BISPECIFIC ANTIBODIES AND USES THEREOF
Methods, compositions and uses are provided for bispecific antibodies comprising one or more unnatural amino acids. The bispecific antibodies may bind to two or more different receptors, co-receptors, antigens, or cell markers on one or more cells. The bispecific antibodies may be used to treat a disease or condition (e.g., cancer, autoimmune disease, pathogenic infection, inflammatory disease). The bispecific antibodies may be used to modulate (e.g., stimulate or suppress) an immune response.
1 . A bispecific antibody comprising:
a. an anti-CD3 antibody or anti-CD3 antibody fragment;
b. a second antibody or antibody fragment,
wherein the anti-CD3 antibody or anti-CD3 antibody fragment is site-specifically connected to the second antibody or antibody fragment.
2 . The bispecific antibody of claim 1 , wherein the anti-CD3 antibody or anti-CD3 antibody fragment is site-specifically connected to the second antibody or antibody fragment by one or more linkers.
3 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment comprises an anti-EGFRvIII antibody.
4 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment comprises an anti-Her2 antibody.
5 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment comprises an anti-CS1 antibody.
6 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment comprises an anti-CLL-1 antibody.
7 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment comprises an anti-CD33 antibody.
8 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment comprises an anti-CD20 antibody.
9 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment is selected from an anti-ROR1 antibody, an anti-CD44v6 antibody, an anti-PVRL4 antibody, an anti-IL13Rα2 antibody and a bscWue1 antibody.
10 . The bispecific antibody of claim 1 , wherein the anti-CD3 antibody or anti-CD3 antibody fragment comprises one or more unnatural amino acids.
11 . The bispecific antibody of claim 1 , wherein the second antibody or antibody fragment comprises one or more unnatural amino acids.
12 . The bispecific antibody of claim 1 , wherein the anti-CD3 antibody or anti-CD3 antibody fragment and the second antibody or antibody fragment comprise one or more unnatural amino acids.
13 . The bispecific antibody of claim 2 , wherein the antibody is of Formula I: X-L1′-Y or Formula IA: Y-L1′-X, wherein:
a. X comprises the anti-CD3 antibody or anti-CD3 antibody fragment;
b. L1′ comprises the one or more linkers; and
c. Y comprises the second antibody or antibody fragment.
14 . The bispecific antibody of claim 13 , wherein X is selected from a human, humanized, human engineered or fully human antibody.
15 . The bispecific antibody of claim 1 , wherein X comprises a chimeric antibody or portion thereof.
16 . The bispecific antibody of claim 1 , wherein X comprises a cross-species reactive antibody or a portion thereof.
17 . The bispecific antibody of claim 16 , wherein the anti-CD3 antibody is cross-species reactive with human and cynomolgus monkey.
18 . The bispecific antibody of claim 13 , wherein X and/or Y comprises one or more Fv, Fc, Fab, (Fab′)2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, alternative scaffold non-antibody molecules, or a combination thereof.
19 . The bispecific antibody of claim 13 , wherein X and/or Y comprises a Fab fragment.
20 . The bispecific antibody of claim 19 , wherein the anti-CD3 antibody fragment is UCHT1.
21 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of a Fab fragment.
22 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an antibody or antibody fragment that binds to an antigen on a hematopoietic cell.
23 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an antibody or antibody fragment that binds to an antigen on a myeloid cell.
24 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an antibody or antibody fragment that binds to an antigen on a lymphocyte.
25 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an antibody or antibody fragment that binds to an antigen on a B-cell or B-cell progenitor.
26 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an antibody or antibody fragment that binds to an antigen on a cancerous cell.
27 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an antibody selected from the group comprising an anti-CD20 antibody, an anti-EGFRvIII antibody, an anti-CS1 antibody, an anti-CLL-1 antibody and an anti-CD33 antibody.
28 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of a Fab fragment of an antibody selected from the group comprising an anti-CD20 antibody, an anti-EGFRvIII antibody, an anti-CS1 antibody, an anti-CLL-1 antibody and an anti-CD33 antibody.
29 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an antibody selected from the group comprising an anti-ROR1 antibody, an anti-CD44v6 antibody, an anti-PVRL4 antibody, an anti-IL13Rα2 antibody and an anti-bscWue1 antibody.
30 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of Fab fragment of an antibody selected from the group comprising an anti-ROR1 antibody, an anti-CD44v6 antibody, an anti-PVRL4 antibody, an anti-IL13Rα2 antibody and a bscWue1 antibody.
31 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of an anti-Her2 antibody.
