Study On Generation And Anti-tumor Effects Of Transferrin Receptor-CAR T Cells

[Background] Transferrin receptor(TfR)belongs to cell membrane-type II transmembrane glycoprotein,and as a major channel for cellular iron uptake,the expression of TfR is closely related to the iron demand of cellular activities.TfR is lowly expressed in normal tissue cells.Due to massive proliferation,the DNA synthesis,cell energy supply,and other life activities of tumor cells are vigorous,and more iron-containing enzymes are required which result in the dramatical increases of iron demand,thus TfR is highly expressed on the surface of rapidly proliferating tumor cells.Clinical studies have reported that upregulation of TfR expression is closely associated with poor prognosis in tumor patients.These results suggested that TfR may be a potential target for Chimeric Antigen Receptor(CAR)T-cell therapy.[Objective] This subject aimed to construct the TfR-directed second generation CAR T cells based on the construction of anti-TfR scFv,bivalent and quadrivalent genetically engineered antibody expression vectors,to investigate the biological properties and anti-tumor effects of TfR-CAR T cells through in vivo and in vitro experiments,and to provide a useful reference for the development of a new target for CAR T cell therapy.[Methods]1.Construction,preparation and identification of TfR-CAR T cells(1)The gene encoding TfR-CAR(TfR scFv-CD8a hinge& transmembrane region-4-1BB costimulatory domain-CD3z)was synthesized and cloned into the p EF1a-T2A-EGFRt lentiviral vector,and then the TfR-CAR lentiviral vector was confirmed by restriction enzyme digestion and sequencing.293 T cells were transfected with the prepared TfR-CAR lentivirus in a ten-fold dilution,and the TfR-CAR lentivirus titer was determined by a flow cytometry-based method.(2)Human Primary T cells were transfected with the TfR-CAR lentivirus to prepare a second-generation TfR-directed CAR-T cell,and the transduction efficiency of TfR-CAR in T cells was detected by flow cytometry.Total protein of TfR-CAR cells was extracted,and the CD3z chain was detected by Western Blot to verify the expression of TfR-CAR in T cells.(3)The CFSE pre-stained Hep G2 cells were co-cultured with TfR-CAR T cells at 4°C for 90 minutes,followed by thoroughly washing coverslips in PBS to discard T cells that were non-specifically attached to the coverslips.After that,mixed cell populations were fixed and stained with DAPI.Then the round coverslips were mounted onto glass slides with antifade reagent and observed using a confocal laser scanning microscope.(4)The cellular activity,immunophenotype,Treg proportions,and memory T cell subsets distribution of TfR-CAR T cells were determined by flow cytometry.2.In vitro anti-tumor effects of TfR-CAR T cells(1)The expression of TfR on U266,Molt4,Kg1 a,and K562 cells was detected by flow cytometry.(2)The Tag-it Violet^TM-labeled TfR+ tumor cells(U266,Molt4,Kg1 a,and K562)were co-cultured with TfR-CAR T cells at the E: T ratios of 1: 1,5: 1,and 10: 1 for 20 hours,and cells were stained with 7-AAD for cytotoxicity evaluation.(3)The Tag-it Violet^TM-labeled TfR+ tumor cells(U266,Molt4,Kg1 a,and K562)were co-cultured with TfR-CAR T cells(E: T= 10: 1)for 20 h.Then cells were harvested and measured for T-cell activation and degranulation indexes(perforin,granzyme B,Fas L,CD25,and CD69)using flow cytometry.(4)The Tag-it Violet^TM-labeled TfR+ tumor cells(U266,Molt4,Kg1 a,and K562)were co-cultured with TfR-CAR T cells at the E: T ratios of 1: 1,5: 1,and 10: 1 for 20 hours,and co-culture supernatants were analyzed for cytokines secretion by the Cytometric Bead Array.3.In vivo anti-tumor effects of TfR-CAR T cells(1)Molt4 tumor cells were transfected with luciferase-expressing lentivirus and were selected by puromycin.Luc-Molt4 monoclonal cell lines were screened by limiting dilution.(2)NPG mice were intravenously inoculated with 1×106 Luc?Molt4 cells.The percentage of hu CD45+ cells in the peripheral blood of mice were determined 48 hours later,and then the mice with hu CD45+ cells> 1% in the peripheral blood were randomly separated into three groups to adoptive transfer of TfR-CAR T cells or NC T cells or saline intravenously and second dosing 6 days later.The tumor progression was dynamically monitored by bioluminescent imaging.If one tumor-bearing mouse exhibited the signs of morbidity,the experiments were terminated.(3)The immunophenotype,Treg proportions,and memory T cell subsets of T cells in the peripheral blood and spleen were determined by flow cytometry.(4)Plasma cytokines were measured by CBA.(5)The tissues of the liver,kidney,and lung sections were stained by H&E to observe the presence of pathological changes.(6)Plasma alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were analyzed using corresponding assay kits.[Results]1.TfR-CAR T cells are successfully established(1)The results of restriction enzyme digestion and gene sequencing confirmed that the Lenti-EF1a-TfR-CAR-EGFRt vector plasmid was successfully constructed.The TfR-CAR T lentivirus titer was 6.28×108 TU/m L(>1×108 TU/m L)which met the experimental requirements.