BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.
- MeSH
- aktivace lymfocytů účinky léků genetika MeSH
- buněčné kultury metody MeSH
- buňky PC-3 MeSH
- chimerické antigenní receptory genetika metabolismus MeSH
- elektroporace MeSH
- genetické vektory MeSH
- HEK293 buňky MeSH
- imunoterapie adoptivní metody MeSH
- interleukin-4 farmakologie MeSH
- interleukin-7 farmakologie MeSH
- interleukiny farmakologie MeSH
- kultivované buňky MeSH
- Lentivirus genetika MeSH
- lidé MeSH
- proteinové inženýrství metody MeSH
- protinádorové vakcíny genetika imunologie MeSH
- receptory antigenů T-buněk genetika metabolismus MeSH
- T-lymfocyty * cytologie účinky léků imunologie metabolismus MeSH
- transdukce genetická metody MeSH
- transpozibilní elementy DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the recent past, it has repeatedly been reported that CD4 cells play an important role in the immunology of chronic myeloid leukaemia. It was therefore of interest to test their activity in an animal model using bcr-abl-transformed cells. BALB/c mice were four times immunized with a DNA vaccine carrying the bcr-abl fusion gene. Two weeks after the last vaccine dose, the animals were challenged with syngeneic bcr-abl-transformed 12B1 cells which form solid tumors after subcutaneous administration. At the time of challenge, animals were treated with antibodies against the CD8+ T cells or CD4+ T cells. The efficacy of the depletion was monitored and found highly effective. All nonimmunized animals developed tumors. All animals untreated with the antibodies as well as those in which CD8+ T cells had been depleted, were fully protected against the challenge. On the other hand, almost all mice treated with anti-CD4+ antibody developed tumors. These results strongly suggested that the CD4+ T cells acted as effectors in the present system.
- MeSH
- antigeny CD95 imunologie metabolismus MeSH
- bcr-abl fúzové proteiny genetika imunologie MeSH
- CD4-pozitivní T-lymfocyty imunologie metabolismus MeSH
- CD8-pozitivní T-lymfocyty imunologie metabolismus MeSH
- DNA vakcíny imunologie MeSH
- histokompatibilita - antigeny třídy II imunologie metabolismus MeSH
- imunizace MeSH
- lidé MeSH
- ligand Fas metabolismus MeSH
- lymfocytární deplece MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nádorová transformace buněk genetika imunologie MeSH
- nádory genetika imunologie mortalita prevence a kontrola MeSH
- protinádorové vakcíny genetika imunologie MeSH
- slezina cytologie imunologie MeSH
- transformované buněčné linie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The development of cervical cancer is associated with infection by oncogenic human papillomaviruses (HPVs), of which type 16 (HPV16) is the most prevalent in HPV-induced malignant diseases. The viral oncoproteins E6 and E7 are convenient targets for anti-tumor immunization. To adapt the corresponding genes for DNA vaccination, their oncogenicity needs to be reduced and immunogenicity enhanced. The main modifications for achieving these aims include mutagenesis, rearrangement of gene parts, and fusion with supportive cellular or viral/bacterial genes or their functional parts. As HPVs are strictly human specific, an animal model of HPV infection does not exist. Therefore, immunization against HPV-induced tumors is most frequently tested in mouse models utilizing transplantable syngeneic tumor cells producing the HPV16 E6/E7 oncoproteins. In this chapter, one such cell line designated TC-1 is characterized and the effect of immunization with the modified E7 fusion gene against TC-1-induced subcutaneous tumors is described. As down-regulation of MHC class I molecules is one of the most important escape mechanisms of cervical carcinoma cells, the TC-1/A9 clone with reversibly reduced MHC class I expression has been developed and, herein, its response to DNA vaccination is also shown and compared with that of the TC-1 cells.
