Designed ankyrin repeat domains with improved stability

Abstract

The present invention relates to designed ankyrin repeat domains with binding specificity for serum albumin, and in particular to such designed ankyrin repeat domains with improved stability. The invention further relates to recombinant binding proteins comprising such designed ankyrin repeat domains, nucleic acids encoding such designed ankyrin repeat domains or proteins, pharmaceutical compositions comprising such proteins, and the use of such proteins or pharmaceutical compositions in the treatment of diseases.

Description

[0001] Cross-reference to related patent applications

[0002] This application claims the benefit of and priority to European Patent Application EP19178282, filed on June 04, 2019, with the European Patent Office. The contents of European Patent Application EP19178282 are incorporated herein by reference in their entirety, including all tables, figures and claims. TECHNICAL FIELD

[0003] The present invention relates to designed ankyrin repeat domains having binding specificity for serum albumin, and in particular to such designed ankyrin repeat domains having improved stability. The present invention further relates to recombinant binding proteins comprising such designed ankyrin repeat domains, nucleic acids encoding such designed ankyrin repeat domains or proteins, pharmaceutical compositions comprising such proteins, and the use of such proteins or pharmaceutical compositions in the treatment of diseases. BACKGROUND

[0004] For biological drug products, where the active ingredient is typically a protein and / or polypeptide, the maintenance of the molecular conformation and thus the biological activity depends on non-covalent forces as well as covalent forces. These products are particularly sensitive to environmental factors such as temperature changes, oxidation, light, ionic content and shear. In order to ensure that the biological activity is maintained, it is critical that the active ingredient of such drug products is subjected to the conditions to which they are exposed during manufacture, transport and storage. Degradation and other forms of molecular change need to be avoided as much as possible. Therefore, the development of stable active ingredients is of paramount importance for the successful development of a commercial product.

[0005] Designed ankyrin repeat domains having binding specificity for serum albumin have been described and they are particularly useful for the production of recombinant binding proteins having an extended terminal half-life (see, e.g., WO2012 / 069654). Such half-life extension in plasma is highly advantageous for therapeutic use compared to proteins not comprising said designed ankyrin repeat domains having binding specificity for serum albumin. For example, WO2016 / 156596 describes a therapeutically useful ankyrin repeat protein comprising a designed ankyrin repeat domain having binding specificity for serum albumin.

[0006] Despite the development of such designed ankyrin repeat domains having binding specificity for serum albumin, there remains a need and challenge to improve the stability properties, and in particular the storage stability properties, of today’s drug products that generally have ankyrin repeat domains having binding specificity for serum albumin. SUMMARY

[0007] We have surprisingly found that the designed ankyrin repeat domains of the present application that have binding specificity for serum albumin provide further improved stability properties and in particular improved storage stability properties. Thus, it was found that the designed ankyrin repeat domains of the present application not only lead to improved storage stability in liquid formulations, but also to advantageous stability properties during heat treatment at elevated temperatures, e.g. at 60°C, in such liquid formulations. The latter is particularly advantageous to allow purification of recombinant binding proteins comprising such designed ankyrin repeat domains. Furthermore, the designed ankyrin repeat domains of the present application show improved stability at elevated temperatures during pH excursions, which is likewise highly advantageous when purifying recombinant binding proteins comprising such designed ankyrin repeat domains. The improvement of stability of the designed ankyrin repeat domains of the present application is particularly evident by a lower degree of identified lower molecular weight species assumed to be degradation products, e.g. after incubation at elevated temperatures and SDS PAGE analysis. In turn, after storage of recombinant binding proteins comprising the designed ankyrin repeat domains of the present application at lower pH, higher molecular weight species are identified by size exclusion chromatography in increased amounts compared to prior art products and thus less of the assumed degradation products. Storage of recombinant binding proteins at lower pH can generally lead to the appearance of undesired lower molecular weight species. Furthermore, it should be noted that the improvement of stability of the designed ankyrin repeat domains of the present application does not impair their binding specificity for serum albumin.

[0008] Thus, in one aspect, the present application provides a designed ankyrin repeat domain having binding specificity for serum albumin, wherein said designed ankyrin repeat domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein said designed ankyrin repeat domain does not have an asparagine (N) at the position corresponding to position 77 of SEQ ID NO: 3 or 4. In another aspect, the present application relates to a designed ankyrin repeat domain having binding specificity for serum albumin, wherein said designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 13.

[0009] In another aspect, the present application provides a recombinant binding protein comprising at least one, typically and preferably one or two, designed ankyrin repeat domains of the present application.

[0010] In another aspect, the present application provides a nucleic acid encoding a designed ankyrin repeat domain or a recombinant binding protein of the present application.

[0011] In another aspect, the present application provides a pharmaceutical composition comprising a designed ankyrin repeat domain of the present application, a recombinant binding protein of the present application, or a nucleic acid of the present application, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0012] In the detailed description, other aspects and embodiments of the application will become apparent. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Sequence alignment of designed ankyrin repeat domains with binding specificity for serum albumin. SEQ ID NOs: 3 and 4 represent designed ankyrin repeat domains with binding specificity for serum albumin and improved stability compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2. Residue numbering is shown above the sequences

[0014] Figure 2 Sequence motif at residue numbers 77 to 79 of different designed ankyrin repeat domains with binding specificity for serum albumin aligned to the sequence motif of SEQ ID NO: 2. The SEQ ID NO of the respective protein is shown at the left side of the alignment.

[0015] Figure 3 SDS-PAGE comparing the stability of designed ankyrin repeat domains with binding specificity for serum albumin. Proteins #2, #3 and #4 (consisting of SEQ ID NOs: 2, 3 and 4, respectively; additionally with SEQ ID NO: 1 at the N-terminus) were prepared as described in Example 2, then incubated at 60°C (-80°C for the reference), pH 7.4, pH 6 and pH 5 for 1 week as described in Example 3, followed by storage stability evaluation using SDS-PAGE as described in Example 4. The three SDS PAGE pictures represent the measurements at pH 7.4, pH 6 and pH 5, as indicated by the respective gels above. Proteins are indicated by their SEQ ID NO. O: -80°C incubated reference sample. I: 60°C incubated sample. Mw: molecular weight marker with kDa values shown at the left side.

[0016] Figure 4LabChip analysis of stability of designed ankyrin repeat domains with binding specificity for serum albumin. Proteins #2, #3, #4, #5 and #6 (comprising SEQ ID NOs: 2 to 6, respectively; all additionally having SEQ ID NO: 1 at the N-terminus) were prepared as described in Example 2, then incubated at 60°C, pH 6, pH 7.4 and pH 8.5 for 1 week as described in Example 3, after which storage stability evaluation was performed using LabChip as described in Example 5. LabChip data are shown in 3 groups for 3 different pH values. The molecular weight marker levels are shown on the left and right of each group (in kDa).

[0017] Figure 5: Size exclusion chromatography of designed ankyrin repeat domains with binding specificity for serum albumin. Proteins #2, #3 and #4 (comprising SEQ ID NOs: 2, 3 and 4, respectively; additionally having SEQ ID NO: 1 at the N-terminus) were prepared as described in Example 2, then incubated at 60°C (for the reference -80°C), pH 7.4, pH 6 and pH 5 for 1 week as described in Example 3, after which storage stability evaluation was performed using size exclusion chromatography as described in Example 6. The chromatogram overlays for pH 7.4, obtained at 60°C and -80°C, are shown in Figure 5a The chromatogram overlays for pH 6 are shown in Figure 5b The chromatogram overlays for pH 5 are shown in. Void volume elutes at 2.18 min, total volume at 5.28 min. OD: optical density at 280 nm (referenced to optical density at 360 nm) in mAu []; T: time in [min]. Figure 5c

[0018] Figure 6: Pharmacokinetic profile of designed ankyrin repeat domains with binding specificity for serum albumin at 1 mg / kg i.v. in mice (a) and cynomolgus monkeys (b). Proteins #2, #3 and #4 (comprising SEQ ID NOs: 2 to 4, respectively; all additionally having SEQ ID NO: 1 at the N-terminus; symbols shown in the figure) were prepared as described in Example 2, and mouse pharmacokinetic profiles were determined as described in Example 9, and cynomolgus monkey pharmacokinetic profiles were determined as described in Example 10. Mean protein concentrations from three mice or two monkeys are shown, including the respective standard deviations. C: concentration in [nM]; T: time in [h].

[0019] ​Figure 7: Pharmacokinetic profile of recombinant binding proteins comprising a designed ankyrin repeat domain with binding specificity for serum albumin in mice dosed intravenously at 1 mg / kg. Proteins #7, #8, #9, #10, #11 and #12 (comprising SEQ ID NOs: 7 to 12, respectively; all additionally having SEQ ID NO: 1 at the N-terminus; symbols shown in the figure) were prepared as described in Example 2 and mouse pharmacokinetic profiles were determined as described in Example 11. Mean protein concentrations from three mice are shown, including standard deviations. For ease of comparison, proteins #7, #9 and #11 (a) and proteins #8, #10 and #12 (b) are shown in two separate figures. C: concentration in [nM]; T: time in [h]. DETAILED DESCRIPTION

[0020] The present application provides designed ankyrin repeat domains with binding specificity for serum albumin, recombinant binding proteins comprising such designed ankyrin repeat domains, and further nucleic acids encoding such designed ankyrin repeat domains and recombinant binding proteins, as well as pharmaceutical compositions comprising the designed ankyrin repeat domains, binding proteins or nucleic acids. The present application also provides the use of such recombinant binding proteins or pharmaceutical compositions in the treatment of diseases.

