Designed ankyrin repeat domains with improved stability

Abstract

The present invention relates to designed ankyrin repeat domains with binding specificity for serum albumin, in particular 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

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to European Patent Application Publication No. 19178282, filed in the European Patent Office on June 4, 2019. The contents of European Patent Application No. 19178282 are incorporated herein by reference in their entirety, including all tables, figures, and claims.

[0002] FIELD OF THE INVENTION The present invention relates to designed ankyrin repeat domains with binding specificity for serum albumin, in particular 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. [Background technology]

[0003] For biological products, whose active ingredients are typically proteins and / or polypeptides, maintaining molecular conformation, and therefore biological activity, relies on non-covalent and covalent bonding forces. These products are particularly sensitive to environmental factors such as temperature changes, oxidation, light, ionic content, and shear. To ensure biological activity, it is important that the active ingredients in such formulations withstand the conditions they are exposed to during manufacturing, transportation, and storage. Degradation and other forms of molecular changes must be avoided as much as possible. Therefore, developing a stable active ingredient is crucial to the successful development of a commercial product.

[0004] Designed ankyrin repeat domains with binding specificity for serum albumin have been described, which are particularly useful for producing recombinant binding proteins with extended terminal half-lives (see, for example, WO 2012 / 069654). Compared to proteins that do not contain a designed ankyrin repeat domain with binding specificity for serum albumin, such an increased half-life in plasma is highly beneficial for therapeutic applications. For example, WO 2016 / 156596 describes therapeutically useful ankyrin repeat proteins that contain a designed ankyrin repeat domain with binding specificity for serum albumin.

[0005] Despite the development of such designed ankyrin repeat domains with binding specificity for serum albumin, there remains a need and challenge to improve the stability properties, particularly the storage stability properties, of today's pharmaceuticals in general and ankyrin repeat domains with binding specificity for serum albumin in particular. Summary of the Invention

[0006] The inventors have surprisingly found that the designed ankyrin repeat domains of the present invention with binding specificity for serum albumin further provide improved stability properties, in particular improved storage stability properties. Thus, the inventors have found that the designed ankyrin repeat domains of the present invention not only provide improved storage stability in liquid formulations, but also beneficial stability properties during heat treatment at elevated temperatures, e.g., 60°C, in such liquid formulations. The latter is particularly beneficial for enabling the purification of recombinant binding proteins comprising such designed ankyrin repeat domains. Furthermore, the designed ankyrin repeat domains of the present invention exhibit improved stability during pH shifts at elevated temperatures, which is also highly beneficial when purifying recombinant binding proteins comprising such designed ankyrin repeat domains. The improved stability of the designed ankyrin repeat domains of the present invention is particularly evident after incubation at elevated temperatures and SDS-PAGE analysis, where a lesser degree of identified lower molecular weight species, presumably degradation products, is identified. Furthermore, compared to prior art products, an increased amount of higher molecular weight species, and therefore a lower amount of potential degradation products, was identified by size exclusion chromatography after storage of recombinant binding proteins comprising the designed ankyrin repeat domains of the present invention at lower pH. Storage of recombinant binding proteins at lower pH can often result in the generation of undesired low molecular weight species. In addition, it should be noted that the improved stability of the designed ankyrin repeat domains of the present invention does not reduce their binding specificity for serum albumin.

[0007] Thus, in one aspect, the invention 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. In another aspect, the invention relates to a designed ankyrin repeat domain with binding specificity for serum albumin, wherein the designed ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 13.

[0008] In a further aspect, the present invention provides a recombinant binding protein comprising at least one, typically and preferably one or two, designed ankyrin repeat domains according to the invention.

[0009] In another aspect, the present invention provides a nucleic acid encoding the designed ankyrin repeat domain or recombinant binding protein of the invention.

[0010] In a further aspect, the present invention provides a pharmaceutical composition comprising a designed ankyrin repeat domain of the invention, a recombinant binding protein of the invention, or a nucleic acid of the invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0011] Further aspects and embodiments of the present invention will become apparent in the detailed description. [Brief explanation of the drawings]

[0012] [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 numbers are indicated in the sequences. [Figure 2]Sequence motifs at residues 77-79 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 NOs of the respective proteins are shown to the left of the alignment. [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, and further comprising SEQ ID NO: 1 at the N-terminus) were produced as described in Example 2 and subsequently incubated at pH 7.4, pH 6, and pH 5 for 1 week at 60°C (-80°C for the reference sample) as described in Example 3, followed by storage stability evaluation using SDS-PAGE as described in Example 4. Photographs of the three SDS PAGEs represent measurements at pH 7.4, pH 6, and pH 5, as indicated above each gel. Proteins are indicated by their SEQ ID NOs. O: Reference sample incubated at -80°C. I: Sample incubated at 60°C. Mw: Molecular weight marker with kDa values ​​indicated on the left. [Figure 4] LabChip analysis of the stability of designed ankyrin repeat domains with binding specificity for serum albumin. Proteins #2, #3, #4, #5, and #6 (consisting of SEQ ID NOS: 2-6, respectively, and all containing SEQ ID NOS: 1 at their N-termini) were produced as described in Example 2 and subsequently incubated at 60°C for 1 week at pH 6, pH 7.4, and pH 8.5 as described in Example 3, followed by storage stability evaluation using LabChip as described in Example 5. LabChip data are displayed in three groups for three different pH values. Molecular weight marker levels (in kDa) are indicated to the left and right of each group. [Figure 5a]Size-exclusion chromatography 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, and further comprising SEQ ID NO: 1 at the N-terminus) were produced as described in Example 2 and subsequently incubated at 60°C (-80°C for the reference sample) at pH 7.4, pH 6, and pH 5 for 1 week as described in Example 3, followed by storage stability evaluation using size-exclusion chromatography analysis as described in Example 6. An overlay of the chromatograms obtained for each protein at 60°C and -80°C is shown for pH 7.4 in Figure 5a. The void volume elutes at 2.18 minutes, and the total volume is 5.28 minutes. OD: optical density at 280 nm (referenced to optical density at 360 nm) [mAu], t: time [min]. [Figure 5b] Size-exclusion chromatography 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, and further comprising SEQ ID NO: 1 at the N-terminus) were produced as described in Example 2 and subsequently incubated at 60°C (-80°C for the reference sample) at pH 7.4, pH 6, and pH 5 for 1 week as described in Example 3, followed by storage stability evaluation using size-exclusion chromatography analysis as described in Example 6. An overlay of the chromatograms obtained for each protein at 60°C and -80°C is shown for pH 6 in Figure 5b. The void volume elutes at 2.18 min, and the total volume is 5.28 min. OD: optical density at 280 nm (referenced to optical density at 360 nm) [mAu], t: time [min]. [Figure 5c]Size-exclusion chromatography 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, and further comprising SEQ ID NO: 1 at the N-terminus) were produced as described in Example 2 and subsequently incubated at 60°C (-80°C for the reference sample) at pH 7.4, pH 6, and pH 5 for 1 week as described in Example 3, followed by storage stability evaluation using size-exclusion chromatography analysis as described in Example 6. An overlay of the chromatograms obtained for each protein at 60°C and -80°C is shown for pH 5 in Figure 5c. The void volume elutes at 2.18 min, and the total volume is 5.28 min. OD: optical density at 280 nm (referenced to optical density at 360 nm) [mAu], t: time [min]. [Figure 6a] Pharmacokinetic profile in mice of designed ankyrin repeat domains with binding specificity for serum albumin given intravenously at 1 mg / kg. Proteins #2, #3, and #4 (consisting of SEQ ID NOs: 2-4, respectively, all further containing SEQ ID NO: 1 at the N-terminus, symbols shown in the figure) were produced as described in Example 2, and their pharmacokinetic profiles in mice were determined as described in Example 9. Mean protein concentrations with standard deviations from three mice or two monkeys, respectively, are shown. C: concentration [nM], t: time [h]. [Figure 6b] Pharmacokinetic profiles in cynomolgus monkeys of designed ankyrin repeat domains with binding specificity for serum albumin given intravenously at 1 mg / kg. Proteins #2, #3, and #4 (consisting of SEQ ID NOS: 2-4, respectively, all further containing SEQ ID NOS: 1 at the N-terminus, and symbols shown in the figure) were produced as described in Example 2, and pharmacokinetic profiles in mice and cynomolgus monkeys were determined as described in Example 10. Mean protein concentrations, including standard deviations, from two monkeys are shown. C: concentration [nM], t: time [hours]. [Figure 7a]Pharmacokinetic profiles in mice of recombinant binding proteins containing engineered ankyrin repeat domains with binding specificity for serum albumin given intravenously at 1 mg / kg. Proteins #7, #9, and #11 (consisting of SEQ ID NOS: 7-12, respectively, all further containing SEQ ID NOS: 1 at their N-termini, and symbols shown in the figure) were produced as described in Example 2, and their pharmacokinetic profiles in mice were determined as described in Example 11. Mean protein concentrations with standard deviations from three mice are shown. For ease of comparison, proteins #7, #9, and #11 are displayed graphically. C: concentration [nM], t: time [h]. [Figure 7b] Pharmacokinetic profile in mice of recombinant binding proteins containing engineered ankyrin repeat domains with binding specificity for serum albumin given intravenously at 1 mg / kg. Proteins #8, #10, and #12 (consisting of SEQ ID NOS: 7-12, respectively, all further containing SEQ ID NOS: 1 at their N-termini, and symbols shown in the figure) were produced as described in Example 2, and their pharmacokinetic profiles in mice were determined as described in Example 11. Mean protein concentrations with standard deviations from three mice are shown. For ease of comparison, proteins #8, #10, and #12 are displayed graphically. C: concentration [nM], t: time [h]. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention 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 invention also provides the use of such recombinant binding proteins or pharmaceutical compositions in the treatment of disease.

