METHOD FOR THE GENERATION OF A MULTIVALENT, MULTISPECIFIC ANTIBODY EXPRESSING CELLS BY TARGETED INTEGRATION OF MULTIPLE EXPRESSION CASSETTES IN A DEFINED ORGANIZATION

Abstract

Disclosed herein, in part, are methods for the expression and production of a trivalent, bispecific antibody using a recombinant nucleic acid comprising multiple, different expression cassettes in a specific and defined sequence, which are stably integrated into the genome of a mammalian cell.by targeted integration.

Claims

1. A method for producing a trivalent, bispecific antibody comprising the steps of: a) cultivating a mammalian cell comprising a deoxyribonucleic acid encoding the trivalent, bispecific antibody, and b) recovering the trivalent, bispecific antibody from the cell or the cultivation medium, wherein, the deoxyribonucleic acid encoding the trivalent, bispecific antibody is stably integrated into the genome of the mammalian cell and comprises in a 5′- to 3′-direction: a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding either the first light chain or the second heavy chain or a second light chain, and a seventh expression cassette encoding the second light chain.

2. A deoxyribonucleic acid encoding a trivalent, bispecific antibody comprising in a 5′- to 3′-direction: a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding either the first light chain or the second heavy chain or a second light chain, and a seventh expression cassette encoding the second light chain.

3. Use of a deoxyribonucleic acid comprising in a 5′- to 3′-direction: a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding either the first light chain or the second heavy chain or a second light chain, and a seventh expression cassette encoding the second light chain, for the expression of a trivalent, bispecific antibody in a mammalian cell.

4. A recombinant mammalian cell comprising a deoxyribonucleic acid encoding a trivalent, bispecific antibody integrated in the genome of the cell, wherein the deoxyribonucleic acid encoding the trivalent, bispecific antibody comprises, in a 5′- to 3′-direction: a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding either the first light chain or the second heavy chain or a second light chain, and a seventh expression cassette encoding the second light chain.

5. A composition comprising a first deoxyribonucleic acid, each of which comprise three different recombination recognition sequences and four expression cassettes, wherein: the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence, and the second deoxyribonucleic acid comprises in a 5′- to 3′-direction a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding either the first light chain or the second heavy chain or a second light chain, a seventh expression cassette encoding the second light chain, and the second recombination recognition sequence.

6. A method for producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a trivalent, bispecific antibody and secreting the trivalent, bispecific antibody, comprising the following steps: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the mammalian cell, wherein the exogenous nucleotide sequence comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and all the recombination recognition sequences are different; b) introducing into the cell provided in a) a composition of two deoxyribonucleic acids comprising three different recombination recognition sequences and at least seven expression cassettes, wherein: the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence, and, the second deoxyribonucleic acid comprises in a 5′- to 3′-direction: a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding either the first light chain or the second heavy chain or a second light chain, a seventh expression cassette encoding the second light chain, and the second recombination recognition sequence, wherein the first to third recombination recognition sequences of the first and second deoxyribonucleic acids are matching the first to third recombination recognition sequence on the integrated exogenous nucleotide sequence, wherein, the 5′-terminal part and the 3′-terminal part of the expression cassette encoding the second selection marker taken together form a functional expression cassette of the second selection marker; c) introducing i) either simultaneously with the first and second deoxyribonucleic acid of b); or ii) sequentially thereafter one or more recombinases, wherein the one or more recombinases recognize the recombination recognition sequences of the first and the second deoxyribonucleic acid; (and optionally wherein the one or more recombinases perform two recombinase mediated cassette exchanges;) and d) selecting for cells expressing the second selection marker and secreting the trivalent, bispecific antibody, thereby producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding the trivalent, bispecific antibody and secreting the trivalent, bispecific antibody.

7-17. (canceled)

18. A method for producing a trivalent antibody comprising the steps of: a) cultivating a mammalian cell comprising a deoxyribonucleic acid encoding the trivalent antibody, and b) recovering the trivalent antibody from the cell or the cultivation medium, wherein the deoxyribonucleic acid encoding the trivalent antibody is stably integrated into the genome of the mammalian cell and comprises in a 5′- to 3′-direction: either, (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, and a fifth expression cassette encoding a second light chain, or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, or (3) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the second light chain, or (4) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the first heavy chain, a sixth expression cassette encoding the second light chain, or (5) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the second heavy chain, a sixth expression cassette encoding the second light chain, a seventh expression cassette encoding the second light chain, or (6) a first expression cassette encoding a first light chain, a second expression cassette encoding a first light chain, a third expression cassette encoding a first heavy chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain.

19. A deoxyribonucleic acid encoding a trivalent antibody comprising in a 5′- to 3′-direction: either (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, and a fifth expression cassette encoding a second light chain, or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, or (3) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the second light chain, or (4) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the first heavy chain, a sixth expression cassette encoding the second light chain, or (5) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the second heavy chain, a sixth expression cassette encoding the second light chain, a seventh expression cassette encoding the second light chain, or (6) a first expression cassette encoding a first light chain, a second expression cassette encoding a first light chain, a third expression cassette encoding a first heavy chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain.

20. Use of a deoxyribonucleic acid comprising in a 5′- to 3′-direction: either (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, and a fifth expression cassette encoding a second light chain, or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, or (3) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the second light chain, or (4) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the first heavy chain, a sixth expression cassette encoding the second light chain, or (5) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the second heavy chain, a sixth expression cassette encoding the second light chain, a seventh expression cassette encoding the second light chain, or (6) a first expression cassette encoding a first light chain, a second expression cassette encoding a first light chain, a third expression cassette encoding a first heavy chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, for the expression of the trivalent antibody in a mammalian cell.

21. A recombinant mammalian cell comprising a deoxyribonucleic acid encoding a trivalent antibody integrated in the genome of the cell, wherein the deoxyribonucleic acid encoding the trivalent antibody comprises in a 5′- to 3′-direction: either (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, and a fifth expression cassette encoding a second light chain, or (2) a first expression cassette encoding the first heavy chain, a second expression cassette encoding the first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the second heavy chain, a fifth expression cassette encoding the second light chain, a sixth expression cassette encoding the second light chain, or (3) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding a second light chain, or (4) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the first heavy chain, a sixth expression cassette encoding the second light chain, or (5) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, a fifth expression cassette encoding the second heavy chain, a sixth expression cassette encoding the second light chain, a seventh expression cassette encoding the second light chain, or (6) a first expression cassette encoding a first light chain, a second expression cassette encoding a first light chain, a third expression cassette encoding a first heavy chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain.

