Methods and means for the production of Ig-like molecules

Patent metadata

Field	Value
Publication number	US-9758805-B2
Application number	US-201514974581-A
Country	US
Kind code	B2
Filing date	Dec 18, 2015
Priority date	Apr 20, 2012
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

The invention provides means and methods for producing one or more Ig-like molecules in a single host cell. Novel CH3 mutations enabling the production of monospecific and/or bispecific Ig-like molecules of interest are also provided.

First claim

The invention claimed is:

1. A method of producing at least two different antibodies in a single host cell, the method comprising:
a. providing a host cell comprising:
(i) a first nucleic acid molecule encoding a 1 st antibody heavy chain comprising at least one substitution of a neutral amino acid residue in the CH3 domain by a positively charged amino acid residue,
(ii) a second nucleic acid molecule encoding a 2 nd antibody heavy chain comprising at least one substitution of a neutral amino acid residue in the CH3 domain by a negatively charged amino acid residue,
(iii) a third nucleic acid molecule encoding a 3 rd antibody heavy chain, and
(iv) a fourth nucleic acid molecule encoding a 4 th antibody heavy chain;

b. culturing the host cell and allowing for expression of the four nucleic acid molecules to produce the 1 st antibody heavy chain, the 2 nd antibody heavy chain, the 3 rd antibody heavy chain and the 4 th antibody heavy chain, wherein the CH3 domain of the 1 st antibody heavy chain preferentially pairs with the CH3 domain of the 2 nd antibody heavy chain, and the CH3 domain of the 3 rd antibody heavy chain preferentially pairs with the CH3 domain of the 4 th antibody heavy chain to produce antibodies containing the 1 st and 2 nd antibody heavy chains and the 3 rd and 4 th antibody heavy chains; and
c. harvesting the at least two different antibodies from the culture.

2. The method of claim 1 , further comprising providing the host cell with a nucleic acid molecule encoding a common light chain.

3. The method of claim 1 , wherein the 1 st antibody heavy chain comprises a substitution of the amino acid residue at position 366 in the CH3 domain according to the EU numbering system by a lysine (K) residue, and wherein the 2 nd antibody heavy chain comprises a substitution of the amino acid residue at position 351 in the CH3 domain according to the EU numbering system by an aspartic acid (D) residue.

4. The method of claim 3 , wherein the CH3-domain of the 1 st antibody heavy chain further comprises a substitution of the amino acid residue at position 351 according to the EU numbering system by a lysine (K) residue.

5. The method of claim 4 , wherein the CH3 domain of the 2 nd antibody heavy chain further comprises amino acid substitution(s) selected from the group consisting of
(i) a substitution of the amino acid residue at position 349 according to the EU numbering system by a glutamic acid (E) residue;
(ii) a substitution of the amino acid residue at position 349 according to the EU numbering system by an aspartic acid (D) residue;
(iii) a substitution of the amino acid residue at position 368 according to the EU numbering system by a glutamic acid (E) residue;
(iv) a substitution of the amino acid residue at position 349 according to the EU numbering system by an aspartic acid (D) residue, and a substitution of the amino acid residue at position 368 according to the EU numbering system by a glutamic acid (E) residue; and
(v) substitution of the amino acid residues at positions 349 and 355 according to the EU numbering system by aspartic acid (D) residues.

6. The method of claim 5 , wherein the CH3 domain of the 2 nd antibody heavy chain comprises the substitution of the amino acid residue at position 368 according to the EU numbering system by a glutamic acid (E) residue.

7. The method of claim 1 , wherein the CH3 domain of the 1 st antibody heavy chain comprises substitutions of the amino acid residues at positions 366 and 351 according to the EU numbering system by a lysine (K) residue, and the CH3 domain of the 2 nd antibody heavy chain comprises a substitution of the amino acid residue at position 351 according to the EU numbering system by an aspartic acid (D) residue and a substitution of the amino acid residue at position 368 according to the EU numbering system by a glutamic acid (E) residue.

8. The method of claim 1 , wherein the CH3 domain of the 3 rd antibody heavy chain comprises the substitution of the amino acid residue at position 356 according to the EU numbering system by a lysine (K) reside and the substitution of the amino acid residue at position 399 according to the EU numbering system by a lysine (K) residue, and the 4 th antibody heavy chain comprises the substitution of the amino acid residue at position 392 according to the EU numbering system by an aspartic acid (D) residue, and the substitution of the amino acid residue at position 409 according to the EU numbering system by an aspartic (D) residue.

9. The method of claim 1 , wherein at least two of the variable regions of the 1 st , 2 nd , 3 rd and 4 th antibody heavy chains recognize different target epitopes.

10. The method of claim 1 , wherein each of the variable regions of the 1 st , 2 nd , 3 rd and 4 th antibody heavy chains recognize different target epitopes.

11. The method of claim 9 , wherein the variable regions of the 1 st and the 2 nd antibody heavy chains recognize different target epitopes, whereas the variable regions of the 3 rd and the 4 th antibody heavy chains recognize the same target epitope.

12. The method of claim 9 , wherein the target epitope recognized by the variable regions of the 3 rd and 4 th antibody heavy chain is the same as one of the target epitopes recognized by the variable region of the 1 st or the 2 nd antibody heavy chain.

13. The method of claim 11 , wherein the target epitope recognized by the variable regions of the 3 rd and 4 th antibody heavy chain is different from the target epitope recognized by the variable region of the 1 st or the 2 nd antibody heavy chain.

14. The method of claim 1 , wherein the variable regions of the 1 st and the 2 nd antibody heavy chains recognize the same target epitope, whereas the variable regions of the 3 rd and the 4 th antibody heavy chains recognize a second target epitope which differs from the target epitope recognized by the 1 st and 2 nd variable regions.

15. The method of claim 9 , wherein the at least two different target epitopes are located on the same target molecule.

16. The method of claim 15 , wherein the target molecule is a soluble molecule.

17. The method of claim 15 wherein the target molecule is a membrane-bound molecule.

18. The method of claim 9 , wherein the at least two different target epitopes are located on different target molecules.

19. The method of claim 18 , wherein the different target molecules are expressed on the same cells.

20. The method of claim 18 , wherein the different target molecules are expressed on different cells.

21. The method of claim 18 , wherein the different target molecules are soluble molecules.

22. The method of claim 18 , wherein one target molecule is a soluble molecule whereas the second target molecule is a membrane bound molecule.

23. The method of claim 1 , wherein
a. the CH3 domain of the 3 rd and 4 th antibody heavy chains are both wild type;
b. the CH3 domain of the 3 rd antibody heavy chain comprises a substitution of the amino acid residues at positions 356 and 399 according to the EU numbering system by a lysine (K) residue, and the CH3 domain of the 4 th antibody heavy chain comprises a substitution of the amino acid residues at positions 392 and 409 according to the EU numbering system by an aspartic acid (D) residue;
c. the CH3 domain of the 3 rd antibody heavy chain comprises a substitution of the amino acid residues at positions 356 and 399 according to the EU

Assignees

• Merus Nv

Inventors

• De Kruif Cornelis Adriaan
• Hendriks Linda Johanna Aleida
• Logtenberg Ton

Classifications

• A61P37/02

  Immunomodulators · CPC title

• A61K39/3955

  against proteinaceous materials, e.g. enzymes, hormones, lymphokines · CPC title

• C12P21/005 Primary

  Glycopeptides, glycoproteins · CPC title

• C07K2317/94

  Stability, e.g. half-life, pH, temperature or enzyme-resistance · CPC title

• C07K2317/31

  multispecific · CPC title