Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70539—MHC-molecules, e.g. HLA-molecules
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

• the present invention is an optimization method of the biophysical properties of proteins of the immunoglobulin (Ig) superfamily. It is thus eminently suitable for application to antibodies. However, it is not limited to these alone, but in principle to all members of the immunoglobulin superfamily, but also on their derivatives such.
• Biomolecules such as proteins, polynucleotides, polysaccharides and the like are gaining increasing commercial importance as drugs, as diagnostics, as additives in foods, detergents and the like, as research reagents and for many other applications.
• the need for such biomolecules can be, for.
• proteins usually no longer satisfy by isolating the molecules from natural sources but requires the use of biotechnological production methods.
• the biotechnological production of proteins typically begins with the isolation of the DNA encoding the desired protein and its cloning into a suitable expression vector. After transfection of the recombinant expression vector into suitable prokaryotic or eukaryotic expression cells and subsequent selection of transfected, recombinant cells, the latter are cultured in bioreactors and the desired protein is expressed. Subsequently, the harvest of the cells or of the culture supernatant and the workup and purification of the protein contained therein takes place.
• Antibodies, in particular the subclass immunoglobulin G (IgG) are among the most important biopharmaceutically produced proteins. You will find a wide range of applications from basic research on diagnostics to a variety of therapies, eg. B.
• Antibodies are complex, glycosylated protein molecules, in the case of IgG composed of two light and two heavy chains (see Figure 1). Antigen recognition and binding take place via two identical antigen-binding sites, so-called paratopes (see Figure 1).
• the target structure of the antibody, the antigen is not only highly specifically recognized, but its binding is coupled to a multitude of so-called effector functions, which are mediated by the Fc fragment (see Figure 1).
• Key effector functions include complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).
• Each protein must undergo a structuring process, called protein folding, in order to perform its inherent function in the defined final structure.
• This multistep structuring process which often involves folding intermediates, can lead to misfolding and aggregation.
• diseases that are due to or associated with protein misfolding because proteins either fail to reach their native folding state or do not remain in that native state. These include z. Alzheimer's, Parkinson's and various amyloidoses. If such protein deficiencies occur in biotechnological production processes, this is at the expense of product titer, yield, quality and / or stability.
• Antibodies belong to the so-called Ig superfamily, which is very widespread in nature.
• Figure 2 shows the typical topology of a member of the Ig superfamily, beta2-microglobulin. Strands B, C, E and F, which are postulated as cited as the core of the folding process for Ig proteins in general, are marked. SUMMARY OF THE INVENTION
• the present invention is a biotechnological process for the production of antibodies or proteins having the immunoglobulin folding pattern, characterized in that an optimization of the natural, helical elements takes place.
• this optimization is carried out by introducing additional helix-internal salt bridges and / or the removal of Helixbrechern or helix-destabilizing residues (proline and / or glycine).
• the invention relates to a biotechnological process for the production of antibodies or proteins having the immunoglobulin folding pattern, characterized in that a transplantation of the natural or optimized helical elements takes place. Preferably, this transplantation occurs in domains that have no or less optimal helical elements.
• a transfer of one or more helical elements from at least one constant domain C L , C H 2 and / or C H 3 into at least one constant C H 1 domain and / or variable domain (eg V L or V H ).
• the invention relates to methods for improving the biophysical properties of proteins having the immunoglobulin folding pattern, characterized in that at least one amino acid in the Ig domain is replaced by another amino acid which increases the formation probability of a HeNx, preferably an ⁇ - Helix, raised.
• the training probability is preferably calculated with an algorithm, in particular with the algorithm AGADIR.
• the exchanged amino acid is preferably in the region between two ⁇ -sheet strands, in particular of the type A and B and / or E and F.
• the exchanged amino acid may be located in a region which already has a helical structure. The aim of such an amino acid exchange in an already existing helical element is then an increase in the helix formation probability of this e- lementes.
• the helix formation can be increased, for example, by the fact that the amino acid to be exchanged in the Ig domain is proline or glycine, preferably if they are at least at the second position (i ⁇ i + 2) after the preceding ⁇ -sheet strand or at most at the last but one position (i ⁇ i-2) in front of the following ⁇ -sheet strand.
• Proline and glycine are replaced by an amino acid which is neither proline nor glycine, preferably by alanine.
• salt bridges by introducing an amino acid having a charged side chain spaced apart (i ⁇ i + 3), (i ⁇ i + 4) or (i ⁇ i + 5) to an amino acid having a sidechain with the opposite charge.
• at least two amino acids are introduced for this purpose which have side chains with a charge of the same name, the distance between the exchanged amino acids being selected so that the side chains can form a salt bridge.
• the exchanged amino acids are separated from one another by 2 (i ⁇ i + 3), 3 (i ⁇ i + 4) or more amino acids (i ⁇ i + 5).
• glutamic acid or aspartic acid can be used, while arginine, lysine, or histidine have positively charged side chains under such conditions.
• the position at which arginine, lysine, or histidine is introduced, or optionally already present is closer to the C-terminus than the position at which glutamic acid or aspartic acid is introduced or may already be present.
• a double salt bridge in which a sequence is generated in which 3 amino acids are located at positions i and i + 3, i + 4 or i + 5 and i + 7, i + 8 or i + 9 in which the amino acids at the positions i and i + 7, i + 8 or i + 9 have side chains with the same charge, the amino acid at the position i + 3, i + 4 or i + 5 has a counterpart thereto.
• 3 corresponding amino acids can be introduced by mutation, if appropriate also less, if corresponding amino acids are already present in the starting sequence.
• a double salt bridge is located at the middle position i + 3, i + 4 or i + 5 preferably aspartic acid, Glutamic acid or arginine.
• Preferred embodiments are characterized in that after exchange the protein has a helical element with the sequence KPKDTLMISR (SEQ ID NO: 8), KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO : 16), and / or SKADYEKHK (SEQ ID NO: 11).
• the present invention relates to the transplantation of suitable helical elements into domains having no or less optimal helical elements, such as the Ig domain of the beta2 microglobulin (SEQ ID NO: 3), the variable domains (V L , V H ) or the constant domain C H 1 of immunoglobulins.
• suitable helical elements such as the Ig domain of the beta2 microglobulin (SEQ ID NO: 3), the variable domains (V L , V H ) or the constant domain C H 1 of immunoglobulins.
• the transplanted elements may originate, for example, from the constant immunoglobulin domains C L , C H 2 or C H 3 or be variants of such elements optimized according to the method of the invention.
• the transplantation is preferably carried out by a method in which 4 to 12 consecutive amino acids (preferably about 10 amino acids) are replaced by an amino acid sequence of equal or greater length, wherein the introduced amino acid sequence has a higher formation probability of a HeNx than the exchanged sequence.
• the introduced sequence is a helical element from the region between the ⁇ -sheet strands A and B and / or E and F of a C L or CH domain of an immunoglobulin.
• Suitable helical elements have, for example, the sequence KPKDTLMISR (SEQ ID NO: 8) from a human C H 2 domain (SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 15) or the optimized KAEDTLHISR sequence (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10) from the murine kappa C L domain (SEQ ID NO: 1), TPEQWKSHRS (SEQ ID NO: 16) from the human lambda C L domain (SEQ ID NO : 13) or also SKADYEKHK (SEQ ID NO: 11) from the human kappa C L domain (SEQ ID NO: 12).
