Chimeric IgG-Fc binding ligand polypeptides for IgG affinity purification and uses thereof

Abstract

The present invention relates to a chimeric IgG-Fc binding ligand polypeptide comprising a protein fragment of SlyD, the IF domain of which is replaced by the affinity peptide Fc-III-4C or its Fc-III-XC variant, and to related uses for IgG affinity purification.

Description

[Technical Field]

[0001] The present invention relates to chimeric IgG-Fc binding ligand polypeptides comprising a protein fragment of SlyD, the IF domain of which has been replaced by the affinity peptide Fc-III-4C or its Fc-III-XC variant, and related uses for IgG affinity purification. [Background technology]

[0002] Background of the Invention With the success of monoclonal antibodies as therapeutic and diagnostic tools, the development of efficient and economical IgG purification procedures became increasingly necessary for both academic and industrial use. In addition to different types of interaction and separation techniques, affinity strategies have been the most common and efficient methodologies to date.

[0003] The growing use of antibodies necessitates the development of appropriate mAb purification strategies, especially for industrial-scale production. Compared to other biotechnology products, in part due to the high dosages and wide range of applications, hundreds of kilograms of bulk drug substance of monoclonal antibodies must be synthesized annually.

[0004] Streptococcal protein A, a natural IgG-Fc binder, remains the gold standard among affinity ligands on a manufacturing scale. Coupled to a suitable matrix, such as agarose beads, these affinity resins represent the first step in the purification chain, isolating antibodies from crude protein mixtures, such as serum, ascites fluid, or cell supernatants, thus making further downstream processing more economical.

[0005] The most commonly used process for downstream purification of monoclonal antibodies today involves a first step in which the production cell culture is harvested, for example by filtration, to remove cells and cell debris and obtain a clarified supernatant suitable for chromatography. The mAb present in the cell culture supernatant is then recovered in a single capture step by applying the filtered fluid to a Protein A chromatography column. Process and product-related impurities are removed by one or two polishing steps, typically incorporating cation or anion exchange chromatography, hydrophobic interaction chromatography, or mixed-mode chromatography.

[0006] Additionally, the process includes several steps to bypass viral contamination, such as viral inactivation and viral filtration steps after Protein A chromatography (e.g., low pH incubation). The purified product is then transferred to the final formulation buffer by ultrafiltration or diafiltration.

[0007] WO 2004 / 076485 A1 describes a method for purifying antibodies by Protein A affinity chromatography.

[0008] Protein A chromatography is highly effective in removing process-related impurities and exhibits other notable features, such as high production yields and ease of operation. However, it has several drawbacks relative to the general requirements mentioned above. First, as a product of bacterial origin, Protein A can contaminate the final product by leaching from the column matrix and act as an immunotoxin. Second, it is relatively expensive: on the one hand, Protein A is obtained in recombinant form from Escherichia coli (E. coli) for resin production, and on the other hand, Protein A is not resistant to common cleaning and disinfecting agents, such as guanidine hydrochloride or urea, for column regeneration, which not only increases costs but also makes disposability an issue. Another significant concern is aggregate formation caused by antibody elution conditions at low pH values, which can lead to loss of biological activity (Arora, I., Chromatographic Methods for the Purification of Monoclonal Antibodies and Their Alternatives: A Review. International Journal of Emerging Technology and Advanced Engineering, 2013.3(10):pp.475-481).

[0009] One of the major drawbacks of conventional IgG purification processes is elution at acidic pH values (pH 2.5–3.5). The high affinity of natural IgG-binding proteins, such as Protein A, for their target leads to high specificity and selectivity of the purification process, but on the other hand, harsh conditions are required to disrupt the strong interaction between the antibody and the ligand. Low pH can promote mAb degradation and lead to contamination of the final product due to leakage of the column ligand.

[0010] To overcome these drawbacks of Protein A-based purification, considerable efforts have been made to improve alternative Ab-binding molecules, particularly in terms of binding properties and pH sensitivity. In addition to Protein A, which is primarily used for Ab isolation due to its high affinity for IgG from different species, other proteins of bacterial origin, such as Protein G, Z, and L, have been investigated (Choe, W., T.A. Durgannavar, and S.J. Chung, Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials (Basel), 2016.9(12)).

[0011] Affinity chromatography techniques using Protein A and G resins remain the most widely used method for mAb capture at manufacturing scale, as they are very well proven techniques and are included in many established platform processes.

[0012] SlyD (susceptible to lysis D; product of the slyD gene) is a metallochaperone composed of two functional units: a peptidyl-prolyl cis / trans isomerase (PPIase) activity in the FK506-binding protein (FKBP) domain and a chaperone function in the 45-amino acid "intraflap insert" (IF) domain (Low, C., et al., "Crystal structure determination and functional characterization of the metallochaperone SlyD from Thermus thermophilus." J Mol Biol, 2010, 398(3):375-90). TtSlyD's excellent biophysical properties, including excellent thermostability and proteolytic stability due to its origin from the thermophilic organism Thermus thermophilus, make it a powerful tool for a wide range of applications. A major advantage of the TtSlyD scaffold is that the IF domain can be easily exchanged without affecting the physicochemical behavior and tertiary structure of the FKBP core region. Furthermore, its simple structure and high solubility allow its recombinant production in bacterial cells such as E. coli. However, therapeutic applications using the TtSlyD scaffold are not feasible in its original composition due to its bacterial origin, which induces an immunological response.

[0013] WO 2003 / 000878 A2 relates to the cloning and expression of heterologous proteins or polypeptides in bacteria such as Escherichia coli. In particular, the invention relates to expression tools comprising an FKBP-type peptidyl-prolyl isomerase selected from the group consisting of FkpA, SlyD and trigger factor, methods for recombinant protein expression, recombinant polypeptides thus obtained, and uses of such polypeptides.

[0014] WO 2014 / 071978 A1 relates to chimeric polypeptide protein scaffolds for engineering polypeptide domains presented by the scaffolds comprising one or more fragments from the FKBP family presenting one or more polypeptides inserted in place of an intraflap domain insert (IF domain), and their use in a method for screening and selecting constrained peptide surrogates exhibiting binding activity to a predetermined target molecule, in particular a polypeptide of the sequence MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRLIPGLEEALEGREEGEAFQAHVPAEKAY-X-GKDLDFQVEVVKVREATPEELLHGHA (SEQ ID NO: 2), where X is an amino acid sequence comprising the variable sequence presented by the Thermus thermophilus SlyD chimeric polypeptide.

[0015] It is an object of the present invention to provide an improved IgG purification procedure comprising a capture system that exhibits rapid complex formation between the ligand and the target analyte and allows dissociation under relatively mild conditions, which method also avoids further drawbacks of the prior art. Other objects and advantages will become apparent to those skilled in the art upon review of this specification of the present invention. Summary of the Invention

[0016] In a first aspect of the present invention, the above-mentioned object is achieved by a chimeric IgG-Fc-binding ligand polypeptide comprising a protein fragment of SlyD, in which the IF domain has been replaced by the affinity peptide Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1) or its Fc-III-XC variant (X1DCAWHLGELVWCTX2, SEQ ID NO: 3), in which X1 is deleted or independently selected from the group of C, D, P, E, and K, and X2 is independently selected from the group of C, Q, P, and E. Preferably, the chimeric IgG-Fc-binding ligand polypeptide according to the present invention comprises SlyD from a Thermus species, such as Thermus thermophilus.

[0017] In a second aspect of the present invention, the above object is solved by a bivalent binder molecule comprising two fused chimeric IgG-Fc binding ligand polypeptides according to the invention, preferably fused head-to-tail to each other.

[0018] In a third aspect of the present invention, the above object is solved by a chimeric IgG-Fc binding ligand polypeptide according to the present invention or a bivalent binder molecule according to the present invention coupled to a solid support, such as a solid matrix material, such as a bead and / or a column matrix.

[0019] In a fourth aspect of the present invention, the above object is solved by a method for producing a chimeric IgG-Fc binding ligand polypeptide according to the present invention, which method comprises recombinant expression of said ligand polypeptide in a suitable host cell such as E. coli, or comprises chemical synthesis of said ligand polypeptide.

[0020] In a fifth aspect of the present invention, the above object is solved by a method for purifying an immunoglobulin, comprising contacting said immunoglobulin with a solid support to which a chimeric IgG-Fc binding ligand polypeptide according to the present invention or a bivalent binder molecule according to the present invention is coupled, and suitably eluting said immunoglobulin from said chimeric IgG-Fc binding ligand polypeptide or bivalent binder molecule, said method preferably comprising fast protein liquid chromatography (FPLC).