22. A composition comprising a first deoxyribonucleic acid and a second deoxyribonucleic acid, each of which comprise three different recombination recognition sequences and five to seven expression cassettes, wherein: the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: either (1) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence; or (2) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence; or (3) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, and a first copy of a third recombination recognition sequence; or (4) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, and a first copy of a third recombination recognition sequence; or (5) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, and a first copy of a third recombination recognition sequence; or (6) a first recombination recognition sequence, a first expression cassette encoding a first light chain, a second expression cassette encoding the first light chain, a third expression cassette encoding a first heavy chain, and a first copy of a third recombination recognition sequence; and, the second deoxyribonucleic acid comprises in a 5′- to 3′-direction: either (1) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, and a second recombination recognition sequence; or (2) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, and a second recombination recognition sequence; or (3) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second light chain, and a second recombination recognition sequence; or (4) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a first heavy chain, a sixth expression cassette encoding a second light chain, and a second recombination recognition sequence; or (5) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding a second light chain, a seventh expression cassette encoding the second light chain, and a second recombination recognition sequence; or (6) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, and a second recombination recognition sequence.

23. A method for producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a trivalent antibody and secreting the trivalent antibody, comprising the following steps: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the mammalian cell, wherein the exogenous nucleotide sequence comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and wherein all the recombination recognition sequences are different; b) introducing into the cell provided in a) a composition of two deoxyribonucleic acids comprising three different recombination recognition sequences and five to seven expression cassettes, wherein: the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: either (1) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence; or (2) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence; or (3) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, and a first copy of a third recombination recognition sequence; or (4) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, and a first copy of a third recombination recognition sequence; or (5) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a second heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding a second light chain, and a first copy of a third recombination recognition sequence; or (6) a first recombination recognition sequence, a first expression cassette encoding a first light chain, a second expression cassette encoding the first light chain, a third expression cassette encoding a first heavy chain, and a first copy of a third recombination recognition sequence; and,  the second deoxyribonucleic acid comprises in a 5′- to 3′-direction: either (1) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, and a second recombination recognition sequence; or (2) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, and a second recombination recognition sequence; or (3) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second light chain, and a second recombination recognition sequence; or (4) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a first heavy chain, a sixth expression cassette encoding a second light chain, and a second recombination recognition sequence; or (5) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding a second light chain, a seventh expression cassette encoding the second light chain, and a second recombination recognition sequence; or (6) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a second light chain, a sixth expression cassette encoding the second light chain, and a second recombination recognition sequence; wherein the first to third recombination recognition sequences of the first and second deoxyribonucleic acids match the first to third recombination recognition sequence on the integrated exogenous nucleotide sequence, wherein, the 5′-terminal part and the 3′-terminal part of the expression cassette encoding the second selection marker taken together form a functional expression cassette of the second selection marker; c) introducing i) one or more recombinases, and ii) the first and the second deoxyribonucleic acid of b); wherein, the recombinase(s) and the deoxyribonucleic acids are introduced either simultaneously or sequentially, and, d) selecting for the cells expressing the second selection marker and secreting the wherein the one or more recombinases recognize the recombination recognition sequences of the first and the second deoxyribonucleic acid; optionally wherein the one or more recombinases performs two recombinase mediated cassette exchanges; and d) selecting for cells expressing the second selection marker and secreting the trivalent antibody, thereby producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding the trivalent antibody and secreting the trivalent antibody.

24-34. (canceled)

35. A method for producing a bivalent, bispecific antibody comprising the steps of: a) cultivating a mammalian cell comprising a deoxyribonucleic acid encoding the bivalent, bispecific antibody, and b) recovering the bivalent, bispecific antibody from the cell or the cultivation medium, wherein the deoxyribonucleic acid encoding the bivalent, bispecific antibody is stably integrated into the genome of the mammalian cell and comprises in a 5′- to 3′-direction: a first expression cassette encoding a first light chain, a second expression cassette encoding a first heavy chain, a third expression cassette encoding a second light chain, and a fourth expression cassette encoding a second heavy chain, wherein the first heavy chain comprises in the CH3 domain the mutation T366W and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V.

36. A deoxyribonucleic acid encoding a bivalent, bispecific antibody comprising in a 5′- to 3′-direction: a first expression cassette encoding a first light chain, a second expression cassette encoding a first heavy chain, a third expression cassette encoding a second light chain, and a fourth expression cassette encoding a second heavy chain, wherein the first heavy chain comprises in the CH3 domain the mutation T366W and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V.

37. Use of a deoxyribonucleic acid comprising in a 5′- to 3′-direction: a first expression cassette encoding a first light chain, a second expression cassette encoding a first heavy chain, a third expression cassette encoding a second light chain, and a fourth expression cassette encoding a second heavy chain, for the expression of the bivalent, bispecific antibody in a mammalian cell, wherein the first heavy chain comprises in the CH3 domain the mutation T366W and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V.

38. A recombinant mammalian cell comprising a deoxyribonucleic acid encoding a bivalent, bispecific antibody integrated in the genome of the cell, wherein the deoxyribonucleic acid encoding the bivalent, bispecific antibody comprises in a 5′- to 3′-direction: a first expression cassette encoding a first light chain, a second expression cassette encoding a first heavy chain, a third expression cassette encoding a second light chain, and a fourth expression cassette encoding a second heavy chain, wherein the first heavy chain comprises in the CH3 domain the mutation T366W and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V.

39. A composition comprising two deoxyribonucleic acids, which comprise in turn three different recombination recognition sequences and four expression cassettes, wherein: the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: a first recombination recognition sequence, a first expression cassette encoding a first light chain, a second expression cassette encoding a first heavy chain, and a first copy of a third recombination recognition sequence, and the second deoxyribonucleic acid comprises in a 5′- to 3′-direction: a second copy of the third recombination recognition sequence, a third expression cassette encoding a second light chain, a fourth expression cassette encoding a second heavy chain, and a second recombination recognition sequence, wherein the first heavy chain comprises in the CH3 domain the mutation T366W and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V.

40. A method for producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a bivalent, bispecific antibody and secreting the bivalent, bispecific antibody, comprising the following steps: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the mammalian cell, wherein the exogenous nucleotide sequence comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and all the recombination recognition sequences are different; b) introducing into the cell provided in a) a composition of two deoxyribonucleic acids comprising three different recombination recognition sequences and four expression cassettes, wherein the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: a first recombination recognition sequence, a first expression cassette encoding a first light chain, a second expression cassette encoding a first heavy chain, and a first copy of a third recombination recognition sequence, and the second deoxyribonucleic acid comprises in a 5′- to 3′-direction: a second copy of the third recombination recognition sequence, a third expression cassette encoding a second light chain, a fourth expression cassette encoding a second heavy chain, and a second recombination recognition sequence, wherein the first to third recombination recognition sequences of the first and second deoxyribonucleic acids are matching the first to third recombination recognition sequence on the integrated exogenous nucleotide sequence, wherein, the 5′-terminal part and the 3′-terminal part of the expression cassette encoding the second selection marker taken together form a functional expression cassette of the second selection marker; wherein the first heavy chain comprises in the CH3 domain the mutation T366W and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V; c) introducing i) either simultaneously with the first and second deoxyribonucleic acid of b); or ii) sequentially thereafter one or more recombinases, wherein the one or more recombinases recognize the recombination recognition sequences of the first and the second deoxyribonucleic acid; (and optionally wherein the one or more recombinases perform two recombinase mediated cassette exchanges;) and d) selecting for cells expressing the second selection marker and secreting the bivalent, bispecific antibody; thereby producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding the bivalent, bispecific antibody and secreting the bivalent, bispecific antibody.

