Method for selecting a single cell expressing a heterogeneous combination of antibodies

• US 20120177637A1
• Filed: 02/14/2011
• Published: 07/12/2012
• Est. Priority Date: 05/30/2003
• Status: Active Grant

• Current Assignee: Merus N.V.
• Original Assignee: Merus Biopharmaceuticals B.V. I.O.
• Inventors: Hoogenboom, Hendricus Renerus Jacobus Mattheus, Logtenberg, Ton
• Granted Patent: US 9,738,701 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/10 from RNA viruses
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/248 IL-6
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/64 : comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: G01N 33/56966 : Animal cells

Abstract

The present invention provides combinations of specific binding proteins, such as immunoglobulins, that are designed to be true combinations, essentially all components of the combination being functional and compatible with each other. The invention further provides a method for producing a composition comprising at least two different proteinaceous molecules comprising paired variable regions, the at least two proteinaceous molecules having different binding specificities, comprising paired variable regions, at least two proteinaceous molecules having different binding specificities, comprising contacting at least three different variable regions under conditions allowing for pairing of variable regions and harvesting essentially all proteinaceous molecules having binding specificities resulting from the pairing.

Claims

1. A method for producing a composition comprising at least two different proteinaceous molecules comprising paired variable regions, wherein the at least two proteinaceous molecules have different binding specificities, said method comprising:

contacting at least three different variable regions under conditions allowing for pairing of variable regions, and

harvesting essentially all proteinaceous molecules having binding specificities resulting from said pairing,

so as to produce said composition.

2. The method according to claim 1, wherein the contacting takes place inside a cell.

3. The method according to claim 1, wherein at least one of said variable regions is produced by expression of a nucleic acid sequence encoding said variable region.

4. The method according to claim 3, wherein all variable regions are produced by one or more nucleic acid sequences encoding these variable regions.

5. The method according to claim 1, wherein at least one variable region is able to functionally pair with more than one other variable region.

6. The method according to claim 5, wherein said at least one variable region able to functionally pair with more than one other variable region, upon pairing, does not significantly contribute to the resulting binding specificity of the resulting proteinaceous molecule.

7. The method according to claim 1, wherein each variable region can only pair with one other variable region.

8. The method according to claim 1, for producing a composition comprising at least three different binding specificities.

9. The method according to claim 8, wherein at least two variable regions are part of one single chain Fv.

10. The method according to claim 1, wherein at least two different variable regions are expressed from one or more nucleic acid sequences encoding these variable regions, wherein expression of said different variable regions is under the direction of different control elements.

11. The method according to claim 10, wherein said different control elements lead to differential expression.

12. The method according to claim 11, wherein said differential expression is different in levels of expression and/or time of expression.

13. The method according to claim 1, wherein said variable regions are derived from heavy chains and/or light chains of immunoglobulins.

14. The method according to claim 1, wherein said proteinaceous molecules are antibodies, fragments and/or derivatives of antibodies.

15. An expression system for carrying out the method according to claim 1, comprising:

nucleic acid sequences encoding variable regions together with all elements required for gene expression and pairing.

16. The expression system of claim 15, which is a recombinant cell.

17. The expression system of claim 16, wherein the recombinant cell is a eukaryotic cell.

18. A collection of expression systems of claim 15, comprising:

a variety of combinations of different specificities.

19. A method for selecting combinations of proteinaceous molecules having specific affinity for at least one target epitope, said method comprising:

contacting the collection of claim 18 with said at least one target epitope, and

selecting combinations showing said specific affinity.

20. A method for selecting combinations of proteinaceous molecules having specific affinity for at least two target epitopes, said method comprising:

contacting the collection of claim 18 with said two target epitopes, and

selecting combinations showing said specific affinity.

21. The method according to claim 20, wherein said two target epitopes are associated with one disease and/or disorder.

22. The method according to claim 21, further comprising:

subjecting a selected combination of proteinaceous molecules to a biological assay indicative of an effect of the combination on the disease and/or disorder.

23. A composition obtainable by the method according to claim 1.

24. The composition of claim 23, comprising:

at least three different paired variable regions, having different binding specificities.

25. The composition of claim 23, wherein said variable regions are derived from immunoglobulin light chains and/or immunoglobulin heavy chains.

26. The composition of claim 23, wherein said proteinaceous molecules are antibodies.

27. The composition of claim 26, wherein said antibodies are directed to at least two target antigens and/or two target epitopes.

28. The composition of claim 27, wherein the target epitopes are associated with one disease and/or disorder.

29. The composition of claim 27, wherein one target antigen is derived from a Rabies virion, is glycoprotein or the ribonucleoprotein of a Rabies virion, or wherein at least one target epitope is present on a Rabies virion.

30. The composition of claim 23, wherein antigenic parts of said proteinaceous molecules originate from one species.

31. The composition of claim 23, wherein antigenic parts of said proteinaceous molecules are derived from human, humanized, or deimmunized antibodies.

32. The composition of claim 23, together with a pharmaceutically acceptable excipient.

33. The composition of claim 23, for use as a pharmaceutical.

34. The composition of claim 23, comprising at least one monospecific antibody and at least one bispecific antibody produced in one cell, together with a pharmaceutically acceptable excipient.

35. The composition of claim 23, comprising at least two monospecific antibodies produced in one cell.

36. A method for producing nucleic acid sequences encoding variable regions for use in the method according to claim 1, said method comprising:

synthesizing nucleic acid sequences encoding variable regions,

expressing said nucleic acid sequences and allowing the expression products to pair, and

selecting nucleic acid sequences encoding variable regions having desired pairing behavior,

so as to produce nucleic acid sequences encoding variable regions.

37. A method for producing nucleic acid sequences encoding variable regions for use in the method according to claim 1, said method comprising:

altering existing nucleic acid sequences encoding variable regions,

expressing said nucleic acid sequences, and

allowing the expression products to pair and selecting nucleic acid sequences encoding variable regions having desired pairing behavior,

so as to produce nucleic acid sequences encoding variable regions.

39. The method according to claim 36, wherein said variable regions comprise at least one element of variable regions of heavy and light chains of antibodies.

40. The method according to claim 39, wherein nucleic acid sequences are produced which encode variable regions having at least one element from both heavy chain variable regions and light chain variable regions.

41. The method according to claim 36, wherein said element comprises at least one complementarity determining region (CDR).

42. The method according to claim 36, wherein a nucleic acid sequence is selected encoding a variable region capable of pairing with more than one different variable region.

43. The method according to claim 36, wherein a nucleic acid is selected encoding a variable region capable of exclusive pairing with one other variable region.

38. A method for producing nucleic acid sequences encoding variable regions for use in a process for producing a composition comprising at least two different proteinaceous molecules comprising paired variable regions, wherein the at least two proteinaceous molecules have different binding specificities, said method comprising:

a combination of

synthesizing nucleic acid sequences encoding variable regions,

expressing said nucleic acid sequences and allowing the expression products to pair, and

selecting nucleic acid sequences encoding variable regions having desired pairing behavior,

with

altering existing nucleic acid sequences encoding variable regions,

expressing said nucleic acid sequences, and

allowing the expression products to pair and selecting nucleic acid sequences encoding variable regions having desired pairing behavior.