WO2010122148A1 - An improved antibody domain and antibody fragments and antibodies based thereon - Google Patents

An improved antibody domain and antibody fragments and antibodies based thereon Download PDF

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WO2010122148A1
WO2010122148A1 PCT/EP2010/055440 EP2010055440W WO2010122148A1 WO 2010122148 A1 WO2010122148 A1 WO 2010122148A1 EP 2010055440 W EP2010055440 W EP 2010055440W WO 2010122148 A1 WO2010122148 A1 WO 2010122148A1
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domain
sequence
seq
chl
wildtype
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Johannes Buchner
Matthias Feige
Moritz Marcinowski
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Boehringer Ingelheim International GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype

Definitions

  • the present invention relates to an improved antibody domain and to antibody fragments and antibodies based thereon. More specifically, it relates to a C H I -domain which has been made more stable by selected mutation of specific residues. The C ⁇ l-domain thus produced is natively folded and stable and therefore contributes to the stability and folding of an antibody fragment or an entire antibody.
  • the present invention also relates to a nucleic acid coding for such an improved antibody domain or antibody fragment or antibody.
  • the present invention also relates to a method of stabilizing a Ciil-domain of an antibody and/or of an antibody fragment comprising a C H I -domain and/or of an antibody comprising a C H I -domain.
  • Antibodies are biotechnologically important proteins. The range of applications extends from fundamental research, diagnostics to therapy, for example in the treatment of malignant diseases. Antibodies are complex glycosylated protein molecules. Antibodies occur in different varieties which are also known as so-called isotypes or classes. In mammals, there are typically five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. These isotypes differ in their biological properties, functional locations and their ability to deal with different antigens. Yet, structurally, they are remarkably similar. As an example, immunoglobulin G (IgG) is the most abundant antibody in the blood.
  • IgG immunoglobulin G
  • the basic structure of an intact antibody is a Y-shaped molecule composed of two heavy (H) and two light (L) chains; in immunoglobulins A and M these molecules are assembled further into larger complexes.
  • the L-chain consists of one variable (V L ) and one constant (C L ) domain, and the IgG H-chains consist of one variable (V H ), and three constant domains (C H I , C H 2, and C H 3). All these domains consist of about 100 residues, are homologous in their primary structure, and are believed to be independent structural units.
  • immunoglobulin domains appear to have essentially the same conformation, designated also as the immunoglobulin fold, consisting of two layers of antiparallel ⁇ -sheet that are usually linked by a disulfide bond.
  • the topology in this immunoglobulin fold typically has a specific connectivity of the individual ⁇ -strands which is also sometimes referred to as the Greek key topology which is named after a pattern found on Greek pottery.
  • the polypeptide chains between the domains are susceptible to proteases to a different extent. Most susceptible is the hinge region linking the two arms to the base of Y. After cleavage at this site, the two arms are released individually which are known as Fab-fragments; the base is known as the Fc-fragment.
  • Each Fab-fragment contains the V L , C L , V H and CHl-domains; the Fc-fragment has two copies of each of the C H 2- and C H 3- domains.
  • Fv- fragments consist of only the V H and VL-domains.
  • the connecting segments between the domains have varying degrees of flexibility and the individual parts of the antibody molecule may undergo considerable motion relative to each other.
  • polypeptide having an amino acid sequence derived from a wildtype sequence of a CHl-domain of an antibody wherein in said wildtype sequence, P 167 and/or P 189 of the wildtype sequence, or its (their) corresponding counterpart(s), is (are) replaced by an amino acid residue independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably A, V, T, and most preferably A, wherein the numbering of residues is with reference to the murine IgGl wildtype heavy chain sequence using the Kabat numbering scheme.
  • said wildtype sequence of said Ciil-domain is a murine sequence, preferably a murine IgGl sequence, more preferably a sequence selected from SEQ ID NO: 1- 8, and 18 even more preferably SEQ ID NO: 4.
  • said wildtype sequence of said C H I -domain is a human sequence, preferably a human IgGl sequence, more preferably a sequence selected from SEQ ID NO: 9- 17, more preferably selected from SEQ ID NO: 13-16, and even more preferably SEQ ID NO: 13.
  • said wildtype sequence of said Ciil-domain is a goat sequence, chicken sequence, rat sequence or rabbit sequence.
  • said wildtype sequence of said C ⁇ l-domain is of an Ig-isotype selected from IgA, IgAl , IgA2, IgD, IgE, IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, and IgGM, preferably a sequence selected from SEQ ID NO: 1-17.
  • said wildtype sequence of said C H I -domain, prior to any replacement of P167, Pl 89 or its (their) counterpart(s) is a sequence selected from SEQ ID NO: 1-18, preferably selected from SEQ ID NO: 4, 13-16 and 18, and more preferably selected from
  • the numbering of residues is with reference to the murine IgGl C H I- domain wildtype sequence, preferably SEQ ID NO:4 or SEQ ID NO: 18, using the Kabat numbering scheme, and wherein said counterparts are residues in Ig C H I -domain wildtype sequences other than murine IgGl CHl-domain wildtype sequence, such as CHl-domain wildtype sequences of murine IgA, IgD, IgE, IgG2a, IgG2b, IgG3, IgM; human IgAl, IgA2, IgG, IgE, IgGl, IgG2, IgG3, IgG4, and IgM, or the same isotypes from other species, such as goat, chicken, rat or rabbit, and wherein said counterparts are determined by sequence homology alignment or structure homology alignment of said murine IgGl CHl-domain wildtype sequence with another CHl-domain wildtype sequence.
  • said counterparts are determined by being aligned with said P167 or P189 in an optimum alignment of sequences, i.e. with the best possible match of residues (highest number of identical and/or homologous residues in aligned positions) between the aligned sequences.
  • said murine IgGl CHl-domain wildtype sequence and said another CHl- domain wildtype sequence have a number of identical and/or homologous residues and their respective positions in common, referred to as "conserved residues", and wherein said sequence homology alignment occurs such that at least 20%, preferably at least 30%, preferably at least 40 %, preferably at least 50 %, preferably at least 60 %, preferably at least 70 %, preferably at least 80 %, preferably at least 90 %, preferably at least 95 %, more preferably at least 96 %, even more preferably at least 98 %, even more preferably at least 99 % of residues are conserved residues; and wherein said structure homology alignment occurs by superpositioning the tertiary structure of said murine IgGl CHl-domain wildtype sequence with the tertiary structure of another C H l-domain wildtype sequence and obtaining maximum overlap of atomic coordinates of non- hydrogen protein atoms of said two wildtype sequences
  • the CHl-domain wildtype sequence is murine IgA (SEQ ID NO: 1), and said corresponding counterparts to P 167 and Pl 89 are P167 and P190, respectively; or the C H l-domain wildtype sequence is murine IgD (SEQ ID NO: 2), and said corresponding counterparts to P167 and P189 are P164 and no second residue, respectively; or the C H I- domain wildtype sequence is murine IgE (SEQ ID NO: 3), and said corresponding counterparts to P167 and P189 are P163 and G180, respectively; or the C ⁇ l-domain wildtype sequence is murine IgG2a (SEQ ID NO: 5), and said corresponding counterparts to P 167 and P189 are P167 and P189, respectively; or the C H I -domain wildtype sequence is murine IgG2b (SEQ ID NO: 6), and said corresponding counterparts to P 167 and P 189 are P166 and P188, respectively; or the CHl-domain wildtype sequence is murine IgG3
  • the terms "no residue” or “no second residue”, when used to denote the “corresponding counterparts”, are meant to indicate that, in such specific case, no such corresponding counterpart residue exists.
  • the statement is made "and said corresponding counterparts to P 167 and P 189 are P 169 and no second residue"
  • this is meant to mean that in the C H I -domain wildtype sequence of human IgE (SEQ ID NO: 12) there is no corresponding counterpart residue to P 189 (of the murine IgGl C H I -domain wildtype sequence) and hence no mutation is to be introduced at such position, for the C H I -domain wildtype sequence of human IgE (SEQ ID NO: 12).
  • the C H I -domain wildtype sequence is murine IgGl as shown in SEQ ID NO: 18, and said corresponding counterparts to P 167 and P189 are P 167 and P 189, respectively.
  • said corresponding counterparts are replaced by an amino acid residue independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably A, V, T, more preferably A.
  • an antibody fragment preferably an Fab-fragment, or an antibody comprising the polypeptide according the present invention.
  • nucleic acid coding for a polypeptide according to the present invention or for an antibody fragment or antibody according to the present invention.
  • the objects of the present invention are also solved by a method of stabilizing a C H I -domain or an antibody fragment, preferably an Fab-fragment, or an antibody, said antibody fragment or said antibody comprising a C H I -domain, said method comprising the step: replacing in a wildtype sequence of a CHl-domain one or two amino acid residue(s) selected from P 167 and P 189, or its (their) corresponding counterpart(s), by an amino acid residue, independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably A, V, T, and most preferably A, wherein the numbering of residues is with reference to the murine IgGl wildtype sequence using the Kabat numbering scheme, and wherein, preferably, said step of replacing occurs by side
  • the polypeptide in accordance with the present invention is no complete heavy chain, but only consists of a sequence of a C H I -domain.
  • a C H I -domain is meant to refer to an amino acid sequence which makes up the first constant domain in the heavy chain, after the variable domain of the heavy chain, when viewed in the direction from the N-terminus to the C-terminus of the heavy chain.
