Peptides promoting cell adherence, growth and secretion

• US 20030162289A1
• Filed: 11/19/2001
• Published: 08/28/2003
• Est. Priority Date: 11/19/2001
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Campbell, Robert L., Heidaran, Mohammad, Wilkins, Jamie H., Spargo, Catherine A., Haaland, Perry
• Granted Patent: US 7,041,506 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 5/06086 with the first amino acid being basic
• CPC Class Codes: C07K 5/0815 with the first amino acid being basic
• CPC Class Codes: C07K 5/0817 the first amino acid being Arg
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids

Abstract

The present invention provides specific peptides identified as having cell adhesion, growth, expression or secretion-enhancing activities. Many of the peptides of the invention may be produced in large quantity by such means as chemical synthesis or recombinant DNA methodology. They may be non-specifically adsorbed, or chemically attached to a surface or, alternatively, formulated in a culture medium to produce the desired effect on cultured cells.

Claims

1. A peptide which enhances cell growth and/or secretion in a cell culture system, wherein said peptide comprises a structure of at least one of (a) xxxkx, (b) xxkxx, (c) xxxxk, (d) xkxxx, and (e) kxxxx, wherein k represents lysine and each x may be the same or different amino acid independently selected from the group consisting of lysine, alanine, isoleucine, phenylalanine, proline, valine, glycine, glutamine, leucine, methionine, asparagine, serine, threonine, tyrosine, aspartic acid, glutamic acid, histidine and derivatives thereof.

2. The peptide of claim 1, wherein the peptide promotes adherence of anchorage-dependent cells on a surface.

3. The peptide of claim 1, wherein the peptide enhances cell growth and is selected from the group consisting of IFFKG (SEQ ID NO:

11), FIKFG(SEQ ID NO;

2), FIFAK(SEQ ID NO;

3), QVVAK(SEQ ID NO;

4), FKFIG(SEQ ID NO;

5), AFFKI(SEQ ID NO;

6), VFPFK(SEQ ID NO;

7), AKIFF(SEQ ID NO;

8), AFKIF(SEQ ID NO;

9), KFAFI (SEQ ID NO;

10) and FAKFI(SEQ ID NO;

11) and combinations thereof.

4. The peptide of claim 1, wherein the peptide enhances cell secretion and is selected from the group consisting of FKLVY(SEQ ID NO:

16), KKKKK(SEQ ID NO;

17), KKKKL(SEQ ID NO;

18), FKKKQ(SEQ ID NO;

19), FKFIG(SEQ ID NO;

5), KKKSK(SEQ ID NO;

20), KKKLK(SEQ ID NO;

21), FKKKK(SEQ ID NO;

22), LKKKK(SEQ ID NO;

23), KKLKK (SEQ ID NO;

24), KKKKT(SEQ ID NO;

25), KKPKK(SEQ ID NO;

26), KKPQY(SEQ ID NO;

27), SKKKK(SEQ ID NO;

28), KVKKK(SEQ ID NO;

29), KNQTY(SEQ ID NO;

30), FKKKV(SEQ ID NO;

31), KPKKK(SEQ ID NO;

32), FFKKK(SEQ ID NO;

33), HKNQT(SEQ ID NO;

34), FKLVG(SEQ ID NO;

35), KKQPK(SEQ ID NO;

36), EKKQT(SEQ ID NO;

37), EKKKK(SEQ ID NO;

38), KKIKQ(SEQ ID NO;

39), KKKKS(SEQ ID NO;

40), KKQKK(SEQ ID NO;

41), KKLNY(SEQ ID NO;

42), DGKKT(SEQ ID NO;

43), KKPTT(SEQ ID NO;

44), KFIFG(SEQ ID NO;

45), FKKMY(SEQ ID NO;

46), FFFKK(SEQ ID NO;

47), KQKKI(SEQ ID NO;

48), HIKKK(SEQ ID NO;

49), DFFHK(SEQ ID NO;

50), AKKKK(SEQ ID NO;

51), AHIKK(SEQ ID NO;

52), AHKKK(SEQ ID NO;

53), LKLVY(SEQ ID NO;

54), PKQKK(SEQ ID NO;

55), AKKKT(SEQ ID NO;

56).

5. The peptide of claim 1, wherein said peptide is introduced into a cell culture system at a concentration of about 500 μ

M to about 6 mM.

