COMPOSITIONS AND METHODS FOR GROWTH FACTOR MODULATION
Provided herein are proteins, antibodies, assays and methods useful for modulating growth factor levels and/or activities. In some embodiments, such growth factors are members of the TGF-β superfamily of proteins.
1 . A recombinant protein comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1-40, 70, 71, 88, 89, 98-109, 116-142, 162-170 and a combination or fragment thereof.
2 . The recombinant protein of claim 1 , wherein said recombinant protein is complexed with a protein comprising one or more sequences selected from the group consisting of SEQ ID NOs: 143-161, 286-294 and a combination or fragment thereof.
3 . The recombinant protein of claim 1 , wherein said recombinant protein is complexed with one or more proteins selected from the group consisting of latent TGF-β binding protein (LTBP)1, LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, glycoprotein A repetitions predominant (GARP,) leucine rich repeat containing (LRRC)33 and a combination or fragment thereof.
4 . The recombinant protein of claim 1 , wherein said recombinant protein comprises one or more detectable labels.
5 . The recombinant protein of claim 4 , wherein said one or more detectable labels comprise at least one biotin label, polyhistidine tag and/or flag tag.
6 . The recombinant protein of claim 1 , wherein said recombinant protein comprises one or more 3C protease cleavage sites and/or one or more secretion signal sequences.
7 . A chimeric protein comprising one or more protein modules from at least two transforming growth factor (TGF)-β-related proteins, wherein said one or more protein modules are selected from the group consisting of a growth factor prodomain complex (GPC), a latency associated peptide (LAP), a LAP-like domain, a straight jacket region, a growth factor domain, a fastener region, a furin cleavage site region, an arm region, a fingers region, an N-terminal region for extracellular association, a latency loop, an alpha 1 helical region, an RGD sequence region, a trigger loop region, a bowtie region, a residue for LTBP association, a residue for GARP association, a bone morphogenetic protein (BMP)/Tolloid cleavage site and a protein module comprising any of the amino acid sequences of SEQ ID NOs: 38-97 or 171-198.
8 . The chimeric protein of claim 7 , wherein said one or more protein modules are selected from one or more vertebrate species.
9 . The chimeric protein of claim 7 , wherein said chimeric protein comprises a GPC.
10 . The chimeric protein of claim 9 , wherein said GPC comprises:
i. at least one LAP or LAP-like domain from a TGF-β family member,
ii. at least one growth factor domain from a TGF-β family member, and
iii. wherein said at least one LAP or LAP-like domain and said at least one growth factor domain are from different TGF-β family members.
11 . The chimeric protein of claim 10 , wherein said at least one LAP or LAP-like domain and said at least one growth factor domain are derived from a TGF-β family member protein selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, growth differentiation factor (GDF)-8, GDF-11 and inhibin beta A.
12 . The chimeric protein of claim 9 , wherein said GPC comprises:
i. at least one N-terminal region from a TGF-β family member,
ii. at least one C-terminal region from a TGF-β family member, and
iii. wherein said at least one N-terminal region and said at least one C-terminal region are from different TGF-β family members.
13 . The chimeric protein of claim 12 , wherein said at least one N-terminal region and said at least one C-terminal region are selected from the group consisting of a TGF-β1 terminal region, a TGF-β2 terminal region, a TGF-β3 terminal region, a GDF-8 terminal region, a GDF-11 terminal region and an inhibin beta A terminal region.
14 . The chimeric protein of claim 9 , wherein said GPC comprises at least one TGF-β family member, said at least one TGF-β family member comprising at least one arm region from an alternative TGF-β family member.
15 . The chimeric protein of claim 9 , wherein said GPC comprises at least one TGF-β family member, said at least one TGF-β family member comprising at least one trigger loop region from an alternative TGF-β family member.
16 . The chimeric protein of claim 9 , comprising a protein module combination, wherein said protein module combination comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-236 and 273.
17 . The chimeric protein of claim 7 , wherein said chimeric protein is complexed with:
i. a protein selected from the group consisting of LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, GARP, LRRC33, perlecan, decorin, elastin and collagen, or
ii. a protein comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153-161, 286-294 and a combination or fragment thereof.
18 . The chimeric protein of claim 7 , wherein said chimeric protein comprises one or more detectable labels.
19 . An antibody capable of binding a recombinant protein according to any of claims 1-6 or a chimeric protein according to any of claims 7-18 .
20 . The antibody of claim 19 , wherein said antibody is a stabilizing antibody and/or inhibiting antibody.
21 . The antibody of claim 19 , wherein said antibody is a releasing antibody and/or activating antibody.