[0014] Thus, in one aspect, the invention 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 wherein 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.

[0015] 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 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.

[0016] 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, 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. 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.

[0017] In one embodiment, 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. 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% amino acid sequence identity, preferably at least 90%, more preferably at least 93%, even more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity, to the amino acid sequence of SEQ ID NO: 3 or 4, wherein 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.

[0018] In one embodiment, the designed ankyrin repeat domain does not have a serine (S) at a 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% amino acid sequence identity, preferably at least 90%, more preferably at least 93%, even more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity, to the amino acid sequence of SEQ ID NO: 3 or 4, wherein the designed ankyrin repeat domain does not have a serine (S) at a position corresponding to position 77 of SEQ ID NO: 3 or 4.

[0019] In one embodiment, 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 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% amino acid sequence identity, preferably at least 90%, more preferably at least 93%, even more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, 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 wherein 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.

[0020] In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence KDFAGKTPLHLAAX1X2G (SEQ ID NO: 13), where X1 represents an amino acid residue selected from the group consisting of A, D and I, and X2 represents an amino acid residue selected from the group consisting of 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% amino acid sequence identity, preferably at least 90%, more preferably at least 93%, even more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity, to the amino acid sequence of SEQ ID NO: 3 or 4.

[0021] In one embodiment, at least one of X1 and X2 is aspartic acid (D). In one embodiment, at least one of X1 and X2 is alanine (A). In one embodiment, at least one of X1 and X2 is 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% amino acid sequence identity, preferably at least 90%, more preferably at least 93%, even more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity, to the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, at least one of X1 and X2 is aspartic acid (D) and the designed ankyrin repeat domain differs from SEQ ID NO: 3 or 4 by at most 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid.

[0022] In one embodiment, the designed ankyrin repeat domain comprises the amino acid sequence KDFAGKTPLHLAAX1X2G (SEQ ID NO: 13), where X1 and X2 are each independently an amino acid residue selected from the group consisting of A and D, and preferably X1 and X2 are 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% amino acid sequence identity, preferably at least 90%, more preferably at least 93%, even more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity, to the amino acid sequence of SEQ ID NO: 3 or 4.

[0023] 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.

[0024] 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% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4, preferably at least 90%, more preferably at least 93%, even more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity.

[0025] In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-6, or preferably selected from the group consisting of SEQ ID NOs: 3 and 4, wherein up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of SEQ ID NOs: 3-6, preferably SEQ ID NOs: 3 and 4, have been substituted with other amino acids. Such amino acid substitutions are preferably such that they do not significantly affect the function of the designed ankyrin repeat domain, preferably its specific binding to serum albumin. Such amino acid substitutions may include, for example, amino acid substitutions in designed ankyrin repeat domains, such as those for capping repeat domains, as described in WO 2012 / 069655. When substituting an amino acid, any other amino acid can be considered as the substituting amino acid. Preferably, such other amino acid is selected from the group consisting of A, D, E, F, H, I, K, L, N, P, Q, R, S, T, V, W, or Y. Preferably, such other amino acids are not C, G, or P. In some embodiments, the substitution is at the K position of a designed ankyrin repeat domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6, or preferably selected from the group consisting of SEQ ID NOs: 3 and 4. D The K value for binding to human serum albumin compared to D The substitutions do not change the amino acid sequence by more than 1000-fold, more than 100-fold, or more than 10-fold. In certain embodiments, the substitutions are conservative substitutions according to Table X. In certain embodiments, the substitutions are made outside the structural core residues of the ankyrin repeat domain, for example, within the beta loop connecting the alpha-helix. In certain embodiments, the substitutions are made within the structural core residues of the ankyrin repeat domain.

[0026] [Table 1]

[0027] In one embodiment, the designed ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 3, and (2) SEQ ID NO: 3, wherein up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of SEQ ID NO: 3 at any position selected from positions 1 to 76 and 79 to 124 of SEQ ID NO: 3 is replaced by another amino acid.

[0028] In one embodiment, the present invention relates to a designed ankyrin repeat domain with binding specificity for serum albumin, wherein the 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 up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of SEQ ID NO: 4 at any position selected from positions 1 to 76 and 79 to 124 of SEQ ID NO: 4 is replaced by another amino acid.

[0029] In all of the above designed ankyrin repeat domains of the invention, the penultimate position may be "A" or "L" and / or the last position may be "A" or "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, 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 optionally wherein A at the penultimate position is substituted with L and / or A at the last position is substituted with 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, 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 optionally, A at the penultimate position is substituted with L and / or A at the last position is substituted with 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, 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 optionally, A at the penultimate position is substituted with L and / or A at the last position is substituted with N.

[0030] In addition, all of the above-described designed ankyrin repeat domains of the invention 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, 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 (ii) further comprises G, S, or GS at its N-terminus. In exemplary embodiments, the designed ankyrin repeat domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 3 or 4, 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 further comprises G, S, or GS at its N-terminus. In another exemplary embodiment, the designed ankyrin repeat domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 4, 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 further comprises G, S, or GS at its N-terminus.In another exemplary embodiment, the designed ankyrin repeat domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 4, 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 optionally wherein A at the penultimate position is substituted with L and / or A at the last position is substituted with N.

[0031] In one embodiment, the designed ankyrin repeat domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0032] In one embodiment, the designed ankyrin repeat domain has a dissociation constant (K D ) binds to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin with a dissociation constant (K) of 90 nM or less. D ) binds to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin with a dissociation constant (K) of 80 nM or less. D ) binds to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin with a dissociation constant (K) of 70 nM or less. D ) binds to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin with a dissociation constant (K) of 60 nM or less. D) binds to human serum albumin in PBS. In one embodiment, the designed ankyrin repeat domain binds to human serum albumin with a dissociation constant (K) of 50 nM or less. D ) and binds to human serum albumin in PBS.

[0033] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO:2.

[0034] In one embodiment, the designed ankyrin repeat domain exhibits fewer resolved 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 resolved 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 at pH 5.0 and 60°C for 1 week.

[0035] In one embodiment, the designed ankyrin repeat domain exhibits fewer high 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 high 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 at pH 5.0 and 60°C for 1 week.

[0036] In one embodiment, the designed ankyrin repeat domain exhibits fewer high 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 high 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 at pH 5.0 and 60°C for 1 week.

[0037] In one embodiment, the designed ankyrin repeat domain (i) exhibits fewer high molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and / or (ii) exhibits fewer high 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 high molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, where SDS-PAGE is performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week, and / or (ii) exhibits fewer high molecular weight peaks in size-exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, where SDS-PAGE is performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week.

[0038] In one embodiment, the designed ankyrin repeat domain exhibits at least 10% fewer high molecular weight bands in 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 high 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 at 60°C at pH 6.0 or 7.4.

