A Preclinical Study Of Anti-TSHR CAR-T Cell Therapy For Differentiated Thyroid Carcinoma

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Evaluation of TSHR expression in healthy tissues and differentiated thyroid carcinomas(DTCs)Purpose: In recent years,the successful application of chimeric antigen receptor T-cell(CART)therapy in hematological malignancies has completely overturned the traditional model of tumor immunotherapy.However,progress of CAR-T therapy in solid tumors remains stagnant,mainly due to the lack of a suitable tumor specific antigen(TSA)as a therapeutic target.The promotion of CAR-T technology to the field of thyroid cancer has unparalleled advantages among other tumors,as most patients have undergone a total thyroidectomy at the time of initial treatment,so thyroid tissue-specific membrane proteins,such as thyroid stimulating hormone receptor(TSHR),can be selected as targets.This part of the study aims to comprehensively evaluate the expression of TSHR in human normal tissues and organs,DTC tissues and cell lines,so as to provide a theoretical basis for the safety and feasibility of TSHR-based CAR-T cells in the treatment of DTC.Methods: We first searched GTEx,GEPIA,HPA,GEO and CCLE databases to assess the expression of TSHR in human health tissues and DTC tissues.Subsequently,we collected 189 cases of DTC primary tumors,57 cases of normal thyroid tissues,23 cases of cervical lymph node metastases,15 cases of radioactive iodine refractory(RAI-R)diseases,15 cases of orbital adipose/connective tissues,3 cases of pretibial connective tissues,as well as a normal tissue microarray,and evaluated TSHR expression by immunohistochemistry.Finally,we detected the expression of TSHR in DTC and thyroid follicular epithelial cell lines by immunofluorescence and flow cytometry.Results: Through bioinformatics and immunohistochemical analysis,we found that TSHR was absent in most normal tissues except thyroid,and was only expressed in gastric mucosal epithelial cells and bladder epithelial cells at low intensity in some samples.As for cancer tissues,the positive rates of TSHR in papillary thyroid carcinoma(PTC),follicular thyroid carcinoma(FTC)and cervical lymph node metastasis were 90.8%,89.2% and 78.2%,respectively.Among the subtypes of PTC,the positive rate of TSHR in conventional and follicular variants was 93.8% and 93.7%,respectively.The positive rates of tall cell variant,hobnail variant,clear cell variant and columnar variant were 85%,90.9%,75% and 50%,respectively.TSHR was also positively expressed in RAIR-DTC local recurrence lesions and cervical lymph nodes,with an overall positive rate of 86.7%.Finally,the results of flow cytometry and immunofluorescence showed that the expression of TSHR in DTC cell lines was generally absent or significantly decreased.Conclusions: The results supported that TSHR was not expressed in most normal tissues except thyroid glands,and was commonly expressed in various pathological subtypes of DTC,which proved the safety and feasibility of TSHR as a CAR-T target for DTC,and provided the premise and theoretical support for the subsequent study of CAR-T therapy targeting TSHR.Part ? Construction and in vitro function experiment of anti-TSHR CAR-T cellsPurpose: Differentiated thyroid cancer(DTC)is the most common pathological type of thyroid cancer,accounting for 95% of all thyroid malignancies.Although the majority of DTC patients have a good prognosis after thyroidectomy,radioactive iodine(RAI)ablation,TSH suppression,but some patients still have disease progression.Due to the insensitivity of DTC to traditional chemotherapy and radiotherapy,and the defects of small molecule inhibitors such as high drug resistance rate and serious side effects,patients with advanced DTC are often faced with the situation of no available medical solution.In recent years,CART cell therapy has attracted attention for its ability to recognize tumor antigens without major histocompatibility complex(MHC)limitations and for its potent antitumor effects by increasing co-stimulatory molecular signaling.Unfortunately,there have been no reports of CAR-T cell therapy for DTC.Therefore,this study aims to develop a CAR-T therapy model targeting DTC,and explore