RESUMEN
Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.
Asunto(s)
Dependovirus , Vectores Genéticos , Dependovirus/genética , Ultracentrifugación/métodos , ADNRESUMEN
Adoptive immunotherapy using genetically engineered patient-derived lymphocytes to express tumor-reactive receptors is a promising treatment for malignancy. However, utilization of autologous T cells in this therapy limits the quality of gene-engineered T cells, thereby inhibiting the timely infusion of the cells into patients. In this study, we evaluated the anti-tumor efficacy and the potential to induce graft-versus-host disease (GVHD) in T cell receptor (TCR) gene-engineered allogeneic T cells that downregulate the endogenous TCR and HLA class I molecules with the aim of developing an "off-the-shelf" cell product with expanded application of genetically engineered T cells. We transduced human lymphocytes with a high-affinity TCR specific to the cancer/testis antigen NY-ESO-1 using a novel retrovirus vector with siRNAs specific to the endogenous TCR (siTCR vector). These T cells showed reduced expression of endogenous TCR and minimized reactivity to allogeneic cells in vitro. In non-obese diabetic/SCID/γcnull mice, TCR gene-transduced T cells induced tumor regression without development of GVHD. A lentivirus-based CRISPR/Cas9 system targeting ß-2 microglobulin in TCR gene-modified T cells silenced the HLA class I expression and prevented allogeneic CD8+ T cell stimulation without disrupting their anti-tumor capacity. This report is the first demonstration that siTCR technology is effective in preventing GVHD. Adoptive cell therapy with allogeneic T cells engineered with siTCR vector may be useful in developing an "off-the-shelf" therapy for patients with malignancy.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Neoplasias , Ratones , Animales , Humanos , ARN Interferente Pequeño/genética , Células Alogénicas/metabolismo , Ratones SCID , Receptores de Antígenos de Linfocitos T , Genes Codificadores de los Receptores de Linfocitos T , Inmunoterapia Adoptiva , Neoplasias/genética , Enfermedad Injerto contra Huésped/prevención & controlRESUMEN
The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αß T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of 'off-the-shelf' CAR-T cell products for successful allogeneic adoptive immunotherapy.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Neoplasias , Profármacos , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/métodos , Difosfonatos , Receptores de Antígenos de Linfocitos T gamma-delta , Profármacos/farmacología , Profármacos/uso terapéutico , Linfocitos T , InmunoterapiaRESUMEN
Adult T cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm caused by infection with human T cell lymphotropic virus type-1 (HTLV-1). Its prognosis remains extremely poor. Tax, the most important regulatory protein for HTLV-1, is associated with the aggressive proliferation of host cells and is also a major target antigen for CD8+ cytotoxic T cells (CTLs). Based on our previous findings that Tax-specific CTLs with a T cell receptor (TCR) containing a unique amino-acid sequence motif exhibit strong HLA-A*24:02-restricted, Tax301-309-specific activity against HTLV-1, we aimed to develop a Tax-redirected T cell immunotherapy for ATL. TCR-É/ß genes were cloned from a previously established CTL clone and transduced into peripheral blood mononuclear cells (PBMCs) of healthy volunteers using a retroviral siTCR vector. Then the cytotoxic efficacy against HTLV-1-infected T cells or primary ATL cells was assessed both in vitro and in vivo. The redirected CTLs (Tax-siCTLs) produced a large amount of cytokines and showed strong killing activity against ATL/HTLV-1-infected T cells in vitro, although they did not have universal activity against ATL cells. Next, in a xenograft mouse model using an HTLV-1-infected T cell line (MT-2), in all mice treated with Tax-siCTLs, the tumor rapidly diminished and finally disappeared without normal tissue damage, although all mice that were untreated or treated with non-gene-modified PBMCs died because of tumor progression. Our findings confirm that Tax-siCTLs can exert strong anti-ATL/HTLV-1 effects without a significant reaction against normal cells and have the potential to be a novel immunotherapy for ATL patients.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T del Adulto , Adulto , Animales , Productos del Gen tax/genética , Genes Codificadores de los Receptores de Linfocitos T , Humanos , Inmunoterapia , Leucemia-Linfoma de Células T del Adulto/terapia , Leucocitos Mononucleares , Ratones , Linfocitos T CitotóxicosRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/genética , Endorribonucleasas/genética , Proteínas de Escherichia coli/genética , Terapia Genética , Infecciones por VIH/terapia , VIH-1/fisiología , Latencia del Virus , Apoptosis , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Infecciones por VIH/genética , Infecciones por VIH/virología , Células HL-60 , Humanos , Activación Transcripcional , Transducción Genética , Replicación ViralRESUMEN
The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1235-243/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.