[0039] In one embodiment the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is (i) exhibiting fewer resolved bands on SDS-PAGE compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, preferably wherein the SDS-PAGE is performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week; (ii) exhibiting fewer high molecular weight bands on SDS-PAGE compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, preferably wherein the SDS-PAGE is performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week; (iii) exhibiting fewer high molecular weight peaks in size exclusion chromatography compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, preferably wherein the size exclusion chromatography is performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week; and (iv) exhibiting at least 10% fewer high molecular weight bands in LabChip analysis compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, preferably exhibiting at least 10% fewer high molecular weight bands in LabChip analysis performed after incubation at 100 micromolar at 60°C at pH 6.0 or 7.4; is selected from the group consisting of:

[0040] In one embodiment the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is (i) exhibiting fewer resolved 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 at pH 5.0 and 60°C for 1 week; (ii) exhibiting fewer high 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 at pH 5.0 and 60°C for 1 week; (iii) exhibiting fewer high 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 at pH 5.0 and 60°C for 1 week; and (iv) exhibiting at least 10% fewer high 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 at 60°C at pH 6.0 or 7.4; is selected from the group consisting of:

[0041] In one embodiment, the designed ankyrin repeat domain with binding specificity for serum albumin has an EC 50 In one embodiment, the designed ankyrin repeat domain with binding specificity for serum albumin binds to human serum albumin in PBST-C with an EC of less than 20 nM. 50 In one embodiment, the designed ankyrin repeat domain with binding specificity for serum albumin binds to cynomolgus monkey serum albumin in PBST-C at an EC of less than 30 nM. 50In one embodiment, the designed ankyrin repeat domain with binding specificity for serum albumin binds to mouse serum albumin in PBST-C with an EC 50 It binds to human serum albumin, cynomolgus monkey serum albumin, and mouse serum albumin in PBST-C at 77°C.

[0042] In one embodiment, the terminal half-life in mice of the designed ankyrin repeat domain is at least 70%, preferably 75%, 80%, 85%, 90%, 95%, 96%, and most preferably 97% of the terminal half-life in mice of the designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the terminal half-life in mice of the designed ankyrin repeat domain differs by less than 30%, preferably 25%, 20%, 15%, 10%, 5%, 4%, and most preferably 3%, from the terminal half-life in mice of the designed ankyrin repeat domain consisting of SEQ ID NO: 2. Preferably, the terminal half-life in mice is determined by administering the designed ankyrin repeat domain at a dose of 1 mg / kg by intravenous injection into the tail vein of Balb / c mice.

[0043] In one embodiment, the terminal half-life of the designed ankyrin repeat domain in cynomolgus monkeys is at least 70%, preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, and most preferably 92% of the terminal half-life in cynomolgus monkeys of the designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the terminal half-life of the designed ankyrin repeat domain in cynomolgus monkeys differs by less than 30%, preferably 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, and most preferably 8%, from the terminal half-life in cynomolgus monkeys of the designed ankyrin repeat domain consisting of SEQ ID NO: 2. Preferably, the terminal half-life in cynomolgus monkeys is determined by administering the designed ankyrin repeat domain at a dose of 1 mg / kg via a 30-minute intravenous injection.

[0044] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is typically and preferably a lower occurrence of high molecular weight products in SDS-PAGE analysis of the designed ankyrin repeat domain upon incubation at 60° C. for 1 week. Preferably, SDS-PAGE is performed after incubation at 100 micromolar at pH 5.0 and 60° C. for 1 week.

[0045] The term "high molecular weight product" in SDS-PAGE analysis typically and preferably refers to a band on a gel that migrates at a higher molecular weight than the expected molecular weight. In one embodiment, high molecular weight product in SDS-PAGE analysis refers to a band on a gel that migrates at a molecular weight greater than 21.5 kDa. In Figure 3, protein #2 incubated at pH 5, 6, or 7.4 for 1 week at 60°C exhibits such a high molecular weight product in SDS-PAGE that migrates at a higher apparent molecular weight than the expected band migrating at approximately 14 kDa. In one embodiment, fewer high molecular weight products in SDS-PAGE analysis refers to a reduction in high molecular weight products of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and most preferably 50% upon incubation at 60°C for 1 week.

[0046] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is typically and preferably a lower occurrence of low molecular weight products in SDS-PAGE analysis of the designed ankyrin repeat domain upon incubation at 60° C. for 1 week. Preferably, SDS-PAGE is performed after incubation at 100 micromolar at pH 5.0 and 60° C. for 1 week.

[0047] The term "low molecular weight product" in SDS-PAGE analysis typically and preferably refers to a band on the gel that migrates at a lower molecular weight than the expected molecular weight. In Figure 3, protein #2 incubated at 60°C for 1 week at pH 5, 6, or 7.4 shows such a low molecular weight product in SDS-PAGE that migrates at a lower apparent molecular weight than the expected band migrating at approximately 14 kDa. In one embodiment, fewer low molecular weight products in SDS-PAGE analysis refers to a reduction in low molecular weight products of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and most preferably 50% upon incubation at 60°C for 1 week.

[0048] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 typically and preferably results in fewer high molecular weight products in LabChip analysis when the designed ankyrin repeat domain is incubated at 60°C for 1 week. In one embodiment, high molecular weight products in LabChip analysis refer to bands migrating at a higher molecular weight than the expected molecular weight on the chromatogram. In one embodiment, high molecular weight products in LabChip analysis refer to bands on the chromatogram migrating at a molecular weight higher than 30 kDa. In Figure 4, protein #2 incubated at 60°C for 1 week at pH 6 or 7.4 shows such high molecular weight products in LabChip analysis migrating at a higher apparent molecular weight than the expected band migrating at approximately 15 kDa to 20 kDa. In one embodiment, fewer high molecular weight products in LabChip analysis refers to a reduction in high molecular weight products of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and most preferably 50% when incubated at 60°C for 1 week.

[0049] In one embodiment, the designed ankyrin repeat domain has improved stability compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2, and the improved stability compared to the designed ankyrin repeat domain consisting of SEQ ID NO: 2 typically and preferably results in fewer low molecular weight products in LabChip analysis when the designed ankyrin repeat domain is incubated at 60°C for 1 week. In one embodiment, low molecular weight products in LabChip analysis refer to bands that migrate at a lower molecular weight than the expected molecular weight on the chromatogram. In one embodiment, low molecular weight products in LabChip analysis refer to bands on the chromatogram that appear at a molecular weight of approximately 7 kDa. In Figure 4, protein #2 incubated at 60°C for 1 week at pH 6 shows such low molecular weight products in LabChip analysis that migrate at a lower apparent molecular weight than the expected band migrating at approximately 15 kDa to 20 kDa. In one embodiment, fewer low molecular weight products in LabChip analysis refers to a reduction in low molecular weight products of at least 5%, 10%, 15%, 20%, and most preferably 25% when incubated at 60°C for 1 week.

[0050] In one embodiment, the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is typically and preferably the occurrence of a lower amount of high molecular weight product in size exclusion chromatography upon incubation at pH 5 and 60°C for 1 week. In one embodiment, the term "high molecular weight product" in size exclusion chromatography typically and preferably refers to a product that elutes before the expected molecular weight product. In Figure 5C, protein #2 incubated at pH 5 for 1 week at 60°C shows a higher molecular weight product eluting at 4.1 minutes in size exclusion chromatography, while the expected molecular weight product elutes approximately 4.6 minutes later. In one embodiment, a lower amount of high molecular weight product in size exclusion chromatography refers to a percentage decrease in the total area under the curve of at least 5%, 10%, 15%, 20%, and most preferably 25% of the total area under the curve upon incubation at 60°C for 1 week.

[0051] In one embodiment, incubation refers to incubation at pH 8.5, 7.4, 6, or 5. In one embodiment, incubation refers to incubation at pH 8.5. In one embodiment, the term "incubation" refers to incubation at pH 7.4. In one embodiment, incubation refers to incubation at pH 6. In one embodiment, incubation refers to incubation at pH 5. In one embodiment, incubation at pH 8.5 refers to incubation in a phosphate / citrate / borate buffer. In one embodiment, incubation at pH 7.4 refers to incubation in PBS. In one embodiment, incubation at pH 6 refers to incubation in a phosphate / citrate buffer pH 6. In one embodiment, incubation at pH 5 refers to incubation in a phosphate / citrate buffer pH 5.

[0052] In another aspect, the present invention further provides a recombinant binding protein comprising at least one designed ankyrin repeat domain of the invention with binding specificity for serum albumin. The preferred embodiments and features described above and herein for the designed ankyrin repeat domain of the invention apply to any and all aspects of the invention, including recombinant binding proteins of the invention comprising at least one designed ankyrin repeat domain of the invention with binding specificity for serum albumin.

