Background: Currently, CAR-T therapy has been successful in the treatment of certain types of hematological malignancy. However, not all patients benefit from this novel therapy due to various resistance mechanisms, such as suppression of immune cell function by inhibitory immune checkpoint signaling. To overcome this, we have developed a novel two-in-one lentiviral vector system that can efficiently induce shRNA-based silencing of PD-1 and TIGIT genes in CAR-T cells while maintaining robust CAR expression. This PD-1 and TIGIT-silenced CD19 CAR-T, CRC01, showed an enhanced anti-tumor activity in vitro and in vivo, compared to conventional CD19 CAR-T. For the development of CRC01 as an investigational new drug, IND-enabling non-clinical studies have been conducted and CRC01 is currently being evaluated in a clinical trial for r/r LBCL patients (NCT04836507). Methods: T cells were enriched from leukapheresis products of r/r LBCL patients and transduced with the two-in-one lentiviral vector. Transduced T cells were expanded within a semi-closed culture system and formulated for cryopreservation. For phase I clinical trial of CRC01, ten batches of CRC01 were manufactured and characterized in quality. Results: In the manufacturing of ten batches of CRC01, manufacturing time was in the range of 8-14 days and total cells expanded 32-53 folds. In the final product of CRC01, T cell percentage (Live CD3+) and viability were not less than 98% and 85%, respectively. The range of transduction efficiency was 11.3%-28.6% and the range of vector copy number was 1.0-3.5 copies/CAR-T. Generally, CRC01(CAR-positive T cells) showed higher CD4/CD8 ratio than leukapheresis product and CAR-negative T cells, and central memory and effector memory T cell subsets were the main populations of CRC01. All batches of CRC01 released IFN-γ in response to CD19-expressing target cells, but the level of IFN-γ production was quite different from batch to batch. When PD-1 and TIGIT expression levels of CRC01 were analyzed after CD3/CD28 stimulation, CAR-positive T cells were found to express PD-1 and TIGIT much lower than CAR-negative T cells, demonstrating an efficient silencing of PD-1 and TIGIT genes in CRC01. Conclusions: Ten batches of CRC01 were manufactured in GMP facility for CRC01 phase I clinical trial. All batches met acceptance criteria for release, showing that CRC01 manufacturing process is a robust one and that silencing of PD-1 and TIGIT genes via shRNAs, a unique feature of CRC01, was successfully implemented in the investigational products.