41-48. (canceled)

49. A method for producing a multivalent, bispecific antibody comprising the steps of: a) cultivating a mammalian cell comprising a deoxyribonucleic acid encoding the multivalent, bispecific antibody, and b) recovering the multivalent, bispecific antibody from the cell or the cultivation medium, wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody is stably integrated into the genome of the mammalian cell and comprises in a 5′- to 3′-direction: either (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding the first light chain, and a sixth expression cassette encoding the first light chain; or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding the first light chain, a seventh expression cassette encoding the first light chain, and an eighth expression cassette encoding the first light chain.

50. A deoxyribonucleic acid encoding a multivalent, bispecific antibody comprising in a 5′- to 3′-direction: either (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding the first light chain, and a sixth expression cassette encoding the first light chain; or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding the first light chain, a seventh expression cassette encoding the first light chain, and an eighth expression cassette encoding the first light chain.

51. Use of a deoxyribonucleic acid comprising in a 5′- to 3′-direction: either (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding the first light chain, and a sixth expression cassette encoding the first light chain; or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding the first light chain, a seventh expression cassette encoding the first light chain, and an eighth expression cassette encoding the first light chain, for the expression of the multivalent, bispecific antibody in a mammalian cell.

52. A recombinant mammalian cell comprising a deoxyribonucleic acid encoding a multivalent, bispecific antibody integrated in the genome of the cell, wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises in a 5′- to 3′-direction: either (1) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding the first light chain, and a sixth expression cassette encoding the first light chain; or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding the first light chain, a seventh expression cassette encoding the first light chain, and an eighth expression cassette encoding the first light chain.

53. A composition comprising two deoxyribonucleic acids, which comprise in turn three different recombination recognition sequences and six or eight expression cassettes, wherein the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: either (1) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence; or (2) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence, and the second deoxyribonucleic acid comprises in a 5′- to 3′-direction: either (1) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a first light chain, a sixth expression cassette encoding the first light chain, and a second recombination recognition sequence; or (2) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding a first light chain, a seventh expression cassette encoding the first light chain, an eighth expression cassette encoding the first light chain, and a second recombination recognition sequence.

54. A method for producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a multivalent, bispecific antibody and secreting the multivalent, bispecific antibody, comprising the following steps: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the mammalian cell, wherein the exogenous nucleotide sequence comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and wherein the first, second and third recombination recognition sequences are different; b) introducing into the mammalian cell provided in a) a composition of a first deoxyribonucleic acid and a second deoxyribonucleic acid, each comprising three different recombination recognition sequences and six or eight expression cassettes, wherein the first deoxyribonucleic acid comprises in a 5′- to 3′-direction: either (1) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence; or (2) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding a first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the first light chain, and a first copy of a third recombination recognition sequence, and the second deoxyribonucleic acid which comprises in a 5′- to 3′-direction: either (1) a second copy of the third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding a first light chain, a sixth expression cassette encoding the first light chain, and a second recombination recognition sequence; or (2) a second copy of the third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, a sixth expression cassette encoding a first light chain, a seventh expression cassette encoding the first light chain, an eighth expression cassette encoding the first light chain, and a second recombination recognition sequence, wherein the first to third recombination recognition sequences of the first deoxyribonucleic acid and the second deoxyribonucleic acid are matching match the first to third recombination recognition sequences on the integrated exogenous nucleotide sequence, wherein, the 5′-terminal part and the 3′-terminal part of the expression cassette encoding the second selection marker taken together form a functional expression cassette of the second selection marker; c) introducing i) one or more recombinases, and ii) the first and the second deoxyribonucleic acid of b), wherein, the recombinase(s) and the deoxyribonucleic acids are introduced either simultaneously or sequentially, wherein, the one or more cre-recombinase is introduced as an mRNA, wherein the one or more cre-recombinases recognizes the recombination recognition sequences of the first and the second deoxyribonucleic acid; and optionally, wherein the one or more cre-recombinases performs two recombinase mediated cassette exchanges; and, d) selecting the cells expressing the second selection marker and secreting the multivalent, bispecific antibody, thereby producing the recombinant mammalian cell comprising deoxyribonucleic acid encoding the multivalent, bispecific antibody and secreting the multivalent, bispecific antibody.

55-63. (canceled)

64. A method for producing a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a polypeptide and secreting the polypeptide comprising the following steps: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the recombinant mammalian cell, wherein the exogenous nucleotide sequence comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, wherein, the first, second and third recombination recognition sequences are different, and wherein, the mammalian cell is free of Cre-recombinase encoding DNA; b) introducing into the mammalian cell provided in a): a composition of two deoxyribonucleic acids comprising the three different recombination recognition sequences and one to eight expression cassettes, wherein the first deoxyribonucleic acid comprises in a 5′- to 3′-direction, the first recombination recognition sequence, one or more expression cassette(s), a 5′-terminal part of an expression cassette encoding ene a second selection marker, and a first copy of the third recombination recognition sequence, and the second deoxyribonucleic acid comprises in a 5′- to 3′-direction, a second copy of the third recombination recognition sequence, a 3′-terminal part of an expression cassette encoding the second selection marker, one or more expression cassette(s), and the second recombination recognition sequence, wherein, the first to third recombination recognition sequences of the first and the second deoxyribonucleic acids match the first to third recombination recognition sequence on the integrated exogenous nucleotide sequence, wherein, the 5′-terminal part and the 3′-terminal part of the expression cassette encoding the second selection marker taken together form a functional expression cassette of the second selection marker, wherein the deoxyribonucleic acids are free of Cre-recombinase encoding DNA; c) introducing the first and the second deoxyribonucleic acid of b) either simultaneously together or sequentially with at least one cre-recombinase, wherein, the source of the at least one cre-recombinase is a Cre-recombinase mRNA, wherein, the Cre-recombinase recognizes the recombination recognition sequences of the first and the second deoxyribonucleic acid; and optionally, wherein the one or more cre-recombinases performs two recombinase mediated cassette exchanges; and, d) selecting the cells expressing the second selection marker and secreting the polypeptide, thereby producing the recombinant mammalian cell comprising the deoxyribonucleic acid encoding the polypeptide and secreting the polypeptide.

65-78. (canceled)

79. Use of a Cre-recombinase mRNA for increasing the number of recombinant mammalian cells comprising: a deoxyribonucleic acid encoding a polypeptide or protein of interest, wherein the deoxyribonucleic acid is stably integrated by targeted integration at a single site in the genome of said recombinant mammalian cell.

80. The use according to claim 79, wherein the recombinant cell further secretes the polypeptide or protein of interest into the cultivation medium.

Description

DESCRIPTION OF THE FIGURES

[2316] FIG. 1: Scheme of a two-plasmid RMCE strategy involving the use of three RRS sites to carry out two independent RMCEs simultaneously.

[2317] FIG. 2: Viability recovery after TI with Cre DNA and Cre mRNA.

[2318] FIG. 3: Exchange efficiency/pool quality after TI with Cre DNA/plasmid; size outer area: 687 AU; size middle area: 132 AU; size inner area: 27 AU.

