Deimmunized lysostaphin and methods of use

1. A de-immunized lysostaphin or fragment thereof, comprising a mutation at one or more of Thr127, Ser126, Ser124, Ser122, Asn121, Val120, Met119, Argil8, Ile99, Lys95, Tyr93, Ser84, Leu83, Val75, Asn72, Ile70, Lys46, Ile41, Asn40, Phe38, Tyr33, Asn13, or Asn12 of SEQ ID NO: 49, wherein the fragment comprises a catalytically active domain of a lysostaphin,

wherein the de-immunized lysostaphin or fragment thereof is at least 90% identical to the catalytically active domain of a lysostaphin of SEQ ID NO: 49.

2. The deimmunized lysostaphin of claim 1 , wherein said lysostaphin is aglycosylated.

3. The deimmunized lysostaphin of claim 1 , wherein the mutation comprises Thr127Ala; Ser126Pro; Ser124Gly; Ser122Asp, Ser122Gly, or Ser122Thr; Asn121Gly; Val120Asp; Met119Arg; Arg118Thr; Ile99Gln; Lys95Glu; Tyr93His; Ser84Tyr or Ser84Gly; Leu83Met; Val75Glu or Val75Gln; Asn72His; Ile70Lys; Lys46His; Ile41Glu; Asn40Thr; Phe38Ser; Tyr33Thr; Asn13His; Asn12Gly; or a combination thereof.

4. The deimmunized lysostaphin of claim 1 , further comprising one or more amino acid substitutions in the C-terminal cell wall binding domain.

5. A pharmaceutical composition comprising the deimmunized lysostaphin of claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5 , further comprising an antibiotic.

7. The pharmaceutical composition of claim 6 , wherein said antibiotic comprises a β-lactam, cephalosporin, aminoglycoside, sulfonamide, antifolate, macrolide, quinolone, glycopeptide, polypeptide or a combination thereof.

8. A method for preventing or treating a microbial infection comprising administering to a subject in need of treatment the pharmaceutical composition of claim 5 , thereby preventing or treating the subject's microbial infection.

9. The method of claim 8 , wherein said infection is a bacterial infection.

10. The method of claim 9 , wherein said bacterial infection is caused by bacteria from the genus Staphylococcus.

11. The aglycosylated lysostaphin of claim 2 , comprising a mutation at Ser126, Thr127, and/or Ser234.

12. The aglycosylated lysostaphin of claim 2 , comprising the mutation Ser126Pro, Thr127Ala, and/or Ser234Lys.

13. The deimmunized lysostaphin of claim 4 , comprising a mutation at one or more of Asn236, Ser234, Asn232, Asn219, Ile200, Val193, Ser191, Arg186, Ala169, Ser168, Ser166, or Tyr160.

14. The deimmunized lysostaphin of claim 4 , wherein the mutation comprises Asn236Asp, Ser234Lys, Asn232Gln, Asn219Tyr, Ile200Thr, Val193Trp, Ser191Ala Arg186Thr, Ala169Gly, Ser168Lys Ser166Asn or Ser166Thr, Tyr160His, or a combination thereof.

15. A deimmunized lysostaphin or fragment thereof, comprising the sequence represented by SEQ ID NO: 218-220.

16. A method for preventing or treating a biofilm comprising administering to a subject in need of treatment the pharmaceutical composition of claim 5 .

17. A vector encoding the deimmunized lysostaphin of claim 1 .

18. A pharmaceutical composition comprising the deimmunized lysostaphin of claim 17 and a pharmaceutically acceptable carrier.

19. The method of claim 8 , wherein the pharmaceutical composition is administered by a route selected from the group consisting of topical, ophthalmic, intranasal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathecal, and inhalation delivery.

20. The method of claim 8 , wherein the pharmaceutical composition is provided on a medical device.

21. The pharmaceutical composition of claim 6 , wherein said antibiotic comprises an inhibitor of cell wall synthesis or protein synthesis.

22. A deimmunized lysostaphin or fragment thereof, comprising a mutation at one or more of Asn236, Asn232, Arg186, Ala169, Ser166, Tyr160, Ser126, Ser124, Ser122, Asn121, Ile99, Lys95, Leu83, Ile41, or Asn12 of SEQ ID NO:49, wherein the fragment comprises a catalytically active domain of a lysostaphin or a cell wall binding domain of a lysostaphin.

23. A de-immunized lysostaphin or fragment thereof where the lysostaphin or fragment thereof comprises a cell wall binding domain of lysostaphin where said cell wall binding domain comprises a mutation at one or more of Asn236, Ser234, Asn232, Asn219, Ile200, Val193, Ser191, Arg186, AIa169, Ser168, Ser166, orTyr160.

24. The deimmunized lysostaphin fragment of claim 22 , wherein the lysostaphin fragment comprises a catalytically active domain of a lysostaphin.

25. The deimmunized lysostaphin fragment of claim 22 , wherein the lysostaphin fragment comprises a cell wall binding domain of a lysostaphin.