Asunto(s)
Inmunidad Celular , Inmunoterapia Adoptiva , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Vacunación , Proteínas WT1/inmunología , Animales , Línea Celular Tumoral , Humanos , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/patología , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Genes Codificadores de los Receptores de Linfocitos T , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicos/terapia , Linfocitos T/metabolismo , Transducción Genética , Proteínas WT1/genética , Traslado Adoptivo , Anciano , Médula Ósea/patología , Femenino , Humanos , Cinética , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Péptidos/farmacologíaRESUMEN
Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells without prior sensitization, and have the potential to exert antibody-dependent cellular cytotoxicity (ADCC). In this clinical trial, we examined the safety and efficacy of the use of NK cells, generated using a novel expansion system, in combination with IgG1 antibodies for the treatment of advanced gastric or colorectal cancers. Treatment consisted of trastuzumab- or cetuximab-based chemotherapy, plus adoptive NK cell therapy. For administration of expanded NK cells, dose escalation with a sequential 3 + 3 design was performed in three steps, at doses of 0.5 × 109 , 1.0 × 109 , and 2.0 × 109 cells/injection (N = 9). After 3 days of IgG1 antibody administration, patients were infused with expanded NK cells three times at triweekly intervals. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D (97.6%) and CD16 (69.6%). The combination therapy was very well tolerated with no severe adverse events. Among six evaluable patients, four presented stable disease (SD) and two presented progressive disease. Of the four SD patients, three showed an overall decrease in tumor size after combination therapy. Immune monitoring suggested that combination therapy enhanced whole blood IFN-γ production and reduced peripheral regulatory T cells (Tregs). In conclusion, this phase I trial provides evidence of good tolerability, induction of Th1 immune responses, and preliminary anti-tumor activity for this combination therapy, in patients with advanced gastric and colorectal cancer that have received previous therapy.
Asunto(s)
Neoplasias Colorrectales/terapia , Inmunoglobulina G/uso terapéutico , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/trasplante , Neoplasias Gástricas/terapia , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cetuximab/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastuzumab/uso terapéuticoRESUMEN
OBJECTIVE: We here describe treatment outcomes in two adenosine deaminase (ADA)-deficiency patients (pt) who received stem cell gene therapy (SCGT) with no cytoreductive conditioning. As this protocol has features distinct from those of other clinical trials, its results provide insights into SCGT for ADA deficiency. PATIENTS AND METHODS: Pt 1 was treated at age 4.7 years, whereas pt 2, who had previously received T-cell gene therapy, was treated at age 13 years. Bone marrow CD34(+) cells were harvested after enzyme replacement therapy (ERT) was withdrawn; following transduction of ADA cDNA by the γ-retroviral vector GCsapM-ADA, they were administered intravenously. No cytoreductive conditioning, at present considered critical for therapeutic benefit, was given before cell infusion. Hematological/immunological reconstitution kinetics, levels of systemic detoxification, gene-marking levels, and proviral insertion sites in hematopoietic cells were assessed. RESULTS: Treatment was well tolerated, and no serious adverse events were observed. Engraftment of gene-modified repopulating cells was evidenced by the appearance and maintenance of peripheral lymphocytes expressing functional ADA. Systemic detoxification was moderately achieved, allowing temporary discontinuation of ERT for 6 and 10 years in pt 1 and pt 2, respectively. Recovery of immunity remained partial, with lymphocyte counts in pts 1 and 2, peaked at 408/mm(3) and 1248/mm(3), approximately 2 and 5 years after SCGT. Vector integration site analyses confirmed that hematopoiesis was reconstituted with a limited number of clones, some of which were shown to have myelo-lymphoid potential. CONCLUSIONS: Outcomes in SCGT for ADA-SCID are described in the context of a unique protocol, which used neither ERT nor cytoreductive conditioning. Although proven safe, immune reconstitution was partial and temporary. Our results reiterate the importance of cytoreductive conditioning to ensure greater benefits from SCGT.