[0021] Thus, in one aspect, the present application provides a designed ankyrin repeat domain with binding specificity for serum albumin, wherein the designed ankyrin repeat domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at a position corresponding to position 77 of SEQ ID NO: 3 or 4 and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at a position corresponding to position 78 of SEQ ID NO: 3 or 4.

[0022] In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at a position corresponding to position 77 of SEQ ID NO: 3 or 4.

[0023] In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have a glutamine (Q) at the position corresponding to position 77 of SEQ ID NO: 3 or 4. Thus, in one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4. In another embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4. In another embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4. In another embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4. In another embodiment, the designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 3 or 4.

[0024] In one embodiment, the designed ankyrin repeat domain does not have a glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4. Thus, in one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, preferably at least 90%, further preferably at least 93%, again further preferably at least 95%, and further preferably at least 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have a glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4.

[0025] In one embodiment, the designed ankyrin repeat domain does not have a serine (S) at the position corresponding to position 77 of SEQ ID NO: 3 or 4. Thus, in one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, preferably at least 90%, more preferably at least 93%, again more preferably at least 95%, and again preferably at least 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have a serine (S) at the position corresponding to position 77 of SEQ ID NO: 3 or 4.

[0026] In one embodiment, the designed ankyrin repeat domain does not have a serine (S) at the position corresponding to position 77 of SEQ ID NO: 3 or 4. Thus, in one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, preferably at least 90%, more preferably at least 93%, again more preferably at least 95%, and again preferably at least 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have a serine (S) at the position corresponding to position 77 of SEQ ID NO: 3 or 4.

[0027] In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence KDFAGKTPLHLAAX1X2G (SEQ ID NO: 13), wherein X1represents an amino acid residue selected from A, D and I; and X2represents an amino acid residue selected from A and D. Preferably, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 90%, further preferably at least 93%, again further preferably at least 95%, and further preferably at least 98%, amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4.

[0028] In one embodiment, at least one of X1and X2is aspartic acid (D). In one embodiment, at least one of X1and X2is alanine (A). In one embodiment, at least one of X1and X2is aspartic acid (D) and the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 90%, further preferably at least 93%, again further preferably at least 95%, and further preferably at least 98%, amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, at least one of X1and X2is aspartic acid (D) and the designed ankyrin repeat domain differs from said SEQ ID NO: 3 or 4 by at most 9, 8, 7, 6, 5, 4, 3, 2, or one amino acid.

[0029] In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence KDFAGKTPLHLAAX1X2G (SEQ ID NO: 13), wherein each of X1and X2is independently an amino acid residue selected from A and D, wherein preferably X1and X2are not equal. Preferably, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 90%, further preferably at least 93%, again further preferably at least 95%, and further preferably at least 98%, amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4.

[0030] In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6. In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 6.

[0031] In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence KDFAGKTPLHLAAADG (SEQ ID NO: 14) or KDFAGKTPLHLAADAG (SEQ ID NO: 15). In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence KDFAGKTPLHLAAADG (SEQ ID NO: 14). In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence KDFAGKTPLHLAADAG (SEQ ID NO: 15). Preferably, the designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, preferably at least 90%, further preferably at least 93%, again further preferably at least 95%, and further preferably at least 98%, amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4.

[0032] In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NO: 3 to 6, or preferably selected from SEQ ID NO: 3 and SEQ ID NO: 4, wherein at most 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids of SEQ ID NO: 3 to 6, preferably of SEQ ID NO: 3 and SEQ ID NO: 4, are replaced by other amino acids. Such amino acid replacements are preferably such that they do not significantly affect the function of the designed ankyrin repeat domain, preferably the specific binding to serum albumin. Such amino acid replacements can for example include amino acid replacements in the designed ankyrin repeat domain, such as amino acid replacements for capping of the repeat domain, as described in WO2012 / 069655. When replacing an amino acid, any other amino acid can be considered as replacement amino acid. Preferably, such other amino acid is selected from A, D, E, F, H, I, K, L, N, P, Q, R, S, T, V, W or Y. Preferably, such other amino acid is not C, G or P. In some embodiments, the replacement does not change the K D value for binding to human serum albumin more than 1000-fold, more than 100-fold or more than 10-fold compared to the designed ankyrin repeat domain comprising an amino acid sequence selected from SEQ ID NO: 3 to 6, or preferably selected from SEQ ID NO: 3 and SEQ ID NO: 4. D In certain embodiments, the replacement is a conservative replacement according to Table X. In certain embodiments, the replacement is made outside of the structural core residues of the ankyrin repeat domain, for example in the beta strands connecting the alpha-helices. In certain embodiments, the replacement is made within the structural core residues of the ankyrin repeat domain.

[0033] Table X: Amino acid substitutions

[0034] Original residue Conservative substitutions Exemplary substitutions Ala (A) Val Val; Leu; lie Arg (R) Lys Lys; Gin; Asn Asn (N) Gin Gin; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gin (Q) Asn Asn; Glu Glu (E) Asp Asp; Gin Gly (G) Ala Ala His (H) Arg Asn; Gin; Lys; Arg lie (I) Leu Leu; Val; Met; Ala; Phe; norleucine Leu (L) lie norleucine; lie; Val; Met; Ala; Phe Lys (K) Arg Arg; Gin; Asn Met (M) Leu Leu; Phe; lie Phe (F) Tyr Leu; Val; lie; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu lie; Leu; Met; Phe; Ala; norleucine

[0035] In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence selected from (1) SEQ ID NO: 3 and (2) SEQ ID NO: 3, wherein at most 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids of SEQ ID NO: 3 at any position selected from positions 1-76 and 79-124 of SEQ ID NO: 3 are exchanged for other amino acids.

[0036] In one embodiment, the present application relates to a designed ankyrin repeat domain with binding specificity for serum albumin, wherein said designed ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 4, and (1) SEQ ID NO: 4, wherein at most 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 4 at any position selected from the group consisting of positions 1-76 and 79-124 of SEQ ID NO: 4 are exchanged for other amino acids.

[0037] In all of the designed ankyrin repeat domains of the application described above, the penultimate position can be an "A" or an "L", and / or the last position can be an "A" or an "N". Thus, in some embodiments, the designed ankyrin repeat domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at the position corresponding to position 77 of SEQ ID NO: 3 or 4, and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4, and wherein optionally, the A at the penultimate position is replaced with an L and / or the A at the last position is replaced with an N. Thus, in one exemplary embodiment, the designed ankyrin repeat domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at the position corresponding to position 77 of SEQ ID NO: 3 or 4, and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4, and wherein optionally, the A at the penultimate position is replaced with an L and / or the A at the last position is replaced with an N. In another exemplary embodiment, the designed ankyrin repeat domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at the position corresponding to position 77 of SEQ ID NO: 3 or 4, and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4, and wherein optionally, the A at the penultimate position is replaced with an L and / or the A at the last position is replaced with an N.

[0038] Further, all of the above designed ankyrin repeat domains of the application can optionally further comprise a "G", "S" or "GS" sequence at their N-terminus. Thus, in some embodiments, the designed ankyrin repeat domain (i) comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at the position corresponding to position 77 of SEQ ID NO: 3 or 4, and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4, and (ii) further comprises a G, S or GS at its N-terminus. In one exemplary embodiment, the designed ankyrin repeat domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at the position corresponding to position 77 of SEQ ID NO: 3 or 4, and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain further comprises a G, S or GS at its N-terminus. In another exemplary embodiment, the designed ankyrin repeat domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at the position corresponding to position 77 of SEQ ID NO: 3 or 4, and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain further comprises a G, S or GS at its N-terminus.In another exemplary embodiment, the designed ankyrin repeat domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain does not have an Asparagine (N) at a position corresponding to position 77 of SEQ ID NO: 3 or 4, and / or the designed ankyrin repeat domain does not have a Glutamic acid (E) at a position corresponding to position 78 of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain optionally further comprises G, S or GS at its N-terminus, and wherein optionally A at the penultimate position is replaced by L and / or A at the last position is replaced by N.

[0039] In one embodiment, the designed ankyrin repeat domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 6. In one embodiment, the designed ankyrin repeat domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the designed ankyrin repeat domain consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the designed ankyrin repeat domain consists of the amino acid sequence of SEQ ID NO: 4. In one embodiment, the designed ankyrin repeat domain consists of the amino acid sequence of SEQ ID NO: 5. In one embodiment, the designed ankyrin repeat domain consists of the amino acid sequence of SEQ ID NO: 6.

[0040] In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 100 nM. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 90 nM. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 80 nM. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 70 nM. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 60 nM. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 50 nM. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 40 nM. D ) to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 90 nM. D ) to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 80 nM. D ) to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 70 nM. D ) to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 60 nM. D ) to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin in PBS with a dissociation constant (Kd) of equal to or lower than 50 nM. D ) to human serum albumin in PBS.

[0041] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2.

[0042] In one embodiment, the designed ankyrin repeat domain exhibits fewer degradation bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the designed ankyrin repeat domain exhibits fewer degradation bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar concentration, 60 °C, and pH 5.0 for one week.

[0043] In one embodiment, the designed ankyrin repeat domain exhibits fewer higher molecular weight peaks in size exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the designed ankyrin repeat domain exhibits fewer higher molecular weight peaks in size exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the size exclusion chromatography is performed after incubation at 100 micromolar concentration, 60 °C, and pH 5.0 for one week.

[0044] In one embodiment, the designed ankyrin repeat domain exhibits fewer higher molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the designed ankyrin repeat domain exhibits fewer higher molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar concentration, 60 °C, and pH 5.0 for one week.

