Peptides for enhanced cell attachment and cell growth

• US 7,074,615 B2
• Filed: 08/15/2003
• Issued: 07/11/2006
• Est. Priority Date: 08/15/2003
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Chaney, Bryce N., Liebmann-Vinson, Andrea, Heidaran, Mohammad A.
• Primary Examiner(s): Carlson, Karen Cochrane
• Assistant Examiner(s): Tsay, Marsha
• Application Number:
  US10/641,286
• Publication Number:
  US 20050036980A1
• Time in Patent Office: 1,061 Days
• Field of Search: 435/325, 435/7.1, 435/402, 435/404, 514/2, 514/17, 530/350, 530/345
• US Class Current: 435/402
• CPC Class Codes: A61K 38/08 Peptides having 5 to 11 amino acids A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: C07K 17/06 attached to the carrier via a bridging agent
• CPC Class Codes: C07K 5/0806 the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C08J 7/12 Chemical modification
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
• CPC Class Codes: C12N 5/0656 Adult fibroblasts

Abstract

The present invention relates to cell adhesion promoting (“CAP”) peptide combinations that promote cell attachment or cell adhesion to culture surfaces that are otherwise cell adhesion resistant “CAR”. The invention provides combination of peptides that, when covalently coupled to a CAR layer such as hyaluronic acid that has been created on a polystyrene surface, promote cell attachment, growth differentiation, and execution of other desired cellular functions in culture.

Claims

1. A cell adhesion promoting (CAP) peptide-modified polymer composition useful for promoting cell attachment or growth, comprising a polymer surface comprising a cell adhesion resistant (CAR) material to which one or more peptides are covalently bound, forming a peptide-modified CAP surface, which peptides/surface promote attachment of cells, which cells substantially do not attach to said CAR surface in the absence of said peptides, wherein each of said one or more peptides comprises:

(a) one or more peptide sequences selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), SEQ ID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

(b) a chemically or recombinantly prepared peptide multimer that includes at least two repeats of any one or a combination of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16;

or

(c) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue, wherein said variant has the ability to promote cell attachment or growth;

wherein said CAR material is hyaluronic acid (HA).

2. The composition of claim 1 wherein said peptide multimer is a chemically prepared peptide multimer having the formula P¹_n, wherein P¹ comprises:

(a) the same or different peptide sequence selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), SEQ ID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

or

(b) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue, wherein said variant has the ability to promote cell attachment or growth;

wherein n=2–

10.

3. The composition of claim 1 wherein said chemically prepared peptide multimer has the formula (P¹—

X_m)_n-P², wherein P¹ and P² comprise;

(a) the same or different peptide sequences selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), SEQ ID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

or

(b) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue wherein said variant has the ability to promote cell attachment or growth;

wherein X is C₁–

C₅ alkyl, C₁–

C₅ alkenyl, C₁–

C₅ alkynyl, or C₁–

C₅ polyether containing up to 4 oxygen atoms, m=0 or 1, and n=1–

7.

4. The composition of claim 1 wherein said recombinantly prepared peptide multimer has the formula (P¹-Gly_z)_n-P², wherein P¹ and P² comprise:

(a) the same or different peptide sequences selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), SEQ ID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

or

(b) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue, wherein said variant has the ability to promote cell attachment or growth;

wherein z=0–

6 and n=1–

10.

5. The composition of claim 1 wherein the composition further comprises a peptide of no more than 30 amino acids having the RGD sequence.

6. The composition of claim 5 wherein the peptide with the RGD sequence comprises the sequence GRGDS (SEQ ID NO:

22).

7. The composition of claim 1 wherein said peptide-modified polymer composition is in the form of a 3-dimensional (3D) scaffold.

8. The composition of claim 1 wherein said polymer surface is in the form of a flexible material.

9. The composition of claim 8 wherein the flexible material is a polydimethyl siloxane (PDMS) or another silicone-based polymer.

14. A method of producing the peptide-modified polymer composition of claim 1 comprising the steps of:

(a) providing a polymer surface;

(b) treating said surface with a CAR material to produce a CAR surface; and

(c) treating said CAR surface with peptides so that said peptides become covalently bound thereto,

thereby producing said peptide-modified polymer composition.

15. A method for attaching cells to a peptide-modified CAR polymer surface comprising:

(a) providing the composition of claim 1;

(b) adding adherent cells to composition; and

(c) allowing said cells to attach to said peptide-modified surface.

10. A composition comprising an isolated peptide of no more than 30 amino acids, which, when covalently bound to a CAR surface alone or in combination with other peptides, promotes cell attachment, wherein said peptide comprises:

(a) one or more peptide sequences selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), SEQ ID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

(b) a chemically or recombinantly prepared peptide multimer that includes at least two repeats of any one or a combination of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16;

or

(c) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue, wherein said variant has the ability to promote cell attachment or growth.