[0053] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the invention. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the invention. In one embodiment, the recombinant binding protein comprises three of the designed ankyrin repeat domains of the invention.

[0054] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 80% sequence identity with 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 invention, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 80% sequence identity with 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 invention, 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 the position corresponding to position 77 of SEQ ID NO: 3 or 4 and / or does not have a glutamic acid (E) at the position corresponding to position 78 of SEQ ID NO: 3 or 4.

[0055] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 90% sequence identity with 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 invention, wherein each of the one or two ankyrin repeat domains independently comprises an amino acid sequence having at least 90% sequence identity with 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 invention, wherein each of the two ankyrin repeat domains independently comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4 is at least 93%, more preferably at least 95%, and again more preferably at least 98%.

[0056] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention, 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 invention, 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 invention, 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.

[0057] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention, wherein each of the one or two ankyrin repeat domains independently comprises 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 invention, wherein each of the one or two ankyrin repeat domains independently comprises 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 invention, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4.

[0058] In one embodiment, the recombinant binding protein comprises one or two of the engineered ankyrin repeat domains with binding specificity for serum albumin of the present invention, and the recombinant binding protein has a dissociation constant (K D In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the engineered ankyrin repeat domains with binding specificity for serum albumin of the present invention, and the recombinant binding protein binds to human serum albumin in PBS with a dissociation constant (K) of 100 nM or less. D In one embodiment, the recombinant binding protein comprises exactly two of the engineered ankyrin repeat domains with binding specificity for serum albumin of the present invention, and the recombinant binding protein binds to human serum albumin in PBS with a dissociation constant (K) of 100 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 90 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 80 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 70 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 60 nM or less.D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 50 nM or less. D ) binds to human serum albumin in PBS.

[0059] In one embodiment, a recombinant binding protein comprises at least two designed ankyrin repeat domains with binding specificity for serum albumin, each of which has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2. SEQ ID NOs: 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 with binding specificity for serum albumin, each of which has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2. SEQ ID NOs: 9 to 12 are examples of such recombinant binding proteins.

[0060] In one embodiment, the recombinant binding protein comprises two, preferably exactly two, designed ankyrin repeat domains with binding specificity for serum albumin, each of which consists of SEQ ID NO: 3. SEQ ID NOs: 9-10 are examples of such recombinant binding proteins. In one embodiment, the recombinant binding protein comprises two, preferably exactly two, designed ankyrin repeat domains with binding specificity for serum albumin, each of which consists of SEQ ID NO: 4. SEQ ID NOs: 11-12 are examples of such recombinant binding proteins.

[0061] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention, 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: 3-6, and the recombinant binding protein has a dissociation constant (K D In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the invention, 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: 3-6, and the recombinant binding protein binds to human serum albumin in PBS with a dissociation constant (K D In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the invention, each of the two ankyrin repeat domains independently comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-6, and the recombinant binding protein binds to human serum albumin in PBS with a dissociation constant (K D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 90 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 80 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 70 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 60 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 50 nM or less. D ) binds to human serum albumin in PBS.

[0062] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the 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 the recombinant binding protein has a dissociation constant (K D In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the 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 the recombinant binding protein binds to human serum albumin in PBS with a dissociation constant (K D In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the invention, each of the two ankyrin repeat domains independently comprising the amino acid sequence of SEQ ID NO: 3 or 4, and the recombinant binding protein binds to human serum albumin in PBS with a dissociation constant (K D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 90 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 80 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 70 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 60 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 50 nM or less. D ) binds to human serum albumin in PBS.

[0063] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the 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 the recombinant binding protein has a dissociation constant (KD ) binds to human serum albumin in PBS, and 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 the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is demonstrated by: (i) showing fewer resolved bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week; and (ii) showing fewer high molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week. (iii) exhibiting fewer high 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 at pH 5.0 and 60°C for 1 week, and (iv) exhibiting at least 10% fewer high 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 at pH 6.0 or 7.4 and 60°C. In one embodiment, the recombinant binding protein comprises exactly one or exactly two of the designed ankyrin repeat domains of the 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 the recombinant binding protein has a dissociation constant (K D) binds to human serum albumin in PBS, and 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 the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 is demonstrated by: (i) showing fewer resolved bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week; and (ii) showing fewer high molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week. (iii) exhibiting fewer high 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 at pH 5.0 and 60°C for one week, and (iv) exhibiting at least 10% fewer high 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 at pH 6.0 or 7.4 and 60°C. In one embodiment, the recombinant binding protein comprises exactly two of the designed ankyrin repeat domains of the invention, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3 or 4, and the recombinant binding protein has a dissociation constant (K D) binds to human serum albumin in PBS, and each of the two ankyrin repeat domains has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2 being characterized by: (i) exhibiting fewer resolved bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after one week of incubation at 100 micromolar at pH 5.0 and 60°C; and (ii) exhibiting fewer high molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after one week of incubation at 100 micromolar at pH 5.0 and 60°C. (iii) exhibiting fewer high 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 at pH 5.0 and 60°C for 1 week, and (iv) exhibiting at least 10% fewer high 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 at pH 6.0 or 7.4 and 60°C. In one embodiment, the recombinant binding protein has a dissociation constant (K D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 80 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 70 nM or less. D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 60 nM or less.D ) in PBS. In one embodiment, the recombinant binding protein binds to human serum albumin with a dissociation constant (K) of 50 nM or less. D ) binds to human serum albumin in PBS.

[0064] 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 with binding specificity for serum albumin is replaced with a designed ankyrin repeat domain consisting of SEQ ID NO: 2. SEQ ID NOs: 9 to 12 are examples of such recombinant binding proteins.

[0065] Recombinant binding proteins consisting of SEQ ID NO: 9 or 11 are examples of such recombinant binding proteins that exhibit improved stability compared to a recombinant binding protein consisting of SEQ ID NO: 7, each comprising two designed ankyrin repeat domains with binding specificity for serum albumin, consisting of SEQ ID NO: 2. Recombinant binding proteins consisting of SEQ ID NO: 10 or 12 are examples of such recombinant binding proteins that exhibit improved stability compared to a recombinant binding protein consisting of SEQ ID NO: 8, each comprising two designed ankyrin repeat domains with binding specificity for serum albumin, consisting of SEQ ID NO: 2.

[0066] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention, 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 invention, 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 invention, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 3.

[0067] In one embodiment, the recombinant binding protein comprises one or two of the designed ankyrin repeat domains of the invention, 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 invention, 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 invention, wherein each of the two ankyrin repeat domains independently comprises the amino acid sequence of SEQ ID NO: 4.

[0068] In one embodiment, the recombinant binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12. 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.

[0069] In one embodiment, the terminal half-life in mouse of the recombinant binding protein is at least 70%, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, and most preferably at least 90% of the terminal half-life in mouse of a recombinant binding protein having the same amino acid sequence except that each designed ankyrin repeat domain with binding specificity for serum albumin is replaced with a designed ankyrin repeat domain consisting of SEQ ID NO: 2. In one embodiment, the terminal half-life in mouse of the recombinant binding protein differs by less than 30%, preferably by less than 25%, 20%, 19%, 18%, 17%, 16%, 15%, and most preferably by less than 10% from the terminal half-life in mouse of a recombinant binding protein having the same amino acid sequence except that each designed ankyrin repeat domain with binding specificity for serum albumin is replaced with a designed ankyrin repeat domain consisting of SEQ ID NO: 2. Preferably, the terminal half-life in mice is determined by applying the recombinant binding protein at a dose of 1 mg / kg by intravenous injection into the tail vein of Balb / c mice.

[0070] In one embodiment, the recombinant binding protein has a terminal half-life in cynomolgus monkeys that is at least 70%, preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, less than 91%, and most preferably 92% of the terminal half-life in cynomolgus monkeys of a recombinant binding protein having an identical amino acid sequence, except that each designed ankyrin repeat domain with 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 in cynomolgus monkeys of the recombinant binding protein differs by less than 30%, preferably by less than 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, and most preferably by less than 8% from the terminal half-life in cynomolgus monkeys of a recombinant binding protein having the same amino acid sequence, except that each designed ankyrin repeat domain with binding specificity for serum albumin is replaced with a designed ankyrin repeat domain consisting of SEQ ID NO: 2. Preferably, the terminal half-life in cynomolgus monkeys is determined by administering the recombinant binding protein at a dose of 1 mg / kg as a 30-minute intravenous injection.