Asunto(s)
Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Agammaglobulinemia/genética , Agammaglobulinemia/terapia , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Adenosina Desaminasa/inmunología , Adenosina Desaminasa/uso terapéutico , Adolescente , Agammaglobulinemia/diagnóstico , Agammaglobulinemia/inmunología , Edad de Inicio , Diferenciación Celular , Preescolar , Activación Enzimática , Terapia de Reemplazo Enzimático , Gammaretrovirus/genética , Expresión Génica , Vectores Genéticos/genética , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunidad , Inmunofenotipificación , Lactante , Recién Nacido , Japón , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Mutación , Inmunodeficiencia Combinada Grave/diagnóstico , Inmunodeficiencia Combinada Grave/inmunología , Transducción Genética , Transgenes , Resultado del TratamientoRESUMEN
T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T-cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH-296, we demonstrated that stimulation via very late Ag (VLA)-4 and VLA-5 in human and BALB/c mouse CD8(+) T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR-transgenic mouse-derived CD8(+) T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma-derived tumor Ag, we showed that stimulation by CH-296 improved the ability of tumor-specific CD8(+) T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3(+) CD4(+) Treg cells in tumors. These results suggest that stimulation via VLA-4 and VLA-5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.
Asunto(s)
Antígenos de Neoplasias/inmunología , Fibrosarcoma/inmunología , Memoria Inmunológica , Integrina alfa4beta1/inmunología , Integrina alfa5beta1/inmunología , Traslado Adoptivo , Animales , Antígenos de Neoplasias/genética , Linfocitos T CD8-positivos , Línea Celular Tumoral , Femenino , Fibrosarcoma/genética , Fibrosarcoma/patología , Fibrosarcoma/terapia , Humanos , Integrina alfa4beta1/genética , Integrina alfa5beta1/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
BACKGROUND: NK cells can destroy tumor cells without prior sensitization or immunization. Tumors often lose expression of MHC molecules and/or antigens. However, NK cells can lyse tumor cells in a non-MHC-restricted manner and independent of the expression of tumor-associated antigens. NK cells are therefore considered ideal for adoptive cancer immunotherapy; however the difficulty of obtaining large numbers of fully functional NK cells that are safe to administer deters its clinical use. This phase I clinical trial seeks to address this obstacle by first developing a novel system that expands large numbers of highly activated clinical grade NK cells, and second, determining if these cells are safe in a mono-treatment so they can be combined with other reagents in the next round of clinical trials. METHODS: Patients with unresectable, locally advanced and/or metastatic digestive cancer who did not succeed with standard therapy were enrolled. NK cells were expanded ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296 induced T cells. Patients were administered autologous natural killer cell three times weekly via intravenous infusions in a dose-escalating manner (dose 0.5 × 10(9), 1.0 × 10(9), 2.0 × 10(9) cells/injection, three patients/one cohort). RESULTS: Total cell population had a median expansion of 586-fold (range 95-1102), with a significantly pure (90.96 %) NK cell population. Consequently, NK cells were expanded to approximately 4720-fold (range 1372-14,116) with cells being highly lytic in vitro and strongly expressing functional markers such as NKG2D and CD16. This NK cell therapy was very well tolerated with no severe adverse events. Although no clinical responses were observed, cytotoxicity of peripheral blood was elevated approximately twofolds up to 4 weeks post the last transfer. CONCLUSION: We successfully generated large numbers of activated NK cells from small quantities of blood without prior purification of the cells. We also determined that the expanded cells were safe to administer in a monotherapy and are suitable for the next round of clinical trials where their efficacy will be tested combined with other reagents. TRIAL REGISTRATION: UMIN UMIN000007527.
Asunto(s)
Neoplasias Gastrointestinales/terapia , Inmunoterapia , Células Asesinas Naturales/citología , Anciano , Femenino , Humanos , Inmunofenotipificación , Células Asesinas Naturales/inmunología , Masculino , Persona de Mediana EdadRESUMEN
Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/ß genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.
Asunto(s)
Traslado Adoptivo , Linfocitos T CD8-positivos/inmunología , Antígeno HLA-A24/inmunología , Leucemia-Linfoma de Células T del Adulto/terapia , Proteínas de Neoplasias/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Telomerasa/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Línea Celular Transformada , Femenino , Antígeno HLA-A24/genética , Antígeno HLA-A24/metabolismo , Humanos , Células K562 , Leucemia-Linfoma de Células T del Adulto/enzimología , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/inmunología , Masculino , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Péptidos/genética , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/genética , Telomerasa/metabolismo , Trasplante HeterólogoRESUMEN
BACKGROUND AIMS: Haplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. METHODS: We investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vß repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. RESULTS: A comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8(+) non-TK cells were also depleted in response to ganciclovir administration. The TCR Vß-chain repertoire of both TK cells and non-TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non-TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. CONCLUSIONS: We demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells.