[0045] In one embodiment, the designed ankyrin repeat domain (i) exhibits fewer higher molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and / or (ii) exhibits fewer higher molecular weight peaks in size exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the designed ankyrin repeat domain exhibits fewer higher molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60°C, and pH 5.0 for one week, and / or (ii) exhibits fewer higher molecular weight peaks in size exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the size exclusion chromatography is performed after incubation at 100 micromolar, 60°C, and pH 5.0 for one week.

[0046] In one embodiment, the designed ankyrin repeat domain exhibits at least 10% fewer higher molecular weight bands in a LabChip analysis compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the designed ankyrin repeat domain exhibits at least 10% fewer higher molecular weight bands in a LabChip analysis compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the LabChip analysis is performed after incubation at 100 micromolar, 60°C, and pH 6.0 or 7.4.

[0047] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is selected from:

[0048] (i) exhibits fewer degradation bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein preferably the SDS-PAGE is performed after incubation at 100 micromolar, 60°C, and pH 5.0 for one week;

[0049] (ii) exhibits fewer higher molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein preferably the SDS-PAGE is performed after incubation at 100 micromolar, 60°C, and pH 5.0 for one week;

[0050] (iii) exhibits less higher molecular weight peaks in size exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein preferably the size exclusion chromatography is performed after incubation at 100 micromolar, 60°C and pH 5.0 for one week; and

[0051] (iv) exhibits at least 10% less higher molecular weight bands in LabChip analysis compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein preferably the LabChip analysis is performed after incubation at 100 micromolar, 60°C and pH 6.0 or 7.4.

[0052] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is selected from:

[0053] (i) exhibits less degradation bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60°C and pH 5.0 for one week;

[0054] (ii) exhibits less higher molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60°C and pH 5.0 for one week;

[0055] (iii) exhibits less higher molecular weight peaks in size exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the size exclusion chromatography is performed after incubation at 100 micromolar, 60°C and pH 5.0 for one week; and (iv) exhibits at least 10% less higher molecular weight bands in LabChip analysis compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the LabChip analysis is performed after incubation at 100 micromolar, 60°C and pH 6.0 or 7.4.

[0056] In one embodiment, the designed ankyrin repeat domain having binding specificity for serum albumin has an EC 50binds human serum albumin in PBST-C. In one embodiment, said designed ankyrin repeat domain with binding specificity for serum albumin has an EC50 of less than 20 nM 50 binds cynomolgus monkey serum albumin in PBST-C. In one embodiment, said designed ankyrin repeat domain with binding specificity for serum albumin has an EC50 of less than 30 nM 50 binds mouse serum albumin in PBST-C. In one embodiment, said designed ankyrin repeat domain with binding specificity for serum albumin has an EC50 of less than 100 nM 50 binds human serum albumin, cynomolgus monkey serum albumin and mouse serum albumin in PBST-C.

[0057] In one embodiment, the terminal half-life of said designed ankyrin repeat domain in mice is at least 70%, preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, most preferably 92% of the terminal half-life of a designed ankyrin repeat domain consisting of SEQ ID NO: 2 in mice. In one embodiment, the terminal half-life of said designed ankyrin repeat domain in mice differs less than 30%, preferably 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, most preferably 8% from the terminal half-life of a designed ankyrin repeat domain consisting of SEQ ID NO: 2 in mice. Preferably, the terminal half-life in mice is determined by intravenous injection of said designed ankyrin repeat domain into the tail vein of a Balb / c mouse at a dose of 1 mg / kg.

[0058] In one embodiment, the terminal half-life of said designed ankyrin repeat domain in cynomolgus monkeys is at least 70%, preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, most preferably 92% of the terminal half-life of a designed ankyrin repeat domain consisting of SEQ ID NO: 2 in cynomolgus monkeys. In one embodiment, the terminal half-life of said designed ankyrin repeat domain in cynomolgus monkeys differs less than 30%, preferably 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, most preferably 8% from the terminal half-life of a designed ankyrin repeat domain consisting of SEQ ID NO: 2 in cynomolgus monkeys. Preferably, the terminal half-life in cynomolgus monkeys is determined by administration of said designed ankyrin repeat domain by intravenous injection over 30 minutes at a dose of 1 mg / kg.

[0059] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is less high molecular weight products in SDS-PAGE analysis, typically and preferably after incubating the designed ankyrin repeat domain at 60 °C for one week. Preferably, the SDS-PAGE is performed after incubation at 100 micromolar, 60 °C and pH 5.0 for one week.

[0060] The term "higher molecular weight products" in SDS-PAGE analysis typically and preferably refers to bands running on the gel with a higher molecular weight than the expected molecular weight. In one embodiment, the higher molecular weight products in SDS-PAGE analysis refers to bands running on the gel with a molecular weight higher than 21.5 kDa. In Figure 3 In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is less high molecular weight products in SDS-PAGE analysis, typically and preferably after incubating the designed ankyrin repeat domain at 60 °C for one week. Preferably, the SDS-PAGE is performed after incubation at 100 micromolar, 60 °C and pH 5.0 for one week.

[0061] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is less high molecular weight products in SDS-PAGE analysis, typically and preferably after incubating the designed ankyrin repeat domain at 60 °C for one week. Preferably, the SDS-PAGE is performed after incubation at 100 micromolar, 60 °C and pH 5.0 for one week.

[0062] The term "lower molecular weight products" in SDS-PAGE analysis typically and preferably refers to bands running on the gel with a lower molecular weight than the expected molecular weight. In one embodiment, the lower molecular weight products in SDS-PAGE analysis refers to bands running on the gel with a molecular weight lower than 21.5 kDa. In Figure 3Protein #2, incubated at 60°C for one week at pH 5, 6, or 7.4, exhibited such a low molecular weight product in SDS-PAGE, running at an apparent molecular weight lower than the expected band running at approximately 14 kDa. In one embodiment, the low molecular weight product in the SDS-PAGE analysis refers to a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and most preferably 50% after one week of incubation at 60°C.

[0063] In one embodiment, the designed ankyrin repeating domain has improved stability compared to the designed ankyrin repeating domain of SEQ ID NO:2, wherein the improved stability compared to the designed ankyrin repeating domain of SEQ ID NO:2 is characterized by fewer high molecular weight products appearing in LabChip analysis, typically and preferably after incubating the designed ankyrin repeating domain at 60°C for one week. In one embodiment, the high molecular weight product in LabChip analysis refers to a band running on the chromatogram with a molecular weight higher than expected. In one embodiment, the high molecular weight product in LabChip analysis refers to a band running on the chromatogram with a molecular weight higher than 30 kDa. Figure 4 Protein #2, incubated at 60°C for one week at pH 6 or 7.4, exhibited such a high molecular weight product in LabChip analysis, running at an apparent molecular weight higher than the expected band running at approximately between 15 kDa and 20 kDa. In one embodiment, the reduced high molecular weight product in LabChip analysis refers to a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and most preferably 50% after one week of incubation at 60°C.

[0064] In one embodiment, the designed ankyrin repeating domain has improved stability compared to the designed ankyrin repeating domain of SEQ ID NO:2, wherein the improved stability compared to the designed ankyrin repeating domain of SEQ ID NO:2 is characterized by fewer low molecular weight products appearing in LabChip analysis, typically and preferably after incubating the designed ankyrin repeating domain at 60°C for one week. In one embodiment, the low molecular weight product in LabChip analysis refers to a band running on the chromatogram with a molecular weight lower than expected. In one embodiment, the low molecular weight product in LabChip analysis refers to a band appearing on the chromatogram at a molecular weight of approximately 7 kDa. Figure 4In particular, Protein #2 incubated at pH 6 for one week at 60°C exhibited such lower molecular weight products in LabChip analysis that ran at lower apparent molecular weights than the expected band running at approximately between 15 kDa and 20 kDa. In one embodiment, the less lower molecular weight products in LabChip analysis refers to at least 5%, 10%, 15%, 20%, most preferably 25% reduction in the percentage of total area under the curve after incubation for one week at 60°C.

[0065] In one embodiment, the designed ankyrin repeat domain has improved stability compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the improved stability compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2 is a lower amount of higher molecular weight products in size exclusion chromatography, typically and preferably after incubation for one week at 60°C at pH 5. In one embodiment, the term "higher molecular weight products" in size exclusion chromatography typically and preferably refers to products eluting before the expected molecular weight product. In one embodiment, the term "lower amount" refers to at least 5%, 10%, 15%, 20%, most preferably 25% reduction in the percentage of total area under the curve after incubation for one week at 60°C. Figure 5c In particular, Protein #2 incubated at pH 5 for one week at 60°C exhibited such higher molecular weight products that eluted at 4.1 minutes in size exclusion chromatography, whereas the expected molecular weight product eluted approximately after 4.6 minutes. In one embodiment, the less higher molecular weight products in size exclusion chromatography refers to at least 5%, 10%, 15%, 20%, most preferably 25% reduction in the percentage of total area under the curve after incubation for one week at 60°C.

[0066] In one embodiment, the incubation refers to incubation at pH 8.5, 7.4, 6, or 5. In one embodiment, the incubation refers to incubation at pH 8.5. In one embodiment, the term "incubation" refers to incubation at pH 7.4. In one embodiment, the incubation refers to incubation at pH 6. In one embodiment, the incubation refers to incubation at pH 5. In one embodiment, the incubation at pH 8.5 refers to incubation in phosphate / citrate / borate buffer. In one embodiment, the incubation at pH 7.4 refers to incubation in PBS. In one embodiment, the incubation at pH 6 refers to incubation in phosphate / citrate buffer at pH 6. In one embodiment, the incubation at pH 5 refers to incubation in phosphate / citrate buffer at pH 5.

[0067] In another aspect, the present application also provides a recombinant binding protein comprising at least one designed ankyrin repeat domain of the present application having binding specificity for serum albumin. Preferred embodiments and features as described above and herein for the designed ankyrin repeat domains of the present application apply to any and all aspects of the present application, including the recombinant binding protein of the present application comprising at least one designed ankyrin repeat domain of the present application having binding specificity for serum albumin.