32 . The bispecific antibody of claim 13 , wherein Y comprises at least a portion of a Fab fragment of an anti-Her2 antibody.
33 . The bispecific antibody of claim 13 , wherein Y is selected form SEQ ID NOs: 3-16.
34 . The bispecific antibody of claim 13 , wherein the bispecific antibody further comprises a second linker.
35 . The bispecific antibody of claim 34 , wherein the bispecific antibody is of Formula II: X-L1-L2-Y or Formula IIA: Y-L2-L1-X, wherein:
a. a L1″ is coupled to X to produce a first intermediate of Formula III: X-L1′ or Formula IIIA: L1′-X, wherein X comprises an anti-CD3 antibody or anti-CD3 antibody fragment; and L1″ comprises a first linker before being coupled to X,
b. L2″ is coupled to Y to produce a second intermediate of Formula IV: Y-L2′ or Formula IVA: L2′-Y; wherein Y comprises at least a portion of a second antibody or antibody fragment and L2″ comprises a second linker before being coupled to Y; and
c. the first intermediate is coupled to the second intermediate.
36 . The bispecific antibody of claim 35 , wherein L1″ and/or L2″ comprise one or more chemical groups selected from an alkoxy-amine, hydrazine, aryl azide, alkyl azide, alkyne, alkene, tetrazine, dichlorotriazine, tresylate, succinimidyl carbonate, benzotriazole carbonate, nitrophenyl carbonate, trichlorophenyl carbonate, carbonylimidazole, succinimidyl succinate, maleimide, vinylsulfone, haloacetamide, cyclooctyne, trans-cyclooctene, cyclopropene, norborene and disulfide.
37 . The bispecific antibody of claim 35 , wherein one terminus of L1″ and/or one terminus of L2″ comprises an alkoxy-amine.
38 . The bispecific antibody of claim 35 , wherein one terminus of L1″ and/or one terminus of L2″ comprises an azide or cyclooctyne group.
39 . The bispecific antibody of claim 35 , wherein X is coupled to L1 by a covalent bond, ionic bond, or non-covalent bond.
40 . The bispecific antibody of claim 35 , wherein Y is coupled to L2 by a covalent bond, ionic bond, or non-covalent bond.
41 . The bispecific antibody of claim 35 , wherein X is coupled to L1 by an oxime.
42 . The bispecific antibody of claim 35 , wherein Y is coupled to L2 by an oxime.
43 . The bispecific antibody of claim 35 , wherein X-L1′ and L2-Y′ are linked through a reaction selected from a Huisgen-cycloaddition, a Diels-Halder reaction, a hetero Diels-Alder reaction and an enzyme-mediated reaction.
44 . The bispecific antibody of claim 43 , wherein the enzyme-mediated reaction comprises a transglutaminase reaction.
45 . The bispecific antibody of claim 35 , wherein X-L1′ and Y-L2′ are linked through a copper-free [3+2] Huisgen-cycloaddition.
46 . The bispecific antibody of claim 35 , wherein X-L1′ and L2′-Y are linked through a disulfide or a maleimide.
47 . The bispecific antibody of claim 35 , wherein the distance between X and Y is less than about 50 angstrom (Å), about 45 angstrom (Å), about 40 angstrom (Å), about 35 angstrom (Å), about 30 angstrom (Å), about 25 angstrom (Å), about 20 angstrom (Å), about 15 angstrom (Å), about 10 angstrom (Å) or about 5 angstrom (Å).
48 . The bispecific antibody of claim 2 , wherein the one or more linkers comprises one or more ethylene glycol subunits.
49 . The bispecific antibody of claim 13 , wherein X and/or Y comprise one or more unnatural amino acids.
50 . The bispecific antibody of claim 49 , wherein one or more unnatural amino acids are site-specifically incorporated into X and/or Y.
51 . The bispecific antibody of claim 49 , wherein the one or more unnatural amino acids of X and/or the one or more unnatural amino acids of Y are genetically encoded.
52 . The bispecific antibody of claim 51 , wherein the one or more unnatural amino acids of X and/or the one or more unnatural amino acids of Y are genetically encoded by a codon selected from a nonsense codon, a stop codon, an umber codon, an ochre codon, an opal codon, a four-base codon and an amber codon.
53 . The bispecific antibody of claim 49 , wherein the one or more unnatural amino acids of X and/or the one or more unnatural amino acids of Y comprise a p-acetylphenylalanine (pAcF).
54 . The bispecific antibody of claim 49 , wherein the one or more unnatural amino acids of X and/or the one or more unnatural amino acids of Y comprise a selenocysteine.
55 . The bispecific antibody of claim 49 , wherein the site-specific connection occurs via the one or more unnatural amino acids of X and/or Y.
