SPLIT CH2 DOMAINS

Abstract

The invention relates to a protein complex comprising at least two polypeptide chains A (PCA) and B (PCB), wherein PCA comprises a heterodimerization domain A (HDA) and PCB comprises a heterodimerization domain B (HDB), wherein HDA and HDB bind to each other and wherein one heterodimerization domain comprises or consists of two N-terminal β-strands (N-β) of an immunoglobulin (Ig) domain and the other heterodimerization domain comprises or consists of two C-terminal β-strands (C-β) of an Ig domain. The invention further relates to polynucleotides encoding one or more polypeptides of the protein complex, expression vectors comprising the polynucleotides and a cell comprising the polynucleotides or the expression vectors.

Claims

1. A protein complex comprising at least two polypeptide chains A (PCA) and B (PCB), wherein PCA comprises a heterodimerization domain A (HDA) and PCB comprises a heterodimerization domain B (HDB) that bind to each other and wherein one heterodimerization domain comprises or consists of two N-terminal ß-strands of an immunoglobulin (Ig) domain (N-ß) and the other heterodimerization domain comprises or consists of two C-terminal ß-strands of an Ig domain (C-ß).

2. The protein complex according to claim 1, wherein a. N-ß comprises a continuous amino acid sequence of an Ig domain comprising at least ß-strand b and c and b. C-ß comprises a continuous amino acid sequence of an Ig domain comprising at least ß-strand e and f.

3. The protein complex according to claim 1 or 2, wherein the Ig domains of N-ß and C-ß are independently selected from an IgA, IgD, IgE, IgG, IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain 2 (CH2), and an IgM or IgE heavy chain constant domain 3 (CH3), and are optionally selected from the same CH2 or CH3.

4. The protein complex according to claim 1, wherein HDA and HDB (i) non-covalently or (ii) non-covalently and covalently bind to each other.

5. The protein complex according to claim 3, wherein N-ß comprises or consists of a continuous amino acid sequence of a CH2 or CH3 domain comprising or consisting of ß-strand a to ß-strand c and C-ß comprises or consists of a continuous amino acid sequence of a CH2 or CH3 domain comprising or consisting of ß-strand e to ß-strand g.

6. The protein complex according to claim 1, wherein the N-ß and C-ß each comprise a non-naturally occurring Cys residue and wherein the Cys residues replace amino acids in the folded N-ß and C-ß, respectively, that naturally have a distance of between 3 to 7.5 Å between their Cα-atoms.

7. The protein complex according to claim 1, wherein HDA comprises or consists of: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSX.sub.1EDPEVX.sub.2FX.sub.3WYVDGVEVHN (SEQ ID NO: 1), wherein X.sub.1 is H or Q, X.sub.2 is K or Q and X.sub.3 is N or K; or a variant thereof with an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% identity to SEQ ID NO: 1; and/or HDB comprises or consists of: NSTX.sub.4RVVSVLTVX.sub.5HQDWLNGKEYKCKVSNKX.sub.6LPX.sub.7X.sub.8IEKTI (SEQ ID NO: 2), wherein X.sub.4 is Y or F; X.sub.5 is L or V; X.sub.6 is A or G; X.sub.7 is K or Q; X.sub.8 is N or K; or a variant thereof with an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% identity to SEQ ID NO: 2, wherein SEQ ID NO: 1 or its variant can heterodimerize with SEQ ID NO: 2 or its variant.

8. The protein complex according to claim 1, wherein the complex comprises one or more antigen binding sites within the PCA and/or the PCB, wherein each of the one or more antigen binding sites is formed by a pair of two domains, wherein one is comprised in the PCA and the other is comprised in the PCB, and wherein the antigen binding site(s) are located N- and/or C-terminally of the HDA or HDB.

9. The protein complex according to claim 1, wherein the PCA and/or the PCB comprises one or more further homo and/or heterodimerization domain C (HDC), wherein the homodimerization domain is selected from the group consisting of a CH3 domain, a CH2-CH3 domain, or a domain where homodimerization is mediated by a an Ig-like fold, a rossmann- or rossmann-like alpha-beta-alpha sandwich fold, an alpha-sandwich fold, a continuous-beta-sheet fold, a beta-sandwich fold, a mixed beta-sheet fold, a 2-helix orientation, an antiparallel alpha-helix-orientation, a parallel alpha-helix orientation, a 4-helix bundle motif, a leucine zipper and a coiled-coil domain and the heterodimerization domain is selected from the group consisting of a knob-into-hole CH3 domain, a knob-into-hole CH2-CH3 domain, a Fc-domain with introduced mutations to force heterodimerization (e.g. charged mutations), a domain of a pair of interchanged domains (such as Fc-one/kappa heterodimerization domain, CL and CH domains), an Ig-like fold with introduced mutations to force heterodimerization, or a domain mediating heterodimerization containing a rossmann- or rossmann-like alpha-beta-alpha sandwich fold, an alpha-sandwich fold, a continuous-beta-sheet fold, a beta-sandwich fold, a mixed beta-sheet fold, a 2-helix orientation, an antiparallel alpha-helix-orientation, a parallel alpha-helix orientation, a 4-helix bundle motif, a leucine zipper and a coiled-coil domain; wherein the antigen binding site(s) are located N- and/or C-terminally of the HDC.