the killing effect,inflammatory cytokine secretion ability and tonic signal activation ability of CAR-T cells targeting TSHR-expression cells in vitro.Methods: Three human TSHR monoclonal antibodies(M22,K1-18 and K1-70)were used to construct the third generation CAR lentivirus(22-CAR,18-CAR,and 70-CAR).The peripheral blood lymphocytes of healthy volunteers were isolated,and T lymphocytes were sorted by magnetic beads.The constructed CAR lentivirus was used to infect T cells.LDH release assay,firefly-luciferase(Fluc)toxicity assay,enzyme-linked immunosorbent assay(ELISA),and flow cytometry were used to evaluate the killing ability,inflammatory cytokine secretion,antigen-specific proliferation,and tonic signal activation of the three CAR-T cells in vitro.Results: We successfully constructed three third-generation CAR vectors targeting TSHR and stably expressed them on the cell surface of T cells by lentivirus.LDH release assay and Fluc toxicity assay showed that all three CAR T cells could effectively kill TSHR-positive DTC cells in vitro and secrete large amounts of IL-2 and IFN-?.Compared with 22-CAR and 18-CAR-T cells,70-CAR-T cells exhibited a relatively lower activation capacity for tonic signaling,manifesting by shorter doubling time after a single round of CD3/CD28 antibody stimulation,faster volume retraction after activation,a lower percentage of apoptotic cells,and possessing more early differentiated CD8+ T cells.Conclusions: Our study demonstrated that third generation CAR T cells based on M22,K1-18 and K1-70 monoclonal antibodies can effectively recognize and lysis TSHR-positive DTC cells in vitro.Compared with 22-CAR and 18-CAR structures,70-CAR showed a much more limited activation status of tonic signaling,which would be conducive to its anti-tumor effect in vivo.Part ? Evaluation of the efficacy and safety of anti-TSHR CAR-T cells in a subcutaneous xenograft model of immunodeficient micePurpose: Although studies in vitro could provide us with information about the short-term killing effect and inflammatory cytokine secreting capacity of CAR-T cells,they cannot mimic the tumor microenvironment in vivo.Therefore,animal experiments are an indispensable means and one of the necessary indicators to evaluate the preclinical efficacy of CAR-T cells.NOD Scid Gamma(NOD.Cg-PrkdcscidIL-2rgtm1Wjl/Sz J,NSG)mice were commonly used to evaluate the efficacy of CAR-T cells in vivo.This strain of mice has a severe immunodeficiency phenotype and is widely used in the evaluation of adoptive cell immunotherapy.Treatment of NSG mice with human tumors with CAR-T cells can be used to analyze the in vivo efficacy,viability,and safety of the treatment.Therefore,this study aims to evaluate the efficacy and safety of anti-TSHR CAR-T cells in NSG mouse subcutaneous xenograft model.Methods: NSG mice aged 6 to 8 weeks were inoculated subcutaneous with K1-TSHR-Luc cells,which were stably overexpressed with Fluc and TSHR proteins.After tumor implantation,70-CAR-T cells or NT cells or PBS were administrated with tail vein injection.In vivo bioluminescence(BLI)imaging was conducted to monitor the changes in tumor size of each group,T cell proliferation in the peripheral blood of mice was detected by flow cytometry,distant metastasis of mice in each group was determined by organ anatomy,followed by ex vivo BLI imaging.The safety of the treatment was evaluated by body weight monitoring,HE staining and immunohistochemistry.Results: Compared with mice injected with NT cells and PBS,70-CAR T cells effectively inhibited the growth of K1-TSHR-Luc in vivo and controlled the formation of lung metastases.The results of flow cytometry showed that human CD3+T cells proliferated with prolonged treatment in the 70-CAR-T group.Finally,the results of HE staining,immunohistochemical staining and body weight monitoring indicated that the 70-CAR T cell therapy exhibited no obvious toxic and side effects on mice.Conclusions: We demonstrate the efficacy,persistence,and safety of 70-CAR-T cell therapy in an immunodeficient mice model.Our proof-of-concept study provides a new therapeutic strategy for DTC patients with relapsing and metastatic lesions postoperatively,especially for those with RAI-R or inoperable tumors.