[0068] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the present application. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present application. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present application. In one embodiment, the recombinant binding protein comprises three of the designed ankyrin repeat domains of the present application.

[0069] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein each of the designed ankyrin repeat domains does not have an Asparagine (N) at a position corresponding to position 77 of SEQ ID NO: 3 or 4 and / or does not have a Glutamic acid (E) at a position corresponding to position 78 of SEQ ID NO: 3 or 4. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein each of the designed ankyrin repeat domains does not have an Asparagine (N) at a position corresponding to position 77 of SEQ ID NO: 3 or 4 and / or does not have a Glutamic acid (E) at a position corresponding to position 78 of SEQ ID NO: 3 or 4. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present application, wherein each of the two ankyrin repeat domains independently comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and wherein each of the designed ankyrin repeat domains does not have an Asparagine (N) at a position corresponding to position 77 of SEQ ID NO: 3 or 4 and / or does not have a Glutamic acid (E) at a position corresponding to position 78 of SEQ ID NO: 3 or 4.

[0070] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present application, wherein each of the two ankyrin repeat domains independently comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4 is at least 93%, further preferably at least 95%, and again further preferably at least 98%.

[0071] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present application, wherein each of the two ankyrin repeat domains independently comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6.

[0072] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present application, wherein each of the two ankyrin repeat domains independently comprises an amino acid sequence of SEQ ID NO: 3 or 4.

[0073] In one embodiment, the recombinant binding protein comprises one or both of the designed ankyrin repeating domains of the present invention that have binding specificity to serum albumin, wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant binding protein binds to human serum albumin in PBS. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeating domains of the present invention that have binding specificity to serum albumin, wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant binding protein binds to human serum albumin in PBS. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeating domains of the present invention that are specific for binding to serum albumin, wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant protein binds to human serum albumin in PBS. In one embodiment, the recombinant protein has a dissociation constant (K0) equal to or less than 90 nM. D The recombinant protein binds to human serum albumin in PBS. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 80 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 70 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 60 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 50 nM.

[0074] In one embodiment, the recombinant binding protein comprises at least two designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the designed ankyrin repeat domains having binding specificity to serum albumin has improved stability compared to the designed ankyrin repeat domain composed of SEQ ID NO:2. SEQ ID NO:9 to 12 are examples of such recombinant binding proteins. In one embodiment, the present invention relates to a recombinant binding protein comprising two, preferably exactly two, designed ankyrin repeat domains having binding specificity to serum albumin, wherein each of the designed ankyrin repeat domains having binding specificity to serum albumin has improved stability compared to the designed ankyrin repeat domain composed of SEQ ID NO:2. SEQ ID NO:9 to 12 are examples of such recombinant binding proteins.

[0075] In one embodiment, the recombinant binding protein comprises two, preferably exactly two, designed ankyrin repeat domains specific for binding to serum albumin, wherein each of the designed ankyrin repeat domains specific for binding to serum albumin is composed of SEQ ID NO:3. SEQ ID NO:9 and 10 are examples of such recombinant binding proteins. In one embodiment, the recombinant binding protein comprises two, preferably exactly two, designed ankyrin repeat domains specific for binding to serum albumin, wherein each of the designed ankyrin repeat domains specific for binding to serum albumin is composed of SEQ ID NO:4. SEQ ID NO:11 and 12 are examples of such recombinant binding proteins.

[0076] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeating domains of the present invention, wherein each of the one or two ankyrin repeating domains independently comprises an amino acid sequence selected from SEQ ID NO:3 to 6, and wherein the recombinant binding protein has a dissociation constant (K) equal to or less than 100 nM. D The recombinant binding protein binds to human serum albumin in PBS. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present invention, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence selected from SEQ ID NO:3 to 6, and wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant binding protein binds to human serum albumin in PBS. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present invention, wherein each of the two ankyrin repeat domains independently comprises an amino acid sequence selected from SEQ ID NO:3 to 6, and wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant protein binds to human serum albumin in PBS. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 90 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 80 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 70 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 60 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 50 nM.

[0077] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeating domains of the present invention, wherein each of the one or two ankyrin repeating domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant binding protein binds to human serum albumin in PBS. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present invention, wherein each of the one or two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant binding protein binds to human serum albumin in PBS. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present invention, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D The recombinant protein binds to human serum albumin in PBS. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 90 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 80 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 70 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 60 nM. In one embodiment, the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) equal to or less than 50 nM.

[0078] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeating domains of the present invention, wherein each of the one or two ankyrin repeating domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the recombinant binding protein has a dissociation constant (K0) equal to or less than 100 nM. D) binds human serum albumin in PBS, and wherein each of the one or two ankyrin repeat domains has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is selected from the group consisting of: (i) exhibits fewer degradation bands on SDS-PAGE after incubation at 100 micromolar, 60 °C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60 °C, and pH 5.0 for one week; (ii) exhibits fewer higher molecular weight bands on SDS-PAGE after incubation at 100 micromolar, 60 °C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60 °C, and pH 5.0 for one week; (iii) exhibits fewer higher molecular weight peaks in size exclusion chromatography after incubation at 100 micromolar, 60 °C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the size exclusion chromatography is performed after incubation at 100 micromolar, 60 °C, and pH 5.0 for one week; and (iv) exhibits at least 10% fewer higher molecular weight bands in LabChip analysis after incubation at 100 micromolar, 60 °C, and pH 6.0 or 7.4 compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the LabChip analysis is performed after incubation at 100 micromolar, 60 °C, and pH 6.0 or 7.4. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the application, wherein each of the one or two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the recombinant binding protein has a dissociation constant (K D) binds human serum albumin in PBS, and wherein each of the one or two ankyrin repeat domains has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is selected from the group consisting of: (i) exhibits fewer degradation bands on SDS-PAGE after incubation at 100 micromolar, 60°C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation; (ii) exhibits fewer higher molecular weight bands on SDS-PAGE after incubation at 100 micromolar, 60°C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation; (iii) exhibits fewer higher molecular weight peaks in size exclusion chromatography after incubation at 100 micromolar, 60°C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the size exclusion chromatography is performed after incubation; and (iv) exhibits at least 10% fewer higher molecular weight bands in LabChip analysis after incubation at 100 micromolar, 60°C, and pH 6.0 or 7.4 compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the LabChip analysis is performed after incubation. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the application, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the recombinant binding protein binds human serum albumin in PBS with a dissociation constant (K D) binds human serum albumin in PBS, and wherein each of the two designed ankyrin repeat domains has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and wherein the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is selected from the group consisting of: (i) exhibits fewer degradation bands on SDS-PAGE after incubation at 100 micromolar, 60°C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60°C, and pH 5.0 for one week; (ii) exhibits fewer higher molecular weight bands on SDS-PAGE after incubation at 100 micromolar, 60°C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60°C, and pH 5.0 for one week; (iii) exhibits fewer higher molecular weight peaks in size exclusion chromatography after incubation at 100 micromolar, 60°C, and pH 5.0 for one week compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the size exclusion chromatography is performed after incubation at 100 micromolar, 60°C, and pH 5.0 for one week; and (iv) exhibits at least 10% fewer higher molecular weight bands in LabChip analysis after incubation at 100 micromolar, 60°C, and pH 6.0 or 7.4 compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the LabChip analysis is performed after incubation at 100 micromolar, 60°C, and pH 6.0 or 7.4. In one embodiment, the recombinant binding protein binds human serum albumin in PBS with a dissociation constant (KD) equal to or lower than 90 nM. In one embodiment, the recombinant binding protein binds human serum albumin in PBS with a dissociation constant (KD) equal to or lower than 80 nM. In one embodiment, the recombinant binding protein binds human serum albumin in PBS with a dissociation constant (KD) equal to or lower than 70 nM. In one embodiment, the recombinant binding protein binds human serum albumin in PBS with a dissociation constant (KD) equal to or lower than 60 nM. In one embodiment, the recombinant binding protein binds human serum albumin in PBS with a dissociation constant (KD) equal to or lower than 50 nM.

[0079] In one embodiment, the recombinant binding protein has improved stability compared to a recombinant binding protein having the same amino acid sequence except that each of the designed ankyrin repeat domains having binding specificity for serum albumin is replaced by a designed ankyrin repeat domain consisting of SEQ ID NO: 2. SEQ ID NOs: 9 to 12 are examples of such recombinant binding proteins.

[0080] A recombinant binding protein consisting of SEQ ID NO: 9 or 11 is an example of such a recombinant binding protein that exhibits improved stability compared to a recombinant binding protein consisting of SEQ ID NO: 7, which recombinant binding protein comprises two designed ankyrin repeat domains having binding specificity for serum albumin, each consisting of SEQ ID NO: 2. A recombinant binding protein consisting of SEQ ID NO: 10 or 12 is an example of such a recombinant binding protein that exhibits improved stability compared to a recombinant binding protein consisting of SEQ ID NO: 8, which recombinant binding protein comprises two designed ankyrin repeat domains having binding specificity for serum albumin, each consisting of SEQ ID NO: 2.

[0081] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present application, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3.

[0082] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the present application, wherein each of the one or two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the present application, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 4.

[0083] In one embodiment, the recombinant binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 to 12. In one embodiment, the recombinant binding protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 9. In one embodiment, the recombinant binding protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10. In one embodiment, the recombinant binding protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 11. In one embodiment, the recombinant binding protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 12.

