SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy is named GBKDYL001_Sequence_Listing.txt, created on Jul. 26, 2021 and is 38,606 bytes in size.

TECHNICAL FIELD

The invention belongs to the field of cell therapy for tumors, and particularly relates to an immune effector cell targeting CLDN18A2, preparation method and application thereof.

BACKGROUND

With the rapid development of biotechnology, immune cell therapy has become the fourth major therapy in the field of cancer treatment.

Cancer immunotherapy mainly includes adoptive cell therapy, cytokines, monoclonal antibodies (mAbs), tumor vaccines, immunology checkpoint block therapy and so on. Chimeric antigen receptor-T (CAR-T) cell immunotherapy is a form of adoptive cell therapy and has provided a dramatically advanced breakthrough as one of the most promising cancer immunotherapies.

CAR-T cells, as a rapidly emerging immunotherapeutic modality, are T cells that are genetically engineered to express an antigen-specific receptor that can recognize a target in a non-MHC restricted manner. This therapy has shown remarkable efficacy in the treatment of acute leukemia and non-Hodgkins lymphoma, and is considered to be one of the most promising cancer treatment modalities.

Trials of CAR-T cells in solid tumors followed soon after those in hematologic cancers. However, the application of CAR-T immunotherapy in solid tumors has many challenges, including immunological toxicity caused by sustained intense activation of the CAR-T cells resulting in a macrophage activation syndrome (MAS) and “On-target off-tumor” toxicity i.e., recognition of the target antigen on normal tissues. It is crucial to choose appropriate antigenic targets to generate high-efficiency CAR-T cells which eliminate tumor cells with minimum toxicity.

In recent years, studies have shown that Claudin proteins are the most crucial components of tight junctions, and play an essential role in maintaining cell polarity, regulating cell permeability and the intercellular ion. There have been 27 members of the family discovered so far and CLDN18A2 is one of the members which is becoming a promising antigen. CLDN18A2 is expressed exclusively on short-lived differentiated gastric epithelial cells in normal tissues and highly expressed on solid tumors such as gastric, pancreas, ovary, gallbladder, and lung adenocarcinoma (Sahin et al. Clinical Cancer Research (2008)). In addition, the exposed extracellular structure of CLDN18A2 allows for antibody binding, which makes it an ideal therapeutic target. Thus, CLDN18A2 is an ideal tumor antigen target of immunotherapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an immune effector cell targeting human CLDN18A2 and the preparation method and application thereof.

In a first aspect, the present invention provides a specific chimeric antigen receptor expressed on the surface of an immune effector cell, wherein the chimeric antigen receptor comprises a sequentially connected extracellular binding region, a transmembrane region and an intracellular signal region, wherein the extracellular binding region comprises a protein which specifically recognizes human CLDN18A2.

In one preferred embodiment, the chimeric antigen receptor which specifically recognizes human CLDN18A2 comprises any one of the amino acid sequences of SEQ ID NOS: 2-5, or comprises any one of the amino acid sequences of the variants having 70%-99% identity with the amino acid sequences shown by the SEQ ID NOS: 2-5.

In some embodiments, the protein specifically recognizing human CLDN18A2 is an antibody or a ligand; preferably, the antibody is a single-chain antibody or single-domain antibody.

In a second aspect, this application provides a chimeric antigen receptor system comprising:(i) a receptor component comprising the guiding peptide, an antigen binding domain, a transmembrane domain; and (ii) an intracellular signaling component comprising one or more of the following intracellular domains: CD28 intracellular domain, 4-1BB intracellular domain, OX40 intracellular domain and CD3 zeta intracellular domain.

In some embodiments, the specific chimeric antigen receptor also comprises a hinge region.

In some preferred embodiments, the chimeric antigen receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain connected in the following sequences:

In one embodiment, the leader sequence is selected from SEQ ID NO: 1.

In some embodiments, the amino acid sequence of human CD8 in the hinge region is selected from SEQ ID NO: 6.

In some embodiments, the amino acid sequence of human CD8 in the transmembrane domain is selected from SEQ ID NO: 7.

In some embodiments, the human CD28 in the transmembrane domain is selected from SEQ ID NO: 8.

In some embodiments, the human 4-1BB intracellular domain is selected from SEQ ID NO: 9.

In some embodiments, the human CD28 intracellular domain is selected from SEQ ID NO: 10.

In some embodiments, the human OX40 intracellular domain is selected from SEQ ID NO: 11.

In some embodiments, the CD3 zeta intracellular domain is selected from SEQ ID NO: 12.

In another preferred embodiment, the chimeric antigen receptor comprises any one of the amino acid sequences of SEQ ID NOS: 13-16.

In a third aspect, this invention provides the nucleic acid encoding the chimeric antigen receptor.

In some embodiments, the nucleic acid encoding the chimeric antigen receptor comprises any one of the nucleotide sequences of SEQ ID NOS: 17-20.

In a fourth aspect, this application provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.

The vector may, for example, be a retroviral vector or a lentiviral vector or a transposon.

In a fifth aspect, this application provides a recombinant virus which expressing a chimeric antigen receptor specifically targeting human CLDN18A2 as according to the second aspect of the invention.

In a sixth aspect, this application provides a cell which expresses a receptor component and a signaling component as defined in the second aspect of the invention.

In some embodiment, the cell may comprise a nucleic acid according to the third aspect of the invention or a vector according to the fourth aspect of the invention.

The cell may be an immune cell such as a T cell, macrophage, human embryonic stem cells or an NK cell.

In a seventh aspect, there is provided a method of preparing a genetically modified immune effector cell targeting CLDN18A2.

In an eighth aspect, there is provided a pharmaceutical composition comprising a plurality of cells according to the sixth aspect of the invention.

In a ninth aspect, the application provides a kit for the treatment or prevention of a disease that contains an immune response cell described in the sixth aspect or a nucleic acid described in the third aspect.

In a tenth aspect, there is provided a method for making a cell according to the sixth aspect of the invention, which comprises the step of introducing a nucleic acid sequence according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a kit according to the seventh aspect of the invention.

In some embodiment, the use of the genetically modified immune effector cells for preparation of a medicine for suppressing tumor, wherein the tumor is CLDN18A2 positive.

In some embodiment, the CLDN18A2 positive cancers include gastric cancer, pancreatic cancer, liver cancer, brain cancer, prostate cancer, lymphoma, leukemia, colon cancer, lung cancer, or breast cancer.

