METHOD FOR PRODUCING MULTISPECIFIC ANTIBODIES

Abstract

Herein are provided a method for producing a multispecific antibody comprising the steps of providing a mammalian cell expressing the antibody, transfecting said mammalian cell with an expression vector comprising an expression cassette encoding a polypeptide of the antibody that has a domain crossover, cultivating the transfected cell and recovering the antibody from the cell or the cultivation medium and thereby producing the multispecific antibody.

Claims

1. A method for producing a multispecific antibody, which comprises at least three different polypeptides, comprising the following steps: cultivating a mammalian cell in a cultivation medium, whereby the mammalian cell has been generated by a) transfecting a mammalian cell with a first expression vector and one, two or three further expression vectors, wherein the first expression vector comprises exactly one nucleic acid sequence encoding a polypeptide of the multispecific antibody, and the one, two or three further expression vectors each comprise at least two nucleic acid sequences each encoding different polypeptide chains of the multispecific antibody, wherein the exactly one nucleic acid sequence of the first expression vector is a nucleic acid sequence encoding a light chain polypeptide of the multispecific antibody, recovering the multispecific antibody from the cell or the cultivation medium, and thereby producing the multispecific antibody.

2. The method according to claim 1, wherein two of the polypeptide chains of the multispecific antibody comprise a domain exchange.

3. The method according to claim 2, wherein the exactly one nucleic acid of the first expression vector encodes a light chain polypeptide with domain exchange of the multispecific antibody.

4. The method according to claim 1, wherein step a) is co-transfecting the first expression vector and the one, two or three further expression vectors.

5. The method according to claim 1, wherein the mammalian cell is transfected first with the one, two or three further expression vectors and transfected thereafter with the first expression vector.

6. The method according to claim 1, wherein the mammalian cell stably expresses the multispecific antibody.

7. The method according to claim 1, wherein the mammalian cell is a CHO cell.

8. The method according to claim 2, wherein the domain exchange is a CH1-CL crossover or a VH-VL-crossover.

9. The method according to claim 1, wherein the multispecific antibody is a bivalent, bispecific antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other.

10. The method according to claim 1, wherein the multispecific antibody is a bivalent, bispecific antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

11. The method according to claim 1, wherein the multispecific antibody is a trivalent, bispecific antibody, comprising a) a first light chain and a first heavy chain of a full length antibody which specifically binds to a first antigen, b) a second heavy chain of a full length antibody which when paired with the first light chain, specifically binds to the first antigen, and c) a Fab fragment, which specifically binds to a second antigen, fused via a peptidic linker to the C-terminus of one of the heavy chains of a) or b), wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

12. The method according to claim 1, wherein the method is for producing a multispecific antibody preparation with low/reduced product-related impurities.

Description

DESCRIPTION OF THE FIGURES

[0411] FIGS. 1A-D: Deglycosylated ESI-MS total ion chromatogram for [0412] 1A transfection 0516; [0413] 1B transfection 0517; [0414] 1C transfection 0518; [0415] 1D transfection 0519; [0416] 1A: second heavy chain with the knob mutations (CrossHC-2-knob) of the IgG1 subclass; 1B: the first heavy chain with the hole mutations (HC-1-hole) of the IgG 1 subclass; 1C: the domain exchanged second light chain (CrossLC-2), 1D: the first light chain (LC-1).

[0417] FIGS. 2A-B: Relative monomer content 2A and 5/6 antibody side product 2B of the reference clone 0131 and the clones obtained with the method as described herein determined by CE-SDS.

EXAMPLES

[0418] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Materials & General Methods

[0419] General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Amino acids of antibody chains are numbered and referred to according to numbering according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

Recombinant DNA Techniques

[0420] Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene Synthesis

[0421] Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The long gene segments, which were flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligating oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to given specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

[0422] DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

[0423] The GCG's (Genetics Computer Group, Madison, Wis.) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

[0424] For the expression of the described bispecific antibodies, expression vectors for transient expression (e.g. in HEK293 cells) based either on a cDNA organization with or without a CMV-intron A promoter or on a genomic organization with a CMV promoter can be applied.

[0425] Beside the antibody expression cassette, the vectors contain: [0426] an origin of replication which allows replication of this vector in E. coli, and [0427] a ß-lactamase gene which confers ampicillin resistance in E. coli.