[0021] In a sixth aspect of the present invention, the above object is solved by the use of a chimeric IgG-Fc binding ligand polypeptide of the present invention or a bivalent binder molecule of the present invention for the purification of immunoglobulins or for the screening and selection of peptide binders to a given target molecule.

[0022] In a main aspect of the present invention, a new type of binder and respective immunoaffinity chromatography column material applicable to IgG purification have been developed. In a particularly preferred aspect, the novel material is suitable for immunoglobulin affinity purification by fast protein liquid chromatography (FPLC). The present invention provides an effective, easy-to-use, and cost-effective alternative to Protein A.

[0023] The present invention provides a chimeric IgG-Fc binding ligand polypeptide comprising a protein fragment of SlyD, in which the IF domain has been replaced with the affinity peptide Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1).

[0024] The Fc-binding ligand used was based on an existing IgG-Fc affinity peptide, designated Fc-III-4C, described by Gong et al. in 2016 (Gong, Y., et al., Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection. Bioconjug Chem, 2016. 27(7):pp.1569-73). Surprisingly, by grafting the peptide onto the FKBP domain of the Thermus thermophilus chaperone SlyD, a highly stable and easily produced affinity ligand, designated TtSlyD-Fc-III-4C, was obtained. Furthermore, a chromatography column was successfully constructed by covalently immobilizing this TtSlyD-Fc-III-4C chimera onto NHS-activated Sepharose resin. The crystal structure of domain B of SpA bound to the IgG-Fc portion is It was elucidated in 1981 and revealed interaction sites with the CH2 and CH3 domains of immunoglobulins. Subunit B contains two alpha-helices, with 11 residues involved in the binding process. Each binding site on the Fc fragment is also a contact point for other natural binding proteins, such as protein G and the neonatal Fc receptor, and is thought to be a favorable point for protein-protein interactions due to its physicochemical properties. This specific interaction site was targeted by DeLano et al. in 2000 (DeLano, WI, et al., Convergent solutions to binding at a protein-protein interface. Science, 2000, 287(5456):1279-83). By screening a cyclic peptide library using phage display, they identified a 13-residue sequence (DCAWHLGELVWCT, SEQ ID NO: 4) that bound to the consensus site in the IgG-Fc region, which was found to compete with natural binders. X-ray crystallography then revealed the alpha-hairpin conformation of selected Fc-III peptides. Despite a structure completely different from known binding motifs and a four-fold smaller size, Fc-III and protein A share many intermolecular interaction points on the surface of the Fc portion, and the binding affinity of the Fc-III peptide was only approximately two-fold weaker.

[0025] In 2006, Dias and colleagues attempted to further stabilize the hairpin conformation by restricting the conformational freedom of the peptide (Dias, R.L., et al., Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptidomimetics. J Am Chem Soc 2006. 128(8):2726-32). They created two main-chain cyclic peptidomimetics based on the original Fc-III. The resulting FcBP-1 and FcBP-2 peptides were generated by grafting Ala3-Trp11 or Asp1-Thr13 onto a hairpin-derived D-Pro-L-Pro template. FcBP-2 showed an 80-fold higher affinity for the Fc domain compared to Fc-III. This was attributed to the constraints imposed by the backbone cyclization and additional disulfide bridges. In contrast, FcBP-1, which lacks the critical disulfide bridge, only weakly interacted with the Fc domain. In 2012, further attempts were made by Gong and colleagues to stabilize the peptide in a double cyclic structure (Gong, Y., et al., Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection. Bioconjug Chem, 2016. 27(7): p. 1569-73).

[0026] Furthermore, to simplify the expression and purification of the TtSlyD-FcIII-4C scaffold, derivatives of the molecule were designed and chemically synthesized peptides were tested for binding to IgG (mAb) in high-throughput microarrays. Both cysteine residues that form disulfide bridges in the stalk of the emerging loop structure were either exchanged or completely removed.

[0027] Due to the difficulty in synthesizing the D-Pro-L-Pro backbone, two prolines were replaced by two cysteine residues at the N- and C-termini, intended to form a second disulfide linkage. The newly generated peptide, designated Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1), was analyzed by surface plasmon resonance (SPR) and showed a 30-fold higher binding affinity for human IgG compared to the original Fc-III. Furthermore, it exhibited strong interactions with various IgGs from different species. Its potential as a ligand for antibody purification was confirmed by selectively capturing IgG from rabbit serum with agarose beads bearing the immobilized affinity peptide.

[0028] The Fc-III-4C peptide beads showed higher binding capacity and reusability than commercially available Protein A beads. The crystal structure of the Fc-III peptide in complex with an IgG-Fc domain shows that the constrained peptide loop targets the same binding site as natural Fc binders. It interacts with similar amino acids found in the interface of commonly used Fc-binding proteins.

[0029] Furthermore, the complex stability of the Fc-III-4C-binding peptide-IgG-Fc complex was altered by systematically manipulating the Fc-III-4C loop insertion. One of the amino acids thought to play a key role in complex formation is Trp11, and each residue was replaced with 12 other amino acids, resulting in different chemical properties. The resulting mutants were expressed, purified, and tested by SPR analysis. The findings were then applied to the TtSlyD scaffold platform, resulting in chimeric IgG-Fc-binding ligand polypeptides according to the present invention, comprising a protein fragment of SlyD, whose IF domain is replaced with the affinity peptide Fc-III-XC (X1DCAWHLGELVWCTX2, SEQ ID NO: 3), where X1 is missing or independently selected from the group consisting of C, D, P, E, and K, and X2 is independently selected from the group consisting of C, Q, P, and E.

[0030] Peptide-based ligands represent a promising new class of binders, successfully developed by various research groups. Their use in place of protein A allows them to be produced at lower cost and is non-immunogenic compared to natural binders. Among this group of binders, cyclic peptides in particular possess several promising features for use as affinity ligands. Compared to their linear counterparts, they exhibit greater enzymatic stability and conformational rigidity, resulting in higher specificity and / or affinity for their target and an entropic advantage in binding. Nevertheless, chemical synthesis of peptides is still required, and aggregation during synthesis can result in low yields.

[0031] Any suitable SlyD scaffold protein can be used for the chimeric IgG-Fc binding ligand polypeptides according to the invention, preferably from bacterial species such as E. coli, and SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. Even more preferred is SlyD from Thermus species, such as Thermus thermophilus.

[0032] More preferred is a chimeric IgG-Fc binding ligand polypeptide according to the invention which comprises MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRLIPGLEEALEGREEGEAFQAHVPAEKAYCDCAWHLGELVWCTCGKDLDFQVEVVKVREATPEELLHGHA (SEQ ID NO: 5).

[0033] The IF domain of the chimeric IgG-Fc binding ligand polypeptide according to the present invention comprises: DCAWHLGELVWCTX2, SEQ ID NO: 6 CDCAWHLGELVWCTX2, SEQ ID NO: 7 DDCAWHLGELVWCTX2, SEQ ID NO: 8 PDCAWHLGELVWCTX2, SEQ ID NO: 9 EDCAWHLGELVWCTX2, SEQ ID NO: 10, and KDCAWHLGELVWCTX2, SEQ ID NO: 11 wherein X2 is independently selected from the group consisting of C, Q, P, and E. and / or X1DCAWHLGELVWCTC 、 SEQ ID NO: 12 X1DCAWHLGELVWCTQ, SEQ ID NO: 13 X1DCAWHLGELVWCTP, SEQ ID NO: 14, and X1DCAWHLGELVWCTE, SEQ ID NO: 15 wherein X1 is absent or independently selected from the group consisting of C, D, P, E and K. Further preferred is a chimeric IgG-Fc binding ligand polypeptide according to the present invention, wherein the Fc-III-XC is replaced by an affinity peptide Fc-III-XC selected from the group consisting of:

[0034] Further preferred is a chimeric IgG-Fc binding ligand polypeptide according to the present invention, wherein the IF domain is replaced by the affinity peptide Fc-III-XC having the sequence EDCAWHLGELVWCTE, SEQ ID NO: 16 (TtSlyD-Fc-III-2C_Hit No. 4).

[0035] Further preferred are chimeric IgG-Fc binding ligand polypeptides according to the present invention which further comprise a C-terminal amino acid tag, such as an 8-tag. Proteins with an affinity tag consisting of polyhistidine residues can be bound to metal ions (Ni) immobilized on a matrix. 2+ ) and the 8x-histidine side chains (see Examples).

