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1. WO2007137760 - DIMERIC MOLECULAR COMPLEXES

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[ EN ]

We claim:
1. A dimeric molecular complex comprising a first and a second fusion protein, wherein each fusion protein comprises from its N to C terminus:
(A) a biological effector moiety selected from the group consisting of:
(1) a single chain antibody;
(2) an Fab fragment;
(3) an extracellular domain of a type I membrane receptor;
(4) a cytokine;
(5) a chemokine;
(6) an enzyme;
(7) a toxin; and
(8) a detectable marker;
(B) a hinge region of an IgG molecule bound to the biological effector moiety;
and (C) a CH4 dimerization domain of an IgE molecule covalently bound to the
hinge region,
wherein the molecular complex comprises a disulfide bond between a cysteine residue in the hinge region of the first fusion protein and a cysteine residue in the hinge region of the second fusion protein.

2. The molecular complex of claim 1 , wherein the biological effector moieties of the first and second fusion proteins are identical.

3. The molecular complex of claim 1, wherein the biological effector moieties of the first and second fusion proteins are different.

4. The molecular complex of claim 1 , wherein the biological effector moieties of the first and second fusion proteins each comprise an antigen binding site.

5. The molecular complex of claim 4, wherein the two antigen binding sites have the same specificity.

6. The molecular complex of claim 4, wherein the two antigen binding sites have different specificities.

7. The molecular complex of claim 1 , wherein the hinge region of both the first and second fusion proteins is from an IgG1 molecule.

8. The molecular complex of claim 7, wherein the hinge region comprises amino acid residues 223 to 243 of SEQ ID NO:25.

9. The molecular complex of claim 7, wherein within the hinge region of at least one of the two fusion proteins, the tetrapeptide VFLF, which occupies positions 240-243 in the IgG, hinge region (SEQ ID NO:25), is replaced with a tetrapeptide selected from the group consisting of DSEY, KSKY1 CSEY, DSCY, DEEY, KRKY, SESE, SDSD, SKSK, SRSR, SESY, SDSY. SKSY, SRSY, DEEY, DDDY, DDEY, DEDY, EEEY, EDDY, EDEY, EEDY, RRRY, RKRY, RRKY1 RKKY, KKKY, KRRY, KRKY, and KKRY.

10. The molecular complex of claim 9, wherein the tetrapeptide is selected from the group consisting of DSEY, KSKY, DEEY, and KRKY.

11. The molecular complex of claim 10, wherein the tetrapeptide is DSEY.

12. The molecular complex of claim 10, wherein the tetrapeptide is KSKY.

13. The molecular complex of claim 9, wherein the tetrapeptide VFLF within the hinge region of both the first and second fusion proteins is replaced with the same tetrapeptide.

14. The molecular complex of claim 13, wherein the replacement tetrapeptide is selected from the group consisting of DSEY, KSKY, DEEY, and KRKY.

15. The molecular complex of claim 9, wherein the tetrapeptide VFLF within the hinge region of each of the first and second fusion proteins is replaced with a different tetrapeptide.

16. The molecular complex of claim 15, wherein the tetrapeptide VFLF within the hinge region of the first fusion protein is replaced with the tetrapeptide DSEY and within the hinge region of the second fusion protein is replaced with the tetrapeptide KSKY.

17. The molecular complex of claim 12, further comprising a moiety covalently bound to a lysine in the hinge region, wherein the moiety is a toxin or a polyglycol.

18. The molecular complex of claim 1 , further comprising an epitope tag at its C terminus.

19. The molecular complex of claim 18, wherein the epitope tag comprises
GAPVPYPDPLEPRAA (SEQ ID NO:23).

20. The molecular complex of claim 1 , further comprising an M2" IgE splice variant having the sequence of SEQ ID NO:26 at the C terminus of the first fusion protein and a different M2" IgE splice variant having the sequence of SEQ ID NO.27 at the C terminus of the second fusion protein.

21. The molecular complex of claim 1 , further comprising an amino acid linker between the biological effector moiety and the hinge region, wherein the linker is covalently bound to the C-terminus of the effector moiety and the N-terminus of the hinge region.

22. The molecular complex of claim 5, wherein both the first and second fusion proteins comprise the amino acid sequence of SEQ ID NO:1.

23. The molecular complex of claim 5, wherein both the first and second fusion proteins are encoded by the nucleotide sequence of SEQ ID NO:2.

24. The molecular complex of claim 22, wherein the amino acid residues at positions 277-281 of

SEQ ID NO:1 (DSEYP) are replaced with amino acid residues KTSG (residues 315-318 of SEQ ID NO:45) or with amino acid residues TKTSG (residues 314-318 of SEQ ID NO:45).

