ABSTRACT
Treatment with chimeric antigen receptor (CAR)-modified T cells targeting CD19 has proved successful in patients with relapsed/refractory B cell malignancies. However, long-term follow-up indicates that remission in a substantial proportion of patients is not sustainable. Most patients that experience recurrence have tumors and lost the CAR-T cells. To maintain the activity of CAR-T cells, Raji-B-NDG mice were treated sequentially with CAR-T-19â¯cells and homologous cells expressing human CD19 to promote expansion of CAR-T cells. Sequential treatment of mice with CAR-T-19â¯cells followed by Raji tumor cells led to marked prolongation of survival. The best case scenario after sequential treatment was a survival time of more than 200 days; the average survival time of mice in the non-sequential treatment group was 80 days. We treated mice with autologous CD19-modified T cells after initial treatment with CAR-T-19â¯cells. The overall survival and recurrence-free survival times of mice receiving sequential treatment were significantly longer. The percentages of CAR+ T cells in peripheral blood increased. Sequential therapy with autologous CAR-T-19 and aT19â¯cells provides a new strategy for generating memory CAR-T cells, which may lead ultimately to increased clinical efficacy.
Subject(s)
Antigens, CD19/immunology , Immunotherapy, Adoptive/methods , Lymphoma, B-Cell/therapy , Neoplasm Recurrence, Local/prevention & control , T-Lymphocytes/transplantation , Animals , Antigens, CD19/genetics , Cell Line, Tumor , Combined Modality Therapy/methods , Disease-Free Survival , HEK293 Cells , Healthy Volunteers , Humans , Immunologic Memory , Longevity/immunology , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/mortality , Mice , Neoplasm Recurrence, Local/immunology , Receptors, Chimeric Antigen/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Remission Induction/methods , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Time Factors , Transduction, Genetic , Transplantation, Autologous/methods , Xenograft Model Antitumor AssaysABSTRACT
Pyrroloquinoline quinone is the third redox cofactor after nicotinamide and flavin in bacteria, and its biosynthesis pathway comprise five steps initiated from a precursor peptide PqqA coded by pqqA gene. Methylovorus sp. MP688 is equipped with five copies of pqqA genes. Herein, the transcription of pqqA genes under different conditions by real-time quantitative PCR and ß-galactosidase reporter genes are reported. Multiple pqqA genes were proved to play significant roles and contribute differently in PQQ synthesis. pqqA1, pqqA2, and pqqA4 were determined to be dominantly transcribed over the others, and correspondingly absence of any of the three genes caused a decrease in PQQ synthesis. Notably, pqqA was up-regulated in low pH and limited oxygen environment, and it is pqqA2 promoter that could be induced when bacteria were transferred from pH 7.0 to pH 5.5. Deletion analysis revealed a region within pqqA2 promoter inhibiting transcription. PQQ concentration was increased by overexpression of pqq genes under control of truncated pqqA2 promoter. The results not only imply there exist negative transcriptional regulators for pqqA2 but also provide us a new approach to achieve higher PQQ production by deleting the target binding sequence.
Subject(s)
Gene Expression Regulation, Bacterial , Genes, Bacterial , Methylophilaceae/genetics , PQQ Cofactor/biosynthesis , PQQ Cofactor/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Deletion , Hydrogen-Ion Concentration , Methylophilaceae/metabolism , Multigene Family , Mutation , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Methylotrophic bacteria are widespread microbes which can use one carbon compound as their only carbon and energy sources. Here we report the finished, annotated genome sequence of the methylotrophic bacterium Methylovorus sp. strain MP688, which was isolated from soil for high-level production of pyrroloquinolone quinone (PQQ) in our lab.
Subject(s)
Genome, Bacterial , Methylophilaceae/genetics , Methylophilaceae/metabolism , PQQ Cofactor/metabolism , Methylophilaceae/isolation & purification , Molecular Sequence Data , Soil MicrobiologyABSTRACT
Pyrroloquinoline quinone (PQQ) was produced by fermentation of the Methylovorus sp. MP688 strain and purified by ion-exchange chromatography, crystallization and recrystallization. The yield of PQQ reached approximately 125 mg/L and highly pure PQQ was obtained. To determine the optimum dose of PQQ for radioprotection, three doses (2 mg/kg, 4 mg/kg, 8 mg/kg) of PQQ were orally administrated to the experimental animals subjected to a lethal dose of 8.0 Gy in survival test. Survival of mice in the irradiation + PQQ (4 mg/kg) group was found to be significantly higher in comparison with the irradiation and irradiation + nilestriol (10 mg/kg) groups. The numbers of hematocytes and bone marrow cells were measured for 21 days after sublethal 4 Gy gamma-ray irradiation with per os of 4 mg/kg of PQQ. The recovery of white blood cells, reticulocytes and bone marrow cells in the irradiation + PQQ group was faster than that in the irradiation group. Furthermore, the recovery of bone marrow cell in the irradiation + PQQ group was superior to that in irradiation + nilestriol group. Our results clearly indicate favourable effects on survival under higher lethal radiation doses and the ability of pyrroloquinoline quinine to enhance haemopoietic recovery after sublethal radiation exposure.