[0036] The chimeric IgG-Fc binding ligand polypeptides according to the present invention have the further advantage over Protein A / G of exhibiting high affinity binding to a wide range of IgG species, e.g. selected from the group consisting of human, rabbit (see Examples), mouse, rat, porcine, goat, horse and bovine IgG.

[0037] Protein ligand multimerization can result in higher alkaline stability and improved binding capacity of affinity column matrices due to the avidity effect. Thus, a bivalent binder with a molecular weight of 29 kDa was generated by head-to-tail linkage of TtSlyD-Fc-III-4C with a second scaffold protein exhibiting the same Fc-III-4C peptide loop. This dual binder exhibited higher affinity for the Fc portion of IgG in SPR interaction assays compared to a single scaffold (KD = 5 nM for rbIgG at 37°C and for human IgG at 25°C). Therefore, another preferred embodiment of the present invention relates to a bivalent binder molecule comprising two fused chimeric IgG-Fc-binding ligand polypeptides according to the present invention, preferably fused head-to-tail to each other.

[0038] A further aspect of the present invention relates to a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent binder molecule according to the invention coupled to, for example, a solid support, such as beads, for example agarose beads and / or a column matrix, preferably an NHS-activated matrix such as Sepharose resin. Preferably, the coupling is via a lysine side chain of SlyD bearing an NHS ester contained in the Sepharose matrix, such as a coupled ligand or binder molecule according to the invention.

[0039] A further aspect of the present invention relates to a solid support material as described above, such as beads, e.g. agarose beads and / or a column matrix, preferably an NHS-activated matrix such as Sepharose resin, coupled with a chimeric IgG-Fc-binding ligand polypeptide according to the present invention or a bivalent binder molecule according to the present invention. The material has improved properties, such as low leakage and high reusability, as described herein. A further aspect of the present invention relates to an immunoaffinity chromatography column comprising a solid support material according to the present invention, which is preferably also applicable for IgG purification as disclosed herein.

[0040] Yet another aspect of the present invention relates to a method for producing a chimeric IgG-Fc-binding ligand polypeptide according to the present invention, comprising recombinant expression of said ligand polypeptide in a suitable host cell such as E. coli, or comprising chemical synthesis of said ligand polypeptide. Each method is known in the art and also disclosed herein. Preferred methods according to the present invention further comprise coupling the chimeric IgG-Fc-binding ligand polypeptide to a solid support, e.g., a solid matrix material such as beads and / or a column matrix, in particular the matrices mentioned above. The matrix can be packed into a suitable column.

[0041] A further aspect of the present invention relates to a method for isolating or purifying an immunoglobulin, comprising contacting said immunoglobulin with a solid support to which a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent binder molecule according to the invention is coupled, and suitably eluting said immunoglobulin from said chimeric IgG-Fc binding ligand polypeptide or bivalent binder molecule. Preferably, the method according to the invention comprises fast protein liquid chromatography (FPLC). Exemplary methods are disclosed herein and in the affinity chromatography literature, e.g., Elliott L. Rodriguez, et al. (Affinity chromatography: A review of trends and developments over the past 50 years, Journal of Chromatography B, Volume 1157, 2020, https: / / doi.org / 10.1016 / j.jchromb.2020.122332; or Huse, Klaus & Boehme, Hans-Joachim & Scholz, Gerhard. (2002). Purification of antibodies by affinity chromatography. Journal of biochemical and biophysical methods. 51.217-31.10.1016 / S0165-022X(02)00017-9). The method can be performed using the matrix materials disclosed herein and can be scaled for industrial-scale production.

[0042] The method according to the present invention is preferred in that the elution conditions for the immunoglobulin are milder than those of the solid support material containing Protein A coupled to the solid support. The fast association and dissociation rates favorable for antibody capture, particularly in the case of variants, allow antibody desorption under relatively mild pH conditions. For Protein A, a low pH, e.g., acetic acid with a pH value of about 3.3, is used as the standard elution procedure (Guelich, S., M. Uhlen, and S. Hober, "Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography." J. Biotechnol., 2000, 76(2-3):pp. 233-44). Low pH can promote degradation of mAbs and lead to contamination of the final product due to leakage of the column ligand. The preferred elution pH range of the present invention is 3.4 to 4.5.

[0043] The method according to the present invention also preferably includes a chemical regeneration step of the solid support material, using more stringent conditions compared to the solid support material to which Protein A is coupled. Successful regeneration involves removing the bound analyte without dissociating the ligand or limiting its activity. Protein A / G binding to the antibody is relatively strong, and regeneration requires washing, e.g., with a glycine buffer, pH 2.7. The preferred pH regeneration range of the present invention is 2.5-2.0.

[0044] Yet another aspect of the present invention relates to the use of a chimeric IgG-Fc binding ligand polypeptide of the invention or a bivalent binder molecule of the invention for the purification of immunoglobulins or for the screening and selection of peptide binders to a given target molecule.

[0045] The chemically synthesized peptide, called Fc-III-4C, is constrained by two disulfide bridges to favor a hairpin conformation capable of binding to its exact complement of targets. The study was based on a cyclic 13-mer peptide identified by phage display, first described by DeLano and colleagues in 2000 (DeLano, W.L., et al., Convergent solutions to binding at a protein-protein interface. Science, 2000. 287(5456): pp. 1279-83) and further optimized for conformational stability and binding affinity (KD = 2.45 nM for human IgG) by Gong et al. in 2016 (Gong, Y., et al., Development of the double cyclic peptide ligand for antibody purification and protein detection. Bioconjug Chem, 2016. 27(7): pp. 1569-73).

[0046] Compared to the two disulfide bridges required for constrained peptide synthesis, scaffold binders require only one functional cross-link, facilitating inexpensive recombinant production in E. coli with high yields. Compared to Protein A, harsher chemical regeneration conditions are possible on affinity columns, allowing for milder elution conditions for antibodies compared to Protein A.

[0047] In this study, the feasibility and efficiency of a new type of immunoaffinity chromatography column applicable to IgG purification was demonstrated for the first time. Grafting the Ig-Fc affinity peptide Fc-III-4C onto the TtSlyD scaffold protein was shown to be feasible while retaining the specificity and affinity of the peptide.

[0048] The advantage of the single Fc-III-4C affinity peptide over Protein A / G is that it exhibits high affinity for many IgG species (human, rabbit, mouse, rat, porcine, goat, horse, and bovine), and Fc-III-4C immobilized on agarose beads as an affinity ligand for mAb purification exhibits extended reusability compared to standard Protein A beads. Furthermore, the TtSlyD-Fc-III-4C scaffold protein coupled to NHS-Sepharose has excellent chemical robustness as evidenced by repeated protein denaturation and refolding, and is highly resistant to alkaline treatment.

[0049] Affinity chromatography columns containing TtSlyD-Fc-III-4C are an economical and efficient alternative to alkaline-sensitive protein A matrices. Furthermore, the protein scaffold provides high conformational stability and enhanced protease resistance of the displayed peptide loop, resulting in less ligand leakage and less contamination of the final product.

[0050] Furthermore, scaffold proteins can be rapidly and cost-effectively produced in large quantities by recombinant expression in E. coli: for all expressed protein variants, approximately 600 mg of biomass and final protein yields of more than 5 mg per 100 mL of bacterial cell culture were obtained.

[0051] Furthermore, protein misfolding and oligomerization, driven primarily by nonspecific intermolecular disulfide bridge formation, are major problems. It was shown that synthesis could be improved by removing the two terminal cysteine residues that constrain the peptide loop. A variant exhibiting only a single disulfide linkage, TtSlyD-Fc-III-XC, was less prone to aggregation compared to the wild-type scaffold with four cysteines.

[0052] Furthermore, to overcome the problem of acidic elution conditions, the binding site of the TtSlyD-Fc-III ligand could be further engineered: the hotspot residue leucine 7 could be modified to obtain the desired association / dissociation properties.

