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Construction and characterization of CD26 specific chimeric antigen receptor T cellsObjective: In recent years,a considerable breakthrough has been “chimeric antigen receptor T cells(CART)immunotherapy”,which is rapidly emerging as a promising novel treatment for malignancies.Compared with the traditional adoptive lymphocyte infusion therapy,CAR T cells can kill tumor cells in a non-MHC-restricted manner.Previous researches have reported that CD26 surface antigen was specifically expressed in the leukemia stem cells(LSCs)of chronic myeloid leukemia(CML),which cannot be eliminated by TKIs.In addition,CD26 was also overexpressed on the surface of a variety of refractory malignant tumor cells,such as T-cell leukemia/lymphoma and malignant mesothelioma.A phase I clinical trial of CD26 monoclonal antibody in the treatment of CD26 positive malignant tumors showed good remission rate and tolerance,while the research of CD26 targeting CAR T cells has not been reported yet.In this part,we plan to construct a novel CAR T cell product with the specificity towards CD26 antigen,and elucidate their biological characteristics.Methods: The single chain fragment variable(VL-linker-VH)sequence of the CD26 CAR derived from YS110(CN 101282994)was encoded using synthetic DNA technology and then cloned into a 2nd generation backbone CAR with a 4-1BB co-stimulatory domain.After being identified by sequencing,lentiviruses were generated by plating 293 T cells transfected with packaging plasmids and the desired CAR lentiviral backbone plasmid,and the lentiviruses titer was determined by q PCR;CD3 positive T cells were selected by magnetic beads,then T cells were activated by Anti-Biotin MACSI Bead Particles;The non-tissue culture plate was coated with Retronectin,then activated T cells and lentivirus at a MOI of 10 were added to transfect activated T cells,and the growth status of BB.z CD26 CAR T cells were observed,and the cell survival,CD26 expression and transfection efficiency were detected by flow cytometry.The BB.z CD26 CAR T cells were co-cultured with autogenous T cells,and cytotoxicity was detected by flow cytometry.The 28.z CD26 CAR T cells with CD28 as costimulatory domain were constructed in the same way;and the transfection efficiency,CD26 expression,CD4/CD8 subsets and memory cell subsets were identified by flow cytometry.The RNA-seq was performed on NT and 28.z CD26 CAR T cells at 7,14,and 28 days post-transduction.Results: It was observed that the BB.z CD26 CAR T cells showed poor viability and failed to expand;The percentage of alive cells among BB.z CD26 CAR T cells was much lower than that of non-transduced or negative control T cells,moreover,the percentage kept declining and was below 20% on day 6 post-transduction,which was detected by flow cytometry;The ability of BB.z CD26 CAR T cells to lyse CFSE-labeled autologous T cells was identified by cytotoxicity assay.In contrast,it was observed that initial expansion of28.z CD26 CAR T cells was delayed because of transient fratricide;however,subsequent expansion was accelerated,and the cells expanded long-term ex vivo;The expression efficiency of 28.z CD26 CAR T cells was 34.24 ± 2.466%,and CAR expression was associated with the downregulation of CD26,which was also discovered in both NCT cells and CD4+/CD8+T cells subsets.Following expansion,28.z CD26 CAR T cells had a low frequency of naive T cells and were enriched for central memory effector and effector-memory phenotypes,in contrast to NT and NCT cells.The functional enrichment analysis of the differently expressed genes(DEGs)in RNA-seq showed that most of the KEGG pathways were related to immune response and immune cell differentiation.SERPINB9,a potent inhibitor of granzyme B and cathepsin B providing resistance to perforin were all upregulated,and perforin was downregulated in 28.z CD26 CAR T cells.Conclusion: BB.z CD26 CAR T cells exhibit self-antigen-driven fratricide,while 28.z CD26 CAR T cells showed limited fratricide and can expand long-term ex vivo.The downregulation of CD26 was discovered in both NCT cells and CD4+/CD8+ 28.z CD26 CAR T cells subsets,indicating that the loss of CD26 was not completely a result of the preferential survival of CD26-negative T cells,and possibly facilitated the expansion of28.z CD26 CAR T cells.Besides,the functional enrichment analysis of the DEGs in RNA-seq was consistent with the occurrence of fratricide and phenotype transformation within the 28.z CD26 CAR T cells.The loss of CD26,upregulation of SERPINB9 and cathepsin B,and the downregulation of perforin may be involved in protecting cells from the damage induced by self-antigen.Therefore,a novel CAR T cell product with the specificity towards CD26 can be produced by utilizing CD28 as costimulatory domain.Part ? Cytotoxic function of 28.z CD26 CAR T cells ex vivo and in vivoObjective: Researches have found that CD26 molecule is a tumor related antigen of many kinds of malignant tumor cells,including CML-LSCs and T-cell leukemia/ lymphoma cells.In the first part of the study,we used CD26 as target and