PRODUCING COMPOSITIONS COMPRISING TWO OR MORE ANTIBODIES

Abstract

The invention relates to means and methods of producing at least two antibodies. Methods may include providing cells with nucleic acid that encodes the antibodies; culturing said cells; collecting the antibodies from the culture; and separating produced antibodies from half antibodies by ion exchange chromatography (IEX). In some embodiments the antibodies exhibit IEX retention times that that deviate by 10% or less from the average of the retention times of the individual antibodies under the IEX conditions used. The invention also relates to compositions of antibodies thus produced. In some aspects the invention relates to compositions comprising 2-10 recombinant antibodies characterized in that the IEX retention times of at least two of said antibodies deviate by 10% or less from the average of the retention times of the individual antibodies under the IEX conditions. It also relates to compositions comprising 2-10 recombinant antibodies characterized in that the pI of at least two of said antibodies differ by 0.4 units or less from the average pI of said at least two antibodies.

Claims

1. A method of producing at least two antibodies comprising providing cells with nucleic acid that encodes the antibodies; culturing said cells; collecting the antibodies from the culture; and separating produced antibodies from half antibodies by ion exchange chromatography (IEX); the method characterized in that the antibodies exhibit IEX retention times that that deviate by 10% or less from the average of the retention times of the individual antibodies under the IEX conditions used.

2. The method of claim 1, wherein the collection of the antibodies from the culture comprises purifying antibody from other proteins by antibody affinity purification, preferably by protein A extraction.

3. The method of claim 2, wherein further comprising subjecting affinity purified antibodies to size-exclusion chromatography (gel-filtration chromatography and/or anion-exchange chromatography.

4. The method of claims 1-3, wherein subsequent to the IEX the collected antibodies are quantitatively analyzed for relative expression levels by hydrophobic interaction chromatography (HIC).

5. The method of claim 4, wherein the specificity of the collected antibodies is verified by ELISA.

6. The method of claims 1-5, wherein the retention times of the respective half antibodies are outside the range spanned by the retention times of the antibodies.

7. The method of claim 6, wherein the cells produce 3 heavy chains.

8. The method of claim 7, wherein said heavy chains comprise domains for efficient heterodimerization of heavy chains.

9. The method of claim 1-8, wherein at least two of said antibodies are bispecific antibodies.

10. The method of claim 1-9, wherein at least two of said antibodies share an identical heavy chain.

11. The method of claims 1-10, wherein said antibodies have isoelectric points (pI) that differ by 0.4 units or less from the average pI of said at least two antibodies.

12. The method of claims 1-11, wherein the antibodies are selected for having heavy and light chain combinations that have retention times that are significantly different from the retention times of the full antibodies under the IEX conditions used.

13. The method of claim 12, wherein the pI of heavy and light chain combinations differ by more than 0.4 units from the average pI of said at least two antibodies.

14. The method of claims 1-13, wherein the heavy chains comprise a CH3 domain that favors heterodimerization of heavy chains.

15. The method of claim 1-14, wherein the heavy chains of said antibodies are IgG heavy chains.

16. The method of claims 7-15, wherein one heavy chain comprises the amino acid substitutions L351K and T366K (EU numbering) in the CH3 region and another heavy chain comprises the amino acid substitutions L351D and L368E in the CH3 region.

17. A method of producing at least two antibodies comprising providing cells with nucleic acid that encodes the antibodies; culturing said cells; collecting the antibodies from the culture; and separating produced antibodies from half antibodies by ion exchange chromatography (IEX): the method characterized in that the antibodies exhibit IEX retention times that that deviate by 10% or less from the average of the retention times of the individual antibodies under the IEX conditions used and wherein subsequent to the IEX the collected antibodies are quantitatively analyzed for relative expression levels by hydrophobic interaction chromatography (HIC) and the specificity of the collected antibodies is verified by ELISA.

