells compared with conventional alphabeta T cells, reminiscent of gammadelta T cells. We propose alphabeta TCR-transduced gammadelta T cells as an alternative strategy for adoptive T-cell transfer.

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

MicroRNAs (miRNAs), which are non-coding RNAs 18-25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering approximately 80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

Adoptive cell transfers of receptor gene-engineered T cells include chimeric antigen receptor-gene transduced T (CAR-T) cell therapy and TCR-gene transduced T (TCR-T) cell therapy. In CD19-CAR-T cell therapy, high incidence of cytokine release syndrome (CRS) is associated with in vivo CAR-T cell proliferation and its clinical efficacy. In human TCR-T cell therapies, there have not been well known about CRS and its association with in vivo T cell kinetics or tumor responses. We have been developing a novel-type affinity-enhanced NY-ESO-1-specific TCR, and an original retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1 TCR is mutated for high affinity with replacements of G50A and A51E in CDR2 region, which is restricted with HLA-A*02:01 and A*02:06. We extensively examined potential cross-reactivities to different antigen-peptides in preclinical studies, and the high-affinity NY-ESO-1 TCR did not recognize analogous peptides. The new generation retroviral TCR-vector provides enhanced expression of transduced tumor-specific TCRs and an inhibition effect of formations of self-reactive TCRs. This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It is designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide with/without fludarabine were administered prior to the TCR-T cell transfer as pre-conditioning. Six patients were treated with the NY-ESO-1 TCR-T cell transfer, and evaluated for the safety and in vivo cell kinetics. The TCR-T cells appeared in peripheral blood with a dose-dependent manner, associated with in vivo proliferation in an early phase. In three patients given 5x108 cells, no toxicities were seen. Two patients receiving 5x109 cells developed early-phase CRS (G2), with elevations of serum IL-6 and IFN-gamma. They were managed the treatment of anti-IL-6 receptor monoclonal antibody, tocilizumab. In a patient who developed CRS, an event of lung injury (G3) occurred, which was associated with marked infiltration of the NY-ESO-1 TCR-T cells. It was successfully treated with steroid. Two synovial sarcoma patients exhibited tumor responses of PRs. In one patient, progression-free survival lasted more than 8 months. In summary, the affinity-enhanced NY-ESO-1 TCR-T cell transfer exhibited CRSs in association with in vivo cell proliferation and sequential tumor responses. Disclosures No relevant conflicts of interest to declare.

In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4(+) T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥ 10(10)) and 100-fold (≥ 5 × 10(9)) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols.

Gene therapy using a Tat-dependent expression system of MazF, an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli, in a retroviral vector appears to be an alternative approach to the treatment of human immunodeficiency virus type 1 (HIV-1) infection. MazF can cleave HIV-1 RNA, since it has more than 240 ACA sequences. Significant inhibition of viral replication, irrespective of HIV-1 strains, was observed in CD4(+) T cells that had been transduced with the MazF-expressing retroviral vector (MazF-T cells). The growth and viability of MazF-T cells were not affected by HIV-1 infection. Interestingly, the infectivity of HIV-1 produced from MazF-T cells was found to be lower than that from control CD4(+) T cells. A long-term culture experiment with HIV-1-infected cells revealed that viral replication was always lower in MazF-T cells than in CD4(+) T cells transduced with or without a control vector for more than 200 days. MazF was expressed and mainly localized in the cytoplasm of the infected cells. Unlike in CD4(+) T cells, the expression level of Tat gradually decreased rather than increased in MazF-T cells after HIV-1 infection. As a consequence, the expression level of MazF appeared to be well regulated and sustained during HIV-1 infection in MazF-T cells. Furthermore, the levels of cellular mRNA were not affected by HIV-1 infection. Thus, the Tat-dependent MazF expression system has great potential for inhibition of HIV-1 replication in vivo without apparent toxicity and may be able to avoid the emergence of resistant strains.

