RESUMO
Autologous chimeric antigen receptor (CAR) T cells targeting the CD19 antigen have demonstrated a high complete response rate in relapsed/refractory B-cell malignancies. However, autologous CAR T cell therapy is not an option for all patients. Here we optimized conditions for clinical-grade manufacturing of allogeneic CD19-CAR T cells using CD45RA-depleted donor memory T cells (Tm) for a planned clinical trial. Tm were activated using the MACS GMP T Cell TransAct reagent and transduced in the presence of LentiBOOST with a clinical-grade lentiviral vector that encodes a 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced T cells were transferred to a G-Rex cell culture device for expansion and harvested on day 7 or 8 for cryopreservation. The resulting CD19-CAR(Mem) T cells expanded on average 34.2-fold, and mean CAR expression was 45.5%. The majority of T cells were CD4+ and had a central memory or effector memory phenotype, and retained viral specificity. CD19-CAR(Mem) T cells recognized and killed CD19-positive target cells in vitro and had potent antitumor activity in an ALL xenograft model. Thus we have successfully developed a current good manufacturing practice-compliant process to manufacture donor-derived CD19-CAR(Mem) T cells. Our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.
Assuntos
Neoplasias , Receptores de Antígenos de Linfócitos T , Humanos , Antígenos CD19/genética , Imunoterapia Adotiva/métodos , Linfócitos T , GMP Cíclico/metabolismoRESUMO
Adeno-associated virus (AAV)-mediated gene therapy is a novel treatment promising to reduce morbidity associated with hemophilia. Although multiple clinical trials continue to evaluate efficacy and safety, limited cost-effectiveness data have been published. This study compared the potential cost-effectiveness of AAV-mediated factor IX (FIX)-Padua gene therapy for patients with severe hemophilia B in the United States vs on-demand FIX replacement and primary FIX prophylaxis, using either standard or extended half-life FIX products. A microsimulation Markov model was constructed, and transition probabilities between health states and utilities were informed by using published data. Costs were aggregated by using a microcosting approach. A time horizon from 18 years old until death, from the perspective of a third-party payer in the United States, was conducted. Gene therapy was more cost-effective than both alternatives considering a $150 000/quality-adjusted life-year threshold. The price for gene therapy was assumed to be $2 000 000 in the base case scenario; however, one of the 1-way sensitivity analyses was conducted by using observed manufacturing, administration, and 5-year follow-up costs of $87 198 for AAV-mediated gene therapy vector as derived from the manufacturing facility and clinical practice at St Jude Children's Research Hospital. One-way sensitivity analyses revealed 10 of 102 scenarios in which gene therapy was not cost-effective compared with alternative treatments. Notably, gene therapy remained cost-effective in a hypothetical scenario in which we estimated that the discounted factor concentrate price was 20% of the wholesale acquisition cost in the United States. Probabilistic sensitivity analysis estimated gene therapy to be cost-effective at 92% of simulations considering a $150 000/quality-adjusted life-year threshold. In conclusion, based on detailed simulation inputs and assumptions, gene therapy was more cost-effective than on-demand treatment and prophylaxis for patients with severe hemophilia B.
Assuntos
Terapia Genética/economia , Hemofilia B/terapia , Adulto , Simulação por Computador , Análise Custo-Benefício , Hemofilia B/economia , Hemofilia B/epidemiologia , Humanos , Cadeias de Markov , Probabilidade , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).
Assuntos
Bussulfano/administração & dosagem , Terapia Genética , Vetores Genéticos , Subunidade gama Comum de Receptores de Interleucina/genética , Lentivirus , Condicionamento Pré-Transplante , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Antígenos de Diferenciação de Linfócitos T/sangue , Linfócitos B/fisiologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulina M/sangue , Lactente , Células Matadoras Naturais , Contagem de Linfócitos , Masculino , Linfócitos T , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologiaRESUMO
Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.
Assuntos
Proteínas do Capsídeo/análise , Dependovirus/química , Eletroforese Capilar/métodos , Dodecilsulfato de Sódio/química , Proteínas do Capsídeo/químicaRESUMO
In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.
Assuntos
Fármacos Anti-HIV/metabolismo , Quimiocinas CC/biossíntese , Infecções por HIV/tratamento farmacológico , HIV/efeitos dos fármacos , Pichia , Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Reatores Biológicos/economia , Reatores Biológicos/normas , Quimiocinas CC/isolamento & purificação , Quimiocinas CC/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Fermentação , Humanos , Concentração Inibidora 50 , Projetos Piloto , Internalização do Vírus/efeitos dos fármacosRESUMO
Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.