  • the term is meant to encompass the wildtype sequences of such C H I -domains, as exemplified by the various C H I -domains in Figures 8 and 9; the term is meant to also encompass variants of such wildtype sequences, wherein P 167 and/or P189 or its (their) corresponding counterparts have been replaced by another amino acid or other amino acids. Such variants are herein also sometimes referred to as being "derived from a wildtype sequence of C H I- domain".
  • a person skilled in the art will be able to determine whether or not a sequence would qualify as a sequence of a C H I -domain, based on sequence alignments made with known C H I -domain sequences, as well as structural determinations of the corresponding sequences using structure determination techniques, such as x-ray crystallography, and nuclear magnetic resonance with and without isotope labelling.
  • the Kabat numbering is used.
  • the amino acid numbering is based on the murine sequence of the heavy chain of IgGl.
  • the terms "pro line residue No. 167", and “proline residue No. 189”, are based on the murine sequence numbering of IgGl but is meant to include and designate also the "corresponding counterpart" residues in other sequences, such as human sequences or sequences of other species or sequences of other Ig isotypes. It is therefore envisaged that, although the polypeptides/antibodies of the present invention are constructed and mutated in the context of the numbering of the murine sequence, the replacements/mutations may also be
  • “transferred” to another sequence This can be done by determining the "equivalent” or “corresponding counterpart” residues between the two sequences, typically based on sequence or structural homology between the sequences of the two C ⁇ l-domains.
  • the amino acid sequence of the first C ⁇ l-domain is directly compared to the sequence of a second Ciil-domain. After aligning the sequences, using one or more of the homology alignment programs well known in the art, such as CLUSTALW; (for example using conserved residues between species), allowing for necessary insertions and deletions in order to maintain alignment (i. e.
  • the residues "equivalent” or "corresponding" to particular amino acid residues in the primary sequence of the first C H I -domain are defined.
  • Alignment of conserved residues preferably should conserve at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, more preferably at least 96%, even more preferably at least 98%, even more preferably at least 99% and most preferably 100% of residues.
  • “Equivalent” or “corresponding counterpart” residues may also be defined by determining structural homology between a first and second C H I- domain, that is at the level of tertiary structure for CHl-domains whose structures have been determined.
  • “equivalent” or “corresponding” residues are defined as those, for which the atomic coordinates of two or more of the mainchain atoms of a particular amino acid residue of the first C H I -domain or precursor (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the proteins.
  • the C H I -domain polypeptides according to the present invention may be constructed into any second C H I -domain which has a significant sequence or structural homology with the first C H I -domain.
  • the "C ⁇ l-domain” of murine IgGl comprises residues 114-214 of the entire heavy chain, using the Kabat scheme.
  • the "CHI -domain” of human IgGl comprises residues 114-
  • residue no. 1 of the CHl-domain of murine IgGl i.e. residue no. 1 in SEQ ID NO: 4
  • residue no. 114 is residue no. 114 with respect to the numbering of the entire heavy chain using the Kabat scheme.
  • residues of the wild type sequence which, in accordance with the present invention, are replaced by another amino acid residue.
  • the polypeptides in accordance with the present invention only consist of the aforementioned amino acid sequences of the respective C H I -domain, with the one or more replacements of amino acid residues, as outlined further above, and no other residues outside of the CHl-domain, such as would for example occur in the regions connecting to and from the C H l-domain within the heavy chain, or in regions of other domains, such as the V H -domain or the C H 2-domain or the C H 3-domain.
  • the C H l-domain may be part of an antibody fragment or an entire antibody.
  • said antibody fragment or antibody is a human or murine antibody fragment or antibody. In another embodiment, said antibody fragment or antibody is a goat, chicken, rat or rabbit antibody fragment or antibody.
  • antibody as used herein, is meant to refer to a complete antibody or complete immunoglobulin, as opposed to a mere fragment thereof, which is herein also sometimes denoted as an "antibody fragment”.
  • the CHl- domain when studied and looked at on its own, i. e. without the other domains of the heavy or light chain present or covalently attached, in fact, is unfolded under native conditions.
  • native conditions as used herein is meant to refer to PBS buffer, pH 7.4, at 25°C.
  • unfolded is meant to refer to a state of the polypeptide chain, wherein the chain does not show any defined structure, as measured by biophysical techniques such as CD (circular dichroism) and nuclear magnetic resonance spectroscopy (NMR).
  • CD circular dichroism
  • NMR nuclear magnetic resonance spectroscopy
  • a "random coil” protein is characterized by the absence of any secondary structure elements such as alpha-helix, ⁇ -strand, ⁇ -sheet, ⁇ -turn 3 10 - helix, let alone the presence of any long range tertiary interactions.
  • secondary and tertiary structural elements or structures or interactions are well known by someone skilled in the art and are used herein in the same sense, as would be understood by someone skilled in the art.
  • the present inventors could detect unexpected structural properties of the C H I- domain which also would have been expected to show such immunoglobulin fold, even if the C H l-domain was looked at in isolation, i. e. without any of the other corresponding domains of the heavy chain or light chain, such as C L , present or attached.
  • the inventors have surprisingly found that the isolated C H l-domain, i. e. without any of the other domains of the heavy chain or light chain present or attached, is unfolded. Contrary to what one would have expected from previous results, the isolated CHl-domain does not have any defined structure.
  • the present invention encompasses variants of the C H l-domain which have improved properties with respect to assembly of the entire antibody and/or secretion of the C H l-domain in cell culture experiments.
  • the present invention also encompasses the stabilized, autonomously folding C H I -domain, as well as larger proteins comprising such stabilized C ⁇ l-domains.
  • larger proteins could,for example be a Fab- fragment, the entire heavy chain or even an entire antibody.
  • the present inventors identified two residues within the C H I -domain, which appear to be pivotal in the autonomy of the C H l-folding. These residues are, with reference to the murine CHl-domain of IgGl P167 and P189.
  • the "corresponding counterpart" residues can be easily determined by optimum sequence homology alignment or structure homology alignment, whereby the maximum degree of identity between the two sequences aligned or between the two structures aligned is achieved.
  • the "corresponding counterpart" residues may also be determined by structural homology, rather than sequence alignments.
  • the C H l-domain in accordance with the present invention i. e. having one or two of the aforementioned residues replaced has improved properties in that it is secreted in higher yields in cell culture media and contributes to a better antibody assembly;
  • the double mutant i.e. the variant in which both residues are replaced is autonomously folding and adopts a folded structure, when looked at in isolated form, i. e. in the absence of any of the other domains of the heavy or light chain, such as C L , V L , C H 2, C H 3 etc.
  • Mutation of any of the residues may occur by means known to someone skilled in the art.
  • a preferred way of producing such mutated C H I -domains on their own or within an antibody is site-directed mutagenesis.
  • the C H I -domains in accordance with the present invention have improved qualities in that they either show improved properties with respect to secretion in cell culture media or with respect to antibody assembly, or they have improved qualities in that they even adopt a folded structure on their own and thus are stable and will therefore aid in the structuring and assembly of an antibody fragment, e. g. Fab-fragment, or an entire antibody.
  • an autonomously folding and stabilized C H I -domain will reduce the requirements of the cellular folding machinery, exemplified by chaperone proteins.
  • Figure 1 shows an antibody of the IgG-isotype.
  • the two light chains are lightly coloured, the heavy chains are represented in dark.
  • the regions responsible for the antigen binding, for glycosylation, as well as those regions mediating effector functions (Fc-fragment) are shown.
  • Figure 2 shows the location of the CHl-domain within the IgG.
  • the heavy chain of the IgG is shown in dark, the light chain is shown lightly coloured.
  • the C H l-domain within the heavy chain is encircled.
  • FIG 3 shows the structural characterization of the C H l-domain using circular dichroism (CD) spectroscopy.
  • the isolated CHl-domain (“C H I”) shows the CD-spectrum of an unfolded protein
  • the isolated CL-domain (“C L ”) shows the typical spectrum of a ⁇ -sheet protein. If both proteins are co-incubated (C H I + C L ), the spectrum shows the typical trace of a ⁇ -sheet protein, and the calculated curve for C H I within the co-incubation also shows such ⁇ -sheet protein trace (C H I in complex, calculated).
  • Figure 4 shows the structuring of the C H I -domain in the presence of the C L -(IO main.
  • Figure 5 shows the folding kinetics of the CHl-domain in the presence of the CL-domain, as followed by the CD-signal at 205 nm. The folding takes several hours to be completed at 25°C in PBS.
  • Figure 6 shows a model of an exemplary CHl-domain (murine from IgGl), with two pro line residues marked which may be replaced in accordance with the present invention.
  • a preferred replacement is by alanine, but other residues are also feasible. Such replacement leads to a folding of the CHl-domain.
  • the coordinates of this model are taken from pdb-f ⁇ le 12E8.
  • Figure 7 shows results of a mutant C H l-domain in accordance with the present invention wherein both proline 167 and proline 189 have been replaced by alanine.
  • the spectrum on the left show the CD-spectrum of a ⁇ -sheet (panel A), and the protein is stable, having a melting point of 50 0 C (panel B).
  • Figure 8 shows a comparison by multiple sequence alignment of CHl-domain by multiple sequence alignment of CHl-domains of various murine Ig-isotypes.
  • the numbering of the C H I -domains is in accordance with the Kabat numbering scheme.