[0053] In the context of the present invention, the Fc-binding portion of the peptide was further modified by QC-PCR, as described below, with the aim of selectively screening protein variants exhibiting the following characteristics: a fast association rate, advantageous for antibody capture and a rapid dissociation rate, allowing antibody desorption under relatively mild pH conditions. Surface plasmon resonance was used to determine the binding affinity of the resulting TtSlyD-Fc-III-4C variants to IgG. To do so, the scaffold was immobilized on a Biacore chip and allowed to interact with IgG in a flow cell, as described below. The analyte (IgG) was applied at five different concentrations, each monitored for one cycle, and the chip surface was regenerated after each run. To identify the best conditions for complete removal of the analyte after each cycle, regeneration scouting with human IgG was performed before kinetic measurements. Regarding the results obtained in this experiment, it can be concluded that grafting the Fc-III-4C peptide onto the TtSlyD domain does not adversely affect the affinity for IgG-Fc. Furthermore, by exchanging the tryptophan residue at position 11, we were unable to detect any protein variants that exhibited altered kinetic properties, such as faster association or dissociation rates. A possible explanation for this could be that the contribution of tryptophan to the binding energy is lower than initially expected. Future studies could shift the focus to other amino acids that exhibit more significant contributions to the binding energy. For example, leucine at position 7 of the Fc-III-4C peptide represents a typical hot spot at the protein-peptide interface. Furthermore, the following experiments demonstrate that the binding strength can be significantly affected by exchanging each residue.

[0054] The four cysteine residues present in the Fc-III-4C peptide loop form a cyclic structure, determining the rigidity and affinity of the binder. However, cysteines pose a major problem for protein expression and purification. Dias et al. have previously demonstrated that removing disulfide bonds in the Fc-III peptide results in a complete loss of binding affinity (Dias, RL, et al., Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptidomimetics. J Am Chem Soc, 2006, 128(8):2726-32). In the present invention, the structural stability of the affinity peptide is preserved by the TtSlyD backbone, and the extra disulfide bonds may not be necessary. In the following experiments, cysteines were replaced with alternative amino acids to investigate whether these bonds are essential for loop stability and the affinity of the protein for IgG-Fc.

[0055] In the context of the present invention, the feasibility and efficiency of a new type of immunoaffinity chromatography column applicable to IgG purification was demonstrated for the first time. Grafting the Ig-Fc affinity peptide Fc-III-4C onto the TtSlyD scaffold protein was shown to be feasible, while at least retaining the specificity and affinity of the peptide.

[0056] The advantage of the single Fc-III-4C affinity peptide over Protein A / G is that it exhibits high affinity for many IgG species (human, rabbit, mouse, rat, porcine, goat, horse, and bovine). As an affinity ligand for mAb purification, Fc-III-4C immobilized on agarose beads exhibits extended reusability compared to standard Protein A beads. Furthermore, the TtSlyD-Fc-III-4C scaffold protein coupled to NHS-Sepharose exhibits excellent chemical robustness, as evidenced by repeated protein denaturation and refolding, and is highly resistant to alkaline treatment. Therefore, affinity chromatography columns containing TtSlyD-Fc-III-4C are an economical and efficient alternative to the alkaline-sensitive Protein A matrix. Furthermore, the protein scaffold provides high conformational stability and enhanced protease resistance of the displayed peptide loop, resulting in less ligand leakage and less contamination of the final product.

[0057] Furthermore, scaffold proteins can be produced in large quantities rapidly and cost-effectively by recombinant expression in E. coli. For all expressed protein variants, approximately 600 mg of biomass and approximately 5 mg of final protein yield per 100 mL of bacterial cell culture were obtained. While the amount produced was sufficient for the screening experiments performed in this paper, it could be further optimized for yield and solubility for potential large-scale production by, for example, reducing the cultivation temperature or the amount of inducer (Marisch, K., et al., Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production. Microb Cell Facts, 2013.12:58). Furthermore, protein misfolding and oligomerization, primarily driven by nonspecific intermolecular disulfide bridge formation, are major problems. It was shown that synthesis could be improved by removing the two terminal cysteine residues that constrain the peptide loop. The variant displaying only a single disulfide linkage, TtSlyD-Fc-III-XC, was less prone to aggregation compared to the wild-type scaffold with four cysteines.

[0058] Protein-ligand multimerization can result in higher alkaline stability and improved binding capacity of affinity column matrices due to the avidity effect. Thus, a bivalent binder with a molecular weight of 29 kDa was generated by head-to-tail linkage of TtSlyD-Fc-III-4C with a second scaffold protein exhibiting the same Fc-III-4C peptide loop. This dual binder exhibited higher affinity for the Fc portion of IgG in SPR interaction assays compared to the single scaffold (KD = 5 nM for rbIgG at 37 °C and human IgG at 25 °C). However, the production and purification of this dual binder is more complicated due to protein aggregation and conjugation.

[0059] Furthermore, to overcome the problem of acidic elution conditions, the binding site of the TtSlyD-Fc-III ligand could be further engineered: the hotspot residue leucine 7 could be further modified to obtain the desired association / dissociation properties. The present invention is further described in the following examples with reference to the accompanying drawings, but is not limited thereto. For purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0060] [Figure 1] FIG. 1 shows the general workflow of the present invention. [Figure 2] Figure 2 shows an example of the coupling process of the binders of the present invention to a matrix. The column used consists of an NHS ester attached to Sepharose HP via a 6-atom spacer arm. The activated ester reacts rapidly with ligands containing primary amino groups, resulting in a highly stable amide linkage. AB = agarose beads. [Example]

[0061] As a general overview, in the context of the present invention, a prokaryotic TtSlyD-Fc-III-4 C expression construct was designed, and a TtSlyD-Fc-III-4 C scaffold molecule and chromatography column were generated. IgG purification experiments were then performed. Subsequently, the Fc-III-4 C binding site was matured, and the loop-flanking cysteines were replaced. After this, kinetic screening of engineered protein variants was performed. Suitable variants were selected.

[0062] Molecular cloning of E. coli expression constructs Cloning of the TtSlyD-Fc-III-4C scaffold affinity peptide chimera was performed in two steps. First, the genes encoding the TtSlyD protein backbone and 8x-histidine tag were inserted into a prokaryotic expression vector using the restriction endonucleases EcoRI-HF® and HindIII-HF®. Subsequently, sequences encoding the appropriate affinity peptides were integrated into the respective insertion sites of the TtSlyD scaffold via BsiWI-HF® and BamHI-HF®. For molecular grafting and generation of the TtSlyD-Fc-III-XC expression construct, double-stranded linear DNA fragments (so-called DNA strings) containing the desired mutations at the appropriate sites in the TtSlyD sequence were designed and directly cloned into the prokaryotic expression vector pQE80-Kan.

[0063] The expression vectors pQE80-Kan-TtSlyD-Fc-III-4C and pQE80-Kan-TtSlyD-Fc-III-XC used in the present invention are derived from the pQE80-Kan vector and contain the following features: KanR: antibiotic resistance to kanamycin; ColE1: replication origin; lacIq: lac repressor; PT5: T5 promoter (derived from Escherichia coli phage T5); MCS: multiple cloning site with restriction sites (i.e., for EcoRI and HindIII).

[0064] The expression system used in this invention relies on the inducible T5-lac system

[65] . In the absence of lactose, the lac repressor protein lacIQ encoded by the expression vector prevents bacterial RNA polymerase (RNAP) from binding to the promoter of the lac operon. Isopropyl-beta-D-thiogalactoside (IPTG), a structurally non-metabolizable analog of allolactose that binds to and inactivates laqIQ, allows RNAP to transcribe sequences downstream of the T5 promoter (PT5). The resulting transcripts can then be translated into recombinant proteins.

[0065] Quick Change PCR (QC-PCR) The Trp11 exchange was performed on the expression construct pQE80-Kan-TtSlyDFc-III-4C via QuickChange polymerase chain reaction (QCPCR, as described by Braman et al. (Braman, GPCGA, Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol, 1996, 57:31-44)) according to the instructions in the QuikChange™ Site-Directed Mutagenesis Kit (Agilent). Therefore, for each exchange, two mutagenic primers were designed complementary to the target plasmid site. Both the forward and reverse primers contained the desired mutation and annealed to the same position on opposite strands of the plasmid. During QC-PCR, the mutagenic primers were extended using non-strand-displacement clone Pfu DNA polymerase (Agilent), resulting in nicked circular strands. The non-mutated parent DNA template, recognized by its methylation site, was digested by adding the restriction enzyme DpnI (NEB) to the final PCR reaction mixture. The nicked dsDNA was transformed into E. coli cells, and the nicks were repaired during replication. Primers were designed according to the instructions of the Agilent kit, with lengths of 25–45 bases (37 bases for the forward primer and 41 bases for the reverse primer) and a melting temperature (Tm) of approximately 78°C. The desired mutation had to be in the center of the primer, with approximately 10–15 bases of the correct sequence on either side. All primers were synthesized by Metabion, Planegg-Steinkirchen.