[0428] The transcription unit of the antibody gene is composed of the following elements: [0429] unique restriction site(s) at the 5′ end [0430] the immediate early enhancer and promoter from the human cytomegalovirus, [0431] the intron A sequence in the case of cDNA organization, [0432] a 5′-untranslated region derived from a human antibody gene, [0433] an immunoglobulin heavy chain signal sequence, [0434] the respective antibody chain encoding nucleic acid either as cDNA or with genomic exon-intron organization, [0435] a 3′ untranslated region with a polyadenylation signal sequence, and [0436] unique restriction site(s) at the 3′ end.

[0437] The fusion genes encoding the antibody chains are generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid sequences are verified by DNA sequencing. For transient transfections larger quantities of the vectors are prepared by vector preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

[0438] For all constructs knob-into-hole heterodimerization technology was used with a typical knob (T366W) substitution in the first CH3 domain and the corresponding hole substitutions (T366S, L368A and Y407V) in the second CH3 domain (as well as two additional introduced cysteine residues S354C/Y349′C) (contained in the respective corresponding heavy chain (HC) sequences depicted above).

Cell Culture Techniques

[0439] Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc., are used.

Transient Transfections in HEK293-F System

[0440] The bispecific antibodies are produced by transient expression. Therefore, a transfection with the respective vectors using the HEK293-F system (Invitrogen) according to the manufacturer's instruction is done. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) are transfected with a mix of the respective expression vectors and 293fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells are seeded at a density of 1.0*10.sup.6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO.sub.2. On the next day the cells are transfected at a cell density of approx. 1.5*10.sup.6 cells/mL with approx. 42 mL of a mixture of A) 20 mL Opti-MEM medium (Invitrogen) comprising 600 μg total vector DNA (1 μg/mL) and B) 20 ml Opti-MEM medium supplemented with 1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucose consumption glucose solution is added during the course of the fermentation. The supernatant containing the secreted antibody is harvested after 5-10 days and antibodies are either directly purified from the supernatant or the supernatant is frozen and stored.

Protein Determination

[0441] The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science 4 (1995) 2411-1423.

Antibody Concentration Determination in Supernatants

[0442] The concentration of antibodies and derivatives in cell culture supernatants was estimated by immunoprecipitation with protein A agarose-beads (Roche Diagnostics GmbH, Mannheim, Germany). Therefore, 60 μL protein A Agarose beads were washed three times in TBS-NP40 (50 mM Tris buffer, pH 7.5, supplemented with 150 mM NaCl and 1% Nonidet-P40). Subsequently, 1-15 mL cell culture supernatant was applied to the protein A Agarose beads pre-equilibrated in TBS-NP40. After incubation for at 1 hour at room temperature the beads were washed on an Ultrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twice with 0.5 mL 2× phosphate buffered saline (2×PBS, Roche Diagnostics GmbH, Mannheim, Germany) and briefly four times with 0.5 mL 100 mM Na-citrate buffer (pH 5.0). Bound antibody was eluted by addition of 35 μl NuPAGE® LDS sample buffer (Invitrogen). Half of the sample was combined with NuPAGE® sample reducing agent or left unreduced, respectively, and heated for 10 min at 70° C. Consequently, 5-30 μl were applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE gel (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.

[0443] The concentration of the antibodies in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies that bind to protein A were applied to an Applied Biosystems Poros A/20 column in 200 mM KH.sub.2PO.sub.4, 100 mM sodium citrate, pH 7.4 and eluted with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted antibody was quantified by UV absorbance and integration of peak areas. A purified standard IgG1 antibody served as a standard.

[0444] Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96 well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) were coated with 100 μL/well biotinylated anti-human IgG capture molecule F(ab′)2<h-Fcγ>BI (Dianova) at 0.1 μg/mL for 1 hour at room temperature or alternatively overnight at 4° C. and subsequently washed three times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). Thereafter, 100 μL/well of a dilution series in PBS (Sigma) of the respective antibody containing cell culture supernatants was added to the wells and incubated for 1-2 hour on a shaker at room temperature. The wells were washed three times with 200 μL/well PBST and bound antibody was detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detection antibody by incubation for 1-2 hours on a shaker at room temperature. Unbound detection antibody was removed by washing three times with 200 μL/well PBST. The bound detection antibody was detected by addition of 100 μL ABTS/well followed by incubation. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).

Preparative Antibody Purification

[0445] Antibodies were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine buffer comprising 150 mM NaCl (pH 6.0). Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at −20° C. or −80° C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

[0446] The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.