26. A deimmunized lysostaphin or fragment thereof, comprising a mutation at

a) Asn232, Arg186, Ala169, Tyr160, Ser126, Ser124, Ser122, Asn121, Ile99, Lys95, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

b) Asn236, Asn232, Arg186, Ala169, Ser166, Tyr160, Ser126, Ser124, Ser122, Asn121, Ile99, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

c) Asn236, Asn232, Arg186, Ala169, Ser166, Tyr160, Ser126, Ser124, Ser122, Asn121, Ile99, Lys95, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

d) Ser234, Asn219, Ile200, Ser122, Asn121, Ser84, Val75, Asn72, Phe38, and Tyr33 of SEQ ID NO:49;

e) Ser122, Val120, Val75, and Ile70 of SEQ ID NO:49;

f) Asn219, Ile200, Ala169, and Ser166 of SEQ ID NO:49;

g) Asn219, Ile200, Ala169, Ser166, Ser122, Val120, Val75, and Ile70 of SEQ ID NO:49;

h) Asn236, Asn219, Ile200, Ala169, Ser166, Ser122, Val120, Ser84, Val75, and Ile70 of SEQ ID NO:49;

i) Asn236, Asn219, Ile200, Ser191, Arg186, Ala169, Ser166, Ser122, Val120, Ser84, Val75, Ile70, and Asn40 of SEQ ID NO:49;

j) Ser122, Ile99, and Ile41 of SEQ ID NO:49;

k) Ser124, Ser122, Lys46, and Ile41 of SEQ ID NO:49;

l) Ser122, Ile99, Lys95, and Ile41 of SEQ ID NO:49;

m) Ser122, Ile99, Lys95, Tyr93, and Ile41 of SEQ ID NO:49;

n) Ser122, Asn121, Ile99, and Lys46 of SEQ ID NO:49;

o) Ser122, Asn121, Ile99, Ile41, and Asn12 of SEQ ID NO:49;

p) Ser124, Ser122, Ile99, Lys95, Ile41, and Asn12 of SEQ ID NO:49;

q) Ser122, Asn121, Ile99, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

r) Ser124, Ser122, Asn121, Ile99, Lys95, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

s) Ser122, Asn121, Ile41, and Asn12 of SEQ ID NO:49;

t) Ser124, Ser122, Asn121, Ile99, Lys95, Tyr93, Ile41, and Asn12 of SEQ ID NO:49;

u) Ser122, Asn121, Ile99, Lys95, Ile41, and Asn13 of SEQ ID NO:49;

v) Ser122, Asn121, Ile99, Lys95, Tyr93, Ile41, and Asn13 of SEQ ID NO:49;

w) Ser122, and Ile41 of SEQ ID NO:49;

x) Ser122, and Lys46 of SEQ ID NO:49;

y) Ser122, Lys95, Tyr93, and Lys46 of SEQ ID NO:49;

z) Ser122, Lys95, Tyr93, Leu83, Lys46, Ile41, and Asn12 of SEQ ID NO:49;

aa) Ser124, Ser122, Ile99, Lys95, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

bb) Ser122, Lys95, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

cc) Ser122, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

dd) Ser122, Lys95, Tyr93, Leu83, Ile41, and Asn12 of SEQ ID NO:49;

ee) Ser122, Ile41, and Asn12 of SEQ ID NO:49;

ff) Ser122, Lys95, Tyr93, Ile41, and Asn12 of SEQ ID NO:49;

gg) Ser122, Leu83, Lys46, and Asn13 of SEQ ID NO:49;

hh) Ser122, Lys95, Tyr93, Leu83, Lys46, and Asn13 of SEQ ID NO:49;

ii) Ser122, Lys46, and Asn13 of SEQ ID NO:49; or

jj) Ser122, Lys95, Tyr93, Lys46, and Asn13 of SEQ ID NO:49;

wherein the fragment comprises a catalytically active domain of a lysostaphin or a cell wall binding domain of a lysostaphin.

27. The deimmunized lysostaphin of claim 26 , wherein the mutation comprises

a) Asn232Gln, Arg186Thr, Ala169Gly, Tyr160His, Ser126Pro, Ser124Gly, Ser122Asp, Asn121Gly, Ile99Gln, Lys95Glu, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

b) Asn236Asp, Asn232Glu, Arg186Thr, Ala169Gly, Ser166Asn, Tyr160His, Ser126Pro, Ser124Gly, Ser122Asp, Asn121Gly, Ile99Gln, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

c) Asn236Asp, Asn232Glu, Arg186Thr, Ala169Gly, Ser166Asn, Tyr160His, Ser126Pro, Ser124Gly, Ser122Asp, Asn121Gly, Ile99Gln, Lys95Glu, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

d) Ser234Lys, Asn219Tyr, Ile200Thr, Ser122Gly, Asn121Gly, Ser84Tyr, Val75Gln, Asn72His, Phe38Ser, and Tyr33Thr of SEQ ID NO:49;

