Dimeric fusion proteins and materials and methods for producing them

1. A polynucleotide encoding a polypeptide fusion, said polypeptide fusion consisting of, from amino terminus to carboxyl terminus, P1-L-D1-(P2) n , wherein:

P1 is a non-immunoglobulin polypeptide, wherein said non-immunoglobulin polypeptide is an extracellular domain of a cell-surface receptor requiring, in its native state, dimerization in order to initiate intracellular signal transduction;

L is a polypeptide linker consisting of from 18 to 32 amino acid residues, wherein from 1 to 8 of said residues are cysteine residues;

D1 is a dimerizing domain selected from the group consisting of an immunoglobulin CH1 domain, a T-cell receptor Cα domain, a T-cell receptor Cβ domain, a major histocompatibility complex class I α3 domain, β2-microglobulin, a major histocompatibility complex class II α2 domain, and a major histocompatibility complex class II β2 domain;

P2 is a linking polypeptide consisting of from 1 to 29 amino acid residues, wherein at least one of said residues is a cysteine residue; and

n is 0 or 1.

2. The polynucleotide of claim 1 , wherein D1 is the immunoglobulin CH1 domain.

3. The polynucleotide of claim 1 , wherein P1 is not a member of the immunoglobulin superfamily.

4. A polynucleotide encoding a polypeptide fusion, said polypeptide fusion consisting of, from amino terminus to carboxyl terminus, P3-L-D2, wherein:

P3 is a non-immunoglobulin polypeptide, said non-immunoglobulin polypeptide being an extracellular domain of a cell-surface receptor requiring, in its native state, dimerization in order to initiate intracellular signal transduction;

L is a polypeptide linker consisting of from 18 to 32 amino acid residues, wherein from 1 to 8 of said residues are cysteine residues; and

D2 is a dimerizing domain selected from the group consisting of an immunoglobulin light chain constant domain, a T-cell receptor Cα domain, a T-cell receptor Cβ domain, a major histocompatibility complex class I α3 domain, β2-microglobulin, a major histocompatibility complex class II α2 domain, and a major histocompatibility complex class II β2 domain.

5. The polynucleotide of claim 4 , wherein D2 is an immunoglobulin κ light chain constant domain or immunoglobulin λ light chain constant domain.

6. The polynucleotide of claim 4 , wherein P3 is not a member of the immunoglobulin superfamily.

7. An expression vector comprising the following operably linked elements:

(a) a transcription promoter;

(b) a DNA segment encoding polypeptide fusion, said polypeptide fusion consisting of, from amino terminus to carboxyl terminus, P1-L-D1-(P2) n , wherein:

P1 is a non-immunoglobulin polypeptide, said non-immunoglobulin polypeptide being an extracellular domain of a cell-surface receptor requiring, in its native state, dimerization in order to initiate intracellular signal transduction;

L is a polypeptide linker consisting of from 18 to 32 amino acid residues, wherein from 1 to 8 of said residues are cysteine residues;

D1 is a dimerizing domain selected from the group consisting of an immunoglobulin CH1 domain, a T-cell receptor Cα domain, a T-cell receptor Cβ domain, a major histocompatibility complex class I α3 domain, β2-microglobulin, a major histocompatibility complex class II α2 domain, and a major histocompatibility complex class II β2 domain;

P2 is a linking polypeptide consisting of from 1 to 29 amino acid residues, wherein at least one of said residues is a cysteine residue; and

n is 0 or 1; and

(c) a transcription terminator.

8. A cultured cell into which has been introduced the expression vector of claim 7 , wherein the cell expresses the DNA segment.

9. An expression vector comprising the following operably linked elements:

(a) a transcription promoter;

(b) a DNA segment encoding polypeptide fusion, said polypeptide fusion consisting of, from amino terminus to carboxyl terminus, P3-L-D2, wherein:

P3 is a non-immunoglobulin polypeptide, said non-immunoglobulin polypeptide being an extracellular domain of a cell-surface receptor requiring, in its native state, dimerization in order to initiate intracellular signal transduction;

L is a polypeptide linker consisting of from 18 to 32 amino acid residues, wherein from 1 to 8 of said residues are cysteine residues; and

D2 is a dimerizing domain selected from the group consisting of an immunoglobulin light chain constant domain, a T-cell receptor Cα domain, a T-cell receptor Cβ domain, a major histocompatibility complex class I α3 domain, β2-microglobulin, a major histocompatibility complex class II α2 domain, and a major histocompatibility complex class II β2 domain; and

(c) a transcription terminator.

10. A cultured cell into which has been introduced the expression vector of claim 9 , wherein the cell expresses the DNA segment.

11. The polynucleotide of claim 1 or the expression vector of claim 7 , wherein P1 is selected from the group consisting of:

an IL-20RA extracellular domain;

an IL-20RB extracellular domain;

an IL-22RA1 extracellular domain;

an IL-21R extracellular domain;

an IL-2 receptor γ common extracellular domain;

a PDGF α receptor extracellular domain; and

a PDGF β receptor extracellular domain.