[2319] FIG. 4: Exchange efficiency/pool quality after TI with and Cre mRNA; size outer area: 812 AU; size middle area: 114 AU; size inner area: 32 AU.

DESCRIPTION OF THE SEQUENCES

[2320] SEQ ID NO: 01: exemplary sequence of an L3 recombinase recognition sequence [2321] SEQ ID NO: 02: exemplary sequence of a 2L recombinase recognition sequence [2322] SEQ ID NO: 03: exemplary sequence of a LoxFas recombinase recognition sequence [2323] SEQ ID NO: 04-06: exemplary variants of human CMV promoter [2324] SEQ ID NO: 07: exemplary SV40 polyadenylation signal sequence [2325] SEQ ID NO: 08: exemplary bGH polyadenylation signal sequence [2326] SEQ ID NO: 09: exemplary hGT terminator sequence [2327] SEQ ID NO: 10: exemplary SV40 promoter sequence [2328] SEQ ID NO: 11: exemplary GFP nucleic acid sequence [2329] SEQ ID NO: 12: anti-human Abeta/human transferrin receptor trivalent, bispecific antibody first heavy chain [2330] SEQ ID NO: 13: anti-human Abeta/human transferrin receptor trivalent, bispecific antibody second heavy chain [2331] SEQ ID NO: 14: anti-human Abeta/human transferrin receptor trivalent, bispecific antibody first light chain [2332] SEQ ID NO: 15: anti-human Abeta/human transferrin trivalent, bispecific receptor antibody second light chain [2333] SEQ ID NO: 16: anti-human CD20/human transferrin receptor trivalent, bispecific antibody first heavy chain [2334] SEQ ID NO: 17: anti-human CD20/human transferrin receptor trivalent, bispecific antibody second heavy chain [2335] SEQ ID NO: 18: anti-human CD20/human transferrin receptor trivalent, bispecific antibody first light chain [2336] SEQ ID NO: 19: anti-human CD20/human transferrin receptor trivalent, bispecific antibody second light chain [2337] SEQ ID NO: 20: Cre-recombinase amino acid sequence [2338] SEQ ID NO: 21: minimal Cre-Recombinase mRNA [2339] SEQ ID NO: 22: lox-site palindromic sequence 1 [2340] SEQ ID NO: 23: lox-site palindromic sequence 2 [2341] SEQ ID NO: 24: core sequence lox-site wild-type [2342] SEQ ID NO: 25: core sequence lox-site mutant L3 [2343] SEQ ID NO: 26: core sequence lox-site mutant 2L [2344] SEQ ID NO: 27: core sequence lox-site mutant LoxFas [2345] SEQ ID NO: 28: core sequence lox-site mutant Lox511 [2346] SEQ ID NO: 29: core sequence lox-site mutant Lox5171 [2347] SEQ ID NO: 30: core sequence lox-site mutant Lox2272 [2348] SEQ ID NO: 31: core sequence lox-site mutant M2 [2349] SEQ ID NO: 32: core sequence lox-site mutant M3 [2350] SEQ ID NO: 33: exemplary nuclear localization sequence [2351] SEQ ID NO: 34: exemplary nuclear localization sequence [2352] SEQ ID NO: 35: exemplary nuclear localization sequence [2353] SEQ ID NO: 36: exemplary nuclear localization sequence [2354] SEQ ID NO: 37: exemplary nuclear localization sequence

EXAMPLES

Example 1

General Techniques

1) Recombinant DNA Techniques

[2355] Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989). The molecular biological reagents were used according to the manufacturer's instructions.

2) DNA Sequence Determination

[2356] DNA sequencing was performed at SequiServe GmbH (Vaterstetten, Germany)

3) DNA and Protein Sequence Analysis and Sequence Data Management

[2357] The EMBOSS (European Molecular Biology Open Software Suite) software package and Invitrogen's Vector NTI version 11.5 or Geneious prime were used for sequence creation, mapping, analysis, annotation and illustration.

4) Gene and Oligonucleotide Synthesis

[2358] Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

5) Reagents

[2359] All commercial chemicals, antibodies and kits were used as provided according to the manufacturer's protocol if not stated otherwise.

6) Cultivation of TI Host Cell Line

[2360] TI CHO host cells were cultivated at 37° C. in a humidified incubator with 85% humidity and 5% CO.sub.2. They were cultivated in a proprietary DMEM/F12-based medium containing 300 μg/ml Hygromycin B and 4 μg/ml of a second selection marker. The cells were splitted every 3 or 4 days at a concentration of 0.3×10E6 cells/ml in a total volume of 30 ml. For the cultivation 125 ml non-baffle Erlenmeyer shake flasks were used. Cells were shaken at 150 rpm with a shaking amplitude of 5 cm. The cell count was determined with Cedex HiRes Cell Counter (Roche). Cells were kept in culture until they reached an age of 60 days.

7) Cloning

General

[2361] Cloning with R-sites depends on DNA sequences next to the gene of interest (GOI) that are equal to sequences lying in following fragments. Like that, assembly of fragments is possible by overlap of the equal sequences and subsequent sealing of nicks in the assembled DNA by a DNA ligase. Therefore, a cloning of the single genes in particular preliminary vectors containing the right R-sites is necessary. After successful cloning of these preliminary vectors the gene of interest flanked by the R-sites is cut out via restriction digest by enzymes cutting directly next to the R-sites. The last step is the assembly of all DNA fragments in one step. In more detail, a 5′-exonuclease removes the 5′-end of the overlapping regions (R-sites). After that, annealing of the R-sites can take place and a DNA polymerase extends the 3′-end to fill the gaps in the sequence. Finally, the DNA ligase seals the nicks in between the nucleotides. Addition of an assembly master mix containing different enzymes like exonucleases, DNA polymerases and ligases, and subsequent incubation of the reaction mix at 50° C. leads to an assembly of the single fragments to one plasmid. After that, competent E. coli cells are transformed with the plasmid.

[2362] For some vectors, a cloning strategy via restriction enzymes was used. By selection of suitable restriction enzymes, the wanted gene of interest can be cut out and afterwards inserted into a different vector by ligation. Therefore, enzymes cutting in a multiple cloning site (MCS) are preferably used and chosen in a smart manner, so that a ligation of the fragments in the correct array can be conducted. If vector and fragment are previously cut with the same restriction enzyme, the sticky ends of fragment and vector fit perfectly together and can be ligated by a DNA ligase, subsequently. After ligation, competent E. coli cells are transformed with the newly generated plasmid.

Cloning Via Restriction Digestion

[2363] For the digest of plasmids with restriction enzymes the following components were pipetted together on ice:

TABLE-US-00009 TABLE Restriction Digestion Reaction Mix component ng (set point) μl purified DNA tbd tbd CutSmart Buffer (10x) 5 Restriction Enzyme 1 PCR-grade Water ad 50 Total 50 

[2364] If more enzymes were used in one digestion, 1 μl of each enzyme was used and the volume adjusted by addition of more or less PCR-grade water. All enzymes were selected on the preconditions that they are qualified for the use with CutSmart buffer from new England Biolabs (100% activity) and have the same incubation temperature (all 37° C.).