Assuntos
Vetores Genéticos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Vetores Genéticos/genética , Terapia Genética , Retroviridae/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Linfócitos TRESUMO
Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products. SIGNIFICANCE: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007.
Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T , Antígenos CD19 , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismoRESUMO
T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.
Assuntos
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Antígenos CD19 , Linfócitos T CD8-Positivos , Efeitos Psicossociais da Doença , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos TRESUMO
Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e. PpPIR1 and PpPIR2, from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes' open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved repeat of PIR yeast block (SQIGDGQIQATT) in both proteins. The PIR yeast block was present eight times in the PpPir1p and four times in the PpPir2p proteins. Both proteins showed conserved glutamine (Q) and aspartic acid (D) in the repeated sequences, indicating a possible alkali-sensitive ß1,3-glucan ester linkage. The fusion constructs of PpPir1-2p and enhanced green fluorescent protein (EGFP) were developed for yeast cell surface display. The immunofluorescence assay showed uniform localization of EGFP protein on the P. pastoris cell surface in all fusion constructs. Furthermore, new vectors were developed for recombinant protein secretion in P. pastoris, utilizing the pre-pro signal of PpPir1p protein. Efficient processing of the signal sequence was observed from EGFP and human α1-antitrypsin (AAT) fusion constructs and recombinant protein secretion was obtained in the culture supernatant.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genética Microbiana/métodos , Proteínas de Membrana/genética , Pichia/genética , Pichia/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , Imunofluorescência , Fluorometria , Genes Reporter , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos , Análise de Sequência de DNARESUMO
A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H(c))], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(H(c)) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(H(c)) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel™), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5-100mL columns and the process was performed at the pilot scale using 0.6-1.6L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H(c))/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(H(c))/kg WCW at the pilot scale. The purified rBoNTC(H(c)) was stable for at least 3 months at 5 and -80°C as determined by reverse phase-HPLC and SDS-PAGE and was stable for 24 months at -80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H(c)) was intact.
Assuntos
Toxinas Botulínicas , Clostridium botulinum/química , Pichia , Proteínas Recombinantes , Sequência de Aminoácidos , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Biotecnologia , Bioterrorismo/prevenção & controle , Toxinas Botulínicas/genética , Toxinas Botulínicas/imunologia , Toxinas Botulínicas/isolamento & purificação , Toxinas Botulínicas/metabolismo , Cromatografia , Clonagem Molecular , Feminino , Fermentação , Camundongos , Dados de Sequência Molecular , Projetos Piloto , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transferência de TecnologiaRESUMO
CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8+ CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes. The CAR T cell post-infusion changes are further characterized by repression of genes (e.g., TCF7 and LEF1) associated with memory potential and a DNA methylation signature (e.g., demethylation at CX3CR1, BATF, and TOX) demarcating a transition toward exhaustion-progenitor T cells. Thus, CD19-CAR T cells undergo exhaustion-associated DNA methylation programming, indicating that efforts to prevent this process may be an attractive approach to improve CAR T cell efficacy.
Assuntos
Antígenos CD19/imunologia , Linfócitos T CD8-Positivos/imunologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Imunoterapia Adotiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Adulto JovemRESUMO
SeVRSV is a replication-competent Sendai virus (SeV)-based vaccine carrying the respiratory syncytial virus (RSV) fusion protein (F) gene. Unmanipulated, non-recombinant SeV is a murine parainfluenza virus type 1 (PIV-1) and serves as a Jennerian vaccine for human PIV-1 (hPIV-1). SeV protects African green monkeys (AGM) from infection after hPIV-1 challenge. The recombinant SeVRSV additionally targets RSV and protects AGM from lower respiratory infections after RSV challenge. The present study is the first to report on the safety, viral genome detection, and immunogenicity following SeVRSV vaccination of healthy adults. Seventeen and four healthy adults received intranasal SeVRSV and PBS, respectively, followed by six months of safety monitoring. Virus genome (in nasal wash) and vaccine-specific antibodies (in sera) were monitored for two and four weeks, respectively, post-vaccination. The vaccine was well-tolerated with only mild to moderate reactions that were also present in the placebo group. No severe reactions occurred. As expected, due to preexisting immunity toward hPIV-1 and RSV in adults, vaccine genome detection was transient. There were minimal antibody responses to SeV and negligible responses to RSV F. Results encourage further studies of SeVRSV with progression toward a clinical trial in seronegative children. Abbreviations: AE-adverse event; SAE-serious adverse event; SeV-Sendai virus; RSV-respiratory syncytial virus; PIV-1-parainfluenza virus-type 1; hPIV-1-human parainfluenza virus-type 1; F-RSV fusion protein; SeVRSV-recombinant SeV carrying the RSV F gene; Ab-antibody; MSW-medically significant wheezing; NOCMC-new onset chronic medical condition, mITT-modified Intent to Treat; ALRI-acute lower respiratory tract infection.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Adulto , Animais , Anticorpos Antivirais , Chlorocebus aethiops , Humanos , Imunogenicidade da Vacina , Vírus da Parainfluenza 1 Humana/genética , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/efeitos adversos , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/genética , Vírus Sendai/genética , Proteínas Virais de Fusão/genéticaRESUMO
BACKGROUND: Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce. RESULTS: Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man5GlcNAc2 N-glycosylation of the secreted proteins is achieved at all specific growth rates tested. CONCLUSIONS: Together, these data illustrate that the GlycoSwitch-Man5 P. pastoris is a robust production strain for homogenously N-glycosylated proteins.