18. A composition comprising 2-10 recombinant antibodies obtainable by a method of claims 1-17.

19. A composition comprising 2-10 recombinant antibodies characterized in that the IEX retention times of at least two of said antibodies deviate by 10% or less from the average of the retention times of the individual antibodies under the IEX conditions.

20. A composition comprising 2-10 recombinant antibodies characterized in that the pI of at least two of said antibodies differ by 0.4 units or less from the average pI of said at least two antibodies.

21. The composition of claims 18-20, characterized in that the IEX retention times and/or the pI are essentially the same for all of the antibodies.

22. The composition of claims 18-21, wherein at least two of the antibodies are bispecific antibodies.

23. The composition of claim 22, wherein at least two of said antibodies share an identical heavy chain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1

[0088] A schematic representation of embodiments wherein the composition comprises two bispecific antibodies that share a common arm. The figure depicts antibodies with heavy chains (1) and light chains (4). The four heavy chains have three different variable regions (5, 6 and 7). The heavy chain that has the shared variable region (5) has one part (3) of a heterodimerization domain. The heavy chains with variable regions (6) and (7) have the compatible part of the heterodimerization domain (2). Preferred pairing of heterodimerization regions (2) and (3) can direct formation of bispecific antibodies.

[0089] FIG. 2

[0090] Panel A: CIEX-profile at 220 nm of bispecific antibody PB4516 production number 8 (p08). Panel B: CIEX-profile at 220 nm of bispecific antibody PB6892 production number 4 (p04).

[0091] FIG. 3

[0092] FIG. 3a: CIEX-profile at 220 nm of bispecific antibody PB4516 production number 10 (p10).

[0093] FIG. 3b: CIEX-profile at 220 nm of bispecific antibody PB11244 production number 1 (p01).

[0094] FIG. 3c: Per box two bispecific antibodies are identified in the column PB (PBXXXX(X)). The heavy chain variable region for PB11244 and PB4516 is indicated in the columns Target 1 and Target 2. The sequence of the light chain is the same for all antibodies and has the amino acid sequence of the common light chain IgKV1*39/jk1 of SEQ ID NO: 26. The pI calculated with the ExPASy. ProtParam tool is indicated for each heavy chain variable region in the columns pI. The pI difference between the two heavy chain variable regions is indicated in the last column, demonstrating that the average VI pI differential between PB11244 and PB4516 is 0.08.

[0095] FIG. 4

[0096] CIEX-profile at 220 nm of an antibody preparation of individual colony cp12 of pool FST2. The CIEX-profile shows a sharp peak of co-eluting antibodies PB4516 and PB11244. The profile shows that the sample contains a limited amount of product related impurities. It also shows the good separation between the co-eluting bispecific antibodies and the separately migrating product-related impurities.

[0097] FIG. 5

[0098] CIEX-profile at 220 nm of an antibody preparation of colony CP07. The colony was picked from a collection of individual colonies of single colony FST2cp09. The second subcloning was done to make sure that the FST2cp09-cp07 cell line was a clonal cell line. The bispecific antibody specific ELISA indicated the presence of 743 sg/ml of the ECFRIHER2 bispecific antibody PB11244 and 1134 μg/ml of the EGFR/HER3 bispecific antibody PB4516.

[0099] FIG. 6

[0100] Retention time of antibody homodimers (PGXXXX) having two identical variable domains. The amino acid sequence of the heavy chain variable region has the sequence indicated for the MF in FIG. 8 and a common light chain IgKV1*39/jk1 of SEQ ID NO: 26.