[2365] Incubation was performed using thermomixers or thermal cyclers, allowing to incubate the samples at a constant temperature (37° C.). During incubation the samples were not agitated. Incubation time was set at 60 min. Afterwards the samples were directly mixed with loading dye and loaded onto an agarose electrophoresis gel or stored at 4° C./on ice for further use.

[2366] A 1% agarose gel was prepared for gel electrophoresis. Therefor 1.5 g of multi-purpose agarose were weighed into a 125 Erlenmeyer shake flask and filled up with 150 ml TAE-buffer. The mixture was heated up in a microwave oven until the agarose was completely dissolved. 0.5 μg/ml ethidium bromide were added into the agarose solution. Thereafter the gel was cast in a mold. After the agarose was set, the mold was placed into the electrophoresis chamber and the chamber filled with TAE-buffer. Afterwards the samples were loaded. In the first pocket (from the left) an appropriate DNA molecular weight marker was loaded, followed by the samples. The gel was run for around 60 minutes at <130V. After electrophoresis the gel was removed from the chamber and analyzed in an UV-Imager.

[2367] The target bands were cut and transferred to 1.5 ml Eppendorf tubes. For purification of the gel, the QIAquick Gel Extraction Kit from Qiagen was used according to the manufacturer's instructions. The DNA fragments were stored at −20° C. for further use.

[2368] The fragments for the ligation were pipetted together in a molar ratio of 1:2, 1:3 or 1:5 vector to insert, depending on the length of the inserts and the vector-fragments and their correlation to each other. If the fragment, that should be inserted into the vector was short, a 1:5-ratio was used. If the insert was longer, a smaller amount of it was used in correlation to the vector. An amount of 50 ng of vector were used in each ligation and the particular amount of insert calculated with NEBioCalculator. For ligation, the T4 DNA ligation kit from NEB was used. An example for the ligation mixture is depicted in the following Table:

TABLE-US-00010 TABLE Ligation Reaction Mix component ng (set point) conc. [ng/μl] μl T4 DNA Ligase Buffer (10x) 2 Vector DNA (4000 bp) 50 50 1 Insert DNA (2000 bp) 125 20 6.25 Nuclease-free Water 9.75 T4 Ligase 1 Total 20

[2369] All components were pipetted together on ice, starting with the mixing of DNA and water, addition of buffer and finally addition of the enzyme. The reaction was gently mixed by pipetting up and down, briefly microfuged and then incubated at room temperature for 10 minutes. After incubation, the T4 ligase was heat inactivated at 65° C. for 10 minutes. The sample was chilled on ice. In a final step, 10-beta competent E. coli cells were transformed with 2 μl of the ligated plasmid (see below).

Cloning Via R-Site Assembly

[2370] For assembly, all DNA fragments with the R-sites at each end were pipetted together on ice. An equimolar ratio (0.05 ng) of all fragments was used, as recommended by the manufacturer, when more than 4 fragments are being assembled. One half of the reaction mix was embodied by NEBuilder HiFi DNA Assembly Master Mix. The total reaction volume was 40 μl and was reached by a fill-up with PCR-clean water. In the following Table an exemplary pipetting scheme is depicted.

TABLE-US-00011 TABLE Assembly Reaction Mix pmol ng conc. component bp (set point) (set point) [ng/μl] μl Insert 1 2800 0.05 88.9 21 4.23 Insert 2 2900 0.05 90.5 35 2.59 Insert 3 4200 0.05 131.6 35.5 3.71 Insert 4 3600 0.05 110.7 23 4.81 Vector 4100 0.05 127.5 57.7 2.21 NEBuilder HiFi DNA 20 Assembly Master Mix PCR-clean Water 2.45 Total 40

[2371] After set up of the reaction mixture, the tube was incubated in a thermocycler at constantly 50° C. for 60 minutes. After successful assembly, 10-beta competent E. coli bacteria were transformed with 2 μl of the assembled plasmid DNA (see below).

Transformation 10-Beta Competent E. coli Cells

[2372] For transformation the 10-beta competent E. coli cells were thawed on ice. After that, 2 μl of plasmid DNA were pipetted directly into the cell suspension. The tube was flicked and put on ice for 30 minutes. Thereafter, the cells were placed into the 42° C.-warm thermal block and heat-shocked for exactly 30 seconds. Directly afterwards, the cells were chilled on ice for 2 minutes. 950 μl of NEB 10-beta outgrowth medium were added to the cell suspension. The cells were incubated under shaking at 37° C. for one hour. Then, 50-100 μl were pipetted onto a pre-warmed (37° C.) LB-Amp agar plate and spread with a disposable spatula. The plate was incubated overnight at 37° C. Only bacteria which have successfully incorporated the plasmid, carrying the resistance gene against ampicillin, can grow on this plates. Single colonies were picked the next day and cultured in LB-Amp medium for subsequent plasmid preparation.

Bacterial Culture

[2373] Cultivation of E. coli was done in LB-medium, short for Luria Bertani, that was spiked with 1 ml/L 100 mg/ml ampicillin resulting in an ampicillin concentration of 0.1 mg/ml. For the different plasmid preparation quantities, the following amounts were inoculated with a single bacterial colony.

TABLE-US-00012 TABLE E. coli cultivation volumes Quantity plasmid Volume LB-Amp Incubation preparation medium [ml] time [h] Mini-Prep 96-well (EpMotion) 1.5 23 Mini-Prep 15 ml-tube 3.6 23 Maxi-Prep 200 16

[2374] For Mini-Prep, a 96-well 2 ml deep-well plate was filled with 1.5 ml LB-Amp medium per well. The colonies were picked and the toothpick was tuck in the medium. When all colonies were picked, the plate closed with a sticky air porous membrane. The plate was incubated in a 37° C. incubator at a shaking rate of 200 rpm for 23 hours.

[2375] For Mini-Preps a 15 ml-tube (with a ventilated lid) was filled with 3.6 ml LB-Amp medium and equally inoculated with a bacterial colony. The toothpick was not removed but left in the tube during incubation. Like the 96-well plate the tubes were incubated at 37° C., 200 rpm for 23 hours.

[2376] For Maxi-Prep 200 ml of LB-Amp medium were filled into an autoclaved glass 1 L Erlenmeyer flask and inoculated with 1 ml of bacterial day-culture, that was roundabout 5 hours old. The Erlenmeyer flask was closed with a paper plug and incubated at 37° C., 200 rpm for 16 hours.

Plasmid Preparation

[2377] For Mini-Prep, 50 μl of bacterial suspension were transferred into a 1 ml deep-well plate. After that, the bacterial cells were centrifuged down in the plate at 3000 rpm, 4° C. for 5 min. The supernatant was removed and the plate with the bacteria pellets placed into an EpMotion. After ca. 90 minutes the run was done and the eluted plasmid-DNA could be removed from the EpMotion for further use.