- MeSH
- biolistika přístrojové vybavení MeSH
- DNA vakcíny aplikace a dávkování chemie genetika imunologie MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory děložního čípku patologie prevence a kontrola MeSH
- proliferace buněk MeSH
- protinádorové vakcíny aplikace a dávkování chemie genetika imunologie MeSH
- vakcinace přístrojové vybavení MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viral CC chemokine inhibitor (vCCI) of the clone P13 vaccinia virus (VACV) strain PRAHA lacks eight amino acids in the signal peptide sequence. To study the influence of vCCI on virus biology, a virus with the vCCI gene coding for a prolonged signal sequence was prepared. We found that secreted vCCI attenuated the virus in vivo, and that it correlated with decreased levels of RANTES, eotaxin, TARC, and MDC in the blood in comparison with the parental virus. We determined the influence of vCCI on the CTL response against VACV E3((140-148)) (VGPSNSPTF) and HPV16 E7((49-57)) (RAHYNIVTF) H-2D(b)-restricted epitopes. The examination of the specific CTL response elicited by immunization with the recombinant VACV-expressing tumor-associated HPV16 E7 antigen by IFN-γ ELISPOT showed that the immunogenicity of the recombinant VACV-producing secretory vCCI was similar to that of the parent virus or deletion mutant in the C23L/B29R locus. Immunization with the secretory vCCI-producing recombinant virus has a lower therapeutic anti-tumor effect against TC-1 tumors. Viral CCI downregulated the E7-specific response induced by gene gun immunization with the DNA vaccines pBSC-SigE7 LAMP and pBSC-vCCI. We also observed that the immune response against vCCI elicited by the DNA vaccine did not affect the multiplication of VACV in vivo.
- MeSH
- buněčné linie MeSH
- chemokin CCL17 krev MeSH
- chemokin CCL5 krev MeSH
- chemokiny CC antagonisté a inhibitory krev MeSH
- cytotoxické T-lymfocyty imunologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové supresorové proteiny krev MeSH
- Papillomavirus E7 - proteiny genetika imunologie MeSH
- proteiny ADAM krev MeSH
- protinádorové vakcíny genetika imunologie MeSH
- sekvenční delece MeSH
- syntetické vakcíny genetika imunologie MeSH
- vakcinace MeSH
- virové proteiny genetika metabolismus MeSH
- virové vakcíny imunologie MeSH
- virus vakcinie genetika imunologie patogenita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.
- MeSH
- bcr-abl fúzové proteiny genetika imunologie MeSH
- časové faktory MeSH
- chronická myeloidní leukemie genetika imunologie patologie prevence a kontrola MeSH
- DNA vakcíny genetika imunologie MeSH
- HL-60 buňky MeSH
- imunizace MeSH
- lidé MeSH
- mapování epitopu MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- peptidové fragmenty imunologie MeSH
- protinádorové vakcíny genetika imunologie MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Recombinant vaccinia viruses (rVACV) expressing various tumor-associated antigens have been shown to elicit anti-tumor effect in numerous experimental models and clinical trials. We tested the hypotheses that rVACV expressing biologically active fms-like tyrosine kinase 3 ligand (Flt3L) would show higher immunogenicity than control viruses expressing only model antigen and that coexpression of Flt3L would influence anti-tumor activity of rVACV in the preventive and therapeutic arrangements of the in vivo experiment. To answer these questions, we took advantage of the well-described model of transplanted tumor cells expressing HPV16 E6 and E7 oncoproteins. To determine the effects of hFlt3L on the induction of anti-tumor immunity, we generated live vaccinia viruses that express human Flt3L regulated by the early H5 or strong synthetic E/L promoter together with fusion protein SigE7LAMP, which is a highly immunogenic form of HPV E7 oncoprotein. We tested Flt3L production in vitro and in vivo. Despite higher expression of Flt3L from the synthetic E/L promoter in vitro, the P13-E/L-FL-SigE7LAMP induced lower levels of Flt3L in the serum of mice than P13-H5-FL-SigE7LAMP. The Flt3L expression under the strong early VACV H5 promoter is able to inhibit expansion of CD11b+Gr-1+ myeloid suppressor cells (MSC) and increase the amount of CD11b+ CD11c+ dendritic cells in the spleen of mice immunized with vaccinia virus. Determination of viral DNA isolated from the ovaries of infected animals did not reveal differences in replication between rVACVs in this organ. Coexpression of Flt3L by replication-competent virus P13-FL-SigE7LAMP induced enhancement of the cellular immune response against HPV16 E7 and VACV E3 proteins as well as increased anti-tumor efficacy in both the protective and therapeutic immunization schemes. On the other hand, the short-time Flt3L coexpression by MVA-H5-FL-SigE7LAMP was not sufficient to enhance anti-tumor effect of immunization.