Background MazF is an endoribonuclease encoded by Escherichia coli that specifically cleaves the ACA sequence of mRNA. In our previous report, conditional expression of MazF in the HIV-1 LTR rendered CD4+ T lymphocytes resistant to HIV-1 replication. In this study, we examined the in vivo safety and persistence of MazF-transduced cynomolgus macaque CD4+ T cells infused into autologous monkeys. Methodology/Principal Findings The in vivo persistence of the gene-modified CD4+ T cells in the peripheral blood was monitored for more than half a year using quantitative real-time PCR and flow cytometry, followed by experimental autopsy in order to examine the safety and distribution pattern of the infused cells in several organs. Although the levels of the MazF-transduced CD4+ T cells gradually decreased in the peripheral blood, they were clearly detected throughout the experimental period. Moreover, the infused cells were detected in the distal lymphoid tissues, such as several lymph nodes and the spleen. Histopathological analyses of tissues revealed that there were no lesions related to the infused gene modified cells. Antibodies against MazF were not detected. These data suggest the safety and the low immunogenicity of MazF-transduced CD4+ T cells. Finally, gene modified cells harvested from the monkey more than half a year post-infusion suppressed the replication of SHIV 89.6P. Conclusions/Significance The long-term persistence, safety and continuous HIV replication resistance of the mazF gene-modified CD4+ T cells in the non-human primate model suggests that autologous transplantation of mazF gene-modified cells is an attractive strategy for HIV gene therapy.

Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naive phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naive phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

Recombinant human fibronectin fragment (FN-CH296, RetroNectin) has been widely used for retroviral gene therapy to enhance gene transfer efficiency. Based on the observation that immobilized FN-CH296 together with anti-CD3 monoclonal antibodies (anti-CD3) enhanced cell proliferation while conserving the naive phenotype of T cells, we used FN-CH296 costimulation to generate engineered T cells. For comparison, human peripheral blood mononuclear cells were stimulated under three kinds of conditions including anti-CD3 only, anti-CD3 and anti-CD28 monoclonal antibodies conjugated with beads (anti-CD3/anti-CD28) and immobilized FN-CH296 together with anti-CD3 (anti-CD3/FN-CH296); all three treatments were followed by retroviral gene transfer. Of all the stimulation methods, the one involving anti-CD3/FN-CH296 produced the most cell expansion with conservation of the naive phenotype. Engineered T cells were transplanted into NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice, and all the mice were killed 14 days later. Transplanted T cells were detected in all the mice; however, mice injected with anti-CD3/FN-CH296-stimulated T cells showed higher transgene expression in organs than mice injected with anti-CD3-stimulated cells. These results demonstrate that the anti-CD3/FN-CH296 stimulation can be an efficient way to generate large numbers of genetically modified T cells that can provide higher and longer lasting levels of transgene expression in vivo and that are suitable for adoptive T-cell transfer therapy.