[2378] For Mini-Prep, the 15 ml tubes were taken out of the incubator and the 3.6 ml bacterial culture splitted into two 2 ml Eppendorf tubes. The tubes were centrifuged at 6,800×g in a table-top microcentrifuge for 3 minutes at room temperature. After that, Mini-Prep was performed with the Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. The plasmid DNA concentration was measured with Nanodrop.

[2379] Maxi-Prep was performed using the Macherey-Nagel NucleoBond® Xtra Maxi EF Kit according to the manufacturer's instructions. The DNA concentration was measured with Nanodrop.

Ethanol Precipitation

[2380] The volume of the DNA solution was mixed with the 2.5-fold volume ethanol 100%. The mixture was incubated at −20° C. for 10 min. Then the DNA was centrifuged for 30 min. at 14,000 rpm, 4° C. The supernatant was carefully removed and the pellet washed with 70% ethanol. Again, the tube was centrifuged for 5 min. at 14,000 rpm, 4° C. The supernatant was carefully removed by pipetting and the pellet dried. When the ethanol was evaporated, an appropriate amount of endotoxin-free water was added. The DNA was given time to re-dissolve in the water overnight at 4° C. A small aliquot was taken and the DNA concentration was measured with a Nanodrop device.

Example 2

Plasmid Generation

Expression Cassette Composition

[2381] For the expression of an antibody chain a transcription unit comprising the following functional elements was used: [2382] the immediate early enhancer and promoter from the human cytomegalovirus including intron A, [2383] a human heavy chain immunoglobulin 5′-untranslated region (5′UTR), [2384] a murine immunoglobulin heavy chain signal sequence, [2385] a nucleic acid encoding the respective antibody chain, [2386] the bovine growth hormone polyadenylation sequence (BGH pA), and [2387] optionally the human gastrin terminator (hGT).

[2388] Beside the expression unit/cassette including the desired gene to be expressed the basic/standard mammalian expression plasmid contains [2389] an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and [2390] a beta-lactamase gene which confers ampicillin resistance in E. coli.

Front- and Back-Vector Cloning

[2391] To construct two-plasmid antibody constructs, antibody HC and LC fragments were cloned into a front vector backbone containing L3 and LoxFAS sequences, and a back vector containing LoxFAS and 2L sequences and a pac selectable marker. The Cre recombinase plasmid pOG231 (Wong, E. T., et al., Nuc. Acids Res. 33 (2005) e147; O'Gorman, S., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 14602-14607) was used for all RMCE processes.

[2392] The cDNAs encoding the respective antibody chains were generated by gene synthesis (Geneart, Life Technologies Inc.). The gene synthesis and the backbone-vectors were digested with HindIII-HF and EcoRI-HF (NEB) at 37° C. for 1 h and separated by agarose gel electrophoresis. The DNA-fragment of the insert and backbone were cut out from the agarose gel and extracted by QIAquick Gel Extraction Kit (Qiagen). The purified insert and backbone fragment was ligated via the Rapid Ligation Kit (Roche) following the manufacturer's protocol with an Insert/Backbone ratio of 3:1. The ligation approach was then transformed in competent E. coli DH5α via heat shock for 30 sec. at 42° C. and incubated for 1 h at 37° C. before they were plated out on agar plates with ampicillin for selection. Plates were incubated at 37° C. overnight.

[2393] On the following day clones were picked and incubated overnight at 37° C. under shaking for the Mini or Maxi-Preparation, which was performed with the EpMotion® 5075 (Eppendorf) or with the QIAprep Spin Mini-Prep Kit (Qiagen)/NucleoBond Xtra Maxi EF Kit (Macherey & Nagel), respectively. All constructs were sequenced to ensure the absence of any undesirable mutations (SequiServe GmbH).

[2394] In the second cloning step, the previously cloned vectors were digested with KpnI-HF/SalI-HF and SalI-HF/MfeI-HF with the same conditions as for the first cloning. The TI backbone vector was digested with KpnI-HF and MfeI-HF. Separation and extraction was performed as described above. Ligation of the purified insert and backbone was performed using T4 DNA Ligase (NEB) following the manufacturing protocol with an Insert/Insert/Backbone ratio of 1:1:1 overnight at 4° C. and inactivated at 65° C. for 10 min. The following cloning steps were performed as described above.

[2395] The cloned plasmids were used for the TI transfection and pool generation.

Example 3

Cultivation, Transfection, Selection, Pool Generation and Single Cell Cloning

[2396] TI host cells were propagated in disposable 125 ml vented shake flasks under standard humidified conditions (95% rH, 37° C., and 5% CO.sub.2) at a constant agitation rate of 150 rpm in a proprietary DMEM/F12-based medium. Every 3-4 days the cells were seeded in chemically defined medium containing selection marker 1 and selection marker 2 in effective concentrations with a concentration of 3×10E5 cells/ml. Density and viability of the cultures were measured with a Cedex HiRes cell counter (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

[2397] For stable transfection, equimolar amounts of front and back vector were mixed. 1 Cre expression plasmid or Cre mRNA was added to 5 μg of the mixture, i.e. 5 μg Cre expression plasmid or Cre mRNA was added to 25 μg of the front- and back-vector mixture.

[2398] Two days prior to transfection TI host cells were seeded in fresh medium with a density of 4×10E5 cells/ml. Transfection was performed with the Nucleofector device using the Nucleofector Kit V (Lonza, Switzerland), according to the manufacturer's protocol. 3×10E7 cells were transfected with 30 μg plasmid. After transfection the cells were seeded in 30 ml medium without selection agents.

[2399] On day 5 after seeding the cells were centrifuged and transferred to 80 mL chemically defined medium containing puromycin (selection agent 1) and 1-(2′-deoxy-2′-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil (FIAU; selection agent 2) at effective concentrations at 6×10E5 cells/ml for selection of recombinant cells. The cells were incubated at 37° C., 150 rpm. 5% CO2, and 85% humidity from this day on without splitting. Cell density and viability of the culture was monitored regularly. When the viability of the culture started to increase again, the concentrations of selection agents 1 and 2 were reduced to about half the amount used before. In more detail, to promote the recovering of the cells, the selection pressure was reduced if the viability is >40% and the viable cell density (VCD) is >0.5×10E6 cells/mL. Therefore, 4×10E5 cells/ml were centrifuged and resuspended in 40 ml selection media II (chemically-defined medium, ½ selection marker 1 & 2). The cells were incubated with the same conditions as before and also not splitted.

[2400] Ten days after starting selection, the success of Cre mediated cassette exchange was checked by flow cytometry measuring the expression of intracellular GFP and extracellular trivalent, bispecific antibody bound to the cell surface. An APC antibody (allophycocyanin-labeled F(ab′)2 Fragment goat anti-human IgG) against human antibody light and heavy chain was used for FACS staining. Flow cytometry was performed with a BD FACS Canto II flow cytometer (BD, Heidelberg, Germany). Ten thousand events per sample were measured. Living cells were gated in a plot of forward scatter (FSC) against side scatter (SSC). The live cell gate was defined with non-transfected TI host cells and applied to all samples by employing the FlowJo 7.6.5 EN software (TreeStar, Olten, Switzerland). Fluorescence of GFP was quantified in the FITC channel (excitation at 488 nm, detection at 530 nm). trivalent, bispecific antibody was measured in the APC channel (excitation at 645 nm, detection at 660 nm). Parental CHO cells, i.e. those cells used for the generation of the TI host cell, were used as a negative control with regard to GFP and trivalent, bispecific antibody expression. Fourteen days after the selection had been started, the viability exceeded 90% and selection was considered as complete.