Asunto(s)
Genes Transgénicos Suicidas , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas , Leucemia/terapia , Linfocitos T/metabolismo , Timidina Quinasa/genética , Femenino , Citometría de Flujo , Ganciclovir/uso terapéutico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/inmunología , Humanos , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Simplexvirus/genética , Linfocitos T/inmunología , Donantes de TejidosRESUMEN
Aurora kinase A (AURKA) is overexpressed in leukemias. Previously, we demonstrated that AURKA-specific CD8(+) T cells specifically and selectively lysed leukemia cells, indicating that AURKA is an excellent target for immunotherapy. In this study, we examined the feasibility of adoptive therapy using redirected T cells expressing an HLA-A*0201-restricted AURKA(207-215)-specific T-cell receptor (TCR). Retrovirally transduced T cells recognized relevant peptide-pulsed but not control target cells. Furthermore, TCR-redirected CD8(+) T cells lysed AURKA-overexpressing human leukemic cells in an HLA-A*0201-restricted manner, but did not kill HLA-A*0201(+) normal cells, including hematopoietic progenitors. In addition, AURKA(207-215)-specific TCR-transduced CD4(+) T cells displayed target-responsive Th1 cytokine production. Finally, AURKA(207-215)-specific TCR-transduced CD8(+) T cells displayed antileukemia efficacy in a xenograft mouse model. Collectively, these data demonstrate the feasibility of redirected T cell-based AURKA-specific immunotherapy for the treatment of human leukemia.
Asunto(s)
Genes Codificadores de los Receptores de Linfocitos T/genética , Terapia Genética , Inmunoterapia , Leucemia/terapia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Linfocitos T/inmunología , Animales , Aurora Quinasa A , Aurora Quinasas , Western Blotting , Estudios de Casos y Controles , Estudios de Factibilidad , Citometría de Flujo , Antígeno HLA-A2/inmunología , Humanos , Leucemia/genética , Leucemia/inmunología , Luciferasas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interleucina-2/fisiología , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T Citotóxicos/inmunología , Transducción Genética , Células Tumorales CultivadasRESUMEN
Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated impressive success in the treatment of patients with hematologic tumors yet achieved very limited efficacy for solid tumors due to hurdles unique to solid tumors. It is also noted that the tumor microenvironment composition varies between tumor type, which again imposes unique set of hurdles in each solid tumor. Therefore, elucidation of individual hurdles is key to achieving successful CAR-T therapy for solid tumors. In the present study, we employed an orthotopic human PDAC xenograft model, in which quantitative, spatial and functional dynamics of CAR-T cells in tumor tissues were analyzed to obtain insights into ways of overcoming PDAC related hurdles. Contrary to previous studies that demonstrated a limited persistency and infiltration of CAR-T cells in many solid tumors, they persist and accumulated in PDAC tumor tissues. Ex vivo analysis revealed that CAR-T cells that had been recovered at different time points from mice bearing an orthotopic PDAC tumor exhibited a gradual loss of tumor reactivity. This loss of tumor reactivity of CAR-T cells was associated with the increased expression of AMP-activated protein kinase and Mitofusin 1/ Dynamin-related protein 1 ratio.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , Receptores de Antígenos de Linfocitos T , Linfocitos T , Xenoinjertos , Inmunoterapia Adoptiva , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19(+) tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19(+) human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.
