METHOD FOR THE GENERATION OF A BIVALENT, BISPECIFIC ANTIBODY EXPRESSING CELL BY TARGETED INTEGRATION OF MULTIPLE EXPRESSION CASSETTES IN A DEFINED ORGANIZATION

Abstract

Herein is reported a method for producing a bivalent, bispecific antibody comprising the steps of cultivating a mammalian cell comprising a deoxyribonucleic acid encoding the bivalent, bispecific antibody, and 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 5′- to 3′-direction either a first expression cassette encoding the first light chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding the second light chain, and a fourth expression cassette encoding the second heavy chain, or a first expression cassette encoding the first light chain, a second expression cassette encoding the second heavy chain, a third expression cassette encoding the second light chain, and a fourth expression cassette encoding the first heavy chain, wherein the first heavy chain comprises from N- to C-terminus a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain, the second heavy chain comprises from N- to C-terminus the first light chain variable domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain, the first light chain comprises from N- to C-terminus a second heavy chain variable domain and a CL domain, and the second light chain comprises from N- to C-terminus a second light chain variable domain and a CL domain, wherein the first heavy chain variable domain and the second light chain variable domain form a first binding site and the second heavy chain variable domain and the first light chain variable domain form a second binding site.

Claims

1. 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 5′- to 3′-direction a first expression cassette encoding the first light chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding the second light chain, and a fourth expression cassette encoding the second heavy chain, wherein the first heavy chain comprises in the CH3 domain the mutation T366W (numbering according to Kabat) and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V (numbering according to Kabat).

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

3. Use of a deoxyribonucleic acid comprising in 5′- to 3′-direction a first expression cassette encoding the first light chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding the second light chain, and a fourth expression cassette encoding the 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 (numbering according to Kabat) and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V (numbering according to Kabat).

4. 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 5′- to 3′-direction a first expression cassette encoding the first light chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding the second light chain, and a fourth expression cassette encoding the second heavy chain, wherein the first heavy chain comprises in the CH3 domain the mutation T366W (numbering according to Kabat) and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V (numbering according to Kabat).

5. 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 5′- to 3′-direction, a first recombination recognition sequence, a first expression cassette encoding the first light chain, a second expression cassette encoding the first heavy chain, and a first copy of a third recombination recognition sequence, and the second deoxyribonucleic acid comprises in 5′- to 3′-direction a second copy of the third recombination recognition sequence, a third expression cassette encoding the second light chain, a fourth expression cassette encoding the second heavy chain, and a second recombination recognition sequence, wherein the first heavy chain comprises in the CH3 domain the mutation T366W (numbering according to Kabat) and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V (numbering according to Kabat).

6. 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 5′- to 3′-direction, a first recombination recognition sequence, a first expression cassette encoding the first light chain, a second expression cassette encoding the first heavy chain, and a first copy of a third recombination recognition sequence, and the second deoxyribonucleic acid comprises in 5′- to 3′-direction a second copy of the third recombination recognition sequence, a third expression cassette encoding the second light chain, a fourth expression cassette encoding the 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 one second selection marker when taken together form a functional expression cassette of the one second selection marker; wherein the first heavy chain comprises in the CH3 domain the mutation T366W (numbering according to Kabat) and the second heavy chain comprises in the CH3 domain the mutations T366S, L368A, and Y407V (numbering according to Kabat); 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.

7. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according to claim 1, wherein one of the heavy chains further comprises the mutation S354C and the respective other heavy chain comprises the mutation Y349C (numbering according to Kabat).

8. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according to claim 1, wherein the second light chain is a domain exchanged light chain VH-CH1 after VH-VL exchange or a domain exchanged light chain VL-CH1 after CH1-CL exchange.

9. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according to claim 1, wherein the first heavy chain comprises from N- to C-terminus a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain, the second heavy chain comprises from N- to C-terminus the first light chain variable domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain, the first light chain comprises from N- to C-terminus a second heavy chain variable domain and a CL domain, and the second light chain comprises from N- to C-terminus a second light chain variable domain and a CL domain, wherein the first heavy chain variable domain and the second light chain variable domain form a first binding site and the second heavy chain variable domain and the first light chain variable domain form a second binding site.

10. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according to claim 1, wherein exactly one copy of the deoxyribonucleic acid is stably integrated into the genome of the mammalian cell at a single site or locus.

11. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according claim 1, wherein the deoxyribonucleic acid encoding the bivalent, bispecific antibody comprises a further expression cassette encoding for a selection marker, wherein the expression cassette encoding for the selection marker is located partly 5′ and partly 3′ to the third recombination recognition sequence, wherein the 5′-located part of said expression cassette comprises the promoter and the start-codon and the 3′-located part of said expression cassette comprises the coding sequence without a start-codon and a polyA signal, wherein the start-codon is operably linked to the coding sequence, wherein the 5′-located part of the expression cassette encoding the selection marker comprises a promoter sequence operably linked to a start-codon, whereby the promoter sequence is flanked upstream by the second expression cassette and the start-codon is flanked downstream by the third recombination recognition sequence; and the 3′-located part of the expression cassette encoding the selection marker comprises a nucleic acid encoding the selection marker lacking a start-codon and is flanked upstream by the third recombination recognition sequence and downstream by the third expression cassette, wherein the start-codon is operably linked to the coding sequence.

12. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according to claim 1, wherein each expression cassette for an antibody chain comprises in 5′-to-3′ direction a promoter, a nucleic acid encoding an antibody chain, and a polyadenylation signal sequence and optionally a terminator sequence and each expression cassette encoding the selection marker comprises in 5′-to-3′ direction a promoter, a nucleic acid encoding the selection marker, and a polyadenylation signal sequence and optionally a terminator sequence, wherein the promoter is the human CMV promoter with intron A, the polyadenylation signal sequence is the bGH polyadenylation signal sequence and the terminator is the hGT terminator except for the expression cassette of the selection marker, wherein the promoter is the SV40 promoter and the polyadenylation signal sequence is the SV40 polyadenylation signal sequence and a terminator is absent.

13. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according to claim 1, wherein the mammalian cell is a CHO cell.

14. The method for producing a bivalent, bispecific antibody or the deoxyribonucleic acid or the use or the recombinant mammalian cell or the composition or the method for producing a recombinant mammalian cell according to claim 1, wherein all cassettes are arranged unidirectional.

Description

DESCRIPTION OF THE FIGURES

[0525] FIG. 1: Scheme of a two-plasmid RMCE strategy involving the use of three RRS sites to carry out two independent RMCEs simultaneously.

DESCRIPTION OF THE SEQUENCES

[0526] SEQ ID NO: 01: exemplary sequence of an L3 recombinase recognition sequence

[0527] SEQ ID NO: 02: exemplary sequence of a 2L recombinase recognition sequence

[0528] SEQ ID NO: 03: exemplary sequence of a LoxFas recombinase recognition sequence

[0529] SEQ ID NO: 04-06: exemplary variants of human CMV promoter

[0530] SEQ ID NO: 07: exemplary SV40 polyadenylation signal sequence

[0531] SEQ ID NO: 08: exemplary bGH polyadenylation signal sequence

[0532] SEQ ID NO: 09: exemplary hGT terminator sequence

[0533] SEQ ID NO: 10: exemplary SV40 promoter sequence

[0534] SEQ ID NO: 11: exemplary GFP nucleic acid sequence

EXAMPLES

Example 1

General Techniques

1) Recombinant DNA Techniques

[0535] 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

[0536] DNA sequencing was performed at SequiServe GmbH (Vaterstetten, Germany)

3) DNA and Protein Sequence Analysis and Sequence Data Management

[0537] The EMBOSS (European Molecular Biology Open Software Suite) software package and Invitrogen's Vector NTI version 11.5 were used for sequence creation, mapping, analysis, annotation and illustration.

4) Gene and Oligonucleotide Synthesis

[0538] 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

[0539] 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

[0540] 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

[0541] 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.

[0542] 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

[0543] For the digest of plasmids with restriction enzymes the following components were pipetted together on ice:

TABLE-US-00003 TABLE Restriction Digestion Reaction Mix component ng (set point) μl purified DNA tbd tbd CutSmart Buffer (10×) 5 Restriction Enzyme 1 PCR-grade Water ad 50 Total 50

[0544] 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.).

[0545] 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.

[0546] 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.

[0547] 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.

[0548] 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-00004 TABLE Ligation Reaction Mix component ng (set point) conc. [ng/μl] μl T4 DNA Ligase 2 Buffer (10×) 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

[0549] 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

[0550] 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-00005 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

[0551] 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

[0552] 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

[0553] 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-00006 TABLE E. coli cultivation volumes Volume LB-Amp Incubation Quantity plasmid 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

[0554] 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.

[0555] 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.

[0556] 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

[0557] 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.

[0558] 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.

[0559] 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

[0560] 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

[0561] For the expression of an antibody chain a transcription unit comprising the following functional elements was used: [0562] the immediate early enhancer and promoter from the human cytomegalovirus including intron A, [0563] a human heavy chain immunoglobulin 5′-untranslated region (5′UTR), [0564] a murine immunoglobulin heavy chain signal sequence, [0565] a nucleic acid encoding the respective antibody chain, [0566] the bovine growth hormone polyadenylation sequence (BGH pA), and [0567] optionally the human gastrin terminator (hGT).

[0568] Beside the expression unit/cassette including the desired gene to be expressed the basic/standard mammalian expression plasmid contains [0569] an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and [0570] a beta-lactamase gene which confers ampicillin resistance in E. coli.

Front- and Back-Vector Cloning

[0571] 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.

[0572] 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 DH5a 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.

[0573] 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 (Sequi Serve GmbH).

[0574] 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.

[0575] The cloned plasmids were used for the TI transfection and pool generation.

Example 3

Cultivation, Transfection, Selection and Pool Generation

[0576] 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).

[0577] For stable transfection, equimolar amounts of front and back vector were mixed. 1 μg Cre expression plasmid was added to 5 μg of the mixture.

[0578] 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.

[0579] 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.

[0580] 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 bivalent, 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). bivalent, 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 bivalent, bispecific antibody expression. Fourteen days after the selection had been started, the viability exceeded 90% and selection was considered as complete.

Example 4

FACS Screening

[0581] 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

[0582] 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. Product quality was determined by Caliper's LabChip (Caliper Life Sciences).

Example 6

Effect of Vector Design

[0583] 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.

[0584] 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-00007 front vector back vector % expression cassettes expression cassettes MP eff. in 5′-to 3′ direction in 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 expression cassettes % eff. in 5′-to 3′ direction in 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 expression cassettes MP eff. in 5′-to 3′ direction in 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