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Publication Number
US-11254747-B2
Publication Date
2022-02-22
Expiration Date
Abstract
The present invention is directed to diabody molecules and uses thereof in the treatment of a variety of diseases and disorders, including immunological disorders, infectious disease, intoxication and cancers. The diabody molecules of the invention comprise two polypeptide chains that associate to form at least two epitope binding sites, which may recognize the same or different epitopes on the same or differing antigens. Additionally, the antigens may be from the same or different molecules. The individual polypeptide chains of the diabody molecule may be covalently bound through nonpeptide bond covalent bonds, such as, but not limited to, disulfide bonding of cysteine residues located within each polypeptide chain. In particular embodiments, the diabody molecules of the present invention further comprise an Fc region, which allows antibody like functionality to engineered into the molecule.
Core Innovation
The invention concerns a diabody molecule capable of binding to an epitope of CD32B and an epitope of CD79B. The diabody comprises a first polypeptide chain and a second polypeptide chain that are covalently bonded to one another, and each chain includes immunoglobulin variable domain binding regions arranged in a defined domain order and connected by intervening linkers.
In the first polypeptide chain, the N-terminal to C-terminal direction includes a light chain variable domain binding region specific for binding the CD79B epitope, followed by a heavy chain variable domain binding region specific for binding the CD32B epitope, and a cysteine residue-containing domain. In the second polypeptide chain, the N-terminal to C-terminal direction includes a light chain variable domain binding region specific for binding the CD32B epitope, followed by a heavy chain variable domain binding region specific for binding the CD79B epitope, and a cysteine residue-containing domain, with intervening linkers constraining the domains from self-assembly and preventing formation of unintended epitope binding sites.
The binding sites are formed by association of complementary domains across the covalently linked chains: the first domain and the fifth domain associate to form a binding site that binds the CD79B epitope, while the second domain and the fourth domain associate to form a binding site that binds the CD32B epitope. The cysteine residues of the third domain and the sixth domain form a disulfide bond between the first and second polypeptide chains, and the first polypeptide chain comprises the light chain variable domain, the intervening GGGSGGGG linker, and the heavy chain variable domain of SEQ ID NO:132, while the second polypeptide chain comprises the light chain variable domain, the same GGGSGGGG linker, and either the heavy chain variable domain of SEQ ID NO:134 or SEQ ID NO:136.
Claims Coverage
The provided material includes one independent claim. The inventive coverage centers on a structurally constrained bispecific diabody that binds CD32B and CD79B by forming two intended binding sites while constraining alternative domain associations.
Covalently bonded diabody with disulfide-linked first and second chains
A diabody molecule capable of binding an epitope of CD32B and an epitope of CD79B, comprising a first polypeptide chain and a second polypeptide chain that are covalently bonded to one another, wherein cysteine residues form a disulfide bond between the first and second polypeptide chains.
Cd79b binding site from vl1/vh1 association across chains
A first polypeptide chain with a first domain comprising a binding region of a light chain variable domain specific for binding the CD79B epitope and a second domain constrained by a linker so they do not self-assemble into unintended binding sites, wherein the first domain and a fifth domain of the second polypeptide chain associate to form a binding site that binds the CD79B epitope.
Cd32b binding site from vh2/vl2 association across chains
A first polypeptide chain including a second domain comprising a binding region of a heavy chain variable domain specific for binding the CD32B epitope and a linker-constrained configuration that prevents unintended binding site formation, wherein the second domain and a fourth domain of the second polypeptide chain associate to form a binding site that binds the CD32B epitope.
Intervening gggsgggg linker constrained from self-assembly
Intervening linker configurations in both polypeptide chains link domain pairs such that the corresponding first and second domains and the fourth and fifth domains are constrained from self-assembly and do not associate to form an epitope binding site; the first polypeptide chain comprises the intervening GGGSGGGG linker between the light chain variable domain and the heavy chain variable domain of SEQ ID NO:132, and the second polypeptide chain comprises the intervening GGGSGGGG linker between the light chain variable domain and either the heavy chain variable domain of SEQ ID NO:134 or SEQ ID NO:136.
The independent claim defines a bispecific diabody with first and second polypeptide chains covalently bonded by a disulfide bond, where linkers constrain domain self-assembly to prevent unintended binding site formation. Two intended binding sites are formed by association across chains for CD79B and CD32B, with specified linker and specified heavy chain variable domain sequences.
Stated Advantages
Inhibition of BCR-mediated signaling in SHIP-deficient mouse B cells, with associated signaling readouts such as calcium mobilization, Ins(1,4,5)P3, Erk phosphorylation, Akt phosphorylation, proliferation and cell cycle progression.
Inhibition of FcγR-mediated phagocytosis/signaling in human monocytes/macrophages, with phagocytic rate and downstream phosphorylation readouts.
Use of binding and effector-function characterization approaches including SPR/BIAcore binding assays and effector-function readouts such as ADCC/phagocytosis/CDC.
Documented Applications
Inhibition of BCR-mediated signaling in SHIP-deficient mouse B cells, assessed using readouts including calcium mobilization and downstream phosphorylation events.
Inhibition of FcγR-mediated phagocytosis/signaling in human monocytes/macrophages, assessed using phagocytic and downstream signaling readouts including SHIP/Akt-associated phosphorylation.
CD16A binding domain selection/design framework for forming CD16A binding diabodies using humanized 3G8-derived VH/VL CDRs and specified combinations.
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