[0101] FIG. 7. Per box two bispecific antibodies are identified in the column PB (PBXXXX(X)). The heavy chain variable region (MFXXXX) of each of the bispecific antibodies is indicated in the columns Target 1 and Target 2. The sequence of the light chain is the same for all antibodies and has the amino acid sequence of the common light chain IgKV1*39/jk1 of SEQ ID NO: 26. The pI calculated with the ExPASy, ProtParam tool is indicated for each heavy chain variable region in the columns pI. The pI difference between the two heavy chain variable regions and the retention time measured are indicated in the last two columns. The measured retention times and the calculated pI and average pI indicate that the couple of bispecific antibodies can efficiently co-elute in CIEX chromatography. The antibodies have IgG1 constant regions and a common light chain. The heavy chain with the shared heavy chain variable region (identical MF) with the CH3 DE heavy chain or the KK heavy chain is indicated. Retentions times are indicated for CIEX chromatography performed on each bispecific, with an exemplary condition for CIEX chromatography described the materials and methods section. Many other CIEX chromatography conditions will result in suitable retention times between listed the bispecific antibody pairs having the provided pI values.

[0102] FIG. 8

[0103] Amino acid sequence of the heavy chain variable regions (MFXXXX) of respective antibodies and CDRs of light chain variable regions and amino acid sequence of common light chain variable regions.

EXAMPLES

Example 1

[0104] Materials and Methods

[0105] Cell Lines

[0106] HEK293 and CHO-K1 were maintained in growth medium.

[0107] Generation of Bispecific Antibodies

[0108] Bispecific antibodies were generated using the proprietary CH3 technology to ensure efficient hetero-dimerization and formation of a bispecific antibody. The CH3 technology uses charge-based point mutations in the CH3 region to allow efficient pairing of two different heavy chain molecules as previously described (PCT/NL2013/050294; published as WO 2013/157954 A1).

[0109] A VH gene was cloned in one of two different backbone IgG1 vectors. Depending on the binding partner the VH was cloned in an IgG1 backbone comprising the CH3 variant with heterodimerization variant. “DE” or in the IgG1 backbone comprising the complementary CH3 heterodimerization variant “KK”. In case of bi- or multispecific antibodies wherein two or more antibodies share a heavy chain. The shared chain preferably has the CH3 heterodimerization variant “DE” (also referred to as the DE-heavy chain) and the two or more unique heavy chains have the CH3 heterodimerization variant “KK” (also referred to as the KK-heavy chains).

[0110] HEK293 cells were transiently transfected with the DNA-FUGENE mixtures and further cultivated. Seven days after transfection, supernatant was harvested and medium was refreshed. Fourteen days after transfection supernatants were combined and filtrated through 0.22 μM. The sterile supernatant was stored at 4° C. Suspension adapted 293F cells were cultivated in T125 flasks at a shaker plateau until a density of 3.0×10e6 cells/ml. Cells were seeded at a density of 0.3-0.5×10e6 viable cells/ml in each well of a 24-deep well plate. The cells were transiently transfected with the individual sterile DNA: PEl-MIX and further cultivated. Seven days after transfection, supernatant was harvested and filtrated through 0.22 μM. The sterile supernatant was stored at 4° C.

[0111] Generation of Stable Cell Line Pools that Co-Express Two Bispecific Antibodies

[0112] CHO cells were transfected with the three heavy chain constructs and a common light chain construct in a molar ratio of common light chain construct (cLC):EGFR heavy chain:HER2 heavy chain:HER3 heavy chain=2.5:2:1:1. Ten pools of stably transfected cells were obtained (A-J). ELISA analysis of anti-EGFR, anti-HER2 and anti-HER3 antibodies was performed on the day 3 and day 6 supernatants of the 10 pools. All 3 species could be detected in all pools.

[0113] Generation of Stable Cell Line Clones that Co-Express Two Bispecific Antibodies.

[0114] The pools were plated in semi-solid medium and allowed to grow for 7-10 days. Single colonies were picked and seeded into 24 well culture plates. Colonies were reseeded prior to collection of antibodies from the supernatant of the cultures.

[0115] Determination of Antibody Titers

[0116] Anti-HER2 antibody titers of samples containing a single bispecific antibody were determined by ELISA against Erbb-2 Fe protein (R&D systems). Anti-HER3 titers of samples containing a single bispecific antibody were determined by ELISA against human Erbb-3-Fe protein (R&D systems). Anti-EGFR antibody titers of samples containing a single bispecific antibody were determined by ELISA against human EGFR ECD-Fc protein (R&D systems). Serial 2-fold dilutions of the antigens were used to coat wells of an ELISA plate, starting at 5 μg/ml.