• the present invention relates to a method of producing a protein having an immunoglobulin folding pattern therewith characterized in that a method as described above for improving the biophysical properties of proteins having the immunoglobulin folding pattern is applied to such a protein and the modified protein obtained thereby is expressed in a host cell.
• the present invention relates to a protein having an immunoglobulin folding pattern prepared by a method of the invention as described above.
• this is an antibody, in particular a complete immunoglobulin containing two light and two heavy chains.
• the present invention relates to a protein having an immunoglobulin folding pattern and at least one variable domain (eg V L or V H ), characterized in that it contains at least one helical element in this variable domain.
• this helical element has the sequence KPKDTLMISR (SEQ ID NO: 8), KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO: 16), or SKADYEKHK (SEQ ID NO: 11).
• the variable domain has the ability to specifically bind to an antigen.
• the present invention relates to a protein having an immunoglobulin folding pattern and at least one constant domain of the type C H 2, characterized in that it contains a helical element in this constant domain which has a higher helix formation probability than a helical element of one naturally occurring in humans C H 2 domain.
• a protein contains a C H 2 domain comprising a helical element having the sequence KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO: 16) or the Sequence contains SKADYEKHK (SEQ ID NO: 11).
• the present invention relates to a protein having an immunoglobulin folding pattern and at least one constant domain of the type C H 1, characterized in that it contains a helical element in this constant domain which has a higher helix formation probability than a helical element of one naturally occurring in humans C H 1 domain.
• a protein contains a C H 1 domain comprising a helical element having the sequence KPKDTLMISR (SEQ ID NO: 8), KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO: 16) or the sequence SKADYEKHK (SEQ ID NO: 11).
• the present invention relates to a modified ⁇ 2-microglobulin having at least one helical element in an Ig domain, preferably a helical element having the sequence KPKDTLMISR (SEQ ID NO: 8), KAEDTLHISR (SEQ ID NO: 9) , TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO: 16), or SKADYEKHK (SEQ ID NO: 11).
• KPKDTLMISR SEQ ID NO: 8
• KAEDTLHISR SEQ ID NO: 9
• TKDEYERH SEQ ID NO: 10
• TPEQWKSHRS SEQ ID NO: 16
• SKADYEKHK SEQ ID NO: 11
• the present invention relates to a protein having an immunoglobulin folding pattern comprising at least one helical element in an Ig domain which has a higher helix formation probability than a helical element present in any one of SEQ ID NO: 1, SEQ ID NO: 12 or SEQ ID NO: 13 (C L WT) or SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 15 (C H 2 WT).
• a protein contains a helical element having the sequence KAEDTLHISR (SEQ ID NO: 9).
• the present invention relates to a protein as described above for medical use.
• the present invention relates to a biotechnological method for modifying the biophysical properties of antibodies or proteins which have the immunoglobulin folding pattern, characterized in that an optimization of the natural, helical elements takes place.
• the present invention relates to a biotechnological method for modifying the biophysical properties of antibodies or proteins having the immunoglobulin folding pattern, characterized in that a transplantation of the natural or optimized helical elements takes place, preferably in domains which have no or have less optimal helical elements.
• the advantages of the present invention are a greater folding efficiency and stability, fewer defects and thus ultimately higher product yield with higher quality proteins, greater flexibility in the cleaning process, a slower rate of unfolding, especially under stress conditions, an improvement in solubility and a lower tendency to Aggregate formation of the proteins according to the invention. Due to the higher robustness for the manufacturing process, this new method is clearly superior to the prior art.
• the present invention is therefore preferably applicable to processes for the production of recombinant antibodies and Fc fusion proteins.
• the present invention can also be applied to other molecules of the immunoglobulin superfamily including their fragments and derivatives or fusion proteins containing domains with homology to immunoglobulin domains.
• FIGURE 1 ANTIBODIES OF IGG SUBKLASSE
• FIGURE 2 BETA2-MICROGLOBULIN AS A REPRESENTATIVE OF THE IG SUPERFAMILY
• ⁇ -sheet strands B, C, E and F of the human beta2-microglobulin (SEQ ID NO: 3) are labeled.
• FIGURE 3 IMMUNOGLOBULIN G TOPOLOGY
• Short helical elements in the Ig topology in the context of an IgG molecule that connect the ⁇ -sheets are labeled dark.
• FIGURE 4 LOCALIZATION OF HELICAL ELEMENTS IN THE IGG1 C L DOMAIN
• the localization of the helical elements in a constant antibody domain using the example of a human IgGI d domain is shown.
• the ⁇ -sheet strands A, B, C, D, E, F and G and the helical elements HeNx 1 and HeNx 2 are designated.
• FIGURE 5 CHARACTERIZATION OF THE CL FIBER INTERMEDIATE DIATH BY NMR SPECTROSCOPY
• the structural elements of the murine kappa d domain (SEQ ID NO: 1) are shown schematically above the peak amplitudes.
• FIGURE 6 STRUCTURING OF THE IGG C L DOMAIN
• FIGURE 7 CD SPECTROSCOPIC INVESTIGATION
• C L with the beta2-microglobulin helices (C L to ⁇ 2m; SEQ ID NO: 2) shows the spectrum of an unfolded protein, all other proteins are signature of a
• FIGURE 8 INFLUENCE OF HELICAL ELEMENTS ON BETA2 MICROGLOBULIN AMYLOID FORMATION
• FIGURE 9 C H 2 DOMAIN OF IGG1 MOLECULAR Localization of the optimized HeNx 1 within the C H 2 domain (A) of a human IgGI molecule (C H 2 HeNx 1 mutant, SEQ ID NO: 6) and optimization of the HeNx 1 by introducing additional salt bridges and removing the helix breaker proline (B) (mutation: KPKDTLMISR (SEQ ID NO: 8) to KAEDTLHISR (SEQ ID NO: 9)).
• FIGURE 10 STRUCTURAL COMPARISON OF THE WILD TYPE C H 2 DOMAIN WITH THE HELIX1-OPTIMIZED MUTANT
• FUV-CD spectra (A) and NUV-CD spectra (B), hence secondary and tertiary structure, are for the IgGI CH2 wild-type domain (dashed line) (C H 2 WT, SEQ ID NO: 5) and the HeNxI mutant (solid line) (C H 2 HeNxI mutant; SEQ ID NO: 6) are almost identical.
• FIGURE 11 THERMAL STABILITY STUDY
• the thermal stability of the wild-type C H 2 domain (dashed line) (C H 2 WT; SEQ ID NO: 5) and the Helixi mutant (solid line) (C H 2 helixi mutant; SEQ ID NO: 6 ) is measured by means of FUV-CD spectroscopy at 218 nm.
• the heating rate is 20 ° C / h.
• the melting point of the wild type is determined to 56.0 0 C, that of the mutant to 60.4 0 C.
• the present invention relates to methods for improving the biophysical properties, in particular the increase in stability, the folding efficiency and the reduction of the aggregation of proteins of the immunoglobulin superfamily, and the thus-modified proteins themselves.
• the immunoglobulin superfamily currently includes more than 760 different proteins. The most economically important group in this group are the immunoglobulins (antibodies).
• immunoglobulins There are several classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW.
• Other members are antigenic receptors on cell surfaces (e.g., T cells).
• Ig domains proteins involved in antigen presentation
• proteins involved in antigen presentation e.g., MHC molecules
• proteins involved in antigen presentation e.g., MHC molecules
• proteins of the immunoglobulin superfamily are characterized by common structural elements, the so-called immunoglobulin domains (Ig domains).
• Ig domains immunoglobulin domains
• IgG antibodies are composed of four subunits, two identical heavy and two identical light chains linked by covalent disulfide bonds to form a ysilon-shaped structure. Each light chain contains two Ig domains, a so-called variable (V L ) and a constant (C L ) Ig domain, each heavy chain containing four such Ig domains (V H , C H 1, C H 2, and C H 3).
• V L variable
• C L constant
• Antibodies of classes IgM and IgE contain an additional constant domain (C H 4).
• Ig domains have a characteristic secondary structure, the immunoglobulin folding pattern (English: "Ig-fold"), a sandwich-like structure with a hydrophobic core formed by two sheets of antiparallel ⁇ -sheet strands (see Figure 4).
• the three-dimensional representation is reminiscent of a folded leaf: the peptide groups lie in the surfaces and the intervening C atoms in the edges of a multiply folded leaf.
• the peptide bonds of several chains interact.
• the hydrogen bonds necessary for stabilization form along the polypeptide backbone, which occur in pairs of two at a distance of about 7.0 A.
• the distance between adjacent amino acids is much larger compared to the much more compact ⁇ -helix: the distance is 0.35 nm compared to 0.15 nm for the HeNx. Since the side groups are still close to each other, larger leaflet areas are usually formed only if the side group residues are relatively small and not all are loaded the same.
• circular dichroism spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and Ramachandran plot frequency random estimation (Ramachandran, GN et al., J. Mol Biol. 7, 95-99, 1963).
• the individual ⁇ -strands are designated according to the order of their appearance in the sequence with A, B, C, D, E, F, G or C, C ", etc.
• interactions of hydrophobic amino acids on the Inside the sandwich hydrogen bonds between the strands and, if present, a highly conserved disulfide bond between cysteine residues of the B and F bonds.
• Strands at. The number of amino acids between the two cysteines can vary and is usually between 55 and 75 amino acids.
• Variable domains of immunoglobulins typically contain 9, constant domains 7 ⁇ strands.
• the sequence regions between the ⁇ -strands are formed by unstructured loops with large sequence variability or, in particular in the constant domains of immunoglobulins, by short helical elements
• An HeNx is a right-handed or left-handed spiral secondary structure in one Protein in which every NH group in the main chain forms a hydrogen bond to a carbonyl group in the main chain
• the distance across which the hydrogen bond spans is four amino acids (i + 4 ⁇ i hydrogen bonding)
• ⁇ helix corresponds to a turn of 3.6 amino acid residues at a height of 1.5A (0.15 nm), so each amino acid is offset by 100 ° .Other helix forms are the 3io-helix (i + 3 ⁇ i hydrogen bond) and the ⁇ - Helix (i + 5 ⁇ i hydrogen bonding)
• the side chains of the amino acids are located outside the HeNx, a typical HeNx in one Protein comprises about 10 amino acids (3 turns), but helical elements of
• Helical secondary structures in proteins can be experimentally determined by methods known per se, for example by X-ray structure analysis or nuclear magnetic resonance spectroscopy (NMR spectroscopy). However, the probability of formation of a HeNx can also be determined using suitable algorithms based on the amino acid sequence (Mu ⁇ oz, V. & Serrano, L. (1997).) Development of the Multiple Sequence Approximation within the Agadir Model of ⁇ -Helix Formation. Bragg and Lifson-Roig Formalisms, Biopolymers 41, 495-509, Lacroix, E., Viguera AR & Serrano, L.
• the present invention is based on the recognition that helical structures are important for the biophysical properties of proteins having immunoglobulin folding patterns.
• biophysical properties can be improved, in particular stability (eg thermal stability, pH stability), folding efficiency and increases solubility and decreases the rate of unfolding, as well as the tendency to misfolding, aggregate or amyloid formation.
• preceding means closer to the N-terminus of the sequence, and “subsequent” closer to the C-terminus of the sequence.
• the present invention is a biotechnological process for the production of antibodies or proteins having the immunoglobulin folding pattern, characterized in that an optimization of the natural, helical elements takes place. Preferably, this optimization is carried out by introducing additional helix-internal salt bridges and / or the removal of Helixbrechern (proline and / or glycine).
• a protein which has the immunoglobulin folding pattern is understood as meaning a protein which has at least one Ig domain of the structure described above. In particular, these are members of the immunoglobulin superfamily, and preferably immunoglobulins.
• the invention also relates to artificial proteins which do not occur in nature but have an Ig domain, eg Fc fusion proteins such as the rheumatic active substance etanercept (TNFR: Fc).
• Fc fusion proteins such as the rheumatic active substance etanercept (TNFR: Fc).
• antibodies are understood not only to be immunoglobulins, as they are also found in nature and obtainable for example by immunizing mammals with an antigen, but also artificial proteins, if they have at least one Ig domain has a paratope and, either alone or together with another Ig domain, specifically binds to an antigen.
• Ig domains are, for example, the variable domains of an immunoglobulin (V H , V L ).
• immunoglobulins which classically consist of two light chains and two heavy chains, are those of the class IgG with heavy chains of the subtypes IgGI, IgG2, and IgG4 are preferred.
• immunoglobulins may be monoclonal or polyclonal, include primates (especially human), rodents or sequences from other mammals, as well as represent chimeras or humanized sequences. Preferred are human or humanized immunoglobulins.
• the immunoglobulins may also have in their domains substitutions, deletions and / or insertions of amino acids which may alter the properties of the molecule. So z.
• effector functions such as complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), apoptosis induction, or FcRn-mediated homeostasis may be modulated.
• CDC complement dependent cytotoxicity
• ADCC antibody-dependent cellular cytotoxicity
• FcRn-mediated homeostasis may be modulated.
• removal of potential deamidation, oxidation and glycosylation sites or deletion of the C-terminal lysine on the heavy chain can reduce the heterogeneity of the molecule.
• fragments of immunoglobulins such as Fab, F (ab ') 2 or Fc fragments, Fc fusion proteins, Fc-Fc fusion proteins, single-chain antibodies consisting of a fusion of the variable domains of a light and a heavy chain (scFv )
• Single domain antibodies consisting only of the heavy or light chain variable domain such as V H V HH or V L dAbs, including the camelid-derived domain antibodies, furthermore minibodies, diabodies, triabodies , as well as fusion proteins of such constructs.
• fragment antigen-binding Fab
• fragment antigen-binding Fab
• fragment antigen-binding Fab
• They can be produced, for example, by treatment with a protease, for example papain, from conventional antibodies or else by DNA cloning.
• Further antibody fragments are F (ab ') 2- Fragments that can be prepared by proteolytic digestion with pepsin.
• the variable region of the heavy and light chain are often linked together by means of a short peptide fragment of about 10 to 30 amino acids, particularly preferably 15 amino acids. In this way, a single polypeptide chain is formed in which V H and V L are linked together by a peptide linker.
• Such antibody fragments are also referred to as a single-chain Fv fragment (scFv). Examples of scFv antibodies are known and described.
• diabody a person skilled in the art refers to a bivalent homodimeric scFv derivative.
• the shortening of the peptide linker in the scFv molecule to 5 to 10 amino acids results in the formation of homodimers by superposing V H / V L - Chains.
• the diabodies can additionally be stabilized by introduced disulfide bridges. Examples of diabodies can be found in the literature.
• minibody refers to a bivalent, homodimeric scFv derivative consisting of a fusion protein which contains the C h 13 region of an immunoglobulin, preferably IgG, particularly preferably IgGI, as the dimerization region, which links the scFv fragments via a hinge region, also from IgG, and a linker region.
• immunoglobulin preferably IgG, particularly preferably IgGI
• fragments designated by the skilled person as mini-antibodies which have a bi-, tri- or tetravalent structure, are likewise derivatives of scFv fragments.
• the multimerization is achieved via di-, tri- or tetrameric "coiled-coil" structures.
• IgNAR new antigen receptor
• Camelidae antibodies from llamas or other animals of the family Camelidae are known, which consist of only two truncated heavy chains, each with a variable and two constant domains (Hamers-Casterman, C. et al., Nature 363, 446-448, 1993).
• the skilled person also knows derivatives and variants of such Camelidae antibodies, which consist only of one or more variable domains of these truncated heavy chains.
• Such molecules are also referred to as domain antibodies.
• Single domain antibodies are also based on sequences from others Species known, for example from mouse and human, or in humanized form (Holt et al., Trends in Biotechnology 21 (11), 484-490, 2003,).
• Variants of these domain antibodies include molecules that consist of multiple variable domains and are covalently linked by peptide linkers. To extend serum half-life, domain antibodies may also be fused to other polypeptide units, such as. B. with the Fc portion of immunoglobulins or with a protein occurring in the blood serum such as albumin.
• helical element and "helix” are used synonymously in the context of the present invention. It is an amino acid sequence of 4 to 12 amino acids, preferably 6 to 12, particularly preferably 8, 9, or 10 amino acids which can form a helix.
• optically is understood to mean a change in the primary structure of a protein which increases the formation probability of a helical element in this protein or creates a helical element in this protein with the aim of improving the biophysical properties of this protein, in particular its folding efficiency, stability, solubility, and propensity for aggregate formation (which is reduced by optimization)
• a preferred method of altering the primary structure of a protein is the mutation of its amino acid sequence, ie, the replacement (substitution), the removal (deletion This is usually achieved by a corresponding modification of the deoxyribonucleic acid (DNA) coding for this amino acid sequence and subsequent expression of this (recombinant) DNA in a host cell n standard methods are available for this.
• the invention relates to a biotechnological process for the production of antibodies or proteins which inhibit immunoglobulin folding.
• a transplantation of natural or optimized, helical elements takes place.
• this transplantation occurs in domains that have no or less optimal helical elements.
• transplantation is understood as meaning the exchange of an amino acid sequence of 4 to 12 amino acids by a different length of the other amino acid sequence.
• a transfer of one or more helical ele- ments from at least one constant domain C L , C H 2 and / or C H 3 into at least one constant C H 1 domain and / or variable domain (V L or V H ).
• the invention relates to methods for improving the biophysical properties of proteins having the immunoglobulin folding pattern, characterized in that at least one amino acid in the Ig domain is replaced by another amino acid which increases the formation probability of a HeNx ,
• the training probability is preferably calculated with an algorithm, in particular with the algorithm AGADIR.
• the exchanged amino acid is preferably located in the region between two ⁇ -sheet strands, in particular of the type A and B or E and F.
• the exchanged amino acid may be present in a region which already has a helical structure. Goal of a
• Amino acid exchange in an already existing helical element is then an increase in the helix formation probability of this element.
• the formation of helix can be increased, for example, by virtue of the fact that the amino acid to be exchanged in the Ig domain is proline or glycine, preferably if it is at least at the second position (i ⁇ i + 2) after the preceding ⁇ -
• the exchanged amino acids are separated from each other by 2 (i ⁇ i + 3), 3 (i ⁇ i + 4) or more amino acids.
• amino acids with negatively charged side chains under physiological conditions glutamic acid or aspartic acid can be used, while arginine, lysine, or histidine have positively charged side chains under such conditions.
• the position at which arginine, lysine, or histidine is introduced, or optionally already present, is closer to the C-terminus than the position at which glutamic acid or aspartic acid is introduced or may already be present.
• a double salt bridge in which a sequence is generated in which 3 amino acids are located at positions i and i + 3, i + 4 or i + 5 and i + 7, i + 8 or i + 9 in which the amino acids at the positions i and i + 7, i + 8 or i + 9 have side chains with the same charge, the amino acid at the position i + 3, i + 4 or i + 5 has a counterpart thereto.
• 3 corresponding amino acids can be introduced by mutation, if appropriate also less, if corresponding amino acids are already present in the starting sequence.
• a preferred embodiment is characterized in that the protein after replacement of a helical element with the sequence KPKDTLMISR (SEQ ID NO: 8) from the human IgG C H 2 domain (SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 15) or the helix sequence KAEDTLHISR (SEQ ID NO: 9) optimized therefrom, the sequence TKDEYERH (SEQ ID NO: 10) from the murine kappa C L domain (SEQ ID NO: 1), the sequence TPEQWKSHRS (SEQ ID NO: 16) from the human lambda C L domain (SEQ ID NO: 13) or the sequence SKADYEKHK (SEQ ID NO: 11) from the human kappa C L domain (SEQ ID NO: 12).
• the present invention relates to the transplantation of suitable helical elements into domains having no or less optimal helical elements, such as the Ig domain of the beta2 microglobulin (SEQ ID NO: 3), the variable domains (V L , V H ) or the constant domain C H 1 of immunoglobulins.
• suitable helical elements such as the Ig domain of the beta2 microglobulin (SEQ ID NO: 3), the variable domains (V L , V H ) or the constant domain C H 1 of immunoglobulins.
• the transplanted elements may originate, for example, from the constant immunoglobulin domains C L , C H 2 or C H 3 or be variants of such elements optimized according to the method of the invention.
• the transplantation is preferably carried out by a method in which 4 to 12 consecutive amino acids (preferably about 10 amino acids) are replaced by an amino acid sequence of equal length or greater length, wherein the introduced amino acid sequence has a higher formation probability of HeNx than the exchanged sequence.
• the introduced sequence is a helical element from the region between the ⁇ -sheet strands A and B and / or E and F of a C L or CH domain of an immunoglobulin.
• Suitable helical elements have, for example, the sequence KPKDTLMISR (SEQ ID NO: 8) from the human IgG C H 2 domain (SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 15) or the optimized helix sequence KAEDTLHISR (FIG.
• SEQ ID NO: 9 the sequence TKDEYERH (SEQ ID NO: 10) from the murine kappa C L domain (SEQ ID NO: 1), the sequence TPEQWKSHRS (SEQ ID NO: 16) from the human lambda C L - Domain (SEQ ID NO: 13) or the sequence SKADYEKHK (SEQ ID NO: 11) from the human kappa C L domain (SEQ ID NO: 12).
• the present invention relates to a method for producing a protein having an immunoglobulin folding pattern, characterized in that a method for improving the biophysical properties of proteins having the immunoglobulin folding pattern as described above is applied to such a protein. and the modified protein thus obtained is expressed in a host cell.