10. The protein complex according to claim 8, wherein PCA and PCB comprise from N- to C-terminus the following elements: (i) PCA: V2-L1-HDA, and PCB: V2-L2-HDB; (ii) PCA: V1-L3-HDA-L4-V2, and PCB: V1-L1-HDB-L2-V2; (iii) PCA: V1-L1-V2-L2-HDA, and PCB: V2-L3-V1-L4-HDB; (iv) PCA: V1-L3-V2-L5-CL-L4-HDA, and PCB: V1-L1-V2-L6-CH1-L2-HDB; wherein L5, CL, L6 and CH1 may be present or absent; or (v) PCA: V1-L4-V2-L5-CL-L6-HDA, and PCB: V2-L1-V1-L2-CH1-L3-HDB; wherein L5, CL, L2 and CH1 may be present or absent; wherein each pair of V1, V2, V3, and V4 comprises a variable domain of a heavy chain and a variable domain of a light chain or a variable domain of an alpha chain and a variable domain of a beta chain and forms an antigen binding site, wherein L1 to L6 are peptide linkers, and wherein the PCA and/or the PCB optionally further comprises a HDC.

11. The protein complex according to claim 10, wherein the PCA and/or the PCB comprises a first HDC, and wherein the complex comprises one or more additional polypeptides comprising one or more antigen binding sites and a second HDC that is covalently or non-covalently bound to the first HDC.

12. One or more polynucleotides encoding one or more polypeptides of the protein complex according to claim 1.

13. One or more expression vectors comprising the one or more polynucleotides according to claim 12.

14. A cell comprising the one or more polynucleotides of claim 12 or one or more expression vectors comprising said one or more polynucleotides.

15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the protein complex according to claim 1, the one or more polynucleotides encoding one or more polypeptides of said protein complex, or one or more expression vectors comprising said one or more polynucleotides.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0211] FIG. 1: Structural basis for splitting the CH2 domain. (A) Cartoon structure of a full-length human antibody (pdb entry 3 S7G). One CH2 domain is highlighted in black. The glycans are depicted as spheres. Note, that the CH2 domain has no direct protein-protein contacts. (B) Structure of the CH2 domain. The split-site is located within a loop connecting the two β-sheets. The N-terminal half is coloured grey, the C-terminal part in black. A disulfide-bond is formed between the two β-sheets which covalently links the N- with the C-terminus in the split CH2 domain architecture. Cartoons were generated with pymol (https://sourceforge.net/projects/pymol/).

[0212] FIG. 2: Schematic structures of Fv-only and Fab-like formats with incorporated split CH2 domain. Upon the CH2 split, a new N- as well as a C-terminus is generated. The N-termini of the different domains are indicated. A his-tag was added in all constructs for purification purposes. (A) Split CH2 domain added to a diabody format (diabody split CH2). (B) DVD-Fab split CH2. (C) CODV-Fab split CH2. (D) Split CH2 domain inserted into a diabody-format (“splite” diabody). In this format different linkers (L1 and L4 respectively) were used at the newly generated N- and C-terminus. The molecules described in this report are numbered consecutively.

[0213] FIG. 3: SDS-PAGE of protein variants (1)-(7) analyzed under non-reducing and reducing conditions. All proteins were purified via their His-tag. Since both protein chains are present under reducing conditions, it is obvious that the connection of the two chains occurs via a disulfide-bridge. Protein bands corresponding to the correctly assembled protein are indicated by an arrowhead.

[0214] FIG. 4: Stability measurements (1)-(7) and CH2-domain (wt). For all proteins the concentration was adjusted to 0.5 mg/mL. (A) Tryptophan fluorescence measurement allows monitoring the melting of a protein. The measured fluorescence ration F350 nm/F330 nm represents the melting curves of the proteins. The calculated T.sub.m (Tab.2) corresponds to the maximum of the first derivative of the F350/F330 curve. (B) Light scattering was used to monitor the aggregation behaviour of the proteins. The calculated T.sub.agg (Tab.2) corresponds to the maximum of the first derivative of the measured scatter intensities. Note, that the CH2-domain (wt) does not start to aggregate despite the protein is unfolded.

