Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.
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ABSTRACT:
SUBMITTER: Sundarasetty BS
PROVIDER: S-EPMC4561294 | biostudies-other | 2015 Sep
REPOSITORIES: biostudies-other
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Project description:BackgroundReactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs ("SmyleDCpp65") that accelerated antigen-specific T cell development.MethodsHere we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed.ResultsUpscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated.ConclusionThese results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
| S-EPMC4511080 | biostudies-literature
Project description:The activation of the stimulating factor of the interferon gene (STING) pathway can enhance the immune response within the tumor. Cyclic diguanylate monophosphate (c-di-GMP) is a negatively charged, hydrophilic STING agonist, however, its effectiveness is limited due to the poor membrane permeability and low bioavailability. Herein, we introduced KL-7 peptide derived from Aβ amyloid fibrils that can self-assemble to form nanotubes to load and deliver c-di-GMP, which significantly enhanced c-di-GMP's effectiveness and then exhibited a robust "in situ immunity" to kill melanoma cells. KL-7 peptide nanotube, also called PNT, was loaded with negatively charged c-di-GMP via electrostatic interaction, which prepared a nanocomposite named c-di-GMP-PNT. Treatment of RAW 264.7 cells (leukemia cells in mouse macrophage) with c-di-GMP-PNT markedly stimulated the secretion of IL-6 and INF-β along with phospho-STING (Ser365) protein expression, indicating the activation of the STING pathway. In the unilateral flank B16-F10 (murine melanoma cells) tumor-bearing mouse model, compared to PNT and c-di-GMP, c-di-GMP-PNT can promote the expression of INF-β, TNF-α, IL-6, and IL-1β. At the same time, up-regulated CD4 and CD8 active T cells kill tumors and enhance the immune response in tumor tissues, resulting in significant inhibition of tumor growth in tumor-bearing mice. More importantly, in a bilateral flank B16-F10 tumor model, both primary and distant tumor growth can also be significantly inhibited by c-di-GMP-PNT. Moreover, c-di-GMP-PNT demonstrated no obvious biological toxicity on the main organs (heart, liver, spleen, lung, and kidney) and biochemical indexes of mice. In summary, our study provides a strategy to overcome the barriers of free c-di-GMP in the tumor microenvironment and c-di-GMP-PNT may be an attractive nanomaterial for anti-tumor immunity.Electronic supplementary materialSupplementary material (synthesis and characterization of KL-7 peptide; the encapsulation rate and cumulative release rate of c-di-GMP-PNT; cytotoxicity of PNT, c-di-GMP, and c-di-GMP-PNT; anti-tumor effect of c-di-GMP-PNT (equivalent to 1 and 5 µg c-di-GMP per mouse); representative immunofluorescence images; and biosafety analysis) is available in the online version of this article at 10.1007/s12274-022-5102-z.
| S-EPMC9643898 | biostudies-literature
Project description:With the emergence of immune checkpoint inhibitors and adoptive T-cell therapies, there is a considerable interest in using personalized autologous dendritic cell (DC) vaccines in combination with T cell-targeting immunotherapies to potentially maximize the therapeutic impact of DC vaccines. Here, we describe the development and optimization of a Good Manufacturing Practice (GMP)-compliant manufacturing process based on tumor lysate as a tumor antigen source for the production of an oxidized tumor cell lysate loaded DC (OC-DC) vaccine. The manufacturing process required one day for lysate preparation and six days for OC-DC vaccine production. Tumor lysate production was standardized based on an optimal tumor digestion protocol and the immunogenicity was improved through oxidation using hypochloric acid prior to freeze-thaw cycles resulting in the oxidized tumor cell lysate (OC-L). Next, monocytes were selected using the CliniMACS prodigy closed system and were placed in culture in cell factories in the presence of IL-4 and GM-CSF. Immature DCs were loaded with OC-L and matured using MPLA-IFN?. After assessing the functionality of the OC-DC cells (IL12p70 secretion and COSTIM assay), the OC-DC vaccine was cryopreserved in multiple doses for single use. Finally, the stability of the formulated doses was tested and validated. We believe this GMP-compliant DC vaccine manufacturing process will facilitate access of patients to personalized DC vaccines, and allow for multi-center clinical trials.
