Improved anti‐solid tumor response by humanized anti‐podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model

Abstract

Recently, research has been conducted with chimeric antigen receptor (CAR)‐T cells to improve efficacy against solid tumors. Humanized CAR improved the long‐term survival of CAR‐T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR‐gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR‐T cells. Previously, an anti‐PDPN CAR was generated from a conventional nonhumanized antibody—NZ‐1, the only anti‐PDPN antibody for which a CAR was produced. In this study, we investigated other anti‐PDPN CARs from the antibody NZ‐27, or humanized NZ‐1, to enhance the therapeutic potential of CAR‐T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T‐cell surface of NZ‐27 CAR‐T cells, which show tumor‐specific cytotoxicity, proinflammatory cytokine production, and anti‐tumor activity against PDPN‐expressing tumor xenografts in mice that were significantly better than those in nonhumanized NZ‐1 CAR‐T cells.

Antitumor activities of a defucosylated anti‑EpCAM monoclonal antibody in colorectal carcinoma xenograft models

“…The applications of anti-EpcAM mAbs in the clinic are still limited, since anti-EpcAM mAbs may generate side-effects by affecting normal tissues. casMabs targeting PdPN (67)(68)(69)(70) and podocalyxin (71) have been previously developed by the authors, which are currently applied to chimeric antigen receptor T-cell therapy in mice models (72)(73)(74). It will be of great value to establish cancer-specific anti-EpCAM mAbs using the casMab method.…”

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas

“…CD44v3-10 ORF was obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. CD44s and CD44v3-10 cDNAs were subcloned into pCAG-Ble-ssPA16 vector possessing signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [ 19 , 49 , 50 , 51 , 52 ], which is detected by NZ-1 [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. CHO/CD44s and CHO/CD44v3-10 were established by transfecting pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3-10 into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc.).…”

Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C₄₄Mab-3 for Multiple Applications against Pancreatic Carcinomas

“…CD44s cDNA was amplified using Hot-Star HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) using LN229 (a glioblastoma cell line) cDNA as a template. CD44v3-10 and CD44 cDNAs were cloned into pCAG-Ble-ssPA16 vector with signal sequence and N-terminal PA16 tag of 16 amino acids (GLEGGVAMPGAEDDVV) [22,[28][29][30][31], which is detected by NZ-1, which was originally developed against human podoplanin [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47]. The pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3-10 vectors were transfected into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc.), which offers an innovative electroporation method that utilizes a proprietary biologically compatible pipette tip chamber to generate a more uniform electric field for a significant increase in transfection efficiency and cell viability.…”