CE-SDS

[0447] Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Therefore, 5 μl of antibody solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer's instructions and analyzed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software.

Analytical Size Exclusion Chromatography

[0448] Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 buffer (pH 7.5) on an Dionex Ultimate® system (Thermo Fischer Scientific), or to a Superdex 200 column (GE Healthcare) in 2×PBS on a Dionex HPLC-System. The eluted antibody was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.

Mass Spectrometry

[0449] This section describes the characterization of the bispecific antibodies with emphasis on their correct assembly. The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact antibody and in special cases of the deglycosylated/limited LysC digested antibody.

[0450] The antibodies were deglycosylated with N-Glycosidase F in a phosphate or Tris buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/ml. The limited LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestions were performed with 100 μg deglycosylated antibody in a Tris buffer (pH 8) at room temperature for 120 hours, or at 37° C. for 40 min, respectively. Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

Example 1

Expression and Purification

[0451] The bispecific antibodies were produced as described above in the general materials and methods section.

[0452] The bispecific antibodies were purified from the supernatant by a combination of protein A affinity chromatography and size exclusion chromatography. The obtained products were characterized for identity by mass spectrometry and analytical properties such as purity by CE-SDS, monomer content and stability.

[0453] The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact antibody and deglycosylated/plasmin digested or alternatively deglycosylated/limited LysC digested antibody as described in the general methods section.

[0454] Additional analytical methods (e.g. thermal stability, mass spectrometry and functional assessment) were only applied after protein A and SEC purification.

Example 2

Determination of Binding to A131-40 Fibers In Vitro by ELISA

[0455] Binding of the bispecific antibodies to fibrillar Aβ is measured by an ELISA assay. Briefly, A13(1-40) is coated at 7 μg/mL in PBS onto Maxisorb plates for 3 days at 37° C. to produce fibrillar Abeta, and then dried for 3 h at RT. The plate is blocked with 1% CroteinC and 0.1% RSA in PBS (blocking buffer) for 1 h at RT, then washed once with wash buffer. Bispecific antibodies or controls are added at concentrations up to 100 nM in blocking buffer and incubated at 4° C. overnight. After 4 wash steps, constructs are detected by addition of anti-human-IgG-HRP (Jackson Immunoresearch) at 1:10,000 dilution in blocking buffer (1 RT), followed by 6 washes and incubation in TMB (Sigma). Absorbance is read out at 450 nm after stopping color development with 1 N HCl.

Example 3

Determination of Binding to Transferrin Receptor In Vitro

[0456] Binding of the bispecific antibodies to murine transferrin receptor is tested by FACS analysis on mouse X63.AG8-563 myeloma cells. If the Aβ antibody shows a certain tendency to non-specifically bind to Ag8 cells, specific binding can be quantified by co-incubation with a 20fold excess of anti-mouse-TfR antibody. Cells are harvested by centrifugation, washed once with PBS and 5×10.sup.4 cells incubated with a 1.5 pM to 10 nM dilution series of the polypeptide fusions with or without addition of 200 nM anti-mouse TfR antibody in 100 μL RPMI/10% FCS for 1.5 h on ice. After 2 washes with RPMI/10% FCS, cells are incubated with goat-anti-human IgG coupled to Phycoerythrin (Jackson Immunoresearch) at a dilution of 1:600 in RPMI/19% FCS for 1.5 h on ice. Cells are again washed, resuspended in RPMI/10% FCS and Phycoerythrin fluorescence measured on a FACS-Array instrument (Becton-Dickinson).

Example 4

Surface Plasmon Resonance-Based Binding Assay for Human TfR-Antibody Interaction

[0457] The binding experiment were carried out on a BIAcore B 4000 (GE Healthcare) equipped with C1 sensor chip (GE Healthcare, cat.no. BR1005-35) pre-treated with anti-human Fab antibody (GE Healthcare, cat.no 28-9583-25) using a standard amine coupling chemistry procedure accordingly to the vendor's manual.

[0458] For kinetic measurements the sample antibody was immobilized applying a contact time of 60 seconds and a flow rate of 10 μL/min in phosphate buffer saline pH 7.4, 0.05% Tween 20 at 25° C. Recombinant His6-tagged human transferrin receptor (“His6” disclosed as SEQ ID NO: 2) (R&D systems, cat.no 2474-TR-050) was applied in increasing concentrations and the signal monitored over the time. An average time span of 150 seconds of association time and 600 seconds of dissociation time at 30 μL/min flow rate was recorded. Data were fit using a 1:1 binding model (Langmuir isotherm).