11. The composition of claim 10 wherein said peptide multimer is a chemically prepared peptide multimer having the formula P¹_n, wherein P¹ comprises:

(a) the same or different peptide sequence selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ lID NO;

3 (EEEKV), SEQ ID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

or

(b) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue, wherein said variant has the ability to promote cell attachment or growth;

wherein n=2–

10.

12. The composition of claim 10 wherein said chemically prepared peptide multimer has the formula (P¹—

X_m)_n—

P², wherein P¹ and P² comprise;

(a) the same or different peptide sequences selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), SEQ lID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

or

(b) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue, wherein said variant has the ability to promote cell attachment or growth;

wherein X is C₁–

C₅ alkyl, C₁–

C₅ alkenyl, C₁–

C₅ alkynyl, or C₁–

C₅ polyether containing up to 4 oxygen atoms, m=0 or 1, and n=1–

7.

13. The composition of claim 10 wherein said recombinantly prepared peptide multimer has the formula (P¹-Gly_z)_n—

P², wherein P¹ and P² comprise;

(a) the same or different peptide sequences selected from the group consisting of SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), SEQ ID NO;

4 (GHK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

8 (AFAFK), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), SEQ ID NO;

12 (AKKMV), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK);

or

(b) a cell adhesion promoting variant of any one or more of SEQ ID NOs;

2–

4, 6, 8, 10–

12, and 14–

16, said variant having a conservative substitution of a single amino acid residue, wherein said variant has the ability to promote cell attachment or growth;

wherein z=0–

6 and n=1–

10.

16. A cell adhesion promoting (CAP) peptide-modified polymer composition useful for promoting cell attachment or growth, comprising a polymer surface comprising a cell adhesion resistant (CAR) material to which at least one combination of peptides is covalently bound, forming a peptide-modified CAP surface, which peptides/surface promote attachment of cells, which cells substantially do not attach to said CAR surface in the absence of said peptides, where the combination is selected from the group consisting of:

(a) SEQ ID NO;

1(FIKFG), SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), and SEQ ID NO;

4 (GHK);

(b) SEQ ID NO;

5 (FIFAK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

7 (AFKIF), and SEQ ID NO;

8 (AFAFK);

(c) SEQ ID NO;

9 (FAKFI), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), and SEQ ID NO;

12 (AKKMV); and

(d) SEQ ID NO;

13 (FKFIG), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK).

17. The composition of claim 16 wherein said combination is SEQ ID NO:

1 (FIKFG), SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), and SEQ LID NO;

4 (GHK).

18. The composition of claim 16 wherein said combination is SEQ ID NO:

5 (FIFAK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

7 (AFKIF), and SEQ ID NO;

8 (AFAFK).

19. The composition of claim 16 wherein said combination is SEQ ID NO:

9 (FAKFI), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), and SEQ ID NO;

12 (AKKMV).

20. The composition of claim 16 wherein said combination is SEQ ID NO:

13 (FKFIG), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK).

21. The composition of claim 16 wherein the peptide combination further comprises a peptide of no more than 30 amino acids having the RGD sequence.

22. The composition of claim 21 wherein the peptide with the RGD sequence comprises the sequence GRGDS (SEQ ID NO:

22).

23. The composition of claim 16 wherein said CAR material is selected from the group consisting of hyaluronic acid (HA), alginic acid (AA), polyethylene glycol (PEG), polyethylene oxide (PEO), and polyhydroxyethyl methacrylate (poly-HEMA).

24. The composition of claim 23 wherein said CAR material is HA.

25. The composition of claim 16 wherein said peptide-modified polymer composition is in the form of a 3-dimensional (3D) scaffold.

26. The composition of claim 16 wherein said polymer surface is in the form of a flexible material.

27. The composition of claim 26 wherein the flexible material is a polydimethyl siloxane (PDMS) or another silicone-based polymer.

28. A composition comprising at least one combination of peptides, which, when covalently bound to a CAR surface alone or in combination with other peptides, promotes cell attachment, where the combination is selected from the group consisting of:

(a) SEQ ID NO;

1 (FIKFG), SEQ ID NO;

2 (EFIKY), SEQ ID NO;

3 (EEEKV), and SEQ ID NO;

4 (GHK);

(b) SEQ ID NO;

5 (FIFAK), SEQ ID NO;

6 (KKLVY), SEQ ID NO;

7 (AFKIF), and SEQ ID NO;

8 (AFAFK);

(c) SEQ ID NO;

9 (FAKFI), SEQ ID NO;

10 (KMLIY), SEQ ID NO;

11 (KSYYY), and SEQ ID NO;

12 (AKKMV); and

(d) SEQ ID NO;

13 (FKFIG), SEQ ID NO;

14 (AFIPV), SEQ ID NO;

15 (KKMYY), and SEQ ID NO;

16 (AIKKK).