[0117] ELISA assays to quantify EFGR×HER2 and EGFR×HER3 bispecific antibodies in compositions comprising mixtures of the two bispecific antibodies were done by coating ELISA plates with EGFR-Fc (R&D systems). After washing plates were incubated with sample. After washing the presence of bound bispecific antibody with one EFGR arm and one HER2 arm was detected by incubating with labelled HER2-Fc. The presence of bound bispecific antibody with one EFGR arm and one HER3 arm was detected by incubating with labelled HER3-Fc.

[0118] IgG Purification

[0119] Purification of IgG was performed using affinity chromatography. Purifications were performed under sterile conditions using vacuum filtration. First the pH of the medium was adjusted to pH 8.0 and subsequently the productions were incubated with protein A Sepharose CL-4B beads (50% v/v) (Pierce) for 2 H at 25° C. on a shaking platform at 600 rpm. Next the beads were harvested by vacuum filtration. Beads were washed twice with PBS pH 7.4. IgG was eluted at pH 3.0 with 0.1 M citrate buffer and the IgG fraction was immediately neutralized by Tris pH 8.0. Buffer exchange was performed by centrifugation using Ultracel (Millipore). The samples ended up in a final buffer of PBS pH 7.4.

[0120] Cation-Exchange Chromatography (CIEX)

[0121] CIEX-HPLC chromatography was done using TSKgel SP-STAT (7 μm particle size, 4.6 mM I.D.×10 cm L, Tosoh 21964) series of ion exchange columns. The columns are packed with non-porous resin particles for speed and high resolution analysis, as well as isolation, of biomolecules. The particles in TSKgel STAT columns contain an open access network of multi-layered ion-exchange groups for loading capacity, while the particle size makes these columns suitable for HPLC and FPLC systems.

[0122] The TSKgel SP-STAT (7 μm particle size, 4.6 mM I.D×10 cm L, Tosoh 21964) is equilibrated using Buffer A (Sodium Phosphate buffer, 25 mM, pH 6.0), after which antibodies are displaced from the column by increasing salt concentration and running a gradient of Buffer B (25 mM Sodium Phosphate, 1 mM NaCl, pH 6.0). Flow rate was set at 0.5 mL/min. The injection sample mass for all test samples and controls (in PBS) was 10 μg and injection volumes 10-100 μl. The chromatograms are analyzed for peak patterns, retention times and peak areas for the major peaks observed based on the 220 nm results.

[0123] Results

[0124] The CIEX profiles of the bispecific antibodies PB4516p08 and PB6892p04 were compared (see FIG. 2). It was observed that the production of PB6892 contained a significant amount of impurities. Also the retention time of the bispecific antibody fraction of PB6892 was significantly lower than the retention time of the bispecific antibody fraction of PB4516. For co-production by the same cell and subsequent co-purification using CIEX the retention time the two bispecific antibodies are preferably closer together. For this reason the variable region of the HER2 arm for PB6892 was replaced by a different variable region. The heavy chain with the variable region MF2032 was selected and used to produce the EGFR×HER2 bispecific antibody PB11244. The CIEX profiles of P14516p10 and PB11244p01 are shown in FIG. 3. The retention times of the bispecific antibody fractions are respectively 16.310 and 16.950. These retention times are sufficiently the same to allow for co-purification using CIEX under the conditions indicated. In addition, the figure shows that the retention times of impurities are sufficiently different to allow efficient separation in analytical and preparative columns. The above bispecific antibody preparations were produced in HEK293 cells.

[0125] For co-production CHO-K1 cells were used. CHO cells were transfected with constructs containing the three heavy chains with the respective variable regions of MF3755 (EGFR), M20:32 (HER2) and MF3178 (HER3) were transfected into CHO-K1 cells together with a construct expression the light chain variable region of SEQ ID: NO: 26. Vector positive cells were selected and pooled. Ten separate pools of transfected CHO-K1 cells (identified A-J) were generated.