[0066] The QC-PCR reaction mixture was combined in a 0.2 mL reaction tube on ice. Cloned Pfu DNA polymerase was added immediately before starting the PCR amplification. Thermal cycling conditions were adjusted to the length of the DNA template and the type of mutation desired. A 10-minute extension time and 16 PCR cycles were selected for a single amino acid exchange in a 4.8 kb plasmid. A negative control without template DNA was prepared for each reaction. After temperature cycling, 2 μL of DpnI was added directly to each amplification reaction to digest the parental plasmid DNA. The samples were incubated at 37°C for 1 hour, followed by a 20-minute restriction enzyme heat inactivation step at 80°C. The PCR samples were then analyzed on a 1% (w / v) agarose gel.

[0067] The desired bands were excised, and the plasmid DNA was purified and transformed into NEB® Express competent E. coli (high efficiency) cells as described. One single colony for each construct was picked and DNA was isolated. Sequence analysis was performed to verify the inserted mutations.

[0068] Small-scale protein test expression Small-scale test expression experiments of various TtSlyD-Fc-III-4C proteins were performed to evaluate the amount of protein produced and the solubility of various variants. Expression (NEB® Express Competent E. coli (High Efficiency), a BL21 derivative) was performed in a 96-well deep-well block (DWB). An overnight starter culture was prepared by inoculating 1.2 mL of LB medium (50 μg / mL kanamycin) from a glycerol stock. The plate was incubated at 37°C and 750 rpm on an orbital shaker for 16 hours. The next day, expression cultures (1.2 mL of fresh Super Broth (SB) medium) were inoculated with 50 μL of the preculture. Upon reaching exponential growth phase (OD600 approximately 1-1.2), expression was induced by adding IPTG to a final concentration of 0.5 mM. After 5 hours of incubation at 37°C, cells were harvested by centrifugation (4000 rcf, 20 minutes). Before inoculation and before cell harvest, samples were taken for subsequent SDS-PAGE analysis (before induction, PreI, and after induction, PostI, respectively). The optical density of the bacterial culture was determined, and the sample volume was adjusted. The cell suspension was centrifuged (8000 rcf, 3 min), and the cells were resuspended in 32.5 μL of 10 mM Tris pH 8.0, 4% SDS, 2% 2-mercaptoethanol to obtain a crude cell extract suitable for SDS-PAGE.

[0069] Mechanical cell disruption The cell pellet was resuspended in 800 μL of bead suspension buffer and transferred to a 2 mL tube filled with 150 mg of 0.1 mm–0.2 mm glass beads. This solution was mixed with 50 μL of a 1:1000 dilution of Antifoam 204 (Sigma-Aldrich), and the cells were mechanically disrupted using a Bead Ruptor 24 (Omni International) according to the following program: To separate the soluble from the insoluble fraction, 100 μL of bacterial lysate was transferred to a 2 mL tube and centrifuged (8000 rcf, 3 min). The insoluble fraction was resuspended in 100 μL of bead suspension buffer before applying it to SDS-PAGE. Protein expression levels and solubility were assessed by SDS-PAGE.

[0070] Intermediate-scale protein expression To obtain sufficient biomass for subsequent protein purification, medium-scale expression cultures of the different TtSlyD scaffold variants were prepared. A 5 mL overnight culture (inoculated from a glycerol stock) of each variant was used to inoculate a 250 mL culture of SB medium containing 50 μg / mL kanamycin. The culture was incubated at 37 °C and 250 rpm in a shaking incubator until an OD600 of 1-1.2 was reached. Expression was induced by adding IPTG to a final concentration of 0.5 mM. After an additional 5 h of culture, cells were harvested by centrifugation (6000 rcf, 20 min). A 2 mL sample of the final cell suspension was taken, and the cells were mechanically disrupted to obtain samples for subsequent SDS-PAGE analysis.

[0071] Chemical cell disruption The cell pellet was lysed by adding 2 mL of B-PER II™ Bacterial Protein Extraction Reagent (Thermo Fisher Scientific) per mg of cells. To prevent proteolysis and reduce lysate viscosity caused by unwanted DNA, the protease inhibitors phenylmethylsulfonyl fluoride (PMSF, 3 μL 0.1 M / mL B-PER II™, Thermo Fisher Scientific) and DNase I (Roche, one spatula tip per mL of B-PER II™, Roche) were added. The suspension was incubated on wet ice for 30 min and filled to a final volume of 20 mL with 20 mM Tris, 150 mM NaCl, pH 7.5 (Buffer A). The lysate was centrifuged at 5000 rcf for 15 min to separate the soluble from the insoluble fraction. The insoluble fraction was resuspended in 20 mL of Buffer A and then sampled for SDS-PAGE. The supernatant containing the soluble proteins was then analyzed by Ni 2+ Further purification was achieved by affinity and size exclusion chromatography.

[0072] Protein purification Immobilized Metal Ion Affinity Chromatography (IMAC) The scaffold proteins were first purified by IMAC, a method first formulated by Porath et al. in 1975 (Porath, J., et al., Metal chelate affinity chromatography, a new approach to protein fractionation. Nature, 1975. 258(5536): pp. 598-9). After cell lysis and centrifugation, the supernatant containing the soluble protein fraction was sterile filtered (0.2 μm) and applied directly to a nickel-loaded nitrotriacetic acid (Ni-NTA) gravity-flow column (Pierce Gravity Flow Column and filter unit packed with Qiagen Superflow Ni-NTA agarose resin; column volume (CV) approximately 1 mL). Proteins bearing affinity tags consisting of polyhistidine residues were bound to metal ions (Ni) immobilized on a matrix. 2+ The protein is captured due to the interaction between the 8x-histidine side chains and the 8x-histidine side chains. The buffers and solutions used for purification are as follows:

[0073] Buffers and solutions used for gravity-flow purification. All buffers and solutions were sterile filtered (0.2 μm) and degassed before use. pH was adjusted with either HCl or NaOH.

[0074] Buffer / solution composition Buffer A (sample buffer) 20mM Tris, 150mM NaCl, pH7.5 Buffer B1 (washing buffer) 20mM Tris, 150mM NaCl, 10mM imidazole, pH 7.5 Buffer B2 (washing buffer) 20mM Tris, 150mM NaCl, 50mM imidazole, pH 7.5 Buffer C (elution buffer) 20mM Tris, 150mM NaCl, 250mM imidazole, pH 7.5 Buffer D (regeneration buffer) 20mM Tris, 150mM NaCl, 500mM imidazole, pH 7.5 Stripping solution: 50mM EDTA, 1% SDS, pH 7.5 Refilling solution 100mM NiSO4 Storage solution 20% ethanol

[0075] Before use, the column was filtered and rinsed with degassed ddH2O. Equilibration was performed with 20 CV of Buffer A, followed by application of the lysate supernatant. Nonspecific and unbound proteins were removed by washing the column with 20 CV of Buffer B1 and 20 CV of Buffer B2. Protein was eluted with 11 CV of Buffer C in 1 mL fractions. The column was regenerated by rinsing with 10 CV of Buffer D. 1 mL samples of the column flow-through after sample loading (flow-through, FT), Buffer B1 (washout, WI), and Buffer B2 (washout II, WII) were collected, respectively. After three purification cycles, the column was stripped and reloaded as follows: first, 20 CV of ddH2O was added, followed by 5 CV of a stripping solution containing the chelator ethylenediaminetetraacetic acid (EDTA) to remove nickel ions. The column was then reloaded with 20 CV of ddH2O, followed by 3 CV of 100 mM NiSO4. The column was prepared for storage by applying 20 CV of ddH2O followed by 20 CV of storage solution.

[0076] Size Exclusion Chromatography (SEC) After Ni-NTA chromatography, the protein solution was further purified by size-exclusion chromatography. This technique allows separation according to molecular size and was applied in this study to remove oligomers and aggregates of the target protein as well as low-molecular-weight components. The system output is shown in a chromatogram, displaying the absorbance intensity, expressed as absorbance units (AU), over the retention volume or retention time (the volume / time required for the protein to elute after injection). The signal intensity is proportional to the concentration of the eluted analyte. In an optimal separation, multiple peaks or shifts on the chromatogram correspond to different components of the separated sample and can be directly assigned to molecular size by comparison with protein standards of known composition. The "Peak Integration" function in the UNICORN 6.3 control software was used to identify and measure several curve characteristics, including peak area, retention time, and peak width. The required baseline was automatically calculated.

