Optimization Of CD19 CAR-T Based On CXCL12/CXCR4 To Enhance The Anti-tumor Effect In Lymphoma

BackgroundChimeric antigen receptor modified T cell(CAR-T)is genetically engineered to express chimeric antigen receptor(CAR)on T cells.CAR-T cells recognize and kill tumor cells in MHC independent manner.CD 19-directed CAR-T cells have achieved great success in patients with relapsed or refractory B-cell lymphoma.The U.S.Food and Drug Administration(FDA)has approved three CD 19-directed CAR-T therapies(Axicabtagene ciloleucel,Tisagenlecleucel,Lisocabtagene maraleucel)for the treatment of relapse/refractory B-cell lymphoma.However,the median progressionfree survival(PFS)of lymphoma patients treated with Axicabtagene ciloleucel,Tisagenlecleucel,and Lisocabtagene maraleucel were only 5.9,2.9,and 6.8 months.Most patients developed disease progression within a short period of time.Therefore,the existing CD 19 CAR-T is needed to be further optimized to increase the efficacy of CAR-T therapy.The lentivirus used in the preparation of CAR-T is derived from HIV virus.CXCR4 is widely expressed on T lymphocytes.CXCR4 as the HIV co-receptor was shown to mediate entry of HIV by endocytosis into cells,resulting in the decreased expression of CXCR4 on the cell surface.The CXCL12/CXCR4 axis is related to the chemotaxis and proliferation of T cells.CXCL12/CXCR4 axis plays a key role in the homing of T cells to bone marrow.Furthermore,the long-term survival of T cells in bone marrow leads to formation of memory phenotypes.On the other hand,the CXCL12/CXCR4 axis plays an important role in B-cell lymphoma.CXCR4 is essential for metastatic spread to organs where CXCL12 is expressed,and thereby forms metastatic lesions.CXCR4 expression granted lymphoma cells a high capacity to seed into bone marrow niches which protect lymphoma cells from chemotherapyinduced apoptosis.CXCL12 itself can formate the inhibitory immune microenvironment which leads to growth of neoplastic cells.Therefore,lymphoma often invades the bone marrow,spleen and lymph nodes that express CXCL12.The CXCL12/CXCR4 signaling pathway mediates the metastasis and progression of lymphoma.Lentivirus used for CAR-T manufacturing may downregulate the expression of CXCR4 on the surface of CAR-T cells and weaken the chemotactic effect of CXCL12/CXCR4 axis on CAR-T.Therefore,the CD19 CAR-T which was constructed by lentivirus was not the optimal.The lymphoma that express CXCL12 cannot attract the CAR-T cells to tumor tissues through CXCL12/CXCR4 axis.In this study,we optimized the conventional second-generation CD 19-targeted CAR-T cells by overexpressing CXCR4.The modified CAR-T cells improve the chemotaxis into tumor tissues with high CXCL12 expression,enhancing the antitumor effect.In addition,the modified CAR-T promote the homing of CAR-T cells to bone marrow,increase the memory phenotypic differentiation of T cells,and prolong the survival time of T cells.Materials and MethodsThe expression of CXCR4 in human lymphoma cell lines Raji,Daudi,Ramos and CD 19 CAR-T cells was detected by flow cytometry.The chemotaxis rate of CD 19 CAR-T was detected by Transwell assay.The expression of CXCL12 gene in the TCGA database and its relationship with prognosis were analyzed by bioinformatics.Immunohistochemical staining was used to detect CXCL12 protein expression in lymphoma and lymph nodes.The sequence of CD 19 CAR containing CXCR4 was constructed by gene synthesis technique.The CD 19 CAR and CD 19 CAR containing CXCR4 were cloned into lentiviral vector pWPXLd by molecular cloning technique.CD3+T cells were isolated in vitro to prepare CAR-T cells.The chemotaxis and CAR expression of CXCR4/CD19 CAR-T and CD 19 CAR-T were compared by Transwell assay and flow cytometry.The infiltration of T cells in tumor,spleen and bone marrow was detected by immunofluorescence staining and flow cytometry.Cell phenotype and proliferative activity of CAR-T cells were detected by flow cytometry.The specific killing activity of CAR-T against target cells in vitro was determined by flow cytometry and enzyme-linked immunosorbent assay(ELISA).Human lymphoma cells Raji-CXCL12 and Ramos-CXCL12 secreting CXCL12 and human lymphoma cells Raji-Luc expressing luciferase were constructed by lentiviral infection screening method.The Raji-CXCL12,Ramos-CXCL12 subcutaneous tumor models and RajiLuc systemic metastatic tumor models were