[0126] Table 1 shows the amounts of bispecific antibody PB34516 (EGFR×HER3) and PB11244 (EGFR×HER2) produced by the respective pools. Also the ratio of the amounts as well as the total amount of IgG produced is shown. Pools F and J were selected for subcloning.

[0127] Table 2 shows the amount of bispecific antibody PB34516 (EGFR×HER3) and PB11244 (EGFR×HER2) produced by the respective clones.

[0128] Antibody produced by clone FST2cp12 was used to analyze the CIEX profile (see FIG. 4). It is clear that the two bispecific antibodies efficiently co-elute in the same CIEX elution fractions.

[0129] Clone FST2cp09 was further subcloned to make sure that the cell line was clonal and a further CIEX profile was determined of the antibodies produced. FIG. 5 shows the CIEX profile. It is clear that the two bispecific antibodies efficiently co-elute in the same CIEX elution fractions. The relative contribution of the two bispecific antibodies in the co-elution is analysed by ELISA and/or by hydrophobic interaction columns. The bispecific antibody specific ELISA indicated the presence of 743 μg/ml of the EGFR/HER2 bispecific antibody PB11244 and 1134 μg/ml of the EGFR/HER3 bispecific antibody PB4516.

Example 2

[0130] Generation of Stable Cell Line Pools that Co-Express Two Bispecific Antibodies

[0131] The cell lines expressing the two by two bispecific antibodies listed in FIG. 7 are produced as follows. CI cells are transfected with three heavy chain constructs and a common light chain construct. The three heavy chains are identified by the heavy chain variable regions (MFXXXX) indicated in the box. The light chain comprises the light chain variable region sequence of IgVk1*39/jk1 of SEQ ID NO: 26. The two bispecific antibodies have one heavy chain in common and one different heavy chain each. For instance, the first couple specified in FIG. 7 share a common heavy chain comprising the same HER3 binding arm comprising a heavy chain variable region (MF3178) and a different second binding arm. PB4528 has an EGFR binding arm with a heavy chain variable region (MF4003) and PB4188 has a HER2 binding arm with a heavy chain variable region (MF3958). The shared heavy chain has the KK CH3 region of the compatible DE/KK heterodimerisation domain. The shared heavy chain arm may also have the DE C13 region. For example, at FIGS. 3-5, two bispecific antibodies are co-purified PB11244 and PB4516. As shown at FIG. 3c, PB11244 and PB4516 share the same EGFR binding arm with a heavy chain variable region (MF3755) and PB11244 has a HER2 binding arm with a heavy chain variable region (MF2032) and PB34516 has a HER3 binding arm with a heavy chain variable region (MF3178). The shared heavy chain arm in this pair of bispecific antibodies have the DE CH3 region, while the different HER2 and HER3 binding arms have the KK C13 region.

[0132] The molar ratio of common light chain construct (cLC):to shared heavy chain construct:to different heavy chain construct 1:different heavy chain construct 2=2.5:2:1:1. Pools of stably transfected cells are obtained. ELISA analysis of the antigens is performed on supernatants collected from the pools. All 3 antigen binding species are detected in the pools. The CIEX retention times of the bispecific antibodies in each couple of FIG. 7 are determined under similar CIEX conditions and indicated in the 8.sup.th column. The deviation from the average retention time is calculated with the formula 100×((A−B)/(A+B)), where A is the retention time of the bispecific antibody with the longest retention time. For example, the deviation for the first couple is 100×((16.46−16.24)/(16.46+16.24))=0.67 or 0.7%.