  • There is a conserved proline residue (underlined) which is rate determining for the folding of the CHl-domain.
  • the two proline residues or their corresponding counterparts (in some cases glycine like for IgM) which have been identified as being detrimental for the proper folding of the C H l-domain are boxed.
  • the present invention envisages replacement of one or several of the boxed residues by amino acid residues, independently selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably selected from A, V, T, and most preferably selected from A.
  • the murine wildtype sequence of the C H I -domain as used as starting point in the present set of experiments has a tryptophan (W) in position 188 (as taken from the MAK33 antibody), whereas other murine IgGl CHl-domain wildtype sequences having Arginine (R) in position 188 are also contemplated (SEQ ID NO: 18).
  • W tryptophan
  • R Arginine
  • Such wildtype sequence can, for example, be found in the SwissProt database as entry IGHG1 MOUSE (status: April 15, 2009) and is also contemplated as wildtype sequence "starting point" into which the respective mutation(s) is (are) introduced, as outlined further above.
  • the "corresponding counterparts" to P 167 and P 189 are P 167 and P 189, respectively.
  • Figure 9 shows a comparison by multiple sequence alignment of the murine IgGl CHl- domain (mlgGl) (from the M AK33 antibody) with C H I -domains of various human IgG- isotypes (hlgAl, hIgA2, etc.). Numbering is in accordance with the Kabat numbering scheme. The proline residues which are found to be detrimental for the folding of the CHl-domain, or their corresponding counter parts, such as glycine at the equivalent positions, are shown boxed.
  • proline and/or glycine residues are replaced by amino acid residues, independently selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably selected from A, V, T, and most preferably selected from A; "m” as prefix denotes murine; "h” as prefix denotes human.
  • Figure 10 shows cell culture experiments to assess the influence of the P189A mutation on the assembly and secretion of complete antibody molecules.
  • a significant increase in assembly and secretion is observed for the single point mutant Pl 89A in comparison to the wt protein.
  • H respectively L denote the heavy or the light chain.
  • the completely assembled antibody corresponds to H 2 L 2 .
  • BiP denotes the Hsp70 molecular chaperone BiP which binds to incompletely assembled antibodies and is thereby co-immunoprecipitated in cell lysates.
  • Lanes with no subscript indicate cell lysates, the subscript m stands for the medium, i.e. secreted protein, " ⁇ " denotes antibodies used for immunoprecipitation which were directed against the IgG heavy chain, HA denotes antibodies used for immunoprecipitation were directed aganist the HA-tag.
  • SEQ ID NO: 1-8 are murine ("m") sequences of the indicated isotypes; e.g. murine IgGl C ⁇ l-domain from the MAK33 antibody is SEQ ID NO:4.
  • SEQ ID NO:9-17 are human ("h") sequences of the indicated isotypes.
  • SEQ ID NO:9 is the sequence of the Ciil-domain of human IgAl
  • SEQ ID NO: 10 is the sequence of the C H I- domain of human IgA2 etc.
  • SEQ ID NO: 18 is murine IgGl C H 1 -domain as found in SwissProt-entry IGHGl MOUSE (status: April 15, 2009).
  • Example 1 Protein production.
  • the C H I domain (Thrl23-Arg215 of the pdb file 1FH5) was amplified from the murine IgGl MAK33 cDNA and inserted into the pET28a vector (Novagen, Gibbstown, NJ, USA) without a tag.
  • the protein was expressed as inclusion bodies as published (Feige et al., 2004).
  • Isotope labeled C H I for NMR experiments was expressed in M9 minimal medium.
  • the wild type CL-domain was essentially purified as published (Feige et al., 2007).
  • Inclusion bodies of the CHl-domain were solubilized in 50 mM Tris/HCl, pH 7.5, 10 mM ⁇ -mercaptoethanol, 10 mM EDTA, 8 M urea and subsequently applied to a Q- Sepharose column equilibrated in 50 mM Tris/HCl, pH 7.5, 10 mM EDTA, 5 M urea.
  • the protein did not bind to the column under these conditions.
  • Refolding was carried out as published (Feige et al., 2007). After refolding, the protein was applied to a Superdex 75pg (26/60) gel filtration column (GE Healthcare, M ⁇ nchen, Germany) equilibrated in PBS. All vectors were sequenced and protein masses were verified by mass spectrometry.
  • Spectra of the C H I domain in the complex were calculated by substraction of the spectrum of the isolated C L domain from the spectrum of the complex, measured after a 4 h equilibration step at 25°C. All spectra were averaged 16 times and buffer corrected. Temperature melts were followed by the change in the far-UV CD signal at 216 nm with heating rate of 10°C/h.
  • NH RDC values were extracted from IPAP- HSQC spectra using Bruker pulse sequences. The sequential information based on the C and C ⁇ chemical shifts as well as the NH RDC values and the crystal structure of the folded C H I domain (pdb code: lORS) served as input for the software MARS (Jung and perspectivestetter,
  • the murine IgGl MAK33 light chain (LC WT ) and heavy chain (HC WT ) CDNAS were obtained with an intact signal sequence for expression in mammalian ER.
  • An HA-epitope tag was engineered at the C-terminus of the wild type light chain for immunoprecipitation purposes.
  • the heavy chain proline exchange mutants was generated by site-directed mutagenesis.
  • Antibodies are modular structures composed of a series of structurally highly homologous domains. These domains can usually be produced and studied separately and represent independent structural units (Goto and Hamaguchi, 1982; Lilie et al., 1995). Analysis of the murine IgGl C H I domain revealed that, in marked contrast to all antibody domains studied thus far (Goto and Hamaguchi, 1982; Thies et al., 1999; Feige et al., 2004; Rothlisberger et al.,
  • the isolated C H I domain is an unfolded protein, irrespective of whether its internal disulfide bridge is formed or not (far-UV CD spectrum in figure 3, data for the reduced protein not shown).
  • iodide fluorescence quenching experiments were carried out. The experiments indicate no significant differences in the burial of tryptophan residues between
  • the CHl-domain with proline 167 and proline 189 mutated to alanine was generated by site directed mutagenesis, recombinantly expressed and purified as the wild type C H I domain (example 1). After size exclusion chromatography the identity of the protein was confirmed by MALDI-MS on a Bruker Ultraflex 2.
  • the double mutant with proline 167 and proline 189 mutated to alanine is folded even in the absence of its cognate partner, the CL-domain.
  • the secondary structure of the mutant C H I domain was analyzed at the physiological temperature of 37°C by CD spectroscopy.
  • the far-UV CD spectrum depicted in figure 7 is typical for a protein consisting of ⁇ -sheets.
  • SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175-182. Goto,Y. and Hamaguchi,K. (1982). Unfolding and Refolding of the Constant Fragment of the Immunoglobulin Light Chain. Journal of Molecular Biology 156, 891-910.

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Abstract

The present invention relates to an improved antibody domain and to antibody fragments and antibodies based thereon. More specifically, it relates to a CH1 -domain which has been made more stable by selected mutation of specific residues. The CH1-domain thus produced is natively folded and stable and therefore contributes to the stability and folding of an antibody fragment or an entire antibody. The present invention also relates to a nucleic acid coding for such an improved antibody domain or antibody fragment or antibody. The present invention also relates to a method of stabilizing a CH1-domain of an antibody and/or of an antibody fragment comprising a CH1-domain and/or of an antibody comprising a CH1 -domain.

Description

An improved antibody domain and antibody fragments and antibodies based thereon
The present invention relates to an improved antibody domain and to antibody fragments and antibodies based thereon. More specifically, it relates to a CHI -domain which has been made more stable by selected mutation of specific residues. The Cπl-domain thus produced is natively folded and stable and therefore contributes to the stability and folding of an antibody fragment or an entire antibody. The present invention also relates to a nucleic acid coding for such an improved antibody domain or antibody fragment or antibody. The present invention also relates to a method of stabilizing a Ciil-domain of an antibody and/or of an antibody fragment comprising a CHI -domain and/or of an antibody comprising a CHI -domain.
Antibodies are biotechnologically important proteins. The range of applications extends from fundamental research, diagnostics to therapy, for example in the treatment of malignant diseases. Antibodies are complex glycosylated protein molecules. Antibodies occur in different varieties which are also known as so-called isotypes or classes. In mammals, there are typically five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. These isotypes differ in their biological properties, functional locations and their ability to deal with different antigens. Yet, structurally, they are remarkably similar. As an example, immunoglobulin G (IgG) is the most abundant antibody in the blood. It is a heterotetrameric glycoprotein assembled from two light and two heavy chains which are comprised of two and four compact Ig-domains, respectively, which are structurally almost identical (see figure 1). Antigen recognition and binding occurs via two identical antigen binding sites, also referred to as paratopes (see figure 1). The antigen which is the target structure of the antibody is not only recognized in a highly specific manner, but its binding is moreover coupled to a plurality of so-called effector functions mediated by the Fc-fragment (see figure 1). Upon antigen binding, macrophages of the organism are stimulated to incorporate the antigen, and a target cell, once recognized as antigen, may be destroyed. Moreover, there also exist methods in which Fab- fragments (see figure 1) of antibodies are used.