6. The peptide of claim 1, wherein said peptide is introduced into a cell culture system at a concentration of about 250 μ

M to about 24 mM.

7. The peptide of claim 1, wherein said peptide is introduced into a cell culture system at a concentration ranging from 3 mM to about 12 mM.

8. The peptide of claim 1, wherein said peptide is present on a surface in the form of a dried film.

9. The peptide of claim 8, wherein said surface is two dimensional or three dimensional.

10. The peptide of claim 1 wherein the cell culture system comprises cells selected from the group consisting of epithelial, endothelial, dermal, neural, tumor, lymphocytic, stem cells, and combinations thereof.

11. The peptide of claim 1 wherein said peptide increases oxygen consumption of said cells.

12. The peptide of claim 1 wherein said peptide enhances growth of said cells in vitro or in vivo.

13. The peptide of claim 1 attached or non-specifically adsorbed to a surface.

14. The peptide of claim 13 wherein said surface is coated with at least one of bovine serum albumin, ovalbumin, keyhole limpet haemocyanin, collagen, fibronectin, laminin, polylysine, a peptide having a cell-surface receptor recognition sequence, an immunoglobulin, a polysaccharide, or a growth factor.

15. The peptide of claim 13 wherein said surface is selected from the group consisting of plastic dishes, plastic flasks, plastic microtitre plates, plastic tubes, sutures, membranes, films, bioreactors, hollow fibers, sacs and microparticles.

16. The peptide of claim 1 covalently linked to a member of the group consisting of extracellular matrix protein, bovine serum albumin, ovalbumin, keyhole limpet haemocyanin, collagen, fibronectin, laminin, an immunoglobulin, a polysaccharide, a growth factor and combinations thereof.

17. The covalently linked peptide of claim 16 wherein said member is adsorbed to a surface.

18. The covalently linked peptide of claim 16 wherein said growth factor comprises at least one of bFGF, GCSF, an ILGF-1, or VEGF.

19. The peptide of claim 1, wherein the peptide is a media constituent.

44. A cell or tissue culture medium including the peptide of claim 1.

48. A method for enhancing cellular growth and/or secretion comprising the step of culturing cells or tissues in the presence of the peptide of claim 1.

20. A peptide which enhances cell growth and/or secretion, the peptide comprising a structure of at least one of (a) xxxkx, (b) xxkxx, (c) xxxxk, (d) xkxxx, and (e) kxxxx, wherein each x may be the same or different hydrophobic or uncharged polar amino acid residue and k represents lysine.

21. The peptide of claim 20, wherein said hydrophobic or uncharged polar residue is selected from the group consisting of Phe, Ile, Ala, Val, Pro, Gly, Gln, Tyr, Thr Leu and derivatives thereof.

22. The peptide of claim 20, wherein the peptide enhances cellular adherence and growth of adherent-type cells and is selected from the group consisting of IFFKG (SEQ ID NO:

1), FIKFG(SEQ ID NO;

2), FIFAK(SEQ ID NO;

3), QVVAK(SEQ ID NO;

4), FKFIG(SEQ ID NO;

5), AFFKI(SEQ ID NO;

6), VFPFK(SEQ ID NO;

7), AKIFF(SEQ ID NO;

8), AFKIF(SEQ ID NO;

9), KFAFI (SEQ ID NO;

10), FAKFI(SEQ ID NO;

11), and combinations thereof.

23. The peptide of claim 20, wherein the peptide enhances cell secretion and is selected from the group consisting of FKFIG(SEQ ID NO:

5), FKLVY(SEQ ID NO;

16), KNQTY(SEQ ID NO;

30), FKLVG(SEQ ID NO;

35), KFIFG(SEQ ID NO;

45), LKLVY(SEQ ID NO;

54), and combinations thereof.

24. A peptide which promotes cellular adherence and/or growth, wherein said peptide is selected from the group consisting of IFFKG (SEQ ID NO:

1), FIKFG(SEQ ID NO;

2), FIFAK(SEQ ID NO;

3), QVVAK(SEQ ID NO;

4), FKFIG(SEQ ID NO;

5), AFFKI(SEQ ID NO;

6), VFPFK(SEQ ID NO;

7), AKIFF(SEQ ID NO;

8), AFKIF(SEQ ID NO;

9), KFAFI (SEQ ID NO;

10), FAKFI(SEQ ID NO;

11), and combinations thereof.