| S-EPMC7157441 | biostudies-literature
Project description:Wilms' tumor 1 antigen (WT1) is overexpressed in acute myeloid leukemia (AML), a high-risk neoplasm warranting development of novel immunotherapeutic approaches. Unfortunately, clinical immunotherapeutic use of WT1 peptides against AML has been inconclusive. With the rationale of stimulating multiantigenic responses against WT1, we genetically programmed long-lasting dendritic cells capable of producing and processing endogenous WT1 epitopes. A tricistronic lentiviral vector co-expressing a truncated form of WT1 (lacking the DNA-binding domain), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) was used to transduce human monocytes ex vivo. Overnight transduction induced self-differentiation of monocytes into immunophenotypically stable "SmartDC/tWT1" (GM-CSF(+), IL-4(+), tWT1(+), IL-6(+), IL-8(+), TNF-α(+), MCP-1(+), HLA-DR(+), CD86(+), CCR2(+), CCR5(+)) that were viable for 3 weeks in vitro. SmartDC/tWT1 were produced with peripheral blood mononuclear cells (PBMC) obtained from an FLT3-ITD(+) AML patient and surplus material from a donor lymphocyte infusion (DLI) and used to expand CD8(+) T cells in vitro. Expanded cytotoxic T lymphocytes (CTLs) showed antigen-specific reactivity against WT1 and against WT1(+) leukemia cells. SmartDC/tWT1 injected s.c. into Nod.Rag1(-/-).IL2rγc(-/-) mice were viable in vivo for more than three weeks. Migration of human T cells (huCTLs) to the immunization site was demonstrated following adoptive transfer of huCTLs into mice immunized with SmartDC/tWT1. Furthermore, SmartDC/tWT1 immunization plus adoptive transfer of T cells reactive against WT1 into mice resulted in growth arrest of a WT1(+) tumor. Gene array analyses of SmartDC/tWT1 demonstrated upregulation of several genes related to innate immunity. Thus, SmartDC/tWT1 can be produced in a single day of ex vivo gene transfer, are highly viable in vivo, and have great potential for use as immunotherapy against malignant transformation overexpressing WT1.
| S-EPMC3696945 | biostudies-literature
Project description:It is often difficult to cure endocarditis, osteomyelitis, and device-associated infections caused by Gram-positive pathogens, despite therapy with clinically appropriate antibiotics. This may be due to antibiotic tolerance or resistance development. Acyldepsipeptides (ADEPs) are a class of bactericidal compounds active against a variety of clinically important Gram-positive bacteria, including staphylococci, streptococci, and enterococci. ADEPs activate caseinolytic protease P (ClpP), killing high-density, nondividing cultures of bacteria that are tolerant to approved classes of antibiotics. Acyldepsipeptide analog 4 (ADEP4) was active against a panel of drug-resistant Gram-positive pathogens in MIC assays, with no preexisting resistance detected. Killing of stationary-phase cultures was observed when ADEP4 was combined with multiple classes of approved antibiotics. Additionally, a hollow-fiber infection model was used to assess the effects of ADEP4 antibiotic combinations on bacterial killing and resistance development. These studies were performed on high-density cultures of methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and vancomycin-resistant Enterococcus faecalis (VRE). None of the approved antibiotics linezolid, ampicillin, and oxacillin tested alone had bactericidal activity under these conditions. ADEP4 initially caused killing, but regrowth of the culture was apparent within 96?h due to resistance. Combinations of ADEP4 with linezolid or oxacillin caused substantially improved killing of MRSA or MSSA cultures, respectively, and no regrowth due to resistance was observed. The combination of ADEP4 and ampicillin eradicated cultures of VRE to the limit of detection within 52?h. These data suggest that combining ClpP activators with traditional antibiotics may be a good strategy to treat complicated Gram-positive infections.
| S-EPMC7187625 | biostudies-literature
Project description:Melanoma is one of the most aggressive and therapy-resistant types of cancer, the incidence rate of which grows every year. However, conventional methods of chemo- and radiotherapy do not allow for completely removing neoplasm, resulting in local, regional, and distant relapses. In this case, adjuvant therapy can be used to reduce the risk of recurrence. One of the types of maintenance cancer therapy is cell-based immunotherapy, in which immune cells, such as T-cells, NKT-cells, B cells, NK cells, macrophages, and dendritic cells are used to recognize and mobilize the immune system to kill cancer cells. These cells can be isolated from the patient's peripheral blood or biopsy material and genetically modified, cultured ex vivo, following infusion back into the patient for powerful induction of an anti-tumor immune response. In this review, the advantages and problems of the most relevant methods of cell-based therapy and ongoing clinical trials of adjuvant therapy of melanoma are discussed.
| S-EPMC9916554 | biostudies-literature
Project description:BACKGROUND:Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. METHODS:Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis. RESULTS:This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7?days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33?±?8.37%), acinar (2.80?±?1.25%) cells, and pancreatic progenitors (5.81?±?2.65%). CONCLUSION:This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.