[0215] FIG. 5: Schematic structures different antibody-like formats with incorporated split CH2 domain. Upon the CH2 split, a new N- as well as a C-terminus is generated. Variants (7)-(9) are based on the Fv-only and Fab-like format constructs (1)-(3) now fused to an IgG Fc-domain generating tetravalent bispecific antibody variants. Variants (11) and (12) are bivalent bispecific antibodies. On one side of the antibody the CH1 and CL domains are replaced by the split-CH2 domain. Since only one light chain is used, this construct design avoids the light chain mispairing problem. Heterodimerization of the heavy chains is achieved by applying knob-into-hole-mutations. (A) Diabody split CH2 tetravalent bispecific antibody. (B) DVD split CH2 tetravalent bispecific antibody. (C) CODV split CH2 tetravalent bispecific antibody. (D, E) Spit CH2 bivalent bispecific antibody.

[0216] FIG. 6: SDS-PAGE of protein variants (8)-(12) analyzed under non-reducing and reducing conditions. All proteins were purified via the Fc-domain on a protein A matrix. Two protein chains are required for variants (8)-(10) whereas four protein chains are required for variants (11) and (12). From the staining intensities of the protein bands in the reduced form it can roughly be estimated that the protein bands are present in equimolar amounts. Note, that for (11) and (12) the two heavy chains have roughly the same molecular weight and are not resolved into distinct bands on the SDS-PAGE.

[0217] FIG. 7: Mass spectrometry analysis of protein variant (1). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0218] FIG. 8: Mass spectrometry analysis of protein variant (2). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0219] FIG. 9: Mass spectrometry analysis of protein variant (3). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0220] FIG. 10: Mass spectrometry analysis of protein variant (4). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0221] FIG. 11: Mass spectrometry analysis of protein variant (5). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0222] FIG. 12: Mass spectrometry analysis of protein variant (6). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0223] FIG. 13: Mass spectrometry analysis of protein variant (7). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0224] FIG. 14: Mass spectrometry analysis of protein variant (8). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0225] FIG. 15: Mass spectrometry analysis of protein variant (9). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0226] FIG. 16: Mass spectrometry analysis of protein variant (10). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0227] FIG. 17: Mass spectrometry analysis of protein variant (11). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0228] FIG. 18: Mass spectrometry analysis of protein variant (12). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0229] FIG. 19: Biacore measurement of protein variant (4) to the human FcRn which was immobilized on the chip.

[0230] FIG. 20: SDS-PAGE of protein variants (13)-(16) analyzed under non-reducing and reducing conditions. Variants (13)-(16) require each two protein chains. Note that for all four variants comprise potential N-glycosylation sites (variant (13): N-terminal split CH2 domain, position 144; variant (14): C-terminal split CH2 domain, position 255; variant (15): N- and C-terminal split CH3 domain, positions 252 and 275, respectively; variant (16) N-terminal split CH2 domain, Pos. 146). These modifications might be the reason for the fuzzy appearance of some protein bands in the SDS-PAGE, especially for protein variants (15) and (16).

[0231] FIG. 21: Mass spectrometry analysis of protein variant (13). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0232] FIG. 22: Mass spectrometry analysis of protein variant (14). (A) Deglycosylated, non-reducing conditions. (B) Deglycosylated, reducing conditions.

[0233] FIG. 23: Stability measurements of protein variants (13)-(16). Tryptophan fluorescence measurement allows monitoring the melting of a protein. The measured fluorescence ration F350 nm/F330 nm represents the melting curves of the proteins. The calculated T.sub.m (Tab.2) corresponds to the maximum of the first derivative of the F350/F330 curve.