25. The peptide of claim 24 attached or non-specifically adsorbed to a surface.

26. The peptide of claim 24, wherein the peptide is a media constituent.

45. A cell or tissue culture medium including the peptide of claim 24.

27. A peptide composition which promotes cellular adherence and/or growth of cells comprising one or more pairs of peptides selected from the group consisting of (a) AFFFQ(SEQ ID NO:

12)/EEEMY(SEQ ID NO;

13) and (b) FIKLM (SEQ ID NO;

14)/FFIPY (SEQ ID NO;

15).

28. The peptide composition of claim 27 attached or non-specifically adsorbed to a surface.

29. The peptide composition of claim 27, wherein said composition is a media constituent.

46. A cell or tissue culture medium including the peptide composition of claim 27.

30. A peptide which enhances cell secretion selected from the group consisting of FKLVY(SEQ ID NO:

16), KKKKK(SEQ ID NO;

17), KKKKL(SEQ ID NO;

18), FKKKQ(SEQ ID NO;

19), FKFIG(SEQ ID NO;

5), KKKSK(SEQ ID NO;

20), KKKLK(SEQ ID NO;

21), FKKKK(SEQ ID NO;

22), LKKKK(SEQ ID NO;

23), KKLKK (SEQ ID NO;

24), KKKKT(SEQ ID NO;

25), KKPKK(SEQ ID NO;

26), KKPQY(SEQ ID NO;

27), SKKKK(SEQ ID NO;

28), KVKKK(SEQ ID NO;

29), KNQTY(SEQ ID NO;

30), FKKKV(SEQ ID NO;

31), KPKKK(SEQ ID NO;

32), FFKKK(SEQ ID NO;

33), HKNQT(SEQ ID NO;

34), FKLVG(SEQ ID NO;

35), KKQPK(SEQ ID NO;

36), EKKQT(SEQ ID NO;

37), EKKKK(SEQ ID NO;

38), KKIKQ(SEQ ID NO;

39), KKKKS(SEQ ID NO;

40), KKQKK(SEQ ID NO;

41), KKLNY(SEQ ID NO;

42), DGKKT(SEQ ID NO;

43), KKPTT(SEQ ID NO;

44), KFIFG(SEQ ID NO;

45), FKKMY(SEQ ID NO;

46), FFFKK(SEQ ID NO;

47), KQKKI(SEQ ID NO;

48), HIKKK(SEQ ID NO;

49), DFFHK(SEQ ID NO;

50), AKKKK(SEQ ID NO;

51), AHIKK(SEQ ID NO;

52), AHKKK(SEQ ID NO;

53), LKLVY(SEQ ID NO;

54), PKQKK(SEQ ID NO;

55), AKKKT(SEQ ID NO;

56), DEETY(SEQ ID NO;

57), HNPPY(SEQ ID NO;

58), GGHMS(SEQ ID NO;

59), AADEG(SEQ ID NO;

60), GGGGS(SEQ ID NO;

61), EEGLS(SEQ ID NO;

62), HHPST(SEQ ID NO;

63), FHHNT(SEQ ID NO;

64), ADELN(SEQ ID NO;

65), KKKK(SEQ ID NO;

66), KKK(SEQ ID NO;

67), KK(SEQ ID NO;

68), OrnOrnOrn(SEQ ID NO;

69), RRR(SEQ ID NO;

70), and combinations thereof.

31. The peptide of claim 30 attached or non-specifically adsorbed to a surface.

32. The peptide of claim 30, wherein said peptide is a media constituent.

47. A cell or tissue culture medium including the peptide of claim 30.

33. A method of modifying a surface in a cell culture system so as to enhance cell growth and/or secretion in said system, comprising the step of applying to said surface a peptide comprising a structure selected from the group consisting of (a) xxxkx, (b) xxkxx, (c) xxxxk, (d) xkxxx, (e) kxxxx and combinations thereof, wherein k represents lysine and each x may be the same or different amino acid independently selected from the group consisting of lysine, alanine, isoleucine, phenylalanine, proline, valine, glycine, glutamine, leucine, methionine, asparagine, serine, threonine, tyrosine, aspartic acid, glutamic acid, histidine and derivatives thereof.