22 . The antibody of claim 19 , wherein said recombinant protein is complexed with:
i. a protein selected from the group consisting of LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, GARP, LRRC33, perlecan, decorin, elastin and collagen, or
ii. a protein comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153-161, 286-294 and a combination or fragment thereof.
23 . The antibody of claim 22 , wherein said antibody is a stabilizing antibody and/or inhibiting antibody.
24 . The antibody of claim 22 , wherein said antibody is a releasing antibody and/or activating antibody.
25 . The antibody of any of claims 19-24 , wherein said antibody is monoclonal antibody.
26 . The antibody of any of claims 19-24 , wherein said antibody is human or humanized.
27 . A method of modulating growth factor activity in a biological system comprising contacting said biological system with the antibody of any of claims 19-26 .
28 . A method of selecting a desired antibody comprising the use of one or more assays, said one or more assays comprising one or more recombinant proteins, said one or more recombinant proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, 70, 71, 74, 75, 88, 90, 98-142, 162-170, 199-236, 273 and a combination or a fragment thereof.
29 . The method of claim 28 comprising the steps of:
i. providing said one or more assays, wherein said one or more assays comprise an antibody binding assay,
ii. contacting said antibody binding assay with one or more candidate antibodies,
iii. obtaining binding data, wherein said binding data indicates the affinity of said one or more candidate antibodies for said one or more recombinant proteins, and
iv. selecting said desired antibody based on said binding data.
30 . The method of claim 29 , wherein said antibody binding assay comprises an enzyme-linked immunosorbent assay (ELISA) and/or a fluorescence-associated cell sorting (FACS)-based assay.
31 . The method of claim 29 , wherein said one or more recombinant proteins are complexed with:
i. a protein selected from the group consisting of LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, GARP, LRRC33, perlecan, decorin, elastin and collagen, or
ii. a protein comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153-161, 286-294 and a combination or fragment thereof.
32 . The method of claim 29 , wherein said one or more recombinant proteins comprise a chimeric protein, said chimeric protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-236 and 273.
33 . The method of claim 32 , wherein said chimeric protein is complexed with:
i. a protein selected from the group consisting of LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, GARP, LRRC33, perlecan, decorin, elastin and collagen, or
ii. a protein comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153-161, 286-294 and a combination or fragment thereof.
34 . The method of claim 28 comprising the steps of:
i. providing said one or more assays, wherein said one or more assays comprise a growth factor activity assay,
ii. contacting said growth factor activity assay with one or more candidate antibodies,
iii. obtaining growth factor activity data, and
iv. selecting said desired antibody based on said growth factor activity data.
35 . The method of claim 34 , wherein said growth factor activity assay comprises a cell-based assay selected from the group consisting of a luciferase-based assay and a proliferation assay.
36 . The method of claim 35 , wherein said cell-based assay comprises:
i. one or more expression cells, wherein said one or more expression cells expresses said one or more recombinant proteins, and
ii. one or more responsive cells, wherein said growth factor activity data comprises gene expression data and/or viability data obtained from said one or more responsive cells.
37 . A composition comprising the recombinant protein of any of claims 1-6 , the chimeric protein of any of claims 7-18 and/or the antibody of any of claims 19-24 and at least one excipient.
38 . The composition of claim 37 , wherein said at least one excipient comprises a pharmaceutically acceptable excipient.
39 . A method of treating a TGF-β-related indication in a subject comprising contacting said subject with a composition according to claim 38 .
40 . The method of claim 39 , wherein said TGF-β-related indication comprises a fibrotic indication selected from the group consisting of lung fibrosis, kidney fibrosis, liver fibrosis, cardiovascular fibrosis, skin fibrosis, and bone marrow fibrosis.
41 . The method of claim 39 , wherein said TGF-β-related indication comprises myelofibrosis.
42 . The method of claim 39 , wherein said TGF-β-related indication comprises one or more types of cancer or cancer-related conditions.
43 . The method of claim 42 , wherein said one or more types of cancer or cancer-related conditions are selected from the group consisting of colon cancer, renal cancer, breast cancer, malignant melanoma and glioblastoma.
44 . The method of claim 39 , wherein said TGF-β-related indication comprises one or more muscle disorders and/or injuries.
45 . The method of claim 44 , wherein said one or more muscle disorders and/or injuries are selected from the group consisting of cachexia, muscular dystrophy, chronic obstructive pulmonary disease (COPD), motor neuron disease, trauma, neurodegenerative disease, infection, rheumatoid arthritis, immobilization, disuse atrophy, sarcopenia, inclusion body myositis and diabetes.
46 . The method of claim 39 , wherein said TGF-β-related indication comprises one or more immune and/or autoimmune disorder.
A kit comprising the composition of claim 37 and instructions for use thereof.