[0084] In one embodiment, the terminal half-life of the recombinant binding protein in mice is at least 70%, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, most preferably at least 90% of the terminal half-life of a recombinant binding protein having the same amino acid sequence, except that each of the designed ankyrin repeat domains having binding specificity for serum albumin is replaced by a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the terminal half-life of the recombinant binding protein in mice differs less than 30%, preferably less than 25%, 20%, 19%, 18%, 17%, 16%, 15%, most preferably less than 10% from the terminal half-life of a recombinant binding protein having the same amino acid sequence, except that each of the designed ankyrin repeat domains having binding specificity for serum albumin is replaced by a designed ankyrin repeat domain consisting of SEQ ID NO: 2. Preferably, the terminal half-life in mice is determined by administering the recombinant binding protein at a dose of 1 mg / kg by intravenous injection into the tail vein of a Balb / c mouse.

[0085] In one embodiment, the terminal half-life of the recombinant binding protein in cynomolgus monkey is at least 70%, preferably less than 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, most preferably 92% of the terminal half-life of a recombinant binding protein having the same amino acid sequence, except that each of the designed ankyrin repeat domains having binding specificity for serum albumin is replaced by a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the terminal half-life of the recombinant binding protein in cynomolgus monkey differs less than 30%, preferably less than 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, most preferably less than 8% from the terminal half-life of a recombinant binding protein having the same amino acid sequence, except that each of the designed ankyrin repeat domains having binding specificity for serum albumin is replaced by a designed ankyrin repeat domain consisting of SEQ ID NO: 2. Preferably, the terminal half-life in cynomolgus monkey is determined by administering the recombinant binding protein by intravenous injection over 30 minutes at a dose of 1 mg / kg.

[0086] In another aspect, the present application relates to a nucleic acid encoding an amino acid sequence of an ankyrin repeat domain or a recombinant binding protein of the present application. In one embodiment, the present application relates to a nucleic acid encoding an amino acid sequence of an ankyrin repeat domain of the present application. In one embodiment, the present application relates to a nucleic acid encoding an amino acid sequence of a recombinant binding protein of the present application. In one embodiment, the present application relates to a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6. In one embodiment, the present application relates to a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 12. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 4. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 9. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 10. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 11. In one embodiment, the present application relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 12. Furthermore, the present application relates to a vector comprising any of said nucleic acids.

[0087] In another aspect, the present application relates to a pharmaceutical composition comprising a recombinant binding protein and / or a designed ankyrin repeat domain of the present application, and / or a nucleic acid encoding a recombinant binding protein and / or a designed ankyrin repeat domain of the present application, and optionally a pharmaceutically acceptable carrier and / or diluent. Pharmaceutically acceptable carriers and / or diluents are known to the person skilled in the art and will be explained in more detail below. Further, a diagnostic composition is provided comprising one or more of the recombinant binding proteins and / or designed ankyrin repeat domains described herein.

[0088] The pharmaceutical composition comprises a binding protein as described above and a pharmaceutically acceptable carrier, excipient or stabilizer, for example as described in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed., 1980. Suitable carriers, excipients or stabilizers known to those of skill in the art include, for example, saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Additional suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition can also be a combined preparation, which comprises an additional active agent, such as an anti-cancer agent or an anti-angiogenic agent.

[0089] Formulations to be used for in vivo administration must be sterile or sterilized. This is readily accomplished by filtration through a sterile filtration membrane.

[0090] The pharmaceutical composition can be administered by any suitable method within the knowledge of the skilled person. The preferred route of administration is parenteral administration. In parenteral administration, the medicament of the present application will be formulated in a unit-dose injectable form in association with a pharmaceutically acceptable excipient as defined above, such as a solution, suspension or emulsion. The dose and mode of administration will depend on the individual to be treated and the particular disease.

[0091] Furthermore, any of the above pharmaceutical compositions are contemplated for use in the treatment of a disease or disorder. The present application also provides methods of treatment. The method comprises administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition or a recombinant binding protein or a designed ankyrin repeat domain of the present application.

[0092] In addition, a method of treating a pathological condition in a mammal, including a human, is provided, the method comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition as described above.

[0093] The present disclosure provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant binding protein or pharmaceutical composition described herein. In certain embodiments, the subject is a human. In certain embodiments, the cancer is a solid tumor. SEQ ID NOs: 9 to 12 are examples of recombinant binding proteins useful in such methods of treating cancer

[0094] In some embodiments, the cancer is brain cancer, bladder cancer, breast cancer, clear cell kidney cancer, cervical cancer, colon and rectum cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, lip and oral cavity cancer, liver cancer, lung squamous cell carcinoma, melanoma, mesothelioma, non-small cell lung cancer (NSCLC), non-melanoma skin cancer, ovarian cancer, oral cavity cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, small cell lung cancer (SCLC), squamous cell head and neck cancer (SCCHN), triple negative breast cancer, or thyroid cancer.

[0095] The present application is not limited to the specific embodiments described in the examples. Other sources can be used and processed according to the outline described below.

[0096] The present specification relates to the multiple amino acid sequences of the amino acid sequence listing named “P5754_Sequence_Protocol.txt” in the present specification, and the amino acid sequences of this sequence protocol are incorporated herein by reference.

[0097] Definitions

[0098] Unless otherwise defined herein, all technical and scientific terms used herein shall have the meaning commonly understood by one of ordinary skill in the art to which this application belongs.

[0099] In the context of the present application, the term “protein” refers to a molecule comprising a polypeptide, wherein at least a portion of the polypeptide has or is capable of acquiring a defined three-dimensional arrangement by forming secondary, tertiary and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. If the protein comprises two or more polypeptide chains, the individual polypeptide chains can be non-covalently or covalently linked, for example by a disulfide bond between two polypeptides. A portion of a protein that has or is capable of acquiring a defined three-dimensional arrangement by forming secondary and / or tertiary structures alone is referred to as “protein domain”. Such protein domains are well known to the person skilled in the art.

[0100] The term "recombinant" as used in the context of recombinant proteins, recombinant polypeptides, etc., refers to the production of the protein or polypeptide by the use of recombinant DNA technology well known to those skilled in the art. For example, a recombinant DNA molecule encoding a polypeptide (e.g., produced by gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), a yeast expression plasmid, a mammalian expression plasmid or a plant expression plasmid, or a DNA capable of expression in vitro. If, for example, such a recombinant bacterial expression plasmid is inserted into appropriate bacteria (e.g., Escherichia coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The corresponding produced polypeptide or protein is referred to as recombinant polypeptide or recombinant protein.

[0101] In the context of the present application, the term "binding protein" refers to a protein comprising a binding domain. A binding protein can also comprise two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein. The binding proteins of the present application comprise ankyrin repeat domains having binding specificity for serum albumin.

[0102] Furthermore, any such binding protein can comprise additional polypeptides (such as, for example, polypeptide tags, peptide linkers, fusions to other protein domains having binding specificity, cytokines, hormones, or antagonists), or chemical modifications well known to those skilled in the art (such as conjugation to polyethylene glycol, toxins (e.g., DM1 from immuno gen), small molecules, antibiotics, etc.).

[0103] The term "binding domain" means a protein domain exhibiting binding specificity for a target. Preferably, the binding domain is a recombinant binding domain.

[0104] The term "target" refers to a single molecule, such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate or any other naturally occurring molecule, including any portion of such single molecule, or a complex of two or more such molecules, or an entire cell or tissue sample, or any non-natural compound. Preferably, a target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein containing a chemical modification (e.g., a naturally occurring or non-natural phosphorylation, acetylation or methylation). For example, in the context of the present application, serum albumin is the target of the disclosed serum albumin-specific binding domains and proteins.

[0105] In the context of the present application, the term "polypeptide" relates to a molecule consisting of a chain of multiple (i.e., two or more) amino acids connected via peptide bonds. Preferably, a polypeptide consists of more than eight amino acids connected via peptide bonds. The term "polypeptide" also includes multiple chains of amino acids connected together by S-S bridges of cysteines. Polypeptides are well known to those skilled in the art.

[0106] The patent application WO 2002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, M. T., Binz, H. K., Pluckthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of repeat protein features and repeat domain features, technologies and applications. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, the repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, the repeat protein can comprise additional non-repeat protein domains, polypeptide tags and / or polypeptide linkers. The repeat domains can be binding domains.

[0107] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein the repeat modules have structural and sequence homology. Preferably, the repeat domain further comprises N-terminal and / or C-terminal capping modules. For the sake of clarity, a capping module can be a repeat module. Such repeat domains, repeat modules and capping modules, sequence motifs and structural and sequence homology are well known to the person skilled in the art from the examples of ankyrin repeat domains (WO 2002 / 020565), leucine-rich repeat domains (WO 2002 / 020565), tetratricopeptide repeat domains (Main, E. R., Xiong, Y., Cocco, M. J., D'Andrea, L., Regan, L., Structure 11 (5), 497-508, 2003) and armadillo repeat domains (WO 2009 / 040338). The person skilled in the art is further well aware that such repeat domains differ from proteins comprising repeat amino acid sequences, wherein each repeat amino acid sequence is able to form a single domain (e.g. the FN3 domain of fibronectin).

[0108] The term "designed" as used in designed repeat proteins, designed repeat domains, etc. refers to the property that such repeat proteins and repeat domains are man-made and do not exist in nature, respectively. The binding proteins of the present application are designed repeat proteins and they comprise at least one designed ankyrin repeat domain.

[0109] The term "target interaction residue" refers to an amino acid residue of a repeat module that contributes to the direct interaction with the target.

[0110] The term "framework residue" refers to an amino acid residue of a repeat module that contributes to the folding topology, i.e., to the folding of the repeat module or to the interaction with an adjacent module. Such contribution can be an interaction with other residues in the repeat module, or an influence on the polypeptide backbone conformation when present in an a-helix or β-sheet, or an amino acid stretch involved in the formation of a linear polypeptide or loop.