Example 5

[0459] Staining of Native Human β-Amyloid Plaques from Brain Sections of an Alzheimer's Disease Patient by Indirect Immunofluorescence Using a Bispecific Antibody as Produced in the Method as Reported Herein

[0460] The bispecific antibodies can be tested for the ability to stain native human 3-amyloid plaques by immunohistochemistry analysis using indirect immunofluorescence. Specific and sensitive staining of genuine human 3-amyloid plaques can be demonstrated. Cryostat sections of unfixed tissue from the temporal cortex obtained postmortem from patients positively diagnosed for Alzheimer's disease are labeled by indirect immunofluorescence. A two-step incubation is used to detect bound bispecific antibody, which is revealed by affinity-purified goat anti-human (GAH555) IgG (H+L) conjugated to Alexa 555 dye (Molecular Probes). Controls can include unrelated human IgG1 antibodies (Sigma) and the secondary antibody alone, which all should give negative results.

Example 6

In Vivo β-Amyloid Plaque Decoration by a Bispecific Antibody as Produced in the Method as Reported Herein in a Mouse Model of Alzheimer's Disease

[0461] Bispecific antibody can be tested in APP/PS2 double transgenic mice, a mouse model for AD-related amyloidosis (Richards, J. Neuroscience, 23 (2003) 8989-9003) for their ability to immuno-decorate β-amyloid plaques in vivo. This enabled assessment of the extent of brain penetration and binding to amyloid-β plaques. The fusion polypeptide can be administered at different doses compared to naked anti-Aβ monoclonal antibody and after 6 days animals are perfused with phosphate-buffered saline and the brains frozen on dry ice and prepared for cryosectioning.

[0462] The presence of the antibodies bound to 3-amyloid plaques can be assessed using unfixed cryostat sections either by single-labeled indirect immunofluorescence with goat anti-human IgG (H+L) conjugated to Alexa555 dye (GAH555) (Molecular Probes) at a concentration of 15 μg/ml for 1 hour at room temperature. A counterstaining for amyloid plaques can be done by incubation with BAP-2, a mouse monoclonal antibody against Aβ conjugated to Alexa 488 at a concentration of 0.5 μg/ml for 1 hour at room temperature. Slides are embedded with fluorescence mounting medium (S3023 Dako) and imaging is done by confocal laser microscopy.

[0463] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Example 7

[0464] Transfection of Stable Cell Line Expressing Bispecific Anti-DR5/FAP Antibody with Expression Vector Comprising an Expression Cassette for the Domain Exchanged Light Chain

[0465] Clone 0131 cells were transfected with the CrossLC expression vector comprising one expression cassette for the light chain with domain crossover. Transfections were performed using linearized DNA in chemically defined medium using nucleofection (Amaxa) and 0.6/1.2/2.4 pM (total) plasmid, leading to 2×3 cell pools which were selected differently.

[0466] The transfected clone pools were selected in chemically defined medium supplemented with 10 mmol/L glutamine and by 250 nM MTX (for DHFR) plus 500 nM and 700 nM Hygromycin B. After three weeks the pools were analyzed by CE-SDS and HIC for reduction of side peaks and increase of main peak.

[0467] Based on these results the three pools which had been selected by 250 nM MTX and 700 NM HygB (0314, 0316, 0318) were chosen for Limited Dilution (LD) and plating of each 3×384w plates with chemically defined medium supplemented with 10 mmol/L glutamine and a MTX-concentration of 250 nmol/L and 700 nM HygB.

[0468] One week later the supernatants of the 3×384w plates were tested for binding to DR5 and FAP by ELISA and a DR5-FAP bridging ELISA. 158 clones with good titers and high reactivity against both antigens were picked and expanded via 24-well plates to 6 wells, where they were evaluated in a four day batch experiment (‘seed train titer’) with regard to target binding by ELISA and bridging ELISA, growth, productivity and side-product profile assessed by CE-SDS. 46 clones thereof with titers up to 830 μg/ml and acceptable product quality were further characterized in 14 days fed-batch cultures in Ambr15 system and analyzed concerning target-binding, growth properties, and side-product profile by CE-SDS and HIC. 20 clones were selected thereof and further tested by mass spectrometry (MS). 10 clones thereof selected were cultivated in shake flasks in chemically defined medium and deposited as PSBs.