CD28 as costimulatory domain to produce a large number of 28.z CD26 CAR T cells via lentivirus transfection,but whether they have specific antitumor ability has not been verified.In this part,we plan to use the anaplastic large cell lymphoma(ALCL)cell line Karpas 299 cells,CD26 overexpressed K562 cells and CML patients derived LSCs as target cells,evaluate the specific anti-tumor ability of 28.z CD26 CAR T cells,and preliminarily evaluate its “on target/off tumor” toxicity towards normal tissues.Methods: K562 and Karpas 299 cells were labeled with CFSE and co-cultured respectively with NT cells or 28.z CD26 CAR T cells for 18-24 hours at different E:T ratio,and then the killing effect was detected by flow cytometry;K562 cells were overexpressed with CD26/GFP by lentivirus transfection,purified by flow sorting,then co-cultured respectively with NT cells or 28.z CD26 CAR T cells for 18-24 hours at different E:T ratio,and the killing effect was detected by flow cytometry;CD34+ cells of CML patients were separated by magnetic beads,and co-cultured respectively with NT cells or 28.z CD26 CAR T cells at an E:T ratio of 1:2,and the killing effect was detected by flow cytometry;The expression of TNF-?,IFN-? and CD107 degranulation assay of the effector cells were detected by flow cytometry;the levels of Th1/Th2 cytokines IL-2,IL-4,IL-6,IL-10,IFN-? and TNF-? in the co-culture supernatant were detected by CBA kit.The killing ability towards Karpas 299 cells in mice was verified by bioluminescence;The killing ability towards CML-LSCs in vivo was verified by PDX model.The “on target/off tumor” toxicity towards normal tissues was evaluated by co-culture assays ex vivo and mice assays in vivo.Results: Compared with NCT or NT cells,28.z CD26 CAR T cells can specifically kill CD26 positive Karpas 299 cells and CD26+ K562 cells,while it has no killing effect on CD26 negative K562 cells;Besides,the killing process is accompanied by the CD107 degranulation and multiple cytokines secretion.In addition,28.z CD26 CAR T cells can specifically kill CD34+CD26+cells from CML patients(including 3 patients at diagnosis and 3 follow-up patients with T315 I mutation),while have no killing effect on CD34+CD26-cells.Compared with NT cells,28.z CD26 CAR T cells limited Karpas 299 tumor progression in nude mice model,and specifically kill CML CD34+CD26+cells implanting in severe immune deficiency mice.However,28.z CD26 CAR T cells also killed the lymphocyte population of healthy donors(the maximum killing rate is less than 60%),while monocytes and granulocytes were almost unaffected.Compared to the NT group,Balb/c mice engrafted with 28.z CD26 CAR T cells exhibited a decline in WBCs,especially lymphocytes(1 ± 0.1155 vs.1.8 ± 0.2082 G/L,P<0.05),but all values were within the normal reference range;RBCs were decreased without statistical significance,and PLTs and liver and kidney function were not affected.There were no fatal events caused by either of the T cell injections.Conclusion: The 28.z CD26 CAR T cells have the ability of killing CD26 positive tumor cells,including T cell lymphoma cell line Karpas 299,CD26-overexpressing K562 cells and primary CML-LSCs,but there was some off-tumor cytotoxicity towards activated lymphocytes.Therefore,these results establish the feasibility of using CD26 as an antigen for CAR T cells to target CD26+ tumor cells,but the off-tumor toxicity must be conquered before clinical transformation.Part ? Mining of CML-LSCs alternative targetsObjective: CD26 is a specific surface biomarker of CML LSCs.In the previous studies,we constructed 28.z CD26-CART cells utilizing CD26 as the target,and verified its specific killing ability on CML derived CD34+CD26+cells.However,28.z CD26-CART cells also had the off-tumor effect on normal activated T cells.To avoid potential off-tumor toxicity,we plan to isolate the CML-LSCs(CD34+CD38-CD26+)and HSCs(CD34+CD38-CD26-),and compared the CML-LSCs with HSCs by global proteomic analysis,then screened the membrane proteins as new alternative targets,which were expressed only in CD34+CD38-CD26+ cells.Methods: CML patients-derived bone narrow samples were collected,and the CD34+CD38-CD26+,CD34+CD38-CD26-and CD34+CD38+ cells were isolated from the CML patients utilizing magnetic and flow sorting,and the global proteomic were performed through high-resolution LC-MS/MS analysis.The membrane proteins which were expressed only in CD34+CD38-CD26+ cells were identified via proteomic analysis,screening the membrane proteins databases and GTEX databases.Results: The purity of CD34+CD38-CD26+ cell was up to 90% through magnetic bead sorting and flow sorting;273 membrane proteins were found to be up-regulated in CD34+CD38-CD26+ cells by proteomic sequencing;then OR4C12,OR6X1,VAMP7,DSG4,LCT,CHRNB4,IL1RAPL1,GRID2,HS3ST5,ADCY8 and GAL3ST2 remained after removing the proteins expressed in 30 normal tissues.Among them,the expression of OR6X1,VAMP7,DSG4 and LCT was significantly upregulated in CML-LSCs cells.Conclusion: OR6X1,VAMP7,DSG4 and LCT may be the potential targets of CML-LSCs. |
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