[0071] In another aspect, the present invention relates to a nucleic acid encoding the amino acid sequence of an ankyrin repeat domain or recombinant binding protein of the present invention. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of an ankyrin repeat domain of the present invention. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of a recombinant binding protein of the present invention. In one embodiment, the present invention 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 invention 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 invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 4. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 9. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 10. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 11. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 12. Furthermore, the present invention relates to a vector comprising any of the nucleic acids.

[0072] In a further aspect, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein and / or designed ankyrin repeat domain of the invention or a nucleic acid encoding a recombinant binding protein and / or designed ankyrin repeat domain of the invention, and optionally a pharmaceutically acceptable carrier and / or diluent. Pharmaceutically acceptable carriers and / or diluents are known to those skilled in the art and are described in more detail below. Furthermore, diagnostic compositions are provided which comprise one or more of the recombinant binding proteins and / or designed ankyrin repeat domains mentioned herein.

[0073] Pharmaceutical compositions can be prepared by combining binding proteins as described above and other methods, such as those described in Remington's Pharmaceutical Sciences 16th The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient, or stabilizer, such as those described in "Therapeutic Agents for the Treatment of Acetaminophen," Osol, A. Ed., 1980. Suitable carriers, excipients, or stabilizers known to those skilled 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. Other 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 acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition may also be a combination drug containing an additional active ingredient, such as an anti-cancer agent or an anti-angiogenic agent.

[0074] Formulations to be used for in vivo administration must be sterile, or must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0075] The pharmaceutical composition can be administered by any suitable method within the knowledge of those skilled in the art. The preferred administration route is parenteral. In parenteral administration, the agent of the present invention is formulated into a unit dose injection form such as a solution, suspension or emulsion together with a pharmaceutically acceptable excipient as defined above. The dose and administration method vary depending on the individual to be treated and the specific disease.

[0076] Furthermore, any of the above pharmaceutical compositions are contemplated for the treatment of diseases or disorders. The present invention further provides a method of treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition or a recombinant binding protein or an engineered ankyrin repeat domain of the present invention to a patient in need thereof.

[0077] Further provided is a method for treating a pathological condition in a mammal, including a human, comprising administering to a patient in need thereof an effective amount of the above-described pharmaceutical composition.

[0078] The present disclosure provides methods for treating cancer, comprising administering a therapeutically effective amount of a recombinant binding protein or pharmaceutical composition described herein to a subject in need thereof. In certain embodiments, the subject is a human. In certain embodiments, the cancer is a solid tumor. SEQ ID NOs: 9-12 are examples of recombinant binding proteins useful in such methods for treating cancer.

[0079] In some embodiments, the cancer is brain cancer, bladder cancer, breast cancer, clear cell renal carcinoma, cervical cancer, colon and rectal cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, lip and oral cancer, liver cancer, lung squamous cell carcinoma, melanoma, mesothelioma, non-small cell lung cancer (NSCLC), non-melanoma skin cancer, ovarian cancer, oral cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, small cell lung cancer (SCLC), squamous cell carcinoma of the head and neck (SCCHN), triple-negative breast cancer, or thyroid cancer.

[0080] The present invention is not limited to the specific embodiments described in the examples, other sources may be used and processed according to the outline below.

[0081] This specification makes reference to a number of amino acid sequences in the amino acid sequence listing herein entitled "P5754_Sequence_Protocol.txt," and the amino acid sequences in the Sequence Protocol are incorporated herein by reference.

[0082] definition Unless otherwise defined herein, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0083] In the context of the present invention, the term "protein" refers to a molecule comprising a polypeptide, wherein at least a portion of the polypeptide has or can assume a defined three-dimensional order by forming secondary, tertiary, and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. When a protein comprises two or more polypeptide chains, the individual polypeptide chains can be linked by non-covalent or covalent bonds, for example, by disulfide bonds between two polypeptides. Portions of a protein that have or can assume a defined three-dimensional order by individually forming secondary and / or tertiary structures are referred to as "protein domains." Such protein domains are well known to those skilled in the art.

[0084] The term "recombinant," as used in reference to recombinant protein, recombinant polypeptide, etc., means that the protein or polypeptide is produced by use of recombinant DNA techniques well known to those of skill in the art. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or into DNA that allows for in vitro expression. For example, when such a recombinant bacterial expression plasmid is inserted into suitable bacteria (e.g., E. coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The polypeptide or protein produced accordingly is referred to as a recombinant polypeptide or recombinant protein.

[0085] In the context of the present invention, the term "binding protein" refers to a protein containing a binding domain. A binding protein may also contain two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein. The binding protein of the present invention contains an ankyrin repeat domain that has binding specificity for serum albumin.

[0086] Furthermore, any such binding protein may comprise additional polypeptides known to those skilled in the art (e.g., polypeptide tags, peptide linkers, fusions to other proteinaceous domains with binding specificity, cytokines, hormones, or antagonists, etc.), or chemical modifications (e.g., coupling to polyethylene glycol, toxins (e.g., DM1 from immunogens), small molecules, antibiotics, etc.).

[0087] The term "binding domain" means a protein domain that exhibits binding specificity for a target. Preferably, said binding domain is a recombinant binding domain.

[0088] The term "target" refers to an individual molecule, such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any portion of such an individual molecule, or a complex of two or more such molecules, or a whole cell or tissue sample, or any non-natural compound. Preferably, the target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein containing a chemical modification, for example, modified by naturally occurring or non-natural phosphorylation, acetylation, or methylation. For example, in the context of the present invention, serum albumin is the target of the disclosed serum albumin-specific binding domains and proteins.

[0089] In the context of the present invention, the term "polypeptide" relates to a molecule consisting of a chain of multiple, i.e., two or more, amino acids linked by peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked by peptide bonds. The term "polypeptide" also encompasses multiple chains of amino acids linked by cysteine ​​S-S bridges. Polypeptides are well known to those skilled in the art.

[0090] Patent application WO2002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Pluckthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of the function of repeat proteins and the function, technology and uses of repeat domains. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, a repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, a repeat protein may comprise further non-repeat protein domains, polypeptide tags and / or peptide linkers. A repeat domain may be a binding domain.

[0091] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as a structural unit, the repeat modules having structural and sequence homology. Preferably, the repeat domain further comprises an N-terminal and / or C-terminal capping module. For clarity, the capping module may be a repeat module. Such repeat domains, repeat modules, and capping modules, sequence motifs, and structural and sequence homology are well known to those skilled in the art from examples such as 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). It is further known to those skilled in the art that such repeat domains are distinct from proteins containing repeated amino acid sequences, all of which can form individual domains (e.g., the FN3 domain of fibronectin).

[0092] The term "designed" as used in designed repeat proteins, designed repeat domains etc. refers to the property that such repeat proteins and repeat domains, respectively, are artificial and do not occur in nature. The binding proteins of the invention are designed repeat proteins, which comprise at least one designed ankyrin repeat domain.

[0093] The term "target interaction residues" refers to amino acid residues of a repeat module that contribute to direct interaction with the target.

[0094] The term "framework residues" refers to amino acid residues of a repeat module that contribute to the folding topology, i.e., contribute to the folding of the repeat module or contribute to interactions with neighboring modules. Such contributions may be interactions with other residues within the repeat module, or influence on the polypeptide backbone structure found in α-helices or β-sheets, or participation in amino acid stretches that form linear polypeptides or loops.

[0095] Such framework and target interaction residues can be identified by analysis of structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known related structural information known to practitioners in structural biology and / or bioinformatics.

[0096] The term "repeat module" refers to the repeated amino acid sequence and structural unit of a designed repeat domain, originally derived from the repeat units of naturally occurring repeat proteins. Each repeat module comprised in a repeat domain is 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 may comprise a "repeat sequence motif" obtained from a repeat domain selected on a target, as described in Example 1, and deduced from homologous repeat modules with the same target specificity.

[0097] Thus, the term "ankyrin repeat module" refers to a repeat module that originally derived from the repeat units of naturally occurring ankyrin repeat proteins. Ankyrin repeat proteins are well known to those skilled in the art.