[0111] Such framework and target interaction residues can be identified by analyzing structural data obtained by physico-chemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known and related structural information well known to the person skilled in the art of structural biology and / or bioinformatics.

[0112] The term "repeat module" refers to a repeating amino acid sequence and structural unit of a designed repeat domain which is originally derived from a repeat unit of a naturally occurring repeat protein. Each repeat module comprised in a repeat domain is originally derived from one or more repeat units of a family or subfamily of naturally occurring repeat proteins, e.g. the ankyrin repeat protein family. Furthermore, each repeat module comprised in a repeat domain can comprise a "repeat sequence motif" which is derived from a homologous repeat module obtained for the repeat domain selected for a target, e.g. as described in Example 1, and has the same target specificity.

[0113] The term "ankyrin repeat module" thus refers to a repeat module which is originally derived from a repeat unit of a naturally occurring ankyrin repeat protein. Ankyrin repeat proteins are well known to the person skilled in the art.

[0114] A repeat module can comprise positions of amino acid residues which have not been randomized in the library for selection of a target-specific repeat domain ("non-randomized positions") and positions of amino acid residues which have been randomized in the library for selection of a target-specific repeat domain ("randomized positions"). Non-randomized positions comprise framework residues. Randomized positions comprise target interaction residues. By "have been randomized" is meant that two or more amino acids are allowed at an amino acid position of a repeat module, e.g. wherein any of the usually twenty naturally occurring amino acids are allowed, or wherein a majority of the twenty naturally occurring amino acids are allowed, such as amino acids other than cysteine, or amino acids other than glycine, cysteine and proline. For the purposes of this patent application, amino acid residues 3, 4, 6, 14 and 15 of SEQ ID NOs: 2 to 6 are randomized positions of an ankyrin repeat module of the present application.

[0115] The term "repeat sequence motif" refers to an amino acid sequence that is derived from one or more repeat modules. Preferably, the repeat modules are from repeat domains having binding specificity for the same target. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of the repeat modules. Likewise, the target interaction residue positions correspond to the positions of the target interaction residues of the repeat modules. Repeat sequence motifs include non-randomized positions and randomized positions.

[0116] The term "repeat unit" refers to an amino acid sequence comprising a sequence motif of one or more naturally occurring proteins, wherein the "repeat unit" is present in multiple copies and exhibits a defined folding topology common to all of the motifs that determine the protein fold. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

[0117] The terms "binding specificity for a target", "bind specifically to a target", "bind to a target with high specificity", "specific for a target", or "target specificity", and the like, mean that the binding protein or binding domain binds to a target in PBS with a lower dissociation constant (i.e., it binds with higher affinity) than it binds to an unrelated protein, such as E. coli maltose binding protein (MBP). Preferably, the dissociation constant ("Kd") of the target in PBS is at least 10 2 -fold lower than the corresponding dissociation constant of MBP; more preferably, at least 10 3 -fold lower; more preferably, at least 10 4 -fold lower; or more preferably, at least 10 5 -fold lower than the corresponding dissociation constant of MBP. Methods to determine the dissociation constant of a protein-protein interaction, such as surface plasmon resonance (SPR)-based techniques (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The measured Kd value for a particular protein-protein interaction can be variable if measured under different conditions (e.g., salt concentration, pH). Thus, preferably, the measurement of the Kd value is performed with a standardized protein solution and a standardized buffer, such as PBS. A typical and preferred determination of the dissociation constant (Kd) of a recombinant binding protein of the application having binding specificity for serum albumin by surface plasmon resonance (SPR) analysis is described in Example 7.

[0118] The term "about" means + / - 20% of the value cited; for example, "about 50" shall mean 40 to 60.

[0119] The term "PBS" means an aqueous phosphate buffered solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KC1 and having a pH of 7.4. This is exemplified in Example 3. The term "phosphate / citrate buffer pH 6" means an aqueous buffer comprising 375 mM Na2HP04*2H20 and 150 mM NaCl, and having a pH of 5.7. This is exemplified in Example 3. The term "phosphate / citrate buffer pH 5" means an aqueous buffer comprising 30 mM citric acid and 30 mM NaH2P04 and having a pH of 4.75. This is exemplified in Example 3. The term "phosphate / citrate / borate buffer" means an aqueous buffer comprising 30 mM citric acid, 30 mM NaH2P04, 30 mM boric acid and having a pH of 8.5. This is exemplified in Example 3.

[0120] The term "mouse serum albumin" refers to UniProt accession number P07724, the term "cynomolgus serum albumin" (i.e., macaca fascicularis) refers to UniProt accession number A2V9Z4, and the term "human serum albumin refers to UniProt accession number P02768.

[0121] Examples

[0122] Proteins used in the Examples:

[0123] Protein #2 (SEQ ID NO: 2, with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0124] Protein #3 (SEQ ID NO: 3, with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0125] Protein #4 (SEQ ID NO: 4, with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0126] Protein #5 (SEQ ID NO: 5, with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0127] Protein #6 (SEQ ID NO: 6, with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0128] Protein #7 (SEQ ID NO: 7, with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0129] Protein #8 (SEQ ID NO: 8 with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0130] Protein #9 (SEQ ID NO: 9 with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0131] Protein #10 (SEQ ID NO: 10 with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0132] Protein #11 (SEQ ID NO: 11 with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0133] Protein #12 (SEQ ID NO: 12 with a His tag (SEQ ID NO: 1) fused to its N-terminus);

[0134] Experiments were performed according to methods well known to the person skilled in the art if not otherwise described. Some example experimental conditions are also described in WO2012 / 069654 and WO2016 / 156596.

[0135] Example 1 : Construction of a designed ankyrin repeat domain with binding specificity for serum albumin and improved stability Protein repeat domains

[0136] Unexpectedly, it was observed that the protein consisting of the amino acid sequence corresponding to SEQ ID NO: 2 (first described in WO2016 / 156596) appeared to be unstable upon incubation at elevated temperatures (see examples 2 to 6), despite the fact that it is a variant with improved stability compared to earlier designed ankyrin repeat domains with binding specificity for serum albumin (WO2012 / 069654). It was therefore an object of the present application to provide variants of SEQ ID NO: 2 that exhibit improved stability upon incubation at elevated temperatures, while maintaining serum albumin binding and pharmacokinetic properties. By a highly iterative process involving several rounds of changes of amino acids at multiple positions (e.g. comparable to an alanine scanning process well known to the person skilled in the art) and in vitro and in vivo characterization of the resulting protein variants, 4 variants were finally obtained that exhibit improved stability properties. Unexpectedly, these variants all involve the amino acid Asp at position 77 and / or 78, which is known to be an amino acid located at the origin of polypeptide chain degradation. It is very surprising that many of these variants comprise the Asp-Gly sequence motif, which is known to be particularly prone to polypeptide chain degradation. Thus, this approach surprisingly led to unexpected variants.

[0137] DNA encoding each of the designed ankyrin repeat domains consisting of SEQ ID NOs: 2 to 6 was cloned into an expression vector based on pQE (QIAgen, Germany) providing a N-terminal His-tag to facilitate simple protein purification as described below. The preparation and characterization of these specifically selected sequences as well as the use is described in the following examples.

[0138] Example 2: Expression and purification of proteins

[0139] Proteins consisting of SEQ ID NOs: 2 to 6, in addition with a His-tag SEQ ID NO: 1 fused to their N-terminus, as well as proteins consisting of SEQ ID NOs: 7 to 12, in addition with a His-tag SEQ ID NO: 1 fused to their N-terminus, were produced in E. coli, purified to homogeneity and stored in PBS buffer. For the sake of clarity, proteins #2 to #6 are designed ankyrin repeat domains with binding specificity for serum albumin, proteins #7 to #12 are recombinant binding proteins comprising a designed ankyrin repeat domain with binding specificity for serum albumin. Proteins #7 and #8 comprise two times SEQ ID NO: 2. Proteins #9 and #10 comprise two times SEQ ID NO: 3. Proteins #11 and #12 comprise two times SEQ ID NO: 4. Protein #7 from SEQ ID NO: 134 of WO2016156596 and protein #8 from SEQ ID NO: 21 of WO2018054971 are well known to the person skilled in the art. Thus, in comparison to protein #7 which is a recombinant binding protein comprising a designed ankyrin repeat domain with binding specificity for serum albumin, proteins #9 and #11 are recombinant binding proteins comprising a designed ankyrin repeat domain with (i) binding specificity for serum albumin and with (ii) improved storage stability. Likewise, in comparison to protein #8 which is a recombinant binding protein comprising a designed ankyrin repeat domain with binding specificity for serum albumin, proteins #10 and #12 are recombinant binding proteins comprising a designed ankyrin repeat domain with (i) binding specificity for serum albumin and with (ii) improved storage stability. Proteins expressed and purified as described in this paragraph were used for the experiments of examples 3 to 12.

[0140] Alternatively, the proteins consisting of SEQ ID NO: 2 to 12 (further with the amino acids GS at the N-terminus) were produced in E. coli, purified to homogeneity and stored in PBS buffer. In case the amino acids GS are located at the N-terminus, the Met residue encoded additionally by the expression vector is efficiently cleaved from the expressed polypeptide in the cytoplasm of E. coli cells because the small Gly residue follows the start Met. In Examples 3 to 12, the proteins consisting of SEQ ID NO: 2 to 12 further with the amino acids GS at the N-terminus show the same results as the proteins consisting of SEQ ID NO: 2 to 12 further with the His tag SEQ ID NO: 1 fused to the N-terminus thereof.