The basic structure of an intact antibody (immunoglobulin) is a Y-shaped molecule composed of two heavy (H) and two light (L) chains; in immunoglobulins A and M these molecules are assembled further into larger complexes. The L-chain consists of one variable (VL) and one constant (CL) domain, and the IgG H-chains consist of one variable (VH), and three constant domains (CHI , CH2, and CH3). All these domains consist of about 100 residues, are homologous in their primary structure, and are believed to be independent structural units. All immunoglobulin domains appear to have essentially the same conformation, designated also as the immunoglobulin fold, consisting of two layers of antiparallel β-sheet that are usually linked by a disulfide bond. The topology in this immunoglobulin fold typically has a specific connectivity of the individual β-strands which is also sometimes referred to as the Greek key topology which is named after a pattern found on Greek pottery.
With respect to the entire antibody structure, i. e. the arrangement of the various domains in the Y-shaped molecule, the polypeptide chains between the domains are susceptible to proteases to a different extent. Most susceptible is the hinge region linking the two arms to the base of Y. After cleavage at this site, the two arms are released individually which are known as Fab-fragments; the base is known as the Fc-fragment. Each Fab-fragment contains the VL, CL, VH and CHl-domains; the Fc-fragment has two copies of each of the CH2- and CH3- domains. Fv- fragments consist of only the VH and VL-domains. The connecting segments between the domains have varying degrees of flexibility and the individual parts of the antibody molecule may undergo considerable motion relative to each other.
Despite the development of advanced technologies for producing antibodies, their manufacture still remains a formidable task, also because of their complex structure and size which requires a complex folding and assembly process. Hence, the production of antibodies even now is still requiring attention and improvement. Antibodies are difficult to produce, and they may be relatively unstable.
Accordingly, it was an object of the present invention to provide for improved polypeptides for use in antibodies. It was also an object of the present invention to provide for improved antibodies. It was also an object of the present invention to provide for antibodies which are easier to produce and/or more stable.
The objects of the present invention are solved by a polypeptide having an amino acid sequence derived from a wildtype sequence of a CHl-domain of an antibody, wherein in said wildtype sequence, P 167 and/or P 189 of the wildtype sequence, or its (their) corresponding counterpart(s), is (are) replaced by an amino acid residue independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably A, V, T, and most preferably A, wherein the numbering of residues is with reference to the murine IgGl wildtype heavy chain sequence using the Kabat numbering scheme.
In one embodiment, only P 167 or its corresponding counterpart is replaced.
In one embodiment only P 189 or its corresponding counterpart is replaced.
In another embodiment P 167 and P 189, or their corresponding counterparts, respectively, are replaced.
In one embodiment said wildtype sequence of said Ciil-domain is a murine sequence, preferably a murine IgGl sequence, more preferably a sequence selected from SEQ ID NO: 1- 8, and 18 even more preferably SEQ ID NO: 4.
In another embodiment, said wildtype sequence of said CHI -domain is a human sequence, preferably a human IgGl sequence, more preferably a sequence selected from SEQ ID NO: 9- 17, more preferably selected from SEQ ID NO: 13-16, and even more preferably SEQ ID NO: 13.
In another embodiment, said wildtype sequence of said Ciil-domain is a goat sequence, chicken sequence, rat sequence or rabbit sequence.
In one embodiment said wildtype sequence of said Cπl-domain is of an Ig-isotype selected from IgA, IgAl , IgA2, IgD, IgE, IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, and IgGM, preferably a sequence selected from SEQ ID NO: 1-17.
In another embodiment said wildtype sequence of said CHI -domain, prior to any replacement of P167, Pl 89 or its (their) counterpart(s) is a sequence selected from SEQ ID NO: 1-18, preferably selected from SEQ ID NO: 4, 13-16 and 18, and more preferably selected from
SEQ ID NO: 4, SEQ ID N0:18 and SEQ ID NO: 13.
In one embodiment the numbering of residues is with reference to the murine IgGl CHI- domain wildtype sequence, preferably SEQ ID NO:4 or SEQ ID NO: 18, using the Kabat numbering scheme, and wherein said counterparts are residues in Ig CHI -domain wildtype sequences other than murine IgGl CHl-domain wildtype sequence, such as CHl-domain wildtype sequences of murine IgA, IgD, IgE, IgG2a, IgG2b, IgG3, IgM; human IgAl, IgA2, IgG, IgE, IgGl, IgG2, IgG3, IgG4, and IgM, or the same isotypes from other species, such as goat, chicken, rat or rabbit, and wherein said counterparts are determined by sequence homology alignment or structure homology alignment of said murine IgGl CHl-domain wildtype sequence with another CHl-domain wildtype sequence. In one embodiment of said sequence homology alignment, said counterparts are determined by being aligned with said P167 or P189 in an optimum alignment of sequences, i.e. with the best possible match of residues (highest number of identical and/or homologous residues in aligned positions) between the aligned sequences.
In one embodiment said murine IgGl CHl-domain wildtype sequence and said another CHl- domain wildtype sequence have a number of identical and/or homologous residues and their respective positions in common, referred to as "conserved residues", and wherein said sequence homology alignment occurs such that at least 20%, preferably at least 30%, preferably at least 40 %, preferably at least 50 %, preferably at least 60 %, preferably at least 70 %, preferably at least 80 %, preferably at least 90 %, preferably at least 95 %, more preferably at least 96 %, even more preferably at least 98 %, even more preferably at least 99 % of residues are conserved residues; and wherein said structure homology alignment occurs by superpositioning the tertiary structure of said murine IgGl CHl-domain wildtype sequence with the tertiary structure of another CHl-domain wildtype sequence and obtaining maximum overlap of atomic coordinates of non- hydrogen protein atoms of said two wildtype sequences.
In one embodiment the CHl-domain wildtype sequence is murine IgA (SEQ ID NO: 1), and said corresponding counterparts to P 167 and Pl 89 are P167 and P190, respectively; or the CHl-domain wildtype sequence is murine IgD (SEQ ID NO: 2), and said corresponding counterparts to P167 and P189 are P164 and no second residue, respectively; or the CHI- domain wildtype sequence is murine IgE (SEQ ID NO: 3), and said corresponding counterparts to P167 and P189 are P163 and G180, respectively; or the Cπl-domain wildtype sequence is murine IgG2a (SEQ ID NO: 5), and said corresponding counterparts to P 167 and P189 are P167 and P189, respectively; or the CHI -domain wildtype sequence is murine IgG2b (SEQ ID NO: 6), and said corresponding counterparts to P 167 and P 189 are P166 and P188, respectively; or the CHl-domain wildtype sequence is murine IgG3 (SEQ ID NO: 7), and said corresponding counterparts to P167 and Pl 89 are no residue and P188, respectively or wherein the CHl-domain wildtype sequence is murine IgM (SEQ ID NO: 8), and said corresponding counterparts to P167 and P189 are P170 and G194, respectively; or the CHI- domain wildtype sequence is human IgAl (SEQ ID NO: 9), and said corresponding counterparts to P167 and P189 are P167 and G193, respectively; or the CHl-domain wildtype sequence is human IgA2 (SEQ ID NO: 10), and said corresponding counterparts to P 167 and P 189 are P167 and G193, respectively; or the CHl-domain wildtype sequence is human IgD (SEQ ID NO: 11), and said corresponding counterparts to P167 and P189 are P168 and G193, respectively; or the CHl-domain wildtype sequence is human IgE (SEQ ID NO: 12), and said corresponding counterparts to P 167 and P 189 are P169 and no second residue, respectively; or the CHl-domain wildtype sequence is human IgGl (SEQ ID NO: 13), and said corresponding counterparts to P167 and P189 are P167 and G190, respectively; or the CHl- domain wildtype sequence is human IgG2 (SEQ ID NO: 14), and said corresponding counterparts to P167 and P189 are P167 and G190, respectively; or the CHl-domain wildtype sequence is human IgG3 (SEQ ID NO: 15), and said corresponding counterparts to P 167 and P189 are P167 and G190, respectively; or the CHl-domain wildtype sequence is human IgG4 (SEQ ID NO: 16), and said corresponding counterparts to P167 and P189 are P167 and G190, respectively; or the CHl-domain wildtype sequence is human IgM (SEQ ID NO: 17), and said corresponding counterparts to P167 and P189 are P170 and G194, respectively.
As used herein, the terms "no residue" or "no second residue", when used to denote the "corresponding counterparts", are meant to indicate that, in such specific case, no such corresponding counterpart residue exists. For example, if, with reference to the CHl-domain wildtype sequence of human IgE (SEQ ID NO: 12), the statement is made "and said corresponding counterparts to P 167 and P 189 are P 169 and no second residue", this is meant to mean that in the CHI -domain wildtype sequence of human IgE (SEQ ID NO: 12) there is no corresponding counterpart residue to P 189 (of the murine IgGl CHI -domain wildtype sequence) and hence no mutation is to be introduced at such position, for the CHI -domain wildtype sequence of human IgE (SEQ ID NO: 12).
In one embodiment, the CHI -domain wildtype sequence is murine IgGl as shown in SEQ ID NO: 18, and said corresponding counterparts to P 167 and P189 are P 167 and P 189, respectively.
In one embodiment said corresponding counterparts are replaced by an amino acid residue independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably A, V, T, more preferably A.
The objects of the present invention are also solved by an antibody fragment, preferably an Fab-fragment, or an antibody comprising the polypeptide according the present invention.
The objects of the present invention are also solved by a nucleic acid coding for a polypeptide according to the present invention or for an antibody fragment or antibody according to the present invention.