(2)Flow cytometric analysis showed the transduction efficiencies measured by TfR-Fc fusion protein-specific,anti-EGFR-specific and murine F(ab)-specific flow cytometric staining were approximately 79.2⁹1.0%,and the expression of TfR-CAR fusion protein with a molecular weight size of 54 k Da in CAR T cells was confirmed by Western blot,indicating that the TfR-CAR T cells were successfully constructed.(3)The confocal results showed the rosette-like configuration was formed by CAR T cells surrounding the Hep G2 cell,while no similar "rosette" configuration was observed in the NC T cells co-incubation group,indicating that TfR-CAR T cells could effectively recognize and bind the TfR+ Hep G2 cells.(4)The cell viability of TfR-CAR T cells was inferior to that of NC T cells.Flow cytometry analysis showed there were no apparent differences in the CD4+/CD8+ subsets and Tregs ratio between the CAR T and NC cells.However,TCM subsets were predominant in TfR-CAR T cells compared to NC T cells.2.TfR-CAR T cells are cytolytic to TfR+ malignant cells(1)Flow cytometric analysis showed these four hematologic tumor cell lines,U266,Molt4,Kg1 a,and K562,were TfR-overexpressing tumor cells.(2)Compared to the NC T cells,TfR-CAR T cells exhibited higher cytotoxicity.Moreover,the cytotoxic capacity of TfR-CAR T cells increased in an E: T ratio-dependent manner.TfR-CAR T cells from different individuals had different killing abilities,and different types of tumor cells responded to the anti-tumor effect of TfR-CAR T cells in different sensitivity and extent.(3)TfR-CAR T cells did not kill TfR-MX-1 more effectively than NC T cells,which indicated the cytotoxic effect of TfR-CAR T cells was antigen-specific.3.TfR-CAR T cells are activated by target cells(1)The flow cytometric analysis showed that after co-incubation with four TfR+ tumor cells for 20 hours,the expression of activation indexes CD25 and CD69 as well as cytotoxicity indexes Fas L,perforin,and granzyme B in TfR-CAR T cells were significantly higher than those in NC T cells,regardless of CD3+ T cells or CD4+ and CD8+ T cell subsets.(2)The CBA analysis showed that both Th1 and Th2 type cytokine secretion levels were higher in TfR-CAR T cells than in NC T cells,especially IFN-g and TNF-a.And there were large differences in cytokine secretion capacity of TfR-CAR T cells prepared from different individuals.4.TfR-CAR T cells effectively controlled the progression of T-ALL in vivo(1)Bioluminescent imaging demonstrated the mice receiving TfR-CAR T cells were significantly protected from rapid tumor progression.The mice receiving NC T cells or saline,by contrast,exhibited malignant progression and had a higher degree of leukemia burden.(2)Compared to the TfR-CAR T group,mice in the saline and NC T groups showed weight loss in varying degrees,while spleen index analysis indicated that TfR-CAR T cells treatment effectively reduced the spleen weight.(3)The CBA analysis showed that compared to NC T cells,TfR-CAR T cells treatment caused a considerable increase in IFN-g,and a trend toward increased IL-2 and IL-10,although not statistically significant.(4)The proportion of CD3+ T cells in peripheral blood and spleen of the TfR-CAR T group was a little bit higher than that in the NC T group overall,but there was no statistical difference.Both in spleen and peripheral blood,TfR-CAR T cells exhibited the clear tendency of increasing CD69 and perforin,granzyme B,Fas L expression when compared with NC T cells.However,the expression of CD25 in TfR-CAR T cells was significantly higher than in NC T cells only in the spleen.Besides,the proportion of PD-1+ T cells and Tregs showed no significant difference between the two groups.(5)The phenotype analysis of T cell differentiation showed that in both the spleen and the peripheral blood,the majority of the T cells from the TfR-CAR T group were prone to differentiation into TEM.(6)H&E staining showed no significant pathological changes in the major organs of mice treated with TfR-CAR T cells compared with saline and NC T groups.Besides,the serum biochemical analysis showed the levels of ALT and AST tended to increase slightly in mice treated with CAR-T cells or NC T cells compared to the saline group,but no significant differences among the groups.[Conclusions]1.We successfully constructed a TfR-CAR lentiviral vector and used the lentiviral transfection system to effectively transfer the TfR-CAR gene into human primary T cells to construct a second-generation TfR-directed CAR-T cell.2.TfR-CAR T cells recognized,bound,and killed TfR+ tumor cells specifically.TfR-CAR T cells from different individuals had different killing abilities,and different types of tumor cells differed in their sensitivity and extent of the response to the anti-tumor effects of TfR-CAR T cells,suggesting that individual differences in immune effector cells and differences of categorie in tumor cells affected anti-tumor effects.3.After co-incubation with TfR+ tumor cells,TfR-CAR T cells were effectively activated by target cells,promoting the expression of T cell activation indexes CD25 and CD69 as well as cytotoxicity indexes Fas L,perforin,and granzyme,while secreting a large number of cytokines to exert anti-tumor effects.4.In the T-ALL mouse xenograft model,TfR-CAR T cells were potent in killing T-ALL cells in vivo and inhibited rapid tumor progression without significant systemic toxicity,indicating that TfR could be an available target for CAR T cells in the treatment of hematological malignancies.