Methods and means for the production of ig-like molecules
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.
1. A method for producing at least two different antibodies in a single host cell, said method comprising,
providing a host cell comprising
a first nucleic acid molecule encoding a 1st antibody heavy chain comprising at least two substitutions of (i) D399R or D399K and (ii) K409E or K409D in the CH3 domain, and
a second nucleic acid molecule encoding a 2nd antibody heavy chain comprising at least one substitution of (i) D399R or D399K and (ii) K409E or K409D in the CH3 domain;
a third nucleic acid molecule encoding a 3rd antibody heavy chain, and
a fourth nucleic acid molecule encoding a 4th antibody heavy chain,
culturing said host cell and allowing for expression of said antibody heavy chains, wherein the CH3 domain of said 1st antibody heavy chain preferentially pairs with the CH3 domain of said 2nd antibody heavy chain, and the CH3 domain of said 3rd antibody heavy chain preferentially pairs with the CH3 domain of said 4th antibody heavy chain to produce antibodies containing said 1st and 2nd antibody heavy chains and said 3rd and 4th antibody heavy chains; and
harvesting said at least two different antibodies from the culture.
2. The method of claim 1 , wherein said 1st antibody heavy chain further comprises the substitution K392D or K392E.
3. The method of claim 1 , further comprising providing said host cell with a nucleic acid encoding a common light chain.
4. The method of claim 1 , wherein each of the encoded CH3 domain-comprising polypeptide chains further comprises a variable region recognizing an epitope.
5. A mixture of at least two different antibodies obtainable by the methods according to claim 1 .
6. A mixture according to claim 5 , wherein said at least two different antibodies comprise at least two monospecific antibodies.
7. A recombinant host cell comprising one or more nucleic acids encoding at least a 1st, a 2nd, a 3rd and a 4th antibody heavy chain, wherein the 1st antibody heavy chain comprises at least two substitutions of (i) D399R or D399K and (ii) K409E or K409D in the CH3 domain, and wherein the 2nd antibody heavy chain comprises at least one substitution of (i) D399R or D399K and (ii) K409E or K409D in the CH3 domain.
8. A recombinant host cell according to claim 7 , wherein said 1st antibody heavy chain further comprises the substitution K392D or K392E.
9. A recombinant host cell according to claim 7 , wherein said host cell further comprises a nucleic acid encoding a common light chain.
10. A pharmaceutical composition comprising a mixture of at least two different antibodies according to claim 5 , and a pharmaceutically acceptable carrier.
11. A pharmaceutical composition comprising at least two different antibodies have been produced by recombinant host cells according to claim 7 .
12. A method for making a host cell for production of at least two different antibodies, the method comprising the step of introducing into said host cell one or more nucleic acids encoding at least a 1st, a 2nd, a 3rd and a 4th antibody heavy chain, wherein the 1st antibody heavy chain comprises at least two substitutions of (i) D399R or D399K and (ii) K409E or K409D in the CH3 domain, and wherein the 2nd antibody heavy chain comprises at least one substitution of (i) D399R or D399K and (ii) K409E or K409D in the CH3 domain.
13. A method according to claim 12 , wherein said 1st antibody heavy chain further comprises the substitution K392D or K392E.
14. A method according to claim 12 , further comprising the step of introducing into said host cell a nucleic acid encoding a common light chain.
15. A culture of recombinant host cells obtained by the method according to claim 12 .