[0077] Prior to application, the protein sample was concentrated to a final volume of approximately 5 mL and manually loaded onto a GE Healthcare HiLoad 60 / 600 Superdex 75 pg column via a capillary loop (5 mL) using an AEKTA™ Avant chromatography system. Before use, the storage solution was removed with 1.5 CV of ddH2O. The column was equilibrated, and the protein was eluted with 1.5 CV of running buffer in 2 mL fractions in a 96-well DWB at a flow rate of 1 mL / min. The run was operated at room temperature (RT) and monitored at a wavelength of 280 nm. The column was then re-equilibrated with ddH2O and storage solution. Protein purity was confirmed by SDS-PAGE, and the molecular weight (MW) was determined based on a calibration curve provided by GE Healthcare. The desired peak fractions were pooled and concentrated. Appropriate aliquots were flash-frozen in liquid nitrogen and stored at -80°C.

[0078] Buffers and solutions used for size exclusion chromatography. All buffers and solutions were sterile filtered (0.2 μm) and degassed before use. pH was adjusted using HCl.

[0079] Buffer / solution composition Running buffer: 20 mM Tris, 150 mM NaCl, pH 7.5 Storage solution 20% ethanol

[0080] Buffer exchange and protein concentration Prior to SEC, the protein sample eluted from the Ni-NTA gravity-flow column was buffer-exchanged and concentrated to remove residual imidazole present in the elution buffer. Buffer exchange was achieved by dialysis. The protein solution was transferred to a Spectra / Por® 3 dialysis membrane (Spectrum Laboratories) with a molecular weight cutoff (MWCO) of 3500 Da and incubated overnight at 4°C in 5 L of running buffer under continuous stirring. The dialyzed protein was applied to a Vivaspin® 6 centrifugal concentration column, 5000 Da MWCO (Sartorius), and centrifuged to obtain a final volume of approximately 5 mL. After SEC, the protein pool was concentrated using a Vivaspin® 20 centrifugal concentration column, 5000 Da MWCO (Sartorius).

[0081] Preparation of immunoaffinity chromatography columns Within the scope of this invention, we developed a novel immunoaffinity column for antibody purification: the aforementioned TtSlyD-Fc-III-4C scaffold affinity peptide chimera served as a capture molecule / ligand for IgG and was permanently coupled to the chromatography column matrix.

[0082] Briefly, for immunoaffinity chromatography, a crude solution containing the desired antibody (e.g., cell culture supernatant) is applied to a column. The antibody is captured by an affinity ligand contained in the column matrix, contaminants are removed by extensive washing, and the immunoglobulin is eluted by adding an appropriate buffer.

[0083] Immobilization of Fc-specific ligands was performed by covalently coupling primary amino groups to N-hydroxysuccinimide (NHS)-activated highly cross-linked agarose beads contained in a HiTrap NHS-activated HP column (1 mL, GE Healthcare). The coupling procedure was performed according to the manufacturer's protocol. The required buffers are listed below. The ligands were dissolved in the standard coupling buffer and concentrated to 1 mL to a final concentration of 0.5–10 mg / mL. The column was washed with ice-cold 1 mM HCl to remove isopropanol present in the manufacturer's storage buffer. 1 mL of the ligand solution was manually injected, and the column was incubated at 25°C for 30 min. The column was then chromatographed. The column was connected to a flow system (AEKTA™ Avant, GE Healthcare), and the ligand solution was washed with 3 CV of standard coupling buffer, and the column flow was collected. To inactivate excess activated groups that were not coupled to the ligand and to wash away nonspecifically bound ligand, 6 CV of buffer A, 6 CV of buffer B, and another 6 CV of buffer A were injected. The washout was collected, and the column was incubated at room temperature for 30 minutes. Then, 6 CV of buffer B, 6 CV of buffer A, and 6 CV of buffer B were injected, and the washout was similarly collected. Finally, the pH was adjusted by applying 10 CV of binding buffer. If the column was not used immediately, it was rinsed with 5 CV of storage buffer and stored at 8 °C.

[0084] Buffers used for preparation of immunoaffinity chromatography columns. a All buffers were sterile filtered (0.2 μm) and degassed before use. pH was adjusted with either HCl or NaOH.

[0085] Buffer composition Standard coupling buffer 0.2M NaHCO3, 0.5M NaCl, pH8.3 Buffer A 0.5 M ethanolamine-HCl, 0.5 M NaCl, pH 8.3 Buffer B 0.1M NaOAc, 0.5M NaCl, pH4 Storage buffer 0.05 M Na2HPO4, 0.1% NaN3, pH7 Binding buffer: 20 mM Tris, 150 mM NaCl, pH 7.5. The binding buffer corresponds to the SEC running buffer. Purified scaffold proteins were dissolved in this buffer for long-term storage.

[0086] To assess the maximum coupling capacity of the column matrix, increasing ligand concentrations from 1.5 to 3 mg / mL were applied. The flow-through containing excess ligand was analyzed by SDS-PAGE, and the coupling efficiency was evaluated as follows: a PD-10 desalting column (GE Healthcare) was equilibrated with 25 mL of 0.1 M NaH2PO4, 150 mM NaCl, pH 7 (equilibration buffer 8), followed by a 0.5 mL washout. Elution was performed in a two-step procedure with the equilibration buffer by first adding 2 mL to remove salts and other low-molecular-weight components, followed by adding 1.5 mL to elute the desired high-molecular-weight protein. The absorbance of the eluted fractions was measured using a NanoDrop™ OneC microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific). The coupling efficiency was calculated. See Figure 2.

[0087] Evaluation of column parameters A chromatographic TtSlyD-Fc-III-4C immunoaffinity column was generated and matched to desired parameters such as affinity, chemical stability, and reusability using commercially available high-purity rabbit immunoglobulin G (rbIg) supplied by Sigma-Aldrich. The affinity column was evaluated using a HPLC-MS / ...

[0088] The buffers and solutions used for rbIgG purification are as follows: All buffers and solutions were sterile filtered (0.2 μm) and degassed before use. pH was adjusted with either HCl or NaOH.

[0089] Buffer / Solution - Composition Equilibration buffer 50mM Tris-HCl, 0.05% Tween®20, pH 7.8 Wash buffer 50mM Tris-HCl, pH6.0 Elution buffer NH4Ac-AcOH, pH3.4 Regeneration buffer NH4Ac-AcOH, pH2.2 CIP solution 100mM NaOH Storage buffer 0.05M Na2HPO4, 0.1%NaN3, pH7 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0090] Protein fractions were analyzed by SDS-PAGE under both reducing and non-reducing conditions. Samples were mixed with the appropriate amount of NuPAGE® LDS sample buffer (4x) (Thermo Fisher Scientific) and NuPAGE® reducing agent (10x) (Thermo Fisher Scientific) (for reducing gels) or water (for non-reducing gels) to obtain a total volume of 50 μL and incubated at 95°C for 10 minutes in a thermocycler. Gels were run on NuPAGE® Bis-Tris 4-12% gels (Thermo Fisher Scientific) using 15 μL / 10 μg sample per lane along with 5 μL of Novex™ Sharp prestained protein ladder (Thermo Fisher Scientific) to estimate the molecular weight of protein bands. Electrophoresis was performed at a constant voltage of 200 V. Gels were stained with Instant Blue™ protein stain (Expedeon) and destained overnight in ddH2O. Images were taken with a ChemiDoc MP™ instrument (Bio-Rad).

[0091] Spectrophotometric determination of protein concentration The concentration of purified protein after SEC was determined spectrophotometrically using a NanoDrop™ OneC microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific). The appropriate dilution buffer was used as a blank. Measurements were performed with a common reference setting based on a 0.1% (1 mg / mL) protein solution (path length 1 cm) producing an absorbance of 1.0 A at 280 nm. Protein concentration was then calculated according to the Beer-Lambert law, taking into account the specific protein absorbance at 280 nm. The corresponding absorbance units (AU) were calculated using Vector NTI (Thermo Fisher Scientific) software.

[0092] Surface Plasmon Resonance (SPR) The affinity and binding kinetics of the generated TtSlyD-Fc-III-4C mutants were evaluated by SPR using a Biacore biosensor system. The Biacore system allows real-time monitoring of molecular interactions between a ligand immobilized on the gold surface of a sensor chip and an analyte free in solution passing over the ligand (Healthcare, G., Biacore™ Assay Handbook 29-0194-00 Edition AA). The interaction of the binding partners results in an SPR signal (response), measured in resonance units (RU), and shown as a plot (sensorgram) against time.