The objects of the present invention are also solved by a method of stabilizing a CHI -domain or an antibody fragment, preferably an Fab-fragment, or an antibody, said antibody fragment or said antibody comprising a CHI -domain, said method comprising the step: replacing in a wildtype sequence of a CHl-domain one or two amino acid residue(s) selected from P 167 and P 189, or its (their) corresponding counterpart(s), by an amino acid residue, independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably A, V, T, and most preferably A, wherein the numbering of residues is with reference to the murine IgGl wildtype sequence using the Kabat numbering scheme, and wherein, preferably, said step of replacing occurs by side- directed mutagenesis, and wherein said wildtype sequence of a CHl-domain is an isolated polypeptide or is part of an antibody fragment or an antibody comprising said CHl-domain wildtype sequence. In one embodiment, the polypeptide in accordance with the present invention is no antibody or Fab-fragment, but consists of only a sequence of a CHI -domain.
In one embodiment, the polypeptide in accordance with the present invention is no complete heavy chain, but only consists of a sequence of a CHI -domain.
The term "a CHI -domain", as used herein, is meant to refer to an amino acid sequence which makes up the first constant domain in the heavy chain, after the variable domain of the heavy chain, when viewed in the direction from the N-terminus to the C-terminus of the heavy chain. The term is meant to encompass the wildtype sequences of such CHI -domains, as exemplified by the various CHI -domains in Figures 8 and 9; the term is meant to also encompass variants of such wildtype sequences, wherein P 167 and/or P189 or its (their) corresponding counterparts have been replaced by another amino acid or other amino acids. Such variants are herein also sometimes referred to as being "derived from a wildtype sequence of CHI- domain". It should be noted that the numbering used herein refers to the numbering of murine IgGl heavy chain using the Kabat scheme (see below), and the "corresponding counterparts" are amino acid residues in other CHI -domains which residues have been identified through sequence homology alignments or structure homology alignments.
A person skilled in the art will be able to determine whether or not a sequence would qualify as a sequence of a CHI -domain, based on sequence alignments made with known CHI -domain sequences, as well as structural determinations of the corresponding sequences using structure determination techniques, such as x-ray crystallography, and nuclear magnetic resonance with and without isotope labelling.
In the context of the present invention, the Kabat numbering is used. In this context, the amino acid numbering is based on the murine sequence of the heavy chain of IgGl. Accordingly, the terms "pro line residue No. 167", and "proline residue No. 189", are based on the murine sequence numbering of IgGl but is meant to include and designate also the "corresponding counterpart" residues in other sequences, such as human sequences or sequences of other species or sequences of other Ig isotypes. It is therefore envisaged that, although the polypeptides/antibodies of the present invention are constructed and mutated in the context of the numbering of the murine sequence, the replacements/mutations may also be
"transferred" to another sequence. This can be done by determining the "equivalent" or "corresponding counterpart" residues between the two sequences, typically based on sequence or structural homology between the sequences of the two Cπl-domains. In order to establish homology, the amino acid sequence of the first Cπl-domain is directly compared to the sequence of a second Ciil-domain. After aligning the sequences, using one or more of the homology alignment programs well known in the art, such as CLUSTALW; (for example using conserved residues between species), allowing for necessary insertions and deletions in order to maintain alignment (i. e. avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues "equivalent" or "corresponding" to particular amino acid residues in the primary sequence of the first CHI -domain are defined. Alignment of conserved residues preferably should conserve at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, more preferably at least 96%, even more preferably at least 98%, even more preferably at least 99% and most preferably 100% of residues. "Equivalent" or "corresponding counterpart" residues may also be defined by determining structural homology between a first and second CHI- domain, that is at the level of tertiary structure for CHl-domains whose structures have been determined. In this case, "equivalent" or "corresponding" residues are defined as those, for which the atomic coordinates of two or more of the mainchain atoms of a particular amino acid residue of the first CHI -domain or precursor (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the proteins. Regardless of how "equivalent" or "corresponding" residues are determined, and regardless of the identity of the first CHI- domain in which the replacement mutations are introduced, what is meant to be conveyed is that the CHI -domain polypeptides according to the present invention may be constructed into any second CHI -domain which has a significant sequence or structural homology with the first CHI -domain.
It should be noted that the numbering adopted in the claims and throughout the description, with the exception of the sequence listing (see below),is the numbering according to the Kabat scheme (Martin; 1996; PROTEINS: Structure, Function and Genetics, 25, 130-133), with reference to the complete heavy chain.
Accordingly, the numbering of residues for each of the sequences referred to herein is with reference to the entire heavy chain starting with residue number 1 at the N-terminus of the heavy chain and ending with residue No. X at the C-terminus of the heavy chain. Since the
CHI -domain is situated within the heavy chain, its residue numbers, as shown in figures 8 and
9, start with a number > 1, usually > 100, given that the CHI -domain is preceded by the variable domain of the heavy chain (VH-domain). Accordingly, the "Cπl-domain" of murine IgGl, as used herein, comprises residues 114-214 of the entire heavy chain, using the Kabat scheme. Likewise, the "CHI -domain" of human IgGl, as used herein, comprises residues 114-
215 of the entire heavy chain. The corresponding starting residue and the ending residue of the various CHI -domain sequences shown in Figures 8 and 9 are as follows: mlgA: 114-212 mlgD: 114-239 mlgE: 114-219 mlgGl : 114-214 mIgG2a: 114-218 mIgG2b 114-219 mIgG3: 114-222 mlgM: 114-221 hlgAl : 114-213 hIgA2: 114-213 hlgD: 114-223 hlgE: 114-216 hlgGl : 114-215 hIgG2: 114-214 hIgG3: 114-215 hIgG4: 114-214 hlgM: 114-223
It should be noted at the same time that within the attached sequence listing and the sequences SEQ ID NO:1-18, which only provide sequences of various CHl-domains, numbering starts with no. 1, despite the fact that the residue denoted thereby is not the first residue to appear in the complete heavy chain. For example residue no. 1 of the CHl-domain of murine IgGl (i.e. residue no. 1 in SEQ ID NO: 4), really is residue no. 114 with respect to the numbering of the entire heavy chain using the Kabat scheme. The same also applies to the residues of the wild type sequence, which, in accordance with the present invention, are replaced by another amino acid residue. For example, with respect to P167 and P189 of the CHl-domain of murine IgGl, the numbers 167 and 189 refer to the respective position of these residues within the entire heavy chain, again using the Kabat scheme whereas in the isolated sequence of the CHI -domain (SEQ ID NO:4), these residues would be numbered as P54 and P76, respectively. Similar considerations apply to all the other CHI -domains shown herein.
In a preferred embodiment, the polypeptides in accordance with the present invention only consist of the aforementioned amino acid sequences of the respective CHI -domain, with the one or more replacements of amino acid residues, as outlined further above, and no other residues outside of the CHl-domain, such as would for example occur in the regions connecting to and from the CHl-domain within the heavy chain, or in regions of other domains, such as the VH-domain or the CH2-domain or the CH3-domain. In another embodiment, the CHl-domain may be part of an antibody fragment or an entire antibody.
In one preferred embodiment, said antibody fragment or antibody is a human or murine antibody fragment or antibody. In another embodiment, said antibody fragment or antibody is a goat, chicken, rat or rabbit antibody fragment or antibody.
The term "antibody", as used herein, is meant to refer to a complete antibody or complete immunoglobulin, as opposed to a mere fragment thereof, which is herein also sometimes denoted as an "antibody fragment".
The present inventors have surprisingly found that contrary to previous assumptions, the CHl- domain, when studied and looked at on its own, i. e. without the other domains of the heavy or light chain present or covalently attached, in fact, is unfolded under native conditions. The term "native conditions", as used herein is meant to refer to PBS buffer, pH 7.4, at 25°C. The term "unfolded" is meant to refer to a state of the polypeptide chain, wherein the chain does not show any defined structure, as measured by biophysical techniques such as CD (circular dichroism) and nuclear magnetic resonance spectroscopy (NMR). The term "unfolded" is also sometimes refereed to as "random coil". A "random coil" protein is characterized by the absence of any secondary structure elements such as alpha-helix, β-strand, β-sheet, β-turn 310- helix, let alone the presence of any long range tertiary interactions. The terms "secondary" and "tertiary" structural elements or structures or interactions are well known by someone skilled in the art and are used herein in the same sense, as would be understood by someone skilled in the art.
The fact that the CHI -domain is unfolded under native conditions is surprising, given that scientific evidence available so far (as an example Goto and Hamagutchi, 1982, JMB, 156:891-910; Feige et al., 2008, PNAS USA, 105 : 13373-13378) demonstrate that the adoption of structure of the Ig-proteins in their folding pathway starts around several hydrophobic amino acids at the core of the β-sheet structure of each domain and is completed in the reshuffling of the structure around this folding core. The evidence so far available shows that all isolated domains such as CHI , CH2, CH3, VH etc. are autonomous structures which are folded and adopt a well defined structure under native conditions, i. e. they show a β-sheet sandwich structure, consisting of a typical fold of two layers of antiparallel β-sheets folded on top of each other to form a sandwich like structure.