EXAMPLE SECTION

Material and Methods

Protein Expression and Purification

[0234] DNA coding for the desired amino acid sequences were synthesized (Thermofisher, Geneart) and cloned into an expression vector under a CMV promoter and a signal sequence required for secretion of the proteins into the cell culture medium. Protein expression was done by transient transfection of FreeStyle HEK293-F-cells (Thermo Fisher Scientific). Cells were cultivated in non-baffled shake flasks (Corning) at 110 rpm, 37° C. and 8% C CO.sub.2. Transfection was done when the cell density reached 1.2×106 cells/mL. DNA was mixed at a ratio of 1:3 with Polyethyleneimine in Optimem I-Medium (Thermo Fisher Scientific). After 20 min incubation at room temperature the transfection mix was added to the cell culture. Cells were further cultivated in FreeStyle F17 medium complemented with 6 mM glutamine for 6 days. Cells were removed from the culture broth by centrifugation (30 min at 4.500 g, 4° C.) and the supernatant was cleared by 0.22 μm sterile filtration. Protein variants (1)-(7) as well as the CH2 domain wt-only construct contain a consecutive stretch of six histidine-residues (His-tag). Protein purification was done by immobilized metal-ion affinity chromatography (IMAC) with a complete His-tag purification column (Roche) on a NGC Discover 100 Pro system (Biorad). The column was equilibrated with 50 mM Tris, 500 mM NaCl, 10 mM histidine. Before loading the culture supernatant, the pH was adjusted to 8.0 by adding 50 mL per L of a 1 M Tris, pH8.0 solution. In addition, 5 mL of a 2 M imidazole, pH 7.5 solution were added per L. The column was washed with equilibration buffer and proteins were eluted with a 35 CV gradient to 50 mM Tris, 300 mM NaCl, 500 mM imidazole. Fractions were analyzed by SDS-PAGE, corresponding fractions were pooled, concentrated by centrifugation (Vivaspin 20) and loaded on a Superdex 200 pg 16/60 (GE Healthcare) gelfiltration column equilibrated in phosphate buffered saline (PBS, Gibco). Fractions containing the desired protein were pooled and concentrated (Vivaspin 20). Protein variants (8)-(12) do not have a His-tag but rather a Fc-domain. For these variants capture was done on a HiTrap Protein A column (GE Healthcare). The column was equilibrated with PBS. After loading the cleared supernatant, the column was washed with PBS (Gibco) and 0.1 M citrate pH 6.0. Proteins were eluted with 0.1 M citrate, pH 3.0 buffer. Eluted fractions were neutralized by adding 10% 1 M Tris pH 8.5. Further purification was done using a Superdex 200 pg 16/69 (GE Healthcare) gelfiltration column equilibrated in PBS (Gibco). Protein concentrations were determined by measuring the absorption at 280 nm using a NanoDrop NT1000 spectrophotometer (Thermo Fisher Scientific). SDS-PAGE analysis was done using 4-12% BisTris gels with IVIES-buffer as running buffer (Invitrogen). For reduced samples, 0.1 M DTT were added to sample buffer (LDS sample buffer, invitroge) and samples were incubated for 5 min at 99° C. Separation was done with constant 200 V for 45 min. The BenchMark protein ladder was used as marker (invitrogen). After running, gels were stained with Coomassie blue (InstantBlue, Expedeon).

Thermal Stability Analysis

[0235] Thermal stability analysis was done with a Prometheus NT Flex device (NanoTemper Technologies) using the nanoDSF technology. The device is equipped with Aggregation Optics, that allows collecting scattering information simultaneously with fluorescence measurements. Analysis was done in the range from 20° C. to 95° C. with a thermal ramp of 1° C./min following the instructions of the manufacturer. All protein samples were dissolved in PBS and protein concentration was adjusted to 0.5 mg/mL. Measurements were done in duplicates. Data analysis was done using the software PR ThermControl V.2.1 (NanoTemper Technologies).

MS-Analysis

[0236] Protein integrity was analyzed by LC-MS. Protein samples were deglycosylated with 12.5 μg of protein diluted to 0.5 mg/ml in ddH2O containing PNGaseF (1:50 v/v) (glycerol free, New England Biolabs) at 37° C. for 15 hours. The LC-MS analysis was done on an Agilent 6540 Ultra High Definition (UHD) Q-TOF equipped with a dual ESI interface and an Agilent 1290/1260 Infinity LC System. Reversed phase (RP) chromatography was done using a PLRP-S 1000A 5 μm, 50×2.1 mm (Agilent) with a guard column PLRP-S 300A 5 μm, 3×5 mm (Agilent) at 200 μL/min and 80° C. column temperature. Eluents were buffer A containing LC water and 0.1% formic acid as well as buffer B containing 90% acetonitrile, 10% LC water and 0.1% formic acid. 1 μg of protein was injected onto the column and eluted using a linear gradient from 0 to 17 minutes with increasing acetonitrile concentration. Data were analyzed using MassHunter Bioconfirm B.06 (Agilent). Molecular masses were calculated based on the amino acid sequences of the proteins using GPMAW software version 10.32 (Lighthouse Data, Denmark).

