Design and development of dual targeting CAR protein for the development of CAR T-cell therapy against KRAS mutated pancreatic ductal adenocarcinoma using computational approaches

Abstract

Mutant KRAS promotes the proliferation, metastasis, and aggressiveness of various cancers including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal adenocarcinoma (CRC) respectively. Mutant KRAS therapeutics are limited, while Sotorasib and Adagrasib were the only FDA-approved drugs for the treatment of KRAS^G12C mutated NSCLC. Chimeric antigen receptor (CAR) T-cell therapy has been emerged as an effective strategy against hematological malignancies and being extended towards solid cancers including PDAC. mesothelin (MSLN) and Carcinoembryonic Antigen (CEA) were reported to be highly overexpressed in KRAS-mutated PDAC. Meanwhile, in clinical trials, several CAR T-cell therapy studies are mainly focused towards these two cancer antigens in PDAC, however, the dual targeting of these two neoantigens is not reported. In the present study, we have designed and developed a novel dual-targeting CAR protein by employing various bioinformatics approaches such as functional analysis (antigenicity, allergenicity, antigen binding sites & signalling cascades), qualitative analysis (physicochemical, prediction, refinement & validation of 2D and 3D structures), molecular docking, and in silico cloning. Our results revealed that the designed CAR protein specifically binds with both MSLN & CEA with significant binding affinities, and was predicted to be stable & non-allergenic. Additionally, the protein-protein interaction network reveals the T-cell mediated antitumor responses of each domain in the designed CAR. Conclusively, we have designed and developed a dual targeting (MSLN & CEA) CAR protein towards KRAS-mutated PDAC using computational approaches. Alongside, we further recommend to engineer this designed CAR in T-cells and evaluating their therapeutic efficiency in in vitro and in vivo studies in the near future.

Keywords: CAR; CAR T-cell therapy; Immunotherapy; KRAS; Precision medicine; Therapeutics.

Fig. 1

Workflow of the present study

Fig. 2

Designing of dual targeting CAR construct against the KRAS-mutated PDAC

Fig. 3

Refined 3D structure of the designed CAR with its domains (A). Ramachandran plots of unrefined (B) and refined model (C), Z-score plots of unrefined (D) and refined model (E) of CAR were also shown respectively

Fig. 4

Antigen binding sites of the designed CAR. Continues (linear) epitope binding regions of MSLN (A, B), Continues (linear) epitope binding regions of CEA (C, E), and discontinuous epitope binding regions of MSLN (F) and CEA (F, G) were depicted respectively

Fig. 5

Molecular interactions of designed CAR with CEA and MSLN. Surface view interaction (A1), total number of interactions (A2), & interacting residues (A3) of CAR-CEA docked complexes, and surface view interaction (B1), total number of interactions (B2), & interacting residues (B3) of CAR-MSLN docked complexes. In the surface view, MSLN scFv was shown in red, (GGGGS)₃ was shown in green, CEA scFv was shown in yellow, CD8α was shown in magenta, CD28 was shown in orange, CD137 was shown in cyan, CD3ζ was shown in blue, and the CEA (A1) and MSLN (B1) were shown in gray respectively

Fig. 6

Protein–protein interaction (PPI) network of MSLN (A), CEA (B), CD8A (C), CD28 (D), CD137 (E), CD3D (F) respectively. Also, the number of nodes, edges and gene ontology enrichment p-values were of each PPI network were provided

Fig. 7

In silico cloning of the codon-optimized CAR protein sequence (4173 bp) shown in red was cloned in the pcDNA3.1+/C-His expression vector (5432 bp) shown in black between restriction sites Kpnl (GGTACC) and EcoR1 (GAATTC), and final cloned vaccine construct was shown (8695 bp)

Fig. 8

Overall summary of the present study. The designed dual targeting CAR has the potential to bind to the CEA and MSLN antigens of the KRAS mutated PDAC, and could exerts T cell mediated cancer cell death