[0098] A repeat module may comprise positions with amino acid residues that are not randomized in the library for the purpose of selecting target-specific repeat domains ("non-randomized positions") and positions with amino acid residues that are randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). Non-randomized positions include framework residues. Randomized positions include target-interaction residues. "Randomized" means, for example, that two or more amino acids are allowed at an amino acid position of a repeat module, and that any of the 20 common naturally occurring amino acids are allowed, or that amino acids other than cysteine, or most of the 20 naturally occurring amino acids are allowed, such as 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 ankyrin repeat modules of the invention.

[0099] The term "repeat sequence motif" refers to an amino acid sequence deduced from one or more repeat modules. Preferably, the repeat modules are derived from repeat domains with binding specificity for the same target. Such a repeat sequence motif comprises framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of a repeat module. Similarly, the target interaction residue positions correspond to the target interaction residue positions of a repeat module. A repeat sequence motif comprises non-randomized positions and randomized positions.

[0100] The term "repeat unit" refers to an amino acid sequence that contains one or more naturally occurring protein sequence motifs, where the "repeat unit" is found in multiple copies and exhibits a defined folding topology common to all motifs that determines protein folding. 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.

[0101] The terms "having binding specificity for a target," "specifically binds to a target," "binds with high specificity to a target," "specific for a target," or "target specificity," etc., mean that a binding protein or binding domain binds to a target in PBS with a lower dissociation constant (i.e., binds with higher affinity) than it binds to an unrelated protein, such as E. coli maltose binding protein (MBP). Preferably, the dissociation constant ("Kd") for the target in PBS is at least 10 times lower than the corresponding dissociation constant for MBP. 2 times, more preferably at least 10 3 times, more preferably at least 10 4 times, or more preferably at least 10 5 fold lower. Methods for measuring the dissociation constant of protein-protein interactions, 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 of a particular protein-protein interaction may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, the measurement of Kd values ​​is preferably performed using a standardized solution of the protein and a standardized buffer solution such as PBS. An exemplary and preferred determination of the dissociation constant (Kd) of a recombinant binding protein of the present invention having binding specificity for serum albumin by surface plasmon resonance (SPR) analysis is described in Example 7.

[0102] The term "about" means + / - 20% of the stated value, for example, "about 50" shall mean 40-60.

[0103] The term "PBS" refers to an aqueous phosphate buffer solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, with a pH of 7.4. This is exemplified in Example 3. The term "phosphate / citrate buffer pH 6" refers to an aqueous solution of 375 mM NaHPO * The term "phosphate / citrate buffer pH 5" refers to an aqueous buffer containing 30 mM citric acid and 30 mM NaH2PO4 and having a pH of 4.75. This is exemplified in Example 3. The term "phosphate / citrate / borate buffer" refers to an aqueous buffer containing 30 mM citric acid, 30 mM NaH2PO4, 30 mM boric acid and having a pH of 8.5. This is exemplified in Example 3.

[0104] The term "mouse serum albumin" refers to UniProt accession number P07724, the term "cynomolgus monkey serum albumin" (i.e., macaca fascicularis) refers to UniProt accession number A2V9Z4, and the term "human serum albumin" refers to UniProt accession number P02768. [Example]

[0105] Proteins used in the examples: Protein #2 (SEQ ID NO: 2, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #3 (SEQ ID NO: 3, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #4 (SEQ ID NO: 4, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #5 (SEQ ID NO: 5, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #6 (SEQ ID NO: 6, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #7 (SEQ ID NO: 7, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #8 (SEQ ID NO: 8, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #9 (SEQ ID NO: 9, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #10 (SEQ ID NO: 10, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #11 (SEQ ID NO: 11, with a His tag (SEQ ID NO: 1) fused to its N-terminus), Protein #12 (SEQ ID NO: 12, with a His tag (SEQ ID NO: 1) fused to its N-terminus).

[0106] Unless otherwise stated, experiments were performed according to methods well known to those skilled in the art. Experimental conditions for some examples are further described in WO 2012 / 069654 and WO 2016 / 156596.

[0107] Example 1: Construction of designed ankyrin repeat domains with binding specificity for serum albumin and improved stability Unexpectedly, we observed that a protein consisting of an amino acid sequence corresponding to SEQ ID NO:2 (originally described in WO 2016156596) did not appear to be stable upon incubation at elevated temperatures, despite the variant having improved stability compared to a previously designed ankyrin repeat domain with binding specificity for serum albumin (WO 2012 / 069654) (see Examples 2-6). Therefore, an object of the present invention is 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. Through a highly iterative process involving several changes in amino acids at multiple positions (e.g., comparable to the alanine scanning process well known to those skilled in the art), we characterized the resulting protein variants in vitro and in vivo, ultimately resulting in four variants exhibiting improved stability properties. Contrary to expectations, all of these variants contain the amino acid Asp at positions 77 and / or 78, an amino acid known to be at the origin of polypeptide chain degradation. Quite surprisingly, many of these variants contain an Asp-Gly sequence motif that is known to be particularly susceptible to polypeptide chain degradation, and thus this process surprisingly yielded unexpected variants.

[0108] DNA encoding each of the designed ankyrin repeat domains consisting of SEQ ID NOs: 2-6 was cloned into a pQE (QIAgen, Germany)-based expression vector to provide an N-terminal His tag to facilitate simple protein purification, as described below. The production, characterization, and use of these specifically selected sequences are described in the Examples below.

[0109] Example 2: Protein expression and purification Proteins consisting of SEQ ID NOS: 2-6 and further having a His-tag SEQ ID NOS: 1 fused to their N-terminus, and proteins consisting of SEQ ID NOS: 7-12 and further having a His-tag SEQ ID NOS: 1 fused to their N-terminus, were produced in Escherichia coli, purified to homogeneity, and stored in PBS buffer. For clarity, proteins #2-#6 are designed ankyrin repeat domains with binding specificity for serum albumin, and proteins #7-#12 are recombinant binding proteins containing designed ankyrin repeat domains with binding specificity for serum albumin. Proteins #7 and #8 contain SEQ ID NOS: 2 twice. Proteins #9 and #10 contain SEQ ID NOS: 3 twice. Proteins #11 and #12 contain SEQ ID NOS: 4 twice. Protein #7 is known to those skilled in the art from SEQ ID NOS: 134 in WO 2016156596, and protein #8 is known to those skilled in the art from SEQ ID NOS: 21 in WO 2018054971. Thus, compared to protein #7, which is a recombinant binding protein containing a designed ankyrin repeat domain with binding specificity for serum albumin, proteins #9 and #11 are recombinant binding proteins containing a designed ankyrin repeat domain with (i) binding specificity for serum albumin and (ii) improved storage stability. Similarly, compared to protein #8, which is a recombinant binding protein containing a designed ankyrin repeat domain with binding specificity for serum albumin, proteins #10 and #12 are recombinant binding proteins containing a designed ankyrin repeat domain with (i) binding specificity for serum albumin and (ii) improved storage stability. The proteins expressed and purified as described in this paragraph were used in the experiments in Examples 3 to 12.

[0110] Alternatively, proteins consisting of SEQ ID NOS: 2-12 and further comprising the amino acid GS at their N-terminus were produced in E. coli, purified to homogeneity, and stored in PBS buffer. When the amino acid GS is at the N-terminus, the Met residue encoded by the expression vector is efficiently cleaved from the expressed polypeptide in the cytoplasm of E. coli because the small Gly residue follows the initiator Met. Proteins consisting of SEQ ID NOS: 2-12 and further comprising the amino acid GS at their N-terminus showed comparable results in Examples 3-12 to proteins consisting of SEQ ID NOS: 2-12 and further comprising the His-tag SEQ ID NOS: 1 fused to their N-terminus.

[0111] Example 3: Storage Stability Incubation The protein of Example 2 was tested for storage stability at various pH values ​​at 60° C. for one week (7 days) at a protein concentration of 100 micromolar. The buffers used were PBS (pH 7.4, 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl), or phosphate / citrate (pH 5.7, 375 mM NaHPO * The buffer solutions were: phosphate / citrate (pH 4.75, 30 mM citric acid and 30 mM NaHPO, pH adjusted using 1 M citrate monohydrate); phosphate / citrate (pH 4.75, 30 mM citric acid and 30 mM NaHPO, pH adjusted using sodium hydroxide); or phosphate / citrate / borate (pH 8.5, 30 mM citric acid, 30 mM NaHPO, 30 mM boric acid). When mixed with the protein, the resulting pH values ​​were pH 7.4, pH 6, pH 5, or pH 8.5, respectively. In parallel with the 60°C incubation, an aliquot of the protein was incubated at -80°C for 1 week (7 days) as a control.

