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1. WO2002018446 - METHODS OF DESIGNING AND PRODUCING COMPOUNDS HAVING IMPROVED BINDING AFFINITY FOR CD154 OR OTHER TRIMERIC PROTEINS

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[ EN ]

CLAIMS

We claim:

1. A multi-molecular, two-dimensional lattice or aggregate comprising:
(a) a plurality of trimeric CD154 molecules
disposed on the extracellular surface of a
cell membrane; and
(b) anti-CD154 monoclonal antibody molecules, or antigen-binding fragments thereof, each
cross-linking said plurality of trimeric
CD154 molecules such that a lattice or
aggregate is formed on the extra-cellular
surface of said cell membrane.

2. A multi-molecular, two-dimensional lattice or aggregate comprising:
(a) a plurality of trimeric CD154 molecules in
solution; and
(b) anti-CD154 monoclonal antibody molecules, or antigen-binding fragments thereof, each
cross-linking said plurality of trimeric
CD154 molecules such that a lattice or
aggregate is formed in solution.

3. The multi-molecular, two-dimensional lattice or aggregate according to claim 1 , wherein said cell membrane is a mammalian cell membrane.

4. The multi-molecular lattice or aggregate according to claim 1 or 2, wherein said anti-CD154 monoclonal antibody molecules, or antigen-binding fragments thereof, interrupt the binding of CD154 to CD40.

5. The multi-molecular lattice or aggregate according to claim 1 or 2, wherein said CD154
monoclonal antibody molecules specifically bind to the 5c8 antigen, which is specifically bound by monoclonal antibody 5c8 (produced by the hybridoma having ATCC Accession No. HB 10916) .

6. A method for improving the binding affinity of a first synthetic molecule for a trimeric protein by generating a second synthetic molecule therewith, comprising the steps of:
(a) providing a first synthetic molecule th'at
binds a trimeric protein with low affinity
and is not capable of cross-linking a
plurality of said trimeric protein and
thereby promoting a plurality of said
trimeric protein on a cell surface to form a lattice or aggregate;
(b) providing a means to generate a second
synthetic molecule comprising said first
synthetic molecule, wherein said second
synthetic molecule is capable of cross- linking a plurality of said trimeric protein and promoting a plurality of said trimeric
proteins on a cell surface to form a lattice or aggregate; and
(c) generating said second synthetic molecule
based on said means.

7. A method for improving the binding affinity of a first synthetic molecule for a trimeric protein by generating a second synthetic molecule therewith, comprising the steps of:
(a) providing a first synthetic molecule that
binds a trimeric protein with low affinity
and is not capable of cross-linking a
plurality of said trimeric protein and
thereby promoting a plurality of said
trimeric proteins in solution to form a
lattice or aggregate;
(b) providing a means to generate a second
synthetic molecule comprising said first
synthetic molecule, wherein said second
synthetic molecule is capable of cross- linking a plurality of said trimeric protein and promoting a plurality of said trimeric
protein in solution to form a lattice or
aggregate; and
(c) generating said second synthetic molecule
based on said means.

8. The method according to claim 6 or 7, wherein said trimeric protein is a protein other than CD154.

9. The method according to claim 8, wherein said trimeric protein is a TNF family member protein.

10. The method according to claim 6 or 7, wherein said trimeric protein is CD154.

11. A synthetic molecule generated by the method according to claim 6 or 7.

12. The synthetic molecule according to claim 11, wherein said molecule is a tethered bivalent molecule.

13. The synthetic molecule according to claim 11, wherein said molecule specifically binds trimeric CD154.

14. The synthetic molecule according to claim 11, wherein said molecule specifically binds trimeric 5c8 antigen, which is specifically bound by monoclonal antibody 5c8 (produced by the hybridoma having ATCC Accession No. HB 10916) .

15. The synthetic molecule according to claim 11, wherein said molecule comprises a pair of binding moieties linked via an organic linker arm.

16. The synthetic molecule according to claim 15, wherein each member of said pair of binding moieties specifically binds trimeric CD154.

17. The synthetic molecule according to claim 15, wherein each member of said pair of binding moieties specifically binds trimeric 5c8 antigen, which is specifically bound by monoclonal antibody 5c8
(produced by the hybridoma having ATCC Accession No. HB 10916) .

18. The synthetic molecule according to claim 11, wherein said molecule inhibits a process selected from the group consisting of T cell dependent B cell activation, B cell dependent T cell activation, humoral immune response and cellular immune response.

19. A method for screening a candidate compound for the property of interrupting CD40:CD154 interaction, comprising the steps of:
(a) providing a cell having trimeric CD154
molecules on the cell surface;
(b) incubating said cell with said candidate
compound, under conditions sufficient for
said compound to promote cross-linking of
said CD154 molecules if said compound has the property of promoting such cross-linking; so as to produce a lattice or aggregate of CD154 molecules on the cell surface;
(c) incubating said cell with a detectable agent that specifically binds to cell surface CD154 molecules; and
(d) detecting that a lattice or aggregate of
cross-linked CD154 molecules has formed on
the cell surface.

20. A method for screening a candidate compound for the property of interrupting CD40:CD154 interaction, comprising the steps of:
(a) providing a solution comprising trimeric
CD154 molecules;
(b) incubating said solution with said candidate compound, under conditions sufficient for
said compound to promote cross-linking of
said trimeric CD154 molecules in solution if said compound has the property of promoting
such cross-linking; so as to produce a
lattice or aggregate of CD154 molecules in
solution; and (c) detecting that a lattice or aggregate of
cross-linked CD154 molecules has formed in
solution.

21. The method according to claim 19, wherein said detectable agent is a monoclonal antibody.

22. The method according to claim 19, wherein said detectable agent is directly linked to a fluorochrome.

23. The method according to claim 19, wherein said detectable agent is indirectly linked to a fluorochrome via a secondary reagent that is linked to a fluorochrome.

24. The method according to claim 19, wherein said cell is a T lymphocyte.

25. The method according to claim 19, wherein said cell is an immortalized T cell.

26. The method according to claim 25, wherein said immortalized T cell is a Dl.l cell derived from ATCC Deposit No. CRL 10915.

27. The method according to claim 19 or claim 20, further comprising the step of:
(e) confirming whether the candidate compound
capable of promoting lattice or aggregate
formation also is capable of interrupting
CD40:CD154 interaction.

28. The method according to claim 27, wherein step (e) is performed using an in vitro assay for T cell activation of B cells.

29. The method according to claim 27, wherein step (e) is performed using an in vitro assay for immunoglobulin production by B cells.

30. The method according to claim 27, wherein step (e) is performed using an in vivo assay for inhibition of a humoral immune response.

31. A method for screening a candidate compound for the property of interrupting CD40:CD154 interaction, comprising the steps of:
(a) providing a cell having trimeric CD154
molecules on the cell surface;
(b) incubating said cell with said candidate
compound, under conditions sufficient for
said compound to promote cross-linking of
said CD154 molecules if said compound is
capable of promoting such cross-linking; so
as to produce a lattice or aggregate of CD154 molecules on the cell surface; and
(c) detecting that a lattice or aggregate of
cross-linked CD154 molecules has formed on
the cell surface by a cell-binding assay.

32. A method for screening a candidate compound for the property of interrupting CD40:CD154 interaction, comprising the steps of:
(a) providing a solution comprising trimeric
CD154 molecules in solution;

(b) incubating said solution with said candidate compound, under conditions sufficient for
said compound to promote cross-linking of
said CD154 molecules if said compound is
capable of promoting such cross-linking; so
as to produce a lattice or aggregate of CD154 molecules in solution; and
(c) detecting that a lattice or aggregate of
cross-linked CD154 molecules has formed on
the cell surface by an assay selected from
the group consisting of gel filtration assay and light scattering assay.

33. The method according to claim 31 or 32, further comprising the step of:
(d) confirming whether the candidate compound
capable of promoting lattice or aggregate
formation also has the property of
interrupting CD40:CD154 interaction.

34. A compound identified by the method of any one of claims 19, 20, 31 or 32.

35. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to claim 34 or a synthetic molecule according to claim 11.

36. A method of attenuating severity of a condition associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.

37. A method of suppressing effects of a condition associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.

38. A method of preventing development of a condition associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.

39. A method of delaying onset of a
condition associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.

40. A method of inhibiting a condition associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.'

41. A method of reversing a condition associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.

42. A method of treating a condition
associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.

43. A method of preventing a condition associated with inappropriate CD154 mediated activation in a subject, comprising the step of administering an effective amount of a pharmaceutical composition according to claim 35 to the subject.

44. The method according to any one of claims 36-43, wherein the subject is a primate.

45. The method according claim 44, wherein said primate is a human.

46. The method according to any one of claims 36-43, wherein the condition is an unwanted immune response.

47. The method according to any one of claims 36-43, wherein the condition is an unwanted inflammatory response.

48. The method according to any one of claims 36-43, wherein the condition is an autoimmune disease.

49. The method according to any one of claims 36-43, wherein the condition is an allergy.

50. The method according to any one of claims 36-43, wherein the condition is an inhibitor response to a therapeutic agent.

51. The method according to any one of claims 36-43, wherein the condition is rejection of a donor organ.

52. The method according to any one of claims 36-43, wherein the condition is a B cell cancer.

53. The method according to any one of claims 36-43, wherein the condition is selected from the group consisting of: systemic lupus erythematosis, lupus nephritis, lupus neuritis, asthma, chronic obstructive pulmonary disease, bronchitis, emphysema, multiple sclerosis, uveitis, Alzheimer's disease, traumatic spinal cord injury, stroke, atherosclerosis, coronary restenosis, ischemic congestive heart failure, cirrhosis, hepatitis C, diabetic nephropathy,
glomerulonephritis, osteoarthritis, rheumatoid
arthritis, psoriasis, atopic dermatitis, systemic sclerosis, radiation-induced fibrosis, Crohn's disease, ulcerative colitis, multiple myeloma and cachexia.