The invention belongs to the technical field of tumor
immunotherapy, and relates to an anti-
Claudin18.2 single-domain
antibody, a multifunctional
fusion protein, a recombinant
natural killer cell (CT-CAR-NK), and preparation and application thereof. Through alpaca immunization and
phage display library construction and panning, the single-domain
antibody VHH-C18.2-1 specifically combined with
Claudin18.2 is obtained, and the
amino acid sequence of the single-domain
antibody VHH-C18.2-1 is SEQ ID NO: 1. The
amino acid sequence of the designed
fusion protein is SEQ ID NO: 3, the
fusion protein sequentially comprises a VHH-C18.2-1, a flexible
Linker, a TGF-beta RII
extracellular domain, a CD8alpha
hinge region, a CD8alpha
transmembrane region, a 4-1BB
intracellular domain and a CD3zeta
intracellular domain from the N end to the C end, and the fusion
protein has the functions of targeting, resisting TGF-beta inhibition and activating signals. The fusion
protein gene transfects human
peripheral blood CD56 + CD3-NK cells through
lentivirus to obtain CT-CAR-NK, in-vitro
verification shows that the CT-CAR-NK still keeps efficient killing in an
immunosuppression environment,
tumor growth can be remarkably inhibited
in vivo, the lifetime can be prolonged, and a safe and efficient scheme is provided for
Claudin18.2 positive
solid tumor treatment.