Affinity Determinations

[0237] Binding of antigens to the antibody constructs was measured using surface plasmon resonance (SPR) on a BIAcore 3000 instrument (GE Healthcare) with HBS-EP buffer (GE Healthcare). As antigens human IL4 (IL004, Millipore) and human IL13 (IL012, Millipore) were used. The capture antibody (human antibody capture kit, His capture kit, Fab capture kit, GE Life Sciences) was immobilized via primary amine groups (11000 RU) on a research grade CMS chip (GE Life Sciences) using standard procedures. The ligands were captured at a flow rate of 10 μl/min with an adjusted RU value that resulted in maximal analyte binding of 30 RU. The antigens human IL4 and human IL13 were used as analytes and injected for 240 sec with a dissociation time of 300 sec at a flow rate of 30 μL/min. IL4 and IL13 were used in a dilution series of 0.1 nM to 3 nM and 0.8 nM to 25 nM, respectively. Chip surfaces were regenerated with 2 min injects of the regeneration buffer provided with the capture kit. Sensorgrams were double referenced with a blank chip surface and HBS-EP buffer blanks. Recombinant human neonatal Fc-receptor (FcRn) protein was immobilized via primary amine groups (200 RU) in the sample flow cell compartment of a research grade CMS chip (GE Life Sciences) using standard procedures. The reference flow cell compartment was activated and deactivated without FcRn immobilization to generate a blank chip surface. For the analysis an assay buffer with pH 6.0 was used (150 mM NaCl, 20 mM Na-phosphate, 0.05% surfactant P20, pH6.0). The antibody was used as analyte at 800 nM dilution in assay buffer and injected over reference and sample flow cells for 240 sec with a dissociation time of 300 sec at a flow rate of 30 μL/min. Chip surfaces were regenerated with 2 min injects of HBS-EP buffer at 30 μl/min. Sensorgrams were double referenced with a the blank chip surface and HBS-EP buffer blanks. All data analysis was done using the BIAevaluation software v4.1.

Results

[0238] It was envisaged to split the IgG1 CH2 domain in two parts with roughly the same size in a similar manner as it had been described for the split-ubiquitin-system. This is in contrast to the split GFP or β-galactosidase approach, where only small parts of the entire protein are sufficient to restore functionality. The structural basis for assessing the split site is shown in FIG. 1. As first attempts to evaluate the reassembly of the split CH2 domain the inventors added two different variable domains with or without the CH1 and CL domains thus creating different formats (FIG. 2). The two different variable domains directed against interleukins IL4 and IL13 are those of a bispecific antibody currently in clinical development and have been described in detail before (Steinmetz et al.; Mabs 2016; 8:867-878). The sequence of the N-terminal part of the split CH2 domain as used in this report consists of 52 amino acids (5.7 kDa) and is characterized by SEQ ID NO: 30. The sequence of the C-terminal part of the split CH2 domain as used in this report consists of 58 amino acids (6.7 kDa) and is characterized by SEQ ID NO: 31. Upon re-assembly of the split CH2 domain, the disulfide-bond between the two parts was expected to form and hence the two protein chains should be covalently connected. Since only one protein chain was fused to a tag for purification only the heterodimeric molecules should be purified. The connection of the two chains by a disulfilde-bond could easily be monitored using SDS-PAGE running under reduced compared to non-reduced conditions (FIG. 3). As apparent from these results, the split CH2 domain was reconstituted and the disulfide-bond connecting the two split CH2 half was formed. Upon reduction of the protein samples, the only disulfide-bond connecting the two protein chains is broken. This disulfide-bond is formed between the split CH2 domain parts. The observed main protein bands migrated in the SDS-PAGE at their predicted size. To further assess whether the produced proteins contain a reassembled CH2 domain the inventors analyzed the protein stability using differential scanning fluorimetry (DSF) technology (FIG. 4, Tab.2). This technique monitors the intrinsic fluorescence of tryptophan residues within a protein. Tryptophan fluorescence is highly sensitive to its immediate environment. Upon protein conformational changes, for example during denaturation, the fluorescence emission maxima is shifted. The inventors anticipate that a distinct melting temperature (T.sub.m) of the protein can be measured if the split-CH2 domain is re-assembled. Simultaneously to the fluorescence measurements light scattering data were recorded that allow calculation of the onset of protein aggregation (T.sub.agg). The obtained T.sub.m and T.sub.agg data are given in Table 2. The measured T.sub.m values are in the range of ˜60° C. or slightly higher and in the range of the T.sub.m of the CH2-domain wt construct. These data suggest that the split CH2 domain is reassembled. Protein variants (2) and (3) have a slightly higher T.sub.m and protein variant (2) has a T.sub.agg that is ˜12° C. higher. The elevated T.sub.m and T.sub.agg are probably due to the presence of the CL and the CH1 domains which further stabilize the protein. Next, the inventors assessed whether it is possible to express the split CH2 domain only and purify the reassembled CH2-domain. However, the inventors could not detect any expression of the split CH2 domain. Probably, a folded fusion partner is required for proper expression and reconstitution of the split CH2 domain. As the expression of the split CH2 domain worked well with the Fv- and Fab only format, the inventors next evaluated the expression of these constructs when incorporated within an IgG-like architecture (FIG. 5A-C). In addition, the inventors used the split CH2-domain to address the light chain pairing problem in a bispecific heterodimeric IgG-like format (FIG. 5 D,E). All variants could be expressed and purified (FIG. 6). All proteins containing the re-assembled split CH2 domain were analyzed by mass spectroscopy under oxidizing and reducing conditions. The presence of the anticipated species and the corresponding protein chains could be demonstrated (FIG. 7-18). The inventors' focus was to show that a split CH2 domain can be in principal incorporated into a larger molecule and is able to reassemble in such an architecture. Correct assembly of the target molecules was demonstrated by evaluating the binding properties to the corresponding antigens. This was checked by surface plasmon resonance (SPR)-measurements (Tab.3, Tab.7-20). Structural changes below the Fv domains might lead to subtle changes in the Fv domains, and as a consequence thereof loss in binding affinity could occur. However, only very minor differences in binding affinity between the various constructs compared to the reference molecule, a bispecific anti-IL4-anti-IL13 CODV-IgG (variant 13) (Steinmetz et al.; Mabs 2016; 8:867-87), were detected. In addition, binding of the reconstituted CH2 domain to human FcRn was analyzed by Biacore analysis. FcRn was immobilized on the chip and variant (4) was used as analyte. The measurement shows binding of variant (4) to the FcRn (FIG. 19).

