Background Chimeric antigen receptor modified T cells (CAR T) have demonstrated remarkable clinical efficacy in the treatment of B cell malignancies and multiple myeloma. Significant challenges restrict their application across solid tumors due to multiple obstacles, including the lack of robust in vivo CAR-T cell expansion and persistence, the immunosuppressive tumor microenvironment, and tumor escape due to heterogeneous tumor cell composition with a potential loss of the targeted tumor antigen.To address these difficulties, we generated CAR T cells using a novel CoupledCAR® technology. Specifically, we engineered CoupledCAR T cells with lentiviral vectors encoding an anti-thyroid stimulating hormone receptor (TSHR) CAR molecule. Immunohistochemistry (IHC) results showed that TSHR was highly expressed in thyroid cancer cells making it an ideal tumor-specific target antigen. In vitro co-culture experiments showed that TSHR CAR T cells specifically recognized and subsequently killed TSHR-positive tumor cells. Animal model experiments showed that TSHR CAR T cells inhibited the proliferation of TSHR-positive tumor cells.
Methods We designed a ‘CoupledCAR’ lentivirus vector containing a single-chain variable fragment (scFv) targeting human TSHR. The lentivirus was produced by transfecting HEK-293T cells with ‘CoupledCAR’ lentiviral vectors and viral packaging plasmids. Patient‘s CD3 T cells were cultured in X-VIVO medium containing 125U/mL 1interleukin-2 (IL-2), and transduced with ‘CoupledCAR’ lentivirus at certain MOI. Transduction efficiency and was evaluated at 7 to 9 days after ‘CoupledCAR’ lentivirus transduction, and quality controls for fungi, bacteria, mycoplasma, chlamydia, and endotoxin were performed. After infusion, serial peripheral blood samples were collected, and the expansion and the cytokine release of CART cells were detected by FACS and QPCR. The evaluation of response level for patients were performed at month 1,month 3,and month 6 by PET/CT.
Results To evaluate the clinical safety and efficacy of anti-TSHR CoupledCAR T cells on refractory or relapsed thyroid cancer, we treated refractory/relapsed post-thyroidectomy thyroid cancer patients according to an IRB approved protocol. We treated two patients using anti-TSHR CoupledCAR T cells and observed the rapid expansion of CAR T cells and enhanced the killing of tumor cells. One patient‘s best response was complete remission, and the other was near complete remission.Patient Profile:Patient 1 Male, 64Y, Papillary Thyroid Carcinoma. In May 2017, Thyroid cancer was diagnosed, bilateral total thyroidectomy, and right cervical lymph node functional dissection were performed in Jun 2018, followed by iodine 131 isotope therapy. In December 2018, bilateral multiple cervical lymph nodes were enlarged, especially on the right side. In February 2019, right neck lymphadenectomy was performed.Patient 2 Female, 60Y, Thyroid Carcinoma. In Aug 2013, a ‘double lobectomy of the thyroid gland’ was performed. From Oct 2013 to Jan 2014, she received iodine 131 isotope therapy. In Sep 2014, she was diagnosed with iodine - resistant thyroid cancer. From Sep to Jan 2016, 5 cycles of chemotherapy were performed. In Jun 2016, she enrolled in the Anlotinib experimental group. In Mar 2019, multiple metastases in both lungs and multiple enlarged lymph nodes in the mediastinum were observed.Observations and Results:Patient 1: One month after infusion (M1), the patient was evaluated as PR: lymph node metastasis became undetectable and the size of the thoracic paratracheal tumor nodules decreased significantly. Three months after infusion (M3), the patient was evaluated as CR, and the tumor tissue was substantially smaller than M1.Patient 2: M1, the patient was evaluated as PR (Partial Response): the tumor volume in the right lower lobe of the lung was reduced by approximately 67.51% (decreased from 65*55 mm to 42*39 mm). Three months after infusion (M3), compared with that before, the tumor volume was reduced by approximately 73.54% and SUV max value decreased from 14.9 to 2.8, therefore, the patient was evaluated as nCR (near complete remission).
Conclusions We show that TSHR is a good target for treating thyroid cancer, and our anti-TSHR CoupledCAR T cells are safe and effective for treating thyroid cancer. Recruitment is ongoing to evaluate the safety and efficacy of our CoupledCAR T cells. Further, since our CoupledCAR® technology is a platform technology, we are developing it to treat other solid tumors using different target tumor markers.
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