Assuntos
Fermentação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Pichia/crescimento & desenvolvimento , Clonagem Molecular , Glicosilação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
With many ongoing clinical trials utilizing adeno-associated virus (AAV) gene therapy, it is necessary to find scalable and serotype-independent primary capture and recovery methods to allow for efficient and robust manufacturing processes. Here, we demonstrate the ability of a hydrophobic interaction chromatography membrane to capture and recover AAV1, AAV5, AAV8, and AAV "Mutant C" (a novel serotype incorporating elements of AAV3B and AAV8) particles from cell culture media and cell lysate with recoveries of 76%-100% of loaded material, depending on serotype. A simple, novel technique that integrates release and recovery of cell-associated AAV capsids is demonstrated. We show that by the addition of lyotropic salts to AAV-containing cell suspensions, AAV is released at an equivalent efficiency to mechanical lysis. The addition of the lyotropic salt also promotes a phase separation, which allows physical removal of large amounts of DNA and insoluble cellular debris from the AAV-containing aqueous fraction. The AAV is then captured and eluted from a hydrophobic interaction chromatography membrane. This integrated lysis and primary capture and recovery technique facilitates substantial removal of host-cell DNA and host-cell protein impurities.
RESUMO
Self-inactivating lentiviral vectors (LVVs) are used regularly for genetic modification of cells, including T cells and hematopoietic stem cells for cellular gene therapy. As vector demand grows, scalable and controllable methods are needed for production. LVVs are typically produced in HEK293T cells in suspension bioreactors using serum-free media or adherent cultures with serum. The iCELLis® is a packed-bed bioreactor for adherent or entrained cells with surface areas from 0.53 to 500 m2. Media are pumped through the fixed bed and overflows, creating a thin film that is replenished with oxygen and depleted of CO2 as media return to the reservoir. We describe the optimization and scale-up of the production of GPRTG-EF1α-hγc-OPT LVV using a stable packaging cell line in the iCELLis Nano 2-cm to the 10-cm bed height low compaction bioreactors (0.53 and 2.6 m2 surface area) and compare to the productivity and efficacy of GPRTG-EF1α-hγc-OPT LVV manufactured under current Good Manufacturing Practice (cGMP) using 10-layer cell factories for the treatment of X-linked severe combined immunodeficiency. By optimizing fetal bovine serum (FBS) concentration, pH post-induction, and day of induction, we attain viral yields of more than 2 × 107 transducing units/mL. We compared transduction efficiency between LVVs produced from the iCELLis Nano and cell factories on healthy, purified CD34+ cells and found similar results.
