Antagonists of MCP proteins
View Patent ↗Novel antagonists of MCP proteins, in particular of MCP-1 protein, can be obtained by generating MCP mutants whose GAG binding site, located at the N-terminal of MCP proteins, is eliminated following non-conservative substitutions. Compounds prepared in accordance with the present invention can be used in the treatment or prevention of diseases related to an undesirable activity of MCP proteins such, such as inflammatory disease, autoimmune diseases, vascular diseases, and cancer.
1. An isolated MCP protein comprising:
a) amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24-99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins; or
b) amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24-99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
2. The isolated MCP protein according to claim 1 , wherein said MCP protein comprises amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24-99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
3. The isolated MCP protein according to claim 2 , wherein said one or more amino acid positions are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine.
4. The isolated MCP protein according to claim 1 , wherein said MCP protein comprises amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24-99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine.
5. The isolated MCP protein according to claim 1 , in which one amino acid residue has been added, deleted, or substituted without interfering with the antagonistic activity of said MCP protein with respect to unaltered MCP proteins.
6. The isolated MCP protein according to claim 1 , further comprising a heterologous amino acid sequence.
7. The isolated MCP protein according to claim 1 , wherein said MCP protein is human MCP-1, human MCP-2, human MCP-3, human MCP-4, or human Eotaxin.
8. The isolated MCP protein according to claim 1 , comprising the sequence of SEQ ID NO: 3.
9. The isolated MCP protein according to claim 1 , further comprising a molecule chosen from radioactive labels, biotin, fluorescent labels, cytotoxic agents, or drug delivery proteins.
10. The isolated MCP protein according to claim 6 , wherein the heterologous amino acid sequence is selected from: extracellular domains of membrane-bound protein, immunoglobulin constant regions, multimerization domains, extracellular proteins, signal peptide-containing proteins or export signal-containing proteins.
11. The isolated MCP protein according to claim 1 , wherein said activity is the recruitment of leukocytes.
12. The isolated MCP protein according to claim 2 , wherein said activity is the recruitment of leukocytes.
13. The isolated MCP protein according to claim 4 , wherein said activity is the recruitment of leukocytes.
14. An isolated nucleic acid encoding a MCP protein comprising:
a) amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins; or
b) amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine. serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
15. An expression vector comprising a nucleic acid encoding a MCP protein comprising:
a) amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins; or
b) amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
16. An isolated host cell transformed with an expression vector comprising a nucleic acid encoding a MCP protein comprising:
a) amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP- 1 , said human mature MCP- 1 corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins; or
b) amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
17. A process of preparing a MCP antagonist comprising culturing a host cell transformed with an expression vector comprising a nucleic acid encoding a MCP protein comprising:
a) amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins; or
b) amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
18. A composition comprising a carrier and a MCP protein comprising:
a) amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins; or
b) amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
19. The composition according to claim 18 , wherein said MCP protein comprises amino acid substitutions at positions 18 and 19 and amino acid substitutions at one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of human mature MCP-1, said human mature MCP-1 corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine and wherein said MCP protein antagonizes an activity of unaltered MCP proteins.
20. The composition according to claim 19 , wherein said MCP protein comprises amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine.
21. The composition according to claim 19 , wherein said MCP protein comprises amino acid substitutions at positions 18 and 19, as numbered on the sequence of human mature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substituted with alanine, glycine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid or glutamine.
22. The composition according to claim 18 , wherein said MCP protein further comprises one amino acid residue that has been added, deleted, or substituted without interfering with the antagonistic activity of said MCP protein.
23. The composition according to claim 18 , wherein said MCP protein further comprises a heterologous amino acid sequence.
24. The composition according to claim 18 , wherein said MCP protein is human MCP-1, human MCP-2, human MCP-3, human MCP-4, or human Eotaxin.
25. The composition according to claim 18 , wherein said MCP protein comprises SEQ ID NO: 3.
26. The composition according to claim 18 , wherein said MCP protein further comprises a molecule chosen from radioactive labels, biotin, fluorescent labels, cytotoxic agents, or drug delivery proteins.
27. The composition according to claim 23 , wherein the heterologous amino acid sequence is selected from: extracellular domains of membrane-bound protein, immunoglobulin constant regions, multimerization domains, extracellular proteins, signal peptide-containing proteins or export signal-containing proteins.
28. The composition according to claim 18 , wherein said MCP protein has amino acids at positions 18 and 19 substituted with alanine.