- MeSH
- analýza rozptylu MeSH
- antigen prezentující buňky imunologie MeSH
- CD8-pozitivní T-lymfocyty imunologie MeSH
- ELISA MeSH
- genetické vektory genetika imunologie MeSH
- lidé MeSH
- membránové proteiny biosyntéza genetika imunologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádory imunologie terapie MeSH
- protinádorové vakcíny genetika imunologie MeSH
- transfekce MeSH
- virus vakcinie genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Therapeutic strategies based on the insertion of cytokine or other immunostimulatory genes into the genome of tumour cells followed by vaccination with the resulting, genetically modified, cytokine-producing vaccines represent a new potential prospect for the treatment of cancer patients. HPV 16 is the aetiological agent of more than 60 percent human cervical carcinomas (CC). At present, two prophylactic vaccines against HPV 16 are available (GlaxoSmithKline "Cervarix" and Merck "Gardasil"). These vaccines can almost completely protect the immunized individuals against both, persistent HPV 16 infection and HPV 16-related pathological findings in cervical cytology. In contrast, no clinically utilizable therapeutic vaccines against CC are available. During the last decade animal models have substantially contributed to the development of the therapeutic vaccines against HPV 16-associated tumours. It has been demonstrated that the HPV 16 E6/E7 oncoproteins can serve as tumour rejection antigens (TRA) and that the HPV 16-associated tumour cells can be genetically modified with DNA encoding immunostimulatory cytokines (IL-2, IL-12, GM-CSF) or other immunostimulatory molecules, used for vaccination, and inhibit tumour growth. To improve the HPV 16 antigen presentation in tumour-bearing individuals, dendritic cell-based vaccines loaded with HPV 16 E6/E7 DNA or hybrids of the dendritic and tumour cells have also been successfully employed. Unfortunately, when these encouraging approaches used in animal models were translated into clinical trials, the results were less optimistic. The problems that are still to be faced before the therapeutic vaccines against high-risk HPV-associated tumours can be approved for clinical purposes are discussed.
- MeSH
- cytokiny genetika imunologie MeSH
- financování organizované MeSH
- infekce papilomavirem imunologie prevence a kontrola virologie MeSH
- lidé MeSH
- lidský papilomavirus 16 genetika imunologie MeSH
- modely nemocí na zvířatech MeSH
- nádory děložního čípku imunologie prevence a kontrola virologie MeSH
- onkogenní proteiny virové genetika imunologie MeSH
- Papillomavirus E7 - proteiny MeSH
- protinádorové vakcíny genetika imunologie MeSH
- racionální návrh léčiv MeSH
- represorové proteiny imunologie MeSH
- vakcíny proti papilomavirům genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- cytokiny genetika MeSH
- finanční podpora výzkumu jako téma MeSH
- financování organizované MeSH
- genetická terapie MeSH
- imunizace MeSH
- lidé MeSH
- myši MeSH
- nádory genetika imunologie terapie MeSH
- preklinické hodnocení léčiv MeSH
- protinádorové vakcíny genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