[2401] In case Cre plasmid and Cre mRNA were used in comparison, after selection, the pool of stably transfected cells was subjected to single-cell cloning by limiting dilution. For this purpose, cells were stained with Cell Tracker Green™ (Thermo Fisher Scientific, Waltham, Mass.) and plated in 384-well plates with 0.6 cells/well. For single-cell cloning and all further cultivation steps selection agent 2 was omitted from the medium. Wells containing only one cell were identified by bright field and fluorescence based plate imaging. Only wells that contained one cell were further considered. Approximately three weeks after plating colonies were picked from confluent wells and further cultivated in 96-well plates. After four days in 96-well plates, the antibody titers in the culture medium were measured with an anti-human IgG sandwich ELISA. In brief, antibodies were captured from the cell culture fluid with an anti-human Fc antibody bound to a MaxiSorp microtiter plate (Nunc™, Sigma-Aldrich) and detected with an anti-human Fc antibody-POD conjugate which binds to an epitope different from the capture antibody. The secondary antibody was quantified by chemiluminescence employing the BM Chemiluminescence ELISA Substrate (POD) (Sigma-Aldrich).

Example 4

FACS Screening

[2402] FACS analysis was performed to check the transfection efficiency and the RMCE efficiency of the transfection. 4×10E5 cells of the transfected approaches were centrifuged (1200 rpm, 4 min.) and washed twice with 1 mL PBS. After the washing steps with PBS the pellet was resuspended in 400 μL PBS and transferred in FACS tubes (Falcon® Round-Bottom Tubes with cell strainer cap; Corning). The measurement was performed with a FACS Canto II and the data were analyzed by the software FlowJo.

Example 5

Fed-Batch Cultivation

[2403] Fed-batch production cultures were performed in shake flasks or Ambr15 vessels (Sartorius Stedim) with proprietary chemically defined medium. Cells were seeded at 1×10E6 cells/ml on day 0, with a temperature shift on day 3. Cultures received proprietary feed medium on days 3, 7, and 10. Viable cell count (VCC) and percent viability of cells in culture was measured on days 0, 3, 7, 10, and 14 using a Vi-Cell™ XR instrument (Beckman Coulter). Glucose and lactate concentrations were measured on days 7, 10 and 14 using a Bioprofile 400 Analyzer (Nova Biomedical). The supernatant was harvested 14 days after start of fed-batch by centrifugation (10 min, 1000 rpm and 10 min, 4000 rpm) and cleared by filtration (0.22 μm). Day 14 titers were determined using protein A affinity chromatography with UV detection.

[2404] Product quality was determined by Caliper's LabChip (Caliper Life Sciences).

Example 6

Effect of Vector Design on the Expression of a Trivalent, Bispecific Antibody

[2405] To examine the effect of expression cassette organization on productivity in the TI host, RMCE pools were generated by transfecting two plasmids (front and back vector) containing different numbers and organizations of the expression cassettes for the individual chains of a trivalent, bispecific antibody with additional Fab fragment with domain crossover/exchange. After selection, recovery, and verification of RMCE by flow cytometry, the pools' productivity was evaluated in a 14-day fed batch production assay.

[2406] The effect of the antibody chain expression cassette organization on expression of different trivalent, bispecific antibodies with additional Fab fragment with domain exchange was evaluated. All had a different targeting specificity.

[2407] For four BS-antibodies the following results have been obtained:

TABLE-US-00013 front vector back vector expression cassettes in expression cassettes in % MP eff. 5′- to 3′ direction 5′- to 3′ direction titer (CE- Titer BS No. 1 2 3 4 1 2 3 4 [g/L] SDS) [g/L] 1 k k xl xl h xl l l 0.8 63 0.50 1 k k xl xl h h l l 0.8 63 0.50 1 k k xl xl h l l — 0.6 61 0.37 1 k xl xl — h xl l l 0.75 46.5 0.35 1 k xl xl — h h l l 0.75 46.5 0.35 1 k xl xl — h l l — 0.7 44.5 0.31 2 k k xl xl h xl l l 1 74.5 0.75 2 k k xl xl h l l — 1 73.5 0.74 2 k xl xl — h l l — 1 53 0.53 3 k k xl xl h l l — 1.36 83 1.13 3 k k xl xl h xl l l 1 90 0.90 3 k xl xl — h l l — 0.95 70.5 0.67 4 k l l — h l — — 0.9 76 0.37 4 k l l — h l l — 0.8 65 0.30 MP = main product, eff. titer = effective titer = titer multiplied by % main product

Example 7

Effect of Vector Design on the Expression of a Bispecific, Trivalent Antibody

[2408] To examine the effect of expression cassette organization on productivity in the TI host, RMCE pools were generated by transfecting two plasmids (front and back vector) containing different numbers and organizations of the individual chains of a trivalent antibody in the TCB format. After selection, recovery, and verification of RMCE by flow cytometry, the pools' productivity was evaluated in a 14-day fed batch production assay. For specific vector organizations an increase in titer compared to the reference pools was observed.

[2409] The effect of the antibody chain expression cassette organization on expression of five different TCBs was evaluated. TCB 1 to 5 all had a different targeting specificity. TCB 3 was tested with 4 different anti-CD3 binding sites.

[2410] For TCB-1 the following results have been obtained; the reference organizations are shaded in grey:

TABLE-US-00014 front vector back vector expression cassettes in expression cassettes in % MP eff. 5′- to 3′ direction 5′- to 3′ direction titer (CE- Titer TCB No. 1 2 3 4 1 2 3 4 [g/L] SDS) [g/L] 1 <xl <xl <k — h l l — 3.75 81 3.04 1 k k xl xl h l — — 1.5 37.5 0.56 1 k xl xl — h l — — 3 75 2.25 1 k h xl l k l — — 2.75 81.5 2.24 1 l xl k h h l l — 2.09 56.3 0.98 1 k h xl l h l l — 2.6 80 2.08 1 k xl xl — h l l — 3.35 63 2.11 1 k h xl l — — — — 1.98 89.6 1.58 ref. 1 k xl — — h l l — 1.9 74 1.41 1 k xl — — h l — — 1 12 0.12 ref. 1 k h xl l l — — — 2.04 89.7 1.63 1 k h xl l — — — — 1.98 89.6 1.58 ref. 1 k xl — — h l — — 1 12 0.12 ref. 1 k h — — xl l — — 1 72 0.72 ref. 1 k h xl l k l — — 2.75 81.5 2.24 1 k h xl l l — — — 2.04 89.7 1.63 1 k h xl l xl — — — 1.75 89.6 1.39 1 k h xl l k — — — 1.72 89.4 1.38 1 k h xl l h — — — 1.96 89.7 1.56 1 k h xl l — — — — 1.98 89.6 1.58 ref. 1 l xl k h h l l — 2.09 56.3 0.98 1 k h xl l — — — — 1.98 89.6 1.58 ref. 1 l xl k h — — — — 1.67 92.5 1.34 ref.