34. The method of claim 33 wherein said peptide enhances cell growth and is selected from the group consisting of IFFKG (SEQ ID NO:

1), FIKFG(SEQ ID NO;

2), FIFAK(SEQ ID NO;

3), QVVAK(SEQ ID NO;

4), FKFIG(SEQ ID NO;

5), AFFKI(SEQ ID NO;

6), VFPFK(SEQ ID NO;

7), AKIFF(SEQ ID NO;

8), AFKIF(SEQ ID NO;

9), KFAFI(SEQ ID NO;

10), FAKFI(SEQ ID NO;

11), and combinations thereof.

35. The method of claim 33, wherein the peptide enhances cell secretion and is selected from the group consisting of FKLVY(SEQ ID NO:

16), KKKKK(SEQ ID NO;

17), KKKKL(SEQ ID NO;

18), FKKKQ(SEQ ID NO;

19), FKFIG(SEQ ID NO;

5), KKKSK(SEQ ID NO;

20), KKKLK(SEQ ID NO;

21), FKKKK(SEQ ID NO;

22), LKKKK(SEQ ID NO;

23), KKLKK (SEQ ID NO;

24), KKKKT(SEQ ID NO;

25), KKPKK(SEQ ID NO;

26), KKPQY(SEQ ID NO;

27), SKKKK(SEQ ID NO;

28), KVKKK(SEQ ID NO;

29), KNQTY(SEQ ID NO;

30), FKKKV(SEQ ID NO;

31), KPKKK(SEQ ID NO;

32), FFKKK(SEQ ID NO;

33), HKNQT(SEQ ID NO;

34), FKLVG(SEQ ID NO;

35), KKQPK(SEQ ID NO;

36), EKKQT(SEQ ID NO;

37), EKKKK(SEQ ID NO;

38), KKIKQ(SEQ ID NO;

39), KKKKS(SEQ ID NO;

40), KKQKK(SEQ ID NO;

41), KKLNY(SEQ ID NO;

42), DGKKT(SEQ ID NO;

43), KKPTT(SEQ ID NO;

44), KFIFG(SEQ ID NO;

45), FKKMY(SEQ ID NO;

46), FFFKK(SEQ ID NO;

47), KQKKI(SEQ ID NO;

48), HIKKK(SEQ ID NO;

49), DFFHK(SEQ ID NO;

50), AKKKK(SEQ ID NO;

51), AHIKK(SEQ ID NO;

52), AHKKK(SEQ ID NO;

53), LKLVY(SEQ ID NO;

54), PKQKK(SEQ ID NO;

55), AKKKT(SEQ ID NO;

56), and combinations thereof.

36. A method of adhering cells to a surface to promote cellular growth comprising the steps of:

(i) providing a surface at least partially coated with a peptide comprising a structure selected from the group consisting of (a) xxxkx, (b) xxkxx, (c) xxxxk, (d) xkxxx, (e) kxxxx and combinations thereof, wherein each x may be the same or different hydrophobic or uncharged polar amino acid residue and k represents lysine; and

(ii) contacting said at least partially coated surface with said cells for a sufficient time to permit cellular attachment thereto.

37. The method of claim 36 wherein said hydrophobic or uncharged polar residue is selected from the group consisting of Phe, Ile, Ala, Val, Pro, Gly, Gln and derivatives thereof.

38. The method of claim 36 wherein said peptide comprises an amino acid sequence selected from the group consisting of IFFKG (SEQ ID NO:

1), FIKFG(SEQ ID NO;

2), FIFAK(SEQ ID NO;

3), QVVAK(SEQ ID NO;

4), FKFIG(SEQ ID NO;

5), AFFKI(SEQ ID NO;

6), VFPFK(SEQ ID NO;

7), AKIFF(SEQ ID NO;

8), AFKIF(SEQ ID NO;

9), KFAFI (SEQ ID NO;

10), FAKFI(SEQ ID NO;

11), and combinations thereof.

39. A cell culture substrate comprising a peptide selected from the group consisting of IFFKG (SEQ ID NO:

1), FIKFG(SEQ ID NO;

2), FIFAK(SEQ ID NO;

3), QVVAK(SEQ ID NO;

4), FKFIG(SEQ ID NO;

5), AFFKI(SEQ ID NO;

6), VFPFK(SEQ ID NO;

7), AKIFF(SEQ ID NO;

8), AFKIF(SEQ ID NO;

9), KFAFI (SEQ ID NO;

10), FAKFI(SEQ ID NO;

11), and combinations thereof.