[0141] Example 3: Incubation for storage stability

[0142] The storage stability of the proteins of Test Example 2 was tested at a protein concentration of 100 micromolar and 60°C for 1 week (7 days) at various pH. The buffers used were PBS (pH 7.4; 137 mM NaCl, 10 mM phosphate and 2.7 mM KC1) or phosphate / citrate (pH 5.7; 375 mM Na2HP04*2H20 and 150 mM NaCl, pH adjusted using 1 M monohydrate citric acid) or phosphate / citrate (pH 4.75; 30 mM citric acid and 30 mM NaH2P04, pH adjusted with sodium hydroxide) or phosphate / citrate / borate (pH 8.5, 30 mM citric acid, 30 mM NaH2P04, 30 mM boronic acid). The resulting pH values were pH 7.4, pH 6, pH 5 or pH 8.5, respectively, when the proteins were mixed with the respective buffers. In parallel to the incubation at 60°C, aliquots of the proteins were incubated at -80°C for 1 week (7 days) as a control.

[0143] The stability during incubation at 60°C and different pH is industrially relevant for the manufacturing of designed ankyrin repeat domains or recombinant binding proteins, because the manufacturing process can comprise one or more process steps during which the polypeptide is exposed to such conditions. In addition, the skilled person determines the storage stability by accelerated storage stability measurements comprising a step of incubation at elevated temperature.

[0144] Example 4: SDS-PAGE of storage stability analysis samples

[0145] The protein samples of Example 3 (10 microgram protein per lane) were analyzed on NuPAGE 4-12% Bis-Tris sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Thermo Fisher) and stained with Instant Blue (Sigma Aldrich). The results are shown in Figure 3 All proteins showed a major band of the expected size of about 14.4 kDa. Some proteins showed additional bands of lower molecular weight (lower molecular weight products) and / or bands of higher molecular weight (higher molecular weight products). Proteins #3 and #4 showed lower amounts of lower molecular weight products at pH 7.4, pH 6, and pH 5 compared to protein #2. The decrease in lower molecular weight products at pH 7.4, pH 6, and / or pH 5 was at least 10% as judged from SDS-PAGE. Similarly, the amount of lower molecular weight products decreased at pH 7.4, pH 6, and / or pH 5 by at least 50% as judged from SDS-PAGE. Similarly, protein #2 showed at least 50% more lower molecular weight products or lower molecular weight product bands at pH 7.4, pH 6, and / or pH 5. Surprisingly, protein #2 showed higher molecular weight bands after incubation at 60°C for one week. Proteins #3 and #4 did not appear to show higher molecular weight bands, i.e., showed improved stability. The decrease in higher molecular weight band product bands was at least 50% at pH 7.4, at least 50% at pH 6, and at least 50% at pH 5 as judged from SDS-PAGE when comparing protein #2 to proteins #3 and #4. Similarly, protein #2 showed an increase in higher molecular weight bands and / or higher molecular weight products that was at least 10% at pH 7.4, at least 20% at pH 6, and at least 50% at pH 5.

[0146] Example 5: LabChip analysis of storage stability analysis samples

[0147] The protein samples of Example 3 were analyzed using a LabChip GXII instrument according to the manufacturer (Perkin Elmer). Briefly, the samples were mixed with denaturing solution and denatured at 70°C for 10 minutes and then analyzed on an HT Protein Express Chip. The runs were analyzed using the software of the instrument. The results are shown in Figure 4Proteins #3 and #4 exhibited lower amounts of degradation products or lower molecular weight products at pH 7.4, pH 6, and pH 5 compared to Protein #2. The decrease in degradation product bands or lower molecular weight product bands at pH 7.4, pH 6, and pH 5 was at least 10% as judged from LabChip data. Protein #2 exhibited higher molecular weight bands after incubation at 60°C for one week, whereas Proteins #3 and #4 did not exhibit higher molecular weight bands. The decrease in higher molecular weight band product bands was at least 10% at pH 7.4 and at least 50% at pH 6 as judged from LabChip data (see Table 1). In this example, for Proteins #2-#4, a band was considered a higher molecular weight band in LabChip analysis if the band was greater than 25 kDa. In this example, for Proteins #2-#4, a band was considered a lower molecular weight band in LabChip analysis if the band was less than 10 kDa. Such lower molecular weight bands can be considered degradation product bands assuming the purity of the protein preparation is high.

[0148] Table 1: Percent higher molecular weight bands detected by LabChip at different pH

[0149]

[0150]

[0151] Proteins #2, #3, #4 in this table represent designed ankyrin repeat domains consisting of the corresponding amino acid sequence of SEQ ID NO: 2 to 4 and an additional N-terminal His-tag (SEQ ID NO: 1).

[0152] Example 6: Size exclusion chromatography analysis of storage stability analysis samples

[0153] Samples of Example 3 were analyzed on a GE Superdex 200 150 / 5 column on an Agilent 1200 HPLC system in PBS at a flow rate of 0.5 ml / min. In each protein, 0.1 ml of the -80°C control sample and each of the 60°C incubated samples were analyzed at 100 micromolar. Size exclusion chromatograms (optical density at 280 nm minus optical density at 360 nm) are shown in Figure 1. Figure 5a (pH 7.4), Figure 5b (pH 6), and Figure 5cProtein #3 and #4 show approximately overlapping chromatograms for the -80°C control sample and the 60°C incubated sample at pH 7.4, pH 6, and pH 5. Protein #2 shows approximately overlapping chromatograms for the -80°C control sample and the 60°C incubated sample at pH 7.4 and pH 6. In contrast, at pH 5, Protein #2 shows a clear increase in higher apparent molecular weight species in the 60°C incubated sample compared to the -80°C control sample. The higher apparent molecular weight species of Protein #2 at pH 5 in the 60°C incubated sample corresponds to 25% of the total area under the curve, whereas the Protein #2 -80°C control sample at pH 5 does not show higher apparent molecular weight species. In this example, the higher apparent molecular weight species is the eluted species with a peak around 4.1 minutes. The expected apparent molecular weight species is the eluted species with a peak around 4.6 minutes. Protein #3 and #4 do not show higher apparent molecular weight species in the 60°C incubated sample at pH 5.

[0154] Example 7: Affinity measurements

[0155] The affinities of the proteins of Example 2 for human serum albumin were determined by SPR measurements in PBS using a ProteOn system (BioRad) according to standard procedures known to the person skilled in the art. The determined affinities are listed in Table 2. All designed ankyrin repeat domains with binding specificity for serum albumin exhibit dissociation constants Kd D <100 nM and dissociation constants in the comparable range.

[0156] Table 2: Dissociation constants (Kd) of designed ankyrin repeat domains binding to human serum albumin

[0157]

[0158] * Protein #2, #3, #4 in this table denote designed ankyrin repeat domains consisting of the corresponding amino acid sequence of SEQ ID NO: 2 to 4 and an additional N-terminal His-tag (SEQ ID NO: 1).

[0159] Example 8: Serum albumin species cross-reactivity

[0160] The protein samples of Example 2 were subjected to ELISA serum albumin cross-reactivity analysis as described in WO2016156596. 100 μΙ_ per well of a 20 nM serum albumin solution in PBS was immobilized in Maxisorp plates (Nunc, Denmark) overnight at 4°C. After 5 washes with 300 μΙ PBST (PBS supplemented with 0.1 % Tween 20), the wells were blocked with 300 μΙ PBST-C (PBST supplemented with 0.25% casein) for 2 hours at room temperature while shaking at 450 rpm on a Titramax 1000 shaker (Heidolph, Germany). After 5 washes as described above, 100 μΙ / well or 50 μΙ / well of protein #2, #3 or #4 (for each protein, a concentration range from 100 nM to 0.01 pM) in PBST-C was applied and incubated for 2 hours at room temperature with 450 rpm shaking. After 5 washes as described above, the binding of the proteins was detected using 100 μΙ of a rabbit anti-designed ankyrin repeat domain monoclonal antibody in PBST-C for 1 hour at room temperature with 450 rpm shaking. After 5 washes as described above, the bound anti-designed ankyrin repeat domain antibody was detected using 100 μΙ / well of a goat anti-rabbit IgG-HRP conjugate in PBST-C for 1 hour at room temperature with 450 rpm shaking. After 5 washes as described above, the ELISA was then developed using 100 μΙ of soluble BM Blue POD substrate (Roche, Switzerland) diluted 1 :4 in water. After 5 minutes, the reaction was stopped using 100 μΙ 1 M H2SO4. OD (OD 450 nm - OD 620 nm) was then recorded. EC 50 values (Table 3) were determined using GraphPad Prism. The analysis showed that human, cynomolgus monkey and mouse serum albumin bound with high affinity. For proteins #2, #3 and #4, the measured EC 50 values for human serum albumin binding were below 1 nM. For proteins #2, #3 and #4, the measured EC 50 values for cynomolgus monkey serum albumin binding were below 20 nM. For proteins #2, #3 and #4, the measured EC 50 values for mouse serum albumin binding were below 30 nM.

[0161] Table 3: EC 50 [nM]

[0162]

[0163] * Protein #2, #3, #4 in this table represent designed ankyrin repeat domains consisting of the corresponding amino acid sequence of SEQ ID NO: 2 to 4 and an additional N-terminal His-tag (SEQ ID NO: 1).