[0239] In contrast to variants (1)-(12), which are all based on the IgG1 split CH2 domain, variants (13)-(16) comprise CH domains of other antibody classes. Variants (13)-(16) have the diabody split CH2 format as used in variant (1), but the IgG1 split CH2 domain is exchanged for one the following split CH domains: variant (13): split IgA CH2; variant (14): split IgD CH2; variant (15): split IgE CH3; and variant (16): split IgE CH2. Tab. 4 indicates the sequences of the variants.

[0240] Experimental procedures were performed as described for variants (1)-(12) above.

[0241] SDS-PAGE analysis of variants (13)-(16) (FIG. 20) demonstrates that all proteins can be produced and assemble. Thermal stability measurements show a distinct melting point in all cases (FIG. 23, Tab. 2). The obtained melting temperature is comparable to the wild-type CH2 domain indicating that the proteins are correctly folded. Variants (13) and (14) were also analyzed by mass spectroscopy (FIGS. 21 and 22, Tab. 5 and 6). In summary, the results for variants (13)-(16) demonstrate that splitting and re-assembly is not restricted to the IgG1 CH2 domain.

TABLE-US-00004 TABLE 2 stability of the reassembled CH2 domain as measured by tryptophan fluorescence. T.sub.m T.sub.agg Variant Description (average) (average) (1) Diabody-added Split CH2 59.01 59.79 (2) DVD-Fab Split CH2 65.33 72.09 (3) CODV-Fab Split CH2 63.88 61.87 (4) Diabody Split CH2 (included) (“Splite”) 59.51 59.94 (5) Diabody Split CH2 (included) (“Splite”) 59.46 59.25 (6) Diabody Split CH2 (included) (“Splite”) 58.97 59.08 (7) Diabody Split CH2 (included) (“Splite”) 58.43 58.88 (13) Diabody-added Split IgA CH2 61.95 63.50 (14) Diabody-added Split IgD CH2 59.01 63.43 (15) Diabody-added Split IgE CH3 60.85 n.d. (16) Diabody-added Split IgE CH2 61.33 n.d. CH2-domain (wt) 59.94 n.d. T.sub.m: melting temperature. T.sub.agg: onset of protein aggregation. Average of two measurements. n.d.: not determined.

TABLE-US-00005 TABLE 3 Antigen affinities for the different protein variants as determined by SPR. Variant Description K.sub.D (IL4) [M] K.sub.D (IL13) [M] CODV-IgG 2.72 × 10.sup.−12 2.45 × 10.sup.−11 (1) Diabody-added Split CH2 1.60 × 10.sup.−11 1.01 × 10.sup.−10 (2) DVD-Fab Split CH2  3.0 × 10.sup.−11 1.05 × 10.sup.−10 (3) CODV-Fab Split CH2 5.48 × 10.sup.−12  6.7 × 10.sup.−11 (4) Diabody Split CH2 (included) 6.48 × 10.sup.−11 2.74 × 10.sup.−11 (“Splite”) (5) Diabody Split CH2 (included) 4.03 × 10.sup.−11 1.46 × 10.sup.−11 (“Splite”) (6) Diabody Split CH2 (included) 8.78 × 10.sup.−11 2.57 × 10.sup.−11 (“Splite”) (7) Diabody Split CH2 (included) 6.85 × 10.sup.−11 4.31 × 10.sup.−11 (“Splite”) (8) Diabody Split CH2 tetravalent 1.43 × 10.sup.−12 4.36 × 10.sup.−11 bispecific antibody (9) DVD-Split CH2 tetravalent 6.09 × 10.sup.−11 3.64 × 10.sup.−11 bispecific antibody (10) CODV-Split CH2 tetravalent 2.22 × 10.sup.−13 3.31 × 10.sup.−11 bispecific antibody (11) Split CH2 bivalent bispecific 1.30 × 10.sup.−12 1.96 × 10.sup.−11 antibody (12) Split CH2 bivalent bispecific 9.22 × 10.sup.−13 8.24 × 10.sup.−12 antibody