e) Ser122Thr, Val120Asp, Val75Glu, and Ile70Lys of SEQ ID NO:49;

f) Asn219Tyr, Ile200Thr, Ala169Gly, and Ser166Thr of SEQ ID NO:49;

g) Asn219Tyr, Ile200Thr, Ala169Gly, Ser166Thr, Ser122Thr, Val120Asp, Val75Glu, and Ile70Lys of SEQ ID NO:49;

h) Asn236Asp, Asn219Tyr, Ile200Thr, Ala169Gly, Ser166Thr, Ser122Thr, Val120Asp, Ser84Gly, Val75Glu, and Ile70Lys of SEQ ID NO:49;

i) Asn236Asp, Asn219Tyr, Ile200Thr, Ser191Ala, Arg186Thr, Ala169Gly, Ser166Thr, Ser122Thr, Val120Asp, Ser84Gly, Val75Glu, Ile70Lys, and Asn40Tyr of SEQ ID NO:49;

j) Ser122Asp, Ile99Gln, and Ile41Glu of SEQ ID NO:49;

k) Ser124Gly, Ser122Asp, Lys46His, and Ile41Glu of SEQ ID NO:49;

l) Ser122Asp, Ile99Gln, Lys95Glu, and Ile41Glu of SEQ ID NO:49;

m) Ser122Asp, Ile99Gln, Lys95Glu, Tyr93His, and Ile41Glu of SEQ ID NO:49;

n) Ser122Asp, Asn121Gly, Ile99Gln, and Lys46His of SEQ ID NO:49;

o) Ser122Asp, Asn121Gly, Ile99Gln, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

p) Ser124Gly, Ser122Asp, Ile99Gln, Lys95Glu, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

q) Ser122Asp, Asn121Gly, Ile99Gln, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

r) Ser124Gly, Ser122Asp, Asn121Gly, Ile99Gln, Lys95Glu, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

s) Ser122Asp, Asn121Gly, Ile41Glu, and Asn 9 12Gly of SEQ ID NO:49;

t) Ser124Gly, Ser122Asp, Asn121Gly, Ile99Gln, Lys95Glu, Tyr93His, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

u) Ser122Asp, Asn121Gly, Ile99Gln, Lys95Glu, Ile41Glu, and Asn13His of SEQ ID NO:49;

v) Ser122Asp, Asn121Gly, Ile99Gln, Lys95Glu, Tyr93His, Ile41Glu, and Asn13His of SEQ ID NO:49;

w) Ser122Asp, and Ile41Glu of SEQ ID NO:49;

x) Ser122Asp, and Lys46His of SEQ ID NO:49;

y) Ser122Asp, Lys95Glu, Tyr93His, and Lys46His of SEQ ID NO:49;

z) Ser122Asp, Lys95Glu, Tyr93His, Leu83Met, Lys46His, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

aa) Ser124Gly, Ser122Asp, Ile99Gln, Lys95Glu, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

bb) Ser122Asp, Lys95Glu, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

cc) Ser122Asp, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

dd) Ser122Asp, Lys95Glu, Tyr93His, Leu83Met, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

ee) Ser122Asp, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

ff) Ser122Asp, Lys95Glu, Tyr93His, Ile41Glu, and Asn12Gly of SEQ ID NO:49;

gg) Ser122Asp, Leu83Met, Lys46His, and Asn13His of SEQ ID NO:49;

hh) Ser122Asp, Lys95Glu, Tyr93His, Leu83Met, Lys46His, and Asn13His of SEQ ID NO:49;

ii) Ser122Asp, Lys46His, and Asn13His of SEQ ID NO:49; or jj) Ser122Asp, Lys95Glu, Tyr93His, Lys46His, and Asn13His of SEQ ID NO:49;

wherein the fragment comprises a catalytically active domain of a lysostaphin or a cell wall binding domain of a lysostaphin.

28. The deimmunized lysostaphin of claim 26 , wherein said lysostaphin is aglycosylated.

29. The aglycosylated lysostaphin of claim 28 , comprising a mutation at Thr127 and/or Ser234.

30. The aglycosylated lysostaphin of claim 28 , comprising the mutation Thr127Ala and/or Ser234Lys.

31. The deimmunized lysostaphin of claim 26 , further comprising a mutation at Ser126 in any one of d)-jj).

32. The deimmunized lysostaphin of claim 31 , comprising the mutation Ser126Pro.

33. A deimmunized lysostaphin or fragment thereof, comprising a mutation at Ser122 of SEQ ID NO:49 and further comprising one or more additional mutations of SEQ ID NO:49.

34. The de-immunized lysostaphin or fragment thereof of claim 23 , where the mutation comprises Asn236Asp, Ser234Lys, Asn232Gln, Asn219Tyr, Ile200Thr, Val193Trp, Ser191Ala, Arg186Thr, Ala169Gly, Ser168Lys, Ser166Asn, orTyr160His or a combination thereof.