[0133] Antibodies in the collected supernatants are first separated from other proteins in the supernatant by protein A extraction followed by acid elution and quick neutralization. The buffer of the collected antibodies is subsequently exchanged for PBS. The samples are subsequently loaded onto CIEX columns and washed and eluted by imparting an increasing salt gradient. The absorption of the eluate is measured at 220 nm and the retention times are calculated from the start of the salt gradient and the observance of the peak(s) for the bispecific antibodies. The bispecific antibodies are collected and the respective bispecific antibodies in the collected eluate is verified by ELISA. The retention times for the respective bispecific antibodies are indicated in the last column. It is clear that many of the couples have retention times that co-elute efficiently in a CIEX column. It is also clear that the CIEX chromatography provides a good separation of the co-eluting bispecific antibodies and the respective homodimers (if any). FIG. 6 lists the retention times of various antibodies with homodimers of heavy chains comprising the heavy chain variable regions present in co-eluting bispecific antibodies. It is clear that the retention time of the homodimers is sufficiently different from the retention times of the respective bispecific antibodies. For instance in the first box of FIG. 7 the homodimers PG3178. PG3958 and PG4003 could be present in a production. Retention times of the respective homodimers are about 22, 12 and 13 (FIG. 6 rows 1-3), whereas the retention times of the bispecific antibodies comprising the heavy chain variable regions are about 19 and 19.5 (FIG. 7, rows 1 and 2).

TABLE-US-00001 TABLE 1 Quantification of EGFR × HER2 and EGFR × HER3 bispecific antibody production in pools of cells. The culture supernatants of ten pools (A-J) were evaluated. The ELISA assays were based on EGFR-Fc coating, binding of antibody produced and detection with either labelled HER2-Fc or labelled HER3-Fc. Pools A-J were analyzed using the two ELISA assays. The bispecific antibodies PB4516 and PB11244 are IgG1 heavy chain antibodies with compatible DE/KK heterodimerization domains. The heavy chains are combined with the common light chain. The bispecific antibodies share the MF3755 heavy chain variable region on one heavy chain and each have a different heavy chain variable region on the other IgG1 heavy chain, MF3178 for PB4516 and MF2032 for PB11244. Ratio Total Pool A 153.6 162.0 1:1.1 315.6 B 120.1 336.3 1:2.8 456.3 C 325.5 741.6 1:2.3 1067.1 D 510.6 856.8 1:1.7 1367.4 E 273.0 526.5 1:1.9 799.6 F 1824.7 1024.9 1:0.6 2849.6 G 704.2 849.8 1:1.2 1554.0 H 951.1 467.5 1:0.5 1418.6 I 450.7 691.0 1:1.5 1141.7 J 1288.9 1572.1 1:1.2 2861.0 indicates data missing or illegible when filed

TABLE-US-00002 TABLE 2 Selected pools were used for single cell cloning. 18 colonies were picked from three pools. Two independent pools F (FST1 and FST2) and one pool J (JST1) were used for single cell cloning. The indication “cp” followed by a number identifies individual colonies of a pool. Picked colonies were grown up and used for the collection of antibodies. Individual colonies from the same pools produced different amounts and different proportions of the respective bispecific antibodies. # HER2 [ug/mL] HER3 [ug/mL] HER2/HER3 FST1cp02 48.7 1449.2 0.03 FST1cp03 103.4 488.9 0.21 FST1cp04 131.4 1675.0 0.08 FST1cp14 259.1 214.6 1.21 FST1cp24 372.3 817.0 0.46 FST1cp26 92.2 706.7 0.13 FST2cp09 1026.2 1509.0 0.68 FST2cp12 725.7 1334.2 0.54 FST2cp13 737.4 1173.0 0.63 FST2cp20 617.6 1759.3 0.35 FST2cp21 993.0 1852.3 0.54 FST2cp23 937.4 1095.5 0.86 JST1cp01 121.3 490.6 0.25 JST1cp04 239.8 383.7 0.62 JST1cp05 187.2 759.0 0.25 JST1cp09 828.6 718.5 1.15 JST1cp13 103.5 175.8 0.59 JST1cp24 481.5 423.7 1.14