[0112] Stability during incubation at 60°C at different pH levels is industrially relevant for the production of designed ankyrin repeat domains or recombinant binding proteins, as the production process may include one or more process steps in which the polypeptide is exposed to such conditions. Alternatively, one skilled in the art may determine storage stability by accelerated storage stability measurements, which include an incubation step at elevated temperatures.

[0113] Example 4: SDS-PAGE of storage stability analysis samples Samples of the proteins from Example 3 (10 micrograms of protein per lane) were analyzed on NuPAGE 4-12% bis-tris-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Thermo Fisher Scientific) and stained with Instant Blue Stain (Sigma-Aldrich). The results are shown in Figure 3. All proteins show a major band at the expected size of approximately 14.4 kDa. Some proteins show additional bands at lower molecular weights (low molecular weight products) and / or bands at higher molecular weights (high molecular weight products). Proteins #3 and #4 show lower amounts of low molecular weight products at pH 7.4, pH 6, and pH 5 compared to protein #2. Judging from SDS-PAGE, the reduction in low molecular weight products is at least 10% at pH 7.4, pH 6, and / or pH 5. Similarly, judging from SDS-PAGE, the reduction in the amount of low molecular weight products is at least 50% at pH 7.4, pH 6, and / or pH 5. Similarly, protein #2 exhibits at least 50% or more lower molecular weight product or bands at pH 7.4, pH 6, and / or pH 5. Surprisingly, protein #2 exhibits higher molecular weight bands upon incubation at 60°C for 1 week. Proteins #3 and #4 appear to exhibit no higher molecular weight bands, thus demonstrating improved stability. Judging from SDS-PAGE, the reduction in higher molecular weight product bands when comparing protein #2 to proteins #3 and #4 is at least 50% at pH 7.4, at least 50% at pH 6, and at least 50% at pH 5. Similarly, protein #2 exhibits an increase in high molecular weight bands and / or products that reaches at least 10% at pH 7.4, at least 20% at pH 6, and at least 50% at pH 5.

[0114] Example 5: LabChip analysis of storage stability analysis samples Samples of the proteins in Example 3 were analyzed using a LabChip GXII instrument according to the manufacturer (Perkin Elmer). Briefly, samples were mixed with a denaturing solution and denatured at 70°C for 10 minutes, followed by analysis on an HT Protein Express Chip. The instrument's software was used to analyze the experiments. The results are shown in Figure 4. Proteins #3 and #4 show lower amounts of degradation or low molecular weight products compared to protein #2 at pH 7.4, pH 6, and pH 5. The LabChip data indicates that the reduction in degradation or low molecular weight product bands is at least 10% at pH 7.4, pH 6, and / or pH 5. Protein #2 exhibits a high molecular weight band upon incubation at 60°C for 1 week, while proteins #3 and #4 do not exhibit a high molecular weight band. The LabChip data indicates that the reduction in high molecular weight product bands is at least 10% at pH 7.4 and at least 50% at pH 6 (see Table 1). In this example, for proteins #2 to #4, a band is considered a high molecular weight band in LabChip analysis if it is larger than 25 kDa. In this example, for proteins #2 to #4, a band is considered a low molecular weight band in LabChip analysis if it is smaller than 10 kDa. Assuming high purity of the protein preparation, such low molecular weight bands can be considered degradation product bands.

[0115] [Table 2]

[0116] Example 6: Size Exclusion Chromatography Analysis of Storage Stability Analytical Samples The samples from Example 3 were analyzed on an Agilent 1200 HPLC system using a GE Superdex 200 150 / 5 column at a flow rate of 0.5 ml / min in PBS. For each protein, 0.1 ml of each of the 100 micromolar -80°C control sample and the 60°C incubation sample was analyzed. Size-exclusion chromatograms (optical density at 280 nm subtracted by optical density at 360 nm) are shown in Figures 5a (pH 7.4), 5b (pH 6), and 5c (pH 5). Proteins #3 and #4 show nearly overlapping chromatograms with the -80°C control sample and the 60°C incubation sample at pH 7.4, pH 6, and pH 5. Protein #2 shows nearly overlapping chromatograms with the -80°C control sample and the 60°C incubation sample at pH 7.4 and pH 6. In contrast, protein #2 showed a clear increase in higher apparent molecular weight species in the 60°C incubation sample compared to the -80°C control sample at pH 5. The higher apparent molecular weight species of protein #2 at pH 5 in the 60°C incubation sample corresponded to 25% of the total area under the curve, while the protein #2-80°C control sample at pH 5 showed no higher apparent molecular weight species. The higher apparent molecular weight species in this example is the species eluting in a peak at approximately 4.1 minutes. The expected apparent molecular weight species is the species eluting in a peak at approximately 4.6 minutes. Proteins #3 and #4 did not show any higher apparent molecular weight species in the 60°C incubation sample at pH 5.

[0117] Example 7: Affinity measurements The affinity of the protein of Example 2 to human serum albumin was determined by SPR measurements using the ProteOn system (BioRad) in PBS according to standard procedures known to those skilled in the art. The determined affinities are listed in Table 2. All designed ankyrin repeat domains with binding specificity to serum albumin have dissociation constants K<100 nM. D and the dissociation constants are in the same range.

[0118] [Table 3]

[0119] Example 8: Serum albumin species cross-reactivity Samples of the proteins from Example 2 were subjected to ELISA serum albumin cross-reactivity analysis as described in WO2016156596. 100 μl of 20 nM serum albumin in PBS per well was immobilized on Maxisorp plates (Nunc, Denmark) overnight at 4° C. After washing five times with 300 μl of PBST (PBS supplemented with 0.1% Tween 20), the wells were blocked with 300 μl of 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 five washes as described above, 100 μl / well or 50 μl / well of protein #2, #3, or #4 (at concentrations ranging from 100 nM to 0.01 pM for each protein) in PBST-C was added and incubated for 2 hours at room temperature with shaking at 450 rpm. After five washes as described above, protein binding was detected using rabbit anti-designed ankyrin repeat domain monoclonal antibody in 100 μl of PBST-C for 1 hour at room temperature with shaking at 450 rpm. After five washes as described above, bound anti-designed ankyrin repeat domain antibody was detected using goat anti-rabbit IgG-HRP conjugate in PBST-C for 1 hour at room temperature with shaking at 450 rpm. After five washes as described above, ELISA was performed using BM soluble blue POD substrate (Roche, Switzerland) diluted 1:4 in 100 μl of water. The reaction was stopped after 5 minutes using 100 μl of 1 M H2SO4. The OD (OD450 nm - OD620 nm) was then recorded. 50 Values ​​were determined using GraphPad Prism (Table 3). Analysis shows that human, cynomolgus monkey, and mouse serum albumin bind with high affinity. Measured EC for human serum albumin binding 50Values ​​are less than 1 nM for proteins #2, #3, and #4. Measured EC for cynomolgus monkey serum albumin binding 50 Values ​​are below 20 nM for proteins #2, #3, and #4. Measured EC for mouse serum albumin binding 50 The values ​​are below 30 nM for proteins #2, #3, and #4.