• Process for the production of proteins by expression of recombinant DNA in host cells and subsequent purification of the desired expressed protein (protein of interest) are well known to those skilled in the art.
• eukaryotic host cells preferably mammalian cells, particularly preferably Chinese hamster ovary (Cricetulus griseus, CHO) cell lines or mouse myeloma cell lines (eg, NSO cells).
• eukaryotic host cells preferably mammalian cells, particularly preferably Chinese hamster ovary (Cricetulus griseus, CHO) cell lines or mouse myeloma cell lines (eg, NSO cells).
• Certain antibody formats such as, for example, domain antibodies can also be advantageously produced in prokaryotic host cells (eg E. coli) or yeast cells.
• the present invention relates to a protein having an immunoglobulin folding pattern prepared by a method of the invention as described above.
• this is an antibody, in particular a complete immunoglobulin containing two light and two heavy chains.
• the present invention relates to a protein having an immunoglobulin folding pattern and at least one variable domain (V L or V H ), characterized in that it contains a helical element in this variable domain.
• V L or V H variable domain
• Naturally occurring variable domains do not contain such helical elements and can be improved in their biophysical properties by introducing such elements.
• such a variable domain contains a helical element having a greater helix formation probability than any amino acid sequence of equal length naturally occurring in a variable domain of an immunoglobulin.
• the variable domains which are set down in the database NCBI GenBank under the accession numbers AAK19936 (IgGI VH) and AAK62672 (IgGI VL).
• variable domain according to the invention contains a helical element having the sequence KPKDTLMISR (SEQ ID NO: 8), KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO: 16) or SKADYEKHK (SEQ ID NO: 11).
• the helical element is located between the folder strands E and F.
• the present invention relates to a protein having an immunoglobulin folding pattern and at least one constant domain of the type C H 2, characterized in that it contains a helical element in this constant domain which has a higher helix formation probability than a helical element of one naturally occurring in humans CH2 domain.
• a domain can here SEQ ID NO: 5 serve.
• such a protein contains a CH2 domain comprising a helical element having the sequence KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), the sequence TPEQWKSHRS (SEQ ID NO: 16) or SKADYEKHK (SEQ ID NO: 11).
• the helical element is preferably located between the pleated sheet strands A and B and / or E and F of the CH2 domain.
• the present invention relates to a protein which has an immunoglobulin folding pattern and at least one constant domain of the type C H 1, characterized in that it contains a helical element in this constant domain which has a higher helix formation probability than a helical element or a helical element
• a protein which has an immunoglobulin folding pattern and at least one constant domain of the type C H 1, characterized in that it contains a helical element in this constant domain which has a higher helix formation probability than a helical element or a helical element
• such a protein contains a C H 1 domain comprising a helical element having the sequence KPKDTLMISR (SEQ ID NO: 8), KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO: 16) or the sequence SKADYEKHK (SEQ ID NO: 11).
• the helical element is preferably located between the pleated sheet strands A and B and / or E and F of the CH1 domain.
• the present invention relates to a modified ⁇ 2-microglobulin having at least one helical element in an Ig domain, preferably a helical element having the sequence KPKDTLMISR (SEQ ID NO: 8), KAEDTLHISR (SEQ ID NO: 9), TKDEYERH (SEQ ID NO: 10), TPEQWKSHRS (SEQ ID NO: 16), or SKADYEKHK (SEQ ID NO: 11).
• the present invention relates to a protein having an immunoglobulin folding pattern comprising at least one helical element in an Ig domain which has a higher helix formation probability than a helical element present in any one of SEQ ID NO: 1, SEQ ID NO: 12 or SEQ ID NO: 13 (C L WT) or SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 15 (C H 2 WT).
• a protein contains a helical element having the sequence KAEDTLHISR (SEQ ID NO: 9).
• the present invention relates to a protein which has an immunoglobulin folding pattern and which contains the sequence SEQ ID NO: 4, SEQ ID NO: 6 and / or SEQ ID NO: 9.
• the present invention relates to a protein as described above for medical use in therapy or diagnostics.
• the medical use of antibodies and other proteins having Ig folding patterns is known to those skilled in the art and a number of such substances are approved as drugs (e.g., rituximab, trastuzumab, etanercept).
• drugs e.g., rituximab, trastuzumab, etanercept.
• the person skilled in the art knows methods of preparing dosage forms of such substances (for example physiologically buffered, aqueous solutions) and of administering such medicaments with a corresponding indication (for example by intravenous injection or infusion).
• the present invention relates to a biotechnological method for modifying the biophysical properties of antibodies or proteins having the immunoglobulin folding pattern, characterized in that an optimization of the natural, helical elements takes place.
• the present invention relates to a biotechnological method for modifying the biophysical properties of antibodies or proteins having the immunoglobulin folding pattern, characterized in that a transplantation of the natural or optimized helical elements takes place, preferably in domains that have no or less optimal helical elements.
• the proteins of the present invention are preferably produced by recombinant expression in a host cell.
• an expression vector is used, which is introduced into the host cell.
• the expression vector contains the "gene of interest" which comprises a nucleotide sequence of any length encoding a product of interest
• the gene product or “product of interest” is typically a protein, polypeptide, peptide or fragment or derivative thereof , It can also be RNA or antisense RNA.
• the gene of interest may be in full length, in truncated form, as a fusion gene or a labeled gene. It may be genomic DNA or preferably cDNA or corresponding fragments or fusions.
• the gene of interest may be the native gene sequence, mutated or otherwise modified. Such modifications include codon optimizations for adaptation to a particular host cell and humanization.
• the gene of interest may e.g. encode a secreted, cytoplasmic, nuclear localized, membrane bound or cell surface bound polypeptide.
• nucleic acid refers to an oligonucleotide, nucleotides, polynucleotides and fragments thereof, and DNA or RNA of genomic or synthetic origin which are present as a single or double strand and the coding or the non-coding Strand of a gene can represent.
• nucleic acid sequence standard techniques, such as site-specific mutagenesis, PCR-mediated mutagenesis or de novo synthesis of oligonucleotide sequences can be used.
• Biopharmaceutically significant proteins / polypeptides in the context of the present invention include e.g. Antibodies or immunoglobulins and other proteins having immunoglobulin folding patterns, e.g. Members of the immunoglobulin superfamily, as well as their derivatives or fragments. In general, these are substances that act as agonists or antagonists and / or can find therapeutic or diagnostic use.
• polypeptides or "proteins” is used for amino acid sequences or proteins and refers to polymers of amino acids of any length. This term also includes proteins that are post-translationally modified by reactions such as glycosylation, phosphorylation, acetylation, or protein processing.
• the structure of the polypeptide may be e.g. by substitutions, deletions or insertion of amino acids, fusion with other proteins, such as the Fc portion of immunoglobulins, while retaining its biological activity.
• the polypeptides can multimerize and form homo- and heteromers.
• expression vectors can in principle be carried out by conventional methods familiar to the person skilled in the art. There is also a description of the functional components of a vector, eg suitable promoters, enhancers, termination and polyadenylation signals, antibiotic resistance genes, selection markers, origins of replication and splice signals.
• Conventional cloning vectors can be used for the production, for example plasmids, bacteriophages, phagemids, cosmids or viral vectors such as baculovirus, retroviruses, adenoviruses, adeno-associated viruses and herpes simplex virus, but also artificial or artificial or mini-chromosomes.