Surprisingly, the present inventors could detect unexpected structural properties of the CHI- domain which also would have been expected to show such immunoglobulin fold, even if the CHl-domain was looked at in isolation, i. e. without any of the other corresponding domains of the heavy chain or light chain, such as CL, present or attached. The inventors have surprisingly found that the isolated CHl-domain, i. e. without any of the other domains of the heavy chain or light chain present or attached, is unfolded. Contrary to what one would have expected from previous results, the isolated CHl-domain does not have any defined structure. Accordingly, this finding led to the assumption that a selected mutation at specific sites within the CHl-domain will lead to a stabilisation of such CHl-domain on its own and will therefore stabilize both the CHl-domain on its own as well as an antibody comprising such CHl-domain within the heavy chain, as well as an antibody fragment, e. g. Fab-fragment containing such CHl-domain. Accordingly, the present invention encompasses variants of the CHl-domain which have improved properties with respect to assembly of the entire antibody and/or secretion of the CHl-domain in cell culture experiments. Moreover, the present invention also encompasses the stabilized, autonomously folding CHI -domain, as well as larger proteins comprising such stabilized Cπl-domains. Such larger proteins could,for example be a Fab- fragment, the entire heavy chain or even an entire antibody.
The inventors have found that in order to cause the unmutated, i. e. wild type CHI -domain to adopt a native structure, i. e. the β-sheet sandwich, as outlined above, the presence of the CL- domain is necessary (see also figures 3 and 4 which show that CHI on its own adopts a random coil structure as shown by the CD-spectrum of figure 3 (trace "CHI")- The CL-domain on its own (trace "CL") shows the typical β-sheet structure thus confirming that the CL- domain is an autonomous folding unit. The same applies to the complex, i. e. the two domains mixed together. The fourth trace in figure 3 is calculated trace wherein the CHI -trace is subtracted from the combined trace "CHI + CL". This calculated trace "CHI in complex" clearly shows that CHI in the presence of CL also adopts a β-sheet sandwich structure.
The same picture emerges from NMR- spectra, wherein the CHI -domain on its own shows the typical non-dispersed 1H-15N HSQC spectrum in the amide region, whereas in the presence of an unlabeled CL-domain, the amide signals are dispersed such as would be expected from a folded protein showing a well-defined structure (see figure 4). If one looks at the kinetics of the CHl-folding process, the inventors have found that this is a rather slow process taking several hours at room temperature, if CHI is folded in the presence of CL (see figure 5).
Based on the aforementioned findings, the present inventors identified two residues within the CHI -domain, which appear to be pivotal in the autonomy of the CHl-folding. These residues are, with reference to the murine CHl-domain of IgGl P167 and P189. In other CHl-domains, the "corresponding counterpart" residues can be easily determined by optimum sequence homology alignment or structure homology alignment, whereby the maximum degree of identity between the two sequences aligned or between the two structures aligned is achieved. As outlined further above, the "corresponding counterpart" residues may also be determined by structural homology, rather than sequence alignments.
The CHl-domain in accordance with the present invention, i. e. having one or two of the aforementioned residues replaced has improved properties in that it is secreted in higher yields in cell culture media and contributes to a better antibody assembly; the double mutant, i.e. the variant in which both residues are replaced is autonomously folding and adopts a folded structure, when looked at in isolated form, i. e. in the absence of any of the other domains of the heavy or light chain, such as CL, VL, CH2, CH3 etc.
Mutation of any of the residues may occur by means known to someone skilled in the art. A preferred way of producing such mutated CHI -domains on their own or within an antibody is site-directed mutagenesis.
The CHI -domains in accordance with the present invention have improved qualities in that they either show improved properties with respect to secretion in cell culture media or with respect to antibody assembly, or they have improved qualities in that they even adopt a folded structure on their own and thus are stable and will therefore aid in the structuring and assembly of an antibody fragment, e. g. Fab-fragment, or an entire antibody. In particular an autonomously folding and stabilized CHI -domain will reduce the requirements of the cellular folding machinery, exemplified by chaperone proteins.
In the following, reference is made to the figures wherein
Figure 1 shows an antibody of the IgG-isotype. The two light chains are lightly coloured, the heavy chains are represented in dark. The regions responsible for the antigen binding, for glycosylation, as well as those regions mediating effector functions (Fc-fragment) are shown.
Figure 2 shows the location of the CHl-domain within the IgG. The heavy chain of the IgG is shown in dark, the light chain is shown lightly coloured. The CHl-domain within the heavy chain is encircled.
Figure 3 shows the structural characterization of the CHl-domain using circular dichroism (CD) spectroscopy. The isolated CHl-domain ("CHI") shows the CD-spectrum of an unfolded protein, whereas the isolated CL-domain ("CL") shows the typical spectrum of a β-sheet protein. If both proteins are co-incubated (CHI + CL), the spectrum shows the typical trace of a β-sheet protein, and the calculated curve for CHI within the co-incubation also shows such β-sheet protein trace (CHI in complex, calculated). Figure 4 shows the structuring of the CHI -domain in the presence of the CL-(IO main. The 1H-
15N HSQC NMR-spectrum in PBS at 25°C of the isolated CHl-domain (shown in light colour) shows no dispersion of the amide region and is thus characteristic for an unfolded protein. Upon addition of the CL-domain, the amide signals shift (spectrum shown in dark colour) and get more dispersed which is indicative of a structuring.
Figure 5 shows the folding kinetics of the CHl-domain in the presence of the CL-domain, as followed by the CD-signal at 205 nm. The folding takes several hours to be completed at 25°C in PBS.
Figure 6 shows a model of an exemplary CHl-domain (murine from IgGl), with two pro line residues marked which may be replaced in accordance with the present invention. A preferred replacement is by alanine, but other residues are also feasible. Such replacement leads to a folding of the CHl-domain. The coordinates of this model are taken from pdb-fϊle 12E8.
Figure 7 shows results of a mutant CHl-domain in accordance with the present invention wherein both proline 167 and proline 189 have been replaced by alanine. The spectrum on the left show the CD-spectrum of a β-sheet (panel A), and the protein is stable, having a melting point of 500C (panel B).
Figure 8 shows a comparison by multiple sequence alignment of CHl-domain by multiple sequence alignment of CHl-domains of various murine Ig-isotypes. The numbering of the CHI -domains is in accordance with the Kabat numbering scheme. There is a conserved proline residue (underlined) which is rate determining for the folding of the CHl-domain. The two proline residues or their corresponding counterparts (in some cases glycine like for IgM) which have been identified as being detrimental for the proper folding of the CHl-domain are boxed. Because the two proline residues or their corresponding counterparts are conserved over antibody isotypes as well as over species (see also figure 9), such replacements in other CHl-domains (other than murine IgGl) will have a similar effect of providing improved properties. The present invention envisages replacement of one or several of the boxed residues by amino acid residues, independently selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably selected from A, V, T, and most preferably selected from A. It should be noted that the numbering used in the sequences in figures 8 and 9 is with respect to the entire heavy chain, despite the fact that only CHI -domain sequences are shown. Hence, for each of the aligned CHI -domains, the respective first residue is denoted herein in the alignments of figures 8 and 9 with a residue number greater than 1 because each of these CHI -domains is preceded by a variable domain, with reference to the entire heavy chain. The "offset" which is thus established is in accordance with the numbering scheme of Kabat as outlined above.
It should be noted that the murine wildtype sequence of the CHI -domain as used as starting point in the present set of experiments has a tryptophan (W) in position 188 (as taken from the MAK33 antibody), whereas other murine IgGl CHl-domain wildtype sequences having Arginine (R) in position 188 are also contemplated (SEQ ID NO: 18). Such wildtype sequence can, for example, be found in the SwissProt database as entry IGHG1 MOUSE (status: April 15, 2009) and is also contemplated as wildtype sequence "starting point" into which the respective mutation(s) is (are) introduced, as outlined further above. In this case, the "corresponding counterparts" to P 167 and P 189 are P 167 and P 189, respectively.
Figure 9 shows a comparison by multiple sequence alignment of the murine IgGl CHl- domain (mlgGl) (from the M AK33 antibody) with CHI -domains of various human IgG- isotypes (hlgAl, hIgA2, etc.). Numbering is in accordance with the Kabat numbering scheme. The proline residues which are found to be detrimental for the folding of the CHl-domain, or their corresponding counter parts, such as glycine at the equivalent positions, are shown boxed. In accordance with the present invention one or several of these proline and/or glycine residues are replaced by amino acid residues, independently selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably selected from A, V, T, and most preferably selected from A; "m" as prefix denotes murine; "h" as prefix denotes human.
Figure 10 shows cell culture experiments to assess the influence of the P189A mutation on the assembly and secretion of complete antibody molecules. A significant increase in assembly and secretion is observed for the single point mutant Pl 89A in comparison to the wt protein. H respectively L denote the heavy or the light chain. The completely assembled antibody corresponds to H2L2. BiP denotes the Hsp70 molecular chaperone BiP which binds to incompletely assembled antibodies and is thereby co-immunoprecipitated in cell lysates. Lanes with no subscript indicate cell lysates, the subscript m stands for the medium, i.e. secreted protein, "γ" denotes antibodies used for immunoprecipitation which were directed against the IgG heavy chain, HA denotes antibodies used for immunoprecipitation were directed aganist the HA-tag.
In the enclosed sequence listing, the sequences are numbered in the order as they appear in figures 8 and 9 (read from the top to the bottom); hence SEQ ID NO: 1-8 are murine ("m") sequences of the indicated isotypes; e.g. murine IgGl Cπl-domain from the MAK33 antibody is SEQ ID NO:4.
SEQ ID NO:9-17 are human ("h") sequences of the indicated isotypes. SEQ ID NO:9 is the sequence of the Ciil-domain of human IgAl , SEQ ID NO: 10 is the sequence of the CHI- domain of human IgA2 etc.