RESUMO
Hu14.18K322A is a humanized anti-GD2 monoclonal antibody with a single point mutation that reduces complement-mediated cytotoxicity, with a maximum tolerated dose (MTD) of 60 mg/m2 daily for 4 days in children with recurrent/refractory neuroblastoma. We report additional results of a Phase 1 trial to determine the MTD and safety profile of hu14.18K322A in patients with osteosarcoma, and of an alternative schedule of weekly hu14.18K322A administration in patients with neuroblastoma or osteosarcoma. Eligible patients with recurrent/refractory osteosarcoma received hu14.13K22A daily x4 every 28 days in a Phase 1 traditional 3 + 3 dose escalation design. Additional patients with osteosarcoma were then enrolled to receive hu14.18K322A once weekly for 4 weeks per course. Patients with recurrent/refractory neuroblastoma were also enrolled on the weekly schedule at 50 mg/m2/dose. Six patients with osteosarcoma treated on the daily schedule received a median of 2 (range 1-6) courses; the recommended daily dose was established as 60 mg/m2. Three patients had stable disease (SD) as best overall response. Five patients (3 neuroblastoma, 2 osteosarcoma) enrolled on the weekly schedule received a median of 1 (1-3) course; 2 achieved SD as best overall response. Pain, fever, hematologic toxicities, hyponatremia, and ocular/visual abnormalities were common toxicities among both schedules. Dose-limiting toxicities attributed to hu14.18K322A included anorexia and fatigue (n = 1). Pharmacokinetic profiles were similar between daily and weekly schedules. The recommended dose for patients with osteosarcoma receiving daily hu14.18K322A x4 is 60 mg/m2. Patients receiving the weekly schedule experienced similar pharmacokinetics and toxicity profile as the daily schedule.
Assuntos
Anticorpos Monoclonais Humanizados , Antineoplásicos Imunológicos , Neoplasias Ósseas , Neuroblastoma , Osteossarcoma , Adolescente , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/farmacocinética , Neoplasias Ósseas/sangue , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Neuroblastoma/sangue , Neuroblastoma/tratamento farmacológico , Osteossarcoma/sangue , Osteossarcoma/tratamento farmacológico , RatosRESUMO
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been the method of choice in the past decades for size-based protein analysis. However, in general it requires the protein concentration in mg/mL level and thus is not practical for trace level protein analysis, not to mention the lengthy labor-intensive procedures. The SDS capillary gel electrophoresis (SDS CGE) method reported herein requires only nanogram-sized proteins loaded onto the autosampler. A sample stacking technique (e.g., head-column field-amplified sample stacking (HC FASS)) was employed, providing three orders of magnitude sensitivity enhancement compared to conventional SDS CGE. This method has been used routinely in purity analysis and characterization of adeno-associated virus (AAV) intermediates and finished gene therapeutics of AAV vectors. The sensitivity achieved is comparable to the currently most sensitive size-based protein assay silver-stained SDS PAGE. The highly sensitive sample stacking SDS CGE can be used for other types of proteins as well.
Assuntos
Proteínas do Capsídeo/análise , Dependovirus/química , Eletroforese Capilar/métodos , Dodecilsulfato de Sódio/química , Análise de DadosRESUMO
A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.
Assuntos
Vacinas Bacterianas/biossíntese , Bancos de Espécimes Biológicos , Toxinas Botulínicas/biossíntese , Microbiologia Industrial , Pichia/citologia , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Vacinas Bacterianas/genética , Bancos de Espécimes Biológicos/normas , Reatores Biológicos/microbiologia , Reatores Biológicos/normas , Toxinas Botulínicas/genética , Toxinas Botulínicas/isolamento & purificação , Simulação por Computador , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Microbiologia Industrial/normas , Modelos Biológicos , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
This chapter provides rational approaches to design and optimize fed-batch and continuous fermentations of both Mut+ and Muts (methanol utilization plus and slow) Pichia pastoris strains. The methods are described in detail for glycerol batch, glycerol fed-batch, transition, and methanol fed-batch/mixed feed/ continuous stirred tank reactor (CSTR) phases of the process based on glycerol and methanol consumption models. Cell density, broth volume, substrate feed rate, and the length of each phase are rationally designed to conduct runs with selected parameters for optimizing a process. The optimization is anchored by the impact of a specific growth rate/dilution time (for CSTRs) on productivity. Equations for simulation of a process with optimal parameters are derived for an optimal process design. This protocol can be used as a practical manual for process development of a P. pastoris recombinant fermentation, and also as a reference for fermentation of other microorganisms.
Assuntos
Fermentação , Engenharia Genética/métodos , Pichia/genética , Glicerol/metabolismo , Metanol/metabolismoRESUMO
Human hookworm infection is one of the most significant parasitic infections, and a leading global cause of anemia and malnutrition of adults and children in rural areas of the tropics and subtropics. Necator americanus secretory protein (Na-ASP1), which is a potential vaccine candidate against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1 protein was expressed extracellulary by employing the leader sequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. The Na-ASP1 steady state mRNA levels of the clones were correlated to their Na-ASP1 gene copy number. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene.