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1. WO2012023053 - PROCÉDÉ DE PRODUCTION D'ANTICORPS MULTISPÉCIFIQUES ET MULTIVALENTS

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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What is claimed is:

1. An isolated monoclonal antibody carrying a different specificity in each combining site and consisting of two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.

2. The isolated monoclonal antibody of claim 1 wherein at least a portion of the first light chain is of the Kappa type and at least a portion of the second light chain is of the Lambda type.

3. The isolated monoclonal antibody of claim 2, wherein the first light chain comprises at least a Kappa constant region.

4. The isolated monoclonal antibody of claim 3, wherein the first light chain further comprises a Kappa variable region.

5. The isolated monoclonal antibody of claim 3, wherein the first light chain further comprises a Lambda variable region.

6. The isolated monoclonal antibody of claim 2, wherein the second light chain comprises at least a Lambda constant region.

7. The isolated monoclonal antibody of claim 6, wherein the second light chain further comprises a Lambda variable region.

8. The isolated monoclonal antibody of claim 6, wherein the second light chain further comprises a Kappa variable region.

9. The isolated monoclonal antibody of claim 2, wherein the first light chain comprises a Kappa constant region and a Kappa variable region, and wherein the second light chain comprises a Lambda constant region and a Lambda variable region.

10. The isolated monoclonal antibody of any one of the preceding claims wherein the constant and variable framework region sequences are human.

11. A method to generate a bispecific antibody of claim 1 comprising:

a. Isolating an antibody or antibody fragment region having a specificity determined by a heavy chain variable domain combined with a first light chain variable domain;

b. Isolating an antibody or antibody fragment region having a different specificity determined by the same heavy chain variable domain as the antibody of step a) combined with a second light chain variable domain; c. Co-expressing in a cell:

i. the common heavy chain variable domain fused to an immunoglobulin heavy chain constant region;

ii. the first light chain variable domain fused either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type; and

iii. the second light chain variable domain fused to a light chain constant domain of a different type than constant domain of the first light chain.

12. The method of claim 11 wherein a Kappa light chain variable domain is fused to a constant region of the Kappa type.

13. The method of claim 11 wherein a Kappa light chain variable domain is fused to a constant region of the Lambda type.

14. The method of claim 11 wherein a Lambda light chain variable domain is fused to a constant region of the Kappa type.

15. The method of claim 11 wherein a Lambda light chain variable domain is fused to a constant region of the Lambda type.

16. The method of any one of claims 11 to 15 further comprising the step of (d) Purifying the bispecific antibodies produced in step c) from the monospecific antibodies produced in step c).

17. The method of claim 16 wherein step d) is an affinity chromatography purification step.

18. The method of claim 17 in which the purification step is performed using Kappa constant domain specific, Lambda constant domain specific or both Kappa constant domain specific and Lambda constant domain specific affinity chromatography media.

19. The method of any one of claims 11 to 18 in which the step a) and b) are facilitated by the use of antibody libraries having a common heavy chain and diversity confined to the light chain variable domain.

20. The method of claim 19 wherein the antibody library is displayed on filamentous bacteriophage, at the surface of yeast, bacteria or mammalian cells or used for ribosome or other type of in vitro display.

21. A method of preparing a bispecific antibody that specifically binds to a first antigen and a second antigen, wherein the first and second antigens are different, the method comprising:

(a) providing a first nucleic acid molecule encoding a first polypeptide comprising a heavy variable chain region of an immunoglobulin polypeptide or fragment thereof that binds the first antigen, coupled to an immunoglobulin constant region;

(b) providing a second nucleic acid molecule encoding a second polypeptide comprising a light chain variable region of the immunoglobulin polypeptide or fragment thereof that binds the first antigen coupled to a first Kappa-type or Lambda-type light chain constant region;

(c) providing a third nucleic acid molecule encoding a third polypeptide comprising a light chain variable region of an immunoglobulin polypeptide or fragment thereof that shares the same heavy variable chain region of the immunoglobulin polypeptide or fragment thereof of step (a) and binds the second antigen, coupled to a second Kappa- type or Lambda-type light chain constant region, wherein the first and second light chain constant domains are different types; and

(d) culturing a host cell comprising the first, second and third nucleic acid molecules under conditions that permit expression of the first, second and third polypeptides.

22. The method of claim 21, further comprising the step of (e) recovering the bispecific antibody.

23. The method of claim 21 , wherein the second nucleic acid encodes a Kappa-type light chain variable domain.

24. The method of claim 23, wherein the second nucleic acid encodes a Kappa-type constant region.

25. The method of claim 23, wherein the second nucleic acid encodes a Lambda-type constant region.

26. The method of claim 21 , wherein the second nucleic acid encodes a Lambda-type light chain variable domain.

27. The method of claim 26, wherein the second nucleic acid encodes a Kappa-type constant region.

28. The method of claim 26, wherein the second nucleic acid encodes a Lambda-type constant region.

29. The method of claim 21 , wherein the third nucleic acid encodes a Kappa-type light chain variable domain.

30. The method of claim 29, wherein the third nucleic acid encodes a Kappa-type constant region.

31. The method of claim 29, wherein the third nucleic acid encodes a Lambda-type constant region.

32. The method of claim 21, wherein the third nucleic acid encodes a Lambda-type light chain variable domain.

33. The method of claim 32, wherein the third nucleic acid encodes a Kappa-type constant region.

34. The method of claim 32, wherein the third nucleic acid encodes a Lambda-type constant region.

35. The method of any one of claims 21 to 34, wherein the bispecific antibody is recovered in a step (e) using an affinity chromatography purification step.

36. The method of claim 35, wherein the purification step is performed using Kappa constant domain specific, Lambda constant domain specific or both Kappa constant domain specific and Lambda constant domain specific affinity chromatography media.

37. An antibody mixture comprising two monospecific antibodies and one bispecific antibody, all having a common heavy chain.

38. A method to generate an antibody mixture of claim 37 comprising:

a. Isolating an antibody or antibody fragment region having a specificity determined by a heavy chain variable domain combined with a first light chain variable domain;

b. Isolating an antibody or antibody fragment region having a different specificity determined by the same heavy chain variable domain as the antibody of step a) combined with a second light chain variable domain;

c. Co-expressing in a cell:

i. the common heavy chain variable domain fused to an immunoglobulin heavy chain constant region;

ii. the first light chain variable domain fused either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type; and

iii. the second light chain variable domain fused to either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type.

39. The method of claim 38 further comprising the step of (d) Purifying the antibody mixture produced in step c) from cell culture supernatant.

40. An antibody mixture comprising three or more monospecific antibodies and three or more bispecific antibodies, all having a common heavy chain.

41. A method to generate an antibody mixture of claim 40 comprising:

a. Isolating an antibody or antibody fragment region having a specificity determined by a heavy chain variable domain combined with a first light chain variable domain;

b. Isolating several antibodies or antibody fragments region having a different specificity determined by the same heavy chain variable domain as the antibody of step a) combined with different light chain variable domains;

c. Co-expressing in a cell:

i. the common heavy chain variable domain fused to an immunoglobulin heavy chain constant region;

ii. all the light chains of the antibodies isolated in step a) and b) fused either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type.

42. The method of claim 41 further comprising the step of (d) Purifying the antibody mixture produced in step c) from cell culture supernatant.