25. The molecular complex of claim 5, wherein both the first and second fusion proteins are encoded by the nucleotide sequence of SEQ ID NO.22.

26. The molecular complex of claim 25, wherein amino acid residues at positions 263 to 267 of SEQ ID NO:20 are replaced with amino acid residues KTSG (residues 315-318 of SEQ ID NO:45) or with amino acid residues TKTSG (residues 314-318 of SEQ ID NO:45).

27. A dimeric molecular complex comprising a first and a second fusion protein, each of which comprise from N to C terminus:
(A) a CH4 dimerization domain of an M2" IgE splice variant;
(B) an amino acid linker which is covalently bound to the CH4 dimerization domain; and
(C) an extracellular domain of a type Il membrane receptor,
wherein the molecular complex comprises a disulfide bond between a cysteine residue within the C terminal M2" IgE splice variant CH4 dimerization domain of the first fusion protein and a cysteine residue within the C terminal M2" IgE splice variant CH4 dimerization domain of the second fusion protein.

28. The molecular complex of claim 27, wherein the amino acid linker comprises glycine.

29. The molecular complex of claim 27, wherein the C terminal M2" IgE splice variant of the CH4 dimerization domain is selected from SEQ ID NO.27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30.

30. The molecular complex of claim 27, wherein the type It membrane receptor is a myeloid

DAP12-associating lectin-1 (MDL-1) receptor.

31. A fusion protein comprising from N to C terminus:
(A) a biological effector moiety selected from the group consisting of:
(1 ) a single chain antibody;
(2) an Fab fragment;
(3) an extracellular domain of a type I membrane receptor;
(4) a cytokine;
(5) a chemokine;
(6) an enzyme;
(7) a toxin; and
(8) a detectable marker;
(B) a hinge region of an IgG molecule bound to the biological effector moiety; and
(C) a CH4 dimerization domain of an IgE molecule covatentty bound to the
hinge region.

32. A fusion protein comprising from N to C terminus:
(A) a CH4 dimerization domain with an M2" IgE splice variant C terminal
extension;
(B) an amino acid linker which is covalently bound to the CH4 dimerization
domain; and
(C) an extracellular domain of a type Il membrane receptor.

33. A nucleic acid molecule which encodes the fusion protein of claim 31 or 32.

34. The nucleic acid molecule of claim 33 which is an expression construct.

35. A host cell comprising the nucleic acid molecule of Claim 34.

36. A pharmaceutical composition comprising:
the dimeric molecular complex of claim 1 or 27; and
a pharmaceutically acceptable vehicle.

37. A method of treating a tumor in a patient in need thereof comprising administering an effective amount of a dimeric molecular complex to the patient, wherein the dimeric molecular complex comprises a first and a second fusion protein, and wherein each fusion protein comprises from its N to C terminus:
(A) a biological effector moiety comprising an antigen binding site for a tumor- associated antigen;
(B) a hinge region of an IgG molecule covalently bound to the tumor associated antigen; and
(C) a CH4 dimerization domain of an IgE molecule covalently bound to the
hinge region,
wherein the molecular complex comprises a disulfide bond between a cysteine residue in the hinge region of the first fusion protein and a cysteine residue in the hinge region of the second fusion protein.

38. The method of claim 37, wherein at least one of the first and second fusion proteins of the dimeric molecular complex is conjugated to a toxin.

39. The method of claim 37 wherein at least one of the first and second fusion proteins is conjugated to a chemotherapeutic agent.

40. A method of imaging a target area of a body in a patient in need thereof comprising administering to a patient a dimeric molecular complex in an amount sufficient to provide a detectable signal at the target area, wherein the dimeric molecular complex comprises a first and a second fusion protein, wherein the first fusion protein comprises from its N to C terminus:
(A) a biological effector moiety comprising an antigen binding site for a target- specific antigen;
(B) a hinge region of an IgG molecule bound to the biological effector moiety;
and
(C) a CH4 dimerization domain of an IgE molecule covalently bound to the
hinge region; and
wherein the second fusion protein comprises from its N to C terminus
(A) a biological effector moiety comprising a detectable label;
(B) a hinge region of an IgG molecule bound to the biological effector moiety; and
(C) a CH4 dimerization domain of an IgE molecule covalently bound to the
hinge region; and
wherein the molecular complex comprises a disulfide bond between a cysteine residue in the hinge region of the first fusion protein and a cysteine residue in the hinge region of the second fusion protein.