40. A cell culture substrate comprising a peptide composition including one or more pairs of peptides selected from the group consisting of (a) AFFFQ(SEQ ID NO:

12)/EEEMY(SEQ ID NO;

13) and (b) FIKLM (SEQ ID NO;

14)/FFIPY (SEQ ID NO;

15).

41. A cell culture substrate comprising a peptide selected from the group consisting of FKLVY(SEQ ID NO:

16), KKKKK(SEQ ID NO;

17), KKKKL(SEQ ID NO;

18), FKKKQ(SEQ ID NO;

19), FKFIG(SEQ ID NO;

5), KKKSK(SEQ ID NO;

20), KKKLK(SEQ ID NO;

21), FKKKK(SEQ ID NO;

22), LKKKK(SEQ ID NO;

23), KKLKK (SEQ ID NO;

24), KKKKT(SEQ ID NO;

25), KKPKK(SEQ ID NO;

26), KKPQY(SEQ ID NO;

27), SKKKK(SEQ ID NO;

28), KVKKK(SEQ ID NO;

29), KNQTY(SEQ ID NO;

30), FKKKV(SEQ ID NO;

31), KPKKK(SEQ ID NO;

32), FFKKK(SEQ ID NO;

33), HKNQT(SEQ ID NO;

34), FKLVG(SEQ ID NO;

35), KKQPK(SEQ ID NO;

36), EKKQT(SEQ ID NO;

37), EKKKK(SEQ ID NO;

38), KKIKQ (SEQ ID NO;

39), KKKKS(SEQ ID NO;

40), KKQKK(SEQ ID NO;

41), KKLNY(SEQ ID NO;

42), DGKKT(SEQ ID NO;

43), KKPTT(SEQ ID NO;

44), KFIFG(SEQ ID NO;

45), FKKMY(SEQ ID NO;

46), FFFKK(SEQ ID NO;

47), KQKKI(SEQ ID NO;

48), HIKKK(SEQ ID NO;

49), DFFHK(SEQ ID NO;

50), AKKKK(SEQ ID NO;

51), AHIKK(SEQ ID NO;

52), AHKKK(SEQ ID NO;

53), LKLVY(SEQ ID NO;

54), PKQKK(SEQ ID NO;

55), AKKKT(SEQ ID NO;

56), DEETY(SEQ ID NO;

57), HNPPY(SEQ ID NO;

58), GGHMS(SEQ ID NO;

59), AADEG(SEQ ID NO;

60), GGGGS(SEQ ID NO;

61), EEGLS(SEQ ID NO;

62), HHPST(SEQ ID NO;

63), FHHNT(SEQ ID NO;

64), ADELN(SEQ ID NO;

65), KKKK(SEQ ID NO;

66), KKK(SEQ ID NO;

67), KK(SEQ ID NO;

68), OrnOrnOrn(SEQ ID NO;

69), RRR(SEQ ID NO;

70), and combinations thereof.

42. A peptide library selected from the group of libraries consisting of:

(a) xxxkx, (b) xxkxx, (c) xxxxk, (d) xkxxx, (e) kxxxx and permutations thereof, wherein k represents lysine and each x may be the same or different amino acid independently selected from the group consisting of lysine, alanine, isoleucine, phenylalanine, proline, valine, glycine, glutamine, leucine, methionine, asparagine, serine, threonine, tyrosine, aspartic acid, glutamic acid, histidine and derivatives thereof.

43. The peptide library of claim 42 wherein said library comprises peptides covalently linked to a member of the group consisting of bovine serum albumin, ovalbumin, keyhole limpet haemocyanin, collagen, fibronectin, laminin, an immunoglobulin, a polysaccharide, a growth factor and combinations thereof.

49. A peptide which enhances cell secretion in a cell culture system, wherein all amino acids of said peptide possess positively charged side chains.

50. The peptide of claim 49 ranging from 2 amino acids to about 20 amino acids.

51. The peptide of claim 49, wherein the peptide enhances cell secretion in said system at a concentration range from about 250 μ

M to about 24 mM.

52. The peptide of claim 49, wherein said peptide is a media constituent.