Cytotoxic ribonuclease variants
This invention relates to cytotoxic variants of human ribonuclease 1 (RNase 1) identified through analysis of the interaction between RNase 1 and the human ribonuclease inhibitor (hRI) as defined by the three dimensional (3-D) atomic structure of the RNase 1 hRI complex. Also disclosed is the 3-D structure of the hRI.RNase 1 complex and methods for designing the RNase 1 variants.
1. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO:2 by at least two amino acid substitutions, which electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, a first amino acid change located within residues 85 to 94 of SEQ ID NO:2, and a second amino acid change being at least one substitution of at least one amino acid residue wherein the at least one amino acid residue is selected from the group consisting of an amino acid residue located at position 7, 11, 31, 32, 38, 39, 41, 42, 66, 67, 71, 111, and 118 of SEQ ID NO:2, wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2, and (iii) has a lower binding affinity for hRI than SEQ ID NO:2.
2. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 4, 38, 39, 67, 88, 89, 91, and 118, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, whereby the amino acid substitutions in SEQ ID NO: 2 causes evasion of hRI relative to the RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for RI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
3. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 2 , wherein the difference consists of the following substitutions: R4C/G38R/R39G/N67R/N88L/G89R/R91G/V118C.
4. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 39, 67, 88, 89, and 91, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
5. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 4 , wherein the difference consists of the following substitutions: R39D/N67D/N88A/G89D/R91D.
6. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 4 , wherein the difference consists of the following substitutions: R39L/N67L/N88A/G89L/R91L.
7. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 39, 67, 89, and 91, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI) than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO:2.
8. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 7 , wherein the difference consists of the following substitutions: R39D/N67A/G89D/R91D.
9. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 39, 67, 88, and 91, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
10. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 9 , wherein the difference consists of the following substitutions: R39D/N67D/N88A/R91D.
11. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 39, 67, 88, and 89, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
12. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 11 , wherein the difference consists of the following substitutions: R39D/N67D/N88A/G89D.
13. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 39, 88, 89, and 91, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
14. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 13 , wherein the difference consists of the following substitutions: R39D/N88A/G89D/R91D.
15. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 38, 39, 67, and 88, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
16. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 15 , wherein the difference consists of the following substitutions: G38R/R39G/N67R/N88R.
17. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO: 2 by amino acid substitutions at amino acid residues 67, 88, 89, and 91, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
18. An engineered human Ribonuclease (RNase 1) polypeptide as defined in claim 17 , wherein the difference consists of the following substitutions: N67D/N88A/G89D/R91D.
19. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO:2 by at least two amino acid substitutions, the first difference being an amino acid substitution at amino acid residue 91 of SEQ ID NO:2, and the second difference being a substitution located at an amino acid residue wherein said amino acid residue is selected from the group consisting of amino acid residues located at position 4, 7, 11, 31, 32, 38, 39, 41, 42, 66, 67, 71, 111, and 118 of SEQ ID NO:2, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 polypeptide has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
20. An engineered human ribonuclease (RNase 1) polypeptide differing in amino acid sequence from an RNase 1 protein comprising SEQ ID NO:2 by at least two amino acid substitutions, the first difference being an amino acid substitution at amino acid residue 88 of SEQ ID NO:2, and the second difference being a substitution located at an amino acid residue wherein said amino acid residue is selected from the group consisting of amino acid residues located at position 4, 7, 11, 31, 32, 38, 39, 41, 42, 66, 67, 71, 111, and 118 of RNase 1, wherein the amino acid substitutions electrostatically repel and/or sterically hinder binding by human Ribonuclease Inhibitor (hRI) to the engineered RNase 1, and wherein the engineered RNase 1 polypeptide has the following biological properties: (i) retains ribonucleolytic activity, (ii) has a lower binding affinity for hRI than that of SEQ ID NO:2, and (iii) exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
21. A method for inhibiting the proliferation of cancer cells, comprising delivering to the cells an effective amount of an engineered human Ribonuclease (RNase 1) polypeptide of claim 1 , wherein the engineered RNase 1 retains ribonucleolytic activity, has a lower binding affinity for hRI than that of RNase 1 (SEQ ID NO:2), and exhibits enhanced cytotoxic activity relative to RNase 1.
22. An isolated protein complex having the three-dimensional atomic crystal structure of a human Ribonuclease Inhibitor (hRI) bound to a human Ribonuclease 1 (RNase 1) in a complex, the hRI-RNase 1 complex being structurally defined by atomic coordinate data deposited in the Protein Data Bank (PDB) as PDB Accession No. 1Z7X and as set forth in Appendix 1, wherein RNase 1 has an amino acid sequence of SEQ ID NO:2 and hRI has an amino acid sequence of SEQ ID NO:4.
23. An engineered human Ribonuclease (RNase 1) polypeptide differing in amino acid sequence from a human RNase 1 protein comprising SEQ ID NO: 2, wherein the difference is selected from the group consisting of the following substitutions:
R4C/G38R/R39G/N67R/N88L/G89R/R91G/V118C;
R39L/N67L/N88A/G89L/R91L;
R39D/N67D/N88A/G89D/R91D;
R39D/N67A/G89D/R91D;
R39D/N67D/N88A/R91D;
R39D/N67D/N88A/G89D;
R39D/N88A/G89D/R91D;
G38R/R39G/N67R/N88R; and
N67D/N88A/G89D/R91D,
wherein said engineered RNase 1 polypeptides have the following biological properties: retain ribonucleolytic activity, exhibit enhanced cytotoxic activity relative to SEQ ID NO: 2, and have a lower binding affinity for human Ribonuclease Inhibitor (hRI) than SEQ ID NO:2.
24. An engineered human Ribonuclease (RNase 1) polypeptide wherein said engineered RNase 1 differs in amino acid sequence from the human RNase 1 protein comprising SEQ ID NO: 2 at the following amino acid residues: R39, N67, N88, G89, and R91, wherein said engineered RNase 1 has the following biological properties: retains ribonucleolytic activity, exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2, and has a lower binding affinity for human Ribonuclease Inhibitor (hRI) than SEQ ID NO:2.