[2411] For TCB-3 the following results have been obtained:

TABLE-US-00015 front vector back vector expression cassettes in expression cassettes in % MP eff. 5′- to 3′ direction 5′- to 3′ direction titer (CE- Titer TCB No. 1 2 3 4 1 2 3 4 [g/L] SDS) [g/L] 3-1 k l l — xl h — — 3.5 95 3.33 3-1 k xl l — xl h — — 3 95 2.85 3-1 k xl l — h l l — 3.7 68 2.52 3-1 k xl xl — h l l — 3 65 1.95 3-2 k l l — xl h — — 2.95 95 2.8 3-2 k xl l — xl h — — 2.6 93 2.42 3-2 k xl l — h l l — 2.5 72 1.8 3-2 k xl xl — h l l — 3.1 70 2.17 3-3 k xl l — xl h — — 2.95 93 2.74 3-3 k l l — xl h — — 2.6 93 2.42 3-3 k xl l — h l l — 2.5 72 1.8 3-3 k xl xl — h l l — 3.15 70 2.2 3-4 k l l — xl h — — 3.35 95 3.18 3-4 k xl l — xl h — — 2.75 95 2.61 3-4 k xl l — h l l — 3.7 69 2.55 3-4 k xl xl — h l l — 3.15 68 2.14

[2412] For TCB-2, -4 and -5 the following results have been obtained:

TABLE-US-00016 front vector back vector expression cassettes in expression cassettes in % MP eff. 5′- to 3′ direction 5′- to 3′ direction titer (CE- Titer TCB No. 1 2 3 4 1 2 3 4 [g/L] SDS) [g/L] 2 k xl l — h l xl — 4.3 73 3.14 2 k xl xl — h l l — 2.7 60 1.62 2 k xl k — l l h — 0.9 74 0.67 4 k xl xl — h l l — 2.16 76 1.64 4 k xl xl — h l — — 1.02 19.5 0.81 5 k xl xl — h l l — 3.6 76 2.74 5 k xl xl — h l — — 1.8 59 1.06

[2413] MP=main product, eff. titer=effective titer=titer multiplied by % main product

Example 8

[2414] Effect of Vector Design on the Expression of a Bivalent, Bispecific Antibody with a Domain Exchange

[2415] To examine the effect of expression cassette organization on productivity in the TI host, RMCE pools were generated by transfecting two plasmids (front and back vector) containing different numbers and organizations of the expression cassettes of the individual chains of a bivalent, bispecific antibody with domain crossover/exchange. After selection, recovery, and verification of RMCE by flow cytometry, the pools' productivity was evaluated in a 14-day fed batch production assay.

[2416] The effect of the antibody chain expression cassette organization on expression yield and product quality in stable transfected cells was evaluated for six different bivalent, bispecific antibodies with domain exchange. All had a different targeting specificity. For some also the effect of different VH/VL pairs had been analyzed. For these ten different antibodies the following results have been obtained.

TABLE-US-00017 front vector back vector expression cassettes in expression cassettes in % MP eff. 5′- to 3′ direction 5′- to 3′ direction titer (CE- Titer mAb No. 1 2 3 4 1 2 3 4 [g/L] SDS) [g/L] 1 xl k — — l h — — 1.5 86 1.29 front vector back vector expression cassettes in expression cassettes in eff. 5′- to 3′ direction 5′- to 3′ direction titer % MP Titer mAb No. 1 2 3 4 1 2 3 4 [g/L] (MS) [g/L] 2 var 1 xl h — — l k — — 2.7 85 2.28 2 var 1 l k — — xl h — — 2.8 89 2.43 2 var 2 xl h — — l k — — 2.9 87 2.52 2 var 2 l k — — xl h — — 3.1 91 2.83 2 var 3 xl h — — l k — — 2.9 82 2.34 2 var 3 l k — — xl h — — 3.2 89 2.80 2 var 4 xl h — — l k — — 2.6 80 2.06 2 var 4 l k — — xl h — — 2.7 82 2.26 front vector back vector expression cassettes in expression cassettes in % MP eff. 5′- to 3′ direction 5′- to 3′ direction titer (CE- Titer mAb No. 1 2 3 4 1 2 3 4 [g/L] SDS) [g/L] 3 var 1 xl h — — l k — — 2.1 94 1.95 3 var 1 l k — — xl h — — 2.3 87 2.02 3 var 2 xl h — — l k — — 2.3 90 2.05 3 var 2 l k — — xl h — — 2.5 91 2.26 4 xl k — — l h — — 3.8 94 3.57 4 xl k xl — l h — — 3 90 2.7 4 xl k xl — l h l — 2.8 93 2.6 4 xl k xl — l h h — 2.6 95 2.47 5 xl k — — l h — — 2.3 92 2.12 6 xl h — — l k — — 1.2 72 0.86 k = heavy chain with knob mutation; h = heavy chain with hole mutations; l = light chain; xl = light chain with domain exchange; var = different binding site sequences

Example 9

Effect of Vector Design on the Expression of a Multivalent, Bispecific Antibody

[2417] To examine the effect of expression cassette organization on productivity in the TI host, RMCE pools were generated by transfecting two plasmids (front and back vector) containing different numbers and organizations of the individual chains of a multivalent, bispecific antibody. After selection, recovery, and verification of RMCE by flow cytometry, the pools' productivity was evaluated in a 14-day fed batch production assay.

[2418] The effect of the antibody chain expression cassette organization on expression of the multivalent, bispecific antibodies was evaluated.

[2419] For the multivalent, bispecific antibody the following results have been obtained:

TABLE-US-00018 front vector back vector expression cassettes in expression cassettes in % MP eff. 5′- to 3′ direction 5′- to 3′ direction titer (CE- Titer 1 2 3 4 1 2 3 4 [g/L] SDS) [g/L] k l l l h l l l 1.95 90.5 1.76 k l — — h l — — 0.6 27.5 0.17 l k — — l h — — 1.6 82 1.31 k l l — h l l — 1.9 91 1.73

Example 10

[2420] CRE mRNA Targeted Integration Results in Increased Number of Positive Clones in CHO Pools

[2421] CHO pools for production of complex antibody formats are generated with either the CRE plasmid or CRE mRNA. Before and after the selection period, the absolute number of clones in the CHO pools is measured using a clone-specific tag. This clone-specific tag is part of the targeted integration technology and read out using deep sequencing enabling identification of the pool size and heterogeneity. After the selection period, the absolute number of clones in the CRE mRNA-generated CHO pools is significantly higher than in the CRE plasmid-generated CHO pools. Thus, by using CRE mRNA instead of CRE plasmid, a CHO pool with greater size and heterogeneity is produced thereby increasing the probability of finding a CHO clone with high titer and product quality. In addition, an increased number of clones from CRE mRNA-generated CHO pools are stable compared to the clones from the CRE plasmid-generated CHO pools