SEQ ID NO: 18 is murine IgGl CH1 -domain as found in SwissProt-entry IGHGl MOUSE (status: April 15, 2009).
Moreover, reference is made to the following examples, which are given to illustrate, not to limit the present invention.
Examples
Example 1 Protein production. The CHI domain (Thrl23-Arg215 of the pdb file 1FH5) was amplified from the murine IgGl MAK33 cDNA and inserted into the pET28a vector (Novagen, Gibbstown, NJ, USA) without a tag. The protein was expressed as inclusion bodies as published (Feige et al., 2004). Isotope labeled CHI for NMR experiments was expressed in M9 minimal medium. The wild type CL-domain was essentially purified as published (Feige et al., 2007). Inclusion bodies of the CHl-domain were solubilized in 50 mM Tris/HCl, pH 7.5, 10 mM β-mercaptoethanol, 10 mM EDTA, 8 M urea and subsequently applied to a Q- Sepharose column equilibrated in 50 mM Tris/HCl, pH 7.5, 10 mM EDTA, 5 M urea. The protein did not bind to the column under these conditions. Refolding was carried out as published (Feige et al., 2007). After refolding, the protein was applied to a Superdex 75pg (26/60) gel filtration column (GE Healthcare, Mϋnchen, Germany) equilibrated in PBS. All vectors were sequenced and protein masses were verified by mass spectrometry.
Optical spectroscopy. A Jasco J-720 spectropolarimeter was used for all CD measurements (Jasco, Gross-Umstadt, Germany). Far-UV CD spectra were recorded in a 0.2 mm quartz cuvette, far-UV kinetics in a 1 mm quartz cuvette. Spectra of the isolated domains were recorded at 45 μM protein concentration, for the spectrum of the complex 15 μM CHI in the presence of 45 μM CL was used. Far-UV CD kinetics were recorded at 10 μM protein concentration of each domain and followed at 205 nm. Spectra of the CHI domain in the complex were calculated by substraction of the spectrum of the isolated CL domain from the spectrum of the complex, measured after a 4 h equilibration step at 25°C. All spectra were averaged 16 times and buffer corrected. Temperature melts were followed by the change in the far-UV CD signal at 216 nm with heating rate of 10°C/h.
NMR spectroscopy. Spectra of the CHI domain in complex with the CL domain were recorded at 25°C on Bruker DMX600 and DMX750 spectrometers (Bruker, Rheinstetten, Germany), whereas spectra for the assignment of the unfolded CHI domain were measured at 12.50C on a Bruker AVANCE900 spectrometer (Bruker, Rheinstetten, Germany). Backbone sequential assignment of the isolated CHI domain was obtained by standard triple resonance experiments implemented with selective proton flip-back techniques for fast pulsing (Diercks et al., 2005). For all measurements of the folded CHI domain in association with CL, a twofold excess of unlabeled CL was added to 15N or 15N, 13C labeled CHI . Prior to steady state measurements, samples were incubated for at least 6 h at room temperature to ensure complete folding of the CHI domain. Backbone sequential assignment of the assembled CHI domain was achieved with standard triple resonance experiments with selective proton flip- back techniques for fast pulsing. The assignment of the carbon chemical shifts was limited to the C and Cα chemical shifts due to the relaxation properties of the whole protein complex. To verify the backbone resonance assignment NH residual dipolar couplings (RDCs) were determined. The sample was prepared as described above and aligned with nonionic liquid crystalline media (Ruckert and Otting, 2000). NH RDC values were extracted from IPAP- HSQC spectra using Bruker pulse sequences. The sequential information based on the C and Cα chemical shifts as well as the NH RDC values and the crystal structure of the folded CHI domain (pdb code: lORS) served as input for the software MARS (Jung and Zweckstetter,
2004b; Jung and Zweckstetter, 2004a).
Cell culture experiments. The murine IgGl MAK33 light chain (LCWT) and heavy chain (HCWT) CDNAS were obtained with an intact signal sequence for expression in mammalian ER. An HA-epitope tag was engineered at the C-terminus of the wild type light chain for immunoprecipitation purposes. The heavy chain proline exchange mutants was generated by site-directed mutagenesis. The recombinant plasmids, along with a pMT vector encoding hamster BiP (Lee et al, 1999) were introduced into COS-I cells (Gluzman, 1981) that were cultured as described (Lee et al., 1999) using FuGENE 6 transfection reagent (Roche, Indianapolis, USA) following the manufacturer's protocol. Metabolic labeling, cell lysis, immunoprecipitation, and visualization of the proteins were performed as described previously (Lee et al., 1999). Anti-rodent BiP antiserum (Hendershot et al., 1995), a monoclonal anti-HA (12CA5) antibody (kindly provided by Dr. Al Reynolds, Vanderbilt University, USA), and a goat anti-mouse Ig K and goat anti-mouse γ antibodies (Southern Biotech, Birmingham, AL, USA) and Protein A Sepharose beads were used for immunoprecipitations. For metabolic labeling experiments, cells were cultured in DMEM lacking methionine and cysteine and labeled with 35S Translabel (MP Biomedicals, Irvine, CA, USA) for the indicated times.
Example 2
Antibodies are modular structures composed of a series of structurally highly homologous domains. These domains can usually be produced and studied separately and represent independent structural units (Goto and Hamaguchi, 1982; Lilie et al., 1995). Analysis of the murine IgGl CHI domain revealed that, in marked contrast to all antibody domains studied thus far (Goto and Hamaguchi, 1982; Thies et al., 1999; Feige et al., 2004; Rothlisberger et al.,
2005), the isolated CHI domain is an unfolded protein, irrespective of whether its internal disulfide bridge is formed or not (far-UV CD spectrum in figure 3, data for the reduced protein not shown). To further characterize the unfolded state of the CHI domain under physiological conditions, iodide fluorescence quenching experiments were carried out. The experiments indicate no significant differences in the burial of tryptophan residues between
CHI in PBS and in 3 M GdmCl (data not shown). Additionally, NMR experiments were recorded. The 1H-15N spectra of the isolated CHI domain are characteristic for an unfolded protein (figure 4). Taken together, these data argue against the presence of a significant amount of stable structure in the isolated CHI domain. However, the pattern changed completely when the CL domain, the cognate association partner of CHI in the antibody, was added. Only then did we observe folding of the CHI domain to a well defined β-sheet structure (figure 3-5). Thus, the CL domain is necessary and sufficient to induce structure formation in CHI . This folding process was observed only if the internal disulfide bridge in the CHI domain was present (data not shown) and had an observable time constant of 50 min in kinetics followed by far-UV CD spectroscopy at 25°C (figure 5).
Example 3
If one of the suggested mutations in the CHI is performed by site directed mutagenesis,, e.g. Pro 189 against alanine, the protein expressed and purified as described above (example 1), the murine CHI domain still remains unfolded (data not shown). It shows only minor differences in the far-UV CD spectrum compared to the wild type protein. Nevertheless, already the single mutation has a significant impact on the overall folding and assembly efficiency of complete antibody molecules. In vivo experiments show that the assembly and secretion efficiency of this single point mutant, P 189 A, is significantly increased (figure 10) highlighting the critical role of the performed mutations.
Example 4
The CHl-domain with proline 167 and proline 189 mutated to alanine was generated by site directed mutagenesis, recombinantly expressed and purified as the wild type CHI domain (example 1). After size exclusion chromatography the identity of the protein was confirmed by MALDI-MS on a Bruker Ultraflex 2.
Unlike the wild type CHI domain or the single proline to alanine mutants P 167 and P 189, respectively (example 3), the double mutant with proline 167 and proline 189 mutated to alanine is folded even in the absence of its cognate partner, the CL-domain. The secondary structure of the mutant CHI domain was analyzed at the physiological temperature of 37°C by CD spectroscopy. The far-UV CD spectrum depicted in figure 7 is typical for a protein consisting of β-sheets. The temperature transition as followed by CD spectroscopy at the wavelength λ = 216 nm reveals that the simultaneous mutation of proline residues 167 and proline 189 transforms the natively unfolded CHI domain into a stably folded Ig domain with a transition midpoint at 500C which is far above the physiological temperature.
Additionally, the CHI domain with proline 167 and proline 189 mutated to alanine is still able to interact with its cognate partner, the CL domain of the light chain as observed by the change in intrinsic fluorescence (data not shown).
The features of the present invention disclosed in the specification, the claims and/or in the drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.
Reference List Diercks,T., Daniels, M., and Kaptein,R. (2005). Extended flip-back schemes for sensitivity enhancement in multidimensional HSQC-type out-and-back experiments. Journal of Biomolecular Nmr 33, 243-259.
Feige,M.J., Hagn,F., EsserJ., Kessler,H., and BuchnerJ. (2007). Influence of the internal disulfide bridge on the folding pathway of the C-L antibody domain. Journal of Molecular Biology 365, 1232-1244.
Feige,M.J., Walter.S., and BuchnerJ. (2004). Folding mechanism of the C(H)2 antibody domain. Journal of Molecular Biology 344, 107-118.
Gluzman,Y. (1981). SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175-182. Goto,Y. and Hamaguchi,K. (1982). Unfolding and Refolding of the Constant Fragment of the Immunoglobulin Light Chain. Journal of Molecular Biology 156, 891-910.