[0093] To complete the procedure, an analyte is injected onto the chip and interacts with the immobilized ligand. Changes in the concentration of molecules at the chip surface result in a change in refractive index, which is monitored as response units (RU). Analyte dissociation is induced by continuous buffer flow, followed by complete removal (chip regeneration) and the initiation of a new analytical cycle. The resulting SPR data were fitted to a mathematical model to derive kinetic parameters, such as the association rate constant ka (M-1s-1) and the dissociation rate constant kd (s-1).

[0094] Interaction kinetics is investigated by monitoring different analyte concentrations over time. Kinetic parameters, such as the association (k) and dissociation rate constants (kd), are evaluated in conjunction with mathematical models. One model applied was the "Langmuir interaction model" (O'Shannessy, DJ, et al., "Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods." Anal Biochem, 1993, 212(2):457-68), which assumes a 1:1 interaction in which one ligand molecule interacts with one analyte molecule. The "bivalent analyte binding" kinetic model assumes a bivalent analyte in which one analyte molecule can bind to one or two ligand molecules. The kinetic analysis experiments performed within this invention were performed as multicycle kinetics, testing each analyte concentration in a single cycle and regenerating the chip surface after each cycle. Successful regeneration means removal of bound analyte without dissociating the ligand or limiting its activity. Therefore, regeneration scouting was performed before kinetic measurements.

[0095] Regeneration Scouting Regeneration scouting was performed using a Biacore 3000 system and a CM5 chip provided by GE Healthcare. The chip displays carboxymethylated dextran covalently attached to a gold surface. Ligands were immobilized on the chip surface via amine coupling, attempting to achieve a ligand density of approximately 3000 RU. Analyte (human IgG, 300 nM) was diluted in sample buffer. Regeneration was tested according to the following protocol: In the first cycle, 300 nM human IgG was injected. Bound antibody was displaced by the addition of system buffer and two 1-minute pulses of 10 mM glycine, pH 2. This cycle was repeated six times, followed by two additional cycles using 10 mM glycine, pH 1.75 and pH 1.5, respectively. Driving program for regeneration scouting [Table 1]

[0096] Kinetic screening Kinetic measurements were performed using a Biacore 3000 and Biacore 8K system (GE Healthcare) equipped with a C1 chip. The chip assembly was similar to the C5 chip, except that it lacked dextran threads. The scaffold was immobilized by amine coupling; that is, the protein was covalently linked to carboxymethyl groups present on the chip surface. To saturate the remaining free binding sites on the chip surface (conditioning), the chip surface was flushed with 270 nM of analyte before measurement and regenerated with glycine buffer (pH 1.75). Briefly, the scaffold protein was immobilized on a Biacore C1 chip via amine coupling. IgG was injected at different concentrations, and the chip was regenerated after each cycle.

[0097] Ligand immobilization The desired ligand was diluted in sample buffer to a final concentration of 5 μg / μL. The chip surface was purged with two 1-min injections of wash buffer and finally primed with system buffer. The reactive groups were activated by applying 40 μL of NHS / EDC (50% mix) at a flow rate of 20 μL / min. The ligand solution was injected at a flow rate of 50 μL / min to achieve an RU response suitable for subsequent performance (an RU value of approximately 100 was not exceeded to avoid steric crowding effects at high analyte concentrations). Excess NHS-activated esters were quenched by exposing the chip to 100 μL of 1 M ethanolamine hydrochloride (EA-HCl) solution (pH 8.5) at a flow rate of 20 μL / min.

[0098] Kinetic screening Analytes were serially diluted with sample buffer to achieve the desired concentrations. IgG (human IgG or rabbit IgG, Sigma-Aldrich) at the indicated concentrations was injected over the chip at 60 μL / min or 40 μL / min and 25°C or 37°C, respectively. At the end of each analysis cycle, the chip surface was regenerated with two 1-min pulses of 10 mM glycine pH 1.75.

[0099] result In the present invention, peptide grafting was achieved by creating a DNA vector construct encoding the FKBP domain of the TtSlyD protein together with the Fc-III-4C peptide. The appropriate sequence was inserted into the prokaryotic expression vector pQE80-Kan, enabling DNA amplification and expression in E. coli. An IgG affinity column was constructed by coupling recombinantly produced TtSlyD-Fc-III-4C to a commercially available NHS-Sepharose matrix. Proof-of-principle purification experiments were performed using a highly purified IgG solution. The chemical stability and reusability of the column matrix were further evaluated by exposure to extreme alkaline conditions and multiple repeated column runs. To facilitate milder elution conditions, maturation of the Fc-binding moiety was achieved by quick change PCR (QC-PCR). Tryptophan 11, which plays a key role in the interaction of the peptide with the Fc portion of IgG, was replaced with 12 amino acids exhibiting different biochemical properties. The expression levels and solubility of different variants were preliminarily tested in a 96-well format.

[0100] Selected variants were expressed in a midscale format (250 mL), and the TtSlyD-FcIII-4C scaffold was purified by immobilized metal ion affinity chromatography (IMAC) and size-exclusion chromatography (SEC). To simplify the expression and purification of potential column ligands, a chemically synthesized Fc-III-4C peptide library lacking the bridging cysteine was generated and screened for high-affinity variants on HT-NimbleGen microarrays. The 10 best binders were identified, grafted onto the TtSlyD scaffold, expressed, and purified. The interaction of the generated protein variants with IgG was assessed by kinetic screening using surface plasmon resonance (SPR) technology.

[0101] Grafting of the Fc-III-4C peptide into the FKBP domain of the Thermus thermophilus SlyD protein was achieved by molecular cloning. Sequence analysis revealed the correct sequence of the final pQE80Kan-TtSlyD-Fc-III-4C expression construct.

[0102] The TtSlyD scaffold carrying the original, unmodified Fc-III-4C insert was expressed at a 250 mL scale and subsequently purified by immobilized metal ion affinity chromatography (IMAC) and preparative size-exclusion chromatography (SEC). In a proof-of-principle experiment, the protein was coupled to a column matrix to evaluate its potential as an affinity ligand for IgG-FPLC purification.

[0103] Ligand-matrix coupling was achieved by covalently coupling primary amine groups present in the scaffold protein to NHS-activated, highly cross-linked agarose beads contained in a HiTrap NHS-activated HP column (1 mL, GE Healthcare). Primary amine groups are found at lysine residues and the N-terminus of proteins. Lysine residues are generally exposed and therefore predisposed as conjugation points. The TtSlyD FKBP domain contains five lysine residues that can potentially react with NHS ester groups. A coupling efficiency of 93% was achieved.

[0104] Attaching an IgG-Fc affinity scaffold to a column via random amine coupling results in a heterogeneous matrix composition. The affinity peptide loop does not necessarily face the analyte solution. Therefore, the binding capacity of the final column may be affected or even impaired. A possible countermeasure would be thiol-directed immobilization by introducing a single cysteine residue into the C-terminal portion of the scaffold protein followed by coupling to a thiol-containing solid matrix (Ljungquist, C., et al., Thiol-directed immobilization of recombinant IgG-binding receptors. Eur J Biochem, 1989. 186(3): pp. 557-61). Furthermore, the potential impact of ligand density must be considered.

[0105] In a proof-of-principle experiment, one of the resulting NHS-Sepharose-TtSlyD-Fc-III-4C columns was tested for its IgG binding capacity. Rabbit IgG purification using the NHS-Sepharose-TtSlyD-Fc-III-4C column showed no ligand leakage. The results are in line with findings from Gong et al. (Gong, Y., et al., Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection. Bioconjug Chem, 2016, 27(7):1569-73), which showed comparable rabbit IgG enrichment efficiency for Fc-III-4C agarose beads to Protein A beads at selected pH levels, successfully demonstrating the competitive IgG binding capacity of the scaffold-affinity chimera compared to commercially available affinity matrices.

[0106] To further evaluate the chemical stability of the ligand and the reusability of the column matrix, the purification process was automated and the protocol was scaled down to perform several cycles consecutively. The yield of eluted antibody was nearly identical for all runs, as confirmed by spectrophotometric measurements of protein concentration and monitored peak areas. Even after 30 cycles, reproducible chromatograms could be recorded without significant peak shifts or broadening.

[0107] To test the improvement of IgG elution conditions by engineering the Fc-III-4C binding site, the Fc binding moiety was altered by QC-PCR with the intention of selectively screening protein variants that exhibited the following characteristics: fast association and dissociation rates, which are advantageous in terms of antibody capture, allowing antibody desorption under relatively mild pH conditions.