• the eukary Ontic expression vectors also typically contain prokaryotic sequences, such as origin of replication, and antibiotic resistance genes, which allow multiplication and selection of the vector in bacteria.
• prokaryotic sequences such as origin of replication, and antibiotic resistance genes, which allow multiplication and selection of the vector in bacteria.
• a variety of eukaryotic and prokaryotic expression vectors containing multiple cloning sites for introducing a polynucleotide sequence are known and some are commercially available from various companies such as Stratagene, La JoIIa, CA, USA; Invitrogen, Carlsbad, CA, USA; Promega, Madison, WI, USA or BD Biosciences Clontech, Paolo Alto, CA, USA.
• Eukaryotic or prokaryotic host cells are transfected or transformed with suitable expression vectors.
• yeast cells and mammalian cells are preferably used as eukaryotic host cells.
• rodent cells such as e.g. Mouse, rat and Hamster cell lines.
• Preferred prokaryotic host cells are bacteria, more particularly Escherichia coli, Bacillus subtilis, Pseudomonas (P. aeruginosa, P.
• Preferred eukaryotic host cells within the scope of the invention are hamster cells such as BHK21, BHK TK “ , CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1 and CHO-DG44 cells or derivatives / derivatives of these cell lines.
• DG44, CHO-DUKX, CHO-K1 and BHK21 cells in particular CHO-DG44 and CHO-DUKX cells
• mouse myeloma cells preferably NSO and Sp2 / 0 cells and derivatives / derivatives of these cell lines, but also derivatives and derivatives thereof
• Cells other mammalian cells, including but not limited to human, mouse, rat Monkeys, rodents, or eukaryotic cells, including, but not limited to, yeast, insect, avian, and plant cells, may also be used as host cells for the production of biopharmaceutical proteins.
• Transfection of the eukaryotic host cells with a polynucleotide or one of the expression vectors according to the invention is carried out by customary methods. Suitable transfection methods are e.g. liposome-mediated transfection, calcium phosphate co-precipitation, electroporation, polycation (e.g., DEAE-dextran) -mediated transfection, protoplast fusion, microinjection, and viral infections.
• Suitable transfection methods are e.g. liposome-mediated transfection, calcium phosphate co-precipitation, electroporation, polycation (e.g., DEAE-dextran) -mediated transfection, protoplast fusion, microinjection, and viral infections.
• prokaryotic host cells with a polynucleotide or one of the expression vectors according to the invention is carried out by customary methods. Suitable methods are for.
• electroporation chemical treatment of cells with, for example, calcium chloride, magnesium chloride, manganese chloride, polyetyhlenglykol or dimethyl sulfoxide, bacteriophage transduction
• a stable transfection is performed wherein the constructs are integrated into either the genome of the host cell or an artificial chromosome / minichromosome or are stably episomally contained in the host cell.
• the transfection method which enables the optimal transfection frequency and expression of the heterologous gene in the respective host cell, is preferred.
• the eukaryotic host cells are preferably established under serum-free conditions, adapted and cultured, optionally in media that are free of animal proteins / peptides.
• media examples include Harn 's F12 (Sigma, Deisenhofen, DE), RPMI-1640 (Sigma), Dulbecco 's Modified Eagle 's Medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma). , Iscove 's Modified Dulbecco 's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-SFMII (Invitrogen), serum-free CHO medium (Sigma).
• yeast media may optionally be supplemented with various compounds, eg, hormones and / or other growth factors (eg insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (eg sodium chloride, calcium, magnesium, phosphate), buffers (eg HEPES), nucleosides (eg adenosine, thymidine), glutamine, glucose or others Equivalent nutrients, antibiotics and / or trace elements
• hormones and / or other growth factors eg insulin, transferrin, epidermal growth factor, insulin-like growth factor
• salts eg sodium chloride, calcium, magnesium, phosphate
• buffers eg HEPES
• nucleosides eg adenosine, thymidine
• glutamine glucose or others
• glucose or others Equivalent nutrients, antibiotics and / or trace elements
• serum-free media are preferred according to the invention, media which have been supplemented with an appropriate amount of serum may also be used to culture the host cells.
• prokaryotic host cells For the cultivation of prokaryotic host cells numerous media are known, which are also commercially available. Examples include LB, TB, M9, SOC, YT and NZ media (Sigma).
• one or more suitable selection agents are added to the medium or suitable "drop-out" media are used which lack essential additives such as amino acids or nucleotides for growth.
• the term "gene expression” or “expression” refers to the transcription and / or translation of a heterologous gene sequence in a host cell.
• the expression rate can be generally determined either on the basis of the amount of the corresponding mRNA present in the host cell or on the basis of the amount of gene product produced which is encoded by the gene of interest.
• the amount of mRNA generated by transcription of a selected nucleotide sequence can be determined, for example, by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization of cellular RNA or by PCR.
• Proteins released from a nucleotide sequence can also be by various methods, such as by ELISA, protein A HPLC, Western blot, radioimmunoassay, immunoprecipitation, detection of the biological activity of the protein, immunostaining of the protein with subsequent FACS analysis or fluorescence microscopy, direct Detection of a fluorescent protein can be determined by FACS analysis or by fluorescence microscopy.
• the proteins of the invention are prepared in a process in which production cells are propagated and used to produce the coding gene product of interest.
• the selected high producing cells are preferably cultured in a serum-free culture medium and preferably in suspension culture under conditions which allow expression of the gene of interest.
• the protein / product of interest is preferably obtained as a secreted gene product from the cell culture medium.
• the gene product can also be isolated from cell lysates. In order to obtain a pure, homogeneous product which is substantially free of other recombinant proteins and host cell proteins, usual purification steps are carried out. To do this, one often first removes cells and cell debris from the culture medium or lysate.
• the desired gene product may then be released from contaminating soluble proteins, polypeptides and nucleic acids, e.g. by fractionation on immunoaffinity and ion exchange columns, ethanol precipitation, reverse phase HPLC or chromatography on Sephadex, silica or cation exchange resins such as DEAE.
• Methods which lead to the purification of a heterologous protein expressed by recombinant host cells are known to the person skilled in the art and are described in the literature.
• AFM atomic force microscopy ß 2 m: beta2-microglobulin bp: base pair
• Chain CHO Chinese Hamster Ovary C L : constant domain of a light Ig chain
• DHFR dihydrofolate reductase
• E. coli Escherichia coli
• EDTA ethylenediamine-N, N, N ', N' tetraacetic acid
• ELISA enzyme-linked immunosorbant assay
• FUV far ultraviolet
• GdmCl guanidine hydrochloride
• GSH glutathione
• GSSG glutathione disulfide
• HSQC heteronuclear single quantum coherence
• HC heavy chain
• HT hypoxanthine / thymidine
• IgG immunoglobulin G kb: kilobase LC: light chain mAb: monoclonal antibody MD: molecular dynamics MTX: methotrexate NMR: nuclear magnetic resonance NPT: neomycin phosphotransferase NUV: near-ultraviolet
• SEAP secreted alkaline phosphatase
• the recombinant E. coli bacteria BL21 DE3 (Stratagene, CA, USA) are cultured overnight in selective LB medium at 37 ° C. and 300 rpm in shake flasks.