Hendershot,L.M., WeiJ.Y., GautJ.R., Lawson,B., Freiden,P.J., and Murti,K.G. (1995). In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum. MoI. Biol. Cell 6, 283-296. Jung,Y.S. and Zweckstetter,M. (2004a). Backbone assignment of proteins with known structure using residual dipolar couplings. Journal of Biomolecular Nmr 30, 25-35.
Jung,Y.S. and Zweckstetter,M. (2004b). Mars - robust automatic backbone assignment of proteins. Journal of Biomolecular Nmr 30, 11-23. Lee,Y.K., Brewer, J. W., Hellman,R., and Hendershot,L.M. (1999). BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Molecular Biology of the Cell 10, 2209-2219.
Lilie,H., Rudolph,R., and BuchnerJ. (1995). Association of antibody chains at different stages of folding: prolyl isomerization occurs after formation of quaternary structure. J. MoI. Biol. 248, 190-201.
Rothlisberger,D., Honegger,A., and Pluckthun,A. (2005). Domain interactions in the Fab fragment: A comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. Journal of Molecular Biology 347, 773-789. Ruckert,M. and Otting,G. (2000). Alignment of biological macromolecules in novel nonionic liquid crystalline media for NMR experiments. Journal of the American Chemical Society 122, 7793-7797.
Thies,M.J.W., MayerJ., AugustineJ.G., Frederick,C.A., Lilie,H., and BuchnerJ. (1999). Folding and association of the antibody domain C(H)3: Prolyl isomerization preceeds dimerization. Journal of Molecular Biology 293, 67-79.

Claims

Claims
1. A polypeptide having an amino acid sequence derived from a wildtype sequence of a Ciil-domain of an antibody, wherein in said wildtype sequence, P167 and/or P189 of the wildtype sequence, or its (their) corresponding counterpart(s), is (are) replaced by an amino acid residue independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, more preferably A, V, T, and most preferably A, wherein the numbering of residues is with reference to the murine IgGl wildtype heavy chain sequence using the Kabat numbering scheme.
2. The polypeptide according to claim 1 , wherein only P 167 or its corresponding counterpart is replaced.
3. The polypeptide according to claim 1 , wherein only P 189 or its corresponding counterpart is replaced.
4. The polypeptide according to claim 1, wherein P 167 and P 189, or their corresponding counterparts, respectively, are replaced.
5. The polypeptide according to any of claims 1-4, wherein said wildtype sequence of said CHI -domain is a murine sequence, preferably a murine IgGl sequence, more preferably a sequence selected from SEQ ID NO: 1-8 and 18, even more preferably SEQ ID NO: 4.
6. The polypeptide according to any of claims 1-4, wherein said wildtype sequence of said CHI -domain is a human sequence, preferably a human IgGl sequence, more preferably a sequence selected from SEQ ID NO: 9-17, more preferably selected from SEQ ID NO: 13-16, and even more preferably SEQ ID NO: 13.
7. The polypeptide according to any of claims 1-6, wherein said wildtype sequence of said CHl-domain is of an Ig-isotype selected from IgA, IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, and IgGm, preferably a sequence selected from SEQ ID
NO: 1-17.
8. The polypeptide according to any of claims 1-6, wherein said wildtype sequence of said Ciil-domain, prior to any replacement of P167, P189 or its (their) counterpart(s) is a sequence selected from SEQ ID NO: 1-18, preferably selected from SEQ ID NO: 4, 13-
16 and 18, and more preferably selected from SEQ ID NO: 4, SEQ ID NO: 18 and SEQ
ID NO: 13.
9. The polypeptide according to any of claims 1-8, wherein the numbering of residues is with reference to the murine IgGl CHl-domain wildtype sequence using the Kabat numbering scheme, and wherein said counterparts are residues in Ig CHl-domain wildtype sequences other than murine IgGl CHI -domain wildtype sequence, such as CHl-domain wildtype sequences of murine IgA, IgD, IgE, IgG2a, IgG2b, IgG3, IgM; human IgAl, IgA2, IgG, IgE, IgGl , IgG2, IgG3, IgG4, and IgM, and wherein said counterparts are determined by sequence homology alignment or structure homology alignment of said murine IgGl CHl-domain wildtype sequence with another CHl- domain wildtype sequence.
10. The polypeptide according to claim 9, wherein said murine IgGl CHl-domain wildtype sequence and said another CHl-domain wildtype sequence have a number of identical and/or homologous residues and their respective positions in common, referred to as "conserved residues", and wherein said sequence homology alignment occurs such that at least 20%, preferably at least 30%, preferably at least 40 %, preferably at least 50 %, preferably at least 60 %, preferably at least 70 %, preferably at least 80 %, preferably at least 90 %, preferably at least 95 %, more preferably at least 96 %, even more preferably at least 98 %, even more preferably at least 99 % of residues are conserved residues; and wherein said structure homology alignment occurs by superpositioning the tertiary structure of said murine IgGl CHl-domain wildtype sequence with the tertiary structure of another CHl-domain wildtype sequence and obtaining maximum overlap of atomic coordinates of non-hydrogen protein atoms of said two wildtype sequences.
11. The polypeptide according to any of the foregoing claims, wherein the CHl-domain wildtype sequence is murine IgA (SEQ ID NO: 1), and said corresponding counterparts to P 167 and P 189 are P 167 and P 190, respectively; or wherein the CHI -domain wildtype sequence is murine IgD (SEQ ID NO: 2), and said corresponding counterparts to P 167 and P 189 are P 164 and no second residue, respectively; or wherein the CHI- domain wildtype sequence is murine IgE (SEQ ID NO: 3), and said corresponding counterparts to P167 and P189 are P163 and G180, respectively; or wherein the CHI- domain wildtype sequence is murine IgG2a (SEQ ID NO: 5), and said corresponding counterparts to P167 and P189 are PP167 and P189, respectively; or wherein the CHI- domain wildtype sequence is murine IgG2b (SEQ ID NO: 6), and said corresponding counterparts to P 167 and P 189 are no residue and P 188, respectively; or wherein the CHl-domain wildtype sequence is murine IgG3 (SEQ ID NO: 7), and said corresponding counterparts to P 167 and P 189 are no residue and Pl 88, respectively; or wherein the CHl-domain wildtype sequence is murine IgM (SEQ ID NO: 8), and said corresponding counterparts to P167 and P189 are P170 and G194, respectively; or wherein the CHl-domain wildtype sequence is human IgAl (SEQ ID NO: 9), and said corresponding counterparts to P 167 and P 189 are P 167 and G 193, respectively; or wherein the CHl-domain wildtype sequence is human IgA2 (SEQ ID NO: 10), and said corresponding counterparts to P 167 and P 189 are P 167 and G 193, respectively; or wherein the CHl-domain wildtype sequence is human IgD (SEQ ID NO: 11), and said corresponding counterparts to P 167 and P 189 are P 168 and G 193, respectively; or wherein the CHl-domain wildtype sequence is human IgE (SEQ ID NO: 12), and said corresponding counterparts to P 167 and P 189 are P169 and no second residue, respectively; or wherein the CHl-domain wildtype sequence is human IgGl (SEQ ID NO: 13), and said corresponding counterparts to P 167 and P 189 are P 167 and G 190, respectively; or wherein the CHl-domain wildtype sequence is human IgG2 (SEQ ID NO: 14), and said corresponding counterparts to P 167 and P 189 are P 167 and G 190, respectively; or wherein the CHl-domain wildtype sequence is human IgG3 (SEQ ID NO: 15), and said corresponding counterparts to P 167 and P 189 are P 167 and G 190, respectively; or wherein the CHl-domain wildtype sequence is human IgG4 (SEQ ID
NO: 16), and said corresponding counterparts to P 167 and P 189 are P 167 and G 190, respectively; or wherein the CHl-domain wildtype sequence is human IgM (SEQ ID NO: 17), and said corresponding counterparts to P 167 and P 189 are P 170 and G 194, respectively, or wherein the CHI -domain wildtype sequence is murine IgGl (SEQ ID
NO: 18), and said corresponding counterparts to P 167 and P 189 are P 167 and P 189, respectively.
12. The polypeptide according to claim 11, wherein said corresponding counterparts are replaced by an amino acid residue independently at each occurrence selected from A, V,
T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably A, V, T, more preferably A.
13. An antibody fragment, preferably an Fab-fragment, or an antibody comprising the polypeptide according to any of claims 1-12.
14. A nucleic acid coding for a polypeptide according to any of claims 1-12 or for an antibody fragment or antibody according to claim 13.
15. A method of stabilizing a Cπl-domain or an antibody fragment, preferably an Fab- fragment, or an antibody, said antibody fragment or said antibody comprising a CHI- domain, said method comprising the step: replacing in a wildtype sequence of a Cπl-domain one or two amino acid residue(s) selected from P 167 and P 189, or its (their) corresponding counterpart(s), by an amino acid residue, independently at each occurrence selected from A, V, T, L, I, S, C, M, D, N, E, Q, K, R, H, F, Y, W, preferably selected from A, V, T, L, I, S, C, M, D, N, E, Q,
K, R, H, more preferably A, V, T, and most preferably A, wherein the numbering of residues is with reference to the murine IgGl wildtype sequence using the Kabat numbering scheme, and wherein, preferably, said step of replacing occurs by side- directed mutagenesis, and wherein said wildtype sequence of a Ciil-domain is an isolated polypeptide or is part of an antibody fragment or an antibody comprising said
CHI -domain wildtype sequence.
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