56 . A method of producing a bispecific antibody, the method comprising connecting a plurality of anti-CD3 antibodies or anti-CD3 antibody fragments to a plurality of second antibodies or antibody fragments to produce a plurality of anti-CD3 bispecific antibodies, wherein at least 60% of the anti-CD3 bispecific antibodies are identical.
57 . A method of producing a bispecific antibody of Formula II: X-L1′-L2′-Y or Formula HA: Y-L2′-L1′-X, comprising:
d. coupling L1″ to X to produce a first intermediate of Formula III: X-L1′ or Formula IIIA: L1′-X, wherein X comprises an anti-CD3 antibody or anti-CD3 antibody fragment; and L1″ comprises a first linker before being coupled to X,
e. coupling L2″ to Y to produce a second intermediate of Formula IV: Y-L2′ or Formula IVA: L2′-Y; wherein Y comprises at least a portion of a second antibody or antibody fragment and L2″ comprises a second linker before being coupled to Y; and
f. linking the first intermediate to the second intermediate, thereby producing the antibody of Formula II or IIA.
58 . The method of claim 57 , wherein coupling L1″ to X comprises site-specific coupling of L1″ to X and/or coupling L2″ to Y comprises site-specific coupling of L2″ to Y.
59 . The method of claim 57 , further comprising incorporating one or more unnatural amino acids into X and/or Y.
60 . The method of claim 59 , wherein coupling L1″ to X occurs at the one or more unnatural amino acids in X and/or coupling L2″ to Y occurs at the one or more unnatural amino acids in Y.
61 . The method of claim 59 , further comprising modifying a nucleic acid encoding X and/or Y to produce one or more amber codons in X and/or Y.
62 . The method of claim 57 , wherein linking the first intermediate to the second intermediate comprises a Huisgen-cycloaddition, a Diels-Halder reaction, a hetero Diels-Alder reaction or an enzyme-mediated reaction
63 . The method of claim 57 , wherein linking the first intermediate to the second intermediate comprises a copper-free [3+2] Huisgen-cycloaddition reaction.
64 . The method of claim 57 , wherein linking the first intermediate to the second intermediate produces an oxime, a tetrazole, a Diels Alder adduct, a hetero Diels Alder adduct, an aromatic substitution reaction product, a nucleophilic substitution reaction product, an ester, an amide, a carbamate, an ether, a thioether, a Michael reaction product, cycloaddition product, a metathesis reaction product, a metal-mediated cross-coupling reaction product, a radical polymerization product, an oxidative coupling product, an acyl-transfer reaction product, or a photo click reaction product.
65 . The method of claim 57 , wherein linking the first intermediate to the second intermediate produces a disulfide bridge or a maleimide bridge.
66 . The method of claim 57 , wherein the distance between X and Y is less than or equal to 50, 45, 40, 35, 30, 25, or 20 angstroms (Å).
67 . The method of claim 57 , wherein the distance between X and Y is greater than or equal to 5 angstroms (Å).
68 . A pharmaceutical composition comprising the bispecific antibody of any of claims 1 - 54 .
69 . The pharmaceutical composition of claim 68 , further comprising a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.
70 . A method for treating a disease or condition in a subject in need thereof, comprising administering the bispecific antibody of any of claims 1 - 55 or the pharmaceutical composition of any of claims 68 and 69 .
71 . The method of claim 70 , wherein the disease or condition is a cancer.
72 . The method of claim 71 , wherein the cancer is a breast cancer.
73 . The method of claim 71 , wherein the cancer is a glioma or glioblastoma.
74 . The method of claim 71 , wherein the cancer is a multiple myeloma.
75 . The method of claim 71 , wherein the cancer is an acute myeloid leukemia (AML).
76 . The method of claim 71 , wherein the cancer is selected from an acute lymphoblastic leukemia (ALL), a B-cell chronic lymphocytic leukemia (B-CLL) and a mantle cell lymphoma (MCL).
77 . The method of claim 70 , wherein the disease or condition is a pathogenic infection.
78 . The method of claim 70 , wherein the disease or condition is an inflammatory disease, an autoimmune disease or a metabolic disease.
79 . The method of claim 70 , wherein the bispecific antibody or pharmaceutical composition is administered by parenteral administration.
80 . The method of claim 79 , wherein parenteral administration comprises intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intravascular administration, intrathecal administration, intravitreal administration, or infusion.
81 . The method of claim 70 , wherein the bispecific antibody is administered by a microneedle device.
82 . The method of claim 70 , wherein the bispecific antibody is administered by topical, oral, or nasal administration.