Antitumor Effect And Mechanism Of Targeting CD105 Chimeric Antigen Receptor T Cells

Background: Malignant tumors are a serious health hazard to humans.Currently,the main traditional treatments for tumors include surgery,radiotherapy and chemotherapy.In recent years,immunotherapy has gradually become the focus of cancer treatment,and adoptive treatment of immune cells has become a research hotspot.Immunotherapy based on T cells modified with a chimeric antigen receptor(CAR)has been demonstrated as a promising strategy for cancer treatment.CAR T expressed receptors that can specifically recognize tumor-specific antigens,eliminate tumor cells by passing the antigen-presenting stage and in a non–major histocompatibility complex-restricted manner.In 2017,the US Food and Drug Administration(FDA)approved the second CAR T cell therapy for the treatment of certain pediatric and young adult patients with B cell acute lymphoblastic leukemia.FDA approved the CAR T cell therapy for the treatment of adult patients with certain types of B cell lymphoma.The application of CAR T to solid tumors is still limited.During CAR T treatment,there are some problems,for example “off-target” effects,insertion mutations caused by genetic engineering methods and cross-reactivity during extracellular segment single-chain antibody binding to antigens.Therefore,seeking for more specific surface antigens of tumor cells,better antibodies and the safe and effective transduction technologies have gradually become the research direction.Endoglin(CD105)is a homodimeric cell membrane glycoprotein with a relative molecular weight of 180 k Da.It was considered to be one of the specific marker molecules of neovascular endothelial cells,expressed on the surface of solid tumor neovascularization and related tumor cells,so the construction of CAR T cells targeting CD105 will provide a safe choice for tumor immunotherapy.Nanobodies consist only of the VHH domain of Camilidae's heavy chain antibody,with no light chains,and these single antigen-binding domains were readily expressed and retained their affinity for specific antigens.Objective: To explore the method of constructing CD105 nanobody CAR T cells and detect the proliferation activity of CD105 CAR T cells and the cytotoxic effect to target cells in vitro,to evaluate the inhibition of CD105-positive hepatocellular carcinoma NOD/SCID mice in vivo.To investigate the anti-tumor mechanism of CD105 CAR T cells and provide new evidence for adoptive immunotherapy of tumors.Method:1.We design g RNAs for the PPP1R12 C gene on the AAVS1 locus of chromosome 19 and construct a CRISPR/Cas9 vector,and use the T7E1 method to verify that whether the designed g RNA has a cleavage effect.Using gene engineering methods,the second-generation CARs molecules targeting CD105 nanobody with co-stimulatory molecule 4-1BB and CD3? signal domain were loaded into the p LVX expression vector and identified by double digestion.The CAR fragment was obtained by PCR and the CAR fragment was ligated to the Puc-Mdonor-AAVS1 vector to construct the homologous group vector Puc-Mdonor-AAVS1-CAR carrying exogenous homologous arms around 1 kb.2.In this study,CRISPR/Cas9 vectors and homologous vectors were introduced into T lymphocytes by electroporation to construct CAR T cells.After the cells are cultured,the DNA of the cells was extracted using the DAN extraction kit,the PCR method was used to verify the CAR fragments and the homologous arms.Fluorescence microscopy was used to observe the expression of GFP,flow cytometry was used to detect the expression rate of fluorescent GFP in T cells and analyzed the ratio of CD4+CD8+ in CAR T cells.3.CD105 CAR T cells,CD19 CAR T cells,Mock T cells were prepared,and non-electroporation T cells as controls,flow cytometry was used to detect the proliferation of CAR T cells stimulated by target cells.Flow cytometry was used to detect phenotype of CAR T cells,including surface active molecule of CD25 and CD69,memory molecule of CD62 L,depleted molecule of PD-1,lysosomal protein(CD107a).4.The cytotoxicity of CD105 CAR T against hepatoma cells Bel7404,Hep G2,SMCC7721 and MHCC97 H was measured by flow cytometry.The cytokines secreted in the supernatant were detected by ELISA(IFN-?,TNF-?,IL-2,IL-10).ELISPOT was used to detect the number of T cells secreting IFN-? after stimulated by target cells.5.NOD/SCID mice hepatocellular carcinoma xenograft tumor model(Bel7404 and MHCC97 H models)was constructed.The Antitumor ability of recombinant CAR T cells in vivo was detected by tail vein injection of recombinant CAR T cells and normal T cells for total of 2 treatments.The size of the tumor and the survival rate of mice was respectively measured and observed to evaluate the anti-tumor effect of CAR T cells in vivo.6.Mice were euthanized when the tumor burden in the control mice reached 15 mm.Xenografts were fixed with formalin,embedded in paraffin,and sections were cut and processed for Immunohistochemical(IHC)staining.IHC staining was used to detect cell proliferation(ki67),vascular density(CD31),expression of apoptotic protein(Bax).Apoptotic cells in tumor tissue were detected by Tunel method.The detection of CD3 content in tumor tissues were detected to assess the infiltration of T cell.Result:1.CRISPR and Donor vectors were successfully constructed.CAR T cells were prepared using CRISPR/Cas9 technology.PCR was used to verify that the CAR fragment and the homologous arm were successfully expressed in the T cell genome.GFP expression was observed by fluorescence microscopy and the expression rate of fluorescent GFP was above 20% by flow cytometry,indicating successful preparation of CAR T cells.2.CD105 CAR T cells can stimulate the proliferation of T cells,the expression of surface activation molecules(CD25,CD69),memory molecules(CD62L)and lysosomal protein CD107 a.The secretion of cytokines IFN-? and TNF-? in CD105 CAR T cells increased,which was higher than other control groups.There was no difference in the secretion of IL-10.3.With different effector-to-target ratios,the killing effect is enhanced with the increase of the effect-target ratio,and the killing efficiency of target cells with higher expression of CD105(Bel7404,Hep G2,SMCC7721)is higher than that with low expression of CD105.The killing effect of cells(MHCC97H)and CD105 CAR T cells was significantly higher than CAR T cells in the control group,but the killing efficiency of MHCC97 H was not significantly different from that of the control group.4.CD105 CAR T cells were not toxic to normal mouse tissues and organs.CD105 CAR T cells had certain therapeutic effects on CD105 high expression and CD105 low expression xenografts.CD105 CAR T cells can inhibit tumor growth and prolong survival time.Highly-expressed xenografts have higher therapeutic efficacy than CD105-low-expressed xenografts.5.Immunohistochemistry showed that CD105 CAR T cells could inhibit tumor cell proliferation,reduce vascular density in tumors,promote expression of apoptotic proteins and increase the number of apoptotic cells.There was a little of T lymphocyte infiltration in mouse tumor tissues to improve the antitumor effect of T cells.Conclusion: The results showed that the CAR T cells have been successfully constructed at the AAVS1 site by using CRISPR/Cas9 technology.CD105 CAR T cells have a certain anti-tumor effect in vivo and in vitro,and the antitumor effect of CD105 CAR T cells is related to the expression of CD105.It provided new strategies for the follow-up treatment of tumors.