TABLE-US-00006 TABLE 4 Polypeptide sequences of variants (1)-(16) Variant Chain 1 Chain 2 Chain 3 Chain 4 1 SEQ ID 11 SEQ ID 12 — — 2 SEQ ID 13 SEQ ID 14 — — 3 SEQ ID 15 SEQ ID 16 — — 4 SEQ ID 17 SEQ ID 18 — — 5 SEQ ID 19 SEQ ID 20 — — 6 SEQ ID 17 SEQ ID 20 — — 7 SEQ ID 19 SEQ ID1 8 — — 8 SEQ ID 11 SEQ ID 21 — — 9 SEQ ID 13 SEQ ID 22 — — 10 SEQ ID 15 SEQ ID 23 — — 11 SEQ ID 24 SEQ ID 27 SEQ ID 26 SEQ ID 25 12 SEQ ID 24 SEQ ID 27 SEQ ID 28 SEQ ID 29 13 SED ID 41 SED ID 42 — — 14 SED ID 43 SED ID 44 — — 15 SED ID 45 SED ID 46 — — 16 SED ID 47 SED ID 48 — —

TABLE-US-00007 TABLE 5 Intact mass, deglycosylated, non-reduced Calculated Measured Variant MW (Da) MW (Da) 1 64996.68 64999.46 2 87366.46 87371.17 3 87646.04 87633.95 4 65283.94 65287.76 5 63563.40 63839.00 6 64509.25 64513.21 7 64338.09 64341.82 8 180567.66 180636.44 9 225309.20 225345.25 10 225868.34 225887.79 11 136953.87 136955.97 12 136953.87 136956.10 13 63461.68 63586.0 14 64410.00 64413.0
Molecular weight were calculated with GPMAW (Vers. 10.32; Lighthouse Data, Denmark) (average mass values, all cysteines are assumed to form disulfid-bridges).

TABLE-US-00008 TABLE 6 Intact mass, deglycosylated, reduced Chain 1 Chain 1 Chain 2 Chain 2 Chain 3 Chain 3 Chain 4 Chain 4 Calc. Meas Calc. Meas. Calc Meas. Calc Meas Variant MW (Da) MW (Da) MW (Da) MW (Da) MW (Da) MW (Da) MW (Da) MW (Da) 1 31970.20 31967.26 33035.57 33033.77 2 41623.89 41619.19 45758.69 45756.17 3 42446.97 42442.30 45215.20 45195.30 4 30192.28 30189.83 35101.73 35100.71 5 29246.44 29243.50 34327.04 34326.06 6 30192.28 30189.70 34327.04 34325.52 7 29246.44 29243.47 35101.73 35100.84 8 31970.20 31967.28 58329.76 58327.34 9 41623.89 41619.61 71052.88 71050.64 10 42446.97 42443.21 70509.39 70404.21 11 48396.95 48391.09 23746.05 23741.88 47461.70 47441.83 17379.40 17377.71 12 48396.95 48390.95 23746.05 23741.78 45224.38 45221.39 19613.70 19597.95 13 31110.19 31108.0 32364.59 32362.0 14 31515.78 31513.0 32904.29 32902.0
Molecular weights were calculated with GPMAW (Vers. 10.32; Lighthouse Data, Denmark) (average mass values, all cysteines are assumed to be reduced (SH))

Biacore-Measurements

[0242]

TABLE-US-00009 TABLE 7 Biacore measurement of protein variant (1) immobilized with the aid of the His-capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KA (1/M) KD (M) Chi2 187 IL4_3.125 nM 7.32E+07 1.17E−03 37 6.24E+10 1.60E−11 1.760 185 IL13_25 nM 1.99E+06 2.00E−04 34 9.93E+09 1.01E−10 0.416