[0108] Tryptophan, one of the main drivers of binding and the strongest interaction between Fc-III-4C and the IgG-Fc portion, was replaced with one of the 12 amino acids: glycine, serine, alanine, arginine, lysine, glutamic acid, lysine, threonine, asparagine, glutamine, tyrosine, or histidine. The introduction of histidine residues has previously been successfully applied to engineer affinity ligands by reducing binding strength (Watanabe, H., et al., Optimizing pH response of affinity between protein G and IgG Fc: how electrostatic modulations affect protein-protein interactions. J. Biol. Chem., 2009, 284(18):12373-83). This facilitates antibody dissociation under relatively mildly acidic conditions due to electrostatic repulsion between the ligand and antibody. Sequence analysis confirmed the successful insertion of all desired mutations.

[0109] For further purification attempts, expression of all protein variants was performed in a 250 mL format as described above. After cell harvesting, the cell pellet was chemically disrupted, centrifuged, and sterile filtered, and the soluble fraction was applied to a Ni-NTA gravity-flow column. The desired protein was eluted by dissolving the polyhistidine tag present in the scaffold protein bound to the nickel column with a fixed concentration of 250 mM imidazole. Monomeric fractions were pooled and used for further analysis. The ratio of the resulting protein monomer to the amount of protein initially loaded ranged from 12% (W-to-A mutation) to 97% (wild type). The results indicate that tryptophan at position 11 is important for the stability of the monomeric protein. The purpose of alternating the Fc binding sites was to generate protein variants characterized by high enough affinity and simultaneously exhibiting fast association / dissociation to enable protein elution under mild pH conditions for the subsequent IgG purification process. Nevertheless, all variants tested demonstrated similar ranges of affinity and association / dissociation rates.

[0110] In additional experiments, the cysteines were replaced with alternative amino acids to determine whether these linkages were essential for loop stability and the affinity of the protein for IgG-Fc.

[0111] The cysteine residues of the Fc-III (DCAWHLGELVWCT, SEQ ID NO: 17) and Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1) peptides were randomized, and the resulting cyclic peptide libraries were tested for binding to mAbs on high-throughput NimbleGen microarrays. For the Fc-III peptide, only one high-affinity variant, the wild-type peptide, could be identified.

[0112] For Fc-III-4C, we identified 299 variants that exceeded the IgG-Fc affinity of the wild-type peptide. However, among all these high-affinity variants, only sequences in which both internal cysteines remained intact were found. All variants lacking the internal cysteines belonged to the low-affinity variants, indicating that the internal cysteines are absolutely essential. In contrast, the terminal disulfide bridge is not required to maintain the peptide's affinity for IgG and can even improve binding strength in some cases.

[0113] We were able to identify several positions where aa exchanges resulted in altered potency and binding affinity. The most notable position was the leucine residue at position 7 in Fc-III-4C. Each replacement with glutamine significantly improved affinity. Ten high-affinity variants were grafted onto the TtSlyD FKBP domain and further investigated for purification profile and IgG-Fc affinity. The variant lacking the extraneous cysteine is designated Fc-III-XC. Only the first 50 variants exhibiting the highest affinity for anti-CD44 antibodies were analyzed. At the 3'-terminus, a clear preference for the acidic amino acids aspartic acid and glutamic acid and the polar amino acid asparagine was observed. However, the most abundant cysteine substitution at the C-terminus of the peptides was proline. Proline has a lower conformational freedom and may therefore stabilize the loop structure, resulting in higher affinity. 5'-substitutions occurred rather randomly, with no obvious trend. In most cases, the cysteines were simply deleted or replaced by the same amino acids as above. Sequencing data revealed the correct sequences for all desired constructs.

[0114] As expected, the TtSlyD-Fc-III-XC mutant is less prone to aggregation compared to variants exhibiting four cysteine residues. The molecular interactions and affinities of the cysteine-deficient protein variants were tested at different temperatures (25°C and 37°C) for both rabbit IgG and human IgG.

[0115] KD values ranging from 8 nM to 43 nM were obtained in interaction analysis with hIgG. For rbIgG, KD values ranged from 15 to 52 nM. According to Gong et al., the KD values of chemically synthesized Fc-III-4C peptide were 2.45 nM for human IgG and 5.67 nM for rabbit IgG, demonstrating higher Fc affinity than natural Protein A binders. TtSlyD-Fc-III-2C_Hit No. 4 was identified as the most promising candidate for IgG purification by FPLC. It exhibited a fast association rate (t / 2diss = 36 min) and a relatively high binding affinity (K A =1.09×10 8 1 / M) (measurement of hIgG at 25° C.) The covalent attachment of an affinity protein to a matrix, such as Sepharose resin, will reveal its full potential as a chromatographic ligand.

Claims

1. A chimeric IgG-Fc binding ligand polypeptide comprising a protein fragment of SlyD comprising an FKBP domain and an IF domain, wherein the IF domain has been replaced with the affinity peptide Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1) or its Fc-III-XC variant (X1DCAWHLGELVWCTX2, SEQ ID NO: 3), wherein X1 is absent or independently selected from the group of C, D, P, E and K, and X2 is independently selected from the group of C, Q, P and E, excluding the case where X1 and X2 are both C.

2. 2. The chimeric IgG-Fc binding ligand polypeptide of claim 1, wherein said SlyD is derived from a Thermus species, including Thermus thermophilus.

3. array MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRLIPGLEEALEGREEGEAFQAHVPAEKAYCDCAWHLGELVWCTCGKDLDFQVEVVKVREATPEELLHGHA (SEQ ID NO: 5) The chimeric IgG-Fc binding ligand polypeptide of claim 1, comprising:

4. The chimeric IgG-Fc binding ligand polypeptide of claim 1, further comprising a C-terminal amino acid tag comprising a His8 tag.

5. 2. The chimeric IgG-Fc binding ligand polypeptide of claim 1, wherein the polypeptide exhibits binding to an IgG species selected from the group consisting of human, rabbit, mouse, rat, pig, goat, horse, and cow.

6. A bivalent binder molecule comprising two fused chimeric IgG-Fc binding ligand polypeptides according to claim 1.

7. A bivalent binder molecule as described in Claim 6, wherein the fusion of two chimeric IgG-Fc-binding ligand polypeptides is achieved by binding the C-terminus of one chimeric IgG-Fc-binding ligand polypeptide to the N-terminus of the other chimeric IgG-Fc-binding ligand polypeptide.

8. 10. The chimeric IgG-Fc binding ligand polypeptide of claim 1 or the bivalent binder molecule of claim 6 coupled to a solid support comprising a solid matrix material including beads and / or column matrices.

9. 9. The coupled chimeric IgG-Fc binding ligand polypeptide or bivalent binder molecule of claim 8, wherein the coupling is via a lysine side chain of the SlyD with an NHS ester contained in a Sepharose matrix.

10. 6. A method for producing a chimeric IgG-Fc binding ligand polypeptide according to any one of claims 1 to 5, comprising recombinant expression of said ligand polypeptide in a suitable host cell, including E. coli, or comprising chemical synthesis of said ligand polypeptide.

11. 11. The method of claim 10, further comprising the step of coupling the chimeric IgG-Fc binding ligand polypeptide to a solid support comprising a solid matrix material including beads and / or a column matrix.

12. 10. A method for purifying an immunoglobulin, the method comprising contacting said immunoglobulin with a solid support to which has been coupled a chimeric IgG-Fc binding ligand polypeptide according to any one of claims 1 to 5 or a bivalent binder molecule according to claim 6, and suitably eluting said immunoglobulin from said chimeric IgG-Fc binding ligand polypeptide or said bivalent binder molecule.

13. 13. The method of claim 12, comprising fast protein liquid chromatography (FPLC).

14. 13. The method of claim 12, wherein the elution conditions for the immunoglobulins are a higher pH than when using a solid support material comprising Protein A coupled to the solid support material.

15. 13. The method of claim 12, comprising a chemical regeneration step of the solid support material, the chemical regeneration step using lower pH conditions compared to the conditions in the chemical regeneration step of a solid support material comprising Protein A coupled to the solid support material.

16. Use of a chimeric IgG-Fc binding ligand polypeptide according to any one of claims 1 to 5 or a bivalent binder molecule according to claim 6 for the purification of immunoglobulins or for the screening and selection of antibodies against a predetermined target molecule.

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