[0120] [Table 4]

[0121] Example 9: Pharmacokinetic profile in mice of designed ankyrin repeat domains with binding specificity for serum albumin Pharmacokinetic analysis was performed in female Balb / c mice using proteins #2, #3, and #4 produced as described in Example 2. Proteins were administered at 1 mg / kg via intravenous injection into the tail vein. Six mice, divided into two groups of three mice each, were used for each protein. For all proteins, blood was collected from mice in one group at 5 minutes, 24 hours, 72 hours, 168 hours, and 360 hours after injection, and from mice in the other group at 4 hours, 48 ​​hours, 144 hours, and 168 hours after injection. The blood samples were left at room temperature, centrifuged, and serum was generated using procedures well known to those skilled in the art. The blood samples were then stored at -80°C until analysis. Serum concentrations of proteins #2, #3, and #4 were measured using a standard curve by sandwich ELISA using a rabbit monoclonal anti-DARPin antibody as the capture reagent and a high RGS-His antibody-HRP conjugate as the detection reagent. Monoclonal anti-DARPin antibodies were generated using conventional rabbit immunization and hybridoma generation techniques well known to those skilled in the art. The binding of the monoclonal antibodies to proteins #2–#16 was verified prior to concentration determination experiments. Briefly, 10 nM goat anti-rabbit antibody (Thermo Scientific) in 100 μl of PBS per well was immobilized on Maxisorp plates (Nunc, Denmark) overnight at 4°C. After washing five times with 300 μl of PBST (PBS supplemented with 0.1% Tween 20), the wells were blocked with 300 μl of 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 five washes as described above, 100 μl / well of 5 nM rabbit anti-DARPin antibody in PBST-C was added for 1 h at room temperature with shaking at 450 rpm. After five washes as described above, different dilutions of serum samples or reference standards diluted in PBST-C were added for 2 h at room temperature with shaking at 450 rpm. After five washes as described above, 50 μl of 100 ng / ml 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 washing five times as described above, ELISA was performed using 50 μl of TMB substrate. The reaction was stopped after 5 minutes using 100 μl of 1 M H2SO4. OD (OD450 nm - OD620 nm) was then recorded. Pharmacokinetic parameters were measured using standard software such as Phoenix WinNonLin (Certara, Princeton, USA) or GraphPadPrism (GraphPad Software, La Jolla, USA) and standard analyses such as non-compartmental analysis (all well known to those skilled in the art). The resulting pharmacokinetic profile is shown in Figure 6a. The pharmacokinetic parameters derived from the measurements, including area under the curve, clearance, volume of distribution, and half-life, are listed in Table 4.

[0122] [Table 5]

[0123] Example 10: Pharmacokinetic profile in cynomolgus monkeys of designed ankyrin repeat domains with binding specificity for serum albumin Pharmacokinetic analysis was performed in two male cynomolgus monkeys at 1 mg / kg of each protein administered via a 30-minute intravenous infusion. For all proteins, blood was collected from all animals at 5 minutes, 6 hours, 24 hours, 72 hours, 120 hours, 168 hours, 336 hours, 408 hours, 504 hours, and 672 hours after injection. Blood samples were left at room temperature, centrifuged, and serum was generated using procedures well known to those skilled in the art. The serum concentrations of proteins #2, #3, and #4 were measured by sandwich ELISA as described in Example 9. Pharmacokinetic parameters were measured using standard software such as Phoenix WinNonLin (Certara, Princeton, USA) or GraphPadPrism (GraphPad Software, La Jolla, USA) and standard analyses such as noncompartmental analysis (all well known to those skilled in the art). The resulting pharmacokinetic profiles are shown in Figure 6b. The pharmacokinetic parameters derived from the measurements, area under the curve, clearance, volume of distribution, and half-life, are listed in Table 5.

[0124] [Table 6]

[0125] Example 11: Generation and characterization of recombinant binding proteins using designed ankyrin repeat domains with binding specificity for serum albumin and improved stability Recombinant binding proteins containing designed ankyrin repeat domains with binding specificity for serum albumin and improved storage stability were produced by recombinant DNA technology. SEQ ID NOs: 7-12 are examples of such recombinant binding proteins. Proteins #7, #8, #9, #10, #11, and #12 (consisting of SEQ ID NOs: 7-12 and having a His-tag SEQ ID NO: 1 fused to their N-termini; see Example 2) were produced as described in Example 3. Similarly, proteins consisting of SEQ ID NOs: 7-12, each further carrying the amino acid MGS at their N-terminus (the N-terminal methionine is efficiently cleaved from the expressed polypeptide in the cytoplasm of E. coli because a small Gly residue follows the initiating Met), can be produced in E. coli and purified using conventional methods.

[0126] The recombinant binding proteins are evaluated for improved stability according to Examples 3 to 6. Recombinant binding proteins consisting of SEQ ID NOs: 9 and 11 exhibit greater stability than the recombinant binding protein consisting of SEQ ID NO: 7. Similarly, recombinant binding proteins consisting of SEQ ID NOs: 10 and 12 exhibit greater stability than the recombinant binding protein consisting of SEQ ID NO: 8.

[0127] Example 12: Pharmacokinetic profile in mice of a recombinant binding protein containing a designed ankyrin repeat domain with binding specificity for serum albumin Pharmacokinetic analyses were performed in mice using proteins #7, #9, and #11, all produced as described in Example 2, and proteins #8, #10, and #12. Studies were performed essentially as described in Example 9, using three mice per protein and drawing blood at 5 minutes, 4 hours, 48 ​​hours, and 96 hours post-injection. Concentration measurements and pharmacokinetic parameter concentration measurements were performed as described in Example 9. For comparison of proteins #7, #9, and #11, pharmacokinetic traces are shown in Figure 7a, and the pharmacokinetic parameters area under the curve, clearance, volume of distribution, and half-life derived from measurements of all proteins are listed in Table 6. For comparison of proteins #8, #10, and #12, pharmacokinetic traces are shown in Figure 7b, and the pharmacokinetic parameters area under the curve, clearance, volume of distribution, and half-life derived from measurements of all proteins are listed in Table 7.

[0128] [Table 7]

[0129] [Table 8]

Claims

1. 1. A designed ankyrin repeat domain having binding specificity for serum albumin, said designed ankyrin repeat domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4, said designed ankyrin repeat domain having (i) an aspartic acid (D) at a position corresponding to position 77 of SEQ ID NO: 3 and an alanine (A) at a position corresponding to position 78 of SEQ ID NO: 3; or (ii) an alanine (A) at a position corresponding to position 77 of SEQ ID NO: 4 and an aspartic acid (D) at a position corresponding to position 78 of SEQ ID NO:

4.

2. 2. The designed ankyrin repeat domain of claim 1, further comprising G, S, or GS at its N-terminus.

3. A designed ankyrin repeat domain according to claim 1 or 2, wherein A at the penultimate position of the C-terminus is replaced by L and / or A at the last position of the C-terminus is replaced by N.

4. The designed ankyrin repeat domain has the amino acid sequence KDFAGKTPLHLAAX １ X ２ G (SEQ ID NO: 13), wherein X １ and X ２ are each independently an amino acid residue selected from the group consisting of A and D, １ and X ２ The designed ankyrin repeat domain of any one of claims 1 to 3, wherein:

5. The designed ankyrin repeat domain according to any one of claims 1 to 4, wherein the designed ankyrin repeat domain comprises SEQ ID NO: 3 or SEQ ID NO:

4.

6. The designed ankyrin repeat domain has a dissociation constant (K Ｄ 6. The designed ankyrin repeat domain of any one of claims 1 to 5, which binds to human serum albumin in PBS at 1000kJ / s.

7. The designed ankyrin repeat domain according to any one of claims 1 to 6, wherein the designed ankyrin repeat domain has improved stability compared to a designed ankyrin repeat domain consisting of SEQ ID NO:

2.

8. 8. The designed ankyrin repeat domain of any one of claims 1 to 7, wherein the designed ankyrin repeat domain shows fewer resolved bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after incubation at 100 micromolar at 60°C, pH 5.0 for 1 week.

9. 9. The designed ankyrin repeat domain of any one of claims 1 to 8, wherein the designed ankyrin repeat domain (i) exhibits fewer high molecular weight bands on SDS-PAGE compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the SDS-PAGE being performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week, and / or (ii) exhibits fewer high molecular weight peaks on size-exclusion chromatography compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, the size-exclusion chromatography being performed after incubation at 100 micromolar at pH 5.0 and 60°C for 1 week.

10. 10. The designed ankyrin repeat domain of any one of claims 1 to 9, wherein the designed ankyrin repeat domain shows at least 10% fewer high molecular weight bands in LabChip analysis compared to a designed ankyrin repeat domain consisting of SEQ ID NO: 2, and the LabChip analysis is performed after incubation at 100 micromolar at 60°C at pH 6.0 or 7.

4.

11. A recombinant binding protein comprising one or two designed ankyrin repeat domains according to any one of claims 1 to 10.

12. 12. The recombinant binding protein of claim 11, wherein the recombinant binding protein has improved stability compared to a recombinant binding protein having an identical amino acid sequence except that each of the designed ankyrin repeat domains with binding specificity for serum albumin has been replaced with a designed ankyrin repeat domain consisting of SEQ ID NO:

2.

13. A nucleic acid encoding a designed ankyrin repeat domain or a recombinant binding protein according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising a designed ankyrin repeat domain or a recombinant binding protein according to any one of claims 1 to 12, or a nucleic acid according to claim 13, and a pharmaceutically acceptable carrier and / or diluent.

Images