TABLE-US-00010 TABLE 8 Biacore measurement of protein variant (2) immobilized with the aid of the His-capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KA (1/M) KD (M) Chi2 291 IL4_3.125 nM 1.42E+07 4.26E−04 46 3.33E+10 3.00E−11 0.997 291 IL13_25 nM 2.03E+06 2.14E−04 40 9.49E+09 1.05E−10 0.395

TABLE-US-00011 TABLE 9 Biacore measurement of protein variant (2) immobilized with the aid of the Fab-capture kit Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KA (1/M) KD (M) Chi2 187 IL4_3.125 nM 1.48E+07 2.59E−04 38 5.73E+10 1.75E−11 1.610 187 IL13_25 nM 1.99E+06 2.12E−04 33 9.39E+09 1.06E−10 0.713

TABLE-US-00012 TABLE 109 Biacore measurement of protein variant (3) immobilized with the aid of the Fab-capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KA (1/M) KD (M) Chi2 185 IL4_3.125 nM 8.26E+07 4.52E−04 43 1.83E+11 5.48E−12 1.310 185 IL13_25 nM 2.03E+06 1.36E−04 33 1.49E+10 6.70E−11 1.370

TABLE-US-00013 TABLE 11 Biacore measurement of protein variant (4) immobilized with the aid of the His-capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 187 IL13_25 nM 5.82E+06 1.60E−04 22 2.74E−11 0.332 187 IL4_3.125 nM 3.46E+06 2.24E−04 26 6.48E−11 0.529

TABLE-US-00014 TABLE 12 Biacore measurement of protein variant (5) immobilized with the aid of the His-capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 331 IL13_25 nM 1.29E+07 1.88E−04 10 1.46E−11 0.223 331 IL4_3.125 nM 4.10E+06 1.65E−04 11 4.03E−11 0.141

TABLE-US-00015 TABLE 13 Biacore measurement of protein variant (6) immobilized with the aid of the His-capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KA (1/M) KD (M) Chi2 195 IL13_25 nM 4.07E+06 1.05E−04 20 3.89E+10 2.57E−11 0.277 195 IL4_3.125 nM 2.97E+06 2.61E−04 30 1.14E+10 8.78E−11 0.328

TABLE-US-00016 TABLE 14 Biacore measurement of protein variant (7) immobilized with the aid of the His-capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KA (1/M) KD (M) Chi2 188 IL13_25 nM 4.14E+06 1.78E−04 21 2.32E+10 4.31E−11 0.326 188 IL4_3.125 nM 3.38E+06 2.32E−04 28 1.46E+10 6.85E−11 0.29

TABLE-US-00017 TABLE 15 Biacore measurement of protein variant (8) immobilized with the aid of the human antibody capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 229 IL13_25 nM 3.46E+06 1.51E−04 35 4.36E−11 0.407 206 IL4_3.125 nM 9.49E+07 1.35E−04 51 1.43E−12 1.23

TABLE-US-00018 TABLE 16 Biacore measurement of protein variant (9) immobilized with the aid of the human antibody capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 226 IL13_25 nM 3.18E+06 1.16E−04 28 3.64E−11 0.324 207 IL4_3.125 nM 1.77E+07 1.08E−04 39 6.09E−12 0.674

TABLE-US-00019 TABLE 17 Biacore measurement of protein variant (10) immobilized with the aid of the human antibody capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 247 IL13_25 nM 2.49E+06 8.26E−05 25 3.31E−11 0.521 225 IL4_3.125 nM 9.19E+08 2.04E−04 32 2.22E−13 0.84

TABLE-US-00020 TABLE 18 Biacore measurement of protein variant (11) immobilized with the aid of the human antibody capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 286 IL4 0.39 nM 6.84E+07 8.91E−05 18 1.30E−12 0.251 290 IL13 25 nM 1.69E+06 3.31E−05 31 1.96E−11 0.589

TABLE-US-00021 TABLE 19 Biacore measurement of protein variant (12) immobilized with the aid of the human antibody capture kit. Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 300 IL4 0.39 nM 2.42E+11 2.23E−01 22 9.22E−13 0.256 301 IL13 25 nM 1.59E+06 1.31E−05 29 8.24E−12 0.320

TABLE-US-00022 TABLE 20 Biacore measurement of protein variant (13) immobilized with the aid of the human antibody capture kit. (anti-IL13, anti-IL4 CODV) Rmax RU 2nd Ab Analyte ka (1/Ms) kd (1/s) (RU) KD (M) Chi2 205 IL13_25 nM 3.86E+06 9.45E−05 27 2.45E−11 0.519 189 IL4_3.125 nM 5.03E+08 1.37E−03 29 2.72E−12 0.576