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Traslado Adoptivo/métodos , Antígenos CD19/inmunología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Linfocitos T/inmunología , Linfocitos T/patología , Animales , Antígenos CD19/genética , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos BALB C , Ingeniería de Proteínas/métodosRESUMEN
Adoptive T-cell therapy for malignancies using redirected T cells genetically engineered by tumor antigen-specific T-cell receptor (TCR) gene transfer is associated with mispairing between introduced and endogenous TCR chains with unknown specificity. Therefore, deterioration of antitumor reactivity and serious autoimmune reactivity are major concerns. To address this problem, we have recently established a novel retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). In this study, to test the clinical application of siTCR gene therapy for human leukemia, we examined in detail the efficacy and safety of WT1-siTCR-transduced T cells. Compared with conventional WT1-TCR (WT1-coTCR) gene-transduced T cells, these cells showed significant enhancement of antileukemia reactivity resulting from stronger expression of the introduced WT1-specific TCR with inhibition of endogenous TCRs. Notably, WT1-siTCR gene-transduced T cells were remarkably expandable after repetitive stimulation with WT1 peptide in vitro, without any deterioration of antigen specificity. WT1-siTCR gene-transduced T cells from leukemia patients successfully lysed autologous leukemia cells, but not normal hematopoietic progenitor cells. In a mouse xenograft model, adoptively transferred WT1-siTCR gene-transduced T cells exerted distinct antileukemia efficacy but did not inhibit human hematopoiesis. Our results suggest that gene-immunotherapy for leukemia using this WT1-siTCR system holds considerable promise.
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Inmunoterapia Adoptiva/métodos , Leucemia/terapia , ARN Interferente Pequeño/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas WT1/genética , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Citotoxicidad Inmunológica/inmunología , Femenino , Terapia Genética/métodos , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Células Jurkat , Células K562 , Leucemia/genética , Leucemia/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , ARN Interferente Pequeño/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Transfección , Células Tumorales Cultivadas , Proteínas WT1/química , Proteínas WT1/inmunología , Proteínas WT1/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adeno-associated virus (AAV) vectors are promising tools for gene therapy. The current AAV vector system produces an abundance of empty capsids that are eliminated before clinical use, leading to increased costs for gene therapy. In the present study, we established an AAV production system that regulates the timing of capsid expression using a tetracycline-dependent promoter. Tetracycline-regulating capsid expression increased viral yield and reduced empty capsids in various serotypes without altering AAV vector infectivity in vitro and in vivo. The replicase expression pattern change observed in the developed AAV vector system improved viral quantity and quality, whereas timing control of capsid expression reduced empty capsids. These findings provide a new perspective on the development of AAV vector production systems in gene therapy.
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Chimeric antigen receptor engineered T cell (CAR-T) therapy has high therapeutic efficacy against blood cancers, but it has not shown satisfactory results in solid tumors. Therefore, we examined the therapeutic effect of CAR-T therapy targeting carcinoembryonic antigen (CEA) in pancreatic adenocarcinoma (PDAC). CEA expression levels on the cell membranes of various PDAC cell lines were evaluated using flow cytometry and the cells were divided into high, medium, and low expression groups. The relationship between CEA expression level and the antitumor effect of anti-CEA-CAR-T was evaluated using a functional assay for various PDAC cell lines; a significant correlation was observed between CEA expression level and the antitumor effect. We created orthotopic PDAC xenograft mouse models and injected with anti-CEA-CAR-T; only the cell line with high CEA expression exhibited a significant therapeutic effect. Thus, the therapeutic effect of CAR-T therapy was related to the target antigen expression level, and the further retrospective analysis of pathological findings from PDAC patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. Therefore, CEA expression levels in biopsies or surgical specimens can be clinically used as biomarkers to select PDAC patients for anti-CAR-T therapy.
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Emerging SARS-CoV-2 Omicron variants are highly contagious with enhanced immune escape mechanisms against the initially approved COVID-19 vaccines. Therefore, we require stable alternative-platform vaccines that confer protection against newer variants of SARS-CoV-2. We designed an Omicron B.1.1.529 specific DNA vaccine using our DNA vaccine platform and evaluated the humoral and cellular immune responses. SD rats intradermally administered with Omicron-specific DNA vaccine via pyro-drive jet injector (PJI) thrice at 2-week intervals elicited high antibody titers against the Omicron subvariants as well as the ancestral strain. Indeed, the Omicron B.1.1.529-specific antibody titer and neutralizing antibody were higher than that of other strains. Longitudinal monitoring indicated that anti-spike (ancestral and Omicron) antibody titers decreased toward 30 weeks after the first vaccination dose. However, neutralization activity remained unaltered. Germinal center formation was histologically detected in lymph nodes in rats immunized with Omicron DNA vaccine. Ancestral spike-specific immune cell response was slightly weaker than Omicron spike-specific response in splenocytes with Omicron-adapted DNA vaccine, evaluated by ELISpot assay. Collectively, our findings suggest that Omicron targeting DNA vaccines via PJI can elicit robust durable antibody production mediated by germinal center reaction against this new variant as well as partially against the spike protein of other SARS-CoV-2 variants.