CD86 ANTAGONIST MULTI-TARGET BINDING PROTEINS
This disclosure provides a multi-specific fusion protein composed of a CD86 antagonist binding domain and another binding domain that is an IL-10 agonist, an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist. The multi-specific fusion protein may also include an intervening domain that separates the other domains. This disclosure also provides polynucleotides encoding the multi-specific fusion proteins, compositions of the fusion proteins, and methods of using the multi-specific fusion proteins and compositions.
1 . A multi-specific fusion protein, comprising a CD86 binding domain linked to a heterologous binding domain by an intervening domain wherein the heterologous binding domain is an IL-10 agonist, an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist.
2 . The multi-specific fusion protein of claim 1 wherein the CD86 binding domain is a CTLA4 ectodomain or a sub-domain of a CTLA4 ectodomain.
3 . The multi-specific fusion protein of claim 1 wherein the CD86 binding domain is a Fab, scFv, a domain antibody, or a heavy chain-only antibody specific for CD86.
4 . The multi-specific fusion protein of claim 3 wherein the CD86 binding domain comprises light and heavy chain variable domains of FUN1 anti-CD86 antibody or a humanized variant thereof.
5 . The multi-specific fusion protein of claim 4 wherein the FUN1 binding domain comprises amino acids 1-258 of SEQ ID NO:187 or 237.
6 . The multi-specific fusion protein of claim 1 wherein the CD86 binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOS:1-6.
7 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises the amino acid sequence provided in any one of SEQ ID NOS:7, 14, 15, 18-22, 25, 26, 29, 32, 33, 39, and 40.
8 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises an IL-10 agonist comprising the amino acid sequence provided in SEQ ID NO:7 or a variant thereof comprising a point mutation at position 87.
9 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises an HLA-G agonist comprising the amino acid sequence provided in SEQ ID NO:14 or 15.
10 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises an HGF agonist comprising the amino acid sequence provided in any one of SEQ ID NO:18-22.
11 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises an IL-35 agonist comprising the amino acid sequence provided in SEQ ID NO:25 or 26.
12 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises a PD-1 agonist comprising the amino acid sequence provided in SEQ ID NO:32 or 33.
13 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises a BTLA agonist comprising the amino acid sequence provided in SEQ ID NO:29.
14 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises a LIGHT antagonist comprising the amino acid sequence provided in SEQ ID NO:29.
15 . The multi-specific fusion protein of claim 1 wherein the heterologous binding domain comprises a GITRL antagonist comprising the amino acid sequence provided in SEQ ID NO:39 or 40.
16 . The multi-specific fusion protein of claim 1 wherein the intervening domain comprises an immunoglobulin constant region or sub-region disposed between the CD86 binding domain and the heterologous binding domain.
17 . The multi-specific fusion protein of claim 16 wherein the immunoglobulin constant region or sub-region is IgG1 CH2CH3.
18 . The multi-specific fusion protein of claim 1 wherein the intervening domain comprises an immunoglobulin constant region disposed between a first and a second linker.
19 . The multi-specific fusion protein of claim 18 wherein the first and second linkers are independently selected from the linkers provided in SEQ ID NOs:43-166, 244, 307, 320, 355-379 and 383-398.
20 . The multi-specific fusion protein of claim 18 wherein the intervening domain comprises a human immunoglobulin Fc region, albumin, transferrin, or a scaffold domain that binds a serum protein.
21 . The multi-specific fusion protein of claim 1 wherein the intervening domain comprises a structure, from amino-terminus to carboxy-terminus, as follows:
-L1-X-L2-
wherein:
L1 and L2 are each independently a linker comprising from two to about 150 amino acids; and
X is an immunoglobulin constant region or sub-region, albumin, transferrin, or another serum protein binding protein.
22 . The multi-specific fusion protein of claim 21 wherein the immunoglobulin constant region or sub-region is an IgG1 CH2CH3.
23 . The multi-specific fusion protein of claim 21 wherein L1 is a human immunoglobulin hinge region, optionally mutated to replace one or more cysteines with other amino acids.
24 . The multi-specific fusion protein of claim 21 wherein X is a human IgG1 Fc domain or at least one CH domain thereof, optionally mutated to eliminate FcγRI-III interaction while retaining FcRn interaction.
25 . The multi-specific fusion protein of claim 1 wherein the intervening domain is a dimerization domain.
26 . The multi-specific fusion protein of claim 1 having the following structure:
N-BD1-X-L2-BD2-C
wherein:
BD1 is a CD86 binding domain that is at least about 90% identical to an ectodomain of CTLA4;
-X- is -L1-CH2CH3-, wherein L1 is the first IgG1 hinge, optionally mutated by substituting the first cysteine and wherein -CH2CH3- is the CH2CH3 region of an IgG1 Fc domain, optionally mutated to eliminate FcγRI-III interaction while retaining FcRn interaction;
L2 is a linker selected from SEQ ID NOs:43-166, 244, 307, 320, 355-379 and 383-398; and
BD2 is the heterologous binding domain.
27 . The multi-specific fusion protein of claim 1 wherein the fusion protein comprises the amino acid sequence provided in any one of SEQ ID NOs: 9, 13, 17, 24, 28, 31, 35, 38, 42, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 237, 239, 252, 254, 256, 258, 260, 262, 266, 276, 302, 330, 334, 336, 338, 340, 350, 352, and 354.
28 . A composition comprising one or more multi-specific fusion proteins according to claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.
29 . The composition of claim 28 wherein the multi-specific fusion protein exists as a dimer or a multimer in the composition.
30 . A polynucleotide encoding the multi-specific fusion protein according to claim 1 .
31 . The polynucleotide of claim 30 wherein the polynucleotide comprises the polynucleotide provided in any one of SEQ ID NOs: 8, 12, 16, 23, 27, 30, 34, 37, 41, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 236, 238, 251, 253, 255, 257, 259, 261, 265, 275, 301, 329, 333, 335, 337, 339, 349, 351 and 353.
32 . An expression vector comprising the polynucleotide according to claim 30 operably linked to an expression control sequence.
33 . A host cell comprising the expression vector according to claim 32 .
34 . A method for treating a subject with a disorder associated with CD86, IL-10, HLA-G, HGF, IL-35, PD-1, BTLA, LIGHT, GITRL or CD40, comprising administering a therapeutically effective amount of a multi-specific fusion protein of claim 1 .
35 . The method of claim 34 wherein the disorder is rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus or solid organ transplant.