[0164] Example 9: Pharmacokinetic profile of a designed ankyrin repeat domain with binding specificity for serum albumin in mice Figure 6a

[0165] Pharmacokinetic analysis was performed in female Balb / c mice using proteins #2, #3 and #4 prepared as described in Example 2. The proteins were administered intravenously into the tail vein at 1 mg / kg. Six mice divided into two groups of 3 mice were used for each protein. For each protein, blood was collected from one group of mice at 5 min, 24 h, 72 h, 168 h and 360 h post injection, and from the other group of mice at 4 h, 48 h, 144 h and 168 h post injection. Blood samples were allowed to stand at room temperature and centrifuged to produce serum using procedures well known to the person skilled in the art, and then stored at -80°C until analysis. Serum concentrations of proteins #2, #3 and #4 were determined by sandwich ELISA using rabbit monoclonal anti-DARpin antibody as capture reagent and anti-RGS-His antibody-HRP conjugate as detection reagent, and using a standard curve. The monoclonal anti-DARpin antibody was generated using routine rabbit immunization and hybridoma generation techniques well known to the person skilled in the art, and binding of the monoclonal antibody to proteins #2 to #16 was verified prior to the concentration determination experiment. Briefly, 100 μΐ of a 10 nM solution of goat anti-rabbit antibody (Thermo Scientific) in PBS per well was immobilized in Maxisorp plates (Nunc, Denmark) at 4°C overnight. After 5 washes with 300 μΐ PBST (PBS supplemented with 0.1% Tween 20), the wells were blocked with 300 μΐ PBST-C (PBST supplemented with 0.25% casein) for 1 h at room temperature while shaking at 450 rpm on a Titramax 1000 shaker (Heidolph, Germany). After 5 washes as above, 100 μΐ / well of a 5 nM solution of rabbit anti-DARpin antibody in PBST-C was added for 1 h at room temperature with shaking at 450 rpm. After 5 washes as above, different dilutions of serum samples or standard references diluted in PBST-C were added for 2 h at room temperature with shaking at 450 rpm. After 5 washes as above, 50 μΐ of a 100 ng / ml solution of mouse anti-RGS-His antibody-HRP conjugate (QIAgen) in PBST-C was added for 30 min at room temperature with shaking at 450 rpm. After 5 washes as above, the ELISA was developed using 50 μΐ TMB substrate. After 5 min, the reaction was stopped using 100 μΐ 1 M H2SO4. The OD (OD 450 nm - OD 620 nm) was then recorded.Pharmacokinetic parameters were determined using standard software such as Phoenix WinNonLin (Certara, Princeton, USA) or GraphPad Prism (GraphPad Software, La Jolla, USA) and standard analyses such as non-compartmental analysis, all well known to the person skilled in the art. The resulting pharmacokinetic profiles are shown in Example 10: Pharmacokinetic profile of a designed ankyrin repeat domain with binding specificity for serum albumin in cynomolgus monkeys Table 3. The pharmacokinetic parameters area under the curve from the measurements, clearance, volume of distribution and half-life are listed in Table 4.

[0166] Table 4: Pharmacokinetic parameters of proteins #2, #3 and #4 in mice

[0167]

[0168] Proteins #2, #3, #4 in this table represent designed ankyrin repeat domains consisting of the corresponding amino acid sequence of SEQ ID NO: 2 to 4 and an additional N-terminal His-tag (SEQ ID NO: 1).

[0169] Figure 6b Example 11 : Generation and characterization of recombinant binding proteins using a designed ankyrin repeat domain with binding specificity for serum albumin and improved stability

[0170] For each protein, pharmacokinetic analysis was performed at 1 mg / kg in two male cynomolgus monkeys by 30 min intravenous infusion administration. For each protein, blood was collected from each animal at 5 min, 6 h, 24 h, 72 h, 120 h, 168 h, 336 h, 408 h, 504 h and 672 h post injection. Blood samples were allowed to stand at room temperature and centrifuged to yield serum using procedures well known to the person skilled in the art, and then stored at -80 °C until analysis. Serum concentrations of proteins #2, #3 and #4 were determined by sandwich ELISA as described in Example 9. Pharmacokinetic parameters were determined using standard software such as Phoenix WinNonLin (Certara, Princeton, USA) or GraphPad Prism (GraphPad Software, La Jolla, USA) and standard analyses such as non-compartmental analysis, all well known to the person skilled in the art. The resulting pharmacokinetic profiles are shown in Example 12: Pharmacokinetic profile of a recombinant binding protein comprising a designed ankyrin repeat domain with binding specificity for serum albumin in mice Table 3. The pharmacokinetic parameters area under the curve from the measurements, clearance, volume of distribution and half-life are listed in Table 4.

[0171] Table 5: Pharmacokinetic parameters of proteins #2 to #4 in cynomolgus monkeys

[0172]

[0173] * Protein #2, #3, #4 in this table represent designed ankyrin repeat domains consisting of the respective amino acid sequence of SEQ ID NO: 2 to 4 and an additional N-terminal His-tag (SEQ ID NO: 1).

[0174] Figure 7a Figure 7b

[0175] Recombinant binding proteins comprising designed ankyrin repeat domains with binding specificity to serum albumin and with improved storage stability were generated by recombinant DNA technology. SEQ ID NO: 7 to 12 are examples of such recombinant binding proteins. Protein #7, #8, #9, #10, #11 and #12 (consisting of SEQ ID NO: 7 to 12, additionally with a His-tag SEQ ID NO: 1 fused to their N-terminus; see Example 2) were prepared as described in Example 3. Similarly, proteins consisting of SEQ ID NO: 7 to 12, each additionally carrying the amino acids MGS at the N-terminus (where the N-terminal methionine is efficiently cleaved from the expressed polypeptide in the cytoplasm of E. coli cells because of the small Gly residue following the initial Met) can be produced in E. coli and purified using conventional methods.

[0176] The recombinant binding proteins were assessed for improved stability according to Examples 3 to 6. The recombinant binding proteins consisting of SEQ ID NO: 9 and 11 exhibited higher stability than the recombinant binding protein consisting of SEQ ID NO: 7. Likewise, the recombinant binding proteins consisting of SEQ ID NO: 10 and 12 exhibited higher stability than the recombinant binding protein consisting of SEQ ID NO: 8.

[0177] ​ ​

[0178] Pharmacokinetic analysis in mice was performed using proteins #7, #9 and #11 and using proteins #8, #10 and #12, all of which were produced as described in Example 2. The study was performed essentially as described in Example 9, using three mice per protein and taking blood at 5 min, 4 h, 48 h and 96 h after injection. Concentration determination and determination of pharmacokinetic parameters were performed as described in Example 9. For comparison of proteins #7, #9 and #11, the pharmacokinetic traces are shown in ​ and the pharmacokinetic parameters area under the curve, clearance, volume of distribution and half-life derived from the measurements for all proteins are listed in Table 6. For comparison of proteins #8, #10 and #12, the pharmacokinetic traces are shown in ​The pharmacokinetic parameters area under the curve from the measurements of all proteins, clearance, volume of distribution, and half-life are listed in Table 7.

[0179] Table 6: Pharmacokinetic parameters of proteins #7, #9, and #11 in mice

[0180]

[0181] Proteins #7, #9, and #11 in this table represent designed ankyrin repeat domains consisting of the corresponding amino acid sequence of SEQ ID NO: 7, 9, and 11 and an additional N-terminal His-tag (SEQ ID NO: 1).

[0182] Table 7: Pharmacokinetic parameters of proteins #8, #10, and #12 in mice

[0183]

[0184] Proteins #8, #10, and #12 in this table represent designed ankyrin repeat domains consisting of the corresponding amino acid sequence of SEQ ID NO: 8, 10, and 12 and an additional N-terminal His-tag (SEQ ID NO: 1).

Claims

1. A designed ankyrin repeat domain having binding specificity for serum albumin, wherein the designed ankyrin repeat domain consists of SEQ ID NO: 3 or 4, and wherein the designed ankyrin repeat domain optionally further comprises a G, S or GS at its N-terminus, and wherein optionally an A at the penultimate position is substituted with an L and / or an A at the last position is substituted with an N.

2. The designed ankyrin repeat domain of claim 1, wherein the designed ankyrin repeat domain binds human serum albumin in PBS with a dissociation constant (K D ) equal to or lower than 100 nM.

3. The designed ankyrin repeat domain according to any one of claims 1 to 2, wherein the designed ankyrin repeat domain has improved stability compared to the designed ankyrin repeat domain consisting of SEQ ID NO:

2.

4. The designed ankyrin repeat domain according to any one of claims 1 to 3, wherein the designed ankyrin repeat domain exhibits less degradation bands on SDS-PAGE compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar concentration, 60 °C and pH 5.0 for one week.

5. The designed ankyrin repeat domain according to any one of claims 1 to 4, wherein the designed ankyrin repeat domain (i) exhibits less higher molecular weight bands on SDS-PAGE compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the SDS-PAGE is performed after incubation at 100 micromolar, 60 °C and pH 5.0 for one week, and / or (ii) exhibits less higher molecular weight peaks in size exclusion chromatography compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the size exclusion chromatography is performed after incubation at 100 micromolar, 60 °C and pH 5.0 for one week.

6. The designed ankyrin repeat domain according to any one of claims 1 to 5, wherein the designed ankyrin repeat domain exhibits at least 10% less higher molecular weight bands in LabChip analysis compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, wherein the LabChip analysis is performed after incubation at 100 micromolar, 60 °C and pH 6.0 or 7.

4.

7. A recombinant binding protein comprising one or two designed ankyrin repeat domains according to any one of claims 1 to 6.

8. The recombinant binding protein according to claim 7, wherein the recombinant binding protein has improved stability compared to a recombinant binding protein having the same amino acid sequence, except that each of the designed ankyrin repeat domains having binding specificity for serum albumin is replaced by a designed ankyrin repeat domain consisting of SEQ ID NO:

2.

9. A nucleic acid encoding the designed ankyrin repeat domain or recombinant binding protein of any one of claims 1 to 8.

10. A pharmaceutical composition comprising the designed ankyrin repeat domain or recombinant binding protein of any one of claims 1 to 8, or the nucleic acid of claim 9, and optionally a pharmaceutically acceptable carrier and / or diluent.

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