Background; Adoptive immunotherapy with chimeric antigen receptor (CAR) gene transduced CD19-CAR T cells, which are engineered to express extracellular single-chain immunoglobulin variable fragments to CD19, linked to cytoplasmic T cell activation domains including CD3-ζ, showed remarkable therapeutic benefits toward CD19+ B cell malignancies including acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma (B-NHL). For clinical setting, the phenotype of manufactured CAR T cells is an important factor; especially less differentiated T cells are anticipated to provide a long-lasting immune reconstitution. Furthermore, in order to avoid the risk of technical error and contamination during T cell manufacturing process, a closed system needs to be established. In this study, we addressed these issues and have established a novel CD19-CAR T cell manufacturing method from a small amount of blood in a closed system for clinical trial to treat patients with B-NHL. Methods; Peripheral blood was obtained from B-NHL patient volunteers and healthy donor volunteers who gave their written informed consents. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of blood using Ficoll-Paque PREMIUM density gradient centrifugation. PBMCs were stimulated in a plastic bag pre-coated with anti-CD3 monoclonal antibody (OKT3) and recombinant fibronectin fragment (RetroNectin®; RN). Following four days of stimulation, stimulated T cells were transferred into a 215 cm2 plastic bag pre-loaded with SFG-1928z retroviral vector (Brentjens et al., Clin Cancer Res. 2007) onto RN-coated substratum with low-temperature shaking (RBV-LTS method; Dodo et al., PLoS ONE, 2014). After one hour incubation, the bag was flipped over to facilitate more efficient utilization of the retroviral vector adsorbed on both top and bottom surfaces of the bag and further incubated. On Day 5, the transduction procedure was repeated, and the cells were transferred into 640 cm2 plastic bags until Day 10-14 for expansion. Closed system liquid handling was managed in all processes of manipulating T cells for stimulation, transduction, expansion and final product formulation. Results; We have previously reported that the fold expansion of T cells under stimulation with RN together with OKT3 enhanced cell proliferation while preserving the naïve phenotype of T cells in comparison to stimulation with OKT3 alone or OKT3 and anti-CD28 monoclonal antibody co-stimulation. Although B-NHL patients’ T cells showed much lower fold expansion compared to healthy donors’ T cells, 4/7 patients’ CAR T cells reached their target dose of 1 x 106 cells/kg from 30 ml of blood on Day 10. For the other 3 patients, 70-150 ml blood was estimated to be required to reach their target dose. The delay in proliferations was marked in B-NHL patients’ T cells compared to healthy donors’ T cells by Day 5, but B-NHL patients’ T cells represented significant catch-up growth, which was superior to healthy donor T cells during Day 7-14. Gene transfer efficiency of patients’ T cells (N = 7, 17.9 ± 4.7%) was equivalent to that of healthy donors’ T cells (N = 5, 22.2 ± 5.3%), and CAR T cells showed potent anti-tumor reactivity with cytokine productions against CD19 positive Raji cells in vitro. Comparing to CD3/CD28 beads stimulation method, RN/OKT3 stimulation method showed equivalent expansion. Furthermore, RN/OKT3 stimulated T cells preserved higher proportion of CD8+/CCR7+/CD45RA+/CD62L+ naïve phenotype T cells (43.6% in healthy donor and 30.9% in patient donor) compared to CD3/CD28 beads stimulated T cells (22.8% in healthy donor and 11.7% in patient donor). Conclusions; With our novel closed system manufacturing method utilizing RN/OKT3 stimulation combined with RBV-LTS transduction from a small amount of blood of B-NHL patients, we are able to manufacture a sufficient number of CAR T cells maintaining higher proportion of naïve phenotype, which is expected to improve the efficacy of adoptive immunotherapy. In our cell manufacturing, patients are not required to undergo leukapheresis, which is more invasive to patients. Based on our results, we employed our novel manufacturing method for the phase I/II clinical trial to treat patients with relapsed/refractory CD19+ B-NHL (clinicaltrials.gov, identifier; NCT02134262). Disclosures Chono: Takara Bio Inc.: Employment. Tahara:Takara Bio Inc.: Employment. Nukaya:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Membership on an entity's Board of Directors or advisory committees. Tsukahara:Takara Bio Inc.: Research Funding. Ohmine:Takara Bio Inc.: Research Funding. Ozawa:Takara Bio Inc.: Research Funding. Takesako:Takara Bio Inc.: Membership on an entity's Board of Directors or advisory committees.

In retroviral gene transduction, the efficiency of viral infection was reduced by the proteoglycans and some other materials secreted by the producer lines. In order to remove these inhibitors we have developed the rFN-CH-296-facilitated protocol. Because the rFN-CH-296 molecule has strong ability to bind a retroviral vector, rFN-CH-296 bound plates are utilized to wash out the unbound putative inhibitors present in a virus supernatant. The gene transduction efficiencies of human CD34(+)CD38(-) BMCs with a GALV-pseudotyped vector and the rFN-CH-296-facilitated protocol were compared with the protocol without a coating plate with CH-296, the mean gene transduction efficiencies being found to be 95.5 and 71.1%, respectively.

In retroviral gene transduction, the efficiency of viral infection is suppressed by the proteoglycans and some other materials secreted by the producer cell lines. In order to remove these inhibitors, we previously developed the rFN-CH-296 (RetroNectin®)-bound virus infection method. In the present study, we further optimized the conditions of gene transfer efficiency into human leukemia cell lines, K562, TF-1, HL-60, and CEM cells utilizing RetroNectin® plates with galv-pseudo-typed or amphotropic retrovirus vector, and applied the optimized protocol to the CD34+ BMCs in a closed system with gas permeable culture bag.