• the recombinant bacteria are cultured in M9 minimal medium (Sigma) with 15 N ammonium chlorohd as sole nitrogen source or optionally additionally 13 C-glucose as sole carbon source.
• the inclusion bodies are then isolated by centrifuging off the bacteria and resuspending in 100 mM Tris / HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, protease inhibitor, the cells are disrupted in a French press, with 2% v / v of Triton X-100 and stirred for 30 min at 4 ° C. Centrifugation (20,000 rpm, 30 min) isolates the inclusion bodies as pellets and then twice in 100 mM Tris / HCl, pH 7.5 , 10 mM EDTA, 100 mM NaCl, protease inhibitor and in each case centrifuged off again (20,000 rpm, 30 min).
• the inclusion body pellet is now in 100 mM Tris / HCl, pH 8.0, 10 mM EDTA, 8 M
• Components are then removed by centrifugation (48000g, 25 min, 20 0 C).
• the supernatant is diluted fivefold in 50 mM sodium phosphate (pH 7.5), 4 M GdmCl and applied to a nickel chelate column (Ni-NTA, Qiagen). After washing with five column volumes, elution is carried out with 50 mM sodium phosphate (pH 4), 4 M GdmCl.
• the refolding by dialysis is carried out in 250 mM Tris / HCl, pH 8.0, 5 mM EDTA, 1 mM oxidized glutathione at 4 ° C overnight. Aggregates are removed by centrifugation (48000g, 25 min, 4 ° C).
• thrombin Novagen
• 20 mM sodium phosphate pH 7.5
• 100 mM NaCl 100 mM EDTA.
• CD spectroscopy CD measurements are performed in a Jasco J-715 spectropolarimeter. Measurements are carried out at 20 ° C. in PBS.
• Remote UV CD spectra are measured from 195-250 nm at a protein concentration of 50 ⁇ M in a 0.2 mm quartz cuvette, near-UV CD spectra are measured from 250-320 nm at a protein concentration of 50-100 ⁇ M in 5 mm Measure quartz cuvettes. Measurements are carried out at 20 ° C. in PBS. Spectra are accumulated 16 times each, averaged and buffer corrected. Temperature transitions are measured at 218 nm (C H 2 WT / mutant) or 205 nm (C L , ⁇ 2 m WT / mutants) in PBS at 10 ⁇ M protein concentration in a 1 mm quartz cuvette at a heating rate of 20 ° C / h. AFM measurements
• a 100 ⁇ M protein solution in PBS 1: 1 is mixed with buffer A (25 mM sodium acetate, 25 mM sodium phosphate, pH 1, 5 or 2.5). The final pH value is thus at pH 1, 5 or pH 3.0.
• the solution is incubated for 7 days with gentle swirling at 37 ° C., then 20 ⁇ l of the solution are applied to fresh mica surfaces, washed three times with sterile filtered water and then analyzed in the AFM.
• the AFM contact mode with a scanning speed of 1.5 ⁇ m / min is used. Measurements are performed on a Digital Instruments Multimode Scanning Probe Microscope and DNP-S20 tips.
• the sequence region for the wild-type CH2 domain and CL domain is determined by PCR from a human IgGI antibody gene or the kappa chain of the murine antibody MAK33 (Augustine, JG et al., J. Biol. Chem. 276 (5 ), 3287-3294, 2001). Introduction of the P35A mutation into the d domain is via PCR mutagenesis using mutagenic primers.
• the sequence regions for the CH2 domain of the helix-optimized CH2 mutant, ß 2 m WT and the ß 2 m mutant are de novo synthesized with the transplanted d helix (www.geneart.com).
• helix mutations are introduced into the wild-type CH2 domain of an IgGI antibody gene via PCR mutagenesis using mutagenic primers.
• the cells CHO-DG44 / dhfr ⁇ / ⁇ are stored permanently as suspension cells in serum-free CHO-S-SFMII medium supplemented with hypoxanthine and thymidine (HT) (Invitrogen GmbH, Düsseldorf, DE) in cell culture flasks at 37 ° C. in a humid atmosphere and 5 % CO2 cultivated.
• HT hypoxanthine and thymidine
• the cell numbers and the viability are determined with a Cedex (Innovatis) and the cells are then seeded in a concentration of 1 - 3 x10 5 / ml_ and passaged every 2 - 3 days.
• CHO-DG44 Lipofectamine Plus reagent (Invitrogen) is used. A total of 1, 0-1 .mu.g of plasmid DNA, 4 .mu.l of lipofectamine and 6 .mu.l plus reagent are mixed per transfection batch according to the manufacturer's instructions and mixed in a volume of 200 .mu.l to 6 ⁇ 10 5 cells in 0.8 ml_ HT. supplemented CHO-S-SFMII medium. After incubation for 3 hours at 37 ° C. in a cell incubator, 2 ml of HT-supplemented CHO-S-SFMII medium are added.
• the transfection batches are either harvested (transient transfection) or subjected to selection. Since one expression vector contains one DHFR and the other an NPT selection marker, the cotransfected cells are transfected for DHFR- and NPT-based selection 2 days after transfection into CHO-S-SFMII medium without added hypoxanthine and thymidine G418 (Invitrogen) was also added to the medium in a concentration of 400 ⁇ g / ml.
• a DHFR-based gene amplification of the integrated heterologous genes is achieved by adding the selection agent MTX (Sigma) in a concentration of 5-2000 nM to an HT-free CHO-S-SFMII medium.
• eukaryotic expression vectors are used which are based on the pAD-CMV vector (Werner, RG et al., Arzneistoff-Forschung / Drug Research 48, 870-880, 1998) and the expression of a terologist gene via the combination CMV enhancer / CMV promoter mediate.
• the first vector pBI-26 contains the dhfr minigene, which serves as an amplifiable selection marker.
• the dhfr minigene is replaced by an NPT gene.
• the NPT selection marker including SV40 early promoter and TK polyadenylation signal, was isolated from the commercial plasmid pBK-CMV (Stratagene, La JoIIa, CA) as a 1640 bp Bsu36I fragment. After a replenishment reaction of the fragment ends by Klenow DNA polymerase, the fragment was ligated with the 3750 bp Bsu36l / Stul fragment of the first vector, which was also treated with Klenow DNA polymerase. Subsequently, the NPT gene was modified. It is the NPT variant F240I (Phe240lle), whose cloning is described in WO2004 / 050884.
• the quantification of the expressed antibodies in the supernatants of stably transfected CHO-DG44 cells is carried out by ELISA according to standard protocols, on the one hand a goat anti human IgG Fc fragment (Dianova, Hamburg, DE) and on the other hand an AP-conjugated goat anti human kappa light chain antibody (Sigma) is used. As a standard purified antibody of the same isotype as the expressed antibodies.
• the SEAP titer in culture supernatants of transiently transfected CHO-DG44 cells is quantified using the SEAP Reporter Gene Assay according to the protocol specifications of the manufacturer (Roche Diagnostics GmbH). ThermoFluor ⁇ method
• a qPCR system (Mx3005P TM, Stratagene) is used, based on the ThermoFluor® method.
• a solvatochromic / ambient fluorescence dye is used as an indicator of small changes in the thermal stability of proteins.
