Antibody variable domains targeting the NKG2D receptor

Inventors

Chang, Gregory P. • Cheung, Ann F. • Grinberg, Asya • Haney, William • LUNDE, Bradley M. • Prinz, Bianka

Assignees

Dragonfly Therapeutics LLC

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Publication Number

US-12264200-B2

Patent

Publication Date

2025-04-01

Expiration Date


Abstract

Antibody heavy chain variable domains that can be paired with antibody light chain variable domains to form an antigen-binding site targeting the NKG2D receptor on natural killer cells are described. Proteins comprising an NKG2D antigen-binding site, pharmaceutical compositions and therapeutic methods thereof, including for the treatment of cancer, are also described.

Core Innovation

The disclosed invention relates to an antigen-binding site that binds NKG2D. The antigen-binding site comprises an antibody heavy chain variable domain with a complementarity-determining region 1 (CDR1) amino acid sequence of SEQ ID NO: 48, a CDR2 amino acid sequence of SEQ ID NO: 30, and a CDR3 amino acid sequence of SEQ ID NO: 71, together with an antibody light chain variable domain with a CDR1 amino acid sequence of SEQ ID NO: 32, a CDR2 amino acid sequence of SEQ ID NO: 33, and a CDR3 amino acid sequence of SEQ ID NO: 34. The NKG2D-targeting antibody variable domains are described as Kabat-determined CDRs and as antigen-binding sites that bind and agonize NKG2D.

The disclosure states that the antibody binding competes with natural ligand binding and exhibits high-affinity binding to NKG2D, with binding affinities reported in the range of approximately 10−62 nM. Binding characterization includes measured binding affinities using surface plasmon resonance (SPR) and epitope binning indicating unique epitopes for certain clones.

The disclosed material further describes optional combinations of the NKG2D-binding variable domains with Fc/constant-region domains and multi-specific formats. The disclosure includes combination into tumor antigen-binding plus CD16-binding Fc-containing proteins such as TriNKETs, and describes TriNKETs activating NK cells and mediating cytotoxicity.

Claims Coverage

The claim coverage centers on one independent claim directed to a specific NKG2D-binding antigen-binding site defined by particular heavy- and light-chain variable domain CDR amino-acid sequences. Dependent claims add sequence-identity constraints, an additional antigen-binding site, Fc/constant-region formats that bind CD16, and specific constant-region substitutions using Kabat EU numbering.

NKG2D-binding antigen-binding site defined by CDR sequences

An antigen-binding site that binds NKG2D, comprising an antibody heavy chain variable domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 30, and CDR3 of SEQ ID NO: 71; and an antibody light chain variable domain comprising CDR1 of SEQ ID NO: 32, CDR2 of SEQ ID NO: 33, and CDR3 of SEQ ID NO: 34.

NKG2D-binding variable domains with sequence-identity constraints

The antibody heavy and light chain variable domains have amino acid sequences at least 95% identical to SEQ ID NO: 7 and SEQ ID NO: 8, respectively, within the antigen-binding site.

Additional antigen-binding site in a protein

A protein that includes the antigen-binding site and an additional antigen-binding site.

CD16-binding complex formed by antibody constant regions

A protein in which first and second antibody constant regions form a complex that binds CD16.

Kabat EU constant-region point substitutions

A protein in which the first antibody constant region includes a Y349C substitution and the second antibody constant region includes an S354C substitution, using Kabat EU numbering.

Overall, the claim coverage centers on a specific NKG2D-binding antigen-binding site defined by particular heavy- and light-chain CDR sequences, and is narrowed and expanded through dependent claim features including sequence-identity constraints, inclusion of an additional antigen-binding site, CD16-binding constant-region complex formation, and specified constant-region substitutions referenced to Kabat EU numbering.

Stated Advantages

Binds and agonizes NKG2D.

Competes with natural ligand binding.

Exhibits high-affinity binding to NKG2D (reported KDs approximately in the 10−62 nM range).

Activates NK cells and mediates cytotoxicity in connection with TriNKETs.

Documented Applications

Cancer treatment and enhancing tumor cell death are described in connection with NKG2D-targeting antibodies and multi-specific formats such as TriNKETs.

Functional evaluation of NK-cell activation and cytotoxicity using assays described in the partial content, including IFN-gamma and CD107a endpoints.

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