constructed.Tumor size and in vivo imaging were measured to observe the treatment efficacy.The safety of treatment was evaluated by weight measurement and H&E staining of vital organs.The proportion of CD3+T cells and the proportion of memory T cells in spleen and bone marrow were determined by flow cytometry 30 days after CAR-T treatment.Transcriptome sequencing was used to explore the effect of bone marrow microenvironment on CART cells.ResultsAfter lentivirus infection,it is found that CXCR4 expression in T cells is downregulated by more than 50%.There is no difference between cell subtypes.The downregulation was mediated by endocytosis of CXCR4 and lasted from 6 h to 7 days after lentiviral infection.The chemotactic rate was monitored on cells with 100 ng/mL CXCL12 for 4 h.The fraction of transmigrating cells was 2.5%in CD19 CAR-T group which is significantly lower than the 5.9%in control T group.Bioinformatics analysis and immunohistochemistry showed that CXCL12 was highly expressed in lymphoma tissues.The high expression of CXCL12 predicted a poor prognosis in lymphoma patients.Immunohistochemical results showed that CXCL12 was expressed in human lymphoma tissue and lymph node tissue.Based on the down-regulation of CXCR4 expression by lentivirus and the high expression of CXCL12 in lymphoma,we optimized the conventional CD 19 CAR-T and successfully produced a CD 19 CAR-T that overexpressed CXCR4(CXCR4/CD19 CAR-T).CD 19 CAR expression was similar in CXCR4/CD19 CAR-T and CD 19 CAR-T,with 60%to 75%in both cases.The chemotactic rates of CXCR4/CD19 CART cells cultured with 100 ng/mL CXCL12 or supernatant containing CXCL12 for 4 h were 6.9%and 2.5%,respectively,which were significantly higher than 2.5%and 1.1%of CD 19 CAR-T cells.In lymphoma subcutaneous tumor models,the mean percentage of CD3+T cells in tumors was 3.21%,5.15%,1%at 1,3,and 5 days after treatment,which were significantly higher than 0.59%,0.47%,0.32%in the CD19 CAR-T group.In the CXCR4/CD19 CAR-T group,the mean percentage of CD3+T cells were 23.69%,7.12%0.9%in spleen and 0.5%,0.67%,1%in bone marrow at 1,3 and 5 days after CAR-T treatment,which were significantly higher than 5.53%,0.8%,0.02%in spleen and 0.08%,0.06%,0.08%in bone marrow in the CD19 CAR-T group.In vitro,overexpression of CXCR4 did not affect the expression of PD-1,Tim-3,and LAG-3 in CAR-T cells.Overexpression of CXCR4 did not affect the memory phenotypic differentiation of CAR-T cells.Overexpression of CXCR4 enhanced the proliferation ability of CAR-T cells.In vitro,CXCR4/CD19 CAR-T showed similar antitumor activity to conventional CD 19 CAR-T.CXCR4/CD19 CAR-T specifically killed CD19-positive lymphoma cells and secreted IL-2,IFN-γ,and TNF-α.In RajiCXCL12 subcutaneous tumor model,1/4 dose of CXCR4/CD19 CAR-T was more effective in tumor control than CD19 CAR-T.In Ramos-CXCL12 subcutaneous tumor model,CXCR4/CD19 CAR-T was effective in tumor control.While the same dose of CD19 CAR-T cannot control the tumor growth.In Raji-Luc metastatic models,CXCR4/CD19 CAR-T effectively controlled metastases in vivo compared to CD 19 CAR-T.No significant weight loss was observed after CAR-T treatment.No pathological changes were observed in vital organs.At 30 days after CXCR4/CD19 CAR-T treatment,CD3+T cells in bone marrow and spleen of mice were 22.7%and 13.9%,which were significantly higher than those in conventional CD19 CAR-T group(8.3%and 6.2%).The proportion of memory T cells in the CXCR4/CD19 CAR T group was 54.3%,significantly higher than that in the conventional CD19 CAR-T group(7.4%).Transcriptome sequencing of T cells in bone marrow showed CXCR4/CD19 CAR-T group increased CXCR4,IL-7,IL-7R,TCF7 expression and decreased PDCD1,TIGIT expression compared to CD 19 CART group.ConclusionLentiviral infection resulted in down-regulation of CXCR4 expression and decreased chemotactic activity of T cells.Overexpression of CXCR4 on the basis of conventional CD 19 CAR-T cells increased the local infiltration of CAR-T cells and enhanced the anti-tumor effect of CAR-T cells.Compared with conventional CD 19 CAR-T,CXCR4/CD19 CAR-T had a higher capacity to home into bone marrow niches and differentiated into memory type in bone marrow microenvironment.CXCR4/CD19 CAR-T cells showed no significant toxic side effects.CXCR4/CD19 CAR-T is currently registered in clinical trials as a safe and effective CAR-T(NCT 04684472).