12. The polynucleotide of claim 4 or the expression vector of claim 9 , wherein P3 is selected from the group consisting of:

an IL-20RA extracellular domain;

an IL-20RB extracellular domain;

an IL-22RA1 extracellular domain;

an IL-21R extracellular domain;

an IL-2 receptor γ common extracellular domain;

a PDGF α receptor extracellular domain; and

a PDGF β receptor extracellular domain.

13. A method of making a dimeric protein consisting of a first polypeptide fusion disulfide bonded to a second polypeptide fusion, the method comprising:

(1) culturing a cell comprising first and second expression units, wherein:

said first expression unit comprises a transcription promoter; a first DNA segment encoding the first polypeptide fusion, wherein said first polypeptide fusion consists of, from amino terminus to carboxyl terminus, P1-L1-D1-(P2) n ; and a transcription terminator;

said second expression unit comprises a transcription promoter; a second DNA segment encoding the second polypeptide fusion, wherein said second polypeptide fusion consists of, from amino terminus to carboxyl terminus, P3-L2-D2; and a transcription terminator;

P1 is a first non-immunoglobulin polypeptide;

L1 is a first polypeptide linker consisting of from 18 to 32 amino acid residues, wherein x of said residues are cysteine residues;

P2 is a linking polypeptide consisting of from 1 to 29 amino acid residues, wherein at least one of said residues is a cysteine residue;

n is 0 or 1;

P3 is a second non-immunoglobulin polypeptide different from P1;

L2 is a second polypeptide linker consisting of from 18 to 32 amino acid residues, wherein y of said residues are cysteine residues;

D1 and D2 are dimerizing domains capable of dimerizing with each other, wherein D1 and D2 are respectively selected from the following dimerizing domain pairs:

(a) an immunoglobulin CH1 domain and an immunoglobulin light chain constant domain;

(b) a TCR Cα domain and a TCR Cβ domain;

(c) a TCR Cβ domain and a TCR Cα domain;

(d) an MHC class I α 3 domain and a β2-microglobulin;

(e) a β2-microglobulin and an MHC class I α 3 domain;

(f) an MHC class II α 2 domain and an MHC class II β 2 domain; and

(g) an MHC class β 2 domain and an MHC class II α 2 domain;

each of P1 and P3 is an extracellular domain of a cell-surface receptor requiring, in its native state, dimerization in order to initiate intracellular signal transduction;

each of x and y is an integer from 1 to 8; and

x =y;

whereby said first and second DNA segments are expressed and the encoded polypeptide fusions are produced as a dimeric protein; and

(2) recovering the dimeric protein.

14. The method of claim 13 , wherein n=1.

15. The method of claim 13 wherein x=2 and y =2.

16. The method of claim 13 , wherein each of P1 and P3 is not a member of the immunoglobulin superfamily.

17. The method of claim 13 , wherein each of L1 and L2 contains exactly two cysteine residues.

18. The method of claim 13 , wherein each of L1 and L2 comprises an immunoglobulin hinge variant wherein the cysteine residue corresponding to residue 103 of SEQ ID NO:1 is replaced by serine.

19. The method of claim 13 , wherein each of L1 and L2 comprises a human γ1 hinge variant wherein the cysteine residue corresponding to residue 103 of SEQ ID NO:1 is replaced by serine.

20. The method of claim 13 , wherein each of L1 and L2 consists of 18 amino acid residues.

21. The method of claim 13 , wherein each of L1 and L2 comprises a plurality of glycine residues.

22. The method of claim 13 , wherein each of L1 and L2 comprises a plurality of serine residues.

23. The method of claim 13 , wherein each of L1 and L2 comprises [Gly-Ser-Gly-Ser] a (SEQ ID NO:2), wherein a is 1 or 2; or [Gly-Gly-Gly-Ser] b (SEQ ID NO:3), wherein b is 1 or 2.

24. The method of claim 13 , wherein each of L1 and L2 comprises a proteolytic cleavage site.

25. The method of claim 13 , wherein:

D1 is an immunoglobulin CH1 domain; and

D2 is an immunoglobulin κ light chain constant domain or immunoglobulin λ light chain constant domain.

26. The dimeric protein of claim 25 , wherein D2 is an immunoglobulin κ light chain constant domain.

27. The method of claim 13 , wherein:

a) one of P1 and P3 is an IL-20RA extracellular domain and the other of P1 and P3 is an IL-20RB extracellular domain;

b) one of P1 and P3 is an IL-22RA1extracellular domain and the other of P1 and P3 is an IL-20RB extracellular domain;

c) one of P1 and P3 is an IL-21R extracellular domain and the other of P1 and P3 is an IL-2 receptor γ common extracellular domain; or

d) one of P1 and P3 is a PDGF α receptor extracellular domain and the other of P1 and P3 is a PDGF β receptor extracellular domain.