• This fluorescent dye which has a low quantum yield in aqueous solution, interacts with hydrophobic, non-native structures of the protein which expands due to temperature increase. The interaction of the dye with already unfolded protein domains results in a significant increase in the fluorescence detected (Cummings, M.D. et al., Journal of Biomolecular Screening 854-863, 2006).
• the measurement of the protein samples in a temperature range from 25 ° C to 95 ° C at intervals of 1 ° C per minute is carried out in a volume of 20 ⁇ l, using 2 ⁇ M protein and 4x SyproOrange (prepared from a 500Ox SyproOrange stock solution; Invitrogen) in be used in each case to be tested buffer.
• the first step is to identify the helical elements or the corresponding loops, if no helices are present, in the immunoglobulin domain used as the target of the optimization. For example, in the case of constant antibody domains, the helices are always between the
• proline and / or glycine residues are replaced by another amino acid, preferably alanine (if not in conflict with the one to be prioritized)
• Point 2 occurs).
• the replacement is only done if it is the residue to be replaced is not the first residue after the previous ⁇ -sheet strand or the last remaining before the subsequent ⁇ -sheet strand.
• Amino acids by amino acids with charged side chains of unlike charge Any combination of arginine, lysine, histidine, aspartate or glutamate can be used.
• the residues to be replaced must be separated by two, three or four amino acids, so that the introduced charged residues can be separated, for example. B. the numbering i and i + 3, i and i + 4 or i and i + 5 will have. All permutations of said residues are possible.
• an introduction of arginine, lysine or histidine is closer to the C-terminus than an introduction of aspartate or glutamate.
• double salt bridges are, if in accordance with the
• EXAMPLE 2 INVESTIGATION OF THE PROTEIN FINDING OF THE C L DOMAIN
• C1_P35A The proteins C L WT (SEQ ID NO: 1) and C L -P135A (SEQ ID NO: 7) are produced recombinantly in E. coli.
• the first four N-terminal amino acids at C L WT result in each case from the selected cloning strategy in the expression vector pET28a and are naturally not present in the CL domain.
• the folding of the CL domain after unfolding in the denaturant GdmCI, can be monitored in real time at low temperatures ( Figure 5 and 6). It turns out that the two short helical ele- ments between strand A and B as well as between strand E and F are already completely patterned in the main folding intermediate ( Figure 5 and 6). Thus, they can be postulated to play an important role in the folding process of these and other antibody domains.
• the rate-determining step in the folding of the C L domain, before which the folding intermediate is promoted is the isomerization of the proline residue 35 from trans to ice. Therefore, this residue is exchanged for alanine, which should always be in trans. This allows the folding intermediate to be stabilized in equilibrium.
• the proteins C L to ⁇ 2 m and ⁇ 2 m to C L and, as a control, the wild-type sequences ⁇ 2 m (SEQ ID NO: 3) and C L (SEQ ID NO: 1) are produced recombinantly in E. coli.
• the first four N-terminal amino acids at C L WT and the first N-terminal amino acid at C L to ⁇ 2 m result in each case from the selected cloning strategy in the expression vector pET28a and are naturally not present in the CL domain.
• EXAMPLE 4 OPTIMIZATION OF THE HUMAN IGG1 C H 2 DOMAIN
• the CH2 domain is the weakest link in terms of stability.
• the Fc fragment may serve as a general platform of IgG.
• Antibodies are considered, so that an optimization of the biophysical properties of the C H 2 domain on the one hand should increase the overall stability of the Fc fragment, on the other hand is a universally applicable optimization.
• the first HeNx of a human IgG1 CH2 domain ( Figure 9A) is chosen for optimization. Within these, additional salt bridges are introduced by targeted mutagenesis ( Figure 9B). Both C H 2 domains, wild-type (C H 2 WT; SEQ ID NO: 5) and Helixi mutant (C H 2 helixi mutant; SEQ ID NO: 6), are expressed in E. coli. The first N-terminal amino acid in the C H 2 Helixi mutant results from the chosen cloning strategy in the expression vector pET28a and does not occur naturally in the CH2 domain.
• IgGI-WT b) pBI-26 / IgG1-HChelix1 and pBI-49 / IgG1-LC, which is responsible for an IgG1 monoclonal antibody in which the first HeNx in the human C H 2 domain is replaced by the sequence region KPKDTLMISR (FIG. SEQ ID NO: 8) is optimized against KAEDTLHISR (SEQ ID NO: 9)
• CHO-DG44 cells For stable transfection of CHO-DG44 cells, co-transfection is performed with the same plasmid combinations as described above. The selection of stably transfected cells is carried out two days after transfection in HT-free medium with the addition of 400 ⁇ g / mL G418. After selection, DHFR-based gene amplification is induced by addition of 100 nM MTX. For material production, the cells are cultured in a 10-day fed-batch process in shake flasks. The purification is identical for the WT or Helixi mutant of the antibody.
• Protein A affinity chromatography (MabSe) lect rProteinA, GE Healthcare) according to the instructions of the manufacturer, using phosphate buffer (20 mM sodium phosphate, 140 mM sodium chloride, pH 7.5, conductivity 16.5 mS / cm) for equilibration and 50 mM acetate pH 3 for elution, 3 is used.
• the eluate is adjusted to a pH of 5.5 by addition of 1 M Tris pH 8.
• the purification profiles for the two antibody variants are comparable.
• the thermal stability of the antibodies is determined by the ThermoFluor® method.
• the thermal stability of the Helixi mutant of the IgGI antibody to the IgG1 -WT antibody can be increased under both basic and acidic buffer conditions.
• the C H 2- domain of the Helixi mutant exhibits a 8 ° C higher melting temperature compared to the CH2 domain of the IgGI-WT.
• 100 mM acetate pH 3.4 it is even increased by 18 ° C. This significant increase in the thermal stability of immunoglobulins is of immense benefit to the biopharmaceutical production of therapeutic proteins.
• the optimization of the natural, helical element of the CH2 domain is achieved by replacing the naturally occurring sequence region KPKDTLMISR (SEQ ID NO: 8) (this sequence region also occurs, for example, in the C H 2 domains of human IgG2 (SEQ ID NO: 14). and IgG4 (SEQ ID NO: 15) against KAEDTLHISR (SEQ ID NO: 9) significantly improved the robustness of the biotechnologically engineered therapeutic proteins.
• the increased temperature and pH stability is particularly advantageous in the process of virus inactivation to increase the product safety of therapeutic proteins since this step is performed at acid pH. Other advantages include greater flexibility in chromatography and protein formulation, less tendency for aggregation, and improved storage stability.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Mycology (AREA)
• Cell Biology (AREA)
• Gastroenterology & Hepatology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Toxicology (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=41105221

Family Applications (1)

Country Status (6)

Citations (1)

Family Cites Families (2)

• 2009
  • 2009-06-30 US US13/000,700 patent/US20110201785A1/en not_active Abandoned
  • 2009-06-30 EP EP09772458A patent/EP2307452A1/de not_active Withdrawn
  • 2009-06-30 WO PCT/EP2009/058225 patent/WO2010000758A1/de active Application Filing
  • 2009-06-30 KR KR1020107025677A patent/KR20110025641A/ko not_active Application Discontinuation
  • 2009-06-30 JP JP2011515444A patent/JP2011526595A/ja active Pending
  • 2009-06-30 CA CA2729591A patent/CA2729591A1/en not_active Abandoned

Patent Citations (1)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events