| S-EPMC7055108 | biostudies-literature
Project description:In the struggle to understand and accurately diagnose Parkinson's disease, radiopharmaceuticals and medical imaging techniques have played a major role. By being able to image and quantify the dopamine transporter density, noninvasive diagnostic imaging has become the gold standard. In the shift from the first generation of SPECT tracers, the fluorine-18-labeled tracer [18F]FE-PE2I has emerged as the agent of choice for many physicians. However, implementing suitable synthesis for the production of [18F]FE-PE2I has proved more challenging than expected. Through a thorough analysis of the relevant factors affecting the final radiochemical yield, we were able to implement high-yielding fully automated GMP-compliant synthesis of [18F]FE-PE2I on a Synthera®+ platform. By reaching RCYs up to 62%, it allowed us to isolate 25 GBq of the formulated product, and an optimized formulation resulted in the shelf life of 6 h, satisfying the increased demand for this radiopharmaceutical.
| S-EPMC8308591 | biostudies-literature
Project description:BACKGROUND:Gallium-68 labeled synthetic somatostatin analogs for PET/CT imaging are the current gold standard for somatostatin receptor imaging in neuroendocrine tumor patients. Despite good imaging properties, their use in clinical practice is hampered by the low production levels of 68Ga eluted from a 68Ge/68Ga generator. In contrast, 18F-tracers can be produced in large quantities allowing centralized production and distribution to distant PET centers. [18F]AlF-NOTA-octreotide is a promising tracer that combines a straightforward Al18F-based production procedure with excellent in vivo pharmacokinetics and specific tumor uptake, demonstrated in SSTR2 positive tumor mice. However, advancing towards clinical studies with [18F]AlF-NOTA-octreotide requires the development of an efficient automated GMP production process and additional preclinical studies are necessary to further evaluate the in vivo properties of [18F]AlF-NOTA-octreotide. In this study, we present the automated GMP production of [18F]AlF-NOTA-octreotide on the Trasis AllinOne® radio-synthesizer platform and quality control of the drug product in accordance with GMP. Further, radiometabolite studies were performed and the pharmacokinetics and biodistribution of [18F]AlF-NOTA-octreotide were assessed in healthy rats using μPET/MR. RESULTS:The production process of [18F]AlF-NOTA-octreotide has been validated by three validation production runs and the tracer was obtained with a final batch activity of 10.8 ± 1.3 GBq at end of synthesis with a radiochemical yield of 26.1 ± 3.6% (dc), high radiochemical purity and stability (96.3 ± 0.2% up to 6 h post synthesis) and an apparent molar activity of 160.5 ± 75.3 GBq/μmol. The total synthesis time was 40 ± 3 min. Further, the quality control was successfully implemented using validated analytical procedures. Finally, [18F]AlF-NOTA-octreotide showed high in vivo stability and favorable pharmacokinetics with high and specific accumulation in SSTR2-expressing organs in rats. CONCLUSION:This robust and automated production process provides high batch activity of [18F]AlF-NOTA-octreotide allowing centralized production and shipment of the compound to remote PET centers. Further, the production process and quality control developed for [18F]AlF-NOTA-octreotide is easily implementable in a clinical setting and the tracer is a potential clinical alternative for somatostatin directed 68Ga labeled peptides obviating the need for a 68Ge/68Ga-generator. Finally, the favorable in vivo properties of [18F]AlF-NOTA-octreotide in rats, with high and specific accumulation in SSTR2 expressing organs, supports clinical translation.
| S-EPMC6989705 | biostudies-literature
Project description:Radiotherapy induces DNA damage, resulting in cell-cycle arrest and activation of cell-intrinsic death pathways. However, the radioresistance of some tumour entities such as malignant melanoma limits its clinical application. The innate immune sensing receptor retinoic acid-inducible gene I (RIG-I) is ubiquitously expressed and upon activation triggers an immunogenic form of cell death in a variety of tumour cell types including melanoma. To date, the potential of RIG-I ligands to overcome radioresistance of tumour cells has not been investigated. Here, we demonstrate that RIG-I activation enhanced the extent and immunogenicity of irradiation-induced tumour cell death in human and murine melanoma cells in vitro and improved survival in the murine B16 melanoma model in vivo. Transcriptome analysis pointed to a central role for p53, which was confirmed using p53-/- B16 cells. In vivo, the additional effect of RIG-I in combination with irradiation on tumour growth was absent in mice carrying p53-/- B16 tumours, while the antitumoural response to RIG-I stimulation alone was maintained. Our results identify p53 as a pivotal checkpoint that is triggered by RIG-I resulting in enhanced irradiation-induced tumour cell death. Thus, the combined administration of RIG-I ligands and radiotherapy is a promising approach to treating radioresistant tumours with a functional p53 pathway, such as melanoma.
| S-EPMC10394996 | biostudies-literature