Humanized DR4 antibody gene having apoptosis-inducing activity and dual-acting chimeric antigen receptor T cell or natural killer cell therapeutic agent using same
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Publication Number
US-11603409-B2
Publication Date
2023-03-14
Expiration Date
Abstract
The present invention relates to a humanized DR4 antibody gene having apoptosis-inducing activity, and a dual-acting chimeric antigen receptor T cell or natural killer cell therapeutic agent, a secretory DR4 scFv antibody recombinant protein synthesized by using a humanized DR4 scFv antibody gene (humanized anti-DR4 scFv) may be used as an anticancer drug specifically targeting DR4 by binding to DR4 expressed on surface of the cancer cells to induce apoptosis of the cancer cells. In addition, the chimeric antigen receptor (CAR) using the humanized DR4 scFv antibody gene is predicted to have a strong anticancer immune effect as a dual-acting cell therapeutic agent, which is capable of simultaneously inducing apoptosis in cancer cells by DR4 and exhibiting a cytotoxic effect for cytotoxic T lymphocytes or natural killer cells by binding to DR4 expressed on the surface of cancer cells, as a DR4-specific CAR-T cell or CAR-NK cell therapeutic agent.
Core Innovation
A gene expression cassette encodes an anti-DR4 antibody fragment recombinant protein, wherein the recombinant protein comprises an anti-DR4 antibody fragment domain having the amino acid sequence of SEQ ID NO: 1 that specifically binds to death receptor (DR4). The recombinant protein further comprises a trimolecular complex forming domain, a secretory inducing domain, a transmembrane domain, and a signaling domain.
The anti-DR4 scFv antibody gene is described as humanized, and a derived secretory (soluble) DR4 scFv recombinant protein binds DR4 on cancer cells to induce apoptosis. The document describes a soluble trimolecular complex concept and includes design elements for a CD8α leader, an isoleucine zipper (ILZ), and tags/hinge in the recombinant protein.
CAR constructs are described that use the humanized DR4 scFv for CAR-T cell dual-acting therapy, including a CD8α transmembrane domain and a 4-1BB/CD3ζ signaling domain. The document describes that CAR-expressing lymphocytes trigger DR4-mediated apoptosis and provide CTL/NK cytotoxicity, and it conceptually references CAR-NK in the same dual-acting therapy context.
Claims Coverage
The document contains one independent claim covering a gene expression cassette that encodes an anti-DR4 antibody fragment recombinant protein with multiple functional domains. Across dependent claims, the inventive structure is further constrained by the specific anti-DR4 sequence, the format of the antibody fragment, implementation as a CAR, the vector type, and the target CAR effector cell type.
Gene expression cassette with anti-DR4 binding domain and functional signaling architecture
The gene expression cassette encodes an anti-DR4 antibody fragment recombinant protein comprising an anti-DR4 antibody fragment domain having the amino acid sequence of SEQ ID NO: 1 that specifically binds DR4, and further comprises a trimolecular complex forming domain, a secretory inducing domain, a transmembrane domain, and a signaling domain.
Specific anti-DR4 binding-domain nucleotide sequence constraint
The gene expression cassette is configured so that the anti-DR4 antibody fragment domain has the nucleotide sequence SEQ ID NO: 2.
CAR implementation of the anti-DR4 antibody fragment recombinant protein
The anti-DR4 antibody fragment recombinant protein in the gene expression cassette is implemented as a chimeric antigen receptor (CAR).
Selected anti-DR4 antibody fragment format
The anti-DR4 antibody fragment is chosen from scFv, (scFv)2, Fab, Fab′, or F(ab′)2.
Vector type selection for the gene expression cassette
The claimed vector is specified as being either a recombinant plasmid vector or a recombinant viral vector.
CAR effector cell type
The chimeric antigen receptor-expressing cell is either a CAR-T cell or a CAR-NK cell.
Overall, the claim set centers on an anti-DR4 gene expression cassette encoding a domain-based recombinant protein that specifically binds DR4 and includes trimolecular complex forming, secretory inducing, transmembrane, and signaling domains. Dependent claims narrow the construct by specifying an additional binding-domain nucleotide sequence, limiting the antibody-fragment format and implementation as a CAR, and by constraining the vector type and whether CAR is expressed in CAR-T or CAR-NK cells.
Stated Advantages
Induces apoptosis by binding DR4 on cancer cells.
Provides dual-acting therapy in CAR-T cells by triggering DR4-mediated apoptosis and providing CTL/NK cytotoxicity.
Documented Applications
Dual-acting therapy using CAR-T cells with an anti-DR4 scFv-based CAR to trigger DR4-mediated apoptosis and provide CTL/NK cytotoxicity.
CAR-NK conceptually described as part of the same DR4-mediated dual-acting therapy context.
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